CLIN. CHEM. 35/7, 1497-1 503 (1989)
Radioimmunoassay of f3-Microseminoprotein, a Prostatic-Secreted Protein Present in Sera
of Both Men and Women
P.-A. Abrahamsson,1 C. Andersson,2 1. BjOrk, P. Fernlund,2’4H. LIIJa,2A. Murne,2 and H. Welber3
We describe a simple radioimmunoassay of /3-microsemino-
protein, one of the three most abundant secretory proteins of
the prostate gland. The detection limit of the assay is 1 j.zg/L,
and its precision, expressed as the total coefficient of variation, is <10% for values between 10 and 150 jzg/L. Using this
assay, we found that /3-microseminoprotein immunoreactivity
was present in sera from both sexes at about the same
concentration. The protein detected had the same molecular
size on gel chromatography as the protein isolated from
seminal plasma, and dilution curves for the sera paralleled
that for the pure protein. The findings suggest that )3microseminoprotein is present in serum of healthy subjects of
both sexes and that it originates in tissue other than the
prostate gland. The range of the serum concentration was
0-10.6 g/L (median 4.1) for 51 healthy adult women and
1.1-14.7 g/L (median 6.2) for 35 healthy adult men not
older than 40 years. In males with prostatic cancer the
concentration in serum was highly variable and often greatly
increased. The concentration of /3-microseminoprotein was
correlated with that of creatinine in serum, suggesting that
the protein is eliminated-at least partly-from the circulation
by glomerular filtration. Little of the protein was present in the
urine of women. In urine from men the concentration was
high and variable, probably because of local contribution
from the prostate gland to the urethral urine.
AddItIonal Keyphrases: fertility
prostatic cancer
reference
seminal plasma
.
gonadotropins
values
renal
19Anhibin
function
urine
The three most abundant proteins in the secretions
produced by the human prostate gland are prostatic acid
phosphatase, prostate-specific antigen, and /3-microseminoprotein (1). All three proteins
are present in seminal
plasma at about 1 gIL each (1-4). /3-Microseminoprotein,
which has an apparent molecular mass of 14 to 16 kDa on
sodium dodecyl sulfate (SDS) electrophoresis (1, 5, 6), has
been called /3-inhibin (7), f3-microseminoprotein
(/3-MSP)
(8), and prostatic secretory protein of 94 amino acids
(PSP94) (5) by various authors.
The amino acid sequence of /3-microseminoprotein
has
been determined, both by protein sequencing methodology
(6, 8, 9) and by complementary DNA sequencing (10). A
94-amino acid peptide containing
10 cysteine residues and
five pairs of adjacent basic amino acids, and lacking carbohydrate, /3-microseminoprotein
shows little structural
similarity to other proteins.
The biological function of J3-microseminoprotein is still
unknown. This protein was originally isolated from semen
as a factor inhibiting the pituitary
release of follitropin
(follicle-stimulating
hormone) (5), but this activity has now
been questioned (11, 12) and several authors have argued
against the name inhibin (13).
The potential of (3-microseminoprotein
as a marker for
prostatic disease (14-16) and also a general interest in its
biological function have prompted us to develop an assay
for this protein. Here we describe the properties of our
radioimmunoassay of /3-microseminoprotein and report the
results of measurements of this protein in serum and urine
of adult humans of both sexes.
Materials and Methods
Materials
The purification of /3-microseminoprotein from seminal
plasma has been described previously (1). The product
obtained was homogeneous,
with an apparent molecular
mass of 16 kDa as judged by SDS/polyacrylamide
gel
electrophoresis,
and its amino acid composition and NH2terminal
sequence were in agreement
data
The concentration
of /3-microseminoprotein
in the stock
solution was determined
by quantitative
amino acid anal-
ysis.
Antiserum
to /3-microseminoprotein
was obtained by
White rabbits with the purified
protein. The antigen (0.1 mg per animal) was injected
subcutaneously on 10 different sites on the back of each
animal. Freund’s complete adjuvant was used the first time
and Freund’s
incomplete adjuvant for repeated injections,
done at monthly intervals. Antibody titers were checked
regularly,
and the antiserum
from the best-responding
animal was used. The antiserum was monospecific when
tested by immunoelectrophoresis
against normal seminal
plasma. The single precipitation
line obtained correimmunizing
New Zealand
sponded to the position for purified
/3-microseminoprotein,
but no precipitation line was obtained with blood serum.
Anti-rabbit IgG antiserum was raised in goats by standard methods (17). A sheep anti-rabbit immunoglobulin
coupled
to a solid phase (Pharmacia
Decanting
Suspension
Ill) was obtained from Pharmacia, Uppsala, Sweden. Purified mouse IgG was kindly provided by Dr. Anders Grubb,
Department
of Clinical Chemistry,
Malm#{246}
General Hospital, Malm#{246},
Sweden. Bovine serum albumin (Cohn Frac-
tion V) was obtained from Armour Pharmaceutical Co.
Ltd., Eastbourne, U.K.; human prolactmn from Hormone
Laboratory,
Departments of’ Urology, 2 Clinical Chemistry, and Surgery,
University of Lund, General Hospital, Malmo, Sweden.
4Address correspondence to this author at: Department of Clinical Chemistry, Malmo General Hospital, S-214 01 Malmo, Sweden.
Received February 28, 1989; accepted April 27, 1989.
with published
(1, 5-10). We dissolved the purified protein in 50 mmol/L
Ti-is HC1 buffer, pH 7.4, containing NaCl, 0.15 molJL (Ths
saline), and stored this stock solution in aliquots at -20 #{176}C.
Aker
Hospital,
Oslo,
Norway;
polyethylene
glycol (mean Mr 6000) from Kebo AB, Stockholm, Sweden;
Chloramine T from Merck AG, Darmstadt,
F.R.G.; Na1251
(17.4 kCilg) from NEN Products, Boston, MA; and porcine
insulin from Novo AS, Copenhagen, Denmark. Blue Dextran 2000, molecular-size
marker proteins for electrophoresis, Sephadex G-25 superfine, and Superose 12 HR1O/30
CLINICAL CHEMISTRY, Vol. 35, No. 7, 1989
1497
were from Pharmacia. Water was purified by an
Elga Spectrum R.O.1 system (The Elga Group, Buckinghamahire, U.K.). All other chemicals used were of analytical grade.
by excluding the antiserum (i.e., using only serum
from nonimmunized rabbits). After the mixture was incubated at room temperature
for 3 h, 100 L of a pooled
specimen of serum from normal humans, all with /3-microseminoprotein
<5 g/L, was added to the standard tubes
and to sample tubes with diluted serum or non-serum
Procedures
fluids. We added 100 j.L of assay buffer to the sample tubes
General methods. SDSlpolyacrylamide
slab-gel electrowith undiluted serum. Immediately
afterwards, we precipphoresis was performed in 100 to 150 g/L gradient gels (18)
itated immunoglobulins
by adding 500 L of a goat antiand immunoelectrophoresis
was carried out with standard
rabbit IgG antiserum diluted 40-fold in assay buffer conmethodology (19). For amino acid analysis we used a
taining 50 g of polyethylene glycol per liter. Alternatively,
Beckman 6300 automatic amino acid analyzer after hydrolwe precipitated the bound tracer with 0.5 mL of a suspenysis of an aliquot of /3-microseminoprotein stock solution in
sion of solid-phase coupled sheep anti-rabbit immunogloban evacuated borosilicate glass tube for 24 h at 110#{176}C
in 6
ulin (Pharmacia Decanting Suspension Ill). After letting
mol/L HC1, with norleucine added as internal standard.
the samples stand for 1 h at room temperature, we centriGel chromatography.
Gel chromatography,
as a test of
ftiged them at 2000 x g and 4 #{176}C
for 15 mm. The supernates
the homogeneity of labeled f3-microseminoprotein
and for
were discarded and the radioactivity in the sediments was
estimating the molecular size of immunoreacting
material,
measured with 70% efficiency in an LKB-Wallac 1277
was performed at room temperature (approximately
20#{176}C)
GammaMaster 10-channel gamma counter (LKB, Bromon a 10 x 310 mm column of Superose 12 HR 10/30. The
ma, Sweden).
elution buffer-sodium
barbital, 75 mmolJL, pH 8.6, conSamples were run in duplicate and results were read
taining 1.0 g of bovine serum albumin and 0.05 g of sodium
from a curve fitted to the counts obtained with the stanaside per liter-was
delivered with a 2150 HPLC-pump
dards (each point in triplicate) by using a smoothed, cubic
(LKB, Bromma, Sweden) at a flow rate of 0.7 mL/min. The
spline function with the standard errors of the means as
column was calibrated with Blue Dextran 2000 (average
weighting factors (20).
molecular mass 2.0 MDa), mouse IgG (150 kDa), bovine
Assay evaluation.
The sensitivity
of the assay was deserum albumin (68 kDa), human prolactin (23 kDa), and
fined as the reading on the standard curve at the mean
porcine insulin (6.0 kDa). The effluent was collected in
counts for the zero-concentration standard minus three
15-drop fractions
and the volume of each fraction was
standard deviations of the counts.
determined by weighing. We monitored the fractions by
We determined
the intra-assay coefficient of variation
radioactivity
measurements when testing tracer homoge(CV) at three different levels by analyzing three different
neity and by radioimmunoassay
in the size-estimation
samples 20 times in one assay run, 10 in the beginning of
experiments.
the series and 10 at the end. The intra-assay CV was also
Radioiodination.
The iodination
was performed at room
estimated
from the duplicate values of samples (covering
temperature. To a 11 x 55 mm glass tube with a small
the range of values between 5 and 175 pg/L) in 20 consecmagnetic stirrer we added 5 L of Na’251 (18.5 MBq) and
utive assays.
50 L of purified f3-microseminoprotein
(35 tg) in TrisWe estimated the total CV (intra-assay + interassay)
by
saline. The reaction was started by adding 10 pL of Chloassaying three different serum pools in 10 consecutive
ramine T trthydrate
dissolved in water (2.5 g/L) and
assays. Aliquots of each sample were kept at -20 #{176}C,
so
stopped after 30s by adding 10 L of sodium metabisulfite
that repeated thawing was avoided. Each result was the
(5 gIL) and 30 L of 75 mmol/L sodium barbital buffer, pH
mean of duplicate values. During the experimental period
8.6, containing
50 mg of sodium aside per liter. The
two different tracer preparations
were used.
iodinated protein was separated from unreacted iodide by
We tested analytical
recovery by adding three different
chromatography
at 20#{176}C
on a 1.0 x 10 cm column of
amounts of purified /3-microseminoprotein
from the stanSephadex G-25 (superfine), equilibrated and eluted with 75
dard stock solution to one serum sample from a woman
mmoIJL sodium barbital buffer, pH 8.6, containing 50mg of
with a low concentration of /3-microseminoprotein (5.5
sodium aside per liter. The flow rate was 0.7 mL/min and
ig/L) and two samples from men, one with a low concenthe eluate was collected in tubes containing 0.5 mL of the
tration (9.0 u.gfL) and the other with a high (73.8 pg/L)
elution buffer supplemented with 20 g of bovine serum
concentration. The amounts added were calculated to inalbumin per liter.
crease the concentration with 47.8, 87.5, and 175 j.g/L,
Radioimmunoassay
procedure. The assay buffer was 75
respectively.
mmol/L sodium barbital,
pH 8.6, containing 2.5 g each of
Assay of serum antibodies to /3-microseminoprotein.
This
disodium EDTA and bovine serum albumin per liter. We
was performed by an immunosorbent
radioassay according
used antiserum
diluted 4000-fold in the assay buffer. Seto Ericsson et al. (21), /3-microseminoprotein
labeled as
rum from nonimmunized
rabbits was included to give a
described above being used as tracer.
concentration of rabbit serum in the working antiserum
Serum creatinine. This was determined
in the routine
solution corresponding
to a 160-fold dilution.
clinical chemistry
laboratory with an alkaline picric acid
Standards
were prepared from the stock solution of
reagent method (22). The normal reference intervals were
purified /3-microseminoprotein by dilution in assay buffer
60-100 .tmoIJL and 80-115 mol/L
for adult women and
to give concentrations ranging from 0 to 350 pg/L. These
men, respectively.
were stored in aliquots at -70 #{176}C.
Gonadotropins.
Follitropin and lutropmn (luteinizing horWe performed
the assay by mixing, in 11 x 55 mm
mone) were measured by radioimmunoassay (23).
polystyrene
tubes, 100 L of standard or sample, 200 .zL of
diluted antiserum,
and 200 L of ‘251-labeled /3-microsemSubjects and Collection of Samples
inoprotein
(diluted in assay buffer to give about 40 000
The reference population consisted of 51 healthy women
counts/mm per 200 ML). Nonspecific binding was detercolumn
1498 CLINICAL CHEMISTRY, Vol. 35, No. 7, 1989
mined
(ages 20-56 years, 10 older than 40) and 43 healthy men
(ages 22-59 years, eight older than 40). Samples were also
obtained from 28 patients (ages 56 to 98 years, median 74)
with prostatic cancer. All patients had tumors in stages T3
or T4 of the Tumor, Node, and Metastasis System of staging
prostate cancers, which includes five stages of increasing
severity (TO to T4) (24).
Blood was collected by venipuncture. We obtained serum
by centrifligation
at 2000 x g for 20 mm after allowing the
blood to clot for 60 mm at room temperature or overnight at
4#{176}C.
To obtain plasma, we sampled blood with EDTA as
anticoagulant.
The serum and plasma samples were stored
at -20 #{176}C.
Urine was collected without additives during 24 h from
24 healthy women (ages 20-56 years) and six healthy men
(ages 20-40 years). The volume was measured and an
aliquot was stored at -20 #{176}C
until analyzed.
Seminal plasma from healthy men was obtained as
described previously
(1). It was stored at -20 #{176}C.
Serum samples for correlation of serum creatinine with
/3-microseminoprotein were selected from samples referred
to the clinical chemistry laboratory for routine tests. They
were selected to give a suitable distribution of serum
creatinine values but with no regard to the sex of the
patient, clinical data, or results of other blood tests. There
were 46 samples from females (ages 14-92 years, median
65) and 55 from males (ages 15-97 years, median 74).
Results
3-Microseminoprotein
Ce
0
100
0
80
60
U
Ce
0
40
Ce
I-
V
20
a
0
0
1/2500
1/10000
1/5000
1/400001/160000
1/20000
1/80000
no
antIserum
Antiserum
dilution
Fig. 1. Antiserum binding of 1251-labeled /3-microseminoprotein
Labeled /3-microseminoprotein
(approximately40000 counts/mm,correspondingto 1 ng of labeled antigen) was incubatedwith various dilutions of
specific rabbit antiserum in a total volume of 0.5 mL at room temperature
(approximately
20#{176}C).
Theindicateddilution of the antiserum refersto thefinal
dilution in the incubationmixture,which also containedserumfrom a nonimmunizedrabbit, at a 400-folddilution.After 3 h, immunoglobulin was
precipitated with a second antibody(see Matenalsarid Methods). The radioactivity was measuredin the precipitate obtained after centrifugation (bound
radioactivity),and the resultwas expressed as percentof total added radioactivity
firmer and more adhesive. The ideal approach
would be to prepare the standard in a serum free from
/3-microseminoprotein,
but we found it difficult to find such
sera. Therefore, we adopted a procedure in which 100 L of
pooled serum with a low concentration of /3-microseminoprotein
was added to the standard tubes immediately
before the precipitation step. To make the volumes equal in
standards and samples, the same volume of buffer was
added to the serum sample tubes.
Separating
system. In the assay procedure adopted for
routine use we separated bound and free tracer by polyethylene glycol-reinforced second-antibody precipitation. Separation with solid-phase-coupled
anti-rabbit immunoglobulin (Pharmacia Decanting Suspension Ill) gave identical
results (not shown) but was not used because of the higher
reagent cost.
Assay performance. A typical standard curve is shown in
Figure 2 together with dilution curves for three different
samples. The sensitivity of the assay was 1 p.g/L. Estimates
of the intra-assay CV obtained with three different samples
in a single run were 3.3% (for a sample with a mean
concentration of 10.7 pgfL), 2.3% (sample mean 81.7 tgfL),
and 2.4% (sample mean 159 g/L), respectively. Estimation
from sample duplicates
in 20 different assay runs gave
3.0%. The estimates of the total CV (i.e., intra-assay plus
inter-assay)
obtained for three different pooled sera in 10
consecutive assays were 6.3% (at the mean concentration of
9.8 g/L),
5.8% (at 80.2 g/L),
and 4.3% (at 156 g/L),
respectively. Analytical recovery of added purified /3-microseminoprotein
varied between 94% and 116% in the
nine recovery experiments described under Materials and
Methods. There was no systematic difference in recovery
between the three test sera used for these experiments.
Sampling.
Values did not differ significantly
between
plasma and serum (Student’s paired two-tailed t-test) according to the results with plasma and serum samples
collected simultaneously from 30 subjects.
precipitates
Assay
Labeling of /3-microseminoprotein.
With the labeling procedure used, between 65% and 85% of the radioactivity (five
labeling experiments) was incorporated
into the material
eluting in the void volume of the G-25 column. Analysis of
this material by gel chromatography
on a Superose 12
column, which has a higher resolving power than the G-25
column, showed that more than 95% of the radioactivity
was eluted in a peak corresponding
to the elution position
of pure f3-microseminoprotein
(determined
in a previous
run on the same column). The specific radioactivity of the
labeled /3-microseminoprotein
was 420 GBq/g. The amount
of tracer used in the assay procedure (approximately
40000
counts/mm)
thus corresponded to about 2 ng of labeled
/3-microseminoprotein per tube.
Antiserum.
Incubation of the labeled (3-microseminoprotein with various dilutions of the antiserum, followed by
precipitation of immunoglobulins with second antibody,
showed a suitable dilution of the antiserum in the incubation mixture to be 10 000-fold (Figure 1), i.e., a 4000-fold
dilution in the working antiserum solution. With antiserum in excess-i.e., a dilution <2500-fold-more
than 90%
of the tracer was precipitated (Figure 1). When no antiserum was included (nonspecific binding), only a few percent
of the tracer was precipitated (Figure 1). An equally low
nonspecific binding was obtained when standards or samples (more than 100 representative samples tested) were
included, making correction for nonspecific binding in the
assay procedure unnecessary.
Standard. The standards we used in our procedure were
prepared in buffer containing bovine serum albumin. Initially we found that the precipitation
with second antibody
was more reproducible with serum samples than with
standards. This phenomenon was probably caused by components (e.g., plasma proteins) present in serum samples,
but not in the buffer used for the standards, that made the
CLINICAL CHEMISTRY, Vol. 35, No. 7, 1989
1499
Dilution factor
5.
1/16
1/4
1/1
1/256
1/64
1/8
1/2
1/128
1/32
0
Normal male serum
-r.rr.,J
20
fi
0
-
.
40
60
80
CS
0
60
5
Normal female serum
0
s,--.
0
U
CS
0
V
CS
J
20
40
40
20
-J
I-
V
a
--r
40
20
60
80
Pmstatic cancer
patient serum
C
a
0
0
a
0
5.5 11
22
44
88175350
Standard concentration (j.tgIL)
Fig. 2. Radioimmunoassaystandardcurve (#{149})
for /3-microseminoprotein and dilution curves for serum from a female (0), serum from
a patient with prostatic cancer (#{149}),
and seminalplasma(A)
Sampleswere diluted in assay buffer and subjectedto the procedure with
addition of pooled serum at the end of the incubation as described in the text.
The seminalplasmawas prediluted 5000-fold
J\N
0
20
0
r
60
40
N
80
C
E
a
a)
0
U
E
U
Si
C)
80
Seminal plasma
40
0
C
Stability of /3-microseminoprotein
in stored serum samples. Thirty-six serum samples (five from healthy men, five
from healthy women, and 26 from men with prostatic
disease) that had been assayed six months previously and
kept at -20 #{176}C
in the meantime were re-assayed. The
values obtained on the first occasion ranged from 5 to 74
g/L. The mean difference between the second and the first
value was plus 4.9%, with a standard deviation of 12.9%.
E
E
0
,
20
p
1
80
4,
-
-‘
40
23
60
80
5
4
4,4, 4,4,
Pure
13-microseminoprotein
40
/3-Microseminoprotein in Serum and Urine
/3-Microseminoprotein
was detected not only in sera from
but also in sera from women and in urine. In Figure 2
are shown the dilution curves for the immunoreactivity in
serum
from a woman and serum from a man, and in
seminal plasma, together with the standard curve obtained
with purified /3-microseminoprotein. The curves are all
parallel. The immunoreactivity
in serum was also investigated by gel chromatography, which separates molecules
according to molecular size. The results obtained with
serum from a normal man, a normal woman, and a patient
with prostatic cancer are shown in Figure 3, which, for
comparison, also shows the chromatograms obtained with
seminal plasma and purified /3-microseminoprotein. In all
cases except for the prostatic-cancer patient the immunoreactivity was eluted as a single peak at a position and with
a width indistinguishable from that of pure f3-microseminoprotein. According to the calibration of the column with
molecular-mass markers, the elution position corresponded
to a molecular mass of about 16 kDa. In the patient with
prostatic cancer the main immunoreactivity eluted as in
the other samples but an extra minor immunoreactive
component was seen eluting before the main component
(Figure 3). The molcular mass of the extra component was
estimated to exceed 150 kDa. Possibly the two other serum
samples contained a similar component, but with a concentration close to the detection limit of the assay (Figure 3).
Reference values. In the reference population, sera from
males tended to have a higher concentration
of /3-microseminoprotein
than sera from females, but the differmen
1500 CLINICAL CHEMISTRY, Vol. 35, No. 7, 1989
i[
0
0
20
40
60
80
Fraction number
Fig. 3. Gel chromatography of immunoreactive /3-microseminoprotein in serum samples and seminal plasma
Serum samples, seminal plasma, and purified p-microseminoprotemn
(0.3 mL
each)were chromatographedon a Superose12 column and the effluent was
assayed for /3-microseminoprotemn
with the radioimmunoassayprocedure(with
additionof pooled serum at the end of the incubation). The sera from the
normal malesand females were undiluted, serum from the prostatic-cancer
patient wasdiluted twofold, and the seminal plasma 5000-fold;the concentrationof the solutionof purified p-microseminoproteinwas 187 igIL. The elution
positions for molecular mass references (determined in a separate run) are
indicatedby numbered arrows:BlueDextran2.0 MDa (1), mouse lgG 150kDa
(2), bovine serum albumin 68 kDa (3), human prolactmn23 kDa (4), and
porcine insulin 6.0 kDa (5)
ence was not great (Figure 4). If subjects older than 40
years were excluded, the difference between males and
females was even less pronounced (Figure 4). The range
was 0-10.6 pgfL (median 4.1) for the women and 1.1-14.7
gfL (median 6.2) for the men not older than 40 years.
There was no correlation in the reference population between age and the concentration of (3-microseminoprotein
in serum, either for women alone (r = 0.079), men alone (r
=
0.224), or for all subjects together (r = 0.154).
The concentration of /3-microseminoprotein was measured in urine from 24 healthy women. Values were below
the detection
limit
(1 jig/L) in 17 of the subjects, and the
a)
200
10
C
5)
2
0.
5
E
100
C,
0,
0
I0
0
0
5
10
15
S-13-Microseminoprotein
25
cb
0
#{149}1
0
l
I
200
-
I
--
400
600
800
1000
S-Creatlnlne (tmoI/L)
(.Lg/L)
Fig. 5. Relation between concentrations of f3-microseminoprotein
and creatinine in serum samples from 46 women (0) and 55 men (#{149})
referred to the clinical laboratory for routine clinical chemistry tests
The subjects were selected only with regard to the creatinine values, so as to
Males
(a
(S
20
includea large range of creatinineconcentrations.The equationof the
regressionline: y 0.063x 2.356 (r 0.49, P <0.001)
10
=
+
=
>
V
C
Discussion
5,
.0
E
z
0
0
mrt
5
10
20
15
S-13-Microseminoorotein
25
(g/L1
Fig. 4. Distribution of values for concentration of /3-microseminoprotein in serum of healthy adults (51 women, 43 men)
Shaded bars represent results for men older than 40 years
highest value measured was 7.7 tg/L. In urmnes from men
the concentration
was much higher and variable.
For six
individuals the range was 12.2 to 223 zgfL (median 81.1
gfL).
Prostatw-cancer
patients.
The concentration of /3microseminoprotein in sera from 28 patients with prostatic
cancer varied greatly. The range was 0 to 486 ug/L (median
14.3 /Lg/L). Fourteen of the patients had values exceeding
the upper reference limit and four had values below the
lower limit. Patients with more severe disease (stage T4)
tended to have higher values than patients in stage T3, a
difference that was statistically
significant
(P = 0.003)
according to the Mann-Whitney
U-test.
Antibodies to /3-Microseminoprotein
in Serum
Sera from 10 healthy women, 10 healthy men (all <40
years old), and 28 patients with prostatic cancer were
tested for the presence of circulating antibodies to (3microseminoprotein.
None contained detectable antibodies.
/3-Microseminoprotein and Serum Creatinine
The relation between the serum concentrations of creatmine and /3-microseminoprotein is shown in Figure 5 for 46
women and 55 men. The low positive correlation between
the two variables (r = 0.49) is nevertheless statistically
significant (P <0.001).
/3-Microseminoprotein and Gonadotropins
Follitropin and lutropin were measured in the serum
samples of the reference subjects. There was no significant
correlation between the concentrations of any of the gonadotropins on the one hand and /3-microseminoprotein on the
other, either in women (n = 51) or men (n = 43).
/3-Microseminoprotein
is present in seminal plasma in a
relatively high concentration. According to evidence obmined with histochemical techniques (1, 14,25), northern
blot analysis (10), and observations in patients lacking
seminal vesicles (1) the /3-microseminoprotein in seminal
plasma originates from the prostate gland. Not unexpectedly, therefore, /3-microseminoprotein may appear in serum of men with prostatic disease, as demonstrated with
previously
developed assays for this protein (5, 26).
Our results with the sensitive assay described in this
work show that immunoreactive
/3-microseminoprotein
is
present in serum of both men and women and at about the
same concentration. Does this immunoreactivity
represent
the same protein that has been isolated from seminal
plasma? Our belief that this is so is supported by two of our
findings: First, the immunoreactivity in serum-both from
men and women-displayed
a dilution curve that paralleled that of the assay standard curve, the latter prepared
with (3-microseminoprotein purified from seminal plasma.
Second, fractionation of the serum immunoreactivity according to molecular size showed that almost all of it
represents a homogeneous component with the same molecular size as (3-microseminoprotein. Although only isolation and analysis of the immunoreactive component could
provide an ultimate proof, the evidence strongly suggests
identity of the immunoreactivity present in sera of women
with /3-microseminoprotein.
The nature of the minor immunoreactive component
with a larger molecular size than (3-microseminoprotein
present in the sample from the patient with prostatic
cancer-and perhaps also in sera of healthy subjects-is
unclear. The component could be an aggregated form of
/3-microseminoprotein or perhaps f3-microseminoprotein in
complex with a high-molecular-mass binder in the patient’s
plasma.
The concentration of /3-microseminoprotein in sera of
women, when measured with our assay, is almost the same
as in healthy men not older than 40 years, an age below
which benign prostatic hyperplasia or prostatic cancer is
rare. Although the mean value for men is statistically
higher than for women, the overlap is large, and one may
conclude that, for healthy subjects, the concentration is
essentially the same in both sexes. This finding suggests
that the prostate gland is not an important
source for
CLINICAL CHEMISTRY, Vol. 35, No. 7, 1989 1501
serum
/3-microseminoprotein
in healthy men, and that
blood of men and women have a common source for this
protein.
In men with prostatic cancer the concentration of (3microseminoprotein
in serum
was often markedly in-
creased, similar to what has been observed with other
prostate-specific markers, such as acid phosphatase and
prostate-specific antigen. This increase most probably represents a direct release of /3-microseminoprotein into blood
from the diseased prostate gland, and it may well be that
the high values occasionally seen in subjectively healthy
men older than 40 years also represent contributions from
the prostate gland.
The release of /3-microseminoprotein into the circulation
as a consequence of a disease process affecting the tissue
producing this protein might stimulate the production of
antibodies directed against (3-microseminoprotein. If present, such antibodies would interfere with our assay and
probably cause falsely increased values. Our failure to
detect antibodies in any of our group of patients with
prostatic disease (many with high values for /3-microseminoprotein in their blood) suggests that this does not happen
frequently.
Although it appears to be somewhat larger in SDS
electrophoresis and gel chromatography (16 kDa), (3-microseminoprotein is a small protein of 94 amino acids with
a calculated molecular
mass of about 11 kDa (1,5,6,8-10).
Proteins of this size are usually more or less freely filtered
in the renal glomeruli. We therefore measured /3-microseminoprotein in urine from females and from males as well. In
females the concentration was low, more than half of the
patients having values below the detection limit of our
assay. Men had much higher and more variable concentrations of /3-microseminoprotein in their urine, and occasionally very high values were encountered. The low values in
women suggest that (3-microseminoprotein, if filtered in the
glomeruli, is effectively reabsorbed by the tubule cells and
that the urine finally is almost free of the protein. The high
values seen for urine from men thus probably represent a
local contribution from the prostate gland to the urethral
urine.
Filtering of /3-microseminoprotein in the renal glomeruli
and subsequent reabsorption in the tubuli might be a major
catabolic route for this protein. One would then expect a
correlation between the concentrations in blood of (3-microseminoprotein and a marker of glomerular ifiration
rate, such as creatinine. Our measurements of f3-microseminoprotein
in a number of patients with various serum
creatinine concentrations showed a statistically significant
positive correlation between these two variables, giving
support to the hypothesis that at least part of the catabolism of /3-microseminoprotein takes place by glomerular
filtration. If the input of /3-microseminoprotein into the
circulation varied little in these patients and glomerular
filtration was responsible for the elimination of the protein,
one would have expected a correlation higher than that
observed. But many of the patients in this study had severe
diseases besides their renal insufficiency, and it is not
unlikely that many of the high values for (3-microseminoprotein seen in these patients were partly caused by an
increased input of (3-microseminoprotein into the circulation as a consequence of a disease affecting an organ
synthesizing (3-microseminoprotein, e.g., the prostate gland
in the men.
The function of (3-microseminoprotein is still unknown.
1502 CLINICAL CHEMISTRY, Vol. 35, No. 7, 1989
Earlier proposals (7) that /3-microseminoprotein
has inhibin activity-i.e.,
inhibition of release of follitropin from
the pituitary-have
been questioned (11, 12). This is consistent with our finding
of a zero correlation between
/3-microseminoprotein
and follitropin.
This work was supported by grants from the Swedish Medical
Research Council (Project No. B88-03X-5913 and B88-13X-7903),
the Faculty of Medicine at the University of Lund, the Albert
PMilsson Foundation, the Greta and Johan Kock Foundation, the
John and Augusta Persson Foundation, the Anna Lisa and SvenErik Lundgren Foundation, and the Cancer Research Fund at
MalmO General Hospital.
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