Download Determination of Neoptenn in Serum and Urine

CLIN. CHEM. 33/1,
62-66
Determination
(1987)
of Neoptenn
in Serum
and Urine
Ernst R. Werner,1 Alfred Blchler,2 G#{252}nter
Oaxenblchler,2 Dlstmar FUChS,’
Relbnegger,’
and Helmut Wachter’
of neoptenn,
a product
of activated
macrophages, in serum from 662 apparently
healthy indMduals
(ages 1 to 97 years, median 22 years) were measured
by
radloimmunoassay
and the results statistically
analyzed.
Concentrations
Consistent
with
prior
investigations
on the
urinary
excretion
of neopterin,
we found no significant sex dependence,
but
values for subjects younger than 18 or older than 75 years
were significantly
higher. Renal clearance
of neopterin
in
nine healthy individuals
was 218 (SD 44) mL/min, which
suggests that the kidneys have an active role in neopterin
excretion.
Results for neopterin concentrations
measured
in
serum by RIA and “high-performance”
liquid chromatography (HPLC)
are consistent,
but for urinary neopterin
the
concordance
between
methods
was weak. Therefore,
the
RIA should be used only for measuring neopterin in serum. In
comparison
of the clinical utility of serum neopterin
and
urinary neopterin/creatinine
concentrations,
in patients with
gynecological
tumors, the latter values (measured
by HPLC)
discriminated
slightly better between patients with favorable
and unfavorable
prognoses.
Lothar
C. Fulth,2
Asno
Hausen,’
Heinz Hetzel,2 GIlbert
favorable
and unfavorable
clinical
course
of disease.
For this
we used patients
with gynecological
tumors
and
the results with those for concentrations
of neoptern in serum and urine
of parenteral
drug
abusers. The
latter
subgroup was chosen because their neopterin
concentrations
are expected
to be less influenced
by changes in
renal function
than for patients with malignant
disorders.
assessment
compared
Materials
and
Methods
Instrumentation.
For HPLC analyses
we used an LC 5500
(Varian,
Palo Alto, CA) controlled
by a Vista 402 data
system
(Varian),
and equipped
with a RF 530 fluorescence
detector
(Shimadzu,
Tokyo, Japan). Radioactivity
was measured with a Gamma
Szint BF 5300 (Berthold
Lab., Wildbad, F.RG.).
Reagents.
Neopterin
RIA kits for serum
and urine
were
obtained
from Henning,
Berlin,
F.R.G.
All other reagents
(analytical
grade) were products
from
Merck,
Darmstadt,
F.R.G.
Neopterin
was a gift of Prof. W. Pfleiderer,
Konstanz,
F.R.G.
Procedures
Addftlonal
Keyphrases:
radioimmunoassay
reversed-phase
age-related
effects
system
reference interval
drug abuse
.
.
chromatography,
cancer
immune
.
Measurement
of neopterin
concentrations
provides
an
sensitive,
reliable
means
to monitor
activation
and macrophages.
In vitro, major
amounts
of
are released
exclusively
by macrophages,
particularly when stimulated
with gamma
interferon
derived
from
activated
T-cells
(1, 2). Thus,
an increase
in neopterin
concentrations
supplies
information
on the activation
state
of cell-mediated
immunity
that is not otherwise
readily
obtainable.
This
information
is particularly
useful
for the
follow-up
of allograft
recipients
(3) for the prognosis
of
patients with cervical
cancer
(4), for staging
patients
with
urological
tumors
(5), and for monitoring
autoimmune
diseases
and diseases
caused
by mycobacteria,
protozoa,
and
viruses
(6). These clinical
studies
have involved
measurement
of urinary
neopterin
by “high-performance”
liquid
chromatography
(HPLC)
(7, 8). More recently,
a radioimmunoassay
(REA) for neopterin
in serum
and another
one for
determinations
in urine
have become
available
(9).
Our aim in this study was to assist clinicians
by determining normal values for neopterin
in serum for different
age
groups
as well as the renal clearance
of neopterin.
In
addition,
we compared
the RIA and the established
HPLC
method for use with urine and serum specimens.
Finally,
we
examined whether
concentrations
of neopterin
in serum and
urine are similarly
suitable
for discriminating
patients
with
immediate,
of T-cells
neopterin
for Medical
Chemistry and Biochemistry, University
A-6020 Innsbruck, Fritz-Pregl-Str.
3, Austria.
of Obstetrics
and Gynecology,
University
Clinic of
‘Institute
of Innabruck,
2J)epartent
Innsbruck,
Received
62
A-6020
June
CLINICAL
Innsbruck,
Anichstr.
35, Austria.
16, 1986; accepted
September
27, 1986.
CHEMISTRY,
Vol. 33, No. 1, 1987
Serum creatinine
was assessed by a modified Jaffe method(1O).
HPLC.
We determined
urinary
neopterin
and creatinine
by reversed-phase
HPLC
as described
elsewhere
(7, 8).
Briefly,
the procedure
was as follows.
We used a C18
reversed-phase
column (4 x 125 mm, 7-tm-diameter
packing, Merck) protected
with
a 4 x 4 mm guard column of
similar
material.
The mobile phase is potassium
phosphate
buffer (15 mmoWL,
pH 6.4), the flow rate 0.8 mL/min.
After
diluting
100 L of untreated
urine specimens with 1.0 mL of
mobile phase containing
2 g of disodium-EDTA
per liter, we
inject a 10-L
sample. Neopterin
is quantified
by its native
fluorescence
(353
nm excitation,
438 urn emission),
the
simultaneously
determined
creatinine
is measured
by ultraviolet absorbance
at 235 nm. The detection
limit of the
method for neopterin
was 40 nmoIJL.
Intraand interassay
CVs for the neopterin/creatinine
ratio were 4.7% and 5.8%,
respectively
(7). Thirty-five
urinary
components
have
been
tested for possible interferences;
furthermore,
during
a fiveyear
period,
200000
specimens
from
various
groups of
patients
have been assayed
and no interfering
substances or
drug metabolites
have been encountered.
To measure
serum
neopterin
by HPLC,
we used a modification of the method of Huber
et al. (11): inject
20 L
of
undiluted
serum onto a 4 x 125 mm C18 reversed-phase
column
(as above), with distilled
water as mobile phase (flow
rate, 0.7 mL/min).
The portion
of effluent that contains
all
the neopterin
(which
is eluted
within
2 mm)
is then
switched onto a second similar C15 column
and eluted
with
sodium dedecyl sulfate, 0.1 g/L in distilled water (adjusted to
pH 4.0 with trifluoroacetic
acid) at a flow rate of 0.7 mLfmin.
The detection limit of this method
is 1 nmol/L.
Intraand
interassay
CVs were 3.7% and 9.3%, respectively.
RIA. The RIA procedures
were as follows: Incubate 50 .L
ofserumwith
100 .tLofneopterinantiserumfor
1 hatroom
temperature,
then add 100 ,uL of I-labeled
neopterin.
Incubate again for 1 h, then add 2 mL of aqueous polyethyleneglycol (PEG) solution (60 g/L). Centrifuge
at 2000 x g for
10 mm, discard the supernate,
and count the radioactivity
of
the pellet with a scintillation
counter.
The detection
limit of
the method is 1 nmollL.
The intra-assay
CV (the mean of the
percentage
deviations
of the duplicates
from their
means,
for 25 randomly
selected
duplicates derived from five different assays) was 1.2%. The interassay
CVs, determined
by
analyzing
two
control
serum
specimens
in 14 different
assays,
was 12% for a mean
concentration
of 4.7 nmol/L,
7.1% for 14.1 nmollL.
Urne
specimens
were diluted
100-fold
with buffer,
then
centrifuged
(5 mm, 2000 x g). We used 20 tL of the diluted
urine
in the RIA procedure
without
extraction
or purification, proceeding
as for the serum method described
above.
The detection
limit was 4 nmol/L.
Intra-assay
variation,
defined
as above, was 1.2%. We could not assess interassay
variation
because
we used only two kits.
assessed
by the Kruskal-Wallis
test, linear
regression
analysis, and Spearman rank correlation
analysis. The reference
ranges were estimated
by a nonparametric
percentile
method (13). For calculations
we used the Cyber 74 computer
(Control
Data Corp., Minneapolis,
MN) with the BMDP
program
package (University
of California
Press, 1983 ed.).
Results
Correlations
To assess
the normal concentrations
of neopterin
in serum,
serum
RIA and serum
specimens
from
662
healthy
individuals,
ages 1 to 97 years (median
22 years). Of these, 263 were children,
ages 1 to 18 years
(median
six years).
We determined
the renal clearance
of neopterin
in nine
the
apparently
healthy
individuals,
five
women
and
four
Subjects
Age and s& dependence
of neopterin
concentrations
in
serum. Figure 1 illustrates
the concentrations
of neopterin
in serum
from
the healthy
individuals.
Because nonpar#{225}metric
statistics
were used, we did not exclude possible
outliers. Three age groups were identified as showing significantly different values for neopterin
(Kruskal-Wallis
test,
p <0.0001,
Table 1), but there was no statistically
significant sex dependence
(Kruskal-Wallis,
p >0.05).
Subjects
between
ages 18 and 75 years showed no significant
age
dependence
of the serum concentrations,
but children (<18
years) and elderly subjects (>75 years) had significantly
higher neopterin
concentrations
than did the middle
group.
We chose the 95th percentiles
as the upper normal
limits.
Renal clearance
of neopterin.
The mean renal clearance of
neopterin in nine healthy
subjects (Table 2) was 218 (SD 44)
Specimens
we used
apparently
in Healthy
mllmin.
Serum
and urinary
neopterin
by both RL4 and HPLC.
Nineteen
concentrations
determined
serum samples
for which
neopterin
was measured
by RIA (y) were also analyzed by
HPLC (x). Linear
regression analysis of the results yielded:
y = 1.02x + 1.04 nmol/L
(r = 0.96). We also measured
neopterin
in 86 urine samples by RIA and by HPLC; results
by the two methods were less well correlated
(Figure 2).
men.
For measurements
in serum we used RIA; for urine, HPLC.
For comparison
of concentrations
of neopterin
in serum as
measured
by RIA and HPLC,
we measured
neopterin
in 19
serum
specimens
obtained
from some of the children
in the
apparently
healthy
group. In these specimens,
neopterin
concentrations
ranged from 2.7 to 28.0 nmoIJL (median
11
nmol/L).
To compare the concentrations
of urinary
neopterin
measured
by RIA
and by HPLC,
we analyzed
86 urinary
specimens
from unselected
apparently
healthy individuals.
To compare
the clinical
significance
of serum
and urinary
neopterin
concentrations,
we collected
83 serum and urine
specimens
from
72 consecutive
patients with gynecological
tumors.
We determined
the neopterin
in serum by RIA and
the neopterinlcreatinine
ratio in urine
by HPLC.
We also
measured
serum
creatinine,
serum
neopterin
(RIA),
and
urinary
neopterinlcreatinine
(HPLC) in another
19 patients
with gynecological
tumors.
Serum neopterin
and urinary
neopterinJcreatinine
values were recorded
from
35 apparently otherwise-healthy
drug
abusers
studied elsewhere
35.
30
C
25
a)
‘r.
a-,
o-
20
cE
E,
15
1)
U,
1:
x,
#{149}
(12).
Specimens were protected from light and stored at -20 #{176}C
until analyzed.
We were careful
not to freeze and thaw
serum samples more than twice.
Statistical
Analyses
Differences
Table
of neopterin
values
1. Nonparametric
groups
in different
Percentile
Estimates
Age, years
-
Group
Median
Median
263
359
40
0-18
12
6.00
19-75
75-97
37
86
4.90
8.50
±
SE
± 0.15
±
0.09
±
0.81
20
40
age
60
80
160
(years)
Fig. 1. Concentrations of neopterin in serum of 662 apparently healthy
subjects, as measured by RIA
were
of Neopterln
individuals
Concentrations
Neopterln,
n
0
of Apparently
Healthy
nmol/L
Percentile
SE
Range
SD
5th
95th
±
0.22
2.7-28.0
±
0.14
1.0-33.6
0.79
2.9-30.0
3.5
2.6
3.7
13.5
±
3.60
2.74
5.01
Mean
6.78
5.34
9.67
In Serum
±
CLINICAL CHEMISTRY,
Vol. 33, No. 1, 1987
8.7
19.0
63
Table 2. Renal Clearance of Neopterin
Apparently
Healthy individuals
in Nine
Neopterin
concn,
Ag., years
nmol/L
Serum
Urine
Vol of 24-h
urine, mL
Glomeruiar filth, rate
for neopterin, mL/mln
950
1560
1096
1720
184
291
2445
1000
265
771
2100
261
695
1953
1756
2315
1272
1600
700
1400
700
900
194
183
189
229
176
Men
28
30
31
26
Women
5.6
4.5
6.4
4.3
22
4.0
20
5.2
23
24
9.0
4.9
25
4.5
observation
represents
physiological
differences,
we collected another
set of urine
and serum specimens
from 13 of
these patients a few days later. Results were identical to the
first observations.
The correlation
of serum
neopterin
(RIA) and urinary
neopterinlcreatinine
(HPLC) results was analyzed by linear
regression
and Spearman’s
rank correlation
method. Including all values, the linear and the Spearman’s
rank correlation coefficients
were 0.92 and 0.65, respectively.
If three
“off-scale” values (4372, 160; 783, 65; 1370,43)
are excluded,
the remaining
80 values (shown in Figure 3) show similar
linear and Spearman’s
rank
correlation
coefficients:
0.52
and 0.60, respectively.
Thus, a moderate
correlation
was
found, which is significantly
different from zero (p <0.001).
In another
19 patients
with gynecological
tumors, the
concentrations
of creatinine
in serum ranged from 6.8 to 15
mg/L (mean 10.8 mg/L). The linear correlation
coefficient
relating urinary
neopterin/creatinine
with serum neopterin
was 0.40; it was 0.36 for urinary
neopterin/creatinine
vs
serum neopterinlcreatinine
ratio. Thus relating serum neopterm to serum creatinine
concentrations
did not change the
correlation
of serum neopterin
to urinary
neopterinlcreatinine values.
We also measured
serum
neopterin
(RIA) and urinary
neopterinlcreatinine
(HPLC)
concurrently
in 35 otherwisehealthy parenteral
drug abusers.
As illustrated
in Figure 3
(right) the correlation
of both methods for this group
is
stronger (linear correlation
coefficient
= 0.73, Spearman’s
rank correlation
coefficient
=
0.72) than for the gynecological cancer patients.
Discussion
Serum
H PLC
Fig. 2. Concentrations
of urinary neoptenn
(pmol/L) in urine of 86
apparently healthy subjects, as measured by RIA and by HPLC
Une of dentity (so/id line) shown for comparison
Correlations
in Cancer
Patients
and
Drug
Abusers
For sample from 72 patients
with gynecological
tumors
we determined
concurrently
neopterin
values for serum
(by
RIA) and urinary
neopterinlcreatinine
values
(by HPLC).
The patients
were classified
into two groups
according
to
their clinical
status: favorable
clinical
course (no evidence
of
disease
or partial
or complete
remission)
and unfavorable
course (evidence
of disease,
progression,
or relapse).
The clinical
status
was compared
with the frequencies
of
neopterin
concentrations
above the upper limits of normal
(95th percentiles
for serum; Table 1; for urine values see ref.
6). Results
for serum
neopterin
were in concordance
with
the clinical
diagnosis in 22/32 (69%) patients
without
evidence of disease
and in 32/40(80%)
patients with evidence of
disease. The corresponding
comparisons
were 24/32 (75%)
and 35/40(88%)
for the urinary neopterin/creatinine
results.
In 52/72 (72%) of patients, the urinary
neopterin/creatinine
and the serum neopterin
results yielded the same classification (data not shown).
In all, 54/72 (75%) patients
are
correctly classified by the results for serum
neopterin
(RIA)
and 59/72 (82%) by the urinary
neopterin/creatinine
findings (HPLC).
Thus,
the discriminatory
power of urinary
neopterinlcreatinine
values appears to be slightly superior
(chi square value 28.87 vs 17.38). To assess whether
this
64
CLINICAL
CHEMISTRY,
Vol.33,
No.1,
1987
Neopterin
in Apparently
Healthy
Individuals
The results
obtained
in this study agree well with data for
1837 blood donors (ages 18-67
years), who had a mean
neopterin concentration
of 5.89 nmolJL, SD 1.78 nmol/L,
and
an upper 99% confidence
limit of 10.5 nmol/L,
estimated
with an assumption
of gaussian
distribution
(14). Except for
that abstract presentation,
the present work represents
the
first study on serum concentrations
of neopterin
in healthy
individuals
that includes
subjects
in virtually
all age
groups. The results
agree well with those obtained
by
urinary neopterinlcreatinine
measurements
in healthy subjects (6, 15) if the known
sex- and age-dependence
of
creatinine
excretion is accounted
for (data not shown).
The
differences in the chosen upper limits of normal for serum
neopterin-10.5
nmoL(L (14) vs 8.7 nmol/L
in this workarise from our use of a nonparametric
percentile
estimation
method
vs the parametric
procedure
that Kern at al. used
(14). Further,
our upper limits of tolerance
are based on the
40
20
C
C
a,
30
15
-
0-..-
0.
E
a,
a,
20
.
V.4.
10
-
- -
10
-
Ec
.5
-
0
0
200
400
arnary
600
aeopterin
800
0
(j,moI/raol
200
400
600
creatinine)
Fig. 3. Concurrently
measured concentrations
of serum neopterin (RIA)
and urinary neopterin/creatinine
(HPLC) for 80 patients with gynecological tumors (left) and 35 parenteral drug abusers (right)
95th percentile,
whereas they used a 99% confidence limit.
For the evaluation
of normal
ranges,
we consider
the
nonparametric
method to be superior
because it requires
no
assumption
that the values follow a special distribution
formula (e.g., normal distribution)
(13).
Renal clearance
of neopterin.
The mean renal clearance of
neopterin,
which was 216 (SD 44) mL/min,
is approximately
1.8 times the standard value of 120 mL/min for the glomerular ifitration
rate. This might be explained
(e.g.) by production of neopterin
by macrophages
of the kidneys,
active
secretion
of the compound
by the kidneys,
or oxidation
processes
occurring
in the kidneys.
However,
the last is not
likely
because
similar
ratios
of native
oxidized
neopterin
compared
with total neopterin
after chemical
oxidation
have
been found in serum
(43%) and urine (45%) (16). Further,
the renal
clearance
of neopterin
measured
after
chemical
oxidation
processes
glomerular
filtration
our results.
in serum and urine was about twice the
rate (17), which is in accordance
with
Comparison
of the RIA and the HPLC
method
for serum
and urinary
neopterin
concentrations.
A good linear correlation was
observed
between
neopterin
concentrations
in
serum
as measured
by RIA and HPLC,
as indicated by the
results of the linear regression
analysis.
In contrast,
the
compared
methods were less well correlated
for urinary
neopterin
values. Particularly,
the observed slope of 0.64
reflects
a considerable
proportional
error of the RIA compared with the HPLC method.
It should be noted that the
established
HPLC method
(7,
is specific,
provides
good
performance
characteristics,
and has been carried
out by
several
laboratories,
yielding
clinically
reliable
results.
Therefore,
it can be concluded that the RIA should not be
used for urinary
neopterin
determination
but yields valid
results for serum neopterin.
8)
Correlations
in Cancer
Patients
and Drug Abusers
Urinary
neopterinlcreatinine
values somewhat better discriminate,
in the case of samples from patients with gynecological tumors, between
those with a favorable and unfavorable prognosis.
Analysis
of another set of urine
and serum
specimens
from a part of the same group of patients
led to
results identical
to the first observations.
Therefore,
e.g.,
values of urinary
neopterin
exceeding
the upper limits
of
normal in the presence of normal
serum neopterin
concentrations in a patient with progressive
disease appear to be a
property
linked to the individual
but are not influenced
by
day-to-day
variations.
The statistical
comparison
of the correlation
between
serum
neopterin
concentrations
and urinary
neopterin/creatinine ratios for samples from 83 patients with gynecological tumors, with and without
exclusion of only three “offscale” values, shows an artifact
of the linear regression
analysis:
the Spearman’s
rank correlation
coefficient
only minor changes upon the dropping of those three
shows
values,
but the linear correlation
coefficient is far more affected.
For patients
with gynecological
tumors the association
between
serum neopterin
concentrations
and urinary neopterin/creatinine
values is not very strong. For drug abusers,
serum neopterin
concentrations
and urinary
neopterinlcreatimne
values
are better
correlated.
The renal
clearance
data might
provide
a possible
explanation
of this observation. Because neopterin
clearance
exceeds glomerular
filtration rate and varies
for different
neopterin
concentrations
and total
tion, but not the neopterinlcreatinine
individuals,
both serum
urinary
neopterin
excrevalues,
are expected
to
be influenced
considerably
by the functional
state of the
consideration
is supported by a study on renal
allograft
recipients
in which the correlation
coefficient between
serum neopterin
concentrations
and urinary
neopterin/creatinine
values was increased
from 0.26 to 0.78 by
relating
serum neopterin
to serum
creatinine
concentrations (Aulitzky
W, et al., in preparation).
Because
patients
with gynecological
tumors
can be assumed to have more affected renal function than is true of
drug abusers generally,
we also determined
creatinine
in
the sera of these patients.
For our tumor patients,
the
correlation
between
serum
neopterin
concentrations
and
urinary
neopterinlcreatinine
values, however,
was similar
to the correlation
between serum neopterin/creatinine
values and urinary
neopterin/creatinmne
values. Further,
the
finding
that several patients
with gynecological
tumors
show increased
urinary
but not increased
serum neopterin
concentrations
cannot be explained
by decreased
glomerular
ifitration
rate. Thus, variables
other than kidney function
also influence
the correlation
between serum concentrations
and urinary
neopterin
values in this case.
The weak correlation
between
serum
neopterin
concentrations (by RIA) and urinary
neopterinlcreatinine
values
(by HPLC)
in gynecological-tumor
patients
indicates
that
clinical conclusions
derived from the two could differ. For
the patient group studied, minor advantages
of measuring
urinary
neopterin
concentrations
were apparent.
The present work and a study on renal allograft recipients (Aulitzky
W, et al., in preparation)
show that the clinical
utility
of
both methods of neopterin
measurement
should be compared separately
for each patient group.
kidney.
This
We are indebted to Dr. Philadelphy
(Laboratorium
fir medizinisch-diagnostische
Untersuchungen)
for generous
presentation
of
serum samples. This work was financially
supported
by the “Jubilaeumsfonds”
of the National
Bank of Austria,
project 2591.
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