Fukuda
et
aL:
Urinary xanthopterin
and
neopterin
in
liver diseases
129
Eur.
J.
Clin.
Chem.
Clin.
Biochem.
Vol.
31,
1993,
pp.
129-134
©
1993
Walter
de
Gniyter
& Co.
Berlin
· New
York
A
Comparative Study
of
Urinary
Xanthopterin
and
Neopterin
in
Liver
Diseases
By
Akira
Fukuda
1
*),
Toshio
Mazda
2
,
William
L.
Gyure
3
,
Teruhiko
lino*,
Hideharu
Harada
1
,
Michiyasu
Yakura
1
,
Hiroshi
Kamitsukasa
1
,
Akira
Ohbayashi
1
,
Teruaki
Oka
5
and
Motoo
Tsusiie
6
1
Department
of
Gastroenterology,
Liver
Unit,
Tokyo
National Sanatorium Hospital, Kiyose,
Tokyo,
Japan
2
Japanese
Red
Cross
Tokyo
Metropolitan Blood Center,
Musashino,
Tokyo,
Japan
3
Seton
Hall
University,
South
Orange,
New
Jersey,
USA
4
Department
of
General Education,
Nihon
University,
Setagaya-ku,
Tokyo,
Japan
5
Department
of
Pathology, Medical School,
Tokyo
University,
Bunkyo-ku,
Tokyo,
Japan
6
Biological
Laboratory, School
of
Liberal Arts
and
Sciences,
Kitasato
University,
Sagamihara,
Kanagawa,
Japan
(Received
September
18,
1992)**)
Summary:
By
adsorption
to
activated charcoal, various pteridine derivatives
in
human urine
are
oxidized
to
xanthopterin.
Following this
oxidation,
xanthopterin
in
urine
from
healthy subjects
and
from
patients
with
liver diseases
was
assayed
by
high performance liquid
chromatography.
The
mean values
for
xanthopterin
in
healthy subjects were
532
±116
μηιοΐ/mol
creatinine (mean
+
SD) in
males
and 585
±153
μπιοΐ/mol
creatinine
in
females;
the
difference
was
statistically significant
(p
<
0.01). Xanthopterin concentrations
in
patients
with liver disease were
significantly
higher
than
those
in
normal subjects. When compared with
urinary neopterin, which
is a
marker
of
activated cell immunity, xanthopterin
was
significantly
increased even
in
fatty liver disease.
These
findings
suggest that increased concentrations
of
urinary xanthopterin
in
liver
diseases
reflect
not
only
the
status
of
activated
cell-mediated immunity,
but
also
injury
to
liver
cells.
Introduction
Many pteridine derivatives have been found
in
human
urine
(1).
Among these
pteridines,
neopterin
has
been
used
as a
biochemical marker
of the
activated state
of
cell-mediated immunity,
and it has
been used
to
monitor
and
screen
for
some clinical
disorders
(2
—
6).
Some current studies show
that
urinary neopterin
concentrations
in
patients
with acute
or
chronic viral
hepatitis
are
higher than
in
normal controls
(7, 8).
Urinary
neopterin
has
also
been utilized
to
distinguish
between chronic
non-A,non-B
hepatitis
and
liver stea-
tosis
(9).
*)
Present address:
First
Department
of
Internal
Medicine,
Osaka Medical
College,
Takatsuki, Osaka, Japan.
**)
Submitted February
10/August
31,
1992
to
"Pteridines",
which
discontinued
with
Vol.
3, No. 3,
1992
On
the
other hand,
little
information
is
available
on
urinary xanthopterin.
An
early report describes
the
occurrence
of the
compound
in
human urine
(1).
Urinary xanthopterin
and
neopterin concentrations
are
found
to be
higher
in
some cancer patients
(10,
11).
Plesner
&
Kalckar
(12)
showed that many
pteri-~
dine
derivatives
in
urine
can be
oxidized,
adsorbed
and
eluted
from
activated
charcoal
to
yield stable
xanthopterin. Because pteridine metabolism takes
place mainly
in
liver
(13),
the
excretion
of
other pter-
idine
derivatives, besides neopterin,
may
possibly
be
an
indicator
of
liver
disease.
In our
preliminary
study,
applying Plesner
&
Kalckar's
extraction method,
we
assayed urinary xanthopterin
in
patients with chronic
non-A,
non-B hepatitis
and in
patients with
fatty
liver
disease
(14).
In the
present paper
we
assayed
urinary
xanthopterin
in
different
types
of
liver
disease.
Uri-
nary
neopterin
was
also determined
for
comparison.
Eur.
J.
Clin, Chem. Clin. Biochem.
/
Vol.
31,1993
/ No. 3
130
Fukuda
et
al.:
Urinary
xanthopterin
and
neopterin
in
liver diseases
Patients
and
Methods
Healthy
individuals
As
controls, urine samples were collected
from
178
apparently
healthy
individuals
(age
21 to 64
years, mean
age
42),
64
were
males
(age
24 to 63,
mean
age 44) and 114
females (age
21 to
64,
mean
age
40).
The
samples
were
collected during routine
medical
examinations
for
health
care workers
at the
Tokyo
National
Sanatorium Hospital.
Patients with liver disease
Urine
samples
were
collected
from
in- and
out-patients
in the
liver
unit, Tokyo National Sanatorium Hospital.
The
patients
were
divided
into
5
groups
as
follows;
1)
acute
viral
hepatitis
8
cases,
2)
chronic
viral
hepatitis
53
cases,
3)
liver
cirrhosis
21
cases,
4)
alcohol-induced
liver
disease
18
cases
and
5)
non-alcoholic
fatty
liver
13
cases.
The
results
of
routine biochemical
liver
tests
in
each group
are
shown
in
table
1.
Among
the
acute viral hepatitis cases
(4
males
and 4
females,
mean
age 37
years),
5
were type
A
(anti-IgM
HAV
antibody
positive),
1 was
type
B
(HBs
antigen
and
anti-IgM
HBc
anti-
body
positive)
and 2
were
type
C
(both above
HAV and HBV
markers
negative
and
anti-clOO-3
positive).
Of the
chronic
viral
hepatitis
cases
(27
males
and 26
females,
mean
age 57
years),
10
were type
B, 33
were type
C and the
other
10
were type
non-A,non-B,non-C.
Among
the
chronic viral hepatitis cases,
histological
features
on
liver biopsy showed chronic persistent
hepatitis
in 20 and
chronic active hepatitis
in 33. Of the
liver
cirrhosis
cases
(16
males
and 5
females,
mean
age 60
years),
12
were
type
B, 2
were
type
C and 7
were type non-A,non-B,non-
C. All the
cases
were
non-alcoholic with biopsy results showing
postnecrotic
cirrhosis.
The
alcohol-induced
liver
disease cases
(all
18
were males, mean
age 52
years)
had a
long history
of
alcohol
abuse, with histological features showing either
fatty
liver
or
alcoholic
fibrosis
with
or
without
fat
deposits.
The
non-
alcoholic
fatty
liver cases
(12
males
and 1
female,
mean
age 43
years)
were
considered
to be due to
obesity and/or metabolic
abnormalities
such
as
hyperlipaemia
or
non-insulin-dependent
diabetes.
Renal
failure
was not
present
in any of the
study subjects,
according
to
urinary
and
serologic tests.
In
addition
no
drugs
such
as
azathiopurine,
corticosteroids
or
interferon were given
in
the 6
months prior
to
this study.
Urine
and
blood
sample
analysjs
After
the
measurement
of
creatinine concentrations
by the
alkaline picrate
method,
urine samples were immediately stored
at
-30°C
in the
dark until used
for the
measurement
of
xanthopterin
and
neopterin.
Blood
samples were taken from
all
test
subjects
at the
Tokyo
National Sanatorium Hospital
at the
time
of
urine
collection.
Serum
tests were performed
for the
following enzymes;
alanine
aminotransferase,
aspartate
aminotransferase,
γ-glutamyl
transferase.
As a
screening test,
the
zinc turbidity test
was
also
performed.
All
analyses were conducted using
a
736-40E multi-channel
autochemicai
analyser
(Hitachi
Co., Ltd., Tokyo,
Japan).
Urinary xanthopterin
assay
Urinary xanthopterin
was
measured applying
the
isolation
method
of
Plesner
&
Kalckar
(12)
and
using high performance
liquid
chromatography
(HPLC)
for
quantitation
as
follows:
To
6
mg
of
activated charcoal
(Norit
A or
Norit
SX-3)
10
μΐ
of
1
mol/1
acetic acid
and 1 ml of
urine were
added
successively,
mixed,
and
allowed
to
stand
at
room
temperature
for 10
min-
utes.
The
mixture
was
centrifuged
at
6700
g for 5
minutes.
The
resulting
precipitate
was
washed once with distilled water, then
resuspended
in 500
μΐ
of
0.05 mol/1 NaOH containing
20
ml/1
pyridine.
This mixture
was
centrifuged
at
6700
g for 5
minutes
and
after
a 10
fold
dilution
of the
supernate
with distilled water,
50
μ!
of the
solution
was
injected into
the
HPLC system.
For
xanthopterin
determinations,
the
Twincle HPLC system
(Japan Spectroscopic Co., Ltd., Tokyo,
Japan)
was
used.
For
fluorescence
detection,
a
Jasco
FP-110
spectre-photometer
(Ja-
pan
Spectroscopic Co., Ltd., Tokyo,
Japan)
was
used with
excitation
at 390 nm and
emission measurement
at 460
nm.
A
cut-off
filter was
used
to
eliminate detection
of
emission wave
lengths
shorter than
460 nm.
Peaks were recorded
on a
D-2000
chromato
integrator (Hitachi Co., Ltd., Tokyo,
Japan).
The
HPLC column
was
packed with
Asahipac
GS
320H
(Asahi
Chemical
Industry Co., Ltd., Kawasaki, Japan).
The
elution
fluid
was
methanol (volume fraction
0.10)
containing
10
mmol/1
potassium
phosphate
buffer
pH 7.0 at a flow
rate
of 1 ml per
minute.
The
main peak, which showed
a
retention time
of
10.5
minutes,
was
identified
as
xanthopterin
by
comparison with
authentic xanthopterin using
the
HPLC system described
above.
The
values were expressed
as
μιηοΐ
xanthopterin
per
mol
cre-
atinine.
Tab.
1.
Biochemical
liver
tests
in
each group
of
liver diseases
Cases
Reference
range
Acute
hepatitis
Chronic hepatitis
active
persistent
Liver
cirrhosis
Alcohol-induced
liver
disease
Non-alcoholic
fatty
liver
(n)
(8)
(33)
(20)
(21)
(18)
(13)
Alanine
aminotransferase
(34-4
IU/1)
mean (range)
1338
(254-3468)
143
(49- 288)
48
(19-
95)
66
(10- 183)
59
(13- 185)
47
(20-
75)
Aspartate
aminotransferase
(31-9 IU/1)
mean
(range)
776
(59-2428)
117
(37- 279)
41
(22-
61)
82
(14- 224)
81
(24- 212)
39
(19-
97)
γ-Glutamyl-
transferase
(33-4
mU/1)
mean (range)
130
(54-193)
92
(19-425)
39
(12-126)
67
(17-253)
198
(24-726)
•
?
74
(13-264)
Zinc
turbidity test
(13-1
Kunkel
U)
mean (range)
11
(6-22)
17
(5-28)
15
(4-24)
17
(7-31)
10
(2-31)
7
(3-13)
Eur.
J.
Clin.
Chem.
Clin.
Biochem.
/
Vol.
31,1993
/ No. 3
Fukuda
et
al.:
Urinary xanthopterin
and
neopterin
in
liver diseases
131
Urinary
neopterin
assay
Urinary
neopterin
was
directly assayed without oxidation
(15)
using
a RIA kit
"IMMUtest
Neopterin" (Henning Berlin
GmbH, Berlin, Germany). Radioactivity
was
counted
by a
programmed gamma-counter
"ANSR"
(Dinabot
Co.,
Ltd.,
To-
kyo,
Japan).
The
values were expressed
as
μπιοί
neopterin
per
mol
creatinine.
Statistical analysis
The
differences
of
xanthopterin values
in
healthy subject
be-
tween
different
age
groups were assessed
by the
Kruskal-Wallis
test. Using urinary xanthopterin
and
neopterin assay values
as
data,
median
values
for
these
pteridines
were determined under
various
conditions
and
evaluated
by the
Mann-Whitney
U-test.
Comparisons
of
abnormal levels
of
xanthopterin
and
neopterin
were
assessed
by the
%
2
-test
for the
different
groups
of
patients
separately.
Tab.
2.
Urinary
healthy
Age
(years)
Males
24-29
30-39
40-49
50-63
Total
Females
21-29
30-39
40-49
50-64
Total
excretion
of
individuals
Number
of
cases
8
18
15
23
64
24
31
27
32
114
xanthopterin
and
Xanthopterin
(μηιοΐ/mol
creatinine)
mean
± SD
527
+ 131
517
+ 90
556
+ 82
529
+ 148
532
+
116
555
± 92
592
± 143
575
+ 176
608
+
178
585
+ 153
neopterin
in
Neopterin
(μπιο1//ιηο1
creatinine)
mean
± SD
98
± 55
82
+ 27
90
+ 42
95
+ 39
91
± 39
105
± 26
117
+ 26
125
+
39
130
+ 44
120
+ 38
Results
Urinary xanthopterin
in
healthy individuals
In
178
healthy subjects,
the
mean values
of
xantho-
pterin were
566
±143
μπιοΐ/mol
creatinine (mean
±
SD).
In 64
males
the
values were
532
±116
μπιοί/
mol
creatinine
and in 114
females
585
±153
μηιοί/
mol
creatinine. Although
a
statistical
difference
in
urinary xanthopterin concentrations
for
males
and
females
was
found
in
this present study
(p
<
0.01),
there were
no
statistically significant
age
group dif-
ferences
(p >
0.05),
as
shown
in
table
2.
Urinary
xanthopterin
in
liver
disease
patients
As
shown
in
table
3,
urinary xanthopterin concentra-
tions
in
patients with liver disease including
non-
alcoholic fatty liver were significantly higher than
those
in
healthy subjects.
The
highest concentrations
of
xanthopterin
were
found
in the
acute hepatitis
patients,
and
xanthopterin concentrations were also
high
in
chronic active hepatitis. Urinary xanthopterin
concentrations
in the
active stages
of
chronic hepatitis
were
statistically higher than
in the
persistent stages
(chronic active hepatitis
vs.
chronic persistent hepa-
titis:
ρ <
0.01).
Comparison with urinary neopterin
Urinary neopterin concentrations
for the
same
sub-
jects
are
also shown
in
table
3.
Neopterin
was
signif-
icantly
elevated
in
cases
of
acute hepatitis, chronic
hepatitis, liver cirrhosis
and
alcohol-induced liver dis-
ease,
when compared with normal subjects.
In
non-
alcoholic
fatty
liver cases, however, there
was no
significant
elevation
of
neopterin.
Urinary concentrations
of
xanthopterin
and
neopterin
in
all
groups
of
liver disease
are
shown
in figure
1.
Tab.
3.
Urinary excretion
of
xanthopterin
and
neopterin
Group
(n)
Xanthopterin
P
(μιηοΐ/mol
creatinine)
Control
Acute
hepatitis
Chronic hepatitis
active
persistent
Liver
cirrhosis
Alcohol-induced
liver
disease
Non-alcoholic
fatty
liver
178
8
33
20
21
18
13
Mean
+
SD
566
+
143
1305
+
579
978
± 174
807
± 261
934
± 251
868
± 248
689
± 183
Range
250
- 902
813
-
2201
564
-
1377
536
-
1462
505-
1325
418
-
1347
435
- 993
Neopterin
(μιηοΐ/ηιοί
creatinine)
P
Mean
+
SD
Range
<
0.001
<
0.001
<
0.001
<
0.001
<
0.001
<0.05
109
+ 40
769
± 571
251
+ 92
224
± 94
227
± 99
154+
48
125
± 42
27-
266
-
91
-
91
-
90-
71
-
74-
232
1791
463
422
452
240
207
<
0.001
<
0.001
<
0.001
<
0.001
<
0.001
NS
NS: not
significant compared with controls
Eur.
J.
Clin.
Chem.
Clin.
Biochem.
/
Vol.
31,1993
/ No. 3
132
Fukuda
et
al.:
Urinary xanthopterin
and
neopterin
in
liver
diseases
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Fig.
1.
Urinary concentrations
of
xanthopterin
(A) and
neopterin
(B) in all
groups
of
liver disease.
Δ:
males
within,
Δ:
males outside
the
range
χ + 2 SD of
healthy male
controls,
o:
females
within,
o:
females
outside
the
range
χ
Η-
2 SD of
healthy
female
controls.
Tab.
4.
Abnormal rates
of
xanthopterin
and
neopterin excre-
tion
in all
groups
of
liver disease.
The
relative
and
absolute
frequency
of
patients having
a
measured value
greater
than
the
mean
+
2 SD of
normal controls
are
shown
Disease
Acute
hepatitis
Chronic
hepatitis
active
persistent
Liver
cirrhosis
Alcohol-induced
liver
disease
Non-alcoholic
fatty
liver
Xanthopterin
%
88
82
25
67
67
31
n
01
8)
(27/33)
(5/20)
(14/21)
(12/18)
(4/13)
Neopterin
% n
100
(8/
8)
79
(26/33)
70
(14/20)
62
(13/21)
39
(7/18)
23
(3/13)
When
the
normal upper limit
of
each pteridine
was
set
at the
mean
+ 2 SD in
healthy controls, then
abnormal rates were highest
in
acute hepatitis
and
chronic
active hepatitis
as
seen
in
table
4.
Mean
+ 2 SD for
xanthopterin
was 764
μηηοΐ/mol
creati-
nine
in
males
and 891
μηιοΐ/mol
creatinine
in
females,
and for
neopterin
it was
169
μιηοΐ/mol
creatinine
in
males
and
196
μηιοΐ/mol
creatinine
in
females. There
was
a
significant
difference
in
xanthopterin concen-
trations between chronic active hepatitis
and
chronic
persistent hepatitis (82%
and
25%,
p <
0.001),
while
neopterin
did not
show this
difference
(79%
and
70%,
p
>
0.05).
Discussion
In
the
present paper
a new
method
of
urinary xan-
thopterin assay
after
treatment with activated
char-
coal
is
presented. Using this
method
we
assayed uri-
nary
xanthopterin
in
healthy individuals
and in pa-
tients with liver disease.
In
healthy subjects
xantho-
pterin
and
neopterin concentrations when expressed
in
μπιοΐ/πιοί
creatinine were higher
in
females
than
in
males,
but
there were
no
significant age-differences
in
this study.
In our
study
populations,
the
concentra-
tions
of
urinary xanthopterin
in all
groμps
with liver
disease were significantly higher when compared with
healthy subjects. Neopterin concentrations were also
raised,
but in
agreement with
Prior
et al.
(9), there
was
no
significant elevation
of
neopterin
in
non-al-
coholic
fatty
liver.
Urinary
or
serum neopterin
concentrations
are
raised
in
infectious
or
malignant disorders with activated
cell-mediated immunity
(4—6).
Neopterin
is
mainly
Eur.
J.
Clin.
Chem.
Clin.
Biochem.
/
Vol.
31,1993
/ No. 3
Fukuda
et
al.:
Urinary
xanthopterin
and
neopterin
in
liver diseases
133
produced
in
human
monocytes/macrophages,
and its
release
is
accelerated upon stimulation
by
interferon-y
(3).
In our
study, urinary neopterin concentrations
were
very high
in
acute hepatitis,
and
high
in
virus-
induced
chronic
liver
diseases.
In
alcoholic liver
dis-
eases, neopterin concentrations were only slightly
higher than
in
controls. This
finding
may
support
the
view
that alcoholic hepatitis successively activates cell-
mediated immunity,
and
that
the
progress
of
liver
damage
in
spite
of
alcohol
abstinence,
is
responsible
for
the
hyperimmune
reactivity
(16,
17).
The
biochemistry
of
xanthopterin production
and ex-
cretion
is
still scarcely known. Nevertheless,
we as-
sayed xanthopterin together with neopterin. Surpris-
ingly,
xanthopterin concentrations were significantly
high
in all the
liver disease groups mentioned above.
The
increase
in
urinary xanthopterin, like that
of
neopterin,
in
liver
diseases
may
also
be
caused
by
activated
cell
immunity, because
the
concentrations
of
both
are
roughly correlated.
However,
abnormally elevated concentrations
of
xan-
thopterin
were found
in 82% of
patients with chronic
active
hepatitis,
but in
only
25% of
patients with
chronic persistent hepatitis
(p
<
0.001). These
two
groups were histologically
differentiated
from
those
with
severe parenchymal destruction. With respect
to
neopterin excretion, however, these
two
groups were
not
significantly
different
(79%
and
70%,
p >
0.05).
Moreover,
the
rate
of
occurrence
of
abnormally
ele-
vated
urinary xanthopterin
in
fatty
liver
and
other
chronic liver diseases
appear
to
parallel
the
mean
values
of
serum
aminotransferase
activities. This
cor-
relation
does
not
exist
for
neopterin excretion. These
findings
indicate
that increased urinary xanthopterin
reflects
liver-cell damage rather than
the
hyperim-
mune
state.
When
solutions
(10~~
5
mol/1)
of
dihydroxanthopterin,
xanthopterin,
dihydroneopterin
or
tetrahydrobiop-
terin were mixed with equal volumes
of
urine
and
assayed
by the
present method,
the
xanthopterin con-
centrations were
found
to be
1.8, 1.5,
1.3 and 1.4
times
the
basal concentration (unpublished data).
However, when neopterin, biopterin
or
dihydrobio-
pterin
were examined
by
this
method,
no
increase
in
urinary
xanthopterin
was
observed. Thus,
the
present
method represents
an
assay
for
dihydroxanthopterin,
dihydroneopterin
and
tetrahydrobiopterin
in
urine.
After
treatment
with
activated charcoal, eluates
ob-
tained
by
this method were
fairly
pure,
and an
HPLC
column
could
be
used
for
hundreds
of
samples
with-
out
column washing.
A
further
convenient
feature
of
this
HPLC method
is
that
it can be
utilized
in
labo-
ratories
where only
a
fluorometer
is
available
(12).
Further studies will
be
undertaken
of the
biochemical
processes leading
to the
raised excretion
of
xantho-
pterin.
Acknowledgements
We
wish
to
thank
Dr. K.
Shibata,
Hoechst Japan
Co., Ltd.,
for
the
generous supply
of RIA
kits;
Mr.
K.
Fukuhara,
Mr.
M.
Nagashima
and his
coworkers
at
Tokyo National Sanatorium
Hospital
for
generous assistance;
and Dr. T.
Katayama,
Director
of the
Tokyo National Sanatorium Hospital,
for
his
help
in
this
study.
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Clin. Chem. Clin.
Biochem.
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Vol.
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