A Simplified Method for the Determination
Pepsinogen in Blood and Urine
Tefsuo Uete, Michiko
Wasa,
of
and Akemi Shimogami
A simplified method for the determination of pepsinogen in blood and urine has been
developed using serum protein as substrate. As crystalline pepsin digests human
serum protein optimally in the region of pH 2, and blood exhibits such proteolytic
activity on its own serum protein in this region, the determination of pepsinogen in
blood is performed at this pH. The proteolytic activity of serum or plasma on its own
(autologous) serum protein is inactivated by alkalinization. The blood of gastrectomized patients showed extremely low proteolytic activity in the region of pH 2 by
the present method, indicating an absence of pepsinogen. The present method has
greater specificity and accuracy than the method employing either hemoglobin or
casein as substrate.
S
Sorenseml (1), Mi.chaelis and Mendelssohn
(2), and Northrop
(3)
found that purified
pepsin
digests
proteins
iii vitro
optimally
in the
region of pH 2, a number
of physiologic
and chemical
studies of pepsin
have been reported.
In 1924, Gottlieb
(4) developed
an assay method
for the determination
of pepsinogen
in blood and urine. In 1932, Anson
amid Mirsky
(5) developed
a method
for the determination
of pepsin
using denatured
hemoglobin
as a substrate.
Since then, a number
of investigators
utilized
Aiison and Mirsky’s
technic for the determination
of pepsimiogen
in urine and blood (6-8).
Recently,
West et at. (9) and
Goodman
et at. (10) have used casein as a substrate
for the determination of urinary
pepsinogen.
Human
dried plasma
is also used as a
substrate
for the determination
of pepsinogen
in blood
(ii).
More
recently,
Chiang
and co-workers
(1.2, 13) developed
a method
for the
determination
of pepsin using N-acetyl-L-phenylalanyl-L-diiodotyrosine,
and others
(14, 15) used radioiodinated
serum albumin
as a substrate.
The significance
of the determination
of urinary
pepsinogen
has been
INCE
Prom the Kitano
Hospital,
Tazuke
Kofukai
City, Japan.
Supported
by a grant from the Tazuke
Kofukai
The authors
are greatly
indebted
to Dr. Ryuzo
for allowing
us to use his gastrectomized
patients
Received
for publication
Oct. 10, 1967; accepted
42
Medical
Research
Institute,
Kita-ku,
Medical
Research
Foundation.
Fusaoka,
Surgeon-In-Chief,
Kitano
for this study.
for publication
June 12, 1968.
Osaka
Hospital,
Vol. 15, No. I, 1969
DETERMINATION
43
OF PEPSINOGEN
recognized
iii considering
various
diagnoses
of gastrointestinal
diseases, such as i)eptic ulcer alid gastric
carcinoma
(6, 16-18).
It is also a
relial)1e differential
test in excluding
pernicious
anemia
(17, 18). In
niacrocytic
anemias
associated
with aclilorhydria,
the level of pepsinogel is not altered
by stimulation
with histamine.
Although
this usefulIICSS is recognized,
the significance
of the determination
of pepsinogen
in 1)100(1 is not well understood,
and only a few studies
concerning
the
levels of pepsinogen
in blood have been published
(4, 7, 8, 11, 18).
The
determination
of pepsinogen
in blood
may be more practical
and may
Sill plify
the diagnosis
of these diseases,
avoiding
the difficulty
of urine
coliectiOll.
In the present
nivestigation,
therefore,
a new accurate,
simple method for the deterniiiiation
of
in blood and urine
was developed.
Materials and Methods
Reagents
Crystalline
Copenhagen,
pepsin
i)enmark)
Specific
trypsin
Specific
Crystalline
activity
activity
1 :10,000
(Kemisk
Fatrike,
1 :250 (Difco)
Human
serum protein
This is inactivated
for endogenous
pepsin
and pepsillogell.
Serum
protein
obtained
from
healthy
subjects
is
incubated
for 1 hr. at pH 2 and at 37#{176},
following
which it is adjusted
to pH 8.
Egg
albumin
Casein
(Difco)
(Hammersten,
Germany)
Phenol
reagent,
Folin-Ciocalteu
Tokyo,
,Jaj)an)
The phenol
reagent
distilled
water.
HC1, tricliloracetic
special grade, Daiichi
(19)
is diluted
(Daiichi
with
Chemical
Co.,
two volumes
of
acid, and NaOH
(Japan
Industrial
Chemical
Co., Tokyo, Japan)
Standard,
Procedures
Pepsinogen in Blood
The determination
of pepsinogen
in blood is carried
out using the
patient’s
own serum protein as a substrate;
thus, the final concentration
of serum protein
in the reaction
system is uniformly
controlled
in each
sample. A suitable
amount of serum specimen
is taken for study, so that
when it is diluted to 4 ml., the final concentration
of serum protein
will
he 1%. In general,
the amount of serum specimen
required
for the study
is less than 1 nil. An aliquot of the serum specimen
is made up to 3.5 ml.
with distilled
water and, after adjusting
the pH to 2 with 8 gm. HC1 per
44
UETE fT AL.
Clinical
Chemistry
100 ml. (w/v), the fiuial volume is made up to 4 ml. by adding distilled
water;
the solution
is then incubated
at 37#{176}
for 24-48 hr. The reaction
is terminated
by the addition
of 1 ml. of 20% (w/v) TCA. To 0.5 ml. of
the TCA filtrate,
0.6 ml. of phenol reagent
and 2.0 ml. of 0.5 N NaOH
are added and mixed.
The absorbance
is measured
at 690 m
after
20 mm. at room temperature.
A tyrosine
standard
is run concurrently,
and the amounts
of tyrosine
liberated
from serum protein
by pepsin
are estimated.
A serum specimen
is similarly
incubated
with 1 ml. of
20% TCA; this serves as the blank. Tile enzyme levels either are expressed
by the amounts
of tyrosine
liberated
from serum protein
per
unit of time per milliliter
of serum,
or they are calculated
by the
simultaneous
incubation
of crystalline
pepsin
with 1% human
serum
protein,
inactivated
for endogenous
pepsin and pepsinogen.
The concentration
of serum
protein
is simply
determined
using a
ref ractrometer
with one drop of serum; the necessary
amount of serum
specimen
for the study is easily calculated.
In order to obtain an increased
sensitivity
of the reaction
system, the amounts
of serum in the
reaction
system can be increased-e.g.,
1.5% of the final protein
concentration.
Pepsinogen in Urine
A urine aliquot of 0.02-0.04 ml. is added to 4 ml. of 1% human serum
protein
(endogenous
pepsin
and pepsinogen
having
been inactivated
and the pH adjusted
to 2 with HC1), and is then incubated
for 24 hr. at
37#{176}.
The reaction
is terminated
by adding
1 ml. of 20% TCA. The
amount
of protein
digested
is determined
as in the methods
for the
determination
of blood pepsinogen,
and the levels of pepsinogen
are
estimated.
Results
Effect of pH
On Proteolytic Action of Crystalline
Pepsin and Trypsin
The effect of pH on the proteolytic
activity
of pepsin upon various
proteins
was studied
(Fig. 1). llJnder the conditions
of the present
experiment,
crystalline
pepsin
digested
human
serum protein,
egg albumin, and casein maximally
in the region of pH 2. On the other hand,
crystalline
trypsin
digested
only casein in the region of pH 8. Serum
protein and egg albumin
were not digested
by trysin.
On Proteolytic Action of Blood and Urine
Since
activity
crystalline
pepsin
digests
serum protein
at pH 2, proteolytic
of blood was studied using its own (autologous)
serum protein
as a substrate
at various
pH levels in order to investigate
whether
or
Vol. 15, No. I. 1969
DETERMINATION
OF PEPSINOGEN
45
not endogenous
pepsin
and pepsinogen
in the blood digests
its own
serum protein.
When serum
from healthy
individuals
was incubated
at varying
pH levels (pH 1-11), serum protein
was digested
between
pH 1 and 4, showing a maximum
peak at pH 2 (Fig. 2). This result was
e
Egg Albumin
2
1,000
asetn
-4
-.4
ii,
500
I
2
3
4
5
6
7
8
9
10
11
pH
Fig. 1. Effect of pIE on proteolvtic
action of pepsill
and trypsin
upon liuniaii serum
protein,
egg albumin,
and casein.
Amount
of enzyme
used:
15 tg. per tube;
concentration
of various
substrate proteins:
lc/( . Incubation
was at 87#{176}
for 4 hr. Proteolytic
activities
of pepsin
and
Ilypsin
on various
sul)strates
are expressed
as.mmiiouitts
of tyrosine
liberated
front substrate
proteins.
I’
200
bO
Serums
150
,
4,
‘5
Serum
+
sein
.
-4
‘-I
10o
UI
50
1
Fig. 2. Effect
of pIE on
2
pi-oteolytic
3
4
5
6
7
action
of blood on
Concentration
of serum
protein
used:
1%. Incubation
was
casein was used as substrate,
1% casein, pH 2, was added.
8
its
at
9
own
370
10
pH
serum protein
amid pH 2 for
and eascin.
24 hr. When
46
UETE fT AL.
in agreement
the finding
in
with serum
When
serum was pretreated
at
then adjusted
to pH 8, the pepsin
pepsin
was
with
incubated
Clinical
the experiment
protein.
pH 2, incubated
and pepsinogen
in which
Chemistry
crystalline
at 37#{176}
for 1 hr., and
appeared
to be com-
pletely destroyed;
this pretreated
serum did not show any proteolytic
action 011 either serum protein
or casein in the region of pH 2.
When 1% casein was added and used as a substrate
for the determination of pepsin
and pepsinogen
ill blood,
the proteolytic
activity
was
observed
in the region of pH 1-4, showing
a maximum
of proteolytic
activity
at pH 3.0-3.4
(Fig. 2); this is in contrast
to the effect of
crystalline
pepsin
on casein.
Nevertheless,
crystalline
pepsin
exerted
its maximum
proteolytic
effect on casein at pH 2. In addition,
blood
digested
casein in the region of pH 8, suggesting
tryptic
activity,
as
seen in Fig. 2.
Similarly,
the effect of pH on the urinary
proteolytic
activity
on
casein, egg albumin,
and serum protein
was studied,
and was shown to
exert a marked
proteolytic
activity
on these substances;
maximum
(ligestion
was observed
at pH 2 in all three (Fig. 3). Urine also had a
proteolytic
activity
effect on casein in the region of pH 8, suggesting
t ryptic
activity.
Proteolytic Action of Crystalline Pepsin
Effect of Substrate Concentration
To investigate
tile optimal
concentration
of substrate
for the determination
of pepsin and pepsinogen,
serum protein
at various
concentrations-0.5,
1.0, 2.0, and 4.0%-was
incubated
with crystalline
pepsui at IJH 2 and 37#{176}.
Tn this experiment,
a correlation
between
the
proteolytic
activity
and the amount
of pepsin
used was observed.
No
signifIcant
difference
in the amounts
of protein
digested
by pepsin with
0.5, 1.0, and 2.0% serum protein
was observed
under the conditions
of
1,000
Sum
3
Protein
800
Egg Albwntn
600
2
400
200
0
5.
.‘
/
Isein
.
/
‘.
.
1
#{149}/
...
-..-..
-.----:
1
Fig.
2
3
4
5
6
7
8
9
13
11
pH
3. Effect
,f pIE on proteolytie
action
of urine
on serum
protein,
egg albumin,
and
taseill . Anmoumit of urine
used:
0.1 ml. per tube;
concentration
of various
substrate
proteins:
1e,. Timeulmatiomi of urine
specimen
was with 4 ml. of substrate
proteins
at 370
and pH 2 for
24 hr. Proteolytic
activity
is expressed
as amount
of tyrosine
liberated
from substrate
proteins.
47
DETERMINATION OF PEPSINOEN
Vol. 15, No. I, 1969
this experiment.
However,
the proteolytic
action of crystalline
pepsin
on serum protein
was reduced with 4% serum protein.
The results
are
shown in Table 1.
Effect of Time
When 0.2, 0.4, and 0.8 g.
4 ml. of 1% serum
protein
of crystalline
(inactivated
pepsin were incubated
for endogenous
pepsin
with
and
pepsinogen)
for 24, 48, and 72 hr. at pH 2 and at 37#{176},
the units of activity
obtained
were linearly
proportional
to the time of incubation
(Fig. 4). However,
when the greater
amounts
of crystalline
pepsine.g., 2, 4, and S p.g.-were
incubated
under
the same conditions,
the
amounts
of serum I)Iotein
digested
were not linearly
proportional
to
the time of incubation
(Fig. 4).
Table
1. EFFECT
Tests
(No.)
---
OF I1ui.Siaui
PROTEIN
CONCENTR.TION
PROTEOLYTIC ACTION OF CRYST.tu.INE
PEPsIN
lib.r,,te,I
T&ro.ine
l’epnn
2
4
S
----
--
24 br.
-
0.5%’
1.0%
244 ±54
386 ± 53
598±74
S
5
S
,j.
(IN
2.0#{176}
4.0%
425 ± 132
231 ± 55
396 ± 53
176 ± 32
231 ± 37
618±
640±82
576 ± 78
262 ±
28
69
Immenbations were carried out at 37#{176}
and p11 2 for 24 h
Concentrations
of se:um protein.
*
200
2000
160
1600
1 200
800
A
40
400
24
Fig. 4. Effect
Crystalline
Proteolytic
pepsin
activity
48
of time
72
on proteolytie
was incubated
is expressed
24
nrs.
action
of crystalline
8 p
4
pepsin
48
on human
with 4 ml. of 1% human
serum protein
at
as amount
of tyrosine
liberated
from human
72
serum
hrs,
protein.
pH 2 amid 370
serum protein.
48
UETE fT AL.
Quantitative
Clinical
Chemistry
Study
When 0.2, 0.4, and 0.8 g. of crystalline
pepsin
were incubated
with
4 ml. of 1% serum protein
at pH 2 and 37#{176}
for 24 and 48 hr., the units
of activity
obtained
were linearly
proportional
to the concentration
of
enzymes
(Fig. 5). However,
when greater
amounts
of crystalline
pepsin
(2, 4, and 8 ,jg.) were similarly
incubated
with 4 ml. of 1% serum
protein,
the units of activity
obtained
were not linearly
proportional
to the concentration
of enzyme
(Fig. 5).
Under
the conditions
crystalline
pepsin below
were linearly
proportional
time of incubation.
of tile present
investigation,
when levels of
1 g. were used, tile units of activity
obtained
to the concentration
of enzyme and to the
Effect on Serum Proteins
Since protein
composition
of human serum specimens
from different
individuals
is variable,
the effect of crystalline
pepsin on various
serum
samples
obtained
from healthy
individuals
and from patients
with
various
diseases
was studied with respect to its proteolytic
action. The
results are shown in Table 2.
Endogenous
pepsin and pepsniogen
in these serums were first inactivated and the following
experiments
were then carried
out: In Experiment 1, the effect of a large quantity
of crystalline
pepsin, 20
was
studied with various
human serum specimens.
In Experiment
2, a small
quantity
of crystalline
pepsin,
5
was incubated
with human serum
200
2O00
160
1,600
I
.9120
1,200
80
800
o - 48 hours
400
__._._...__-
-
Pepsin
0,2
Fig.
protein.
Crystalline
pH 2. Protcolytic
0.6
0.4
5. Quantitative
pepsin
activity
study
of
0.8
proteolytic
24
hour*
Pests
pg
4
2
action
of
crystalline
was incubated
with 4 ml. of 1%
is expressed
as amount
of tyrosino
pepsin
on
human
serum
human
serum protein
at 37#{176}
amid
liberated
from serum protein.
DETERMINATION
Vol. 15, No. I, I969
49
OF PEPSINOGEN
lii both ills! ailces, tile uiiial c()Ilcentratioll
of total serum
protein of each salnple was i.
Under the con(litions
of the present
study,
no significant
difference
ill the proteolytic
action of crystalline
pepsin
on various
human serum
specimens
was observed,
and the amounts
of
tyrosine
liberated
from the digestion
of these serum protein
specimens
by crystalline
pepsin
did not differ significantly,
(average
deviations
were 0.7-4.3%),
despite
tile difference
in the composition
of serum
proteins
in each sample. The total protein concentrations
of the original
serum specimens
were from 6.8 to 7.8%. The albumin :globulin
ratios
of these serum
specimens
ranged
from 1.1 to 3.8. This experiment
strongly
supports
the accuracy
of the present
method for the determination of blood pepsin
and pepsinogen
using autologous
serum protein.
SpeCimells.
Serum Pepsinogen Determination
Since the concentration
not significantly
influence
Using Autologous Serum Protein as Substrate
(0.5-2.0%)
the
of serum
protein
as substrate
proteolytic
activity
of pepsin
when
did
tile
incubation
was carried
out at pH 2 and 37#{176}
for 24 and 48 hr., a study
was carried
out concerning
the relationship
between
the amount
of
serum
used and its own proteolytic
activity,
with the final concentration
of serum protein
in the reaction
system
below 2.0%. In this experiment, 0.3, 0.6, and 0.9 ml. of serum obtained
from a healthy
male was
200
48 hrs
160
Fig.
amounts
protein.
pressed
erated
6. Proteolytic
action
of various
of serum on autologous serum
Proteolytic
activity
is exas amount
of tyrosine
libfrom
serum
so
5-
120
0
4.
protein.
24hrs.
40
Serum
0.3
0.6
0.9
ml
made up to the final volume of 4 ml., distilled
water was added, and the
pH was adjusted
to 2. The final concentration
of serum protein
of these
samples
was, respectively,
0.5, 1.0, and 1.5%. These serum specimens
were incubated
at pH 2 and 37#{176}
for 24 and 48 hr. The results are shown
UETE fT AL.
50
in Fig. 6. Tile
and the serum
tions : twice as
as with that of
linear relationship
protein
(ligested
much as protein
24 hr.
Clinical
between
the
amounts
Chemistry
of serum
used
is seen in 1)0th 24- and 48-hr. incuhawas digested
wit-h the 48-hr. incubation
Effect of Time on Proteolytic Action of Serum on Autologous Serum Protein
When the activities
of pepsin and pepsinogen
in blood of 2 healthy
individuals
were determined
under the conditions
of the present
method
using 24-, 48-, and 72-hr. incubation
times, the levels of activities
obtamed were linearly
proportional
to the times of incubation
(Fig. 7).
Specificity of Blood Pepsinogen Method
\Viien the blood pepsinogen
levels of totally gastrectomized
patients
were determined
by tile present
method,
extremely
low levels were
observed,
indicating
that the method
is specific for pepsinogen,
as is
shown in Table 3.
Optimal Conditions for Urinary Pepsinogen Method
Ill
were
all instances
used
when
as substrates
human
for
serum
the
protein,
determination
Serum
B
Serums
A
casein,
and
of pepsinogen
egg albumin
in urine,
200
160
52
5-
Fig. 7. Effect
tion
120
of serum
of time
on autologous
Serum
specimens
conditions
of the
on proteolytic acserum
protein.
incubated
under
method
for determination
of pepsinogen
in blood at various time intervals.
Proteolytic
activity
of
serum is expressed
as amount
of tyrosimie
liberated
from serum protein.
0
5.
80
24
48
were
present
72 hrs
the optimal
pH was 2. Of all the protein
SUi)Strate used, human serum
protein
was digested
by urine, as well as by crystalline
pepsin,
at the
highest
rate (Fig. 1 and 3).
When the shorter
incubation
time (e.g., 2-4 hr.) is used for the
Vol. 15. No. I, 1969
Table 2. PROTEOLYTIC
SPECIMENS
ORTAINED
ACTION OF CRYSTALLINE
FROM HEALTHY
INDIVIDUALS
Composition
Serum
specimen
Total
(gm!
100 ml.)
(No.)
Albumi,,
(% of
total)
7.4
7.8
7.5
7.7
PEPSIN
Globulin
A: G
ratio
---
-y
1 (wITH
20 pG.
l)IsEbSES
liberated
by pepsin
7.6
10.4
7.3
5.3
7.1
7.9
5.9
4.6
2 (wITH
5 pG.
Ar.
g/.24
deriatmon
(%)
hr.
PEPSIN)
20.3
26.2
18.1
9.3
1.7
1.2
2.0
3.8
1110
1030
1096
1020
1064
+4.3
-3.2
+3.0
-4.1
AVERAGE
EXP.
SERUM
(% of total)
----
2.5
1.2
1.4
2.0
HuM.N
WITH V.%RIous
Tyrosine
sit
62.5
54.3
67.3
78.8
ON \ARIOUS
AND PATIENTS
of serum prokin
-----
EXP.
2
3
4
51
DETERMINATION OF PEPSINOGEN
PEPSIN)
1
2
7.2
7.3
53.2
59.3
2.8
1.7
10.9
10.3
8.1
8.6
25.0
20.1
1.1
1.4
418
412
+0.7
-0.7
3
4
5
7.4
6.8
7.5
57.3
68.7
57.6
2.1
2.1
1.4
8.9
6.3
9.1
8.0
5.6
7.S
23.7
17.3
24.1
1.3
2.2
1.4
420
402
422
+1.2
-3.1
+1.7
415
AVERAGE
Incubations
were
carried
Table 3.
omit at 37#{176}
amid p11 2 for 24 hr.
ACTIVITY OF Bmoon OF
TOTALLY GASTRECTOMIZED
PATIENTS
PROTEOLYTIC
HEALTHY
AT
Subject
Condition
Blood
Sex
Healthy
Healthy
Healthy
Healthy
Healthy
Ga.st-rectomized
Gastrectomized
2
3
4
5
6
7
specimnelis
were
SUBJECTS
ol)taiued
before
Age (yr.)
M
1\E
M
38
20
60
\[
37
M
F
55
41
26
ANI)
2
Digestion
-
No.
H
of serum protein:
tyrosine
)g./ml./4
liberated
hr.)
103.2
81.0
65. 1
120.6
7.0
3.5
breakfast
determination
of urinary
pepsinogen,
the preincubation
of urine at
pH 2-3 and 37#{176}
is necessary
to convert
pepsinogen
to pepsin in order
to obtain accurate
results,
similar
to the method
of Peak et al. (17).
Otherwise,
activity
obtained
is not linearly
proportional
to the time of
incubation,
since the conversion
of pepsirlogen
to pepsin in urine requires an incubation
of 1-2 hr. at pH 2 and 37#{176}.
In this case, 0.1-0.2 ml.
of activated
urine specimen
is incubated
with 4 ml. of 1% serum protein
(inactivated
for endogenous
pepsin and pepsinogen)
for 2-4 hr.
When
a longer
incubation
time (e.g., 24-48 hr.) is used, the preincubation
may be omitted.
Aliquots
(0.02-0.04
ml.) of urine specimens
were incubated
at 37#{176}
and pH 2 for 24-48 hr. with 4 ml. of 1% serum
UETE fT AL.
52
Clinical
Chemhtry
protein
that had been inactivated
for endogenous
pepsin and pepsinogen. In this case, the activity
obtained
was linearly
proportional
to the
time of incubation
(Fig. 8). However,
with a larger
aliquot
of urine,
linearity
was not obtained
when activity
and time of incubation
were
plotted.
200
‘60
02
Fig.
rJ.
atjon
120
8. Effect
of time
on proteolytic
of urine
on human
serum
pro-
tein. Urine
specimens
were incubated
with 4 ml. of 1% serum
protein
at
pH 2 and 370
Proteolytic
activity
is
expressed
as amount
of tyrosine
lib-
30
erated
from
serum
protein.
40
24
48
hrs.
72
Proteolytic Activity of Blood at pH 2
A preliminary
study
of the proteolytic
activities
of various
serum
samples
on autologous
serum protein
at pH 2 in healthy
individuals
and gastrectomized
patients
was carried
out using the present
method.
The results are shown in Table 3.
Discussion
A number of methods
for the determination
in urine and blood have been developed
for
function.
substrates.
Many
Tile
of them
utilize
denaturated
method
of Mirskv
et a!. (7),
of pepsin and pepsinogen
the evaluation
of gastric
hemoglobin
or
casein
as
which utilizes
denaturated
used for the determination
as a substrate,
has been widely
of blood pepsin
and pepsinogen.
According
to these investigators,
when
denaturated
hemoglobin
was used for the determination
of the proteolytic
activity
in blood,
the hemoglobin
was maximally
digested
at
pH 3-4. This fact is of interest,
since crystalline
pepsin
digested
lienloglobin
maximally
at pH 2 under tile same conditions.
Furthermore,
hemoglobin
these
authors
natiirated
reported
hemoglobin
that
at- ph
tile
proteolytic
action
2-4 was not modified
of
serum
on
by tot-al gastrectomy.
de-
Vol. 15. No. I, 1969
DETERMINATION
OF PEPSINOGEN
53
This fact strongly
suggests
that this proteolytie
action
of serum
on
hemoglobin
in the region of pH 3-4 possibly may not be due to the action
of pepsin
or pepsinogen
in blood. However,
if the proteolytic
activity
is measured
at pH 1.5, the activity
determined
represents
the activity
of pepsinogen
in blood (7). In the present
investigation,
crystalline
pepsin digested
casein maximally
in the region of pH 2. However,
serum
from healthy
subjects
or patients
with various
diseases
digested
casein
maximally
at pH 3-4, as in the case with hemoglobin.
From the present
results,
it is not possible
to say that this proteolytic
activity
of serum
on casein at pH 3-4 is due mainly to pepsin
and pepsinogen
in blood.
Whether
or not the determination
of the activity
of pepsin and pepsinogen in blood using hemoglobin
or casein as substrate
at pH 1-4 quantitatively
represents
accurate
levels of pepsinogen
in blood is questionable.
In -the present
ulvestigatioll,
crystalline
pepsin also digested
human
serum protein
maximally
in the region of pH 2. When the proteolytic
activity
of blood was determined
using autologous
serum
protein
as
substrate,
tile serum protein
was digested
maximally
in the region of
pH 2, as was the case with crystallille
pepsin action on serum protein.
This is in contrast
to tile determination
of proteolytic
activities
in blood
using denatured
hemoglobin
or casein. This finding strongly
indicates
that human serum protein is the preferable
substrate
for the determination of blood pepsin and pepsinogen.
Although
Edwards
et l. (ii) have
used dried human plasma as a substrate
for the determination
of blood
pepsinogen,
this is unnecessary
since the patient’s
own protein
can be
used.
When the levels of pepsin and pepsinogen
in blood of gastrectomized
patients
were determined
by the present
method,
extremely
low proteolytic activities
in the region
of pH 2 were obtained.
In addition,
the
proteolytic
action of serum or plasma
on its own serum protein
in the
region of pH 2 was inactivated
by alkalinization.
Therefore,
it appears
highly
probable
tilat -the proteolytic
activity
in blood, determined
by
the present
method,
is pepsinogen
that is secreted
into the circulation
by the peptic cells of the stomach,
and that with acidification
in vitro,
an autocatalytic
conversion
to pepsin ensues. The levels of pepsinogen
in blood and tile blood volume used for the determination
in the present
study were well correlated.
The activity
of enzyme obtained
was linearly
proportional
to time under tile conditions
of our experiments.
Although
in the present
method
the specimens
of serum protein
obtained
from
the different
individuals
were used as substrate,
the proteolytic
effects
of crystalline
pepsin
on these serum
protein
substrates
did not significantly
differ, indicating
the accuracy
of the method.
54
IJETE fT AL.
rfhese
findings
show
that
the
present
Clinical
method
is more
Chemistry
simple
and
specific than the method employing
henioglobin
or casein as substrates.
The simultaneous
determination
of pepsii
and pepsinogen
in blood
and urine would be of value in clarifying
the physiologic
role of pepsin
and pepsinogen
in the body under various
conditions.
Tile fact that pepsin, secreted
by gastric
peptic cells into the circulation, is inactivated
at the p11 of blood is well known (17, 18). Therefore,
the proteolytic
activity
of blood at pH 2 is due to pepsinogen,
following
an autocatalytic
conversion
to pepsin
with acidification
of serum
in
vitro. In the present
investigation,
both serum and urine showed
no
proteolytic
activity
during
a period of 1 hr. of incubation
at pH 2 and
37#{176}.
After the activation
of serum and urine by 1-2 hr. of incubation
at pH 2 and 37#{176},
the proteolytic
activity
of serum
and urine
was
observed.
This is the time required
for tile autocatalytic
conversion
of
pepsinogen
to pepsin
in blood and urine.
This consideration
is important
to obtain accuracy
in the determination
of pepsinogen.
In the
present
study,
therefore,
a longer
incubation
time was selected
for
the routine
use.
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