CLIN. CHEM. 27/4,
586-593
(1981)
CentrifugalAnalysisfor PlasmaKallikreinActivity,with Use of the
ChromogenicSubstrateS-2302
Ralph Ito and Bernard E. Statland
The amidolytic activity of activated kallikrein in plasma can
be measured by use of the chromogenic substrate, S-2302
(H-D-Pro-Phe-Arg-pNA). Plasma prekallikrein was activated
to kallikrein by exposure to 50 mg/L dextran sulfate in
acetone/water
(35/65 by vol) at 0 #{176}C
for 15 mm. The acetone slows anti-kallikrein
activity and increases the kallikrein activity by 30%. The 37 #{176}C
reaction mixture contained 0.54 mmol of S-2302 substrate per liter of Tris
buffer (pH 7.5 at 37 #{176}C).
We monitored the change in absorbance at 405 nm for 60 s. The specificity of the substrate for kallikrein was demonstrated by using plasma
deficient in prekallikremn (Fletcher trait) diluted with pooled
normal human plasma. We recommend collecting blood
specimens with sodium citrate as the anticoagulant and
with use of a double-syringe
technique
and all-plastic
containers. Plasma kallikrein activity with Chromozym-PK
(Bz-Pro-Phe-Arg-pNA) as substrate (y-axis) compared with
S-2302 as substrate (x-axis) gave the relation: y = O.28x
+ 0.82 (r = 0.94). Day-to-day analytical variation was
2.4% for a pooled plasma with a mean value of 85.9
tkat/L The mean (and 2 SD) for 50 healthy adults was 86.4
(32.4) J2kat/L.
AddItional Keyphrases: blood coagulation
kreln
enzyme activity
reference interval
enzyme assay
variation, sources of
prekallikinetic
Measurement
of kallikrein
(EC 3.4.21.8) in plasma is of
great physiological
importance
because of the pivotal role
kallikrein
plays in several functions:
an early step in the
coagulation
cascade, the maintenance
of blood pressure, and
the influence on complement
consumption
and the fibrinolytic
system.
In earlier evaluation
of the kinetic activity of kallikrein
in
plasma, amino acid esters such as a-benzoyl-L-arginine
ethyl
ester and a-tosyl-L-arginine
methyl ester were used as the
substrates.
Various methods for measuring esterolysis of these
substrates
were used, such as measurement
of the liberated
alcohol (1, 2), differential
ultraviolet
absorption
of acids and
esters, and the radioactive-methanol
method
(3). Because
digestion of these substrates
is also catalyzed
by trypsin (EC
3.4.21.4), soybean trypsin inhibitor
was used to eliminate
interference
by endogenous
trypsin in the assay mixture. This
technique
gave some informative
results, but the use of radioactive isotopes may not be desirable,
and esterolytic
substrates
lack specificity
for the amidolytic
enzyme
kallikrein.
There currently
are three non-radioactive
amidolytic
substrates available commercially
for use in the kinetic analysis
for plasma kallikrein (PK). Two are chromogenic,
S-2302 and
Chromozym-PK.
The third is a dipeptide
fluorogenic
subDepartment of Pathology, University of California, Davis, Medical
Center, 2315 Stockton Boulevard, Sacramento, CA 95817.
Address correspondence
to this author.
Received June 4, 1980; accepted Feb. 3, 1981.
588
CLINICAL #{212}IEMISTRY,
Vol. 27, No. 4, 1981
strata with the fluorescent
compound
7-amino-4-methylcoumarin linked to an arginine group. Amundsen
et al. (4) first
described
a method for assaying plasma kallikrein
with use
of the chromogenic
substrate
Chromozym-PK,
and later a
modification
of this method was reported
by Stormorken
et
al. (5). A very extensive evaluation
of PK activity as measured
with the same substrate
was reported
by Kluft et al. (6, 7).
Their approach
was to activate plasma prekallikrein
(PPK)
with dextran
sulfate at 0 #{176}C
and then measure
with a double-beam spectrophotometer
the rate of p-nitroaniline
release
at 37 #{176}C.
Friberger
et al. (8) and Claeson et al. (9) using the
substrate
S-2302, presented
similar methods.
PK analysishas some unique problems:
activation
of PPK
to PK must be complete and PK activity decrease by natural
inhibitors
in plasma must be prevented.
We address both of
these problems
in the present method by use of a mixture of
dextran sulfate and acetone in the activation step. In addition,
we chose the substrate
S-2302 for its greater sensitivity
and
used a centrifugal
analyzer to measure the rate of substrate
degradation.
Materials and Methods
Apparatus
Plasma kallikrein
activity was determined
kinetically
by,
monitoring
the change in absorbance
at 405 nm as measured
in the centrifugal
analyzer
(CentrifiChem
System 400 Analyzer; Union Carbide Corp., Tarrytown,
NY 10591).
Reagents
The chromogenic
substrate
S-2302 (H-D-Pro-Phe-ArgpNA. 2HC1, M = 611.6; courtesy
of KABI Group
Inc.,
Greenwich,
CT 06830) was dissolved
in de-ionized
water to
make a 6.0 mmol/L stock solution, which was stable for at least
two months at 2 to 8 #{176}C.
The 0.6 mmol/L working substrate
was prepared
by diluting
the stock solution
10-fold with
tris(hydroxymethyl)methylamine
(Tris) buffer (50 mmolfL,
pH 7.8 at 24 #{176}C)
containing
12 mmol of NaC1 per liter. Chromozym-PK
(Bz-Pro-Phe-Arg-pNA
. HC1, Mr = 679.3; Pentapharm
AG, Basle, Switzerland)
working substrate
was dissolved in Tris buffer to a concentration
of 0.6 mmol/L.
pNitroaniline,
50 mol/L
aqueous standard,
was a gift from Dr.
Lam Mellstam
(AB KABI Peptide Research,
Molndal,
Sweden). Dextran sulfate (M = 500000; Pharmacia
Ltd., Uppsala, Sweden) was prepared
by dissolving
10 mg in 30 mL of
de-ionized water, then adding 70 mL of acetone. The solution
was stable for at least three months
at 2 to 8 #{176}C.
Fletcher
(prekallikrein)-deficient
plasma was kindly supplied
by Dr.
Charles Abildgaard
(University
of California,
Davis Medical
Center).
Hageman
(Factor XII)-deficient
plasma was purchased from Dade Diagnostics,
Inc., Miami, FL 33152. Normal
human plasma was collected
and pooled according
to the
procedure
outlined
below, from volunteer
laboratory
personnel. Plasma from a minimum
of 10 healthy
adults was
pooled and 200-1L aliquots were stored at -50 #{176}C
in plastic
containers.
Acetone
and all other reagents
were of AR
grade.
Specimen
Collection
70
and Preparation
Blood was collected by venipuncture,
with 0.1 mol/L sodium
citrate as anticoagulant
(one part sodium citrate to nine parts
blood), with use of a double-syringe
collection technique.
In
this technique
two syringes are used. The needle is inserted
into the vein and 2 to 4 mL of blood is drawn. The syringe is
removed,
leaving the needle in place. The second syringe is
attached
to the needle and the specimen
is collected.
The
purpose of the first syringe is to remove “endogenous”
tissue
factors
that may contaminate
the specimen
during venipuncture.
The contents of second syringe are presumed
to be
free of such contamination.
All syringes and tubes are plastic.
We centrifuged
the specimens
within 1 h of collection
(at
15-24 #{176}C,
1500 X g, 10 mm), removed the plasma with a plastic
pipette,
and stored it in plastic containers.
The specimens
either are maintained
at room temperature
and assayed within
3 h or stored at -50 #{176}C
for future analysis.
The effects of spontaneous
cold activation
of PPK were
investigated
by collecting specimens
and storing plasma allquota from two subjects at 4#{176}C.
The aliquots were assayed for
PK activity with and without activation
with dextran sulfate
and 35 mL/dL acetone.
Procedure
Kallikrein
for Activation
and Analysis
of Plasma
Frozen specimens
are thawed at room temperature
until
clear. Aliquots of Tris buffer (50 mmolfL) and dextran sulfate
solutions are chilled in an ice bath before use. After 13 X 100
mm glass tubes are placed in the ice bath, 50 zL of test plasma
is added to each tube. Plasma prekallikrein
is activated to PK
by adding 50 tL of the dextran sulfate solution, mixing, and
incubating
for 15 mm at 0 #{176}C.
Then 1300 iL of cold buffer is
added and, without delay, 40 JLL of this mixture is pipetted
into the sample well of the CentrifiChem
transfer disc, which
contains
350 j.tL of S-2302 working substrate
solution in the
adjacent
reagent well.
The CentrifiChem
Analyzer
is programmed
as follows:
initial reading time = 3s, reading interval = 1 mm, temperature = 37#{176}C,
filter = 405 nm, and blank set to “auto.” Plasma
kallikrein
activity is measured
as the change per minute in
absorbance
at 405 nm (.A/min).
The results are calculated
by multiplying
AA/min times a factor derived from the molar
absorptivity
of p-nitroaniline
(e,#{176}’= 9.20 X 106 L-mol’
cm’)
and expressed
in tkat/L
(1 kat
= 60 U). We assume
a hematocrit
of 40 mL/dL. Given our conditions,
the resulting
plasma volume in the final assay mixture would be 1.19 uL.
Then
Activity,
U/L =
=
mm
reaction
sample
A
390 L
106
mm
1.19 iL
9.20 X 106
(.i
=
min)
vol
vol
60
30
V
40
30
20
O.I371,
5,.
22X104,rn,oI/L
10
VMAX
-
-.
-‘
#{163} i
5
5
1)
LV
IS)
Fig. 1. Reciprocal of activity of plasma kalllkrein in pooled
normal human plasma as a function of reciprocal of substrate
(S-2302) concentration in Tris buffer, pH 7.5 at 37 #{176}C
mixture for PK stability
was determined
by constructing
a
gradient of acetone concentrations
(0-40 mL/dL),
each containing 50 mg of dextran
sulfate per liter, as the activation
solutions.
Plasma pool was activated
in duplicate
for 15 mm
with each solution in an equivolume
ratio at 0#{176}C,
then diluted
with Tris buffer. The activated
plasma pools were brought to
23 #{176}C
for various timed intervals to allow response of viable
PK inhibitors.
Then samples under each test condition
were
assayed, to determine
the optimum
acetone concentration
to
use for PK stability
with time.
Reference
Values
Plasma specimens
assayedto determine
done during several
from 50 ostensibly
healthy adults were
the reference interval. These assays were
days on 17 men (age 19-48 years) and 33
0.700
ACTIVITY
AABS4,./MIN
kot/L
A 0.035
B 0.082
C 0.110
20.8
D 0.170
101.0
E
0.210
48.7
65.3
124.7
E
U)
106 umol/mol
e.light path in cm
z
L
0
U,
(35623 umol/L)
or
Activity,
ikatfL
= activity,
=
U/L
35623 umol
!.!I
L
60s
mm
=
11 min
z.A
594 umol
mm
L
Plasma Kallikrein
Activity with Various Acetone
Concentrations
in the Activation
Mixture
The optimum
acetone
concentration
in the PPK
activation
REACTION TIME, SECONDS
Fig. 2. Absorbance at 405 nm as a function of time for each of
five plasma dilutions assayed for kalllkreln activity
CLINICAL CHEMISTRY,
Vol. 27, No. 4, 1981
587
240
C,
z
z
200
‘-I.-
-J
0
160
I-
120
5
IU
PERCENTACETONEIN
F
80
40
0.7
.5
2.1
1.4
p.L POOLEDPLASMAIN REACTIONVOLUME
Fig. 3. LInearity of method for plasma kallikrein activity as assessed from dilutions of plasma specimens
women (age 20-60 years). Of the women in the study, three
were taking oral contraceptives
and three were pregnant (first
or early second trimester).
In a longitudinal study, five women (24, 25, 27, 28, and 31
years of age) had specimens drawn periodically
during 28 days,
and theirrespective dates of menstruation
were noted. All the
specimens
collected from a particular
subject were frozen and
thawed and then assayed in one batch,to eliminate day-to-day
analytical
variation. One subject (number 5) reported taking
oral contraceptives
during the course of this study.
27%
35%
A-A
I
I
I
linear slope based on the values at 3 and 63s. The relationship
of initial activity (3-63 s) to volume of plasma was linear
(Figure 3).
Table 1. ActivatIon Conditions: Effects of
Temperature and Acetone (35 mL/dL)
_______________
PKacty.
M#{149}an Rango
Temp.,
Ac.ton.
0
0
a Results
kat/L
presnt
Yes
No
Yes
No
23
23
Fig. 4. Effects of various acetone concentrations (mLIdL) on
activity of plasma kallikrein remaining after Intervals at 23 #{176}C
Activation
of Prekallikrein
to Kallikrein
We recommend
activating PPK at 0#{176}C
with dextran sulfate
and acetone for 15 mm. Dextran sulfate was used to initiate
the activation
process. Acetone and low temperatures
(0#{176}C)
were used to prevent PK inhibition (Table 1). Figure 4 demonstrates that 35 mL of acetone per deciliter of activation
mixture is the optimum concentration
for stabilizing the PK
activity, because with lower concentrations
the activity decays
faster. Activation
of PPK with and without
acetone in the
assay mixture, based on five split-sample
trials, is compared
in Figure 5. Figure 6 demonstrates that PK activity was about
30% greater when acetone was used. The optimum time for
maximum PPK activation with acetone was 15 mm (see Figure
We examined
Conditions
We adjusted the Tris buffer, as suggested by Friberger et
al. (7) and Claeson et al. (8), to pH 7.8 at room temperature.
Figure 1 presents the relationship of initial activity (3-63 s)
to substrate concentration at pH 7.5 in the assaymixture. The
Km determined
from the Lineweaver-Burk
plot is 2.2 X iO
mol/L. The absorbance
readings at 405 nm at pH 7.5 and
substrate concentration
of 0.54 mmolfL are presented
at four
timed values, i.e., 3,63, 123, and 183 s for each of five dilutions
of plasma specimens (Figure 2). With higher activities there
was a tendency for absorbances
to deviate from the initial
588
20%
o-o
5).
Results
ec
a-a
5
10
15
20
25
ELAPSED TIME AT 23#{176}C
PRIOR TO MEASURING
ACTIVITY, MINUTES
0
Assay
ACTIVATION VOL.UME
#{149}.__-.
0%
89.7
73.6
88.5-90.3
72.5-74.8
12.6
12.3
12.3-12.8
11.6- 13.1
basedon fourreplicate determinations.
CLINICAL CHEMISTRY,
Vol. 27, No. 4, 1981
the possibility
14
_______
during
a
>-
I-
>
I-
0’
5% ACETONEIN ACTIVATIONVOLUME
#{149}
OS ACETONE IN ACTIVATION VOWUE
acty.
as compar.d to
method of choice
14
activation
-J
P.rc.nt
100
82
of PPK
specimen collection (Table 2). Specimens were collected from
four subjects under conditions simulating those that may
occur in routine collections.
As noted in Table 2 for either the
single- or double-syringe
technique, for storage up to 60 mm
or 180 mm, at 23#{176}C
or at 4#{176}C,
the activity when glasswas used
(Conditions I-P) varied from 68 to 79% of that for the proposed method (Condition A). When plastic was used for these
4
6
8
10
12
14
16
18
20
22
24
26
TIME OF INCUBATIONAT 0#{176}C,
MINUTES
FIg. 5. Mean activity
and range of activities of plasma kallikrein
after various intervals of incubation with dextran sulfate and
either no acetone or 35 mL of acetone per deciliter
Table 2. Effects of Blood-Collection Conditions and Short-Term Storage Conditions on Plasma
Kaliikreln Activity
Percent of activity under condition A
Conditions
A
E
F
0, P, 23, 60
D, P, 23, 180
S, P, 23, 60
S, P, 23, 180
D, P, 4,60
D, P, 4, 180
G
S, P, 4,60
H
S.
0,
D,
S,
S,
B
C
D
J
K
L
M
N
0
P
Subject
P, 4, 180
G, 23, 60
G, 23, 180
G, 23, 60
G, 23, 180
0, G, 4,60
D, G, 4, 180
S, G, 4,60
S,G,4,180
SubJect
Subject 2
1
3
Subject 4
Mean
.j00b
bob
100b
100b
100
109
111
111
108
118
109
111
87
78
79
69
82
107
98
107
115
113
108
115
83
81
85
77
85
107
93
99
112
120
106
101
84
74
77
73
79
102
93
96
103
105
91
95
62
57
59
52
60
106
#{149}99
103
110
114
104
106
79
73
75
68
77
72
75
73
56
69
77
69
90
78
74
71
57
53
75
68
‘0. double-syrInge technique; 5, single-syringe technique; P, plastic contaIners;0. glass containers; 23, 23 #{176}C;
4, 4#{176}C;
60, 60 mm; 180, 180 mm.
(ikat/L)
for Condition A: Subject 1, 79.7; Subject 2, 53.5; Subject 3, 85.6; Subject 4, 72.0.
variations (Conditions
99 to 114% of the proposed
B-H), the activity varied from
method. In the case of plastic
same
containers,
the value at 180-mm storage often seemed somewhat greater than at 60-mm storage. In addition, storage at
4#{176}C
for the plastic containers
seemed to result in a somewhat
higher value than at 23 #{176}C
storage.
However,
we did note
spontaneous
cold (4 #{176}C)
activation
of PPK on longer storage
in specimens
from two of the subjects. Table 3 presents the
plasma kallikrein
activity with and without dextran sulfateacetone activation
at 1.5, 24, and 36 h of storage at 4 #{176}C
for
each of two subjects. The PPK was activated to PK in the cold
before the 24th hour for Subject 1 and between 24 and 36 h for
Subject 2, possibly demonstrating
4#{176}C.
spontaneous
activation
at
Reliability of the Method
2’s..
Normal human pooled-plasma
-20 or -50 #{176}C
and were assayed
aliquots were stored at either
with and without acetone in
the activation mixture during the 30 succeeding days. The
results (Figure 7) indicate that normal human plasma stored
at -50 #{176}C
and assayed with acetone produced the most con-
70
C
i-;
60
P1
n#{176}
26
#{149}I.
y’ 0.68 + 0.79
r’ 0.91
30
201‘I
0(1/ 40
50
60
70
I
I
sistent results. No loss of activity was observed during the
month. Twenty-six aliquots of normal plasma stored at -50
were assayed, one per day, during 30 days. The values
ranged from 81.1 to 90.0 ikatfL
(mean, 85.9 zkat/L; SD, 2.1
tkatfL; CV, 2.4%).
The method was tested for PK specificity by diluting pooled
plasma with various volumes of Fletcher-deficient
plasma and
assaying for PK. The results (Table 4) demonstrate
that
Fletcher-deficient
plasma showed essentially no activity and
that the other results compared well with the expected
values.
The necessity for Factor XII activation
of PPK to PK was
tested by using Factor XII (Hageman
trait) -deficient plasma
to dilute normal human plasma, thereby comparing
PK activity for a range of Factor XII values. The results (Figure 8)
show the critical level for Factor XII to lie between 25 to 33%
of that contained in normal human plasma. When Factor XII
concentrations
were decreased, the time necessary for peak
activation was observed to increase (Figure 9).
In Figure 10 we compare PK activities with S-2302 and
Chromozym-PK
as substrates. The final concentration
for
both substrates were equal, i.e., the reaction volume concentration contained 0.54 mmol of each substrate per liter of Tris
buffer (pH 7.5 at 37 #{176}C).
There is excellent correlation (r =
0.94), but the activity with S-2302 was about 3.5 times that
with Chromozym-PK.
Reference Intervals for Healthy Adult Subjects
The distribution of our results for healthy adults is plotted
in Figure 11. The three women taking oral contraceptives
all
50
40
P$(value
I
I
80 90 100 110
ACTIVITY AFTER ACTIVATIONWITH 35%
ACETONE, A. kat /L
Fig. 6. Plasma kallikreln activity compared with and without
acetone (35 mL/dL) in the dextran sulfate activation mixture
Table 3. Effects of Cold Storage on Plasma
KallikreIn Activity Assayed with and without
Dextran Sulfate/Acetone Activation
Specimen stored
Subject 1
Activation No activation
at 4 #{176}C
for
(hours)
kat/L
1.5
24
36
106.5
33.9
19.5
0.9
9.0
8.5
CLINICAL CHEMISTRY,
Subject
2
Activation No activation
gkat/L
106.7
100.8
38.0
0.4
1.2
10.6
Vol. 27, No. 4, 1981 589
0
STORAGEAT -20C, ASSAYPERFORMED
WITHOUT ACEtONE ACTIVATION
STORAGE AT-50C. ASSAY PERFORMED WITHOUT ACETONE ACTIVATION
#{149}
STORAGEAT - SOC.
z
l00
ASSAY PERFORMED WITh 35% ACETONE IN ACTIVATION
VOLUME
k
#{176}FI
E
z
hJdHII ________
60
Ii I
#{176}fHI
soil I
2468
Ill
0
12 14
n
1#{128}
IS
24
26
28
20
40
>
GE0
P-I-
Fig. 7. Plasma kallikrein activity
TIME OF STORAGE,
of pooledCYS
plasma under various
0
AFTER
15 MINUTES
ACTIVATION
WITH
SULFATE
IN
35% DEXTRAN
ACETONE
ATO#{176}C
25
50
75
PERCENTOF FACTOR [
storage conditions
had significantly
greater PK values than did the other women
in the group. There was no significant
difference
between PK
values for women not taking oral contraceptives
and those for
the men. The mean value for these 50 individuals is 86.4(2 SD
32.4) 1zkatfL. Figure 12 shows the day-to-day
variation in PK
values in five healthy women during 28 days. Their individual
means and day-to-day
variations
are presented
in Table 5.
Subject number 5 was taking oral contraceptives
during the
study.
100
IN TEST PLASMA
Fig. 8. Percent conversion of plasma prekallikrein to plasma
kallikrein, based on the measured plasma kallikrein activity as
a function of Factor XII in the test plasma
A plasmadeficient In Factor XII was dIluted in various ratios wIth normal human
pooled plasma to construct the range of Factor XII values
kaolin, ellagic acid, and acetone were compared
as PPK activators by Kluft et al. (6, 7) and Friberger
et al. (8). Low activation temperatures
(0 #{176}C)
were shown to decrease the influence of PK inhibitors (13) and have been used by many
Discussion
investigators
(1,6-8). High dilution ratios of activated plasma
Major problems in setting up an assay for plasma kallikrein to buffer were used to decrease the concentration
of inhibitors
include (a) collecting and preserving the specimen without
(6-8,9)
and acetone was used to denature them (3,6). Using
activating
PPK to PK until it is deliberately
activated
by
a combination
of these methods,
we describe an activation
dextran sulfate; (b) activating PPK to PK as completely
as
procedure
most suitable for an automated
PK assay.Dextran
possible without
allowing combination
with naturally ocsulfate was selected as the activator because it is water soluble
curring inhibitors; and (c) developing an automated, rapid,
and therefore problems of insoluble activators (such as kaolin)
and reliable assay that is specific for PK.
were avoided in the centrifugal
analysis. Acetone was added
First, to avoid prior activation of PPK to PK, we used the
to the activation
step because it reportedly
increases PK acdescribed
double-syringe
collection
technique.
Glass contivity and stability by decreasing
the effect of the inhibitors
tainers should not be used, because glassmay cause premature
(3, 6). We show that 35 mL/dL
acetone
in the activation
activation
of PPK and the subsequent
combination
of PK
mixture is optimum
for PK stability;
concentrations
greater
with various naturally
occurring
inhibitors.
The specimens
than 35 mL/dL cause an increase in protein precipitation
and
stored at 4 #{176}C
maintained greater activity than those at 23 #{176}Cturbidity
in the activation
mixture and do not appear to induring the first 3 h after collection. However, we used 23 #{176}Ccrease PK activity or stability. This acetone concentration
in
for specimen processing before analysis since possible sponthe activation mixture increased results by about 30% over the
taneous cold activation of PPK may occur via Factor XII activation (6, 10, 11, 12). We have observed this phenomenon
(Table 2) and therefore suggest separating plasma from cells
and assaying
specimens
within
3 h, or storing
at -50
#{176}C
for
future analysis.
The second problem-activation
of PPK to PK without
combination with in vivo inhibitors-has
been dealt with in
various ways by other workers (1,3,5-9,
13). Current proce-
range tested as compared
with no acetone.
Thus, the three
advantages
of acetone use are: (a) greater PK activity,
(b)
increased
PK stability at peak activation, and (c) a smaller
range of variability
at peak activation. Because PK inhibitors
are both time- and temperature-dependent
(1,6, 14), we ac-
dures for PPK activation
include descriptions
of the effectiveness
of various PPK activators
as well as methods
to
minimize
the influence
of PK inhibitors.
Dextran
sulfate,
Table 4. Activity of Activated Plasma Kailikreln in
Various Mixtures of Fietcher(Prekailikrein)Deficient Plasma and Normal Human Pooled
Plasma (NHPP)
NHPP, ml per dL
Activity of mixture,
of NHPP and PP$C-d.ficlent
kat/L
plasma mixture
Mean
Range
100
50
33
20
0
‘Based
590
86.7
43.1
29.0
18.8
0.6
Activity as
percent of PlC activity
of NHPP
83.2-90.2
39.0-47.2
24.1-33.9
18.5-19.1
0.0-1.2
on three replicate determinations.
CLINICAL CHEMISTRY,
OF PLASMA PREKAU.IKREIN
WITH PLASWA CONTAINING
25% OF
FACTOR FOUNDIN NORMALPOOL
ACTIVATION
Vol. 27, No. 4, 1981
0#{149}
35%
100.0
49.7
33.4
21.7
0.7
ACETONE
IN
ACTIVATION
VOLUME
#{149}
0% ACETONE IN
ACTIVATiON
VOLUME
0
INCUBATIONTIME,MINUTES
Fig. 9. Plasma kallikrein activities compared with and without
acetone (35 mL/dL) in the activation mixture at various incubation times with a plasma containing 25% of the Factor XII expected to be present in pooled normal human plasma
-j
n ‘23
30’
S.,
+ 0.82
r ‘ 0.94
S
25
S
NJ
0
S
20
0
;
.
‘
-IS
0
F-.
-10
-5
0
+5
+10
+15
+20
+25
DaysBefas Menstruation
DaysAfter Menstruation
DAYOFSPECIMENCOLLECTION
15
>
Fig. 12. Day-to-day variation in values for activated plasma
kallikrein in each of five women subjects during a menstrual
S
cycle
IC-)
Subject number 5 was taking oral contraceptives
10
V
40
“30
50
I
60
I
70
80
_t
90
I
tOO
ACTIVITY, S-2302,,.tkat/L
Fig. 10. Plasma kallikrein activity as measured with Chromozym-PK as the substrate and with S-2302 as the substrate (same
specimen)
For in vitroprocedures, three elements are necessary for the
activation of PPK to PK: negatively charged surface particles,
kininogen
of high molecular
mass, and Factor XII (15, 16).
Factor XII in normal plasma was diluted by adding Factor XII
(Hageman)-deficient
plasma.
We found that the critical
amount for Factor XII lies between 25% to 33% of that contained in normal plasma when 15-mm activation
was used
(Figure 8). For this activation time and 25% Factor XII in
normal plasma, only 5% to 10% of the total PK activity was
observed
(Figure
9). When incubation
of this activation
mixture was prolonged,
the PK activity seen at 15 mm was
tivated PPK in the cold (0#{176}C)
to retard the effects of PK inhibitors. The results (Table 3) show the effects of temperature
and acetone
addition
on the activation
of PPK in NHPP.
From the evidence, we conclude that use of dextran sulfate
and 35 mL/dL acetone activation
at low temperature
(0 #{176}C)
results in the most suitable conditions
for PPK activation and
stability.
w
We determined
the Michaelis-Menton
constant (Km) at
pH 7.5 to be 2.2 X 10 mol/L for the substrate
S-2302 and for
normal pooled human plasma as the enzyme source. This
value is in close agreement
with that reported by Friberger et
z
al. (Km = 1.8 X i0
molfL), who used purified kallikrein
in
uJ
Ia similar system (8), and Claeson et al. (Km = 2.0 X 10
molfL) (9). The Km, as determined
from Figure 1, indicates
jJ
the desirable
S-2302
concentration
to be 2.2
that 10 times the Km were to be used, while
by making various dilutions of Fletcher (PPK)-deficient
plasma with pooled normal human plasma (Table 2). These
results indicate that no other plasma enzyme showed significant activity for the substrate.
14
0
lZ
0
0
10
0
0
0
0
0
0
0
0
0
2
0
E8
A
A
A
A
0
A
0
0
A
A
A
A
A
A
A
A
A
A
69
79
89
99
4
a
<4O
0
0
.5
S
90’
80
.
____________________
-
e
0
-I
30
MALE
0.
5
FEMALE
5’
FEMALE ON ORAL
20
FEMALEPREGNANT
CONTRACEPTIVES
10
‘-
5
0
0
40-
50- 60- 70- 80- 90- tOO-
0
A
S
S
110- 120-130-140- >150
49
59
119
0
109
0
4o
0
S
S
#{149}
‘SPECIMENSPROPERLY
PROCESSED
AND SlOPED
S’SPECIMENS IMPROPERLYPROCESSED
OR STORED
70
o
A’
tl.50
Z6
lOOl
substrate
mmol/L, assuming
we used 0.54 mmol/L substrate
or about 2.5 times the Km.
This was done to decrease substrate
consumption,
because its
cost is relatively
high. This concentration
did not appear to
affect the assay if the rate of reaction was measured within the
first 60 s after the reaction is initiated
(see Figure 2).
The specificity
of the S-2302 substrate
for PK was verified
-
110
129
139 149
Activated PlasmaKallikrein Activity, kot/L
Fig. 11. Activated plasma kalllkrein activity in each of 50 healthy
adults
0
I
I
I
I
I
I
I
5
tO
IS
20
25
30
35
PLASMA KALLIKREIN ACTIVITYWITHOUTACTIVATION,.IIot/L.
FIg. 13. Plasma kallikrein activity as measured in specimens
with and without dextran sulfate/acetone (35 mI/di) activation,
to determine possible premature activation of plasma prekallikreln to plasma kallikrein owing to Improperly processed or
stored specimens
CLINICAL CHEMISTRY,
Vol.
27. No. 4,
1981
591
Table 5. Mean and Day-to-Day Variability of Plasma Kallikrein Activity In Each of Five Healthy Women
as Monitored during 28 Days
1
2
3
4
5
No. sp.clm.ns
collscisd
y.ars
Ag.,
31
27
25
28
24
10
9
12
10
10
about fivefold greater at 30 mm. The extended
lag phase apparently resultedfrom the decreased
quantity of Factor XII.
This decreasedactivity would probably be interpreted
as a low
PPK concentration
in the usual 15-mm activation period.
Thus, low PK activity in the clinical
setting may be due to: (a)
low PPK, (b) decreased
Factor XII, (c) decreased high-molecular-mass kmninogen, or (d) premature activation of PPK
to PK before analysis. The first three conditions may be tested
by mixing the test plasma
with an equal volume of normal
plasma.
This will either
restore
PK activity,
suggesting that Factor XII or high-molecular-mass
therefore
kininogen
is deficient,
indicating
decreased
or will not restore
concentrations
Premature
normal
activity,
of PPK.
PPK activation,
the fourth condition,
is deter-
mined by testing the plasma for PK activity without dextran
sulfate/acetone
activation.
In a study in progress, for example,
we noted the reference interval for PK activity in pregnant
women to be 70-130 1zkat/L. We had first noticed that these
data included
some very low results,
10-60 jkatfL.
These
results were produced when too little care was given to specimen preparation.
Blood specimens
collected
with sodium
citrate were placed in a refrigerator
(4 #{176}C)
for 8-16 h before
being centrifuged.
We speculate that the low results were the
result of spontaneous cold activation of PPK to PK and the
subsequent
inactivation
by anti-kallikreins
as described
by
Kluft (6). We could determine which specimens were improperly processed by assaying each specimen with and
without dextran sulfate/acetone (35 mL/dL) activation. Figure
13 represents
data on 10 cases with low PK activity (11-59
zkat/L) after dextran sulfate/acetone
activation.
It was noted
that “endogenous”
PK activity
without dextran sulfate/acetone
of these same specimens
treatment
was high (8-30
zkat/L),
suggesting
prior activation
of PPK. The properly
processed
specimens
had low “endogenous”
activity, usually
<5 1zkat/L. Once having instituted
strict collection procedures
without refrigeration
of specimens before analysis, we no
longer observed PK activity (after treatment with dextran
sulfate/acetone)
less than 70 ikat/L for pregnant women.
In our calculation of the plasma kallikrein
activity we made
at least two important
assumptions.
The first assumption
relates to the molar absorptivity of p-nitroaniline,
the second
to the assumed value of the hematocrit.
The molar absorptivity
for p-nitroaniline
that we used was
based upon the determination
of the absorbance
of a 50
smol/L aqueous standard.
The hematocrit plays a direct role in the calculation of
plasma
kallikrein
activity,
because
it affects the initial dilution
of plasma. In our method whole blood is diluted 10-fold with
sodium citrate solution. In our calculations, we assume a hematocrit
of 40 mL/dL to be rather typical of the specimens
that one would assay in the clinical
setting.
Because the citrate
added to blood remains in the plasma, not penetrating the
blood cells, if the hematocrit is 40 mL/dL the net effect would
be to dilute the plasma six volumes of plasma with one volume
592
CLINICAL CHEMISTRY,
Vol. 27, No. 4, 1981
Usan plasma
kalllkrsln
acty., Mkat/L
Day-to-day
(comblnsd
an&ytlcal and
physIologIcal)
varIatIon
SD, gikat/L
CV,%
86.0
89.3
107.8
105.4
118.4
3.5
5.0
6.6
4.3
6.3
4.1
5.6
6.1
4.1
5.3
of sodium citrate solution. For a hematocrit
of 35 mL/dL we
would have 6.5 volumes of plasma diluted with one of sodium
citrate; for a hematocrit of 50 mL/dL it would be five volumes
of plasma with one of citrate. To put it another way, given a
hematocrit
of 35 mL/dL, because we assumed a hematocrit
of 40 mL/dL, we would have overestimated
the kallikrein
activity by 2%; given a hematocrit
of 50 mL/dL, we would have
underestimated
the kallikrein
activity by about 4%.
In summary, this procedure appears to be an excellent way
to assay PK activity in many specimens, rapidly and efficiently, and with a high degree of precision and reproducibility. The adaptability
of centrifugal
this instrument
an excellent apparatus
on the use of chromogenic
analyzers
also makes
for other assays based
substrate.
We thank Dr. Yoshio Hojimi, National Institute of Health, Bethesda, MD, for bringing to our attention the use of acetone for activating plasma prekallikrein.
References
1. Colman, R. W., Mattler, L., and Sherry, S., Studies on the prekaflikrein (kallikreinogen)-kallikrein
enzyme system of human
plasma. II. Evidence relating the kaolin-activated
arginine esterase
to plasma kallikrein. J. Clin. Invest. 48, 23-32 (1969).
2. Trautschold,
I., and Werle, E., Spektrophotometrische
Bestimmung des Kailikreins und seiner Inhibitoren.
Hoppe-Seylers
Z.
Physiol. Chem. 325,48-59 (1961).
3. Toshio, I., Tokio, K., Hesanobu, Y., et al., Radiochemical assays
for human urinary,
salivary,
and plasmakallikreins.
InChemistry and
Biology of the Kallikrein-Kinin
Sy8tem in Health and Disease. J.
J. Pisanoand F. K. Austen, Eds., Fogartyhit.Center Proc. 27, DHEW
Publications
No. (NIH) 76-791, Washington, DC, 1974, pp 205213.
4. Amundsen, E., Svendsen, L., Vennerod, A. M., and Laake, K.,
Determination of plasmakallikrein
witha new chromogenic tripeptide
derivative. Ibid., pp 215-220.
5. Stormorken,
H., Baklund, A., Gallimore, M., and Ritland, S.,
Chromogenic substrate assays of plasma prekallikrein with a note on
its site of biosynthesis. Haemostasis
7,69-75 (1978).
6. Kluft, C., Determination
of prekallikrein in human plasma: optional conditions for activating prekallikrein. J. Lab. Clin. Med. 91,
83-95 (1978).
7. Kluft, C., Trumpi-Kalshoven,
M. M., and Jie, A. F. H., Crucial
conditions for the determination of prekallikrein
levels in plasmawith
chromogenic
substrates,
In Chromogenic
Peptide
Substrates:
Chemistry and Clinical Usage. M. D. Scully and V. V. Kakkar,Eds.,
Churchill Livingstone, Edinburgh,
London and New York, 1979, pp
84-92.
8. Friberger, P., Eriksson, E., Gustavsson, S., and Glaeson, G., Determination of prekallikrein in plasma by means of a chromogenic
tripeptide substrate for plasmakallikrein
In Kinins II: Biochemistry,
Pathophysiology,
and Clinical Aspects, S. Fujiii, H. Moriya, and T.
Suzuki, Eds., Plenum, New York, NY, 1979, pp 67-81.
9. Claeson, G., Friberger, P., Kn#{246}s,
M., and Eriksson, E., Methods
Factor VII in human plasma. Acta Pharmacol. Toxicol. 33,229-240
of prekallikrein in plasma, glandular kallikrein,
(1973).
urokinase. Haemostasis 7, 76-78 (1978).
10. Gjonnaess, H., Cold promoted activation of Factor VII. III.
14. Gallimore, M. J., Ainundsen, E., Larsbraaten, M., et al., Studies
on plasma inhibitors of plasmakallikrein using chromogenic peptide
Relation to the kallikrein system. Thromb. Diath. Haemorrh. 28,
182-193 (1972).
substrate assays. Thromb. Res. 16,695-703 (1979).
11. Gjonnaeas, H., and Stormorken, H., Blood clotting, plasma kinin
15. Mandle, R. J., Colman, R. W., and Kaplan, A. P., Identification
and fibrinolysis. Thromb. Diath. Haemorrh. 24, 308-310 (1970).
of prekallikrein and high molecular weight kininogen as a complex
in human plasma. Proc. NatI. Acad. Sci. USA 73, 4179-4183
12. Armstrong, D., and Mills, G. L., The reversible cold-induced activation of the human plasma kinin-forming system between 37 #{176}C (1976).
and 0#{176}C.
J. Physiol. 179, 89-90 (1965).
16. Murano, G., The Hageman connection: Interrelations
of blood
13. Laake, K., Gjonnaess, H., and Fagerhol, M. K., Components of
coagulation, fibrino(geno)lysis,
kinin generation, and complement
the kallikrein-kinin
system and the spontaneous cold activation of
activation. Am. J. Hematol. 4,409-417 (1978).
for determination
CLINICALCHEMISTRY,Vol. 27, No. 4, 1981 593