Clinical Chemistry 46: 928-933, 2000;
(Clinical Chemistry. 2000;46:928-933.)
© 2000 American Association for Clinical Chemistry, Inc.
Total and Pancreatic Amylase Measured with 2-Chloro-4-nitrophenyl-4-O-ß-D-galactopyranosylmaltoside
Yoshitaka Morishita1,
Yoshitsugu Iinuma1,a,
Nobuo Nakashima1,
Keiichi Majima2,
Katsuhiko Mizuguchi2 and
Yoshihisa Kawamura2
1
Department of Clinical Laboratory, Nagoya University Hospital, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan.
2
Tsuruga Institute of Biotechnology, Toyobo Co., Ltd.,
10-24 Toyo-cho, Tsuruga 914-0047, Japan.
a Author for correspondence. Fax 81-52-744-2613;
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Abstract
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Background: Many different methods have been used to assay amylase
activity, using nitrophenylated oligosaccharides as substrate; however,
the hydrolysis steps in these methods are complex.
Methods: We developed a new continuously monitoring assay for
amylase activity in biological fluids, using
2-chloro-4-nitrophenyl-4-O-ß-D-galactopyranosylmaltoside
(GalG2CNP) as the substrate; this assay was used with anti-human
salivary amylase monoclonal antibodies for specific determination of
the pancreatic isoenzyme. Amylase converted GalG2CNP into
ß-D-galactopyranosylmaltose and
2-chloro-4-nitrophenol, which was measured at 405 nm.
Results: GalG2CNP was cleaved between 2-chloro-4-nitrophenol and
ß-D-galactopyranosylmaltose and did not undergo transfer
reactions. The within-assay CVs (n = 20) for total amylase (T-AMY)
and pancreatic amylase (P-AMY) were 0.6–1.6% and 0.5–2.5%,
respectively; and day-to-day CVs (n = 10) for T-AMY and P-AMY were
0.8–3.7% and 0.6–4.1%, respectively. T-AMY and P-AMY activities in
serum or urine obtained by the proposed method correlated well with
those determined by the 2-chloro-4-nitrophenyl
4-O-ß-D-galactopyranosyl-ß-maltotetraoside
method or the modified IFCC method.
Conclusions: This novel assay for T-AMY and P-AMY measures both
activities stoichiometrically, directly, and easily, and may be
suitable for routine procedures.
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Introduction
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Assaying the activity of 
-amylase
(1,4--D-glucan-4-glucanohydrolase; EC 3.2.1.1),
especially that of pancreatic amylase, in human serum is important for
accurate diagnosis of pancreatic disorders. Methods using various
oligosaccharides covalently bound to 2-chloro-4-nitrophenol
(CNP)1
or 4-nitrophenol as substrate and auxiliary enzymes such as
-glucosidase or glucoamylase for the measurement of total (T-AMY)
and pancreatic (P-AMY) amylase activities have been investigated
(1)(2)(3)(4)(5)(6). Most of the substrates need a bulky modification of
the glucose residue at the nonreducing end to prevent substrate
degradation by the auxiliary enzymes. The main problem with many of
these methods is multiple hydrolysis. Recently, methods using
2-chloro-4-nitrophenyl--D-maltotrioside
(G3CNP) as a substrate have been developed (7)(8)(9)(10); in these
methods, the chromophore is released without auxiliary enzymes.
However, partial polymerization of liberated glucose with the substrate
complicates the reaction (8).
A currently available immunoinhibition method that uses specific
monoclonal antibodies (11)(12)(13) against salivary amylase
(S-AMY) shows no cross-reactivity with P-AMY. Thus, we used
2-chloro-4-nitrophenyl-4-O-ß-D-galactopyranosylmaltoside
(GalG2CNP; Fig. 1
), which has D-galactose at the
nonreducing end of the D-glucosyl group, as the
substrate and two anti-human S-AMY monoclonal antibodies, Tu66C7 and
Tu88E8, for the isoenzyme determination. GalG2CNP requires no auxiliary
enzymes and is cleaved only at the aglycone bond by amylase. Transfer
of glucose to GalG2CNP does not occur; therefore, the hydrolysis of the
substrate proceeds stoichiometrically.
We describe here the measurement of total and pancreatic amylase
activity. This proposed method has also been evaluated and compared
with other widely established methods. The proposed method is based on
the reaction:
where GalG2 is ß-D-galactopyranosylmaltose. The rate
of CNP release is followed at 405 nm. P-AMY activity is measured by the
same method as T-AMY after S-AMY activity is inhibited by anti-human
S-AMY monoclonal antibodies.
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Materials and Methods
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apparatus
The proposed method and the conventional methods were performed
with a Hitachi 7170 automated analyzer. Each aglycone fragment
generated from GalG2CNP by amylase hydrolysis was examined with a
Shimadzu SPD-M6A HPLC equipped with a Tosoh TSKgel ODS-120T (4.5
x 250 mm) column.
chemicals
GalG2CNP was purchased from Yoshitomi Fine Chemicals. NaCl,
CaCl2, and KSCN were from Nacalai Tesque,
and MES buffer was from Dojindo Labs. G3CNP was from Oriental
Yeast, and 2-chloro-4-nitrophenyl--D-maltoside (G2CNP)
and 2-chloro-4-nitrophenyl--D-glucoside (G1CNP) were
from Toyobo. Anti-human S-AMY monoclonal antibodies Tu88E8 and Tu66C7
were purchased from Nippon Roche. Calibzyme AMY [P] and [S] came
from International Reagents. L-Ascorbic acid,
D-glucose, sucrose, and -D-fructose were
purchased from Wako Pure Chemical Industries; bilirubin was from Sigma;
and Intra-lipid 10% was from Kabi Vitrum.
reaction reagents, assay procedure, and calculation
Reagent 1 (R1-TAMY and R1-PAMY) and reagent 2 for assaying T- and
P-AMY activities are given in Table 1
(11)(12)(13). The assay protocol is shown in Table 2
. The activities obtained from R1-TAMY are the T-AMY activity
(T). The catalytic concentrations after the antibody
reaction using R1-PAMY, which contained anti-human S-AMY antibodies,
indicate the residual amylase activity (R), i.e., 100% of
P-AMY and 5% of S-AMY activity because 95% of pure human salivary
amylase (Calibzyme AMY [S]) activity is inhibited and pure human
pancreatic amylase (Calibzyme AMY [P]) is not. Therefore, P-AMY
activity is calculated by using the equations: P-AMY activity =
[R - (0.05 x T)]/0.95; and
S-AMY = T-AMY - P-AMY.
chromatographic analysis
Calibzyme AMY [P] or [S] (0.03 U) was added to 1.0 mL of
reagent solution (750 µL of R1-TAMY + 250 µL of reagent 2), and the
mixture was incubated for 0, 15, 30, 60, 120, 180, or 360 min at
37 °C. Ten microliters of the mixture was then injected onto the
HPLC column; each sample was eluted with methanol-acetic acid-water
(30:1:69, by volume) at a flow rate of 1 mL/min, and monitored at 280
nm. Each peak on the chromatogram was identified by comparison with
reference materials.
comparison method
We also measured the activities of T-AMY and P-AMY in patients’
sera and urines by
2-chloro-4-nitrophenyl-4-O-ß-D-galactopyranosyl-ß-maltotetraoside
(GalG4CNP) methods [Ref. (2); Diacolor Liquid AMY kit
and Diacolor Liquid P-AMY kit from Toyobo] and the modified IFCC
method (6), which uses 4,
6-ethylidene-4-nitrophenyl--D-maltoheptaoside
(Liquitec AMY EPS kit and Liquitec P-AMY EPS kit from Nippon Roche).
specimens
Serum specimens were collected from patients in Nagoya University
Hospital. We obtained informed consent from patients for this study.
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Results
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product analysis by hplc
The release of CNP from GalG2CNP hydrolyzed by P-AMY over 6 h
is shown in Fig. 2
. The rate of increase of CNP corresponded to the decrease of
GalG2CNP; the other aglycone fragments (G2CNP and G1CNP) did not
appear. Polymerized products of GalG2CNP produced by the
enzyme-catalyzed hydrolytic cleavage were not observed. The products
from S-AMY-hydrolyzed GalG2CNP were similar to those of P-AMY.
optimization studies
Two pooled human sera with T-AMY activities of 95 and 356 U/L,
respectively, and Calibzyme AMY [P] (P-AMY, 258 U/L), [S] (S-AMY,
212 U/L) were used for the optimization studies of this proposed
method.
Effect of pH.
The effects of pH on T-AMY activity were
examined in 50 mmol/L MES buffer at various pH values (Fig. 3
A). The maximum activity of T-AMY was observed at pH 5.50,
decreasing at pH 6.0 or above. The reaction of S-AMY with anti S-AMY
antibody was inhibited at a pH <6.0; therefore, we chose MES buffer,
pH 6.0.
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Figure 3. Effects of pH (at 37 °C; A) and KSCN
concentration (B) on T-AMY activity.
Two pooled human sera ( , 356 U/L T-AMY; •, 95 U/L T-AMY),
Calibzyme AMY [P] ( ; 258 U/L P-AMY), and Calibzyme AMY [S] ( ;
212 U/L S-AMY) were used. (A), 150 mmol/L KSCN, 300
mmol/L NaCl, 5.0 mmol/L CaCl2, 5.0 mmol/L GalG2CNP.
(B), pH 6.0 (37 °C), 300 mmol/L NaCl, 5.0 mmol/L
CaCl2, 5.0 mmol/L GalG2CNP.
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Effect of KSCN.
The T-AMY activities of specimens increased
with increasing concentrations of KSCN, almost reaching the maximum at
150 mmol/L or above (Fig. 3B
), whereas the reactivity of anti-S-AMY
antibody decreased with increasing KSCN concentrations: 95.4%, 94.4%,
89.0%, and 77.8% of S-AMY activity was inhibited at 150, 300, 600,
and 900 mmol/L KSCN, respectively. Therefore, we chose 150 mmol/L KSCN.
Effects of CaCl2, NaCl, and GalG2CNP.
We examined
the effects of 0.5–10.0 mmol/L CaCl2 and 50–500
mmol/L NaCl, as activators, and 2.4–12.0 mmol/L GalG2CNP on T-AMY
activity. The T-AMY activities of the specimens increased with
increasing concentrations of CaCl2, NaCl, and
GalG2CNP. We chose 5.0 mmol/L CaCl2, 300 mmol/L
NaCl, and 5.0 mmol/L GalG2CNP to obtain the maximum T-AMY activity.
Effect of anti-S-AMY antibody.
S-AMY activity decreased with
increasing concentrations of Tu88E8 and Tu66C7 antibodies, whereas the
P-AMY activity was not affected (Fig. 4
). We added 15.0 mg/L Tu88E8 antibody and 2.0 mg/L Tu66C7
antibody to reagent 1 for P-AMY measurement because S-AMY was inhibited
95% at these concentrations.
Measurement interval.
After reagent 2 was added, the reaction
was linear with time for sera, whereas the reagent blank did not
increase (Fig. 5
). We chose the measurement interval from 7.35 to 10.0 min after
start.
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Figure 5. Time course of the reaction.
Two patients’ sera [T-AMY, 397 U/L (•) or 101 U/L ()] and 154
mmol/L NaCl ( ; reagent blank) were used. R1, reagent
1; R2, reagent 2. Arrows indicate the
addition of the reagents.
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assay evaluation
Precision.
The within-run and day-to-day CVs of the proposed
assay were established with sera of low (serum L; 38.2 U/L T-AMY, 24.7
U/L P-AMY), middle (serum M; 99.7 U/L T-AMY, 56.0 U/L P-AMY), and high
(serum H; 398.0 U/L T-AMY, 173.2 U/L P-AMY) amylase concentrations. The
data (Table 3
) showed excellent reproducibility.
Analytical recovery.
Pooled human serum (55 U/L T-AMY, 28 U/L
P-AMY) supplemented with 107, 215, 308, or 412 U/L Calibzyme AMY [P]
and 87, 175, 253, or 338 U/L Calibzyme AMY [S] showed recoveries of
95.3–99.2% (mean, 96.7%) and 94.2–99.3% (mean, 97.3%) for T-AMY.
The respective recoveries for the P-AMY assay supplemented with AMY
[P] were 96.5–101.2% (mean, 98.8%).
Linearity.
The T-AMY assay was linear up to at least 1100 U/L
for serum.
Critical limit.
We examined the critical limit of this T-AMY
assay by assaying 154 mmol/L NaCl 10 times. The result was 0.40 ±
0.516 U/L (mean ± SD). The critical limit (mean + 3 SD) was 1.95
U/L.
Interferences.
Various substances were examined for their
potential effects on the T-AMY and P-AMY determination. One volume of
each examined substance was mixed with nine volumes of pooled human
serum (116 U/L T-AMY, 84 U/L P-AMY). We found no interference with
T-AMY and P-AMY activities from bilirubin up to 0.342 mmol/L, ascorbic
acid up to 1.14 mmol/L, Intra-lipid up to 0.2%, hemoglobin up to 4.0
g/L, glucose up to 10 g/L, fructose up to 1.0 g/L, and sucrose up to
1.0 g/L.
Stability of reagents.
Reagent 1 (R1-TAMY and R1-PAMY) and
reagent 2 were examined with two human pooled sera (T-AMY, 250 and 115
U/L; P-AMY, 182 and 70 U/L, respectively). Identical activities and
constant reagent blanks were observed after storage for 3 months at
4 °C.
Comparison of reactivities of GalG2CNP and G3CNP.
The amylase
activity using GalG2CNP or G3CNP was compared at 0–900 mmol/L KSCN
with 258 U/L Calibzyme AMY [P] and 212 U/L Calibzyme AMY [S]. Good
reactivity of GalG2CNP was observed; at 0–900 mmol/L KSCN, the P-AMY
and S-AMY activities obtained using GalG2CNP were higher than those
using G3CNP (Fig. 6
).
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Figure 6. Comparison of reactivities of GalG2CNP and G3CNP.
Amylase activities of Calibzyme AMY[P] (; 258 U/L) and Calibzyme
AMY[S] (; 212 U/L) were assayed using 5.0 mmol/L GalG2CNP (—–)
or G3CNP (- - - -) at 0–900 mmol/L KSCN [in 300 mmol/L NaCl, 5.0
mmol/L CaCl2, 50 mmol/L MES, pH 6.0 (37 °C)].
Activities of P-AMY and S-AMY are shown as relative amylase activity
(%) compared with P-AMY and S-AMY activities at 900 mmol/L KSCN using
G3CNP, which were assigned as 100%, respectively.
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Sensitivity.
The sensitivity of this T-AMY assay, although
less optimal, was good, with a mean sensitivity comparable to
that of the other methods: the changes in absorbance
(
A/min) for this method, the GalG4CNP method, and the
modified IFCC method at an arbitrary amylase activity of 100 U/L were
0.022, 0.040, and 0.013 at uniform sample volume ratios and light
pathlengths.
Correlation.
The correlation between the proposed method and
the conventional methods was examined. The T- and P-AMY activities in
patients’ sera and urines obtained by the proposed method correlated
well with those determined by the GalG4CNP method or the modified IFCC
method (Table 4
).
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Discussion
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Different methods have been reported for the assay of amylase
activity using maltooligosaccharides of defined chain length coupled to
a chromophore as substrates; however, the hydrolysis process frequently
is complex (1)(3)(4)(5)(6). We developed a new
continuous monitoring assay for measuring amylase activity in
biological fluids with GalG2CNP as the substrate, which requires no
auxiliary enzymes to release the chromophore and can be used with
anti-human S-AMY monoclonal antibodies for the determination of the
pancreatic isoenzyme. This new substrate, GalG2CNP, was cleaved into
only two degradation products,
ß-D-galactopyranosylmaltose and CNP; other
fragments were not produced. This is similar to G3CNP
(7)(8)(9)(10), which had been considered recently as a substrate
for the method by the IFCC (10) but was not adopted. G3CNP
is polymerized to GnCNP (n >3) by a transfer reaction of the
glucose residue liberated from G3CNP by amylase to the nonreducing end
of G3CNP (8). However, GalG2CNP is resistant to
transglycosylation because the 4-position of the nonreducing end is
modified by a D-galactose. Therefore, the amylase
reaction using GalG2CNP proceeds stoichiometrically and more
cleanly than that of G3CNP. On the other hand, maximum amylase
activity is almost reached at KSCN concentrations 150 mmol/L in this
method, which exceeded the amylase activity toward G3CNP, the substrate
proposed by the IFCC (10), at 900 mmol/L KSCN.
Because the inhibitory effect of S-AMY antibodies Tu88E8 and Tu66C7 was
impaired by KSCN concentrations >300 mmol/L, the GalG2CNP test is more
suitable for the measurement of P-AMY than G3CNP.
We investigated the optimized conditions for assaying both T-AMY and
P-AMY; however, the conditions for maximal possible release of CNP were
not obtained because of the mutual dependence of pH, KSCN
concentration, and antibody reaction in promoting reaction velocity.
Thus, we determined the optimized conditions for the P-AMY assay with
monoclonal antibodies. However, this method is also suitable for the
assay of T-AMY, although less optimally, because the sensitivity of
this assay is not inferior to those of other methods, such as the
GalG4CNP method and modified IFCC method. Furthermore, GalG2CNP
exhibited sufficient affinity and almost equal reactivity to P-AMY and
S-AMY because the Michaelis-Menten
(Km) values for P-AMY and S-AMY were
0.86 and 0.64 mmol/L, respectively (not shown), and these
Km values are similar to those for
G3CNP [0.35 and 1.01 mmol/L (7)],
3-ketobutylidene-ß-2-chloro-4-nitrophenyl-maltopentaoside [0.318 and
0.377 mmol/L (1)], and GalG4CNP [0.173 and 0.216 mmol/L
(2)].
This method, which does not need auxiliary enzymes, will also lower
reagent costs in contrast to conventional methods that incorporate an
auxiliary enzyme. Good precision, reasonable analytical recovery, good
linearity and critical limit, and good correlation with conventional
methods (modified IFCC method and GalG4CNP method) were obtained with
this method; in addition, various substances did not interfere with
this method. This novel method for the assay of T-AMY and P-AMY
activities in serum or urine can be performed stoichiometrically,
directly, and easily on the Hitachi 7170 automated analyzer, which is
available for routine procedures in clinical diagnosis.
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Footnotes
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1 Nonstandard abbreviations: CNP, 2-chloro-4-nitrophenol; S-, P-, and T-AMY, human salivary, pancreatic, and total amylase; G3CNP, 2-chloro-4-nitrophenyl--D-maltotrioside; GalG2CNP, 2-chloro-4-nitrophenyl-4-O-ß-D-galactopyranosylmaltoside; G2CNP, 2-chloro-4-nitrophenyl--D-maltoside; G1CNP, 2-chloro-4-nitrophenyl--D-glucoside; and GalG4CNP, 2-chloro-4-nitrophenyl-4-O-ß-D-galactopyranosyl-ß-maltotetraoside. 
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Abstract
Introduction
, G2CNP; 