Note: Descriptions are shown in the official language in which they were submitted.
~068586
Back~round Of The Invention
a-Amylase levels in samples can be of considerable
importance, particularly in biological fluids such as serum
or urine. High levels of serum a-amylase usually signify
disorder of the pancreas. a-amylase hydrolyzes the ~ 4
;~ glucan linkages of polysaccharides and oligosaccharides in
a random fashion. The final produc~s are mainly maltose and
some glucose. Limit dextrins are also produced when branch-
chained starch is the substrate. Currently, a-amylase activity
is measured by one of the following methods: 1) Decrease in
viscosity of the starch solution; 2) Decrease in the ~-
turbidity of starch suspension; 3) Decrease in the intensity
of the starch-iodine complex; 4) Increase in the amount of
reducing ends (saccharogenic assay); 5) Formation of glucose
in a-glucosidase-coupled reaction; or 6) Intensity of chromagen
; in solution after cleavage of stained insoluble starch.
All of these current methods have one or several of
; the following disadvantages: Poor sensitivity; Long lag time;
Insoluble substrate; High background reading, especially when
glucose is present; Multi-step procedure; or Short linearity.
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I Summary Of The Invention
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-1 The method described herein concerns an assay for
the enzyme a-amylase (a-1,4-glucan-4-glucanohydrolase, EC
3.2.1.1) especially that of serum or urine origin, and has
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~068586
particular application in clinical diagnosis. The method -
involves 1) the use of a modified starch which contains
~lockages to the action of the exo-acting enzymes (Marshall, J.J.
and Whelan, W.J., 1971, Anal. Biochem., 43, 316-321; Marshall,
J.J., 1970, Anal. Biochem., 2, 132-145) such as phosphorylase
and 2) the coupling of the reaction to a polysaccharide
phosphorylase so that glucose-l-phosphate is formed when ~-
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a-amylase is present. Phosphorylase attacks the starch from ;~
the nonreducing ends. Thus it cannot utilize the modified
10 starch (with blocked nonreducing ends) as a substrate. ~-
a-Amylase, on the other hand, is an endo-acting enzyme which
hydrolyzes the internal a-l--~ 4 bonds of the starch. Therefore,
the modified starch can serve as a substrate for a-amylase. The
hydrolytic action of a-amylase results in the revealing of
nonreducing ends which can then be utilized by phosphorylase
to form glucose-l-phosphate. The overall reaction sequence is
as follows:
Modified (blocked) starch a-amylase ~ Nonreducing ends
(unblocked starch)
Nonreducing ends inorganic phosphate (Pi) phosphorylase b
plus adenosine 5'-monophosphate (AMP) ~ or phosphorylase ~ -
= ~ or maltodextrin phosphorylase ~ glucose-l-phosphate
Glucose-l-phosphate phospho~lucomutase > glucose-6-phosphate
Glucose-6-phosphate nicotinamide adeninedinucleotide (NAD~,
glucose-6-phosphate dehydro~enase, ~r reduced nicotinamide
adeninedinucleotide plus 6-phosphogluconate. `
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1 068586
; The rate of increase of absorbance at 340 nm. due to NADH
formation can be followed by a photometer to monitor the
enzymatic activity of a-amylase. With the addition of a
tetrazolium dye, 2-p-iodophenyl-3-nitrophenyl-5-phenyl
tetrazolium chloride (INT) and an electron carrier, the assay
~ can be converted into a colorimetric one: ~ -
; INT plus NADH diaphorase or phenazine methosulfate INTH
(Absorbance peak = 500 nm.) plus NAD. --
Compared with current methods, the method described
herein has the following advantages:
1) Since the reaction is coupled through the
formation of glucose-l-phosphate and glucose-
6-phosphate, which are not present at
appreciable levels in body flulds, the assay
is free from lnterference by glucose and other
reducing sugars. Consequently, a sample blank -
l, need not be performed.q 2) The coupling enzymes used in the reaction,
, namely, phosphorylase, phosphoglucomutase, and
glucose-6-phosphate dehydrogenase, all have
high activity at pH 7, the optimal pH for
a-amylase. Hence the assay can be performed
~') under the optimal reaction conditions. In
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~ other methods where the reaction is coupled
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to a-glucosidase or glucoamylase, either a
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lower pH or a large amount of the coupling
enzyme is required.
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068586
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3) Each bond hydrolyzed by a-amylase immediately
results in the formation of glucose-l-phosphate.
Therefore, this reaction has little or no lag -
time. The a-glucosidase-coupled reaction has
a long lag time since a build-up of maltose is `
necessary prior to detection.
4) The activity of a-amylase can be measured in
a slngle operation in a continuous manner.
Other tests which utilize iodometric, visco-
metric, saccharogenis principles or dyed
substrates require separate steps. ` `
5) The assay offers high sensitivity and long
linearity. The viscometric, turbidimetric, ~-
and dyed-substrate approaches suffer from --
; either low sensitivity or short linearity ~ -~
~with respect to both the amount of a-amylase
and time).
In one particular a3pect the present invention provides
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a method of assaying for a-amylase in a sample which comprises ``
incubating said sample with a modified starch substrate con-
taining blocked nonreducing ends in the presence of a suitable
- phosphorylase, phosphoglucomutase, and glucose-6-phosphate
dehydrogenase as the coupling enzymes for the measurement of ~he
liberated nonreducing ends due to the action of a-amylase.
In another particular aspect the present invention provides
a reagent for use in assaying for a-amylase in a sample com-
prising, in association:
phosphate buffer,
i pH range of 6-8 20-100 mmoles/liter
Mg salts 1-5 mmoles/liter
adenosine 5'-mono-
phosphate 0.1-0.5 mmoles/liter
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~068586
~ nicotinamide
adeninedinu-
cleotide 1-5 mmoles/liter
glucose-l,
6-diphosphate 0.1-5 U/mmoles/liter
NaCl 2-100 mmoles/liter
4% periodate .
oxidized, NaBHb
reduced starch 2-5 g/liter reagent
Phosphorylase b
(rabbit muscle) 8-30 U/ml reagent
Phosphoglucomutase 1-10 Utml reagent
Glucose-6-phosphate
dehydrogenase 5-50 U/ml reagent
Detailed Description of the Invention .
The preferred .substrate is a modified branch-chained : ;
polysaccharide, preferably modified to the extent of 2-10%.
The substrate used in the following description is a modified
amylopectin which is prepared by limited oxidation with
NaI04, followed by reduction by NaBH4, then by treatment with
- an exo-acting enzyme such as gluco-amylase, ~-amylase, or
phosphorylase to remove the unreacted nonreducing ends.
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- ` 10685~6 ~ ~ ~
: However, other methods of modification can be -
used. Starch glycollate and modified glycogen, amylose or
soluble starch, for instance, can serve as substrates in
; this reaction, though they are not as desirable. :.
The extent of oxidation also affects the efficiency
of the substrate. The substrate employed herein is 4%
oxidized amylopectin, but starches modified by lower or higher
percentage of oxidation (2-8%) have been shown to be good
substrates.
A single reagent, containing all of the ingredients .
for the determination of a-amylase, is represented by the
following formula:
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In~redient Concentration .
::~ Phosphate buffer, :: -
:~ 15 pH 7.0 20-100 mmoles/liter :
MgC12 1-5 mmoles/liter :
. AMP 0.1-0.5 mmoles/liter .
.; NAD 1-5 mmoles/liter -
Glucose-l, 6-
.~ 20 diphosphate 0.1-5 umoles/liter
NaCl 2-100 mmoles/liter
4% oxidized,
reduced starch 2-5 g/liter reagent
Phosphosylase b 8-30 U/ml reagent
--. 25(rabbit muscle)
Phosphoglucomatase 1-10 U/ml reagent
Glucose-6-phosphate
. dehydrogenase 5-50 U/ml reagent
: Assay temperature = 37C
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~68586
It should be emphasized that phosphorylase a -
or phosphorylase from other mammalian organs or from plant
or microbial sources can be used to replace phosphosylase b.
Maltodextrin phosphosylase (Schwartz, M. and Hofnung, M.,
1967, European J. Biochem; ~ 132-145) can also be used. In
addition, a mixture of several phosphorylases can be
employed to achieve higher efficiency. An analog may be
used in place of AMP. In colorimetric assay the following
ingredients are added:
INT 0.4g/liter reagent
Diaphorase 0.5U/ml reagent
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Any electron acceptor such as PMS can be used to replace
diaphorase.
As described, the method involves the use of a
modified starch in conjunction with a phosphorylase as the
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key coupling enzyme to produce an ultraviolet (UV) or a
` colorimetric test. Changes in the type of the modified
starch or the source of phosphosylase used are considered to
be within the scope of the invention. The glucose-6-phosphate
dehydrogenase can be obtained from yeast, leuconostoc or
other sources. The electron transfer agent can be an enzyme
such as diaphorase, phenazine methosulfate or other suitable
electron carrier.
The body fluid to be assay for ~-amylase can be
a serum or a urine sample. When human serum is the sample,
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1068586
20 ul. of serum is added to 1.0 ml of the above described
assay mixture and the reaction is followed at 340 nanometers
(nm) (or 500 nm in the colorimetric test) for 5 minutes at
37 with a suitable spectrophotometer. The procedure has
been adapted to the Abbott Bichrometic Analyzer 100, using
the following settings:
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37 C.
340/380 filter (500/600 filter for -
colorimetric test)
5 minute cycle
FRR (First revolution
reading) mode
Rate-up
1:51 syringe plate (1:101 plate for
lS color test)
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When the previously described assay mixture and
procedure are used, the test is linear to 0.3 absorbance
unit per minute at 340 nm, equivalent to 6000U a-amylase/
liter serum. This method correlates very well with the
established iodometric method of Street and Close (Street,
H.V., and Close, J.R., 1956, Clin. Chim. Acta., 1, 256).
One Street and Close unit is equivalent to about five
international units.
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