Note: Descriptions are shown in the official language in which they were submitted.
BACKGROUND OF THE_INVENTION
l. Field of the Invention: This lnvention relates
to amylase assays and to a reagent test kit for use in
such assays. More particularly, it relates to amylase
assays in which a polysaccharide is used as the amylase : .-
substrate.
2. Discussion of the Prior Art: a-Amylase is an
enzyme which hydrolyzes the ~[l t4] linkages between the
glucose units in starch and the lower polymers and oligomers
of glucose. This enzyme is produced in the human body,
primarily in the pancreas and in the salivary glands, and
its concentration in various body ~luids is a useful
diagnostic tool ~or physicians. For example, in healthy
i.ndi~iduals, serum ~-amylase le~els are relatively constant,
15 but they rise in response to pathological conditions, such
as acute pancreatitis.
U.S. Patent 3,879,263, issued ~pril 22, 1975,
and U.S. Patent 4,000,042 issued December 28, 1976,
disclose a process and reagent tes-t kit for use in deter-
mining the ~-amylase content of a sample using the deined
oligosaccharides maltotetraose, maltopentaose or malto-
hexaose as the amylase substrate. The reaction between
~-amylase and these substrates, preerably in the presence
; of a maltase, produces a specific amount of glucose which
.~ 25 can be measured by any conventional glucose detection
: system. The additional glucose detection step is an
incon~enience. Furthermore, if glucose is present in the ~ :
sample, it must either be removed or compensated for.
Although this can be done by conventional techniques, ;.:
~ ', '
-2- ~ :
~ .
:
'~ : . , .
.
it is an extra step in the process which is a disadvantage.
A. P. Jansen and P. ;. A. B. Wydeveldl Nature,
182, 525 (1958) postulate that ~ nitrophenyl)maltoside
could he a substrate for an amylase assay. However, this
paper shows that the authors never identified the active
agent responsible for their observations. They reported:
(1) Incubation of human urine or saliva samples with a-
(p-nitrophenyl)maltoside at 37 for 16 hours produces 4-
nitrophenol, identified spectrophotometrically by mixing
the hydrolyzate with 0. 02N sodium hydroxide. (2) I'he
hydrolysis was inhibited by protein precipitants such as
10% trichloroacetic acid and 0.5N silver nitrate. (3) The
hydrolysis was pH-dependent, being most effective at pH
5.~-7Ø They state that this was evidence for "the
possible existence of an unidentified carbohydrase". a-
(4-Nitrophenyl)maltoside is not believed to be useful for
human amylase assay because the cleavage of this compound
by ~-amylase is extremely slow.
SUMMARY OF THE INVENTI_N
; 20 Accordlng to the present invention, there is
provided a method for determining the amylase content of
a sample comprising the steps of:
(a) adding to a solution containing a me~sured amount
of the sample a defined pol~saccharide substrate having
the folIowing formula:
_ _ .
~i ~ ~C~2oH CH~Oll --
r ; ~ ~1
O~ ~ _ o ~ OH ~ _ O ~ OII
IO ~ ~ ~ ~ ~ //OR
; ~C)ll ~01~ ~
: ~ .
~ 3
.
1: .
.
.. : . : : ,
9~2
~ '
: where n is an integer in the range of from 1 to 10, and
R is a substituted aromatic radical which, as a detach~d
aglycone exhibits a different sp~ctral absorbance than
the substrate; and
(b~ monitoring the spectral absorbance of the solution.
In the preferred embodiment, n is 2, 3 or 4
and R is a substituted aromatic radical selected from
the group consisting of
~} ~J and ~ ~
. ':,
: 10 in which X and Y are individually selected rom the group
consisting of
H, NO2, halogens, alkyls of from 1 to 4 carbon atoms,
OR' or CO2R' :
where R' is an alkyl group of from 1 to 6 carbon atoms,
,
and at least one o~ X and Y is NO2.
In the preferred embodiments, the substrates are ~:
glycosides of maltotetraose, maltopentaose or maltohexaose :
in which the terminal glycoside unit has an aromatic radical
attached to it. In the most preferred embodiment, the ter-
: 20 minal glycoside unit is an ~-(4-nitrophenyl) glycoside, and
a maltase is also added to the solution.
. A reagent test kit is also provided~ This test - :~
kit contains one o the substrates listed above and a
maltasc.
:,
:
~.
,
.
DETAILEO DESCRIPTION OF TH~ INVENTION
The following disclosure, and the invention it
describes, is restricted to polymers and oligomers of
glucose which are ~[1--~4] linked and which have a sub-
5 stituted aromatic radical attached to the terminal (reducing)glucose unit. Such compounds are represented by the
general formula
CH2CH CH~OII CH2OH
I ~ Q~ H ~ \H
~1 ~ o ~ o ~ 0~1 ,1 . ~
~ OH n
ln where n is an integer, R is the substituted aromatie
radieal, and the remaining portion of the compound is
the glycosyl residue. When detaehed from the glycosyl
residue by hydrolysis, the radical R becomes a phenol,-
; ROH,or an anion of that phenol, RO ,(depending upon the
eondition of the solution) both of which are normallyreferred to as an aglycone.
This eompound, which functions as a substrate
for amylase, is a de~ined polysaccharide, or when n<8
a defined oligosaccharide. The term "defined" as applied
to polysaccharides has, in the past, been used in a loose
sense, often referring to any mixture of polysaccharides
in whieh the relative percentages of the various polymers
and oligomers is known. As used herein, however, the
term "define~ polysaccharide" shall mean a substanee con-
taining at least 90~O of a polysaccharide with a givenchain length, i.e., where n is a given inte~er.
-5-
:........ .. . .
l~L6?~
~ -Amylase acts as a catalyst in the hydrolysis
of polysaccharides into small chain polysaccharides and
eventually into maltose. It has been found and reported
in the patents listed above that from among all po]y-
saccharides, maltotetraose (G4), maltopentasoe (G5) andmaltohexaose (G6) are preferred for use as a substrate in
an amylase assay. The G nomenclature is a convenient
shorthand for n ~ 4] linked glucose units.
These three substrates are preferred for kinetic
and stoichiometric reaso~s. The binding constant of ~-
; ; amylase to polysaccharides increases as the number of
a[l--~4~ bonds increases, up to about G6 where it levels
o~f. For homologs lower than G4, the binding constant is
too small to give reasonable reaction rates. For homologs
higher than G6, even though the reaction proceeds rapidly,
the results are not stoichiometric. Unproductive reactions
occur so that n glucose units are not formed when ~-amylase ~-
reacts with Gn. Furthermore, the maximum velocity of sub-
- strate release from ~-amylase decreases with decreasing n.
.. . .
Where the detection system involves glucose and Gn is used
as the substrate, then, within a reasonable time, n glucose
units should be produced for every a-amylase interaction
with Gn. Otherwise, the percentage of the total glucose
; units released compared to those available must be esti-
mated and this leads to error. These factors make G4,
G5, and G6 the pre~err~ed substrates.
In the~present system, where the detection
~system depends upon the release o~ a substituted phenol
ROH and not glucose formation, stoichiometric considera-
~` 30 tions~are not quite as critical and n can be in the range
.. .
l ~ -6~
. .
l~q~
of 1 to 10~ However, it is still preferred that the
polysaccharide be an oligosaccharide, with n in the range
of 1 to 8.
As explained in U.S~ Patent 3,879,2~3, ~he use
of a maltase such as ~-glucosidase is not necessary in the
measurement of either pancreatic or total N-amylase. This
is true when the substrates used are the substrates of the
present invention as well as G4, G5 and G6. Furthermore,
since the present invention is not dependent upon glucose
detection, maltase does not appear to even be necessary
in assays ~or salivary a-amylase using the substrates of
the present invention. However, the use oE a ~altase does
increase the reaction rate in all circumstances. It is
particularly use~ul to achieve a truly stoichiometric
reaction, because the reaction rate of the maltase with
the lower oligosaccharides is greater than the reaction
rate of ~-amylase with those substrates. a~Amylase acts
to hydrolyze the substrate into smaller fractions, and
the maltase acts to complete the hydrolysis to glucose
units, 50 that the release of the substituted phenol
OCCUl-S stoichiometrically. For this reason, oligo-
saccharides of the formula given above, with n = 2, 3
or 4 are the most preferred substrates for the present
invention. The discussion which follows, therefore,
will be limited to those substrates, particularly those
where n is 2 or 3. This limitation, however, is for
convenience and is not intended to limit the disclosure.
When a maltase, such as a-glucosidase is used,
a side reaction which gives rise to a blank rate occurs
because of the reactivity of the maltase with the sub-
strate. This means that even ln the absence of ~-amylase,
.
: '
there will be release of the phenol. Since the maximum
velocity of product release from maltase decreases with
increasing n, the growth of a blank rate is slower as n
increases. One would expect, then, that the blank r~te
5 for G5 would be less than that for G4. This is verified
by experimentation. However, an additional factor is
involved in the choice between higher and lower oligo-
saccharides (i.e., G4 or G5) as the substrate. In all
reactions of the substrate with amylase and reactions of
maltase with the substrate, the rate increases, as a
function of substrate concentration, to an optimum, at
which point it levels off. The substrate concentration
at which optimization occurs appears to increase with
increasirlg n so that more of the substrate (and the
maltase) must be used to optimize (linearize) the
standard curve. For expensive chemicals, this is an
important consideration.
The substrates of the present invention are the
defined polysaccharides covered by the formula given above
in which n is an integer between 1 and 10 and R is a sub-
stituted aromatic radical which, when detached from the
polysaccharide in the form of a phenol or a phenolate
anion, exhibits a di.fferent spectral absorbance than the
substrate. There are a large number of such radicals.
Chief among them, however, are those radicals selected
from the group consisting of
y
.
~ .
. :
in which X and Y are individually selected from the group
consistiny of H, NO2, halogens, alkyls of from 1 to 4
carbon atoms, OR' or CO2R'; where R' is an alkyl of from
1 to 6 carbon atoms, and at least one of X and Y is NO2.
The anions of the phenols formed when these radicals are
separated from the glycosyl residue have a maximum
absorbance ~max of be-tween about 290 and about 600 nm~
The details of the procedures for preparing these
preferred compounds is set forth in Canadian Patent Appli- -
cations Serial No. 282,407 (R.C. Burns et al) and Serial
No. 282,406 tW.B. Farnham et al), filed on the same day as
this application.
Amon~ the preferred embodiments, two compounds
are particularly preferred; those in which n is 2 or 3 and
R is 4-nitrophenyl. These compounds, ~-(4-nitrophenyl~
maltotetraoside (G4pNp) and ~-(4-nitrophenyl) maltopenta- ~
oside (G5pNp), are used to determine the amylase content o~ ~ -
a sample, such as blood serum or urine, accordin~ to the
following reaction scheme
~ .
: ': ''
' ,
" '
. ~, ," '''
:, ' .:
4~
CH2011 - CH20H CH20H
H~e~o~ o~O
OH OH n OH
)l ~Ax 290-305 nm
R -. 4 ~ 2 ~ C6H 4 n: 2 n - (4 - NITROPHEN~L ) !~IALTOTETRAOSIDE
n: 3 ~ -t4-NITROPHE~YL) I.~ALTC~ENTAOSIDE
Al~YLASE
~ '
CH20H CH20HCtJ20H
~ H ~ /~ \ H~ ~0\ :
62 'r G3 L Bo~OJ~
R-4-02N~G~J4 n:O a-(4-NITROP~IENYL~ ~.lALTOSIDE
- MAL~ASE
4 ~r5 Gl ~ HO g~NO2
4- NI~ROPHENOL
~,o~
-O~NO2 : ~ .
.
4-NlTROrlJENOlATE ANI()N A~ X 410nm
.
The defined oligosaccharide substrate is added
to a solution containing a measured amount of the sample
to be tested; and the spectral absorbance of the solution
is monitored, either as an end point determination or a
rate determination using conventional techniques. Usually,
as in all enzyme reactions, the reaction solution is main- .
tained at a substantially constant p~ and a substantially
constant temperature. When these substances are used, it
~':
: ::
: . ,
. .
is desirable to perform the assay in a solution which has
had its p~I adjus-ted to -the basic range in order to enhance
the absorbance at 410 nm. For example~ G4pNp and G5pNp
(~max 290-305 nm) and 4-nitrophenol (~max 313 nm~ have a
low extinction coefficient at 410 nm compared to 4-nitro-
phenolate anion (~max 410 nm).
To best accomplish this, a reagent test kit
containing the defined substrate disclosed above and a
maltase is used. One exemplary test kit is disclosed in
U.S. Patent 3,476,515. This test kit can be used in the
analyzer described in U.S. Patent 3,770,382.
EXAMPLE 1
A sample o~ ~-(4-nitrophenyl) maltotet~aoside
(G~pNp) prepared in accorclance with Example l~I of Canadian
Patent Application Serial No. 282,407 (R.C. Burns et al~/
filed on the same da~ as this application, was dissolved in
66.7 mM sodium phosphate buffer, pH 6~5, to provide various ~-
substrate concentrations ranging from 2 to 8 mg~3 ml. As
described in the aforementioned Canadian Patent Application
Serial No. 282,407 (R.C. Burns et al), this substrate sample
has been purified using a Sephadex~ LH-20 Chromatographic
Column. ~-Glucosidase of various concentrations rang:ing
from 2.5 to 12.5 International Units per three millilite~s
of solution (IU/3 ml) was then added to the substrate solu-
tion and the volume of the solution was hrought up to 3.0
ml. The solution was incubated at 37C. for l to 10 minutes~
After the blank xate was measured at 41Q nm;
using a Gil~ord spectrophotometer, the reaction was
initiated by adding 0.1 ml of an Elevated Enzyme Control i
Product sold by the E. I. du Pont de Nemours and Company
'
~1~ ~ / ï .
'
., ,, , ,, , ~ ~
(1150 Somogyi Units per deciliter (SU/dl) amylase~ diluted
1:1 with Du Pont Enz~me Diluent. This level of amylase
is approximately six times the upper normal serum level.
The total reaction rate was then measured using the
Gilford spectrophotometer, and by subtracting the blank
rate from the total rate, the net reaction rate was
obtained.
A two-variable statistical optimi~ation for the
substrate and the ~-glucosidase was run. I`he results of
this evaluation are given in Table I in arbitrary Absor-
bance units (A) per minute.
TABLE I
,005l .012 .018
12.5 .0912 .113 .llO
,~ .o863 .101 .092
P~ .004.008 .011
7.5 .090.llO .lOg
.086.102 .Og8
o
~ .001.008 .002
~ 2.5 .079.091 .0~0
078.083 .078
2.05.0 8.0
G4pNp mg/3 ml
l. Blank rate (A/min)
2. Total rate (A/min)
3. Net rate (A/min)
From this evaluation, it can be seen that the blank rate
increases as the concentrations of both the G~pNp and the
a-glucosidase increase, that the optimum concentration of
G4pNp is approximately 4.0 mg/3 ml, and that the optimum
-12-
concentration of a-glucosidase is approximately 7.5 IU/3
ml.
Using these optimum values of G4pNp and a-glucosi-
dase in a reaction solution of 3 ml,'the reaction rates or
various amylase sample concentrations were measured and
a standard curve was generated. From this curve, the
sensitivity in m~/min/SU/dl was measured. The standard
curve for this sample is given in Figure l; the blank
rate and sensitivity are given for this and other examples
; 10in Table II.
TABLE II
Example Blank Rate Sensitivity
(mA/minj (mA/min/SU/dl)
1 5.0 0.115
2 not measured 0.231
3 10.3-13.3 0.110
4 3.0 0.116 -
EXAMPLE 2
A small amount of the substrate sample used in
Example 1 was further purified by High Performance Liquid
Chromatography (HPLC) which is a standard puriication
techni~ue, well known to those skilled in the art. Using
the optimum values for G4pNp and a-glucosidase obtained in
Example 1, and the amylase sample of Example 1, a standard
~25 curve was generated using this purified substrate. The
sensitivity was also obtained, as described in Example 1.
The standard curve is given in Figure 1, the sensitivity
is given in Table II. As can be seen rom Table II, the
sensitivity o the assay was increased markedly by the
purification, indicating that the substrate sample of
Example 1 contained some inhibltor.
-13~
'; ' ~'
'' ' ' ' ' :' ' ~ ' "', ' ' ''
E~AMPLE 3
The ~-(4-nitrophenyl) maltotetraoside (G~pNp)
sample used in this Example was obtained by deacetylation
of the HPLC purified acetate of Example lD oE t,he afore-
mentioned Canadian Patent Application Serial No. 282,407
(R.C. Burns et al). In particular, to a sample of this
acetate, a solution of sodium methoxide and methanol was
added and the solution was stirred at room temperature in
a closed vessel for 18 hours. The methanol was then removed
under reduced pressure.
The G4pNp so formed was dissolved in 66.7 mM
sodium phosphate bu~er, pH 6.5, to provide a substrate
concentration of 4 mg/3 ml. Then 7.5 IU/3 ml of ~-glucosi-
dase was added to the subskrate solution and the volume o~ '
the solution was brought up to 3.0 ml. The solution was
incubated at 37C. for one to ten minutes~
After the blank rate was measured, as described
in Example 1 above, the reaction was incubated by adding ',~
0.1 ml of Du Pont Ele~ated Enzym~ Control Product, diluted
1:1 with Du Pont Enzyme Diluent. The reaction rates for
various amylase sample concentrations were measured as
discussed in Example 1 above, and a standard curve was
generated. From this curve, the sensitivity ln m~min~SU/dl
was measured. The standard curve ~or this substrate'is
given in Figure l; the blank rate and sensitivity are given
in Table II.
This is a crude sample; one that has not been
purified by chromatographic sepaxation techniques. As
a result, the blank rate is very hi,~h,, ran~in~ from 10.3
to 13~3 m~/min, but the sensitivity is equivalent to that
o~ the substrate reported in E~ample 1 ~here ini,tial
14
purification was accomplished using a Sephadex~ LH-20
column.
Another series of reactions were run us~ng the
conditions descri~ed above, except that five minutes after
it was initiated, the reaction was quenched by adding a
l.5 ml aliquot of the sample solution in-lo either l.5 ml
of 0.2 M Na2CO3 or 5 ml of 0.002 N NaOH. At pH 6.5 r the
extinction coefficient o~ the 4-nitrophenol is relatively
low because the 4-nitrophenol is no-t all ionized. The
increase in pH caused by the quenching is suffIcient to
completely ionize the 4-nitrophenol to 4-nitrophenylate
anion, thereby increasing the extinction coefficient. This
giVes rise to an l'end point" determination for which the
standard curves were non-linear, probably because the system
was optimiæed ~or a rate and not an end-point approach.
However, a Pive to ten fold lncrease in sensitivity was
obseryed.
EXAMPLE 4
A sample of a-(4-nitrophenyl) maltotetxaoside~
prepared in accordance with Example lG of the aforementioned
Canadian Patent Application Serial No. 282,407 (R.C. Burns
et al), was dissolved in 66.7 m~ sodium phosphate buffer,
pEI 6.5, to provide a substrate concentration of 4 mg~3 ml.
Then 7.5 IU/3 ml of ~-glucosidase was added to the substrate
solution and the volume of the solution was brought up to
3.Q ml. The solution was incubated at 37~C. for one to ten
,: , ,
; minutes.
After the blank rate was measured in a Gilford ~ ~`
spectrophotometer at 4l0 n~, the reaction was initiated by
addin~ O.l ml of the Du Pont Elevated Enzyme Control Product,
diluted l:l with Du Pont Enzy~e Di`luent. The reaction rates
..
.,
::
for the various amylase sample concentrations were measured,
using -the Gilford spectrophotometer, and a standard curve
was generated. From this curve, the sensitivity in
m~/min/SU/dl was measured. The standard curve for this
sample is given in Figure l; -the blank rate and sensitivity
are given in Table II.
This again is a substrate that was purified by
using a Sephadex~ LH 20 column. The sensitivity of the
assay using the substrate of this Example is equivalent to
that o~ the assay reported in Examples 1 and 3. The blank
rate, however, is somewhat lower than that of Example 1 and ;~
considerably lower than that o~ Example 3.
EX~MPLE 5
A sample of ~-(4-nitrophenyl) maltopentaoside
(G5pNp), prepared in accordance with Example 2E of the
aforementioned Canadian Patent Application Serial No. 282,407
(R.C. Burns et al), was dissolved in 6Ç.7 mM sodium phosphate
. buf~er, pH 6.5, to provide various substrate concentrations
ranging ~rom 4.0 to 12.0 mg/3 ml~ a-Glucosidase of various
activity ran~ing from 15 to 45 IU/3 ml was then added to the ;:
substrate solution and the volume o~ the solution was brought
up to 3.0 ml. The solution was incubated at 37C~ for one
to ten minutes.
Initial tests were conducted using 4.0 m~/3 ml
G5pNp and three ~-glucosidase concentratlons, 7.0, 14.0,
and 28.0 IU/3 ml. For each of these three: concentrations,
as set forth in Example 1~ the standard curves were pro-
duced usin~ the amylase sample identi~ied in Example 1~
In each case, the blan.k rate was 3~Q mA~min. The standard
curve for the three ~-glucosid~se concentrations a.re given
in Figure 2. A11 curves were non-linear which made a
--1~--
.,~ .
determination o the sensitivity difficulto Sensitivity/
however, is estimated to be greater than .160 mA/min/IU/dl.
Linearit~ increased as a-glucosidase concentration increased
indicating that, the ~~~lucosidase concentration was sub-
optimal.
A two-variable optimization was performed as
described in Example 1. The results of this optimization
are given in Table III.
TABLE III
,0031 ~00~ .005
.128 .139 .145 '"""'' '
.1253 .135 .140
H
~ .003 .00~ .012
o~
~ 30 .122 .13S .133
.,.
u~
o .119 .131 .121
, . .
.003 .005 007
.110 .119 .114
4.0 8.0 12.0
G5pNp my/3ml -
1, 2, 3 see Table I
From this evaluation, it can be seen that there is little increase
in the blank rate as G5pNp or ~-glucosidase concentrations are
increased. This is consistent with the situation w1th G5
25 as explained above. This analysis also indicates that the , , ,
optimum value for either G5pNp or ~-~lucosidase has not
been reached at 12.0 m~j3ml or 45 IU/3ml, respectivcly. ~-
In the sense that G5pNp has a lower or at least
a stable blank rate as a unction o substrate and ~-
~luco~idase concentrations, it is a preferred substrate.
-17-
However, the large concentrations of G5pNp and ~-glucosidase
required for the assay decreases its preferred status.
The disclosure above is intended to instruct
those skilled in the art, and is not intended to limit
the scope of the invention. Many modiEications well
within the skill of the art are intended to he included
~ith the scope of the invention as set forth in the
appended claims.
