Method for determination of components

The present invention relates to a method for quantitative determination of NAD(P)H derived from a specific component, which comprises converting NAD(P)H present in a sample or formed from components other than the objective component by reactions into NAD(P) by the action of glutathione of oxidation type and glutathione reductase; decomposing the remaining glutathione of oxidation type by the action of .gamma.-glutamyl transpeptidase in the presence or in the absence of a mercapto compound; forming NAD(P)H from the component to be determined in the sample utilizing an NAD(P)H-forming reaction system; and quantitatively determining NAD(P)H. According to the method of the present invention, amylase activity, etc. in vital components containing maltose and glucose can be determined accurately.

INDUSTRIALLY APPLICABLE FIELD 
The present invention relates to a method for quantitative determination of 
a component in a sample by utilizing a reaction system which forms 
NAD(P)H, wherein NAD(P)H present in the sample or derived from components 
other than the objective component by reactions is eliminated prior to the 
quantitative determination of the objective component by utilizing the 
system which forms NAD(P)H. 
BACKGROUND ART 
Several methods for determining a component in a sample by utilizing 
oxidoreductase which catalyzes a reaction involving NAD(P)-NAD(P)H are 
known. 
The methods are advantageous in stoichiometrical accuracy, small influence 
of other components, and the like. However, when NAD(P)H derived from 
components other than the objective component (hereinafter referred to as 
other components) is added during the determination, such NAD(P)H must be 
avoided or removed. 
To avoid or remove such NAD(P)H, there is a method in which a reagent blank 
test is separately carried out and after determination of other components 
(so called blank test of a sample), the sum of NAD(P)H derived from the 
other components and the objective components is determined and the amount 
of the objective component is determined by calculation. 
For example, in the case of determination of amylase activity in blood, the 
enzyme reaction described below is carried out and the rate of formation 
of NAD(P)H is determined, whereby the amylase activity can be determined. 
However, as maltose and glucose are contained in blood, NAD(P)H must be 
determined after they are removed or decomposed. 
##STR1## 
In the quantitative determination of NAD(P)H by colorimetry of the reaction 
solution, the absorbancy of the objective component is added to those of 
the other components, and the determinable range becomes narrow, depending 
upon the amount of glucose or maltose in a sample. The reason is that the 
absorbancy which is detectable with a spectrophotometer according to the 
currently employed technique is limited to a certain range; that is, it is 
impossible to detect an absorbancy of 3.0 ABS or more with an ordinary 
spectrophotometer. In view of this aspect, a region in which a measurement 
value is low is preferred. Further, in the case of reagent blank test with 
a high absorbancy, reproducibility is poor. Assuming that blank solutions 
have absorbancies of, for example, A and B (A&lt;&lt;B) respectively, the 
absorbancy E(E&lt;&lt;B) appearing as the result of reaction is added to form 
A+E and B+E (A+E&lt;&lt;B+E). When this run is repeated with measurements, the 
respective variations referred to as As and Bs are obtained (variations in 
the blank are similarly referred to as Ab and Bb, respectively). From the 
nature of a spectrophotometer as an apparatus, the percent transmission (T 
%) is logarithmically converted to absorbancy. Thus, a difference based on 
the logarithmic function appears on the variation in absorbancy in the 
case of low absorbancy (high percent transmission) and on the variation in 
absorbancy in the case of high absorbancy (low percent transmission), and 
even though the variations in percent transmission are identical, As&lt;Bs 
(Ab&lt;Bb) (absorbancy=log I.sub.0 /I, wherein I is transmittance). From the 
foregoing, the difference E is calculated for each of the blanks A and B. 
With respect to A: 
EQU (A+E).+-.As-A.+-.Ab=E.+-.As.+-.Ab 
With respect to B: 
EQU (B+E).+-.Bs-B.+-.Bb=E.+-.Bs.+-.Bb 
From As&lt;Bs and Ab&lt;Bb is led, As+Ab&lt;Bs+Bb; that is, when the blank has a 
large value, the variation becomes large, so that the coefficient of 
variation obtained by division by E becomes large. 
An object of the present invention is to provide a method for quantitative 
determination of the objective component utilizing an NAD(P)H-forming 
reaction system without performing a blank test of the sample, by 
eliminating NAD(P)H formed from the other components or present in the 
sample by reactions. 
DISCLOSURE OF THE INVENTION 
According to the present invention, the objective component in a sample can 
be quantitatively determined by converting NAD(P)H present in the sample 
or formed from components other than the objective component into NAD(P) 
by the action of glutathione reductase (referred to as GR) in the presence 
of glutathione of oxidation type [hereinafter referred to as G(OX)] (first 
reaction); decomposing G(OX) by the action of .gamma.-glutamyl 
transpeptidase (hereinafter referred to as .gamma.-GTP) in the presence of 
glycine, glycylglycine or an equivalent thereof, or decomposing G(OX) to 
glutathione of reduction type [hereinafter referred to as G(OH)] with a 
mercapto compound and at the same time decomposing G(OX) and G(OH) with 
.gamma.-GTP (second reaction); and then quantitatively determining the 
objective component utilizing the reaction system forming NAD(P)H. The 
objective component includes both of a substrate and an activity of an 
enzyme which participate in a reaction led to the NAD(P)H-forming reaction 
system.

The present invention is described in detail below. 
In the first reaction, G(OX) and glutathione reductase are added to a 
sample, if necessary, together with a substrate or an enzyme necessary for 
the reaction for forming NAD(P)H from the other components during the 
course of forming NAD(P)H from the objective component. The mixture is 
subjected to reaction in an appropriate buffer solution to convert NAD(P)H 
present in the sample or formed from the other components into NAD(P). 
G(OX) is used in an amount equivalent to that of NAD(P)H to be eliminated 
or more, preferably more than 1.5 times the amount of NAD(P)H. Glutathione 
reductase and the buffer solution are used in concentrations of 0.1 to 20 
U/ml and 0.001 to 2M, respectively. The reaction is carried out at a 
temperature of 25.degree. to 50.degree. C. at pH 6 to 9 and is completed 
in several minutes. 
Examples of the buffer are 3-(N-morpholino)propanesulfonic acid (MOPS), 
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), 
Tris-hydrochloride, phosphate, borate, oxalate and Good's buffers. 
In the second reaction, G(OX) present in the reaction solution after the 
first reaction is decomposed by .gamma.-GTP. The decomposition of G(OX) 
can be accelerated by allowing the mercapto compound to exist. The 
mercapto compound converts G(OX) into G(OH) and G(OH) is readily 
decomposed by .gamma.-GTP. The decomposition of G(OX) and G(OH) by 
.gamma.-GTP is carried out in the presence of glycine, glycylglycine or an 
equivalent thereof. Examples of the reactions are shown by the reaction 
formulae (6) and (7) below. The equivalent mentioned above refers to a 
compound that can react with G(OX) and G(OH) by the action of .gamma.-GTP 
and thereby can be converted into a substance which does not affect the 
subsequent quantitative determination of NAD(P)H. Glycylglycine, etc. are 
used in an amount equivalent to that of G(OX) or more, usually in an 
amount of 3 to 10 equivalents based on G(OX). The reaction is carried out 
at a temperature of 25.degree. to 50.degree. C. at pH 6 to 9. The mercapto 
compound is added in an amount equivalent to that of G(OX) or more, 
preferably in an amount of more than 1.5 equivalents based on G(OX). 
.gamma.-GTP is used in an amount of 0.1 to 500 U/ml. As the mercapto 
compound, any compound having SH group and which does not inhibit the 
enzyme used is usable. Examples of the mercapto compound include 
dithiothreitol (DTT), 2-mercaptoethanol, cysteine or salts thereof, 
N-acetylcysteine or salts thereof, homocysteine or salts thereof, 
cysteamine or salts thereof, 2-mercaptoethanesulfonate, 
3-mercapto-1,2-propanediol, 2-mercaptopropionate, 3-mercaptopropionate, 
mercaptosuccinate, thiomalate, 1-thioglycerine and dithioerythritol. 
In cases where the formed NAD(P)H is quantitatively determined by measuring 
the absorption at 340 nm, it is preferred to use the mercapto compound 
having no absorption at 340 nm. 
As the third reaction, when the objective component is a substrate, the 
substrate is decomposed to lead to the NAD(P)H-forming reaction and the 
formed NAD(P)H is quantitatively determined in a conventional manner. 
When the objective component is an enzyme, a substrate for the enzyme is 
added and, if necessary, other enzymes are further added to lead to the 
NAD(P)H-forming reaction, and the rate of formation of NAD(P)H is 
determined. 
Any known methods and methods which will be developed in the future are 
applicable to the quantitative determination of NAD(P)H formed by the 
reaction, unless they are unsuitable for the object of the present 
invention. 
There are known, for example, a method in which the absorption is measured 
at 340 nm (UV method); the fluorescence method in which the absorption is 
measured at 365 nm or 460 nm; a method which comprises subjecting NAD(P)H 
to reaction with diaphorase or phenazine methosulfate, and tetrazolium 
salt, and measuring the absorption in a visible region; a method which 
comprises subjecting NAD(P)H to reaction with a chromogen in the presence 
of peroxidase or thiol oxidoreductase, and diaphorase, and quantitatively 
determining the dye formed; and a method which comprises converting 
NAD(P)H into a signal of hydrogen peroxide and quantitatively determining 
the signal (Japanese Published Unexamined Patent Applications Nos. 
43398/85, 180600/85, 205999/84, 106299/84 and 32867/84). 
In cases where the sample is a vital component or the like, a variety of 
surface active agents (for example, nonionic, anionic and cationic surface 
active agents such as polyoxyethylene higher aliphatic ethers or esters 
thereof, polyoxyethylene sorbitan higher fatty acid esters, 
polyoxyethylene-polyoxypropylene copolymer, polyoxyethylene higher 
aliphatic phenyl ethers, higher fatty acid sodium salts and sodium higher 
aliphatic benzenesulfonates) and lipases (for example, lipoprotein lipase) 
are used as lipid-solubilizing agents, etc. In addition, chelating agents 
(for example, EDTA or salts thereof, GEDTA, CYDTA, TTHA and IDA) and 
enzyme activators (for example, magnesium salts, calcium salts and zinc 
salts) are appropriately used. Furthermore, if necessary, specific enzyme 
inhibitors, for example, amylase inhibitor (made from wheat), lipase 
inhibitor, enzyme inhibitory antibody (monoclonal or polyclonal), and 
glutathione derivatives which are inhibitors of glutathione reductase may 
be used in order to terminate the enzyme reaction. 
In the case of the aforesaid amylase determination, which is taken as an 
example of the determination of a component that achieves excellent 
results by the method of the present invention, addition of NAD(P), ATP, 
glucokinase (or hexokinase), G6PDH, maltose phosphorylase, glutathione 
reductase, G(OX) and phosphate to a sample results in formation of NAD(P)H 
derived from glucose and maltose in the sample and the formed NAD(P)H is 
converted into NAD(P). 
The reactions described above are represented by the reaction formulae (1) 
through (4) given hereinabove and additionally the following formula: 
##STR2## 
Then, by addition of maltopentose which is a substrate for amylase, OTT, 
.gamma.-GTP and glycylglycine, the remaining G(OX) is converted into G(OH) 
by the reducing power of DTT and at the same time, G(OX) is converted into 
glutamylglycylglycine and cystine by .gamma.-GTP and G(OH) is converted 
into glutamylglycylglycine and cysteinylglycine. 
##STR3## 
The added maltopentose is decomposed by amylase and NAD(P)H is formed 
according to the reaction formulae (1) through (4) shown above. By 
measuring the absorbancy of the reaction solution at 340 nm, NAD(P)H can 
be quantitatively determined and accordingly, amylase can be 
quantitatively determined. 
Other examples of the determination of components which achieves excellent 
results by applying the present invention are shown below. 
(1) Quantitative determination of amylase (A) (elimination of maltose and 
glucose which are originally present) 
Maltose which is originally present is decomposed by maltose phosphorylase 
(MP) in the presence of phosphate. The formed glucose and glucose which is 
originally present are decomposed by glucose dehydrogenase in the presence 
of NAD(P), and then the formed NAD(P)H is eliminated. After G(OX) is 
decomposed, maltopentose is added and decomposed by amylase in the sample. 
The formed maltose is quantitatively determined. 
(2) Quantitative determination of amylase (B) (elimination of maltose which 
is originally present) 
Maltose which is originally present is decomposed in the same manner as in 
(1). The formed glucose-1-P is converted into glucose-6-P by 
phosphoglucomutase. Glucose-6-P is acted on by glucose-6-P-dehydrogenase 
(G6PDH), and the formed NAD(P)H is eliminated. After G(OX) is decomposed, 
maltopentose is added and the amylase activity is measured. 
(3) Quantitative determination of maltose (elimination of glucose which is 
originally present) 
Glucose which is originally present is decomposed by glucose dehydrogenase 
in the presence of NAD(P), and the formed NAD(P)H is eliminated. After 
G(OX) is decomposed, MP is added and maltose is quantitatively determined. 
(4) Lipase activity (elimination of free glycerol which is originally 
present) 
Free glycerol which is originally present is decomposed by glycerol 
dehydrogenase (G.DH), and the formed NADH is eliminated. After G(OX) is 
decomposed, glycerol ester is added and the formed glycerol is 
quantitatively determined, whereby the lipase activity is determined. 
(5) Quantitative determination of glycerol ester (elimination of free 
glycerol which is originally present) 
Free glycerol is decomposed by G.DH, followed by elimination of the formed 
NADH. After G(OX) is decomposed, lipase is added and the formed glycerol 
is quantitatively determined, whereby glycerol ester is determined. 
(6) Choline esterase activity (elimination of free choline which is 
originally present) 
Free choline is decomposed by choline dehydrogenase and betaine aldehyde 
dehydrogenase, and the formed NADH is eliminated. After G(OX) is 
decomposed, choline ester is added and the rate of formation of choline is 
measured, whereby the choline esterase activity is determined. 
(7) Lipase activity (elimination of free fatty acids which are originally 
present) 
Free fatty acids are converted into acyl CoA by CoA and acyl CoA 
synthetase. The acyl CoA is decomposed by 2-enoylacyl-CoA hydrolyase, 
L-3-hydroxyacyl-CoA, NAD oxidoreductase, acyl-CoA: acetyl 
CoA.C-acetyltransferase complex enzyme (HDT) and acyl CoA oxidase, 
followed by elimination of the formed NADH. After G(OX) is decomposed, 
glycerol ester is added and the formed fatty acids are quantitatively 
determined, whereby the lipase activity is determined. 
As illustrated above, in cases where the component (.alpha.) formed during 
the course of leading the objective component into the NAD(P)H-forming 
reaction is originally contained in a sample, the objective component in 
the sample can be determined accurately by leading the component (.alpha.) 
originally contained in the sample into the NAD(P)H-forming reaction; 
eliminating the formed NAD(P)H by the method of the present invention; 
leading the objective component into the NAD(P)H-forming reaction system; 
and then determining the formed NAD(P)H. 
Certain embodiments of the present invnetion are illustrated by the 
following examples. 
EXAMPLE 1 (DETERMINATION OF AMYLASE ACTIVITY) 
Reagent A 
______________________________________ 
MOPS buffer solution (Dojin Chemical 
0.1 M 
Research Institute) pH: 7.5 
Dipotassium phosphate 10 mM 
ATP 2.5 mg/ml 
Magnesium chloride 2 mg/ml 
Triton X-100 2 mg/ml 
EDTA 0.1 mg/ml 
Maltose phosphorylase 10 U/ml 
Glucokinase 5 U/ml 
NADP 1 mg/ml 
GP6DH 5 U/ml 
GR 3 U/ml 
G(OX) 0.5 mg/ml 
______________________________________ 
Reagent B 
______________________________________ 
MOPS (pH: 7.5) 0.1 M 
Maltopentose 4 mg/ml 
DTT 5 mg/ml 
.gamma.-GTP 10 U/ml 
Glycylglycine 5 mg/ml 
______________________________________ 
Reagent C 
The same composition as that of Reagent A except that GR and G(OX) are not 
contained. 
Reagent D 
The same composition as that of Reagent B except that DTT is not contained. 
When Reagents A and B are used, NADPH derived from glucose and maltose in a 
sample is eliminated, followed by measurement of the amylase activity, in 
which maltopentose acts as a substrate. 
When Reagents C and D are used instead of Reagents A and B, NADPH derived 
from glucose and maltose is not eliminated but remains. 
Test 1. 
Reagent A (1.5 ml) was placed in a cell for a spectrophotometer and heated 
at 37.degree. C. for 5 minutes. Then, 0.02 ml each of samples respectively 
containing the substances shown in the table was added to the cell, 
followed by heating for further 5 minutes. Separately, 1.5 ml of Reagent B 
was heated at 37.degree. C. and added to the mixture. Absorption of the 
reaction solution at 340 nm was traced. 
Test 2. 
In the procedure of Test 1, Reagents C and D were used instead of Reagents 
A and B, respectively. The test results show the absorption values of the 
reaction solutions. 
______________________________________ 
Sample Test 1 
No. (ABS/min) Test 2 
______________________________________ 
1 Glucose 10 mg/ml 
constant at 0 
constant at 2.2 
2 Maltose 10 mg/ml 
constant at 0 
constant at 1.17 
3 Amylase 100 mu/ml 
increased with 
same as in Test 1 
time, 0.0041 
4 Glucose 10 mg/ml 
increased with 
increased with 
Amylase 100 mu/ml 
time, 0.0041 
time from 2.3 
5 Maltose 10 mg/ml 
increased with 
increased with 
Amylase 100 mu/ml 
time, 0.0041 
time from 1.15 
6 Serum (containing 
increased with 
increased with 
6.24 mg/ml glucose) 
time, 0.0025 
time from 1.5 
______________________________________ 
The absorption values of Sample Nos. 1 and 2 in Test 2 result from glucose 
and maltose. 
In Sample No. 3, neither glucose nor maltose is originally present and so 
NADPH to be eliminated is not formed. 
In the case of Sample Nos. 4 and 5, after elimination of glucose or maltose 
which is originally present, maltopentose is decomposed by amylase in the 
samples and the absorption due to the formation of NADPH increases in Test 
1. In Test 2, NADPH derived from glucose and maltose is included from the 
beginning. The results similar to those with Sample Nos. 4 and 5 are 
obtained with Sample No. 6. 
The procedures of Test 1 and Test 2 were carried out using Sample No. 3 
with a variety of the amylase activity, i.e. 10 mu/m.+-., 20 mu/ml, 50 
mu/ml, 100 mu/ml and 200 mu/ml. Reagent B or D was added thereto and the 
absorbancy was measured after 10 minutes to prepare calibration curves. 
The calibration curves are shown in FIG. 1 (Test 1) and FIG. 2 (Test 2). 
With respect to the test using Sample No. 6, the absorption was measured 5 
minutes after the addition of Reagent B or D (initiation of the absorption 
due to the amylase reaction) and 10 minutes after the addition and the 
mean rate of change (.DELTA.E) was calculated to determine the amylase 
activity. Test 1 and Test 2 were repeated 10 times, respectively, and the 
coefficients of variation (CV %) obtained are shown in the table below. 
TABLE 1 
______________________________________ 
Number Test 1 (U/l) 
Test 2 (U/l) 
______________________________________ 
1 58.6 71.8 
2 60.7 48.7 
3 62.5 73.7 
4 59.8 62.6 
5 61.6 48.2 
6 61.8 59.4 
7 64.0 61.6 
8 59.5 71.2 
9 62.5 62.5 
10 61.4 51.7 
Mean Value 61.2 61.1 
CV (%) 2.5% 14.5% 
______________________________________ 
As is clear from the table, Test 1 provides results much superior to those 
of Test 2 in reproducibility. 
EXAMPLE 2 
The same experiment as in test 1 of Example 1 was carried out using Sample 
No. 6, except that 12 U/ml glucose dehydrogenase was used instead of ATP, 
glucokinase, and G6PDH. The results are shown below. 
TABLE 2 
______________________________________ 
Number Amylase value (mu/ml) 
______________________________________ 
1 59.2 
2 61.9 
3 62.1 
4 61.4 
5 60.2 
6 61.5 
7 62.3 
8 60.0 
9 61.0 
10 63.1 
Mean 61.3 mu/ml 
CV = 1.84% 
______________________________________ 
EXAMPLE 3 
The same experiment as in Test 1 of Example 1 was carried out using Sample 
No. 6 supplemented with 20 mg/ml maltose, except that 0.1 mg/ml 
glucose-1,6-diphosphate and 5 U/ml phosphoglucomutase were used instead of 
ATP and glucokinase. The results are shown in Table 3. 
TABLE 3 
______________________________________ 
Number Amylase value (mu/ml) 
______________________________________ 
1 60.4 
2 59.3 
3 60.6 
4 59.5 
5 63.2 
6 58.0 
7 60.1 
8 63.1 
9 60.8 
10 63.0 
Mean 60.8 mu/ml 
CV = 2.77% 
______________________________________ 
EXAMPLE 4 
The same experiment as in Test 1 of Example 1 was carried out using Sample 
No. 6, except that 5 U/ml hexokinase was used instead of glucokinase. The 
results are shown in Table 4. 
TABLE 4 
______________________________________ 
Number Amylase value (mu/ml) 
______________________________________ 
1 60.8 
2 60.7 
3 61.7 
4 61.0 
5 60.6 
6 61.2 
7 61.3 
8 61.4 
9 61.4 
10 61.2 
Mean 61.2 mu/ml 
CV = 0.51% 
______________________________________ 
EXAMPLE 5 
Reagent A 
______________________________________ 
TES buffer solution (pH 6.75) 
0.04 M 
Pluronic F-68 1 mg/ml 
1,2-Dilinolein 1.33 mM 
Co-A 2 mM 
Co-lipase 0.1 mg/ml 
CaCl.sub.2 1.17 mM 
NAD 8.17 mM 
ATP 1 mM 
Acyl CoA synthetase (ACS) 
1.7 U/ml 
Acyl CoA oxidase (ACO) 20 U/ml 
HDT 45 U/ml 
MgCl.sub.2 1.17 mM 
GR 5 U/ml 
G(OX) 0.8 mg/ml 
______________________________________ 
Reagent B 
______________________________________ 
TES buffer solution (pH 6.75) 
0.04 M 
Pluronic F-68 1 mg/ml 
Deoxycholic acid 16.5 mM 
Dithioerythritol 5 mg/ml 
.gamma.-GTP 10 U/ml 
Glycylglycine 8 mg/ml 
______________________________________ 
Serum (0.02 ml) having the lipase content of 55.4 mu/ml as measured with 
the lipase assay kit lipase UV (Toyo Jozo Co., Ltd.) was added to 1.5 ml 
of Reagent A. After reaction at 37.degree. C. for 3 minutes, Reagent B was 
added to the reaction mixture. Two minutes after the addition, the rate of 
increase in absorption of the reaction solution at 340 nm was measured to 
determine the lipase activity. The mean value of 10 measurements was 56 
U/ml with CV of 1.8%. 
EXAMPLE 6 
The same procedure as in Example 5 was carried out, except that 
1-monolinolein was used instead of 1,2-dilinolein and glycerol 
dehydrogenase was used instead of ACS, ACO, HDT and CoA. Test 1 gave the 
result of 55.1 U/l with CV of 2.7%.