Method for determination of components

• US 5,302,513 A
• Filed: 07/09/1991
• Issued: 04/12/1994
• Est. Priority Date: 06/29/1988
• Status: Expired due to Fees

• Current Assignee: Kyowa Medex Co., Ltd. (Resonac Holdings Corp.)
• Original Assignee: Kyowa Medex Co., Ltd. (Resonac Holdings Corp.)
• Inventors: Miike, Akira, Tatano, Toshio
• Primary Examiner(s): Wityshyn, Michael G.
• Assistant Examiner(s): Leary, Louise N.
• Application Number:
  US07/727,369
• Time in Patent Office: 1,008 Days
• Field of Search: 435/25, 435/15, 435/26, 435/16, 435/14, 435/23, 435/4
• US Class Current: 435/15
• CPC Class Codes: C12Q 1/008 for determining co-enzymes or co-factors, e.g. NAD, ATP
• CPC Class Codes: C12Q 1/40 involving amylase
• CPC Class Codes: C12Q 1/48 involving transferase

Abstract

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 γ-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.

Claims

1. In a method for determination of amylase activity in a biological sample, which comprises the steps of:

(1) mixing the sample with NAD(P), ATP, glucokinase, glucose-6-P-dehydrogenase, maltose phosphorylase and phosphate to form a first reaction solution;

(2) subjecting the first reaction solution to reaction with maltopentose to form a second reaction solution; and

(3) measuring the absorption of the second reaction solution at 340 nm, the improvement comprising;

carrying out the reaction of step (1) at 25°

to 50°

C. at pH 6 to 9 in the presence of glutathione reductase and glutathione of oxidation type; and

carrying out the reaction of step (2) at 25°

to 50°

C. at pH 6 to 9 in the presence of dithiothreitol, γ

-glutamyl transpeptidase and glycylglycine.

2. In a method for determination of amylase activity in a biological sample, which comprises the steps of:

(1) mixing the sample with NAD(P), phosphate, maltose phosphorylase, β

-phosphoglucomutase, glucose-6-P-dehydrogenase and at least one of glucose-1,6-diphosphate or fructose-1,6-diphosphate to form a first reaction solution;

(2) subjecting the first reaction solution to reaction with maltopentose to form a second reaction solution; and

(3) measuring the absorption of the second reaction solution at 340 nm, the improvement comprising;

carrying out the reaction of the step (1) at 25°

to 50°

C. at pH 6 to 9 in the presence of glutathione reductase and glutathione of oxidation type; and

carrying out the reaction of step (2) at 25°

to 50°

C. at pH 6 to 9 in the presence of dithiothreitol, γ

-glutamyl transpeptidase and glycylglycine.

3. In a method for quantitative determination of NAD(P)H formed by an enzymatic reaction in which an objective enzyme or substrate in a sample participates, the improvement comprising, prior to said quantitative determination, carrying out the steps of:

(1) converting to NAD(P) the NAD(P)H present in a sample or formed by an enzymatic reaction in which the objective enzyme or substrate does not participate using glutathione reductase at 25°

to 50°

C. at pH 6 to 9 and glutathione of oxidation type; and

(2) decomposing the remaining glutathione of oxidation type using γ

-glutamyl transpeptidase at 25°

to 50°

C. at pH 6 to 9 and glycine or glycylglycine, in the optional presence of a mercapto compound.

4. A method according to claim 3, wherein said quantitative determination of NAD(P)H is effected by measuring the ultraviolet absorption of a solution containing NAD(P)H at 340 nm.

5. A method according to claim 3, wherein said objective enzyme or substrate in a sample is a member selected from the group consisting of amylase, maltose, lipase, glycerol ester and choline esterase.

6. A method according to claim 3, wherein said mercapto compound is a member selected from the group consisting of dithiothreitol (DTT), 2-mercaptoethanol, cysteine or salts thereof, N-acetylcysteine or salts thereof, homocysteine or salts thereof, cysteamine or salts thereof, 2-mercapto-ethanesulfonate, 3-mercapto-1,2-propanediol, 2-mercaptopropionate, 3-mercaptopropionate, mercaptosuccinate, thiomalate, 1-thioglycerine and dithioerythritol.

7. A method for quantitatively determining NAD(P)H comprising the steps of:

(a) oxidizing a sample using oxidized glutathione and glutathione reductase;

(b) decomposing the oxidized glutathione remaining in the sample using γ

-glutamyl transpeptidase at 25°

to 50°

C. at pH 6 to 9 in the presence of glycine, glycylglycine or an equivalent thereof;

(c) forming NAD(P)H in a reaction using a component present in the sample;

(d) quantitatively determining any NAD(P)H formed in said reaction; and

(e) correlating the amount of component present in the sample from the amount of NAD(P)H determined in step (d).

8. A method according to claim 7, wherein the NAD(P)H formed in step (c) is quantitated by measuring the ultraviolet absorption of the sample at 340 nm.

9. A method according to claim 7, wherein the component of step (c) is selected from the group consisting of amylase, maltose, lipase, glycerol ester and choline esterase.

10. A method according to claim 7, wherein the decomposing step is conducted in the presence of a mercapto compound selected from the group consisting of dithiothreitol, 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-propandiol, 2-mercaptopropionate, 3-mercaptopropionate, mercaptosuccinate, thiomalate, 1-thioglycerine and dithioerythritol.