JPH04234997A - Quantitative analysis of polyamine - Google Patents

Abstract

PURPOSE:To quantitatively analyze a polyamine readily and high sensitively by acting a polyamine oxidase on a sample, then acting catalase thereon in the presence of an alcohol, subsequently acting a dehydrogenase in the presence of NAD and measuring the generated NADH. CONSTITUTION:A polyamine oxidase (e.g. putrescine oxidase) is acted on a polyamine (e.g. putrescine)-containing sample to convert the polyamine to hydrogen peroxide and an aminoalkylaldehyde. Catalase is acted on the generated hydrogen peroxide in the presence of an alcohol (e.g. ethanol) to convert the alcohol to aldehydes and aminoalkylaldehyde dehydrogenase is subsequently acted on the generated aldehyde and aminoalkylaldehyde in the presence of NAD and NADP. The generated NADH or NADPH is analyzed, e.g. by measuring the absorption at 340nm using a spectrometer, thus quantitatively analyzing the objective polyamine.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for quantifying polyamines in biological samples. The amount of polyamine in a biological sample is closely related to the occurrence and progression of cancer, and its quantification has high clinical significance as it provides useful information for cancer diagnosis. In addition, a relationship with diabetic nephropathy has recently been reported, and it is attracting attention as a marker for diabetes.

0002

BACKGROUND OF THE INVENTION As methods for quantifying polyamines, liquid chromatography, electrophoresis, and, as a simpler method, enzyme method have been proposed. The conventional enzymatic method involves applying an enzyme that oxidizes polyamines to the sample.
A method in which catalase is allowed to act on the generated hydrogen peroxide in the presence of an aminoalkyl alcohol and the generated aldehyde is measured (see JP-A-61-274698),
The polyamine in the sample is converted to aminoalkyl aldehyde, the generated aminoalkyl aldehyde is exposed to the presence of NAD or NADP, and aminoalkyl aldehyde dehydrogenase is applied to generate NADH or NADPH.
There is a known method for quantifying
(See Publication No. 00).

0003

[Problems to be Solved by the Invention] However, none of these methods is satisfactory in terms of sensitivity, etc., and a highly sensitive method for quantifying polyamines has been desired. An object of the present invention is to provide a simple and highly sensitive method for quantifying polyamines.

0004

[Means for Solving the Problem] The gist of the present invention is to convert a polyamine-containing sample into hydrogen peroxide and aminoalkyl aldehyde by allowing an enzyme that oxidizes the polyamine to act on the sample, and to convert the polyamine into hydrogen peroxide and aminoalkyl aldehyde. In the presence of alcohol, catalase is activated to convert it into aldehyde, and NA is added to the generated aldehyde and the above aminoalkyl aldehyde.
NADH or NADP produced by the action of aminoalkyl aldehydrogenase in the presence of D or NADP
The present invention relates to a method for quantifying polyamines characterized by measuring H.

In this case, if necessary, the acyl form of the polyamine may be reacted with acyl polyamine amidohydrolase (APAH) in advance or simultaneously to liberate the polyamine. To measure NADH or NADPH of the present invention, it can be directly quantified by absorption at 340 nm, or it can be measured by guiding it to a formazan coloring system or a luminescent system using reductase and luciferase in the presence of an electron carrier.

[0006] The polyamine oxidizing enzyme used in the present invention includes putrescine oxidase, polyamine oxidase, etc., and may be of any origin.
Some polyamine oxidases are produced by microorganisms such as the genus Nocardia and the genus Pseudomonas, and polyamine oxidases may be of any origin, but some are produced by microorganisms such as the genus Penicillium, the genus Aspergillus, and the genus Streptomyces.

The aminoalkyl aldehydrogenase used in the present invention is not particularly limited as long as it acts on at least one of formaldehyde, acetaldehyde, propionaldehyde, and butyraldehyde, and its origin is not particularly limited. , for example, aminobutyraldehydrogenase (ABAL-DH) produced by microorganisms such as Pseudomonas sp.
(Agri.Biol.Chem.50(
8) (1986) 2009-2016). Catalase used in the present invention is not particularly limited, and commercially available products may be used.

[0008] There are no particular restrictions on the concentrations of various enzymes;
Measurement time can be freely set depending on reaction conditions, economic conditions, etc.

[0009] The alcohol used in the present invention includes methanol, ethanol, propanol, and butanol, but ethanol is preferably used. The concentration of alcohol may be as long as it does not inhibit the entire enzyme reaction system, but is preferably 0.05 to 2.0%. Furthermore, when commercially available catalase is an ethanol solution, it is not necessary to add alcohol.

The NAD concentration may be any concentration that can maximize the reaction of aminoalkyl aldehyde dehydrogenase, such as ABAL-DH, and that does not inhibit the entire enzyme reaction system, but is preferably 0. 1-20mM is suitable.

[0011] When polyamines in a sample are measured using the above enzymes, coenzymes, and alcohol, it is preferable to carry out the measurement in an appropriate buffer. There are no particular limitations on the type of buffer as long as it can maintain the pH within the range of 4 to 10. Further, the reaction temperature may be any temperature as long as the reagent acts, but it is preferably around 20 to 40°C.

[0012] In the present invention, the enzymatic reaction may be carried out in one step or in two or more steps.

[0013]

[Examples] Example 1 The following reaction mixture was used to quantify putrescine at a known concentration. Note that the measurement was performed at 25° C. by changing the absorbance at a wavelength of 340 nm. 0.2M potassium phosphate buffer pH 8.0 1.1ml 30mM
NAD aqueous solution
0.1 3.0U/ml ABAL
-DH 0.2
25U/ml putrescine oxidase
0.1 75KU/ml
Catalase*
0.4 10% ethanol 0.
2 water

0.3
total
2.4ml *Commercial product (manufactured by Boehringer:
A 10% ethanol solution) was used after removing ethanol. The reaction to putrescine was as shown in Figure 1(b), and the reaction was terminated within 3 minutes. Further, the calibration curve at this time was shown by the straight line in FIG. 2, b, and the sensitivity was twice as high as that of the comparative example shown later.

Comparative Example A known concentration of putrescine was quantified in the same manner as in Example 1 using the following reaction mixture. 0.2M potassium phosphate buffer pH 8.0 1.1ml 30mM
NAD aqueous solution
0.1 3.0U/ml ABAL
-DH 0.2
25U/ml putrescine oxidase
0.1
water
0.9
total
The reaction to 2.4 ml putrescine was as shown in Figure 1(a), and the reaction was terminated within 3 minutes. In addition, the calibration curve at this time is shown in Figure 2, a.
It was like a straight line.

[0015]

Effects of the Invention According to the present invention, the conventional polyamine U
It is possible to obtain twice the sensitivity of the quantitative method using the V system, and more accurate quantitative determination is possible.

[Brief explanation of the drawing]

Figure 1: Quantification of putrescine at final concentrations of 0 to 120 μM.
The time course of NADH production at 0 nm is shown. In the figure, (a) shows the conventional method, and (b) shows the method of the present invention.

FIG. 2 shows calibration curves for method b of the present invention and conventional method a.

[Explanation of symbols]

1. When putrescine concentration is 0 μM

Claims (2)

[Claims] Claim 1: Applying an enzyme that oxidizes polyamines to a sample containing polyamines to convert the polyamines into hydrogen peroxide and aminoalkyl aldehyde, and applying catalase to the generated hydrogen peroxide in the presence of alcohol; A method for quantifying polyamines, which comprises converting the aldehyde into an aldehyde, reacting the generated aldehyde and the aminoalkyl aldehyde with aminoalkyl aldehydrogenase in the presence of NAD or NADP, and measuring the generated NADH or NADPH. [Claim 2] The aminoalkyl dehydrogenase is
The method for quantifying polyamines according to claim 1, which is an aminoalkyl dehydrogenase that acts on at least one of formaldehyde, acetaldehyde, propionaldehyde and butyraldehyde.