JPS592700A - Method for measuring total polyamine - Google Patents

Abstract

PURPOSE:To measure the total polyamines rapidly with ease, by oxidatively decomposing ascorbic acid in a sample, oxidizing a free and a conjugation type polyamines with a specific enzyme, reacting the resultant H2O2 with a chromogen, and optically measuring the resultant substance. CONSTITUTION:0.01-0.20ml sample is added to a solution prepared by dissolving 0.1-50U/ml ascorbic acid oxidase in 0.5-20ml buffer solution of 5.5- 7.5pH and 0.02-0.10 M concentration, and reacted at 20-40 deg.C for 3min or longer to decompose oxidatively the ascorbic acid in the sample. A solution containing 20-50U/ml enzyme, e.g. polyamine oxidase, capable of oxidizing free and conjugation type polyamines to form H2O2 in 1.0-4.0ml buffer solution of 7.0-9.0pH and 0.05-0.2 M concentration, and 0.1-50U/ml enzyme having the peroxidase-like activity and a chromogen, e.g. homovanilic acid, is added thereto to oxidize the free and conjugation type polyamines and react the formed H2O2 with the chromogen. The resultant substance is then colorimetrically measured.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for enzymatically directly measuring polyamines and their acetyl forms in a sample.

Polyamines are non-protein, low molecular weight aliphatic basic compounds that are one of various physiologically active amines, are widely distributed in living tissues and microorganisms, and are deeply involved in biological reactions. Putrescine, cataverine, spermidine, spermine, and their conjugated forms (acetyl forms) are mainly present in the body fluids of mammals including humans.

Incidentally, it has recently been known that total polyamines in body fluids are a useful indicator of human cancer. In other words, the total polyamines in body fluids such as urine, gastric fluid, blood, ascites, and pleural effusion of cancer patients clearly increase compared to normal subjects, and decrease when the affected area is removed or physical/chemical therapy is performed. Therefore, it is thought that it can be applied to the diagnosis of cancer, the determination of therapeutic effects, and the follow-up of prognosis. Moreover, since the increase in total polyamines in body fluids is not limited to specific cancers, if it can be measured quickly, easily and accurately, it has great promise as a screening test for cancer.

The conventionally known method for measuring total polyamines in biological samples is to thermally decompose various conjugated polyamines in the sample at 100°C for about 6 hours under strong acidity to convert them to free polyamines. , protein removal is required before that), and the extract is subjected to electrophoresis, dansyl derivative-thin skin chromatography, gas chromatography, amino acid analyzer method, or high performance liquid chromatography. This is something that will be analyzed by law. Shigashi,
All of these methods require complicated and time-consuming procedures for sample pretreatment, and require special equipment and skilled techniques for analysis, so they have not been put into practical use as routine clinical tests.

In order to correct these drawbacks, the present inventor has developed a technique for enzymatically measuring total polyamines as a result of research (Japanese Patent Publication No. 56-36918). This method uses polyamine oxidase and peroxidase extracted from germinated soybeans, and first the sample is acid-treated in the same way as before.
The conjugated polyamine is converted into a free form by thermal hydrolysis, and then passed through a cation exchange column to remove reducing substances such as ascorbic acid that coexist in the sample, and the polyamic acid adsorbed in the column is eluted. After the addition of polyamine oxidase, peroxidase, and a chromogen, total polyamines are measured by colorimetry or fluorescence. This method is still inconvenient because it requires pretreatment of the specimen, such as hydrolysis treatment and removal of coexisting substances using a cation exchange column, and is therefore of little use as an actual clinical test. Moreover, the process for removing coexisting substances not only takes time and effort, but also has the disadvantage that it is difficult to completely separate and elute polyamines, which may cause errors.

The present invention was made in view of the current situation, and is a quick and simple method that does not require any special technology or equipment.
The purpose of the present invention is to provide a method for measuring total polyamines in body fluids that is accurate enough to be used as a cancer screening test.

In order to achieve this objective, the present inventors have conducted extensive research and found that currently known polyamine oxidases extracted from animals and bacteria oxidize only specific free polyamines; It was discovered that polyamine oxidase extracted from leguminous plants oxidizes not only various free polyamines but also conjugated polyamines, and using these polyamines eliminates the need for hydrolysis treatment to convert conjugated polyamines into free forms. Thought. However, in polyamine oxidase extracted from leguminous plants, the Km value (Michaels constant) when using a conjugated polyamine as a substrate is extremely large compared to that of the free type. Larger amounts (high activity units) in the enzymatic analysis method found in No. 36918
It has been found that using such polyamine oxidases requires long reaction times. Therefore, we continued our research and found that ascorbic acid oxidase oxidizes ascorbic acid, an interfering substance coexisting in samples, and oxidizes both free and conjugated (acetyl) polyamines to generate hydrogen peroxide. The chromogen is oxidized by the action of an enzyme system with peroxidase-like activity that uses the generated hydrogen peroxide as a substrate, and the resulting color-forming substance or fluorescent substance We have completed a method for measuring total polyamines by colorimetric or fluorescent methods.

According to this method, free and conjugated polyamines can be oxidized simultaneously, so there is no need to hydrolyze the sample.
In addition, since ascorbic acid, which is a reducing substance that coexists with ascorbic acid oxidase, is oxidized and decomposed without generating hydrogen peroxide, there is no need to use a cation exchange column, so total polyamines can be used extremely quickly and easily. Can be measured accurately.

Therefore, it is useful in clinical tests aimed at diagnosing cancer, determining therapeutic efficacy, detecting metastasis/recurrence, and observing prognosis.

The total polyamine measurement method according to the present invention uses ascorbate oxidase, an enzyme that oxidizes both free and conjugated polyamines, an enzyme system that exhibits peroxidase-like activity, a chromogen, a pH buffering agent, and the like. Furthermore, various additives such as preservatives, stabilizers, reaction inhibitors, and reaction accelerators can be added as necessary.

Ascorbic acid oxidase can be used as long as it is an enzyme that has the effect of oxidizing ascorbic acid and converting it into dehydroascorbic acid.
s sp) Yaka Irf: Chi 4? (Cucurbi
Specific examples include those originating from taceae sp). Enzymes that oxidize both free and conjugated polyamines are mainly putrescine, cataverine, spermidine,
Any material may be used as long as it oxidizes spermine and its acetyl forms to produce hydrogen peroxide, such as those originating from germinated soybeans, alfalfa, or clover. However, JP-A-55-96093, JP-A-56-92787, or JP-A-56-92788
Polyamine oxidases originating from microorganisms such as those shown in the above issue cannot be used in the present invention because they have the effect of oxidizing only free spermidine and spermine.

Any enzyme system with peroxidase-like activity may be used as long as it can oxidize the chromogen by decomposing hydrogen peroxide as a substrate. In addition to these enzymes, iodide and molybdate,
Compounds such as metal porphyrin and iron ferrocyanide can also be used because they have similar functions.

As the chromogen, any substance or substance group that can be optically detected by being oxidized by an enzyme system exhibiting peroxidase-like activity and hydrogen peroxide can be used. Among these, when the optical detection means is a colorimetric method, orthodianisidine, 2
, 2-azinodi(3ethylbenzothiazoline)-6-sulfonic acid (abbreviated as ABTS), varadiphenylamine sulfonate, 4-aminoantipyrine (hereinafter 4-A
A) and phenol, orthotolidine, 3-methyl-2-benzothiazoline hydrazone and N,N-dimethylaniline, 4-AA and diazo red RC, 4-AA and 3
．． 5-dimethoxy-N-ethyl-N(2-hydroxy-
3-Sulfopropyl) Aniline Natriu! (DA
OS), 4-AA and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-meta-toluidine.
Sodium salt (abbreviated as ToO8) and the like are used. In the case of the fluorescence method, tyramine hydrochloride, 4-hydroxy-
3-methoxyphenylacetic acid, homovanillic acid, etc. are used. Preferred examples include 4-AA and TOO5.

4-AA, DAO8 and homovanillic acid are mentioned because of their high sensitivity. Any pHfl buffer can be used as long as it can provide optimal pH conditions for each enzyme reaction, such as phosphate buffer, Tris buffer, borate buffer, etc.

Therefore, in order to measure the total polyamines in body fluids using the method of the present invention, an appropriate amount of the sample is added to a solution in which ascorbic acid oxidase, polyamine oxidase, and a chromogen are dissolved in pHwI buffer, and the amount of the sample is kept constant until the reaction is completed. temperature and measure the reaction system optically (colorimetrically or fluorometrically).
The total amount of polyamine is determined based on a calibration curve determined in advance. However, since the details of the measurement method differ depending on the type of body fluid, a detailed explanation will be given below as an example.

First, when measuring total polyamines in urine, pH 5.5 ~
75. pf of 0.02-0.10 M (buffer 0.5-
2.0- ascorbic acid oxidase 0.1-50U
Sample amount 0.01 to 0.20+ is added to the solution in which /d is dissolved.
+tl! at a constant temperature (20-40'
The reaction in C) oxidizes and decomposes ascorbic acid coexisting in urine. Next pH7.0-9.0.0.05-0.2
M pH 1.0-4. A solution containing 20-50 U/yptl of polyamine oxidase and peroxidase was added to OWLl, and the reaction solution was kept at a constant temperature (20-40'C) until the reaction was completed (about 30 minutes or more). Measure colorimetrically. In addition, the polyamine oxidase used in the above case is 100 to 5.
000 U, but it is preferable to use 2000 or more, particularly 600 U or more.

This is because polyamine oxidase completes the reaction with free polyamines in a relatively short time (about 5 minutes) even if the unit is small, but it is difficult to react with conjugated polyamines (acetyl polyamines). . For example, Figure 1 shows a urine sample from a normal person (the same person) (the total amount of polyamines is approximately 170n).
This shows the relationship between absorbance and reaction time when polyamine oxidase of various units is applied to mode/at. It may not be possible to complete the process completely, or even if it is possible, it will take a long time.
For U it takes 60 minutes and for 100 U it takes more than 70 minutes).

On the other hand, Fig. 2 shows 1 d of urine samples from multiple subjects.
This shows the relationship between absorbance and reaction time when using 600 U of enzyme per enzyme, and shows the total amount of polyamines and the proportion of acetyl forms in both normal subjects (curves ■ and ■) and cancer patients (curves ■ to ■). Although various patterns are drawn depending on the
．． QOOn mob e/me (see Figure 3).

Therefore, when water washing is used for cancer screening tests, the shorter the time required (for example, 30 to 60 minutes), the better.
, Therefore, polyamine oxidase should be as high unit as possible (
For example, it is preferable to use 600 U or more), but there are economic limitations. Therefore, even if the reaction does not completely complete in about 60 minutes, it is practical to use about 100 to 200 U, which is necessary to complete the reaction. I can't stand it. Incidentally, in the method of Japanese Patent Publication No. 56-36918, which precedes the present invention, about 0.5 to 100 urine samples were used per 1 to 1 of urine specimens.

Next, when quantifying total polyamines in serum, the concentration of total polyamines in the groove is extremely low, so a fluorescent reagent is used as a chromogen. That is, 0.1 to 1 of serum
rttl was taken, and a supernatant liquid of 0.05 to 1. Using Orttl as a specimen, it is operated in the same manner as in the case of urine, and the fluorescence intensity of the final reaction solution is measured. However, in the case of serum, the concentration of total polyamines is several tens of times lower than that in urine, so polyamine oxidase is also in relatively low units. For example, it is practical if the amount is about 150 U or more per 14 specimens.

Furthermore, when quantifying the total polyamines in red blood cells, a fluorescent reagent is also used. Since total polyamines are present in the red blood cell sample as well as in urine, it is preferable that the number of polyamine oxidase units be the same as in the case of urine.

Incidentally, body fluids other than urine, serum, and red blood cells, such as gastric fluid, ascites, brachial fluid, semen, and even total polyamines in living tissues, can be similarly treated with polyamine oxidase and peroxidase after simple pretreatment such as deproteinization. This can be easily and reliably obtained. However, for cancer screening tests, etc., it is desirable to use urine and blood, which can be easily collected, as samples, so the present invention will be described in more detail below with reference to examples for urine, serum, and red blood cells. However, the technical idea and scope of the present invention are not limited in any way by this example. In addition to those described above and those shown in this example, various compounds and drugs may be used within the scope of the technical idea of the present invention, and various additives may also be used for the purpose of preservatives, stabilization, reaction control/promotion, etc. Example 1 Determination of total polyamines in urine (reagent) Reagent ascorbate oxidase 4
U/mlD A OS
O,2tny/d0.05M-phosphate buffer (PH6,5) Reagent B Polyamine oxidase 30
tJ/d peroxidase
2U/nt14- A A
0.2my/lnln100I phosphoric acid solution (pH 8, o) standard solution (1ρ00n moire
7-l) Spermidine trihydrochloride
25.46rnf! purified water
Total volume of 100 liters (600 U of polyamine oxidase per 1 mg of urine) Specimen, standard, and blind absorbance were determined (S) and (St), respectively.

(BJ,) Then, the total polyamine concentration in the specimen is calculated using the following formula.

Incidentally, the relationship between the concentration of the standard solution and its diluted solution and the absorbance obtained by subtracting the blind absorbance from each absorbance is shown in FIG.

(Reproducibility test) A reproducibility test was conducted on the urine of three people, and the results were as shown in Table 2.

Table 2 Reproducibility test (unit: n-mole/m1) (Correlation with other units) The method disclosed in Japanese Patent Publication No. 56-36918 by the inventor of the present application (hereinafter referred to as the "hydrolysis mini-column method") We investigated the correlation with the mainstream. The result number was the next one (Table 3 and Figure 4) 0 Table 3 Relationship with other streams [Unit - is molle/1tt13 Correlation coefficient 0946 Regression line Y = 1.063 + 7. 20 Example 2 Determination of total polyamines in serum (reagent) Protein removal reagent (0.33M perchloric acid aqueous solution) 60% perchloric acid 55.25f purified water Total amount 1000m/1% sodium hydroxide solution Sodium hydroxide 1f purified
Water All ff1loOd Coloring Solution Polyamine Oxidase 5'/
ytl ascorbate oxidase
1'/mA' peroxidase 0.5 u
/me Homovanillic acid 0.01~
/-〇, IM-phosphate buffer standard solution (!in mole /ntl) spermidine 1.27 mg purified water
Total amount: 1000 d (measurement procedure) 0.5 d of serum and protein removal reagent 2. Add Otrtl, mix well, and centrifuge at 3000 rpm for 10 minutes, and use the supernatant as a sample.

Table 4 (Polyamine oxidase 250U per 1mA of serum)
The fluorescence intensities of the specimen, blinded specimen, standard solution, and blinded test were determined respectively [
S], (S - Bl x (St), (Bu), the total polyamine concentration in the sample is calculated using the following formula.

(Measurement results) Measured using 10 normal human serum samples: 0.7-1
．． 3nmoj! Got one shift of e/rttl.

Table 5 Example 3 Quantification of red blood cell layer polyamine (reagent) Other than setting the concentration of polyamine oxidase to 115,
Same as Example 2.

(Measurement procedure) Collect blood into a heparin blood collection tube, mix thoroughly, and
Centrifuge for 10 minutes at Orpm. After removing the plasma and the upper layer of white blood cells and platelets using an aspirator, several doses of physiological saline are added and thoroughly mixed and washed. After centrifugation at 3.00 rpm, the upper layer is removed. The red blood cells are further washed twice with physiological saline and centrifuged to remove the upper layer to obtain a red blood cell layer. Take 0.10++tA' from this red blood cell layer into a test tube, add 2:0ml of protein removal solution, mix thoroughly, and then 8ρ0
The supernatant obtained by centrifugation at 0 rpm for 10 minutes is used as the sample.

/''' Table 6 Composition of reaction solution (420 U of polyamine oxidase per red blood cell)
) If the fluorescence intensities of the sample, blinded sample, standard solution, and blinded sample are (S), (S-Br), (St), and (Bj), respectively, the total polyamine concentration in the sample is calculated using the following formula. be done.

(Measurement results) Measured using 10 samples of normal human red blood cells.
A value of nmole/- was obtained.

Table 7 As detailed above, the present invention provides a method for measuring the total amount of polyamines in a sample using an enzyme, in which ascorbic acid is decomposed by using ascorbic acid oxidase, and free polyamines as well as conjugated polyamines are decomposed. Because it oxidizes both substances, special equipment and techniques are not required for separation and analysis, and cation exchange columns can be used to hydrolyze the sample and remove coexisting interfering substances. It can be easily measured optically. Therefore, the measurement of total polyamines, which conventionally required special and advanced skills, can be performed quickly and accurately with simple operations.
Since it is possible to easily analyze a large amount of samples, it will make a great contribution to the field of clinical cancer testing.

[Brief explanation of the drawing]

Figure 1 is a graph showing the relationship between reaction time and absorbance when the number of polyamine oxidase units per 1n of urine sample from normal subjects is changed. Figure 2 is a graph showing the relationship between normal subjects (curves ■ and ■) and cancer patients (curves ■ and ■). Graph showing the color development time course of urine samples (■～■), Figure 3 is for polyamine 1ρ00n mode.
Figure 4 is a graph showing the correlation between the measured values of the hydrolysis mini-column method and the method of the present invention. Bundle of Thoughts Relationship between absorbance and reaction time per 1 ml of absorbance and reaction time Figure 2 Keihan Sai's Reiseki time course (Namafu e I ml this 'J 100ul! 60p
Ll) No. 4riJ-556

Claims (1)

[Claims] 1. Ascorbic acid oxidase oxidizes ascorbic acid coexisting in the sample, and free and conjugated polyamines are oxidized by an enzyme that has the function of oxidizing free and conjugated polyamines to generate hydrogen peroxide,
A method for measuring total polyamines, which comprises reacting the generated hydrogen peroxide and a chromogen by the action of an enzyme system having peroxidase-like activity, and optically measuring the obtained substance.