WO2004111635A1

WO2004111635A1 - Composition for aldehyde detection

Info

Publication number: WO2004111635A1
Application number: PCT/JP2003/007470
Authority: WO
Inventors: Akihiko Kimura; Yoshinari Hirota; Xiaohong Wu; Toshitaka Okada; Fumiko Suzuki; Yasuo Inagaki
Original assignee: Toyo Hakko Co., Ltd.
Priority date: 2003-06-12
Filing date: 2003-06-12
Publication date: 2004-12-23
Also published as: JPWO2004111635A1; AU2003242311A1

Abstract

A composition comprising a basic fuchsine, sodium hydrogen sulfite, and phosphoric acid. It is for use in detecting aldehydes, e.g., malondialdehyde (MDA), contained in a biological sample. The aldehydes are an index to lipid peroxidation or active oxygen.

Description

Specification

Composition for aldehyde detection

The present invention relates to a composition for detecting an aldehyde in a biological sample as an indicator of lipid peroxidation or active oxygen. Background art

When an oxidative stress disorder is given to a living body, the first target is the unsaturated fatty acids that make up the biological membrane of cells. Various low molecular aldehydes are produced from unsaturated fatty acids by a chain reaction caused by reactive oxygen attack. The generated low molecular aldehydes are highly reactive and attack biological components such as proteins, nucleic acids, and lipids, and damage the living body by modifying them.

As the above aldehyde, malondialdehyde (MDA) is known. MDA is metabolized by the body's antioxidant mechanism and excreted in the urine via the blood. Thus, by measuring the concentration of MDA in a biological sample, such as urine, the level of active oxygen production can be estimated. You can also judge the balance of your daily diet and antioxidant levels.

Acrolein is another known aldehyde. Acrolein is a highly reactive aldehyde generated by heating cooking oil and burning tobacco, and has been reported to have strong cytotoxicity and mutagenicity. In recent years, it has been demonstrated that acrolein is produced by the peroxidation of lipids in vitro, and its presence has been confirmed in atherosclerotic lesions in humans, and is useful as a marker for lipid oxidation damage in vivo. Sex is expected.

Lipids are also more susceptible to oxidation than nucleic acids and proteins, producing hydroperoxide. Hydroperoxide also reacts with nucleophilic sites on proteins to form complex modifications that cause protein degradation. Therefore, hydroveloxide has also attracted interest as an oxidized stress marker in recent years.

Therefore, by measuring the concentration of MDA in a biological sample, You can know the level of oxidation.

As a conventional method for detecting aldehydes in biological samples, there is a method based on instrumental analysis such as HPLC GC-MAS, but it requires expensive equipment and advanced technology, and has the problem of complicated operation. .

Simple methods for detecting aldehydes in biological samples include Schiff reagents.

(Including basic fuchsin and sodium bisulfite and, optionally, hydrochloric acid) are known.

Known Schiff reagents are obtained by decolorizing Magiening, which has been used as a dye for a long time, through a sulfurous acid gas, and used for the detection of aldehyde or the periodic acid Schiff staining method (PAS).

However, known Schiff reagents have very low or no reactivity with biological samples, such as urine, and may detect formaldehyde (HCHO) or malondialdehyde (MDA). Could not.

Therefore, development of an inexpensive composition that can be used for a simple method for detecting aldehydes in a biological sample has been awaited.

The present inventors have surprisingly found that by using phosphoric acid having a buffering capacity instead of hydrochloric acid, the stability of the composition can be increased and the detection sensitivity of aldehydes can be improved. The present invention has been completed. Disclosure of the invention

The present invention relates to a composition for detecting aldehyde, comprising basic fuchsin, sodium bisulfite, and phosphoric acid.

More specifically, the present invention relates to a composition for detecting aldehyde, comprising 0.1 to 0.5% by weight of basic fuchsin, 0.5 to 5% by weight of phosphoric acid, and 1 to 5% by weight of sodium bisulfite. About.

Known Schiff reagent compositions include basic fuchsin and sodium bisulfite, and optionally, hydrochloric acid. On the other hand, the composition of the present invention is characterized by containing basic fuchsin, sodium bisulfite and phosphoric acid.

Known Schiff reagents include formaldehyde (HCHO) or malondialdehyde. Very low reactivity to MDA. In addition, whereas the composition of the present invention has high reactivity to HCHO and MDA, while having low or no reactivity to biological samples, for example, urine, For example, it has reactivity to urine and its sensitivity is high.

Phosphoric acid was used in place of hydrochloric acid used in known Schiff reagents for the following reasons.

The condensation of the carbonyl compound and RNH ₂ derivative _{(C = OH + H 2 N_R-} C = N one R + H ₂ 0), usually requires acidic catalysts.

The rate of this condensation reaction depends on the acid concentration, and as the pH is increased, the reaction rate reaches a maximum at a certain pH. The value of p H of the reaction rate is maximized, greatly depends on 1 the nature of R- 1 ^ 11 _2.

The maximum in rate is understood by the equilibrium between RNH ₂ and the carbonyl compound (RNH ₂ + H ⁺ = RNH ₃ ⁺ ). Here, if a proton is attached to the lone pair of the nitrogen of RNH ₂ , the carbon of the carbonyl group cannot be attacked. On the other hand, when the carbonyl group is protonated, the reactivity with the nucleophile increases (RC = O ⁺ H + RC = OH +).

Therefore, the optimum pH is the pH at which the conjugate acid, a carboxylate compound, is at an appropriate concentration and RNH ₂ is not completely RNH ₃ + but the reaction proceeds.

Therefore, it is considered that phosphoric acid (pKa = 2.14) promotes this reaction system rather than concentrated hydrochloric acid (pKa = -8).

Further, phosphoric acid has a buffering ability and can maintain stability against the pH value of the solution. In addition, it can be adapted in biological samples.

The aldehyde detected by the composition of the present invention is preferably malondial. The composition of the present invention can be obtained, for example, by the following method.

Dissolve 30 fmg of basic fuchsin in 10 ml of water, add 3. Og of sodium bisulfite, and shake until completely dissolved. Then add 2. Og of 85% phosphoric acid. Activated carbon (1.0 g) was added to this solution and filtered. The compositions of the present invention can be used for detecting aldehydes in biological samples. You.

The biological sample is preferably urine.

The composition of the present invention can be used for measuring lipid peroxide and active oxygen. The present invention also relates to a container containing the above-described aldehyde detection composition and an aldehyde detection kit including instructions.

The composition of the present invention can easily measure the oxidation state in the body and can provide an important index for maintaining health.

By using the composition of the present invention, aldehydes such as MDA excreted in urine, which could not be detected by the conventional Schiff reagent, can be detected simply and quickly.

The data obtained by using the composition of the present invention can be used as an index of the state of oxidative stress in a living body, and can be used as a guide for nutrition intake and as a guide to the auxiliary effect of an antioxidant. .

BRIEF DESCRIPTION OF THE FIGURES

Figures 1A and B show the relationship between lifestyle and MDA levels.

FIGS. 2A and B show the absorbance intensity obtained using the composition of the present invention and the FRT kit.

FIG. 3A shows the correlation between the absorbance value obtained using the composition of the present invention and the MDA value contained in urine.

Figure 3B shows absorbance values obtained using the FRT kit and MD contained in urine.

The correlation with the A value is shown. Example

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

1. Reactivity to standard substances MDA and HCHO

1-1. Preparation of standard

MDA was obtained by hydrolyzing 1,1,3,3-tetraethoxypropane (TEP) with HC1. Using purified water, 0.05, 0.1 and ImM in MDA Was prepared and used as an MDA standard solution.

HCHO was prepared by diluting a commercially available reagent, formaldehyde, with purified water to prepare HCHO solutions of 0.05, 0.1, and ImM, which were used as HCHO standard solutions.

1-2. Detection with FRT solution

The FRT solution was obtained from Nippon Shokuyo Kagaku Co., Ltd., the country of origin and the United States. The reaction was performed at a ratio of FRT solution: MDA or HCHO standard solution = 1: 1.

The results are shown below.

1-3. Detection by the composition of the present invention

The method was carried out at a ratio of the composition of the present invention: MDA or HCHO standard solution: 0. The results are shown below.

Table 2

The reactivity to MDA showed a very low coloration with the FRT solution, while the present invention composition showed a high coloration result. In addition, it was observed that the reactivity of the composition of the present invention to MDA tended to increase depending on the concentration of MDA.

2. Reactivity to biological urine

2- 1. Reactivity by FRT

Morning urine (the first urine after waking up) from healthy humans (n = 20) was collected. The reaction was performed at a ratio of FRT: urine = 1: 10, and the color reaction was detected.

The results are shown below. Table 3

No. 11 12 13 14 15 16 17 18 19 20 Level ++ »++++》 ++++ ++ IIII》 ++++》 ++++

OD value 0.416 0.166 6.004 7.67 5.358 0.49 2.009 1.645 5.166 2.402

The OD value was determined by reacting at room temperature with the ratio of urine fluid: FRT kit = 10: 1. After 5 minutes, 150 µΐ of the reaction solution was quickly placed in a 96-well plate, and the wavelength was 532 nm using a microplate reader. Is the value obtained by measuring the absorbance.

Lebenore Visual method OD range

Urine color (yellow)

：: slight color development 0.05 <± ≤ 0.1

+: Light pink 0.1 <+ ≤ 0.4

+ + Slightly red 0.4 <+ + ≤ 0.7

Red 0.7 <+ + + ≤ 1.2

Dark red: .2

As shown in the above table, the color development for each individual using the FRT kit for 5 minutes was extremely high, and it was not possible to detect diversity for each individual.

As is well known, in vivo (blood and urine) MDA is an oxidative degradation product of an unsaturated fatty acid which is a biological component. The normal value of MDA generated in vivo is still unknown, but at present the value differs depending on the measurement method.

The literature states that in normal humans, the amount of MDA in blood is about 1 OnmolMDA or less. The reaction of the FRT kit to urine is very strong and develops a deep color. However, it is questionable whether it accurately reflects the amount of MDA in urine, and the credibility of the reaction results is considered to be very low.

2-2. Reactivity of the composition of the present invention with human urine

Morning urine (the first urine after getting up in the morning) of healthy humans (n = 20) was collected. The reaction is The composition of the present invention: urine = 10 was performed, and the color reaction was detected.

The results are shown below.

Table 4

The OD value was determined by reacting at room temperature with urine: composition of the present invention at a ratio of 10: 1. This is a value obtained by measuring the absorbance at 532 nm.

Lebenore Visual method OD range

Urine color (yellow)

Soil: slight color development 0.05 <± ≤ 0.1

+: Light pink 0.1 <+ ≤ 0.4

+ + Slightly red 0.4 <+ + ≤ 0.7

Red 0.7 <+ + + ≤ 1.2

Dark red .. 2

The color development for 5 minutes in each individual using the composition of the present invention was distributed at various levels, was very diverse, and was considered to be more reliable than the FRT kit.

3. Relationship to lifestyle of each individual

Early morning urine was collected from 20 healthy subjects, 10 smokers and drinkers, and 10 non-smokers and non-drinkers, and measured using the composition of the present invention. The results are shown in FIG. Measurement result

Smoker + Drinker Non-smoker + Non-drinker

(Visual method)

0 0

Sat 0 1

+ 1 3

+ + 4 2

5 3

0 1 In populations with smokers and drinking habits, 50% of humans have a high level of red coloring (+++), 40% have a medium level of coloring (++), and 40% have a low level of coloring.

The percentage of (+) is 10%, whereas the percentage of the highest level (+ + + +) is 10% in the population without smoking and drinking habits, and the next highest level (+ + The percentage of +) is 30%, the percentage of the intermediate color level (++) is 20%, and the low color level

The percentage of (+) was 30% and the percentage of humans with very low color development levels or near no color development (soil) was 10%.

Also, if the coloration degree is below 10 and the low level is 10 and above and 10 and above, the high level is 10% for smokers and drinkers, and the high level is 90% for smokers and drinkers. Accounted for.

In contrast, low levels accounted for 40% of non-smokers and non-drinkers, while high levels accounted for 60%. It was revealed that the results of using the composition of the present invention were higher in the group having smoking and drinking habits than in the group of individuals having no smoking and drinking habits. Oxidative stress has been shown to occur in smoking, drinking, and excessive exercise. Smoking contributes to the production of Reactive Oxygen Species (ROS) and may induce oxidative damage to DNA, proteins and lipids associated with diseases such as cancer and cardiovascular disease.

4. Correlation between absorbance obtained using the composition of the present invention and FRT kit and MDA value In order to compare the correlation between the absorbance value obtained using the composition of the present invention and the FRT kit and the MDA value contained in urine, a method similar to the above was carried out with the addition of 15 more samples. The absorbance of each sample was measured using the composition of the present invention and the FRT kit.

In addition, urinary MDAi can be obtained from literature (W.G.Niehaus, Jr. and B. Samuelesson, Eur.

J. Biochem. 6, 126 (1968); ED Wills, Biochem. J. 113, 315 (1969)).

As the measured value, MDA was an equivalent value when TEP was used as a standard. The correlation between the absorbance obtained for each and the urinary MDA value was determined.

As a result, when the inventive composition is used, the coloring level (+++), (+ ten),

The number of specimens belonging to (10) and (P) was 15 (43%), 12 (34%), 7 (20%), and 1 (3%), respectively.

In contrast, when the FRT kit was used, the number of samples belonging to the coloring levels (+ + +), (+ tens), (ten), (1 1%), 1 (3%) and 0 (0%) samples.

When the FRT kit was used, the color reaction was very strong and no diversity could be detected in each sample. On the other hand, when the composition of the present invention was used, the degree of coloration (range of the obtained OD value) was determined by the levels “+++”, “ten”, “ten”, and “earth”. The ratios belonging to “1” and “1” indicate that the detection is rich in diversity, highly concentration-dependent, and enables more reliable detection (Fig. 2).

The correlation coefficient between the absorbance obtained using the composition of the present invention and the FRT kit and the MDA value was determined. As a result, the correlation coefficient between the absorbance obtained using the composition of the present invention and the MDA value was 0.45, whereas the correlation coefficient between the absorbance obtained using the FRT kit and the MDA value was found. The relationship number was 0.31, indicating that the composition of the present invention had a higher correlation than the FRT kit (FIG. 3).

Claims

The scope of the claims

1. A composition for detecting aldehydes, comprising basic fuchsin, sodium bisulfite, and phosphoric acid.

2. The composition according to claim 1, wherein the aldehyde is formaldehyde or malondialdehyde.

3. The composition according to claim 1 or 2, which is used for measurement in a biological sample.

4. The composition according to any one of claims 1 to 3, wherein the biological sample is urine.

5. The composition according to any one of claims 1 to 4, which is used for measuring lipid peroxide.

6. The composition according to any one of claims 1 to 4, which is used for measuring active oxygen.

7. A container for containing the composition according to any one of claims 1 to 6, and a kit for detecting aldehydes containing instructions.