JP2000310625A - Method for measuring oxidative dna damaged product - Google Patents

Abstract

PROBLEM TO BE SOLVED: To accurately grasp to what active oxygen an organism is actually exposed to by passing urine through a gel filtration column for fractionation, passing a fraction through an inverse-phase column, and measuring the amount of an oxidative DNA damaged product or a carcinogen-DNA adduct in an eluate from the inverse-phase column. SOLUTION: Urine is passed through a gel filtration column (multi-mode column) with the characteristics of an inverse-phase column and an ion-exchange column for fractionation to obtain a fraction containing an oxidative DNA damaged product or a carcinogen-DNA adduct. As a separation mode, an inverse-phase mode, an ion-exchange mode, and a gel filtration mode simultaneously operate for separation. In the inverse-phase mode, operation is made by using polyvinyl alcohol as a support material. Also, for operating the ion- exchange mode, a carboxyl group is contained in the solid phase of the support material of the multi-mode column. By using the multi-mode column, salt content, protein, and low-molecule impurities in the urine can be simultaneously eliminated.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring the urinary concentration of an oxidative DNA damage product or a carcinogen-DNA adduct (adduct).

[0002]

2. Description of the Related Art In recent years, attention has been paid to the action of active oxygen (oxygen radicals) in vivo, and several facts suggesting involvement of various diseases and aging are known. That is,
1) radiation, which is a carcinogen, causes the generation of reactive oxygen species, resulting in DNA damage, and 2) the frequency of spontaneous cancers in animals is correlated with the oxygen consumption of that species.
3) Many carcinogens and tumor promoters generate active oxygen. As described above, if active oxygen is closely related to the development of human cancer, it is necessary to prove by some means that antioxidants (so-called radical scavengers) can be taken to prevent cancer and prevent cancer. It will be possible to come up with an effective strategy. The generation of active oxygen is generated by oxygen metabolism, chemical carcinogens and radiation as described above, but also shows an important function in metabolic systems such as arachidonic acid cascade and in apoptosis which are important in inflammation.

As described above, it can be easily presumed that the generation of active oxygen having various functions in a living body is involved in the carcinogenesis process, but active oxygen itself is unstable and can be directly detected in cells. It is difficult at present. The most important thing at the moment is to find a lot of direct evidence that active oxygen is actually involved in human carcinogenesis, in which case it is simple and easy to measure active oxygen directly or indirectly Establishment of a method is required.

A product of DNA damage caused by reactive oxygen, 8-hydroxyguanine (hereinafter also referred to as 8-OH-Gua), is one of the oxidative DNA damage markers discovered by Kasai et al. GC by base pairing →
It is known to cause TA transversion mutations and appears to play an important role in the carcinogenesis process.

At present, 8-hydroxydeoxyguanosine (hereinafter, also referred to as 8-OH-dG) is widely used as a marker for oxidative DNA damage, and an electrochemical detector (HPLC) connected to high performance liquid chromatography (HPLC) is used. (ECD) (HPLC-ECD method).

[0006] 8-OH-dG can be measured using blood-derived cells such as organs, cultured cells and peripheral blood leukocytes, but it has also been attempted to measure the amount in urine. (Kasai et al., Mutation Res., 387, 147-163, 199
7). To measure 8-OH-dG as an index for assessing the risk of carcinogenesis of individuals, diagnosing reactive oxygen-related diseases, and assessing the degree of aging, it is simple and practical to measure urinary amounts It is. Urinary 8-OH-d reported so far
G analysis methods are roughly classified into three types. 1) 8-OH-
The fraction purified on the affinity column to which the antibody against dG is bound is analyzed by HPLC-ECD (Park et al., Pro
c. Natl. Acad. Sci. USA, 89, 3375-3379, 1992).
2) Connect three columns and use column switching
8-OH-dG is separated and detected by an electrochemical detector (ECD) (Loft et al., Carcinogenesis, 13, 2241-2247, 199
2). 3) Direct analysis of urine by ELISA (Erholaet a)
l., FEBS Lett., 9, 287-291, 1997).

However, each method has its own problems. The method 1) cannot be generally used because an affinity column is not commercially available.
It is also necessary to calculate urinary concentrations using radioactive internal standards with low recovery rates. In the method 2), the pretreatment is complicated and the recovery rate is not clear. Also, 8-OH-dG
In many cases, contaminants appear in the vicinity of the peak, and the measured values vary considerably from laboratory to laboratory. The method 3) has a problem in specificity, and the measured value is reported to be eight times higher than that of the HPLC-ECD method, and the data has a large variation (Prieme et a).
l., Natural antioxidants and food quality in ather
osclerosis and cancer prevention (Kumpulainen et a
l., eds.), The Royal Society of Chemistry, pp78-8
2, 1996).

Further, since the above methods 1) and 3) are detection methods using an antibody column or ELISA,
Oxidatively damaged nucleosides other than -OH-dG cannot be measured. In the method 2), 8-OH-dG can be measured, but 8-OH-Gua produced by repairing 8-OH-dG cannot be measured. There is a wide variety of oxidatively damaged nucleosides in urine other than 8-OH-dG. In addition, a carcinogen-DNA adduct (adduct) for monitoring the risk of carcinogenesis may be present. However,
Conventional methods cannot detect these trace substances.

Further, an antibody affinity column was used.
Only one case of simultaneous analysis of 8-OH-dG and 8-OH-Gua has been reported (Park et al., Proc. Natl. Acad. Sci. USA, 89, 3
375-3379, 1992). However, the recovery rate is poor, and urine is mixed with an internal standard substance labeled with a radioactive isotope ¹⁴ C to calculate the urinary concentration by the isotope dilution method. In addition, an affinity column to which an antibody is bound is not commercially available, and 8-OH-dG and 8
Since the binding ability of -OH-Gua changes, this method is unlikely to be a general method in that it lacks convenience and reproducibility.

In addition to 8-OH-dG, 2-hydroxydeoxyadenosine (2-OH-dA), 5-hydroxydeoxycytidine (5-OH-dC), 5-formyldeoxyuridine (5-C)
Although several tens of oxidative DNA damages are known, such as HO-dU), no analysis has been made on biological samples.

[0011]

SUMMARY OF THE INVENTION An object of the present invention is to provide a highly sensitive and simple method for measuring oxidative DNA damage products or carcinogen-DNA adducts (adducts) produced by DNA oxidative damage present in urine. It is to provide a method.

[0012]

SUMMARY OF THE INVENTION The present inventors have developed a highly sensitive and sensitive method for detecting a wide variety of oxidative DNA damage products present in urine and carcinogen-DNA adducts (adducts) for monitoring the risk of carcinogenesis. In order to establish a simple measurement method, urine samples were directly purified by using two types of columns sequentially, impurities in urine were removed, and an electrochemical detector (E
CD).

That is, the gist of the present invention is as follows. (1) A method for measuring the urinary concentration of an oxidative DNA damage product or a carcinogen-DNA adduct, comprising the following steps: 1) A step of fractionating urine through a gel filtration column having characteristics of a reversed phase column and an ion exchange column to obtain a fraction containing an oxidative DNA damage product or a carcinogen-DNA adduct. 2) passing the fraction through a reversed phase column. 3) measuring the amount of oxidative DNA damage product or carcinogen-DNA adduct in the eluate from the reverse phase column. (2) The method according to (1) above, wherein the urinary concentration of the oxidative DNA damage product is measured. (3) The method according to (2) above, wherein the oxidatively damaged DNA product is an oxidatively damaged nucleoside and / or an oxidatively damaged base. (4) The method according to (3), wherein the oxidatively damaged nucleoside is 8-hydroxideoxyguanosine and the oxidatively damaged base is 8-hydroxyguanine.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

Oxidative DNA damage products measured by the measurement method of the present invention are produced as a result of DNA damage by active oxygen (oxygen radicals) or the like in a living body,
It is excreted in urine. Specific examples include oxidatively damaged nucleosides and oxidatively damaged bases. Examples of oxidatively damaged nucleosides include 8-hydroxydeoxyguanosine (8-OH-dG), 2-hydroxydeoxyadenosine (2-OH-dA), 5-hydroxydeoxycytidine (5-OH-dC), and 5-formyl Deoxyuridine (5
-CHO-dU). Oxidatively damaged bases include:
Examples include the base portion of the oxidatively damaged nucleoside, and specific examples include 8-hydroxyguanine (8-OH-Gua). As shown in the examples, in the method of the present invention, 8-hydroxideoxyguanosine (8-OH-d
G) and oxidatively damaged nucleosides such as 8-hydroxyguanine (8-OH-Gua) and oxidatively damaged bases can be measured simultaneously.

The carcinogen-DNA adduct (adduct) measured by the measuring method of the present invention is a carcinogen such as benzopyrene added to DNA and excreted in urine. Specific examples include a benzpyrene-dG adduct.

In the first step of the measurement method of the present invention, the urine is fractionated through a gel filtration column having characteristics of a reversed phase column and an ion exchange column, and the fraction containing an oxidative DNA damage product or a carcinogen-DNA adduct is fractionated. This is the step of obtaining the minutes.
A gel filtration column having the characteristics of a reversed-phase column and an ion-exchange column (hereinafter, also referred to as a multi-mode column) is used as a separation mode in which a reverse-phase mode, an ion-exchange mode, and a gel-filtration mode operate simultaneously to perform separation. Refers to a column.

The reverse phase mode of the present multi-mode column works by using polyvinyl alcohol as a filler. In addition, a carboxyl group is contained in the solid phase of the filler of the present multi-mode column in order to operate the ion exchange mode. For the gel filtration mode to work, the pore size ranges from about 50 ° to about 1000 °, and the exclusion limit molecular weight ranges from about 500 to about 40,000. The particle size of the filler may range from about 2 μm to about 50 μm.

As a specific example of the present multi-mode column, Sh
odex ^™ Asahipak ^™ GS-320 HQ (Shokotsu Trading) and Sho
dex ^™ Asahipak ^™ GS-320 7G (Shokotsu Trading Co., Ltd.). These columns use polyvinyl alcohol as a filler and contain carboxyl groups. The exclusion limit molecular weight of these columns is about 40,000. The eluent has a pH of about 2 to about 1
Although an aqueous solution of 2 is possible, for the analysis of 8-OH-dG
A 0.1% acetic acid solution is most suitable. By using the present multi-mode column, salt, protein, and low molecular impurities in urine can be removed almost at once.

In the second step of the measuring method of the present invention, the oxidative DNA damage product or carcinogen-D obtained in the first step is used.
In this step, a fraction containing an NA adduct is passed through a reversed-phase column.
As the reversed phase column, for example, a standard column used for analysis of 8-hydroxydeoxyguanosine (8-OH-dG) can be used. Specifically, for example, YMC-Pack ODS
-AM (S-5 μm) (YMC).

In the third step of the measuring method of the present invention, the amount of the oxidative DNA damage product or the carcinogen-DNA adduct in the eluate from the reversed phase column in the second step is determined by an electrochemical detector (ECD). Or it is measured using a liquid chromatography mass spectrometer (LCMS) or the like.

FIG. 1 shows a conceptual diagram of the detection system. Two columns, a multi-mode column (HPLC-1 in FIG. 1) and a reversed-phase column (HPLC-2 in FIG. 1), were connected via an auto-sampling injector, and the fraction obtained by HPLC-1 was analyzed. Stir automatically and allow a fixed amount to be injected into HPLC-2. At this time, it is preferable to set the temperature of the column to 25 ° C. The solution eluted from HPLC-2 is directly analyzed by, for example, ECD.

Specifically, for example, a pump 1 (CCPM-I) having a built-in deaerator (SD-8022) such as a Tosoh HPLC system
0.5 ml of urine corresponding to I) is injected into HPLC-1 using an autosampler (AS-950-10, manufactured by JASCO Corporation). As a column to be used, for example, Asahipak ^™ GS-320 7G (7.6x300 mm)
(Shokotsusho) or Asahipak ^™ GS-320 HQ (7.6x300m
m) (Shokotsu Trading Company) can be used. The eluent is
It is sent to the sampling injector by the pump 2. As the sampling injector, for example, an auto sampling injector M231XL manufactured by Acom can be used. At this time, the separation pattern is confirmed with a UV detector (290 nm). The preparative range is determined for each column using a standard sample to be measured. When the sampling range is determined, the sampling injector automatically sorts (approximately
10 ml), and after automatic stirring, set to inject 100 μl into HPLC-2. Next, it is further fractionated by HPLC-2 and analyzed by, for example, ECD. In addition, when analyzing a urine sample continuously by automatic operation, urine may be injected using an autosampler every 165 minutes.
The -1 column may be set to be washed with 100% methanol. The HPLC-2 column can be used repeatedly without washing.

The measurement method of the present invention is characterized in that a fraction containing a target component is determined in advance from a separation pattern of HPLC-1 using a standard sample, and the elution conditions of HPLC-2 are appropriately controlled. The ability to measure various oxidative DNA damage products or carcinogen-DNA adducts, including 8-hydroxydeoxyguanosine (8-OH-dG). For example, 5-hydroxydeoxycytidine (5-OH-dC) and its base, 5-hydroxycytosine, can be analyzed using ECD. One type of this oxidative DNA damage, 5-OH-dC, is mutagenic and may be involved in carcinogenesis, but has been shown to be detected by ECD and can be detected in urine by this method. is there. In addition, various oxidatively damaged nucleosides including, for example, 2-OH-dA and 5-CHO-dU can be measured by using a liquid chromatography mass spectrometer (LCMS), which is not detected by ECD but has recently made remarkable progress. it can. 2-OH-dATP in the nucleotide pool has a higher mutagenicity than 8-OH-dGTP, and it is thought that the amount of production is higher, so human urine
Measurement of 2-OH-dA is considered to be highly useful as a predictor of carcinogenesis and a health index. In addition, it is considered that about 20 kinds of oxidative DNA damage products are present, and the method of the present invention can be applied to the measurement thereof. Further, the present invention can be applied to the analysis of a component added to DNA by a carcinogen.
For example, a benzpyrene-dG adduct can be used, and can be used for predicting carcinogenesis, and is highly useful.

[0025] In addition, it is considered that the risk of carcinogenesis is very high in the case of a patient lacking a repair enzyme as compared with the case of having a normal function. By utilizing the measurement method of the present invention, for example, diseases in which the repair enzyme activity is deleted or reduced by examining the ratio of abundances of 8-OH-dG and 8-OH-Gua, for example, familial highly carcinogenic diseases Can be used for diagnosis.

[0026]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Separation of urine by a multi-mode column A multi-mode column (Asahipak ^™ GS-320 7G; 7.6 × 50)
(0 mm). Urine was dispensed into tubes in 1.0 ml portions and stored frozen. Thawed urine for analysis 1.
0.5 ml of pure water was added to 0 ml, and 45 μl of acetic acid was further added and stirred well. After centrifugation at 15,000 rpm for 5 minutes, 0.75 ml of the supernatant was injected into a multi-mode column using an autosampler. The eluent was 0.1% acetic acid, and the column temperature was 25 ° C. The flow rate was 1.0 ml / min. FIG. 2 shows an example of separation. In the case of the column used in this example, 8-OH-dG and 8-OH-Gu
As a result of examining the elution fraction of the standard sample of a, it was found that the fraction of 51 to 60 minutes should be collected. Similar separation patterns were obtained by using Asahipak ^™ GS-320 HQ (7.6 × 300 mm) instead of Asahipak ^™ GS-3207G (7.6 × 500 mm) as a multimode column.

Example 2 Separation of Standard Sample by Reversed Phase Column A standard sample was separated by a reversed phase column (YMC-pack ODS-AM, S-5 μm; 4.6 × 250 mm). Standard samples of 8-OH-dG and 8-OH-Gua were injected on the reversed-phase column. The eluent was a solution (pH 7.25) containing 45 ml 1N NaOH, 10 ml 1N acetic acid, 25 ml 1N sodium acetate, 12.5 ml 1N citric acid, and 50 ml methanol per liter. The column temperature was set at 25 ° C. and eluted at a flow rate of 0.8 ml / ml. The detection of 8-OH-dG and 8-OH-Gua was performed using ESA Coulochem II of Coulochem. Detection conditions are: guard cell 350 mV, E1 100 mV, E2 300 mV
The detection signal was used. The measurement results are shown in FIG. 8-
The peak positions of OH-dG and 8-OH-Gua were far apart, indicating that both components can be measured with low background.

Example 3 Separation of urine fraction by reversed-phase column Fractionation of urine collected in Example 1 for 51 to 60 minutes (10 ml) using the same column separation conditions and measurement conditions as in Example 2 Of which
100 μl was applied to a reverse phase column. The result is shown in FIG. As in FIG. 3, 8-OH-dG and 8-OH-Gua were separated as a single component. In particular, the peak of 8-OH-dG had a low background and was a good one in which no contaminants were detected in the vicinity. The figure shows the elution pattern of 8-OH-dG with the UV range changed.

[0030]

According to the present invention, the urine concentration of oxidatively damaged nucleosides such as 8-OH-dG, which has been measured so far, as an indicator of the generation of active oxygen can be measured with high sensitivity and ease. became. Also, oxidatively damaged nucleosides and their repair products, oxidatively damaged bases, such as 8-OH-G
By simultaneously measuring ua and 8-OH-dG, it is possible to not only accurately determine how much active oxygen the living body is exposed to, but also to check for diseases in which repair enzyme activity is lost or reduced. It became possible. Furthermore, carcinogens-
A technique that enables the measurement of DNA adducts (adducts) has been established, and can greatly contribute to future cancer prevention measures.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of the connection of an analytical instrument for measuring oxidative DNA damage products or carcinogen-DNA adducts in urine.

FIG. 2 shows a multi-mode column Asahipak ^™ GS-
FIG. 3 shows an example of separation when urine is fractionated using 320 7G (7.6 × 500 mm). The 51-60 minute fraction injected into the reversed phase column is shown in the figure.

FIG. 3 shows a reversed-phase column YMC-Pack ODS-AM (S-5μ,
This is an example of separation when 8-OH-dG and 8-OH-Gua were analyzed using a standard sample. Peak 1 is 8-OH-Gua
And peak 2 is 8-OH-dG.

FIG. 4 shows 51-6 when passed through a multi-mode column.
This is an example of separation when fractions at 0 minutes are collected and 100 μl thereof is analyzed by a reverse phase column. Peak 1 is 8-OH-Gua, Peak 2
Is 8-OH-dG. The peak 2 when the UV range was doubled is shown in the figure (x2).

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 30/48 G01N 30/48 G 30/80 30/80 F 33/493 33/493 A 33/50 33 / 50 P

Claims (4)

[Claims] 1. Oxidative DNA comprising the following steps:
A method for measuring the urinary concentration of a damaged product or a carcinogen-DNA adduct. 1) A step of fractionating urine through a gel filtration column having characteristics of a reversed phase column and an ion exchange column to obtain a fraction containing an oxidative DNA damage product or a carcinogen-DNA adduct. 2) passing the fraction through a reversed phase column. 3) measuring the amount of oxidative DNA damage product or carcinogen-DNA adduct in the eluate from the reverse phase column. 2. The method according to claim 1, wherein the urinary concentration of the oxidative DNA damage product is measured. 3. The method according to claim 2, wherein the oxidatively damaged DNA product is an oxidatively damaged nucleoside and / or an oxidatively damaged base. 4. The method according to claim 3, wherein the oxidatively damaged nucleoside is 8-hydroxideoxyguanosine and the oxidatively damaged base is 8-hydroxyguanine.