JP4569959B2 - Method and kit for use as an index of chronic hepatitis and cirrhosis caused by hepatitis C virus - Google Patents

Description

  The present invention relates to a method for detecting, evaluating or diagnosing viral liver disease and a diagnostic kit.

  Viral infections such as hepatitis C virus (HCV), hepatitis B virus (HBV), and hepatitis A virus (HAV) are major causes of liver diseases such as chronic hepatitis and cirrhosis.

  When left untreated, these diseases gradually shift from chronic hepatitis to cirrhosis due to liver fibrosis. Advanced viral liver disease increases the risk of developing complications such as ascites, jaundice, varicose veins, encephalopathy, and liver failure, while also significantly increasing the risk of hepatocellular carcinoma.

  Viral liver disease can be cured or delayed if detected and treated early, but it is difficult to treat effectively even if it is discovered after the disease has progressed. Discovery is important. On the other hand, since it is difficult to detect and diagnose viral liver disease, a method for diagnosing it by a simple test is required.

  An object of the present invention is to provide a method and kit for appropriately diagnosing, detecting, and evaluating viral liver disease, particularly chronic hepatitis and cirrhosis.

  As a result of repeated investigations in view of the above problems, the present inventor correlates the levels in the samples of HODEs and 7-FCOHs with the type and severity of viral liver disease, and examines the levels in these samples. We have found that it is possible to diagnose viral liver disease.

An object of the present invention is to provide the following diagnostic method and diagnostic kit.
1. Viral liver disease characterized by measuring at least one selected from the group consisting of hydroxyoctadecadienoic acid (HODE) and 7-hydroxycholesterol (7-FCOH) in a sample derived from a mammal Diagnosis method.
2. Item 2. The diagnostic method according to Item 1, wherein the HODE is at least one selected from the group consisting of 9-HODE, 10-HODE, 12-HODE and 13-HODE.
3. Item 3. The diagnostic method according to Item 2, wherein the HODE is at least one selected from the group consisting of 9-HODE and 13-HODE.
4). Item 2. The diagnostic method according to Item 1, wherein the 7-FCOH is at least one selected from the group consisting of 7α-hydroxycholesterol (7α-FCOH) and 7β-hydroxycholesterol (7β-FCOH).
5). Item 5. The diagnostic method according to any one of Items 1 to 4, wherein the viral liver disease is at least one selected from the group consisting of chronic hepatitis and cirrhosis.
6). Item 6. The method according to any one of Items 1 to 5, wherein hydroxyoctadecadienoic acid (HODE) in a sample derived from a mammal is measured to distinguish and diagnose viral chronic hepatitis and cirrhosis. Diagnosis method.
7). Item 1 wherein the viral liver disease is chronic hepatitis and cirrhosis caused by hepatitis C virus
6. The diagnostic method according to any one of 6.
8). A sample derived from a mammal is treated with a reducing agent, and then treated with an alkali to measure at least one selected from the group consisting of HODEs and 7-FCOHs. Item 8. The diagnostic method according to any one of Items 1 to 7.
9. Item 9. The diagnostic method according to Item 8, wherein the reducing agent is NaBH ₄ .
10. Item 9. The diagnostic method according to Item 8, wherein the alkali treatment is performed using an alkali metal hydroxide.
11. A diagnostic kit for diagnosing viral liver disease by measuring at least one selected from the group consisting of HODEs containing a reducing agent and an alkali treating agent and 7-FCOHs.

  ADVANTAGE OF THE INVENTION According to this invention, the diagnostic method and diagnostic kit of viral liver disease can be provided, and the diagnosis according to the chronic hepatitis and the cirrhosis according to the differential diagnosis and severity is possible.

The present invention is useful as a method for detecting or diagnosing viral liver disease in mammals (particularly humans). Diagnosis or evaluation of viral liver disease in a non-human mammal can be applied to, for example, a screening system for a therapeutic drug for viral liver disease.
Each disease and severity of viral liver disease is evaluated, detected or diagnosed by measuring or quantifying hydroxyoctadecadienoic acid (HODE) or 7-hydroxycholesterol (7-FCOH) in a sample. can do.
In the present invention, the sample is derived from mammals such as human, cow, horse, pig, sheep, monkey, dog, cat, rat, mouse, etc., preferably human.
Samples include blood (including plasma, serum, blood cells (red blood cells, white blood cells)), urine, cerebrospinal fluid, tears, semen, saliva, lymph, and part of tissue (obtained by biopsy, etc.). Preferably, blood (including plasma, serum, blood cells), particularly plasma or serum is exemplified.

Viral liver diseases that can be diagnosed by the present invention are chronic hepatitis and cirrhosis. These can be diagnosed by measuring levels in samples such as HODEs and 7-FCOHs. Hepatitis viruses that cause viral liver disease include HCV, HBV, HAV, and the like, preferably HCV.

  Examples of HODEs include octadecadienoic acid having a hydroxyl group, and two double bonds of the HODE are (E, E), (E, Z) or (Z, E). Any or a mixture thereof may be used. Specific examples of HODE include 9-hydroxyoctadecadienoic acid (9-HODE), 13-hydroxyoctadecadienoic acid (13-HODE), 10-hydroxyoctadecadienoic acid (10-HODE), 12-hydroxyoctadecadienoic acid (12-HODE). These may be any of (E, E) isomer, (E, Z) isomer, and (Z, E) isomer. Preferred HODEs for measurement of diagnostic methods are 9- (E, E) -HODE, 13- (E, E) -HODE, 9- (E, Z) -HODE, 13- (Z, E) -HODE, 10- (E, Z) -HODE and 12- (Z, E) -HODE. HODEs include 9- (E, E) -HODE, 13- (E, E) -HODE, 9- (E, Z) -HODE, 13- (Z, E) -HODE, 10- (E, It is preferred to measure the total HODE including Z) -HODE and 12- (Z, E) -HODE.

When linoleic acid (two double bonds are Z, Z (cis type)) is oxidized, it isomerizes to ZE or EZ or EE HODEs. In the HODEs at this time, the total amount of (Z, E) and (E, Z) isomers and the ratio (R) of (E, E) isomers are
R = {total amount of (Z, E) and (E, Z) isomers} / (E, E) isomers. The lower the ratio (R) of HODEs, the higher the possibility of suffering from viral liver disease, and the higher the ratio (R), the higher the possibility that the viral liver disease is severe.

  Examples of 7-hydroxycholesterol (7-FCOH) include 7α-hydroxycholesterol (7α-FCOH) and 7β-hydroxycholesterol (7β-FCOH). A preferred 7-FCOH is 7β-FCOH.

  The structural formulas of preferred HODEs that can be used in one embodiment of the present invention are shown below.

  HODEs and 7-FCOHs can be quantified as follows, for example.

  Preferably, the sample is first treated with a reducing agent to lead the reaction product with active oxygen such as hydroperoxide, peroxide, etc. to its reductant and to prevent further decomposition. For example, linoleic acid, which is a raw material of HODE, reacts with active oxygen to become hydroperoxide.

When this is converted to a reduced form, it is converted to 9-HODE or 13-HODE. HODE reacts with active oxygen in the mammalian body and is further reduced to 9-HODE, 13-HODE, etc.
Upon reaction with singlet oxygen, it can be converted to 10-HODE or 12-HODE.

Hydroperoxide can be reduced as a reducing agent for reducing the biological sample to lead the peroxide to stable HODEs and 7-FCOHs, but it can be reduced by carbon-carbon double bond, carboxyl (COOH) group, ester group For example, a reducing agent that does not reduce is used, and examples thereof include sodium borohydride (NaBH ₄ ) and sodium cyanoborohydride (NaCNBH ₃ ).

The reduction reaction proceeds advantageously by using a reducing agent from 1 equivalent to an excess amount and reacting in an alcohol such as methanol or ethanol or a hydrous alcohol at a temperature of about 0 ° C. to 50 ° C. for about 1 minute to 5 hours.

Since 7-FCOHs also go through hydroperoxide (OOH) or peroxide (OO) as in HODEs, further decomposition is suppressed by reduction treatment, and these can be measured more accurately.

HODEs such as 9-HODE, 10-HODE, 12-HODE, 13-HODE, 7α-FCOH, 7β-FCOH and 7-FCOH produced by the above reduction treatment are relatively stable compounds. The decomposition of is suppressed.

  Although the reduced form obtained above exists in the fatty acid type of HODEs, many exist as glycerin esters such as phospholipids such as phosphatidylcholine, cholesterol esters, triglycerides, and diglycerides. Therefore, in order to hydrolyze this ester into a higher fatty acid, it is reacted with an alkali treating agent, preferably an alkali metal hydroxide such as sodium hydroxide, potassium hydroxide or lithium hydroxide to lead HODE to a fatty acid.

  The 7-hydroxy cholesterol ester in which the hydroxyl group at the 3-position is esterified is converted to the corresponding 7-hydroxy cholesterol by alkali treatment.

Alkaline hydrolysis advantageously proceeds in the presence of a base in the presence of a base at a temperature at which the solvent boils for about 10 minutes to 24 hours. The base is not particularly limited as long as it can hydrolyze the ester by making the solution alkaline. For example, alkali metal hydroxide (NaOH, KOH, LiOH, etc.), alkaline earth metal hydroxide (Ca (OH)) 2, Mg (OH) 2, Ba (OH) 2)), alkali metal hydrogen carbonates (NaHCO3, KHCO3, LiHCO3), alkali metal carbonates (Na2CO3, K2CO3, Li2CO3) and the like. Preferred bases are alkali metal hydroxides such as NaOH, KOH, LiOH.

  Hydrolysis may be performed in water, or a solvent such as tetrahydrofuran, acetonitrile, ethanol, methanol, isopropanol, butanol, acetone, DMF, DMSO, or N-methylpyrrolidone may be mixed with water.

  By such hydrolysis, it is possible to measure oxides (HODEs, 7-FCOHs) in the ester form as well as in the free form.

  The mechanism by which HODE is generated in vivo is shown below.

(In the formula, _one of R ₁ and R ₂ represents (CH ₂ ) ₄ CH ₃ and the other represents (CH ₂ ) ₇ COOH. Hpo represents hydroperoxide.)
By the reducing agent treatment, the peroxide or carbonyl compound of cholesterol and cholesterol ester is led to 7α-hydroxycholesterol or 7β-hydroxycholesterol as shown in the following formula.
Formation mechanism of 7-FCOHs

The sample after the alkali treatment is separated by a usual separation means such as HPLC, and a substance such as HODEs and 7-hydroxycholesterols is quantified by using a usual analysis means such as GC / MS. The diagnosis and severity of the disease can be assessed.

  The present invention further relates to a kit for measuring HODEs and 7-hydroxycholesterols to detect, diagnose or evaluate viral liver disease.

  The kit reduces a sample from a mammal (particularly preferably human), reduces the peroxide such as hydroperoxide to alcohol, and saponifies the ester-type hydroperoxide into a free carboxylic acid. An alkali treatment agent.

  The reducing agent contained in the kit is preferably contained in an excessive amount for reducing the peroxide of the sample, and for example, about 0.1 to 100 mg (or 3 micromol to 3 mmol) is used per sample. A preferred reducing agent is NaBH4.

  The alkali treating agent is not particularly limited as long as it is an amount capable of hydrolyzing the ester.

  The kit of the present invention may further contain a solvent used for the reduction reaction in which a reducing agent is added to the sample. Preferred solvents include alcohol solvents such as methanol, ethanol, n-propanol and isopropanol.

Hereinafter, the present invention will be described in more detail with reference to examples.
Example 1
Blood was collected from 55 patients with hepatitis C (average age 58.9 years SD12.7 years) and 27 patients with hepatitis C (mean age 65.0 years SD14.6 years) and immediately Plasma was collected with a centrifuge (3500 rpm × 10 minutes). 2 ml of plasma was accurately measured, and 50 ng of 8-iso-prostaglandin F _2a -d ₄ and 9-hydroxyoctadecadienoic acid-d ₄ were added as internal standards. After adding 1 ml of methanol, 0.01 g of sodium borohydride was added under a nitrogen stream, and the mixture was vigorously stirred for 1 minute, and then allowed to stand at room temperature for 4 minutes. Subsequently, 1 ml of a methanol solution of potassium hydroxide adjusted to 1 mol / l under nitrogen was added and sealed, and then stirred at 40 ° C. for 30 minutes. Subsequently, the mixture was centrifuged at 3000 G for 10 minutes at 4 ° C., and the supernatant was collected, and then the pH was adjusted to 3 with an aqueous hydrochloric acid solution. After further centrifugation at 3000 G for 10 minutes at 4 ° C., the supernatant was filled into Sep-Pak C18 (Waters, volume 1 ml) preconditioned with methanol and pH 3 water (2 ml each), each with 10 ml pH 3 water and Washed with acetonitrile / water (15/85, volume) mixture. Subsequently, the target component was eluted with 4 ml of a mixed solution of hexane / ethyl acetate / isopropyl alcohol (30/65/5, volume). The eluted solution was charged into Sep-Pak NH2 (Waters, volume 1 ml) preconditioned with 5 ml of hexane, and washed with 5 ml of hexane / ethyl acetate (3/7, volume) and acetonitrile, respectively. Subsequently 5 ml of ethyl acetate / methanol / acetic acid (10 /
(85/5, volume) The target component was eluted with the mixture. The solvent was distilled off from the eluted liquid under a nitrogen stream. 30 μl of N, O-bis (trimethylsilyl) trifluoroacetamide (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the evaporated and dried components and reacted at 60 ° C. for 1 hour. 3 μl of the obtained TMS sample was analyzed by gas chromatography (Agerent 6890N) equipped with a mass spectrometer (Agelent 5973) to obtain total hydroxyoctadecadienoic acid as the plasma concentration. It was. On the other hand, the plasma of 50 healthy subjects with no disease history (average age 55.1 years SD14.9 years) was measured in exactly the same manner. As shown in FIG. 1, as a result of statistical analysis by ANOVA-Duncan, it was found that total hydroxyoctadecadienoic acid had significantly higher values in patients with chronic hepatitis and cirrhosis than in healthy individuals. In addition, statistical analysis of total hydroxyoctadecadienoic acid in patients with chronic hepatitis and cirrhosis was performed by t-test between two groups, and it was found that patients with cirrhosis were significantly higher than those with chronic hepatitis. Thus, by measuring the total hydroxyoctadecadienoic acid in plasma, it has become possible to easily diagnose cirrhosis patients that could not be confirmed without a biopsy.
Example 2
Blood was collected from 13 patients with hepatitis C (average age: 65.5 years old, SD: 14.7 years old), and plasma was immediately collected using a centrifuge (3500 rpm × 10 minutes). 0.5 ml of plasma was accurately measured, and 50 ng of 16 hydroxyhexadecanoic acid was added as an internal standard substance. After adding 1.0 ml of methanol, 0.01 g of sodium borohydride was added under a nitrogen stream and stirred vigorously for 1 minute, and then allowed to stand at room temperature for 4 minutes. Subsequently, 2 ml of a methanol solution of potassium hydroxide adjusted to 1 mol / l under nitrogen and 2 ml of diethyl ether were added and sealed, and then stirred at 40 ° C. for 30 minutes. After adding 2 ml of 20 volume% acetic acid aqueous solution to stop the hydrolysis reaction, 3 ml of hexane was added. After vigorous stirring for 1 minute, the mixture was centrifuged at 3000 G for 10 minutes at 4 ° C., 3.0 ml was collected from the upper hexane and diethyl ether layers, and the solvent was distilled off under a nitrogen stream. 30 μl of N, O-bis (trimethylsilyl) trifluoroacetamide (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the evaporated and dried components and reacted at 60 ° C. for 1 hour. 3 μl of the obtained TMS sample was analyzed by gas chromatography (Agerent 6890N) equipped with a mass spectrometer (Agelent 5973), and total 7-hydroxycholesterol (ionic strength at M / Z = 456) Was quantified. As shown in FIG. 2, the statistical analysis t-test between the two groups revealed that the total 7-hydroxycholesterol (T-FCOH) value of chronic hepatitis patients was significantly higher than that of healthy subjects.

The result of Example 1 is shown. The result of Example 2 is shown.

Claims (10)

A hepatitis C virus characterized by measuring at least one selected from the group consisting of hydroxyoctadecadienoic acid (HODE) and 7-hydroxycholesterol (7-FCOH) in a sample derived from a mammal A method for using as an index of chronic hepatitis and cirrhosis caused by the disease. The method according to claim 1, wherein the HODEs are at least one selected from the group consisting of 9-HODE, 10-HODE, 12-HODE and 13-HODE. The method according to claim 2, wherein the HODEs are at least one selected from the group consisting of 9-HODE and 13-HODE. The method according to claim 1, wherein the 7-FCOH is at least one selected from the group consisting of 7α-hydroxycholesterol (7α-FCOH) and 7β-hydroxycholesterol (7β-FCOH). Measured hydroxy octadecadienoic diethyl Roh dichroic acid (HODE) such in a sample from the mammal, and wherein distinguish to Rukoto viral chronic hepatitis and cirrhosis, any one of claims 1-4 The method described in 1. It distinguishes based chronic hepatitis and cirrhosis caused by hepatitis C virus on the total HODE, method according to any one of claims 1-5. After treatment the samples from mammals with a reducing agent, and measuring at least one selected from the group consisting of HODE acids and 7-FCOH acids and alkali treatment, either of claims 1-6 1 The method according to item . The method of claim 7 the reducing agent is NaBH _4. The method according to claim 7 , wherein the alkali treatment is performed using an alkali metal hydroxide. A kit for diagnosing chronic hepatitis and cirrhosis caused by hepatitis C virus by measuring at least one selected from the group consisting of HODEs and 7-FCOHs, comprising a reducing agent and an alkali treating agent.

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