JP5642922B2 - Evaluation method of liver injury using multiple liver injury markers as indicators - Google Patents

Description

  The present invention uses a plurality of specific enzymes as liver damage markers, a method and a kit for evaluating the degree of liver damage, and the accuracy of liver damage evaluation by using these specific enzymes as liver damage markers It relates to a method for making it happen.

  It is extremely important to ensure the safety of substances directly taken into the living body, such as foods and drinks and pharmaceuticals. Therefore, in the development of foods and drinks and pharmaceuticals, it is necessary to subject them or their raw materials to many safety tests including liver damage tests.

  There are many biomarkers for monitoring liver damage. Among them, aspartate aminotransferase (AST: this enzyme is also called GOT) and alanine aminotransferase (ALT: this enzyme is also called GPT) are widely used as markers of liver damage in clinical and research settings. The measured enzyme activities are used as indicators of various liver disorders (Non-patent Document 1).

  However, in some types of liver disease, the activity of these enzymes is only at normal levels, which can lead to a false determination that no hepatocyte damage has occurred. Furthermore, these enzymes are not expressed specifically in the liver, but are also expressed in other organs other than the liver such as heart, muscle and kidney. Therefore, even if the liver itself is not damaged, the levels of these enzymes can show abnormally high values.

Therefore, in order to accurately evaluate liver damage, a more reliable liver damage marker or liver damage evaluation method is required.
Clinica Chimica Acta 375 (2007), 63-68

  Accordingly, an object of the present invention is to provide a means for evaluating liver damage with high accuracy.

  The present inventor conducted intensive studies to find a highly reliable liver injury marker or liver injury evaluation method. Specifically, the time course of various enzyme concentrations in the liver injury model was investigated, and it was clarified that liver injury can be evaluated with high accuracy by using a combination of specific enzymes, and the present invention was completed. .

That is, the present invention
(1) A method for evaluating the degree of liver injury, comprising measuring at least the level of α-GST and the level of OCT or GLDH in a blood-derived sample collected from an animal;
(2) The method according to (1), further comprising measuring the level of ARG in the sample;
(3) The method according to (1) or (2), wherein the liver disorder is acute liver disorder or drug-induced liver disorder;
(4) A method for evaluating the degree of liver injury, comprising means for measuring at least the level of α-GST and the level of OCT or GLDH in a blood-derived sample collected from an animal. kit;
(5) The kit according to (4), further comprising means for measuring the level of ARG in the sample;
(6) The method according to (4) or (5), wherein the liver disorder is acute liver disorder or drug-induced liver disorder;
(7) Inducing liver injury of a test substance, characterized by measuring at least α-GST level and OCT or GLDH level in a blood-derived sample collected from an animal given the test substance How to evaluate the effect;
(8) The method according to (7), further comprising measuring the level of ARG in the sample;
(9) The method according to (7) or (8), wherein the liver disorder is acute liver disorder or drug-induced liver disorder;
(10) A method for improving the accuracy of liver injury evaluation, characterized by using at least α-GST and OCT or GLDH in combination as a marker;
(11) Furthermore, ARG is used as a marker, The method of Claim 10 characterized by the above-mentioned;
(12) The method according to (10) or (11), wherein the liver disorder is acute liver disorder or drug-induced liver disorder;
It is.

  According to the present invention, it is possible to capture a liver disorder that cannot be captured by a currently widely used evaluation method with high accuracy. Therefore, for example, by using the present invention for a safety test in the development of foods and drinks or pharmaceuticals, it is possible to accurately determine a material having a risk of liver damage. Since it is important to prevent the occurrence of liver damage after launch, the technical contribution of the present invention to this field is great.

  In addition, since finding a liver disorder with high accuracy leads to early treatment or treatment, the present invention is also useful in the clinical field.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention uses a combination of α-GST and OCT or GLDH as a marker of liver damage, optionally with ARG.
Liver disorder As used herein, the term “liver disorder” refers to a condition in which a histological or functional abnormality has occurred in the liver. Based on the cause, drug-induced liver disorder, alcoholic liver disorder It is classified as viral hepatic disorder. Furthermore, these liver disorders are classified into acute liver disorders and chronic liver disorders, respectively.

The marker combination glutathione S-transferase (GST) includes four classes (α, μ, θ, and π) that transfer glutathione to drugs and various endogenous nucleophilic compounds and conjugate with glutathione. Give rise to Of these, α-glutathione S-transferase (α-GST) is an enzyme with high liver specificity.

  As shown in the examples described below, the present inventor showed that the blood level of this enzyme rises significantly early after administration of the hepatotoxic substance (about 4 hours after administration in rats), but then Was confirmed to decrease to the same level as AST or ALT. Therefore, although α-GST shows the occurrence of liver damage more sensitively than AST or ALT, it is considered that there is a period when this enzyme alone cannot sufficiently reflect the degree of liver damage.

  Ornithine carbamoyltransferase (OCT) is an enzyme of the urea production system, and catalyzes a reaction for producing citrulline and phosphate from ornithine and carbamoyl phosphate. This enzyme is known to be a liver-specific marker that is expressed exclusively in the mitochondria of liver cells.

  As shown in the examples described below, the present inventor has shown that the blood level of this enzyme rises to a very high level after a certain time (about 24 hours in the rat) after administration of the hepatotoxic substance. However, it was found that it is not necessarily higher than AST or ALT at the previous time point. Therefore, OCT can also be a more sensitive liver injury marker than AST or ALT, but it is considered that there is a time when this enzyme alone cannot sufficiently function as a liver injury marker.

  Glutamate dehydrogenase (GLDH) catalyzes the reaction of oxidative deamination of glutamate to convert it to 2-oxoglutarate. And it is known that this enzyme is overwhelmingly contained in the liver.

  As shown in the examples described later, the blood level of this enzyme rises markedly after a certain period of time (about 24 hours in the rat) after administration of the hepatotoxic substance, but before that time, It was confirmed that the blood level was not high compared to AST or ALT. Therefore, this enzyme can also be a liver injury marker with higher sensitivity than AST or ALT, but it is considered that there is a time when this enzyme alone cannot function sufficiently as a liver injury marker.

  Arginase (ARG) is one of the enzymes constituting the urea cycle in hepatocytes that catalyzes the reaction of hydrolyzing L-arginine to produce urea and ornithine. And most of this enzyme is known to be localized in the liver.

  As shown in Examples described later, it was confirmed that the blood concentration of ARG markedly increased during liver injury. However, the time at which the enzyme exhibits high blood levels can vary from test to test. Therefore, ARG can be a highly sensitive liver injury marker, but when this enzyme is used alone as a marker, there may be a period when liver injury cannot be sufficiently detected.

  Thus, α-GST, OCT, GLDH, and ARG are all highly sensitive to liver damage, but there is a period when they cannot function as a liver damage marker alone. The present inventor is dependent on the timing by appropriately combining these enzymes based on the results of original experiments regarding changes in blood concentrations of α-GST, OCT, GLDH, and ARG over time. It was found that the high sensitivity of these enzymes to liver damage can be utilized.

Such a combination is specifically α-GST and OCT or GLDH. Preferably, ARG is used in combination with the combination.
Measurement of enzyme level and evaluation of liver damage In the present invention, animals, particularly human and non-human mammals (rodents such as mice, rats, guinea pigs, hamsters and gerbils, cats, dogs, rabbits, goats, sheep, The level of each enzyme present in a blood-derived sample obtained from mammals, including pigs, monkeys, etc.) is measured. The sample to be measured is, for example, whole blood, serum, plasma or the like.

  Various methods for measuring the level of the enzyme in the sample are known. In the present invention, any of these known methods may be used without any particular limitation. Alternatively, any enzyme measurement method developed in the future may be used. Typical methods are a method for measuring enzyme activity and an immunological method using an antibody against the enzyme molecule (for example, ELISA, immunochromatography). In particular, the immunochromatography method does not require the acquisition of special techniques, and according to this method, the target marker can be measured qualitatively or semi-quantitatively simply and rapidly. Therefore, the immunochromatography method is particularly useful.

Actually, specific operations for measuring an enzyme used as a marker in the present invention are described in several known documents. For example, JP-A-11-503235 describes in detail an immunological method for quantification of α-GST. Clinica Chimica Acta, 368 (2006) 125-130 describes an ELISA method for the quantification of OCT. As for OCT, a quantitative method using enzyme activity is also known (Japanese Patent Laid-Open No. 62-232398). About the measuring method of GLDH, description is described in the attached document of GL441 (Randox Laboratories Ltd., United Kingdom) of Landox Co., Ltd. which is a commercial measuring kit for measuring enzyme activity. Regarding ARG, Non-Patent Document 1 describes a method using ELISA. An immunological method for quantifying ARG is also described in WO 96/28733. Based on the descriptions in these documents, the enzyme used as a marker in the present invention can be easily measured.

  As a result of the measurement, the degree of liver injury can be evaluated by comparing the level of the target enzyme in the test sample with the level of the target enzyme in the control sample. If the level of at least one target enzyme in the test sample is significantly increased compared to the control sample, it is determined that the liver of the animal from which the sample is derived is impaired. The evaluation of the degree of liver damage includes evaluation of the presence or absence of liver damage in this way. Levels that can be used as a basis for determining that an animal has liver damage are, for example, about 2.5 times or more the blood concentration in a control for α-GST, OCT, GLDH, and ARG, respectively, in rats ( Preferably 3.0 times or more), about 1.6 times or more (preferably 5.0 times or more), about 3.8 times or more (preferably 5.7 times or more), about 2.3 times or more (preferably 5.4 times or more).

  In addition, measuring the level of each marker in a blood-derived sample collected after administering a test substance to an animal and evaluating the degree of liver damage leads to the evaluation of the liver damage-inducing action of the test substance. . Therefore, the method of the present invention can also be used as a method for evaluating the liver injury-inducing action of a substance.

Kit The kit used in the present invention comprises at least a means such as a reagent or tool for measuring the level of α-GST and the level of OCT or GLDH. The kit may further comprise a means for measuring the level of ARG.

  For example, when the enzyme activity is measured for enzyme quantification, the kit of the present invention includes reagents such as a substrate for the reaction catalyzed by the target enzyme and a buffer. A specific example of such a kit is Landox GL441 for the measurement of GLDH. When using an immunoassay, for example, an ELISA method, the kit of the present invention includes an antibody immobilized on a target enzyme (eg, a support such as a plate coated with a polyclonal or monoclonal antibody), Western Another antibody to which a labeling enzyme such as horseradish peroxidase is bound, a substrate for the labeling enzyme, and the like are included. A specific example of such a kit is Biotrin's Biotrin Rat Alpha GST EIA for the determination of α-GST. When using an immunochromatography method, the kit includes a membrane on which a specific antibody against the target enzyme is solidified.

  In the present invention, a plurality of kits for measuring one enzyme may be used in combination, but one or more kits capable of measuring a plurality of enzymes may be used. For example, in an immunochromatography method, a plurality of separate solid layers (supports) to which a plurality of antibodies are immobilized may be used, or a single solid layer (support) to which a plurality of antibodies are immobilized is used. Also good.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this invention is not limited to these Examples.
Example 1 Evaluation of acute drug-induced liver injury by acetaminophen (APAP) administration
SD male rats (6 weeks old) were administered 500, 1000, or 1500 mg / kg, po of acetaminophen (solvent: 0.5% CMC), which is a hepatic lobular central disorder drug. Moreover, the solvent was administered once to the control group.

  Blood was collected from the tail vein under awakening 4 hours after administration, and serum was collected by centrifugation. After 24 hours, blood was collected from the abdominal aorta under Nembutal anesthesia, and serum was collected by centrifugation. From the time of drug administration to 24 hours after administration, the rats were fasted.

Using these sera, the levels of AST and ALT, which are generally known as liver injury markers, and the levels of α-GST, OCT, GLDH and ARG as novel liver injury marker candidates were measured. AST, ALT and GLDH levels were measured by measuring enzyme activity. Specifically, for the enzyme activity of AST and ALT, use measurement kit L-type Wako GOT · J2 (Wako Pure Chemical Industries, Ltd.) and L-type Wako GPT · J2 (Wako Pure Chemical Industries, Ltd.), respectively. The concentration of NAD ⁺ produced by a series of reactions involving AST or ALT was measured with a Hitachi automatic analyzer (type 7070) (reaction conditions and measurement conditions were as described in the package insert of the measurement kit). Regarding GLDH, a concentration of NAD ⁺ produced by a reaction involving GLDH was measured using Landox kit GL441 (reaction conditions and measurement conditions were as described in the package insert of the measurement kit). The levels of α-GST, ARG, and OCT were measured by enzyme immunoassay. Specifically, α-GST was quantified using a measurement kit Biotrin Rat Alpha GST EIA (Biotrin International Ltd., Ireland) (detailed experimental procedures and measurement conditions were as described in the kit attachment). ) ARG was quantified by the method according to the document Clinical Chimica Acta 375 (2007) 63-68, and OCT was quantified by the method according to the document Clinical Chimica Acta 368 (2006) 125-130. That is, both are plates coated with antibodies to the target enzyme; F (ab ′) ₂ fraction of monoclonal IgG to the target enzyme bound to the enzyme HRP; and HRP substrate (3,3 ′, 5,5′− Quantification was performed using tetramethylbenzine) or the like.

  The enzyme level indicated by the test group at the corresponding time point, with the average level of AST, ALT, α-GST, OCT, GLDH, ARG in the control group (APAP non-administered group) at each time point (4 and 24 hours) as 1. The relative value (average value) of was obtained. GLDH 4 hours after APAP administration could not be measured due to insufficient blood collection. The results are shown in Table 1.

From the results of Table 1, it is clear that the blood levels of α-GST, OCT, GLDH, and ARG all increase due to liver damage to values higher than the conventionally used indexes (AST, ALT). It was.
Also, in the anatomical examination 24 hours after APAP administration, abnormal findings were observed in the liver of the group of 1000 mg / kg or more. Therefore, it is considered that liver damage can be detected or evaluated with higher sensitivity than before by using these enzymes.

  In particular, the blood concentration of α-GST increased to a higher level earlier (after 4 hours) than AST and ALT. Therefore, when α-GST is used as a marker, it is considered possible to detect liver damage that cannot be detected when AST or ALT is used as a marker.

Example 2 Acute drug by administration of glutathione depleting agent (BSO) and allyl alcohol (AA)
Of sexual liver disease
L-Buthionine (S, R) -sulfoximine (BSO, 400mg / kg, ip) that induces glutathione depletion is administered to SD male rats (7 weeks old), and 1 hour later, it is a hepatotoxic substance Allyl alcohol (AA) was administered once (10, 20, or 30 mg / kg, po).

By depleting glutathione in this way, the effect of glutathione detoxification can be eliminated.
Blood was collected from rats by the same procedure as in Example 1, and the levels of AST, ALT, α-GST, OCT, GLDH, and ARG were measured for the obtained serum. The kit and method used for the measurement were the same as those described in Example 1 (in order to express the effect of BSO more strongly, rats were fasted until 4 hours after drug administration).

  The relative values of the respective enzyme levels with the average value of AST, ALT, α-GST, OCT, GLDH, and ARG in the control group (AA non-administered group) as 1 are shown below.

  The results of this example were similar to those in Table 1. That is, α-GST, OCT, GLDH and ARG all increase in blood concentration to a level higher than the conventionally used indicators (AST, ALT), and among them, the blood level of α-GST is It rose to a higher value earlier (after 4 hours) than AST and ALT. However, the blood level of ARG increased early (4 hours), and this result was different from the result of Example 1.

In the anatomical examination 24 hours after AA administration, abnormal findings were observed in the liver of the group of 20 mg / kg or more.
From the results of Examples 1 and 2, it is possible to evaluate liver damage with higher accuracy than in the case of using AST or ALT by using α-GST in combination with OCT or GLDH (and optionally combining ARG). Conceivable.

Example 3 Evaluation Example of Beverage Drinking water (tap water) 30 parts by volume, 60 parts by volume and 100 parts by volume of distilled water were mixed to prepare 100 parts by volume of water, respectively, and SD male rats (6 A single dose of 15 ml / kg, po was administered at week). In the control group, distilled water was administered at 15 ml / kg, po.

  Blood was collected from rats by the same procedure as in Example 1, and the levels of AST, ALT, α-GST, OCT, GLDH, and ARG were measured for the obtained serum. The kit and method used for the measurement were the same as those described in Example 1.

  As a result, the level of any marker did not rise significantly. Therefore, it was confirmed that tap water has no acute drug-induced liver injury.

Claims (5)

Measure at least the level of α-glutathione S transferase (α-GST), the level of ornithine carbamoyltransferase (OCT) or glutamate dehydrogenase (GLDH), and the level of arginase (ARG) in a blood-derived sample collected from an animal. And comparing the level of the enzyme in the sample with the level of the enzyme in a control sample,
A method of measuring enzyme levels to assess the extent of liver damage.   The method according to claim 1, wherein the liver disorder is acute liver disorder or drug-induced liver disorder. Means for measuring at least the level of α-GST and the level of OCT or GLDH in a blood-derived sample taken from an animal; and
Comprising means for measuring the level of ARG in the sample ,
A kit for evaluating the degree of liver damage. Measuring at least α-GST level, OCT or GLDH level, and ARG level in a blood-derived sample collected from an animal given the test substance; and the enzyme in the blood-derived sample Comparing the level of said enzyme with the level of said enzyme in a control sample,
A method for evaluating the liver injury-inducing action of a test substance. Measuring at least the level of α-GST, the level of OCT or GLDH and the level of ARG in a blood-derived sample taken from an animal, and determining the level of the enzyme in the sample in the control sample It is characterized by comparing with the level of
A method to improve the accuracy of liver damage assessment.