Patent Application
20090111136
The present invention relates to a method for detecting a hepatic disorder of a subject, in which the amount of a mitochondrion-derived protein contained in a blood sample from the subject is measured, and a hepatic disorder of the subject is detected on the basis of the measurement thereof. The present invention relates to a method for detecting a hepatic disorder derived from metabolic syndrome and/or non-alcoholic fatty liver disease; in particular, for example, non-alcoholic steatohepatitis, hepatic fibrosis, cirrhosis, or liver cancer.
The present invention also relates to, for example, a method for predicting progress from metabolic syndrome or non-alcoholic fatty liver disease to non-alcoholic steatohepatitis, hepatic fibrosis, cirrhosis, or liver cancer.
Fatty liver diseases which are not caused by excessive alcohol intake are called “non-alcoholic fatty liver disease” (hereinafter may be abbreviated as “NAFLD”), which encompass metabolic-syndrome-induced fatty liver disease described hereinbelow. Among NAFLD, a pathological condition further involving inflammation of the liver is called “non-alcoholic steatohepatitis” (hereinafter may be abbreviated as “NASH”). NASH is a chronic liver disease which may gradually progress to hepatic fibrosis, cirrhosis, or liver cancer by any factor such as oxidative stress.
Metabolic syndrome, also referred to as abnormal metabolic regulation, which is a combination of visceral fat accumulation with two or more of hyperglycemia, hypertension, and hyperlipidemia, is generally considered a risk factor for inducing arteriosclerotic diseases or fatty liver diseases, and thus is required to be prevented or treated.
NASH is considered the hepatic manifestation of metabolic syndrome. Patients having metabolic syndrome or diabetes are considered to have a high risk to develop NASH in combination with such a pathological condition.
Although NASH is described above as a pathological condition further involving inflammation of the liver among NAFLD, NASH is not clearly defined at present. For example, in some cases, NASH may progress to hepatic fibrosis, cirrhosis, or liver cancer, although inflammatory cells do not infiltrate the liver. Some researchers have considered that hypertrophy of the hepatocyte (which is called “ballooning”), rather than inflammation of the liver, plays an important role in progress of NASH to hepatic fibrosis, cirrhosis, or liver cancer. In addition, it has been reported that progress to cirrhosis is not observed in most of NASH patients. Therefore, as used herein, “NASH” collectively refers to the state where the liver develops a hepatic disorder (e.g., inflammation, ballooning, fibrosis, cirrhosis, or cancer), or the state where the liver may induce such a pathological condition, and “NASH” is distinguished from “simple steatosis”; i.e., a condition in which fat is simply accumulated in the liver, and which does not progress to another hepatic-disorder-developing condition.
As indicated by its name, NASH is not caused by alcohol intake. However, NASH exhibits symptoms similar to those of alcoholic hepatitis. In advanced countries, NASH is considered the most frequent liver disease and one of severe diseases. Therefore, patients with NASH are required to undergo vigorous treatment, and accurate discrimination between simple steatosis and NASH is very important in the diagnosis thereof.
Conventionally, NASH has been diagnosed through diagnostic imaging (e.g., abdominal echography or computed tomography), or through a blood test for examining change in amount of a marker for diagnosis of hepatic disorders (hereinafter the marker may be referred to as a “hepatic injury marker”) (e.g., glutamic-oxaloacetic transaminase (GOT) or glutamic-pyruvic transaminase (GPT)). However, the level of the aforementioned hepatic injury marker does not necessarily fall within an abnormal range in all NASH patients, and thus the definite diagnosis of NASH must be established by liver biopsy.
However, liver biopsy is not suitable as a general examination technique, since liver biopsy is highly invasive to patients and requires an intricate process, and the pathological results must be determined by skilled experts. Therefore, demand has arisen for development of a highly reliable examination method for diagnosis of NASH in place of the aforementioned examination methods.
Hitherto, there have been reported many studies on change in hepatic injury marker blood levels by use of animal models. Most of these studies are experiments employing rats or mice fed with a choline-deficient diet or a methionine- and choline-deficient diet, or rats or mice to which a drug inducing symptoms similar to those of NASH has been administered. These experiments generally employ GPT as a hepatic injury marker. In these animal models, necrosis occurs in the liver, and an increase in GPT level is observed. However, it has been known that, in human NASH patients, NASH may progress to cirrhosis without an increase in GPT level. Therefore, GPT is not necessarily suitable as a hepatic injury marker for diagnosis of NASH.
Also, there have been reported rat experiments in which, for example, ornithine carbamoyltransferase (OCT) is employed as a hepatic injury marker other than GPT, and the concentration of OCT is measured (see, for example, Non-Patent Documents 1 and 2).
Non-Patent Document 1: Biosci. Biotechnol. Biochem. 2001; 65 (4): 935-938
Non-Patent Document 2: Toxicol. Lett. 2000; 114 (1-3): 163
Since OCT is localized in mitochondria, leakage of OCT into blood requires passing through the cell membrane and also the two mitochondrial membranes, and thus OCT leaks into blood slower than, for example, GPT, which is derived from cytoplasm. Therefore, OCT has not been considered a more effective marker than GPT, and no data have been reported on such applicability of OCT. As has been reported so far, OCT is used merely for the purpose of supporting occurrence of a disorder in the liver by utilizing high liver-specificity of OCT, or for the purpose of determining whether or not the disorder is so severe that it affects mitochondria. Thus, it has not hitherto been described that the mitochondrion-derived protein contained in blood is a highly detectable and excellent marker as compared with GPT. That is, GPT has been recognized as a marker for detecting hepatic disorders such as NASH at high sensitivity. Therefore, almost all of the related papers, etc. which have been reported hitherto disclose use of GPT as a hepatic injury marker.
Patent Document 1 discloses a method for diagnosing the presence or severity of hepatic fibrosis on the basis of the presence or level of α-2-macroglobulin, hyaluronic acid, or metalloproteinase-1 inhibitor, which serves as a hepatic injury marker.
Patent Document 2 discloses a method for the differential diagnosis of NASH by determining phospholipid contained in peripheral blood and the fatty acid composition of the phospholipid.
Patent Document 3 discloses a method for diagnosing NASH by quantifying, in a hepatic tissue sample, the level of a protein selected from among apolipoprotein A1, mitochondrial ATPase β subunit, leukotriene A4 hydrolase, keratin 18, guanidinoacetate N-methyltransferase, superoxide dismutase, albumin, antioxidant protein 2 (isoform 1), prohibitin, methionine adenosyltransferase, long-chain acyl-CoA dehydrogenase, selenium-binding protein, antioxidant protein 2 (isoform 2), and combinations thereof.
Patent Document 4 discloses a method for diagnosing NASH by determining the type III procollagen N-terminal peptide level.
Patent Document 4: Japanese Patent Application Laid-Open (kokai) No. 2006-29919
As described above, a blood test employing, as an index, the level of GOT or GPT, which is a hepatic injury marker, is not satisfactory in that NASH may fail to be detected when the index represented by such a hepatic injury marker indicates negative or weakly positive.
There has also been reported a test method for the diagnosis of NASH employing a hepatic injury marker other than GOT or GPT. The test method requires an intricate measuring process and, in some cases, requires measurement of a plurality of markers. Thus, the test method is not necessarily convenient means.
When the level of such a hepatic injury marker in a patient falls within an abnormal range, liver biopsy may be carried out. However, liver biopsy is highly invasive to patients and requires an intricate process, and the pathological results must be determined by skilled experts. Therefore, liver biopsy is not suitable as a general examination technique, and even when the patient is suspected of having NASH, liver biopsy is not carried out in some cases.
In view of the foregoing, an object of the present invention is to provide a method in which the mitochondrion-derived protein level of a blood sample from a subject is measured, and whether or not the subject has a hepatic disorder (in particular, NASH) is determined on the basis of the measured mitochondrion-derived protein level.
In order to achieve the aforementioned object, the present inventor has conducted extensive studies, and as a result has found that, for example,
(A) in a rodent animal model fed with a choline-deficient diet or a methionine- and choline-deficient diet, and developing symptoms similar to those of NASH, GPT level does not significantly increase as compared with a control group;
(B) in many human NASH patients, GPT level is observed to fall within a normal range;
(C) in an NASH case; in particular, a mild NASH case with no or slight increase in GPT level, there is a significant increase in blood level of a mitochondrion-derived protein such as ornithine carbamoyltransferase (OCT) or glutamate dehydrogenase (GDH); and
(D) measurement of a mitochondrion-derived protein is useful for detecting not only NASH but also metabolic syndrome (including diabetes). The present inventor has first found that the amount of leakage of a mitochondrion-derived protein into blood increases in an NASH case with no or only a slight increase in GPT level. In connection therewith, the present inventor has found that NASH, which is a hepatic disorder, can be examined by determining the mitochondrion-derived protein blood level. The present invention has been accomplished on the basis of this finding. Accordingly, the present invention provides the following.
(1) A method for detecting a hepatic disorder, comprising measuring the level of mitochondrion-derived protein in a blood sample from a subject, and detecting a hepatic disorder of the subject on the basis of the measured mitochondrion-derived protein level.
(2) A detection method as described in (1), wherein the hepatic disorder of the subject is a hepatic disorder occurring via a mechanism in which glutamic-pyruvic transaminase level is less likely to increase, or a hepatic disorder at such a degree that glutamic-pyruvic transaminase level does not increase.
(3) A detection method as described in (1), wherein the hepatic disorder of the subject is derived from metabolic syndrome and/or non-alcoholic fatty liver disease.
(4) A detection method as described in (1), wherein the hepatic disorder of the subject is non-alcoholic steatohepatitis, hepatic fibrosis, cirrhosis, or liver cancer.
(5) A method for determining a hepatic disorder, comprising measuring the level of mitochondrion-derived protein level in a blood sample from a subject; comparing the measured mitochondrion-derived protein level with an averaged value of mitochondrion-derived protein levels of blood samples from healthy volunteers; and, when the mitochondrion-derived protein level of the subject is higher than the averaged value of mitochondrion-derived protein levels of the healthy volunteers, determining that the subject has a hepatic disorder derived from metabolic syndrome and/or non-alcoholic fatty liver disease.
(6) A method for predicting a hepatic disorder of a patient with metabolic syndrome and/or non-alcoholic fatty liver disease, the method comprising employing a mitochondrion-derived protein as a hepatic injury marker.
(7) A method for predicting progress of a hepatic disorder, comprising tracing change over time in level of mitochondrion-derived protein in a blood sample from a patient with metabolic syndrome and/or non-alcoholic fatty liver disease.
(8) A method for determining a therapeutic effect on a hepatic disorder, comprising measuring the level of mitochondrion-derived protein level in a blood sample from a patient with metabolic syndrome and/or non-alcoholic fatty liver disease, and determining a therapeutic effect on a hepatic disorder on the basis of the measured mitochondrion-derived protein level.
(9) A method as described in any of (1) to (8), wherein the mitochondrion-derived protein is a protein which exists specifically in the liver and/or a protein which exists abundantly in the liver.
(10) A method as described in any of (1) to (8), wherein the mitochondrion-derived protein is ornithine carbamoyltransferase and/or glutamate dehydrogenase.
(11) A method as described in any of (1) to (8), wherein the mitochondrion-derived protein level is determined by measuring the concentration or activity of the protein.
(12) A method as described in any of (1) to (8), wherein the mitochondrion-derived protein level is measured through an immunological technique or an enzymatic technique.
According to the present invention, a hepatic disorder derived from NAFLD can be detected, and NASH can be accurately discriminated from simple steatosis. Also, the present invention is useful as a definite diagnosis of NASH or as an auxiliary examination for determining whether a definite diagnosis (e.g., liver biopsy) is needed or not.
Furthermore, according to the present invention, progress of NASH to hepatic inflammation, hepatic fibrosis, cirrhosis, liver cancer, or the like can be predicted by tracing change over time in mitochondrion-derived protein level of a blood sample from an NAFLD patient. Also, the present invention can be applied to determination of a therapeutic effect on a hepatic disorder.
In addition, the present invention can detect not only a hepatic disorder derived from NAFLD, but also a hepatic disorder derived from metabolic syndrome. Therefore, the present invention can also be applied to detection of metabolic syndrome itself, or prediction of progress of a metabolic-syndrome-derived hepatic disorder such as NASH (i.e., hepatic inflammation, hepatic fibrosis, cirrhosis, or liver cancer).
No particular limitation is imposed on the test sample for measurement, so long as the test sample is a blood-derived sample such as blood, serum, or plasma.
A characteristic feature of the method of the present invention resides in that the mitochondrion-derived protein level is measured for detection of a hepatic disorder such as NASH. Examples of the mitochondrion-derived protein include OCT and/or GDH.
The OCT or GDH level of a blood sample may be determined on the basis of the concentration or activity of the protein, which may be measured through a known method. For example, when the level of OCT or GDH is measured through an immunological technique such as ELISA, there may be employed, for example, the highly sensitive OCT assay method developed by the present inventor (Patent Document 5: WO 2006/73073) or a known method (Enzyme Protein (1994-95) 48: 10-17).
When the level of OCT or GDH is measured through an enzymatic technique, there may be employed, for example, a commercially available kit (e.g., OCT-Test Wako, product of Wako Pure Chemical Industries, Ltd.) or a known method (Clin. Chim. Acta (1976) 67: 145-152, Anal. Lett. (1993) 26: 475-486, or Eur. J. Clin. Chem. Clin. Biochem. (1992) 30: 493-502).
The mitochondrion-derived protein level of a blood sample from a subject is measured through any of the aforementioned methods or by use of the aforementioned commercially available kit, and the thus-measured level is compared with an averaged value of mitochondrion-derived protein levels of blood samples from healthy volunteers. When the mitochondrion-derived protein level of the subject is higher than the average of the an averaged value of mitochondrion-derived protein levels of the healthy volunteers, the subject is determined to be suspected of having a hepatic disorder; specifically, a hepatic disorder (e.g., NASH) derived from metabolic syndrome and/or NAFLD. In contrast, when the mitochondrion-derived protein level of the subject is equal to or lower than the averaged value of mitochondrion-derived protein levels of the healthy volunteers, the subject is determined not to have a hepatic disorder (e.g., NASH).
When the OCT or GDH concentration is determined through an immunological technique such as ELISA, an antibody to OCT or GDH is employed. The antibody may be prepared through a customary method (see, for example, Patent Document 5), and may be a monoclonal or polyclonal antibody. Alternatively, the antibody may be an antibody fragment having a binding ability to OCT or GDH (e.g., Fab or F(ab′)2).
The present invention will next be described by way of examples, which should not be construed as limiting the invention thereto.
Sixteen male SD rats (8-week old) were divided into two groups. SD rats of one group were fed with a choline-deficient (CD) diet, and SD rats of the other group were fed with a choline-supplemented (CS) diet. Four weeks later, blood was collected from each SD rat under anesthesia. In addition, a portion of the liver of each SD rat was collected, and fixed in formalin, followed by hematoxylin-eosin staining. Thus, the degree of a hepatic disorder was histopathologically examined. SD rats fed with the CD diet for eight weeks were also examined in a manner similar to that described above.
Tables 1 and 2 show the results of measurement of the serum level and activity of mitochondrion-derived proteins (OCT and GDH) and cytoplasm-derived proteins (GPT, GOT, guanase (GU), isocyanate dehydrogenase (ICD), sorbitol dehydrogenase (SDH), and lactate dehydrogenase (LDH)). The levels of GPT, GOT, GU, ICD, SDH, LDH, and GDH were measured through an enzymatic technique, and the OCT level was measured through ELISA.
As shown in Table 1, in the CD diet group, the levels of OCT and GDH, which are mitochondrion-derived proteins, significantly increased. In contrast, as shown in Table 2, there was no significant difference in level of each cytoplasm-derived protein between the CD diet group and the CS diet group. These data indicate that NASH is effectively detected on the basis of the blood mitochondrion-derived protein level.
Through the histopathological examination, four weeks after initiation of feeding of the CD diet, accumulation of fat was observed in the liver of the rats of the CD diet group, but no marked hepatic disorder was observed. However, eight weeks after initiation of feeding of the CD diet, hepatic fibrosis was observed in the rats of the CD diet groups. These data indicate that the liver of SD rats is damaged by feeding of the CD diet, and a mitochondrion-derived protein (OCT or GDH) is useful as a hepatic injury marker.
Mitochondrial disorder or oxidative stress associated therewith is thought to be involved in the mechanism of development of NASH. As has been known, even in human, a drug which induces mitochondrial disorder may cause a lesion similar to NASH. This lesion is also called “drug-induced NASH.” In Example 2, there was performed measurement over time of the blood hepatic injury marker levels of rats with an acute hepatic disorder caused by administration of tetracycline, as well as the amount of thiobarbituric acid reactive substance (TBARS) (which is considered an index of oxidative stress) contained in liver tissue.
Male Wistar rats (8-week old) were employed, and tetracycline hydrochloride (192 mg/kg) was intraperitoneally administered to each rat. Subsequently, blood was collected from each rat under anesthesia, followed by removal of the liver. 20 mM Phosphate buffer (4 mL) was added to liver tissue (1 g), followed by homogenization, to thereby prepare a liver tissue sample. The thus-prepared sample was subjected to measurement of TBARS.
As shown in
A KK-Ay mouse is an obese and diabetic mouse established by transduction of the obese gene Ay into KK mouse, which has a genetic background that makes it more likely to develop diabetes. As has been reported, when KK-Ay mice are fed with a high-fat diet, the adiponectin (i.e., good adipokine) level decreases, and the TNF-α ((i.e., bad adipokine) level increases. Therefore, a KK-Ay mouse fed with a high-fat diet is considered an animal model of metabolic syndrome.
In Example 3, the blood level of a mitochondrion-derived protein was measured in KK-Ay mice fed with a high-fat diet.
Ten male KK-Ay mice were divided into two groups (five mice each). Mice of the two groups were respectively fed with a control diet and a high-fat diet for three weeks, followed by measurement of blood OCT and GPT levels. The results are shown in Table 3.
As shown in Table 3, in the high-fat diet group, only OCT level significantly increased. These data indicate that OCT is more preferred than GPT as an index for detecting NASH, which is a hepatic disorder associated with metabolic syndrome. An increase in OCT level in KK-Ay mice fed with the high-fat diet (i.e., model of metabolic syndrome) supports the relationship between metabolic syndrome and NASH. Thus, it is shown that the blood mitochondrion-derived protein level is effectively employed not only for diagnosis of NASH, but also for diagnosis of metabolic syndrome.
These data indicate that the method employing a mitochondrion-derived protein (e.g., OCT or GDH) as a hepatic injury marker can more sensitively detect mitochondrial disorder or oxidative stress associated therewith (which is considered to be involved in the mechanism of development of NASH), as compared with the case where a cytoplasm-derived protein (e.g., GPT or GOT) is employed; i.e., the method is effective for diagnosis of NASH.
NASH is considered to be the hepatic manifestation of metabolic syndrome, and 80 to 90% of NASH patients have visceral obesity. Most of diabetic patients have hepatic steatosis, and are considered to have a high risk to develop NASH, as compared with healthy people. Ob/ob mouse, which is a mouse model of type 2 diabetes, has extreme obesity and hepatic steatosis, and exhibits sensitivity to hepatotoxicity such as lipopolysaccharide or alcohol intake. Therefore, ob/ob mice were employed as a model of NASH. In Example 4, the blood mitochondrion-derived protein level of ob/ob mice was evaluated.
Five male ob/ob mice and five male control mice were employed, and blood OCT and GPT levels were measured from 4-week old to 8-week old. The results are shown in
As has been known, in ob/ob mice (i.e., diabetic mouse model with high obesity), an increase in blood glucose level, which is an index of diabetes, is less likely to be observed. An increase in blood hepatic injury marker level of the ob/ob mice indicates that metabolic syndrome with obesity or hepatic steatosis may progress to a hepatic disorder, even in the case of mild diabetes. Thus, it is shown that measurement of the blood mitochondrion-derived protein level of the ob/ob mice is effective for detecting not only NASH but also metabolic syndrome (including diabetes).
In the ob/ob mice having visceral obesity, the rate of increase in blood level of OCT (i.e., a mitochondrion-derived protein) over time is higher, as compared with the case of GPT, which is generally widely used as a hepatic injury marker. This suggests that OCT is more effective than GPT for diagnosis of NASH.
The present invention has first realized use of a mitochondrion-derived protein as a hepatic injury marker for predicting a hepatic disorder of a patient with metabolic syndrome and/or non-alcoholic fatty liver disease.
According to the present invention, progress of a hepatic disorder can be predicted by tracing change over time in mitochondrion-derived protein level of a blood sample from a patient with metabolic syndrome and/or non-alcoholic fatty liver disease.
In addition, according to the present invention, a therapeutic effect on a hepatic disorder can be determined on the basis of the mitochondrion-derived protein level of a blood sample from a patient with metabolic syndrome and/or non-alcoholic fatty liver disease.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2006-092169 | Mar 2006 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2007/000323 | 3/29/2007 | WO | 00 | 9/29/2008 |
