Methods for aiding in the detection of nonalcoholic steatohepatitis

Abstract

Provided are a method for assisting in the detection of non-alcoholic steatohepatitis (NASH) with extremely low invasiveness compared to liver biopsy, without relying on the skill of a technician, by a simple operation; and a method for assisting in the determination of the degree of progression of a condition associated with NASH. Provided is a method for assisting in the detection of non-alcoholic steatohepatitis, the method comprising: measuring the presence of LDL-TG and / or ApoE-rich HDL-C contained in a blood sample isolated from an organism. Also provided is a method for assisting in the determination of the degree of progression of a condition associated with non-alcoholic steatohepatitis, the condition being at least one selected from the group consisting of steatosis, inflammation, ballooning degeneration, and fibrosis, the method comprising: measuring the presence of LDL-TG and / or ApoE-rich HDL-C contained in a blood sample isolated from an organism.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for assisting in the detection of nonalcoholic steatohepatitis, and a method for assisting in the determination of the degree of progression of a condition associated with nonalcoholic steatohepatitis. BACKGROUND

[0002] Nonalcoholic fatty liver disease (hereinafter also referred to as "NAFLD") is a condition in which fatty liver is confirmed by histological diagnosis or imaging diagnosis, and viral hepatitis, autoimmune hepatitis, alcoholic liver disorder, and the like are excluded, and is rapidly increasing worldwide with the increase in the number of obese people. Nonalcoholic fatty liver disease (NAFLD) is classified into nonalcoholic fatty liver (hereinafter also referred to as "NAFL") in which the condition is considered to hardly progress, and nonalcoholic steatohepatitis (hereinafter also referred to as "NASH") which is progressive and also becomes a cause of liver cirrhosis or liver cancer. As characteristic conditions of nonalcoholic steatohepatitis (NASH), there are steatosis, ballooning degeneration, inflammation, fibrosis, and the like.

[0003] In order to discriminate various conditions starting from steatosis of the liver, or to distinguish NAFL and NASH, a definitive diagnosis based on liver biopsy is required. However, liver biopsy is an invasive examination and is expensive, and thus the patient is consumed in various aspects. In addition, in liver biopsy, sampling errors and the like sometimes occur with a certain probability, and are left to the power of the technician who performs, and the same is true for the diagnosis of the collected sample. Therefore, in order to receive a certain level of medical care, the patient needs to visit a specific institution, and there is a problem that the burden on the patient is large.

[0004] On the other hand, as clinical examination items generally performed with respect to liver disease, there are AST, ALT, AST / ALT ratio, or TNF-a, high-sensitivity CRP, ferritin, and the like as markers of inflammation / cytokines, cytokeratin 18 fragment as a marker of apoptosis, hyaluronic acid, collagen type 4 7S, and the like as markers of fibrosis (Patent Documents 1 and 2). However, there is also no biomarker like a biomarker determined to be suspected NASH as detected in various conditions associated with NASH. That is, a biomarker that comprehensively reflects conditions associated with NASH has not been found so far.

[0005] PRIOR ART DOCUMENTS

[0006] PATENT DOCUMENTS

[0007] Patent Document 1: Japanese Patent Application Publication No. 2010-94131

[0008] Patent Literature 2: Japanese Patent Application Publication No. 2018-80943 SUMMARY

[0009] PROBLEMS TO BE SOLVED BY THE INVENTION

[0010] An object of the present application is to provide a method for assisting in the detection of non-alcoholic steatohepatitis (NASH) with extremely low invasiveness compared to liver biopsy, without relying on the skill of a technician, by a simple operation, and a method for assisting in the determination of the degree of progression of a condition associated with NASH.

[0011] MEANS FOR SOLVING THE PROBLEMS

[0012] As a result of intensive studies by the present inventors, it has been found that by measuring the amount of LDL-TG and / or ApoE-rich HDL-C contained in a blood sample isolated from a living body, even without performing liver biopsy, the detection of NASH can be assisted by a simple operation, and the degree of progression associated with NASH can also be assisted in the determination, thereby completing the present application.

[0013] That is, the present application is as follows.

[0014] [1] A method for assisting in the detection of non-alcoholic steatohepatitis, the method comprising: measuring the amount of LDL-TG and / or ApoE-rich HDL-C contained in a blood sample isolated from a living body.

[0015] [2] The method according to [1], wherein the amount of LDL-TG in the above-mentioned blood sample is used as an index, and the amount of LDL-TG is more than that in a blood sample of a patient with non-alcoholic fatty liver, indicating a high possibility of the onset of non-alcoholic steatohepatitis.

[0016] [3] The method according to [1] or [2], wherein the LDL-TG / LDL-C ratio in the above-mentioned blood sample is used as an index, and the LDL-TG / LDL-C ratio is higher than that in a blood sample of a patient with non-alcoholic fatty liver, indicating a high possibility of the onset of non-alcoholic steatohepatitis.

[0017] [4] The method according to any one of [1] to [3], wherein the ApoE-rich HDL-C / HDL-C ratio in the above-mentioned blood sample is used as an index, and the ApoE-rich HDL-C / HDL-C ratio is lower than that in a blood sample of a patient with alcoholic fatty liver, indicating a high possibility of the onset of non-alcoholic steatohepatitis.

[0018] [5] A method for aiding in the determination of the progression of at least one of steatosis, inflammation, ballooning degeneration and fibrosis in relation to non-alcoholic steatohepatitis, comprising: determining the presence of LDL-TG and / or ApoE-rich HDL-C in a blood sample isolated from an organism.

[0019] Invention Effects

[0020] According to the present invention, liver biopsies, which impose a significant burden or risk on patients, are minimized, and the expertise of the technicians performing the biopsy is not required. A safe and simple procedure can be used to assist in the detection of NASH, or to help determine the progression or presence of NASH-related conditions. As a result, treatment for NASH can be initiated at an early stage. Furthermore, by understanding the risk of progression, treatment plans can be determined, preventing or delaying progression to critical illness or death. Attached Figure Description

[0021] [ Figure 1 [Image] is a graph showing the results of measuring the total TG level in the blood collected from the subject in Comparative Example 1.

[0022] [ Figure 2 [Image] is a graph showing the results of measuring the amount of LDL-C in the blood collected from the subject in Comparative Example 2.

[0023] [ Figure 3 [This is a graph showing the results of calculating the total TG / TC ratio in the blood collected from the subject in Comparative Example 3.]

[0024] [ Figure 4 [Image] is a graph showing the results of measuring the amount of LDL-TG in the blood collected from the subject in Example 1.

[0025] [ Figure 5 [ ] is a graph showing the results of calculating the LDL-TG / LDL-C ratio in the blood collected from the subject in Example 2-1.

[0026] [ Figure 6 [This is a graph showing the results of calculating the LDL-C / LDL-TG ratio in the blood collected from the subject in Example 2-2.]

[0027] [ Figure 7 [Image] is a graph showing the results of measuring the amount of HDL-C in the blood collected from the subject in Comparative Example 4.

[0028] [ Figure 8 [This is a graph showing the results of calculating the ratio of ApoE-rich HDL-C to HDL-C in the blood collected from the subject in Example 3-1.]

[0029] [Figure 9 FIG. 3-2 is a graph showing the results of calculating the HDL-C / ApoE-rich HDL-C ratio in blood collected from the subject in Example 3-1.

[0030] [ Figure 10 FIG. 6-1 is a graph showing the results of measuring the LDL-TG amount in blood collected from the NAFLD patient group in Example 6-1, and comparing in the steatosis stage group.

[0031] [ Figure 11 FIG. 6-2 is a graph showing the results of calculating the LDL-TG / sdLDL-C ratio in blood collected from the NAFLD patient group in Example 6-2, and comparing in the steatosis stage group.

[0032] [ Figure 12 FIG. 6-3 is a graph showing the results of calculating the sdLDL-C / LDL-TG ratio in blood collected from the NAFLD patient group in Example 6-3, and comparing in the steatosis stage group.

[0033] [ Figure 13 FIG. 7-1 is a graph showing the results of calculating the ApoE-rich HDL-C / HDL-C ratio in blood collected from the NAFLD patient group in Example 7-1, and comparing in the steatosis stage group.

[0034] [ Figure 14 FIG. 7-2 is a graph showing the results of calculating the HDL-C / ApoE-rich HDL-C ratio in blood collected from the NAFLD patient group in Example 7-2, and comparing in the steatosis stage group.

[0035] [ Figure 15 FIG. 8 is a graph showing the results of measuring the LDL-TG amount in blood collected from the NAFLD patient group in Example 8, and comparing in the inflammation stage group.

[0036] [ Figure 16 FIG. 9 is a graph showing the results of measuring the ApoE-rich HDL-C amount in blood collected from the NAFLD patient group in Example 9, and comparing in the inflammation stage group.

[0037] [ Figure 17 FIG. 10-1 is a graph showing the results of measuring the LDL-TG amount in blood collected from the NAFLD patient group in Example 10-1, and comparing in the ballooning stage group.

[0038] [ Figure 18 FIG. 10-2 is a graph showing the results of calculating the LDL-TG / LDL-C ratio in blood collected from the NAFLD patient group in Example 10-2, and comparing in the ballooning stage group.

[0039] [ Figure 19 ] is a graph showing the results of calculating the LDL-C / LDL-TG ratio in blood collected from the NAFLD patient group in Example 10-3, comparing the stage groups of ballooning.

[0040] [ Figure 20 ] is a graph showing the results of calculating the LDL-TG / LDL-C ratio in blood collected from the NASH patient group in Example 11-1, comparing the stage groups of fibrosis.

[0041] [ Figure 21 ] is a graph showing the results of calculating the LDL-C / LDL-TG ratio in blood collected from the NASH patient group in Example 11-2, comparing the stage groups of fibrosis.

[0042] [ Figure 22 ] is a graph showing the results of calculating the LDL-TG / sdLDL-C ratio in blood collected from the NASH patient group in Example 11-3, comparing the stage groups of fibrosis.

[0043] [ Figure 23 ] is a graph showing the results of calculating the sdLDL-C / LDL-TG ratio in blood collected from the NASH patient group in Example 11-4, comparing the stage groups of fibrosis.

[0044] [ Figure 24 ] is a graph showing the results of calculating the ApoE-rich HDL-C / HDL-C ratio in blood collected from the NASH patient group in Example 12-1, comparing the stage groups of fibrosis.

[0045] [ Figure 25 ] is a graph showing the results of calculating the HDL-C / ApoE-rich HDL-C ratio in blood collected from the NASH patient group in Example 12-2, comparing the stage groups of fibrosis. DETAILED DESCRIPTION

[0046] The present application relates to a method for assisting in the detection of nonalcoholic steatohepatitis (NASH) by measuring the amount of LDL-TG and / or ApoE-rich HDL-C contained in a blood sample separated from a living organism, and a method for assisting in the determination of the degree of progression of a condition associated with nonalcoholic steatohepatitis (NASH).

[0047] In the present application, LDL-TG refers to triglyceride (TG) in low-density lipoprotein (hereinafter, referred to as LDL), and ApoE-rich HDL-C refers to cholesterol (C) in high-density lipoprotein containing a large amount of apolipoprotein E (hereinafter, referred to as ApoE-rich HDL).

[0048] In the present application, since a blood sample is used as a test sample, the invasiveness is extremely low compared to liver biopsy, and the condition of the entire liver can be grasped in total, not depending on a specific sample obtained by taking a part. In addition, the collection of the test blood sample is easy, and if a determination kit corresponding to an automatic analysis device is used in the determination of the presence amount of LDL-TG and / or ApoE-rich HDL-C contained in the blood sample, the determination can be performed by a simple operation to the same extent as routine medical treatment or health diagnosis, etc. As the blood sample in the present application, whole blood, plasma, and serum can be exemplified.

[0049] (Method for assisting in the detection of nonalcoholic steatohepatitis (NASH))

[0050] Hereinafter, a specific method for assisting in the detection of nonalcoholic steatohepatitis (NASH) in the present application will be described.

[0051] (1) Presence amount of LDL-TG

[0052] By determining the presence amount of LDL-TG (also referred to as the amount of LDL-TG) in the test blood sample and using it as an index, in the case where the amount of LDL-TG in the test blood sample is significantly more than the amount of LDL-TG in the blood sample of a nonalcoholic fatty liver (NAFL) patient (NAFL patient), it can be judged that the possibility of NASH is high (see Reference Example 1, Reference Example 4, Figure 4 ). As for the amount of LDL-TG in the blood sample of a nonalcoholic steatohepatitis patient (NASH patient), the sensitivity was 82.9% and the specificity was 55.6% when the cutoff value was set to 15.1 mg / dL, and the sensitivity was 65.7% and the specificity was 88.9% when the cutoff value was set to 17.6 mg / dL. Further, the sensitivity was 94.3% and the specificity was 22.2% when the cutoff value was set to 12.1 mg / dL, and the sensitivity was 48.6% and the specificity was 100% when the cutoff value was set to 20.2 mg / dL. Here, the cutoff value for determining a NASH patient can be appropriately set within a range corresponding to the sensitivity and the specificity sought by a person using the present application, for example, within a range of 12.1 to 21.2 mg / dL of the cutoff value of the amount of LDL-TG, preferably within a range of 17.6 mg / dL ± 20%, and the best value is 17.6 mg / dL. The amount of LDL-TG in the blood sample of a NAFL patient can be specified by analyzing blood samples collected in advance from a group of NAFL patients.

[0053] (2) LDL-TG / LDL-C ratio

[0054] By measuring the amount of LDL-TG and the amount of LDL-C in the blood sample to be examined, the LDL-TG / LDL-C ratio is calculated and used as an index, and in the case where the LDL-TG / LDL-C ratio in the blood sample to be examined is significantly higher than the LDL-TG / LDL-C ratio in the blood sample of a healthy person or a NAFL patient, it can be determined that the possibility of the onset of NASH is high (see Example 2-1, Example 5-1, Figure 5 ). Here, the amount of LDL-C refers to the amount of cholesterol (C) in low-density lipoprotein (LDL). Regarding the LDL-TG / LDL-C ratio in the blood sample of a NASH patient, the sensitivity was 100% and the specificity was 55.6% when the cutoff value was set to 0.103, the sensitivity was 94.3% and the specificity was 66.7% when the cutoff value was set to 0.133. Further, the sensitivity was 82.9% and the specificity was 77.8% when the cutoff value was set to 0.149, and the sensitivity was 31.4% and the specificity was 100% when the cutoff value was set to 0.203. Here, the cutoff value for determining a NASH patient can be appropriately set within a range corresponding to the sensitivity and the specificity calculated using a person to whom the present application is applied, for example, within a range of 0.103 to 0.203 of the LDL-TG / LDL-C ratio, preferably within a range of ± 20% of 0.133, and the optimal values are 0.149 or 0.133.

[0055] Further, by calculating the LDL-C / LDL-TG ratio in the blood sample to be examined and using it as an index, in the case where the LDL-C / LDL-TG ratio is lower than the LDL-C / LDL-TG ratio in the blood sample of a healthy person or a NAFL patient, it can also be determined that the possibility of the onset of NASH is high (see Example 2-2, Example 5-2, Figure 6 ). Regarding the LDL-C / LDL-TG ratio in the blood sample of a NASH patient, the sensitivity was 100% and the specificity was 55.6% when the cutoff value was set to 9.75, the sensitivity was 94.3% and the specificity was 66.7% when the cutoff value was set to 7.50. Further, the sensitivity was 82.9% and the specificity was 77.8% when the cutoff value was set to 6.70, and the sensitivity was 31.4% and the specificity was 100% when the cutoff value was set to 4.93. In this case, the LDL-C / LDL-TG ratio is inversely proportional to the LDL-TG / LDL-C ratio. Here, the cutoff value for determining a NASH patient can be appropriately set within a range corresponding to the sensitivity and the specificity calculated using a person to whom the present application is applied, for example, within a range of 4.93 to 9.75 of the LDL-C / LDL-TG ratio, preferably within a range of ± 20% of 7.50, and the optimal values are 7.50 or 6.70.

[0056] (3) HDL-C / HDL-C ratio rich in ApoE

[0057] By measuring the amount of ApoE-rich HDL-C and the amount of HDL-C in the blood sample to be examined, the ApoE-rich HDL-C / HDL-C ratio is calculated and used as an index, and in the case where the ApoE-rich HDL-C / HDL-C ratio in the blood sample to be examined is significantly lower than the ApoE-rich HDL-C / HDL-C ratio in the blood sample of the NAFL patient, it can be determined that the possibility of the onset of NASH is high (see Example 3-1, Example 13-1, Figure 8 ). Here, the amount of HDL-C refers to the presence amount of cholesterol (C) in high-density lipoprotein (HDL). Regarding the ApoE-rich HDL-C / HDL-C ratio in the blood sample of the NASH patient, the sensitivity is 80.0% and the specificity is 66.7% when the cutoff value is set to 0.0912, and the sensitivity is 74.3% and the specificity is 88.9% when the cutoff value is set to 0.0899. Further, the sensitivity is 42.9% and the specificity is 100% when the cutoff value is set to 0.0840, and the sensitivity is 91.4% and the specificity is 11.1% when the cutoff value is set to 0.0971. Here, the cutoff value for determining the NASH patient can be appropriately set within a range corresponding to the sensitivity and specificity calculated using the person of the present application, for example, within a range of 0.0899 ± 20% of the ApoE-rich HDL-C / HDL-C ratio, preferably within a range of 0.0840 to 0.0971, and the optimal value is 0.0899.

[0058] Further, by calculating the HDL-C / ApoE-rich HDL-C ratio in the blood sample and using it as an index, in the case where the HDL-C / ApoE-rich HDL-C ratio is significantly higher than the HDL-C / ApoE-rich HDL-C ratio in the blood sample of a healthy person or a NAFL patient, it can also be determined that the possibility of the onset of NASH is high (see Example 3-2, Example 13-2, Figure 9). As for the HDL-C / ApoE-rich HDL-C ratio in the blood sample of the NASH patient, the sensitivity was 80.0% and the specificity was 66.7% when the cutoff value was set to 10.97, and the sensitivity was 74.3% and the specificity was 88.9% when the cutoff value was further set to 11.12. The sensitivity was 91.4% and the specificity was 11.1% when the cutoff value was further set to 10.30, and the sensitivity was 42.9% and the specificity was 100% when the cutoff value was set to 11.90. In this case, the HDL-C / ApoE-rich HDL-C ratio is inversely proportional to the above-mentioned ApoE-rich HDL-C / HDL-C ratio. Here, the cutoff value for determining the NASH patient can be appropriately set within a range corresponding to the sensitivity and the specificity sought by using the human of the present application, for example, within a range of 11.12 ± 20% of the cutoff value of the HDL-C / ApoE-rich HDL-C ratio, preferably within a range of 10.30 to 11.90, and the optimal value is 11.12.

[0059] As described above, the method for determining the high possibility of the onset of NASH using the presence amount of LDL-TG, the LDL-C / LDL-TG ratio, and the ApoE-rich HDL-C / HDL-C ratio in the blood sample of the subject was described, and these indexes can sufficiently assist the detection of NASH when any one of them is used alone, but the detection of NASH can be more effectively assisted when a plurality of indexes are used in combination.

[0060] By measuring the presence amount of LDL-TG and / or ApoE-rich HDL-C contained in the blood sample of the subject as described above and using the indexes using these measured values, the differentiation between NASH and NAFL, which has conventionally required liver biopsy, can be easily assisted by a simple operation.

[0061] (Method for assisting determination of the progression degree of the condition related to nonalcoholic steatohepatitis)

[0062] Hereinafter, a specific method for assisting determination of the progression degree of the condition related to nonalcoholic steatohepatitis (NASH) selected from at least one of steatosis, ballooning degeneration, inflammation, and fibrosis in the present application will be described.

[0063] (1) Presence amount of LDL-TG

[0064] By measuring the amount of LDL-TG in the blood sample of the subject and using it as an index, it can be determined that the more the amount of LDL-TG in the blood sample of the subject, the higher the progression stage of steatosis, inflammation, or ballooning degeneration.

[0065] Specifically, in the case where the amount of LDL-TG in the blood sample under examination is 12.2 mg / dL or more, further 13.7 mg / dL or more, particularly 14.2 mg / dL or more, it is extremely likely that the stage of steatosis has progressed to 1 or more (see Example 6-1, Figure 10 ).

[0066] Further, in the case where the amount of LDL-TG in the blood sample under examination is 15.1 mg / dL or more, further 16.9 mg / dL or more, it is likely that the stage of inflammation has progressed to 1 or more, and in the case where the amount of LDL-TG in the blood sample under examination is 22.0 mg / dL or more, further 22.5 mg / dL or more, it is likely that the stage of inflammation has progressed to 3 or more (see Example 8, Figure 15 ).

[0067] Further, in the case where the amount of LDL-TG in the blood sample under examination is 15.1 mg / dL or more, further 17.2 mg / dL or more, particularly 19.5 mg / dL or more, it is extremely likely that the stage of ballooning has progressed to 1 or more (see Example 10-1, Figure 17 ).

[0068] (2) LDL-TG / sd LDL-C ratio

[0069] By measuring the amount of LDL-TG and the amount of sd LDL-C in the blood sample under examination, and taking the LDL-TG / sd LDL-C ratio as an index, it is determined that the lower the LDL-TG / sd LDL-C ratio in the blood sample under examination, the higher the stage of progression of steatosis. Here, the amount of sd LDL-C refers to the amount of cholesterol (C) present in small particle low density lipoprotein (sd LDL). Specifically, in the case where the LDL-TG / sd LDL-C ratio in the blood sample under examination is 1.11 or less, further 0.1 to 1.06, it is likely that the stage of steatosis has progressed to 1 or more (see Example 6-2, Figure 11 ).

[0070] In addition, it can be determined that the higher the LDL-TG / sd LDL-C ratio in the blood sample under examination, the higher the progression stage of fibrosis. Specifically, in the case where the LDL-TG / sd LDL-C ratio in the blood sample under examination is 0.39 or higher, further 0.60 or higher, it can be determined that the progression stage of fibrosis is extremely likely to be 2 or higher, and in the case where the LDL-TG / sd LDL-C ratio in the blood sample under examination is 0.61 or higher, further 0.72 or higher, in particular 0.88 or higher, it can be determined that the progression stage of fibrosis is likely to be 4 or higher (see Example 11-3, Figure 22 ).

[0071] Further, by using the sd LDL-C / LDL-TG ratio in the blood sample under examination as an index, it is also possible to determine the progression stage of steatosis or fibrosis. In this case, the sd LDL-C / LDL-TG ratio is inversely proportional to the LDL-TG / sd LDL-C ratio described above Figure 12 , Figure 23 ).

[0072] (3) ApoE-rich HDL-C / HDL-C ratio

[0073] By measuring the amount of ApoE-rich HDL-C and the amount of HDL-C in the blood sample under examination, and using the ApoE-rich HDL-C / HDL-C ratio as an index, it can be determined that the higher the ApoE-rich HDL-C / HDL-C ratio in the blood sample under examination, the higher the progression stage of steatosis. Specifically, in the case where the ApoE-rich HDL-C / HDL-C ratio in the blood sample under examination is 0.089 or higher, further 0.09 or higher, it can be determined that the progression stage of steatosis is likely to be 1 or higher (see Example 7-1, Figure 13 ).

[0074] In addition, by using the ApoE-rich HDL-C / HDL-C ratio in the blood sample under examination as an index, it can be determined that the lower the ApoE-rich HDL-C / HDL-C ratio in the blood sample under examination, the higher the progression stage of fibrosis. Specifically, in the case where the ApoE-rich HDL-C / HDL-C ratio in the blood sample under examination is 0.095 or lower, further 0.094 or lower, in particular 0.092 or lower, it can be determined that the progression stage of fibrosis is likely to be 2 or higher, and in the case where the ApoE-rich HDL-C / HDL-C ratio in the blood sample under examination is 0.094 or lower, further 0.086 or lower, in particular 0.084 or lower, it can be determined that the progression stage of fibrosis is likely to be 4 or higher (see Example 12-1, Figure 24 ).

[0075] Further, by taking the HDL-C / ApoE-rich HDL-C ratio in the blood sample under examination as an index, the progression stage of steatosis or fibrosis can also be determined. In this case, the HDL-C / ApoE-rich HDL-C ratio is inversely proportional to the above-mentioned ApoE-rich HDL-C / HDL-C ratio Figure 14 , Figure 25 ).

[0076] (4) ApoE-rich HDL-C amount

[0077] By measuring the ApoE-rich HDL-C amount in the blood sample under examination and taking it as an index, it can be determined that the less the ApoE-rich HDL-C amount in the blood sample under examination, the higher the progression stage of inflammation. Specifically, in the case where the ApoE-rich HDL-C amount in the blood sample under examination is 5.5 mg / dL or less, further 4.6 mg / dL or less, particularly 4.4 mg / dL or less, it can be determined that the stage of inflammation has progressed to 1 or 2 with a high probability (see Example 9, Figure 16 ).

[0078] (5) LDL-TG / LDL-C ratio

[0079] By measuring the LDL-TG amount and the LDL-C amount in the blood sample under examination, calculating the LDL-TG / LDL-C ratio and taking it as an index, it can be determined that the higher the LDL-TG / LDL-C ratio in the blood sample under examination, the higher the progression stage of ballooning degeneration. Specifically, in the case where the LDL-TG / LDL-C ratio in the blood sample under examination is 0.15 or more, further 0.18 or more, particularly 0.19 or more, it can be determined that the stage of ballooning degeneration has progressed to 1 or more with a high probability (see Example 10-2, Figure 18 ).

[0080] Further, by taking the LDL-TG / LDL-C ratio in the blood sample under examination as an index, it can be determined that the higher the LDL-TG / LDL-C ratio in the blood sample under examination, the higher the progression stage of fibrosis. Specifically, in the case where the LDL-TG / LDL-C ratio in the blood sample under examination is 0.14 or more, further 0.17 or more, it can be determined that the stage of fibrosis has progressed to 2 or more with a high probability (see Example 11-1, Figure 20 ).

[0081] Further, by taking the LDL-C / LDL-TG ratio in the blood sample under examination as an index, the progression stage of ballooning degeneration or fibrosis can also be determined. In this case, the LDL-C / LDL-TG ratio is inversely proportional to the above-mentioned LDL-TG / LDL-C ratio Figure 19 , Figure 21).

[0082] As described above, the method for assisting in determining the progression degree of the condition related to NASH using the amount of LDL-TG, the LDL-TG / sd LDL-C ratio, the ApoE-rich HDL-C / HDL-C ratio, the amount of ApoE-rich HDL-C, and the LDL-TG / LDL-C ratio in the LDL-TG contained in the blood sample to be examined as indexes has been described. These indexes have sufficient effects of assisting in the determination even if any one of them is used alone, but the determination of the progression degree of the condition described above can be more effectively assisted if a plurality of indexes are used in combination.

[0083] By measuring the amounts of LDL-TG and / or ApoE-rich HDL-C contained in the blood sample to be examined as described above and using indexes using these measured values, the determination of the progression stage of the condition related to NASH, which has conventionally required a liver biopsy, can be easily assisted by a simple operation.

[0084] In the present application, as a method for measuring the amounts of LDL-TG, ApoE-rich HDL-C, HDL-C, LDL-C, and sd LDL-C contained in a blood sample, a method known in the art can be used. For example, a method in which, after subject lipoproteins are separated by a fractionation separation operation such as an ultracentrifugation method, an electrophoresis method, or a high-performance liquid chromatography method, triglyceride or cholesterol is quantified by a quantitative operation; or a method in which, without performing a fractionation operation, triglyceride or cholesterol is removed from all lipoproteins other than the subject lipoproteins by Step 1, and then triglyceride or cholesterol in the subject lipoproteins is measured by Step 2 can be used.

[0085] Specifically, as a method for measuring the amount of LDL-TG, the method described in WO2013 / 157642 can be used, as a method for measuring the amount of HDL-C, the method described in WO98 / 26090 can be used, as a method for measuring the amount of LDL-C, the method described in WO98 / 47005 can be used, and as a method for measuring the amount of sd LDL-C, the method described in WO2009 / 048143 can be used. Example

[0086] Hereinafter, the present application will be specifically described according to examples, but the present application is not limited to the following examples.

[0087] <Comparative Example 1>

[0088] The total TG amount in blood collected from a total of 80 people consisting of 36 healthy people (healthy), 9 NAFL patients, and 35 NASH patients was measured, and the groups were compared. The total TG amount was measured using an automatic analysis device used on the spot of clinical examination, using TG-EX "Jikken" (enzymatic method) (manufactured by Denka Seiken Co., Ltd.) as a reagent for triglyceride measurement. The graph after comparison is shown in Fig. 1. Figure 1 .

[0089] As a result, the total TG amount of the NASH group was significantly higher than that of the healthy people group (p<0.0001), but there was no difference from the NAFL group. The total TG amount of the NAFL group was significantly higher than that of the healthy people group (p<0.05).

[0090] <Comparative Example 2>

[0091] The LDL-C amount in blood collected from a total of 80 people consisting of 36 healthy people (healthy), 9 NAFL patients, and 35 NASH patients, which were the same as in Comparative Example 1, was measured, and the groups were compared. The LDL-C amount was measured using an automatic analysis device used on the spot of clinical examination, using an automatic analysis reagent "Jikken" LDL-EX (N) (direct method) (manufactured by Denka Seiken Co., Ltd.) as a reagent for LDL-cholesterol measurement. The graph after comparison is shown in Fig. 2. Figure 2 .

[0092] As a result, the LDL-C amount of the NASH group showed a tendency to be lower than that of the NAFL group, but there was no difference from the healthy people group. In addition, the LDL-C amount of the NAFL group showed a tendency to be higher than that of the healthy people group.

[0093] <Comparative Example 3>

[0094] The total TG amount and the total TC amount in blood collected from a total of 80 people consisting of 36 healthy people (healthy), 9 NAFL patients, and 35 NASH patients, which were the same as in Comparative Example 1, were measured, and the total TG / TC ratio was calculated, and the groups were compared. The total TG amount was measured by the same method as in Comparative Example 1, and the TC amount was measured using an automatic analysis device used on the spot of clinical examination, using an automatic analysis reagent "Jikken" T-CHO (S) (enzymatic method) (manufactured by Denka Seiken Co., Ltd.) as a reagent for cholesterol measurement. The graph after comparison is shown in Fig. 3. Figure 3 .

[0095] As a result, the total TG / TC ratio of the NASH group was significantly higher than that of the healthy people group (p<0.0001), but there was no difference from the NAFL group. In addition, the total TG / TC ratio of the NAFL group was not different from that of the healthy people group.

[0096] <Example 1>

[0097] The amount of LDL-TG in blood collected from a total of 80 people consisting of 36 healthy people (healthy) identical to those of Comparative Example 1, 9 NAFL patients, and 35 NASH patients was measured, and each group was compared. The amount of LDL-TG was measured using LDLTG-EX "SEIKEN" (manufactured by Denka Seiken Co., Ltd.) as a reagent for LDL-triglyceride measurement, using an automatic analysis device used on the site of clinical examination. The graph after comparison is shown in Figure 4 .

[0098] As a result, the amount of LDL-TG in the NASH group was significantly higher than that in the NAFL group (p<0.05), and was also significantly higher than that in the healthy person group (p<0.0001). In addition, the amount of LDL-TG in the NAFL group was significantly higher than that in the healthy person group (p<0.05).

[0099] Example 2-1

[0100] The amount of LDL-TG and the amount of LDL-C in blood collected from a total of 80 people consisting of 36 healthy people (healthy) identical to those of Comparative Example 1, 9 NAFL patients, and 35 NASH patients were measured using the same method as in Example 1 and Comparative Example 2, the ratio of LDL-TG / LDL-C was calculated, and each group was compared. The graph after comparison is shown in Figure 5 .

[0101] As a result, the ratio of LDL-TG / LDL-C in the NASH group was significantly higher than that in the NAFL group (p<0.05), and was also significantly higher than that in the healthy person group (p<0.0001). In addition, there was no difference in the ratio of LDL-TG / LDL-C between the NAFL group and the healthy person group.

[0102] Example 2-2

[0103] The amount of LDL-TG and the amount of LDL-C in blood collected from a total of 80 people consisting of 36 healthy people (healthy) identical to those of Comparative Example 1, 9 NAFL patients, and 35 NASH patients were measured using the same method as in Example 1 and Comparative Example 2, the ratio of LDL-C / LDL-TG was calculated, and each group was compared. The graph after comparison is shown in Figure 6 .

[0104] As a result, the ratio of LDL-C / LDL-TG in the NASH group was significantly lower than that in the NAFL group (p<0.05), and was also significantly lower than that in the healthy person group (p<0.0001). In addition, there was no difference in the ratio of LDL-C / LDL-TG between the NAFL group and the healthy person group.

[0105] According to the <Comparative Example 1>, <Comparative Example 2>, <Comparative Example 3>, it was not possible to clearly distinguish NASH and NAFL by measuring the total TG amount or the LDL-C amount or the TC amount. However, according to the <Example 1> of the present application, it was possible to clearly distinguish NASH, NAFL and healthy people by measuring the LDL-TG amount. In addition, according to the <Example 2-1>, <Example 2-2> of the present application, it was possible to further clearly distinguish NASH and NAFL by calculating the ratio of LDL-TG to LDL-C.

[0106] <Comparative Example 4>

[0107] The HDL-C amount in blood collected from a total of 80 people consisting of 36 healthy people (healthy), 9 NAFL patients, and 35 NASH patients, which were the same as in Comparative Example 1, was measured, and the groups were compared. The HDL-C amount was measured using an automatic analysis device using the reagent "Shin Nippon" HDL-EX (direct method) (manufactured by Denka Seiken Co., Ltd.) as a reagent for HDL-cholesterol measurement. The graph after comparison is shown in Figure 7 .

[0108] As a result, there was no difference in the HDL-C amount among the NASH group, the NAFL group, and the healthy people group.

[0109] <Example 3-1>

[0110] The ApoE-rich HDL-C amount and the HDL-C amount in blood collected from a total of 50 people consisting of 6 healthy people (healthy), 9 NAFL patients, and 35 NASH patients were measured, the ApoE-rich HDL / HDL-C ratio was calculated, and the groups were compared. The ApoE-rich HDL-C amount was measured using an automatic analysis device used in the field of clinical examination using an ApoE-rich HDL-cholesterol measurement reagent, and the HDL-C amount was measured using the same method as in Comparative Example 4. The ApoE-rich HDL-cholesterol measurement reagent was implemented according to the method of Japanese Patent Application Publication No. 2014-030393. The graph after comparison is shown in Figure 8 .

[0111] As a result, the ApoE-rich HDL-C / HDL-C ratio of the NASH group was significantly lower than that of the NAFL group (p < 0.05). In addition, there was no difference in the ApoE-rich HDL-C / HDL-C ratio between the NAFL group and the healthy people group.

[0112] <Example 3-2>

[0113] The amount of ApoE-enriched HDL-C and the amount of HDL-C in blood collected from a total of 50 people consisting of 6 healthy people (healthy), 9 NAFL patients, and 35 NASH patients were measured by the same method as in Example 3-1 and Comparative Example 4, the ratio of HDL-C / ApoE-enriched HDL-C was calculated, and each group was compared. The graph after comparison is shown in Figure 9 .

[0114] As a result, the ratio of HDL-C / ApoE-enriched HDL-C in the NASH group was significantly higher than that in the NAFL group (p<0.05). In addition, there was no difference in the ratio of HDL-C / ApoE-enriched HDL-C between the NAFL group and the healthy people group.

[0115] According to Comparative Example 4, even if only the amount of HDL-C is compared, it is not possible to distinguish between NASH and NAFL, but according to Example 3-1 and Example 3-2, by calculating the ratio of ApoE-enriched HDL-C to HDL-C, it is possible to clearly distinguish between NASH and NAFL.

[0116] Example 4

[0117] The amount of LDL-TG in blood collected from a total of 44 NAFLD consisting of 9 NAFL patients and 35 NASH patients was measured, and when ROC analysis was performed, the AUC was 0.78 (95% CI: 0.63-0.92), which was good.

[0118] When the cutoff value of this group was set to 15.1 mg / dL, the sensitivity and specificity were 82.9% and 55.6%, respectively, and when it was set to 17.6 mg / dL, the sensitivity and specificity were 65.7% and 88.9%, respectively. Further, when the cutoff value was set to 12.1 mg / dL, the sensitivity and specificity were 94.3% and 22.2%, respectively, and when it was set to 20.2 mg / dL, the sensitivity and specificity were 48.6% and 100%, respectively.

[0119] Example 5-1

[0120] The amount of LDL-C in blood of the same group as in Example 4 was measured, and the ratio of LDL-TG / LDL-C was calculated, and when ROC analysis was performed, the AUC was 0.86 (95% CI: 0.70-1.02), which was good.

[0121] The sensitivity and specificity of the cut-off value of the LDL-TG / LDL-C ratio of the group was set to 0.13, and the sensitivity and specificity were 100%, 55.6%, respectively. The sensitivity and specificity of the cut-off value set to 0.133 were 94.3%, 66.7%, respectively. Further, the sensitivity and specificity of the cut-off value set to 0.149 were 82.9%, 77.8%, respectively, and the sensitivity and specificity of the cut-off value set to 0.203 were 31.4%, 100%, respectively.

[0122] Example 5-2

[0123] The LDL-C / LDL-TG ratio was calculated from the amount of LDL-TG in blood and the amount of LDL-C in blood of the same group as Example 5-1, and when ROC analysis was performed, the AUC was 0.86 (95% CI: 0.70-1.02), which was good.

[0124] The sensitivity and specificity of the cut-off value of the LDL-C / LDL-TG ratio of the group was set to 9.75, and the sensitivity and specificity were 100%, 55.6%, respectively. The sensitivity and specificity of the cut-off value set to 7.50 were 94.3%, 66.7%, respectively. Further, the sensitivity and specificity of the cut-off value set to 6.70 were 82.9%, 77.8%, respectively, and the sensitivity and specificity of the cut-off value set to 4.93 were 31.4%, 100%, respectively.

[0125] Example 6-1

[0126] The amount of LDL-TG in blood collected from the NAFLD patient group (n=37) determined to be in the stage of steatosis was measured using the same method as Example 1, and the comparison was made in each stage group. The graph after comparison is shown in Figure 10 .

[0127] As a result, the stage 1, 2, and 3 groups were significantly higher than the stage 0 group (p<0.05, p<0.05, p<0.05). In addition, the average values of each stage in stages 0, 1, 2, and 3 were 12.2 mg / dL, 18.1 mg / dL, 18.5 mg / dL, and 20.8 mg / dL, respectively.

[0128] Example 6-2

[0129] The LDL-TG amount and sd LDL-C amount in blood collected from the NAFLD patient group (n=37) determined to be in the stage of steatosis were measured, the ratio thereof was calculated, and comparison was made among the stage groups. The LDL-TG amount was measured by the same method as in Example 1, and the sd LDL-C amount was measured using an automatic analyzer by using sd LDL-EX "Seisan" (manufactured by Denka Seiken Co., Ltd.) as a reagent for sd LDL-cholesterol measurement. The graph after comparison is shown in Fig. 6. Figure 11 .

[0130] As a result, in terms of the LDL-TG / sd LDL-C ratio, the stage 2 group was significantly lower than the stage 0 group (p<0.05), and the stage 3 group showed a tendency to be lower than the stage 0 group. In addition, the median values in the stages 0, 1, 2, and 3 were 1.120, 0.604, 0.576, and 0.426, respectively.

[0131] Example 6-3

[0132] The LDL-TG amount and sd LDL-C amount in blood collected from the NAFLD patient group (n=37) determined to be in the stage of steatosis were measured, the ratio thereof was calculated, and comparison was made among the stage groups. The graph after comparison is shown in Fig. 7. Figure 12 .

[0133] As a result, in terms of the sd LDL-C / LDL-TG ratio, the stage 2 group was significantly higher than the stage 0 group (p<0.05), and the stage 3 group showed a tendency to be higher than the stage 0 group. In addition, the median values in the stages 0, 1, 2, and 3 were 0.893, 1.655, 1.735, and 2.349, respectively.

[0134] According to Examples 6-1 to 6-3 of the present application, by evaluating the LDL-TG amount or the ratio of LDL-TG to sd LDL-C, it is possible to infer the progression of steatosis.

[0135] Example 7-1

[0136] The ApoE-rich HDL-C amount and HDL-C amount in blood collected from the NAFLD patient group (n=37) determined to be in the stage of steatosis were measured, the ratio thereof was calculated, and comparison was made among the stage groups. The graph after comparison is shown in Fig. 8. Figure 13 .

[0137] As a result, the stage 3 group was significantly higher than the stage 1 group in the ApoE-rich HDL-C / HDL-C ratio (p<0.05).

[0138] Example 7-2

[0139] Using the same method as in Example 3-1 and Comparative Example 4, the amount of ApoE-rich HDL-C and the amount of HDL-C in blood collected from a group of NAFLD patients (n=37) determined to be in the stage of steatosis were measured, the ratio thereof was calculated, and the stages were compared. The graph after comparison is shown in Figure 14 .

[0140] As a result, the stage 3 group showed a tendency to be lower than the stage 0 group in the HDL-C / ApoE-rich HDL-C ratio.

[0141] According to Example 7-1 and Example 7-2 of the present application, the progression of steatosis can be inferred by evaluating the ratio of ApoE-rich HDL-C to HDL-C.

[0142] Example 8

[0143] Using the same method as in Example 1, the amount of LDL-TG in blood collected from a group of NAFLD patients (n=38) determined to be in the stage of inflammation was measured, and the stages were compared. The graph after comparison is shown in Figure 15 .

[0144] As a result, the stage 3 group was significantly higher than the stage 0, 1, and 2 groups in the amount of LDL-TG (p<0.001, p<0.05, p<0.05), the stage 2 group was significantly higher than the stage 0 group (p<0.05), and the stage 1 group was significantly higher than the stage 0 group (p<0.0001).

[0145] Example 9

[0146] Using the same method as in Example 3-1, the amount of ApoE-rich HDL-C in blood collected from a group of NAFLD patients (n=38) determined to be in the stage of inflammation was measured, and the stages were compared. The graph after comparison is shown in Figure 16 .

[0147] As a result, the stage 1 group was significantly lower than the stage 0 group in the amount of ApoE-rich HDL-C (p<0.05).

[0148] According to the <Example 8>, <Example 9> of the present application, by evaluating the amount of LDL-TG or the amount of ApoE-rich HDL-C, the progression of inflammation can be inferred.

[0149] <Example 10-1>

[0150] Using the same method as in Example 1, the amount of LDL-TG in blood collected from a group of NAFLD patients (n=37) judged to be in the stage of ballooning was measured, and each stage was compared. The graph after comparison is shown in Figure 17 .

[0151] As a result, in terms of the amount of LDL-TG, the group obtained by combining stages 1 and 2 was significantly higher than the stage 0 group (p<0.01).

[0152] <Example 10-2>

[0153] Using the same method as in Example 1 and Comparative Example 2, the amount of LDL-TG and the amount of LDL-C in blood collected from a group of NAFLD patients (n=37) judged to be in the stage of ballooning were measured, and the ratio thereof was calculated, and each stage was compared. The graph after comparison is shown in Figure 18 .

[0154] As a result, in terms of the ratio of LDL-TG / LDL-C, the group obtained by combining stages 1 and 2 showed a tendency to be higher than the stage 0 group.

[0155] <Example 10-3>

[0156] Using the same method as in Example 1 and Comparative Example 2, the amount of LDL-TG and the amount of LDL-C in blood collected from a group of NAFLD patients (n=37) judged to be in the stage of ballooning were measured, and the ratio thereof was calculated, and each stage was compared. The graph after comparison is shown in Figure 19 .

[0157] As a result, in terms of the ratio of LDL-C / LDL-TG, the group obtained by combining stages 1 and 2 was significantly lower than the stage 0 group (p<0.05).

[0158] According to the <Example 10-1>, <Example 10-2>, <Example 10-3> of the present application, by evaluating the amount of LDL-TG or the ratio of LDL-TG to LDL-C, the progression of ballooning can be inferred.

[0159] <Example 11-1>

[0160] The amount of LDL-TG and the amount of LDL-C in blood collected from a group of NASH patients (n=27) determined to be in a stage of fibrosis were measured using the same method as in Example 1 and Comparative Example 2, the ratio thereof was calculated, and the groups were compared for each stage. The graph after comparison is shown in Figure 20 .

[0161] As a result, in terms of the LDL-TG / LDL-C ratio, the stage 2, 3, and 4 groups were significantly higher than the stage 1 group (p<0.05, p<0.05, p<0.01).

[0162] Example 11-2

[0163] The amount of LDL-TG and the amount of LDL-C in blood collected from a group of NASH patients (n=27) determined to be in a stage of fibrosis were measured using the same method as in Example 1 and Comparative Example 2, the ratio thereof was calculated, and the groups were compared for each stage. The graph after comparison is shown in Figure 21 .

[0164] As a result, in terms of the LDL-C / LDL-TG ratio, the stage 2, 3, and 4 groups were significantly lower than the stage 1 group (p<0.005, p<0.005, p<0.0005).

[0165] Example 11-3

[0166] The amount of LDL-TG and the amount of sd LDL-C in blood collected from a group of NASH patients (n=27) determined to be in a stage of fibrosis were measured using the same method as in Example 1 and Example 6-2, the ratio thereof was calculated, and the groups were compared for each stage. The graph after comparison is shown in Figure 22 .

[0167] As a result, in terms of the LDL-TG / sd LDL-C ratio, the stage 4 group was significantly higher than the stage 1 group (p<0.05), and showed a tendency to be higher than the stage 3 group. In addition, the stage 2 group showed a tendency to be higher than the stage 1 group.

[0168] Example 11-4

[0169] The amount of LDL-TG and the amount of sd LDL-C in blood collected from a group of NASH patients (n=27) determined to be in a stage of fibrosis were measured using the same method as in Example 1 and Example 6-2, the ratio thereof was calculated, and the groups were compared for each stage. The graph after comparison is shown in Figure 23 .

[0170] As a result, the stage 4 group was significantly lower than the stage 1 and 3 groups in the sd LDL-C / LDL-TG ratio (p<0.005, p<0.05). In addition, the stage 2 group showed a tendency to be lower than the stage 1 group.

[0171] According to the <Example 11-1 to 4> of the present application, by evaluating the LDL-TG / LDL-C ratio or the LDL-TG / sd LDL-C ratio, the progression of fibrosis can be inferred.

[0172] <Example 12-1>

[0173] The amount of ApoE-enriched HDL-C and the amount of HDL-C in the blood collected from the NASH patient group (n=27) determined to be in the stage of fibrosis were measured by the same method as in Example 3-1 and Comparative Example 4, the ratio thereof was calculated, and comparison was made among the stage groups. The graph after comparison is shown in FIG. 6. Figure 24 .

[0174] The stage 4 group was significantly lower than the stage 1 and 2 groups in the ApoE-enriched HDL-C / HDL-C ratio (p<0.005, p<0.05), respectively.

[0175] <Example 12-2>

[0176] The amount of ApoE-enriched HDL-C and the amount of HDL-C in the blood collected from the NASH patient group (n=27) determined to be in the stage of fibrosis were measured by the same method as in Example 3-1 and Comparative Example 4, the ratio thereof was calculated, and comparison was made among the stage groups. The graph after comparison is shown in FIG. 6. Figure 25 .

[0177] The stage 4 group was significantly higher than the stage 1 and 2 groups in the HDL-C / ApoE-enriched HDL-C ratio (p<0.01, p<0.05), respectively, and showed a tendency to be higher than the stage 3 group.

[0178] According to the <Example 12-1> and <Example 12-2> of the present application, by evaluating the ApoE-enriched HDL-C / HDL-C ratio, the progression of fibrosis can be inferred.

[0179] <Example 13-1>

[0180] The amount of ApoE-enriched HDL-C and the amount of HDL-C in the blood collected from the same collective as in Example 4 were measured, and the ApoE-enriched HDL-C / HDL-C ratio was calculated. When ROC analysis was performed, the AUC was 0.80 (95% CI: 0.65-0.94), which was good.

[0181] The sensitivity and specificity of the cut-off value of the HDL-C / ApoE-rich HDL-C ratio of this group was set to 0.0912, 80.0%, 66.7%, and the sensitivity and specificity was set to 0.0899, 74.3%, 88.9%. Further, the sensitivity and specificity was set to 0.0840, 42.9%, 100%, and the sensitivity and specificity was set to 0.0971, 91.4%, 11.1%.

[0182] Example 13-2

[0183] The HDL-C / ApoE-rich HDL-C ratio was calculated from the amount of ApoE-rich HDL-C and the amount of HDL-C in the blood of the same group as Example 13-1, and when ROC analysis was performed, the AUC was 0.80 (95% CI: 0.65-0.94), which was good.

[0184] The sensitivity and specificity of the cut-off value of the HDL-C / ApoE-rich HDL-C ratio of this group was set to 10.97, 80.0%, 66.7%, and the sensitivity was set to 11.12, 74.3%, and the specificity was set to 88.9%. Further, the sensitivity and specificity was set to 10.30, 91.4%, 11.1%, and the sensitivity and specificity was set to 11.90, 42.9%, 100%.

Claims

1. Use of a reagent for LDL-TG measurement in the manufacture of a kit for aiding in the detection and determination of non-alcoholic steatohepatitis, which reagent is used to measure the amount of LDL-TG contained in a blood sample taken from a subject isolated from a living organism, and the amount of LDL-TG in the above-mentioned blood sample taken from a subject is used as an index to determine the likelihood of the onset of non-alcoholic steatohepatitis.

2. The use of claim 1, wherein, The amount of LDL-TG is greater than the amount of LDL-TG in a blood sample from a patient with non-alcoholic fatty liver, indicating a high likelihood of the onset of non-alcoholic steatohepatitis.

3. Use of a reagent for HDL-C and ApoE-rich HDL-C measurement in the manufacture of a kit for aiding in the detection and determination of non-alcoholic steatohepatitis, which reagent is used to measure the amount of HDL-C and ApoE-rich HDL-C contained in a blood sample taken from a subject isolated from a living organism, and the ratio of ApoE-rich HDL-C / HDL-C in the above-mentioned blood sample taken from a subject is used as an index to determine the likelihood of the onset of non-alcoholic steatohepatitis.

4. The use of claim 3, wherein, The ratio of ApoE-rich HDL-C / HDL-C is lower than the ratio of ApoE-rich HDL-C / HDL-C in a blood sample from a patient with non-alcoholic fatty liver, indicating a high likelihood of the onset of non-alcoholic steatohepatitis.

5. Use of a reagent for LDL-TG and LDL-C measurement in the manufacture of a kit for aiding in the detection and determination of non-alcoholic steatohepatitis, which reagent is used to measure the amount of LDL-TG and LDL-C contained in a blood sample taken from a subject isolated from a living organism, and the ratio of LDL-TG / LDL-C in the above-mentioned blood sample taken from a subject is used as an index to determine the likelihood of the onset of non-alcoholic steatohepatitis.

6. The use of claim 5, wherein, The ratio of LDL-TG / LDL-C is higher than the ratio of LDL-TG / LDL-C in a blood sample from a patient with non-alcoholic fatty liver, indicating a high likelihood of the onset of non-alcoholic steatohepatitis.

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