阅读说明：本技术 有关非酒精性脂肪性肝病的组织学严重程度诊断或预后测量的信息提供方法 (Method for providing information on histological severity diagnosis or prognostic measurement of non-alcoholic fatty liver disease ) 是由 金元 于 2019-10-24 设计创作，主要内容包括：本发明涉及有关非酒精性脂肪性肝病的组织学严重程度诊断或预后测量的信息提供方法,当鞘磷脂的含量为正常对照组的1.3倍以上时,可以认为与非酒精性脂肪性肝病的严重程度相关的危险度增加,从而可将此有效地用于严重程度诊断或预后测量。(The present invention relates to a method for providing information on the histological severity diagnosis or prognostic measurement of non-alcoholic fatty liver disease, and when the content of sphingomyelin is 1.3 times or more as high as that of a normal control group, it can be considered that the risk associated with the severity of non-alcoholic fatty liver disease increases, and thus this can be effectively used for the severity diagnosis or prognostic measurement.)

1. A method for providing information on the diagnosis of histological severity or prognostic measurement of non-alcoholic fatty liver disease, characterized by comprising a measurement step of measuring the sphingomyelin content in a biological sample isolated from a specimen.

2. The method of claim 1, wherein the sphingomyelin is saturated sphingomyelin.

3. The method according to claim 2, further comprising a risk assessment step of assessing that the risk associated with the severity of the non-alcoholic fatty liver disease in the specimen is increased if the content of the saturated sphingomyelin in the measurement step is 1.3 times or more as high as that in the normal control sample.

4. The method of claim 1, wherein the body mass index of the specimen is less than 25.

5. The method of claim 1, wherein the specimen is asian.

6. The method of claim 1, wherein the biological sample is serum or plasma.

Technical Field

The present invention was made under the support of korea department of health and welfare, and was carried out under the heading No. 1465025699, where the special institution of research and management was the happy institute of the korean health care industry, the title of the research project was "development of disease-overcoming technology", the title of the research subject was "analysis of microbiome and serum metabolites in feces based on the opinion of the liver disease organization of large-scale korean non-alcoholic fatty liver transformed population", the competent institution was the Boramae hospital of korea special city, and the research period was 2018, 01/2018, 12/31/2018.

The present application claims priority from korean patent application No. 10-2018-0129190, which was filed in korean patent office at 26.10.2018, the disclosure of which is incorporated herein by reference.

The present invention relates to a method for providing information on histological severity diagnosis or prognostic measurement of non-alcoholic fatty liver disease, and more particularly, to the following method: whether the risk associated with the severity of non-alcoholic fatty liver disease is increased is confirmed by measuring the content of saturated sphingomyelin.

Background

NAFLD (nonalcoholic fatty liver disease) is caused by abnormal accumulation of hepatic Triglycerides (TG), known as hepatic steatosis, and sometimes develops into fibrosis associated with NASH (nonalcoholic steatohepatitis). Although excessive accumulation of hepatic triglycerides is known to cause non-alcoholic fatty liver disease, the effects of lipid changes on the specific type, number of lipid classes accumulated in fatty liver and the progression of non-alcoholic fatty liver disease have not been fully characterized. Since most lipids have important biological activities, the lipidological proximity method provides useful information that is helpful in understanding the cause of non-alcoholic fatty liver disease.

Obesity and insulin resistance are major risk factors for metabolic syndromes such as non-alcoholic fatty liver disease (NAFLD) and type II diabetes. However, a significant number of non-alcoholic fatty liver disease patients have a Body Mass Index (BMI) < 25kg/m²Exhibit a lower body mass index and maintain lower insulin resistance. Since non-alcoholic fatty liver disease is closely related to obesity, previous metabonomics research focuses on body mass index of more than or equal to 30kg/m²The obese adult male of (1).

However, the overall mortality rate of lean non-alcoholic fatty liver patients with a healthier metabolic profile is still increased compared to non-alcoholic fatty liver patients of overweight or obese people. Therefore, further knowledge of metabolic differences in non-obese and obese patients with non-alcoholic fatty liver disease is crucial to the development of sophisticated medical and customized therapies based on the variety of phenotypes and the progressive content of non-alcoholic fatty liver disease.

Although it is known to propose a body mass index < 30kg/m²The study of the blood metabolite profiles of non-obese non-alcoholic fatty liver disease patients of western people, but the human health organization Expert Consultation (WHO Expert consensus) suggested that a reference value different from the international classification should be used for the body mass index (WHO BMI) of asian people.

Although non-obesity (body mass index ≦ 25 kg/m) was found²) Is suitable for obesity (body mass index is greater than or equal to 30 kg/m)²) There is a clear difference between serum metabolites, but there is a need to further confirm the histologically confirmed non-alcoholic fatty liver disease (nonalcoholic fatty liver; NAFL) and nonalcoholic steatohepatitis.

Therefore, it is necessary to comprehensively investigate changes in blood lipid distribution according to the histological severity of non-alcoholic fatty liver disease using serum obtained from non-obese and obese sub-adult patients who have been demonstrated in biopsy results.

Disclosure of Invention

Technical problem

To this end, the present inventors confirmed that the histological severity can be diagnosed by measuring the Sphingomyelin (SM) content in the serum of a non-obese non-alcoholic fatty liver disease patient.

Accordingly, an object of the present invention is to provide a method for providing information on the diagnosis of histological severity or prognostic measurement of non-alcoholic fatty liver disease, comprising a measurement step of measuring the sphingomyelin content in a biological sample isolated from a specimen.

Another object of the invention relates to the use of information on the histological severity diagnosis or prognostic measurement of non-alcoholic fatty liver disease for sphingomyelin measurements.

Technical scheme

The present invention relates to a method for providing information on the diagnosis of the histological severity or the prognostic measurement of non-alcoholic fatty liver disease, which can confirm whether the risk associated with the severity of non-alcoholic fatty liver disease is increased by measuring the sphingomyelin content in the serum of a patient.

The present inventors have derived that when the content of saturated sphingomyelin is 1.3 times or more higher than that of normal serum, this indicates an increase in the risk associated with the severity of non-alcoholic fatty liver disease.

Hereinafter, the present invention will be described in more detail.

One embodiment of the present invention is a method for providing information on the diagnosis of histological severity or prognostic measurement of non-alcoholic fatty liver disease, comprising a measurement step of measuring the content of sphingomyelin in a biological sample isolated from a specimen.

In the present invention, the sphingomyelin may be saturated sphingomyelin.

In the present invention, the method may further include a risk level determination step of determining that the risk level associated with the severity of the non-alcoholic fatty liver disease in the specimen is increased if the measured content of the saturated sphingomyelin is 1.3 times or more as high as that in the normal control sample.

In the present specification, a specimen is classified as non-obese when the body mass index of the specimen is less than 25, and is classified as obese when the body mass index of the specimen is 25 or more.

In the present invention, the body mass index (BM) of the specimen may be less than 25. This is because, in one example of the present invention, in the case of the non-obese group having a body mass index of less than 25, the specific saturated sphingomyelin plays a large role in the occurrence of non-alcoholic fatty liver disease and the progression to non-alcoholic steatohepatitis, and this phenomenon cannot be confirmed in the obese group having a body mass index of 25 or more.

In the present invention, the specimen may be an asian person. This is because, although it is known to study the blood metabolite profiles of western non-obese non-alcoholic fatty liver disease patients, the world health organization suggests that a reference value different from the international classification should be used for the reference value of the world health organization body mass index of asian.

In the present invention, "asian person" means a person originated from china, mongolia, taiwan region of china, singapore, korea, japan, vietnam, cambodia, laos, burma, thailand, malaysia, indonesia, and philippine native soil.

In the present invention, the biological sample may be serum or plasma.

In one example of the present invention, in serum isolated from a non-obese asian patient with a body mass index of less than 25, an increase in the risk associated with the severity of non-alcoholic fatty liver disease can be judged when the measured content of saturated sphingomyelin is increased more than 1.3-fold over that of a normal control sample.

ADVANTAGEOUS EFFECTS OF INVENTION

The present invention relates to a method for providing information on the histological severity diagnosis or prognostic measurement of non-alcoholic fatty liver disease, and when the content of sphingomyelin is 1.3 times or more as high as that of a normal control group, it can be considered that the risk associated with the severity of non-alcoholic fatty liver disease increases, and thus it can be effectively used for the severity diagnosis or prognostic measurement.

Drawings

FIG. 1a is a graph showing fold change in lipid class for non-alcoholic fatty liver disease (no-NAFLD)/NAFL in non-obese patients.

FIG. 1b is a graph showing the fold-change of lipid types in nonalcoholic steatohepatitis (NASH)/nonalcoholic fatty liver in a non-obese patient.

Figure 1c is a graph showing fold-change in lipid class for non-alcoholic fatty liver/non-alcoholic fatty liver disease in non-obese patients.

FIG. 1d is a graph showing fold change in lipid class for non-alcoholic steatohepatitis/non-alcoholic fatty liver disease in obese patients.

FIG. 2a is a graph showing the change in the content of Diglycerides (DAG) according to the length of an acyl chain (acyl chain) and the degree of unsaturation.

Fig. 2b is a graph showing the change in Triglyceride (TAG) content according to the length of the acyl chain and the degree of unsaturation.

Fig. 3 is a graph showing changes in sphingomyelin content between non-alcoholic fatty liver/non-alcoholic fatty liver disease and non-alcoholic steatohepatitis/non-alcoholic fatty liver disease in relation to the non-obese group and the obese group.

FIG. 4 is a thermal map of the Spearman correlation showing the correlation between sphingomyelin content and metabolic risk factors and the severity of hepatic histology.

Fig. 5a is a graph showing the content of saturated sphingomyelin at different levels of steatosis in the non-obese group.

Fig. 5b is a graph showing the content of saturated sphingomyelin at different leaflet inflammation levels in the non-obese group.

Fig. 5c is a graph showing the content of saturated sphingomyelin at different balloon-like varying levels in the non-obese group.

Fig. 5d is a graph showing the content of saturated sphingomyelin at different fibrosis levels in the non-obese group.

Fig. 6a is a graph showing the predicted likelihood of hepatic histology of steatosis using a combination of saturated sphingomyelins.

Figure 6b is a graph showing the predicted likelihood of liver histology of lobular inflammation using a combination of saturated sphingomyelins.

Fig. 6c is a graph showing the likelihood of a hepatic histology prediction using ballooning of a combination of saturated sphingomyelins.

FIG. 7 is a graph showing the ability to diagnose the histological severity of non-alcoholic fatty liver disease by the combination of sphingomyelin d36:0, sphingomyelin d38:0, and sphingomyelin d40:0 with aspartate transaminase (AST), alanine transaminase (ALT), and gamma-glutamyl transpeptidase (GGT) in the non-obese (blue curve) and obese (red curve) groups.

Detailed Description

The present invention relates to a method for providing information on the diagnosis of histological severity or prognostic measurement of non-alcoholic fatty liver disease, which comprises a measurement step of measuring the sphingomyelin content in a biological sample isolated from a specimen.

Modes for carrying out the invention

The present invention will be described in more detail below with reference to the following examples. These examples are merely illustrative of the present invention, and the scope of the present invention is not limited to these examples.

Example 1: basic characteristics of study participants ]

As shown in table 1 and table 2, subjects were classified into non-obese (< 25) and obese (≧ 25) (DM (diabetes mellitus), MS (metabolic syndrome), hsCRP (high sensitivity C-reactive protein), FPG (fasting plasma glucose), TC (total cholesterol), HDL-C (high density lipoprotein cholesterol)) according to body mass index.

TABLE 1

TABLE 2

From tables 1 and 2, it was confirmed that the contents of serum glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase and γ -glutamyltranspeptidase were increased in the non-obese group and the obese group in accordance with the histological severity of the non-alcoholic fatty liver disease.

As a result of biopsy, among 361 (mean age 53 ± 14 years, 48.5% for men), 295 was non-alcoholic fatty liver disease, and 66 was a control group (non-alcoholic fatty liver disease group) which was metabolically healthy. The non-alcoholic fatty liver disease group had no clinical, biochemical, radiological or histological evidence of fatty liver. Among the non-obese patients, 82 were non-alcoholic fatty liver disease, and 48 were control groups. Among the obese group, 213 were non-alcoholic fatty liver diseases, and 18 were control groups.

Based on body mass index and liver histology recommendations (non-alcoholic fatty liver disease, non-alcoholic fatty liver, and non-alcoholic non-fatty hepatitis), subjects were divided into the following six groups: non-obese/non-alcoholic fatty liver disease; non-obese/non-alcoholic fatty liver; non-obese/non-alcoholic non-steatohepatitis; obesity/non-alcoholic fatty liver disease; obese/non-alcoholic fatty liver and obese/non-alcoholic non-steatohepatitis. The more the histological severity of non-alcoholic fatty liver disease increases, the more the frequency of body mass index, waist circumference, visceral fat area (visceral fat mass), adipose tissue insulin resistance index, glycated hemoglobin, fasting plasma glucose, triglycerides (neutral fat), high density lipoprotein cholesterol, diabetes incidence and metabolic syndrome increases in the non-obese group and the obese group (table 1).

Example 2: serum lipid mass spectrum based on ultra-high performance liquid chromatography and tandem quadrupole time-of-flight mass spectrometer (UPLC/Q TOF-MS) ]

Will be confirmed by biopsy as non-alcoholic fatty liver disease and body mass index less than 25kg/m²The subject of (1) (non-alcoholic fatty liver patient population of Boramae hospital (NCT 02206841)) was defined as a non-obese non-alcoholic fatty liver disease subject. For solid liver masses suspected of being hepatoadenoma or focal nodular hyperplasia by evaluating liver biopsies during liver transplantation from live donors according to abdominal imagingCharacteristic analysis was performed to determine that the control group had no non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease is defined as the presence of 5% or more of vesicular steatosis as confirmed by histological examination. Non-alcoholic steatohepatitis is diagnosed on the basis of the histological basis of the NASH-CRN (clinical research network) of non-alcoholic steatohepatitis. The overall pattern of histological liver injury consisting of steatosis, lobular inflammation, ballooning, or fibrosis was graded according to a non-alcoholic fatty liver disease activity scoring system.

For the quantification of the amount of visceral fat, the visceral adipose tissue area (VAT) was measured. The systemic insulin resistance index (HOMA-IR) and the pancreatic islet beta cell function index (HOMA-beta) were evaluated using the homeostatic model (HOMA-B), and the adipose tissue insulin resistance index (adipo-IR) was calculated. Metabolic syndrome is defined according to the international cholesterol education program criteria of the 3 rd report on adult treatment group (NCEP-ATP III).

A global lipid profile was performed on 361 subjects' sera using ultra high performance liquid chromatography coupled with a tandem quadrupole time-of-flight mass spectrometer technique and 224 lipid metabolites were identified using various standard substances and online databases such as the Human Metabolome Database (HMDB, Human Metabolome Database, www.hmdb.ca), METLIN (METLIN. script. edu) and lipid metabolite and metabolic pathway research strategies (LIPID MAPS (www.lipidmaps.org)).

Sphingomyelin-grade solvents for High Performance Liquid Chromatography (HPLC) for ultra-High performance liquid chromatography combined with tandem quadrupole time-of-flight mass spectrometry (UPLC-MS) analysis were purchased from the Seimer Feishell Scientific, Walser, Mass., USA. Ammonium acetate and lipid standards were purchased from Sigma-Aldrich (st. louis, missouri, usa).

The lipidology profile for all lipid grades includes: free Fatty Acids (FFA), glycerolipids (diglycerides and triglycerides), phospholipids (lysophosphatidic acid; LPA), Lysophosphatidylcholine (LPC), Lysophosphatidylethanolamine (LPE), Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylinositol (PI)), and sphingolipids (ceramide and sphingomyelin). The fold change (fold changes) of lipid types in non-alcoholic fatty liver/non-alcoholic fatty liver disease and non-alcoholic steatohepatitis/non-alcoholic fatty liver in non-obese and obese patients is shown in fig. 1.

As can be seen from fig. 1, in non-obese and obese adult non-alcoholic fatty liver patients, several lipid types such as diglycerides, triglycerides, sphingomyelin show characteristic patterns of change depending on the histological severity of non-alcoholic fatty liver disease.

In particular, although the blood glycerolipid content (concentration) ratio between non-alcoholic fatty liver disease and normal liver (non-alcoholic fatty liver disease) was significantly higher in the obese group than in the non-obese group, there was no significant difference in the blood glycerolipid content ratio between non-alcoholic steatohepatitis and non-alcoholic fatty liver disease. In contrast, in the case of a fraction of specific saturated sphingomyelin, the ratio of the amount of sphingomyelin in blood of non-alcoholic fatty liver to that of normal liver (non-alcoholic fatty liver disease) and the ratio of sphingomyelin in non-alcoholic steatohepatitis to that of non-alcoholic fatty liver were significantly increased only in the non-obese group, while showing no statistical difference in the obese group.

From this, it was confirmed that in the obesity group, glycerolipids in blood play an important role in the occurrence of non-alcoholic fatty liver disease, but do not play a significant role in the progression to non-alcoholic steatohepatitis, or, on the contrary, play a preventive protective role in the case of triglycerides.

In contrast, in the non-obese group, saturated sphingomyelin plays a great role not only in the development of non-alcoholic fatty liver disease but also in the progression to non-alcoholic steatohepatitis, but this phenomenon was not confirmed in the obese group.

Example 3: changes in diglycerides and triglycerides based on the severity of non-alcoholic fatty liver disease ]

50 μ L of each serum sample was mixed with 500 μ L of chloroform/methanol (2: 1, v/v). After centrifugation at 13000rpm for 20 minutes at 4 ℃ 300. mu.L of the lower lipid phase were collected and the solvent was removed under a gentle stream of nitrogen at room temperature. The dried extract was reconstituted using 250. mu.L of an isopropanol/acetonitrile/water mixture (2: 1, v/v/v). After centrifugation at 13000rpm for 10 minutes at 4 ℃ for lipid analysis, high performance liquid chromatography (ACQUITY) in combination with a quadrupole time-of-flight high resolution mass spectrometer (QTOF-MS) was used^TMUPLC system, Waters corporation, manchester, uk) transferred the supernatant to a vial and was measured using a quadrupole time-of-flight high resolution mass spectrometer.

The column oven and the autosampler were maintained at 40 ℃ and 4 ℃ respectively. The samples were eluted and separated using an Acquity UPLC BEH C18 chromatography column (2.1 μm × 1.7mm particles, Waters). The Liquid Chromatography (LC) mobile phase consisted of adding 10mM ammonium acetate solvent (solvent A) to an acetonitrile/water mixture (4: 6, v/v) and 10mM ammonium acetate solvent (solvent B) to an acetonitrile/isopropanol mixture (1: 9, v/v). Gradient elution started at 40% B, increased to 65% B after 5 minutes, and then continued until the B component reached 99% after 10 minutes.

The composition was kept in 99% B for 2 minutes. After 17 minutes, the reaction solution was returned to the initial state and kept for 3 minutes until the state became an equilibrium state. The flow rate was set at 350. mu.L/min. 5 microliters of lipid extract was injected into a high performance liquid chromatography/quadrupole time-of-flight high resolution mass spectrometer system.

All samples were collected in the same amount to generate Quality Control (QC) samples. To evaluate analytical reproducibility, solvent voids and injections of quality control samples were performed between 12 samples. All cations and anions were applied to each sample using a Triple TOF 5600(AB Sciex, Concord, canada) equipped with a hybrid quadrupole time-of-flight tandem mass spectrometry (QTOF) device and a DuoSpray ion source. The mass range is set to m/z 50-1500.

The following parameters were used for operation: the ion spray voltage was 5500V, the ion source temperature was 500 ℃, the nebulizer pressure was 50psi, the dry gas pressure was 60psi, the air curtain gas was 30psi, the declustering voltage was 90V, and Information-dependent acquisition (IDA) was used in order to obtain MS/MS spectra of the ions concerned.

To obtain MS/MS spectra, the collision energy and collision energy spread were adjusted to 40V and 15V, respectively. Quality accuracy was maintained by the DuoSpay ion source and an interfaced auto-calibrated delivery system (AB Sciex, Inc.). Lipid metabolites were confirmed from online databases (DB, HMDB, METLIN, LIPID MAPS) and available information (correct mass, fragment ions and/or retention time) that could be consistent with data for standard compounds.

A significant increase in glycerolipids including the diglyceride and triglyceride classes was observed in both the non-obese and obese groups, with a gradual increase from non-alcoholic fatty liver to non-alcoholic non-fatty hepatitis. The difference in acyl chain length, degree of unsaturation, fold change, and diglyceride and triglyceride content with p-value was visualized using a bubble chart.

Bubble plots show the relationship between acyl chain length, degree of unsaturation, fold change, and p-value for both diglycerides and triglycerides by comparing the histological subclasses. The y-axis indicates the degree of unsaturation and the position of the bubble relative to the x-axis corresponds to the length of the acyl chain. The color of the bubbles indicates the fold change, while the size of the bubbles indicates the p-value obtained in the Mann-Whitney U-test using bonferoni (Bonferroni) calibration.

From FIG. 2a, it can be confirmed that the change in diglyceride content shows a distinctive pattern between the various histological subgroups. Independently of obesity, the content of diglycerides having relatively short chains and low unsaturation degree is statistically increased in non-alcoholic fatty liver/non-alcoholic fatty liver disease. As a control, in non-alcoholic steatohepatitis/non-alcoholic fatty liver disease in the obese group, the content of diglycerides having long chains and a high degree of saturation was significantly reduced.

From fig. 2b, it can be confirmed that the change in the triglyceride content shows a similar tendency to the change in the diglyceride content. Although the statistical significance and fold change was reduced, the chain length and saturation varied slightly.

Glycerolipids, including both di-and tri-glyceride species, exhibit a characteristic pattern of change that is influenced by the length of the acyl chain and the degree of unsaturation. Also, the pattern of change in glycerolipids based on histological severity was not different between the non-obese group and the obese group. Finally, the rate of circulating diglycerides and triglycerides, classified according to histological severity, varies greatly in obese non-alcoholic fatty liver patients compared to non-obese non-alcoholic fatty liver patients.

In particular, although the blood glycerolipid content (concentration) ratio between non-alcoholic fatty liver disease and normal liver (non-alcoholic fatty liver disease) was significantly higher in the obese group than in the non-obese group, there was no significant difference in the blood glycerolipid content ratio between non-alcoholic steatohepatitis and non-alcoholic fatty liver disease.

From this, it was confirmed that glycerolipids play an important role in the occurrence of non-alcoholic fatty liver disease in the non-obese group, but do not play a significant role in the development of non-alcoholic steatohepatitis, or, in the case of triglycerides, which are biologically inactive substances, are harmless lipids that play a preventive protective role.

Example 4: correlation of changes in metabolic syndrome due to severity of non-alcoholic fatty liver disease with liver tissue and histology ]

The difference in the intensity of metabolic syndrome according to the histological severity of non-alcoholic fatty liver disease was shown in both the non-obese group and the obese group. Significant differences are indicated in the histograms by the number (Mann-Whitney U-test with appropriate bonpfoni (bonpnoni) correction), p < 0.05, p < 0.01 and p < 0.001).

As can be seen from fig. 3, interestingly, the content of saturated sphingomyelin including SM d36:0, SM d38:0, and SM d40:0 was significantly increased to 1.3 times or more in the non-alcoholic fatty liver/non-alcoholic fatty liver disease group of the non-obese group. As a control, in the non-alcoholic fatty liver/non-alcoholic fatty liver disease group, the content of sphingomyelin having a long chain of 42 carbon atoms or more was decreased regardless of obesity.

In the obese group, although the content of saturated sphingomyelin such as SM d34:0, SM d36:0, SM d38:0, and SM d40:0 was significantly increased in the non-alcoholic steatohepatitis/non-alcoholic fatty liver group, it was not significantly increased in the non-alcoholic steatohepatitis/non-alcoholic fatty liver disease group.

Differences in sphingomyelin content in saturated states associated with the severity of non-alcoholic fatty liver disease and obesity have also been identified in the correlation between metabolic risk factors and liver histology. Spearman correlation heatmaps show the correlation between metabolic syndrome risk factors or hepatic histology and sphingomyelin in non-obese and obese groups. Statistical significance is indicated by the numbers (VAT for visceral adipose tissue; HbA1c for glycated hemoglobin; adipose tissue-infrared for insulin resistance; HOMA-IR for an assessment of an homeostasis model for insulin resistance; HOMA-beta for an assessment of an homeostasis model for islet beta cell function; LI for lobular inflammation).

From fig. 4, it was confirmed that the contents of SM d36:0, SM d38:0, and SM d40:0 showed a significant amount correlation with the visceral adipose tissue region, the adipose tissue insulin resistance index, and the insulin resistance index in the non-obese group, and showed a significant amount correlation with the adipose tissue insulin resistance index and the insulin resistance index in the obese group. In the non-obese group in particular, the content of saturated sphingomyelin shows a strong correlation with the severity of steatosis, ballooning and lobular inflammation. However, in the obese group, only steatosis showed quantitative correlation with saturated fat concentration.

Then, it was confirmed whether the content of the saturated sphingomyelin species including SM d36:0, SM d38:0, and SM d40:0 was changed to the presence and severity of steatosis, lobular inflammation, and ballooning. In steatosis (A; 0-3), lobular inflammation (B; 0-3), balloon-like lesions (C; 0-2) and non-obese subjects, the intensity of SM D36:0, SM D38:0 and SM D40:0 was not fibrotic (D; 0-4). Data are presented as mean ± standard deviation. Significant differences are indicated by the number (J-T test (Jonckheere-Terpstra test),. p < 0.05,. p < 0.01 and. p < 0.001).

From fig. 5, it can be confirmed that, as shown in the heat map, the content of sphingomyelin in a saturated state is significantly increased particularly in non-obese patients according to the severity of steatosis, lobular inflammation, ballooning and fibrosis of each grade. In contrast, except for the cases where steatosis grade was measured, the saturated state sphingomyelin content of obese patients did not escalate in relation to the measurement of the histological severity of non-alcoholic fatty liver disease. Independently of obesity, the fibrosis stage is also not progressively associated with saturated sphingomyelin content.

The content of the saturated sphingomyelin is more useful for improving the diagnostic performance of the non-alcoholic fatty liver disease and the non-alcoholic steatohepatitis of the non-obese patient compared with the non-alcoholic fatty liver disease and the non-alcoholic steatohepatitis of the obese patient, and can prevent unnecessary examination, especially liver biopsy, of the non-obese adult non-alcoholic steatohepatitis patient.

Example 5: prediction of non-obese non-alcoholic fatty liver disease using circulating saturated sphingomyelin content

To predict the histological severity of non-alcoholic fatty liver disease by evaluating the diagnostic performance of the combination of the above-mentioned saturated sphingomyelin content (SM d36:0, SM d38:0 and SM d40:0), the area under the receiver operating characteristic curve (AUROC) with a 95% confidence interval (95% CI) was divided into a non-obese group and an obese group and is shown in table 3 and fig. 6.

TABLE 3

As can be seen from fig. 6a, in the non-obese group, the areas under the receiver operating characteristic curves of steatosis 1, steatosis 2, and steatosis 3(S1-S3) were 0.720, 0.768, and 0.804, respectively. In the obese group, the areas under the receiver operating characteristic curves of S1, S2, and S3 were 0.722, 0.656, and 0.733, respectively.

As can be seen from fig. 6b, in the case of lobular inflammation, the areas under the receiver operating characteristic curves of lobular inflammation were 0.613 (score 1) and 0.821 (score 2) in the non-obese group, and the areas under the receiver operating characteristic curves were 0.541 and 0.614 in the obese group.

From fig. 6c, it can be confirmed that, in the ballooning case, the areas under the receiver operating characteristic curve of leaflet inflammation were 0.709 (fraction 1) and 0.784 (fraction 2) in the non-obese group. In the obese group, the receiver operation characteristic curve for score 1 and score 2 had an area under the curve of 0.567 and 0.551, respectively.

Overall, the receiver operational characteristic curve based on the histological severity of the non-obese group exhibited a significantly higher area under the receiver operational characteristic curve than the obese group.

From fig. 7, it was confirmed that in order to effectively distinguish subjects having nonalcoholic steatohepatitis or nonalcoholic fatty liver disease among patients having no nonalcoholic steatohepatitis, the area under the receiver operation characteristic curve was adjusted to the combination of the contents of glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, and γ -glutamyl transpeptidase (model 1) or the contents of saturated sphingomyelin (SM d36:0, SM d38:0, and SM d40:0) and the contents of glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, and γ -glutamyl transpeptidase (model 2). The DeLong test was used to provide a p-value for a paired (pairwise) comparison of the area under the receiver operating characteristic curve.

TABLE 4

As can be seen from table 4, the areas under the receiver operation characteristic curves of model 1 and model 2, which compare non-alcoholic fatty liver disease with non-alcoholic fatty liver disease, were 0.720 and 0.833 in the non-obese group (when the areas under the receiver operation characteristic curves are compared, p is 0.011). When serum saturated sphingomyelin is added to the combination of serum glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase and gamma-glutamyl transpeptidase, the diagnosis performance for distinguishing nonalcoholic fatty liver disease from nonalcoholic steatohepatitis in a non-obese group is obviously improved.

The areas under the receiver operating characteristic curves for model 1 and model 2 comparing non-alcoholic steatohepatitis to non-alcoholic steatohepatitis were 0.823 and 0.914 (when comparing the areas under the receiver operating characteristic curves, p is 0.033). In the obese group, the areas under the receiver operating characteristic curves for model 1 and model 2, which compared non-alcoholic fatty liver disease to non-alcoholic fatty liver disease, were 0.785 and 0.781 (when comparing the areas under the receiver operating characteristic curves, p is 0.866). The areas under the receiver operating characteristic curves of model 1 and model 2, which compare non-alcoholic steatohepatitis with non-alcoholic steatohepatitis, were 0.928 and 0.960 (when the areas under the receiver operating characteristic curves were compared, p ═ 0.059)

[ industrial applicability ]

The present invention relates to a method for providing information on histological severity diagnosis or prognostic measurement of non-alcoholic fatty liver disease, and more particularly, to the following method: whether the risk associated with the severity of non-alcoholic fatty liver disease is increased is confirmed by measuring the content of saturated sphingomyelin.