RU2768466C1 - Method for prediction of progressive clinical course of early forms of non-alcoholic fatty liver disease - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to hepatology. TNF-α, interleukin-6, cytokeratin-18, alkaline phosphatase, gamma-glutamyl transpeptidase, glucose and fasting insulin levels are determined in peripheral blood in vitro. Further, the following syndromes are assessed: systemic inflammation, hepatocyte apoptosis, intrahepatic cholestasis and insulin resistance. In the presence of any two or more of the evaluated syndromes, the risk of progression of early forms of non-alcoholic fatty liver disease (NAFLD) is predicted, in the presence of only one of the analyzed syndromes or in the absence of each of them, the absence of a risk of progression of non-alcoholic fatty liver disease is determined.

EFFECT: invention makes it possible to simplify and reduce economic costs for assessment of NAFLD course prognosis, to improve quality of diagnostics, reducing the number of personnel involved for examination, justifying the use of a more active approach in patients with early forms of NAFLD with a high risk of progression.

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Description

Purpose of the invention.

The method relates to medicine, namely to the field of hepatology, and is intended for screening detection of patients with early forms of non-alcoholic fatty liver disease (NAFLD) who have signs of a progressive course of the disease with a likelihood of transformation into active hepatitis and liver cirrhosis.

The method is planned to be used among the adult population - outpatients and inpatients, patients of dispensaries, outpatient clinics, clients of private clinics and laboratories.

The level of world technology.

From the level of world technology known method of differential diagnosis of steatohepatitis of alcoholic and non-alcoholic etiology [1]. This method consists in determining the concentration of uroporphyrin (UP) and coproporphyrin (CP) in the urine, as well as the content of CP and protoporphyrin (PP) in the feces in a patient with an established diagnosis of "steatohepatitis". Depending on the combination of their values, alcoholic steatohepatitis (ASH) or non-alcoholic steatohepatitis (NASH) is diagnosed. Using the method allows to differentiate steatohepatitis of alcoholic and non-alcoholic etiology. The disadvantage of this method is that as a result only the etiology of steatohepatitis is established, but no conclusions can be drawn about the prognosis and recommendations for the management of patients.

A known method for the diagnosis of non-alcoholic steatohepatitis, which allows to detect metabolic changes in the body, including the study of the patient's saliva, characterized in that a drop of crystal-forming substance - 3% alcohol solution of copper chloride is added to the drop of the patient's saliva, the resulting preparation is kept for 24 hours in a horizontal position at a temperature 20-25 ° C, humidity 50-70%, away from direct sunlight and heating devices and examined under a microscope, while the presence and severity of non-alcoholic steatohepatitis is determined by the presence and amount of amorphous dark yellow masses in the microscopic preparation superimposed on crystals in the form “panicles” or “bunches”, and the absence of non-alcoholic steatohepatitis is determined by the presence of crystals in the form of “panicles” or “bunches” in a microscopic preparation without imposing amorphous dark yellow masses [2]. The disadvantage of this method is that it requires specific reagents and a microscope, a study with the participation of specially trained personnel, and the result of the study can be obtained no earlier than a day later.

From the level of world technology, a method for the differential diagnosis of liver steatosis and steatohepatitis by biochemical research is known, characterized in that phospholipase A2, nitric oxide and endotoxin are determined, and with a value of phospholipase A2 of 199.7-252.5 ng/ml, nitric oxide 93-94, 6 nmol/l and endotoxin 2.2-2.6 EU/ml diagnose liver steatosis, and with a value of phospholipase A2 412.5-576.5 ng/ml, nitric oxide 137.5-168.5 nmol/l and endotoxin 3.32-4.18 EU/ml diagnose steatohepatitis [3]. The disadvantage of this method is the need to study three specific laboratory parameters that are not used in routine healthcare.

A known method of ultrasound diagnosis of non-alcoholic fatty liver disease, which consists in the fact that during ultrasound scanning of the liver, a fixed image is obtained - a transverse scan of the liver, including an image of the hepatic veins so that longitudinally oriented hepatic veins, their mouths and the inferior vena cava are visualized on one section. the level of confluence of the hepatic veins into it, post-processor image processing is used, zones of interest (ROI) are identified on the echogram in the area of the liver parenchyma (ROIp) and in the area of the lumen of the inferior vena cava without capturing the vessel wall (ROInv), histograms of the distribution of brightness levels for quantitative assessment of echogenicity are obtained , determine the median of the distribution on the obtained histograms of brightness levels, calculate the hepatic venous index (HVI) as the ratio of the median of the liver echo intensity to the median of the echo signal intensity in the lumen of the inferior vena cava and with PVI values of 3.64, as well as with the exception of the patient p by collecting an anamnesis of factors indicating the presence of hepatitis of various etiologies, alcohol consumption (in quantities of more than 30 g / day. for men and more than 20 g / day. for women) diagnose the presence of NAFLD [4]. The disadvantage of this is the need for a specific instrumental study with additional software with the participation of a specially trained narrow specialist.

From the level of world technology known method of non-invasive diagnosis of non-alcoholic fatty liver disease in obese children [5]. This method consists in the fact that obese children undergo ultrasound of the organs of the hepatobiliary system and polyhepatography. The data of changes in liver echogenicity, depletion of the vascular pattern, hemodynamic disturbances are analyzed, the body mass index and the duration of the disease are assessed, after which the presence/absence of the disease is diagnosed according to the original calculation formula. The incidence rate D0 is determined. If D0>0, the presence of non-alcoholic fatty liver disease is diagnosed. EFFECT: method provides accurate and early diagnosis of NAFLD in obese children due to simultaneous assessment of changes in liver echogenicity, depletion of the vascular pattern, hemodynamic disorders, body mass index and disease duration. The disadvantages of this method are the development only for pediatric patients, the need for a combined specific instrumental study using an ultrasound machine, with the participation of a specially trained specialist. It indicates only the presence of structural changes in the liver without differentiation of inflammation and fibrosis.

A known method for determining mitochondrial dysfunction in patients with non-alcoholic fatty liver disease [6]. This method consists in a clinical and laboratory study in which DNA is isolated from venous blood leukocytes of a patient with NAFLD, followed by a polymerase chain reaction using standard primer pairs, followed by determining the length of the terminal sections of chromosomes of peripheral blood leukocytes, and with the obtained values of this indicator higher reference determine mitochondrial dysfunction in patients with NAFLD, in particular to hepatology and clinical laboratory diagnostics. The disadvantages of this method are the need for high-tech laboratory research using expensive equipment and the participation of specially trained personnel, which is not available in primary health care.

From the level of world technology known method for determining atherosclerotic lesions of the vascular wall in patients with abdominal obesity and non-alcoholic fatty liver disease [7]. When using this method, diagnostics of pathological changes in the liver is carried out - determination of the value of 10 Steatoscreen markers. At the same time, the average thickness of the intima-media of the carotid artery in a patient is determined by the formula Y=0.64+0.34 Steatoscreen, and if Y≥0.86 mm, then early signs of atherosclerosis are determined in the patient. Also proposed is a diagnostic method using the peak systolic blood flow velocity in the internal carotid artery in a patient. EFFECT: group of inventions makes it possible to diagnose liver pathology and the development of atherosclerotic lesions of the vascular wall without the use of invasive techniques. The disadvantages of this method are the need to perform instrumental research using special equipment and highly specialized trained medical personnel.

There is a known method for diagnosing liver damage in atherogenic dyslipidemia by biochemical blood tests, characterized in that they additionally conduct a morphological study of liver biopsy specimens and determine the presence of anisokaryosis of hepatocytes, and when the content of hepatocytes with vacuoles is 33% or more, the presence of hepatocyte necrosis, intralobular infiltrates and fibrosis is judged on activity of chronic hepatitis [8]. The disadvantage of this is the need for an invasive study, namely a biopsy of the liver tissue, which is performed strictly in a round-the-clock hospital, requires the participation of highly qualified medical personnel, has a number of potentially dangerous complications, is economically costly and it is impossible to perform this study repeatedly to assess the dynamics of the pathological process. , moreover, it depends on the place where the biopsy material is taken, which may not reflect the pathological process in the liver as a whole.

From the level of world technology known method for predicting the outcome of hepatitis in cirrhosis [9]. With this method, contrast-enhanced ultrasound is performed. There are 4 criteria by analyzing the quantitative and qualitative parameters of contrast-enhanced ultrasound: 1) an increase in the time of onset of the porto-venous phase by 20% or more of the norm; 2) an increase in the transit of the contrast agent by 15% or more from normal values; 3) the presence of heterogeneity in the excretion of the contrast agent in the IV-V-VI-VII segments of the liver in the delayed phase at 4-6 minutes with the formation of a focal pattern; 4) the presence of deformation of segmental and intrasegmental vessels in the form of a tortuous structure in the arterial phase. In the presence of 2 or more criteria, an unfavorable outcome is predicted. If there is only 1 criterion, it is defined as an unreliable result, which requires further observation. In the absence of these criteria, a favorable outcome is predicted. The disadvantages of this method are the need for instrumental research using specialized equipment and the participation of a specially trained specialist.

From the level of world technology there is a method for assessing the severity of the course of the pathological process in chronic diffuse liver lesions [10]. When using this method, the levels of liver-specific enzymes (PSF) are determined in blood plasma: urokinase (UK), histidase (GIS), fructose-1-phosphate aldolase (F-1-F), serine dehydrotase (L-SD), threonine dehydratase (L -TD). Products of lipid peroxidation: diene conjugates (DC), malondialdehyde (MDA). The state of parameters of nonspecific immunity is determined: immunoregulatory index (IRI) as the ratio of T-helpers and T-suppressors, circulating immune complexes (CIC). Additionally, the state of regional blood flow is assessed using rheohepatography (RGG), a microhemocirculation system using conjunctival biomicroscopy (CB) with the determination of the intravascular index (CI). In the case of an increase in the level of UC, GIS up to 0.5 μmol / ml / h, F-1-F, L-SD, L-TD, lipid peroxidation products, CEC up to 1.5 times, an increase in IRI up to 2 at a rate of 1.0 -1.5, changes in indicators of regional blood flow, an increase in intravascular CI up to 2 points at a rate of not more than 1 point, a mild degree of the process is diagnosed. With an increase in the level of UA, GIS up to 0.75 µmol/ml/h, F-1-F, L-SD, L-TD, lipid peroxidation products, CEC by 1.5-2 times, an increase in IRI up to 2.5, a change indicators of regional blood flow, an increase in intravascular CI up to 3-4 points, the average degree of the process is diagnosed. With an increase in the level of UA, GIS over 0.75 µmol/ml/h, P-1-P, L-SD, L-TD, lipid peroxidation products, CEC by 2 or more times, an increase in IRI over 2.5, a change in regional blood flow, an increase in intravascular CI up to 5 points or more points, a severe degree of the process is diagnosed. This method is closest to the defined parameters to the proposed method, and was taken as a prototype. This method for assessing the severity of the course of the pathological process in chronic diffuse liver diseases is pathogenetically justified. Unlike existing analogues, it allows assessing the severity of "breakdown" at the organ level by determining liver-specific enzymes, the intensity of lipid peroxidation and correlating the results with disorders that have arisen in the circulatory and immune systems during the development of chronic diffuse liver diseases. However, when using this method, it is necessary to determine a large number of specific laboratory markers (7 indicators) that are not determined in practical health care institutions, as well as additional instrumental studies - rheohepatography and conjunctival biomicroscopy, which also requires additional special equipment and trained medical personnel.

Disclosure of the invention.

The technical result of the proposed method consists in simplifying the assessment of the prognosis in early forms of NAFLD, reducing the economic costs of assessing the prognosis of the course of NAFLD, improving the quality of diagnosis, and reducing the number of personnel involved in the examination. In addition, this method allows to justify the use of more active tactics, including medication, in patients with early forms of NAFLD with a high risk of progression, even in the absence of cytolysis syndrome. Also, this method is unified, that is, it can be used by any medical institution with the possibility of enzyme immunoassay and does not require the involvement of narrow specialists, instrumental research methods. The proposed method is non-invasive and can be performed multiple times to assess changes in risk over time in the same patient.

The technical result is achieved due to the fact that a venous blood sample is taken from the patient, in which the levels of TNF-α, interleukin-6, cytokeratin-18, alkaline phosphatase, gamma-glutamyl transpeptidase, glucose and insulin are determined on an empty stomach, characterized in that patients are assessed for the presence of the following syndromes : systemic inflammation, hepatocyte apoptosis, intrahepatic cholestasis, and insulin resistance; while systemic inflammation is diagnosed by an increase in the following parameters above the upper limit of normal: TNF-α more than 5 pg/ml and interleukin-6 more than 3.5 pg/ml; apoptosis of hepatocytes is detected with an increase in the level of cytokeratin-18 more than 100 U/l; intrahepatic cholestasis is determined by an increase in the following parameters above the upper limit of normal: alkaline phosphatase more than 270 U / l and / or gamma-glutamyl transpeptidase more than 32 U / l in women and 50 U / l in men; insulin resistance - with an increase in the HOMA-IR index value of more than 2.6, when this index is calculated using the level of fasting insulin and glucose according to the following formula: [fasting glucose (mmol / l) x fasting insulin (mcU / ml) / 22.5] ; and in the presence of any two or more of the evaluated syndromes, the risk of progression of early forms of non-alcoholic fatty liver disease is predicted, in the presence of only one of the studied syndromes or in the absence of each of them, the absence of the risk of progression of non-alcoholic fatty liver disease is determined.

Description of the implementation of the invention.

The proposed method includes the following operations: taking a sample of venous blood, conducting an enzyme immunoassay using appropriate kits to determine the levels of TNF-α, interleukin-6, cytokeratin-18, insulin, determining the levels of glucose, alkaline phosphatase, gamma-glutamyl transpeptidase using standard laboratory methods, calculating the HOMA index -IR, counting the number of parameters (out of all determined) that this particular patient has, forming, based on the parameters obtained, a conclusion about the presence of a high or unlikely risk of progression of early forms of NAFLD. This method allows examining large groups of people of different age groups (from 18 to 70 years old), therefore, a method is needed that would allow, in a short time and without additional labor and economic resources, to determine the circle of those patients who are at risk of NAFLD progression to overt hepatitis and cirrhosis of the liver. A method for predicting the progressive course of NAFLD by assessing laboratory parameters has been proposed, which has the following advantages:

• The result can be obtained within a few hours.

• The method can be used on a large number of patients.

• Evaluates immediately all the main links in the pathogenesis of NAFLD (apoptosis of liver cells, inflammation, cholestasis, insulin resistance).

• The method does not require additional prior training.

• No contraindications for the method.

• The method can be used both for patients with liver steatosis and for patients with low activity steatohepatitis, that is, regardless of the presence of cytolysis syndrome.

• No instrumental studies are required.

• A conclusion on the result of the method can be made by a primary care physician, so the involvement of narrow specialists is not required.

As patients to whom this method is applicable, adults with early forms of NAFLD - hepatic steatosis and steatohepatitis - patients, i.e. with obesity, which is established in the presence of BMI ≥30 kg / m ² , proatherogenic dyslipidemia (in the absence of drug correction lipid profile), a diffuse increase in liver echogenicity according to abdominal ultrasound, in the absence of other causes of liver damage. The proposed method includes the assessment of biomarkers of the main pathophysiological processes in NAFLD: inflammation, hepatocyte apoptosis, cholestasis and insulin resistance, which allows a comprehensive assessment of the existing risk of NAFLD progression.

Characteristics of the studied laboratory markers of the progression of early forms of NAFLD.

TNF-α and IL-6. In obesity, excessive accumulation of lipids in adipose tissue and liver is accompanied by neoangiogenesis and damage to hypertrophied cells - adipocytes and hepatocytes. As a result, activated macrophages migrate to them, secrete cytokines and initially activate delayed-type immune reactions - systemic inflammation develops, which is one of the mechanisms of NAFLD progression [11, 12, 13, 14, 15]. Expression of TNF-α and IL-6 significantly increases in adipose tissue and liver in patients with steatohepatitis compared with healthy individuals [16, 17, 18, 19]. The level of these cytokines correlates with the degree of fibrosis, dyslipidemia, markers of cytolysis and cholestasis [20, 21, 22, 23, 24, 25, 26, 27, 28]. In FH, the level of IL-6 is closely associated with indicators of abdominal obesity and IR, as well as with the degree of inflammation and fibrosis [29, 30], the level of ACT, highly sensitive CRP [31, 32]. TNF-α leads to insulin resistance [33, 17, 34]. TNF-α increases the resistance of cells to the action of insulin intracellularly by activating stress proteases [23]. This is confirmed by data on the positive correlation of TNF-α with the HOMA index [35; 36]. TNF-α is a ligand to cell death receptors, which initiates apoptosis via an external pathway [37, 38, 39]. TNF-α can also activate the internal pathway of hepatocyte apoptosis by shifting the equilibrium in favor of proapoptotic proteins [40, 41].

Cytokeratin-18. This marker is the main protein of intermediate filaments of cells (including hepatocytes) and appears in the blood when cells are at the final stage of apoptosis, when the cell cytoskeleton is destroyed [42, 43]. A direct relationship has been confirmed between the serum level of CK-18 and the histological sign of apoptosis - Mallory bodies [44]. Uptake of apoptotic bodies by hepatic stellate cells and myofibroblasts increases the production of type 1 collagen, transforming growth factor-β1; nucleotides contained in apoptotic bodies additionally stimulate collagen production through special stellate cell receptors, leading to fibrosis, the association with which CK-18 has been confirmed by clinical data [42, 45, 46, 47, 41, 48]. A relationship between apoptosis and cytolysis, neoangiogenesis, TNF-α, IR, dyslipidemia, and intrahepatic cholestasis has also been found [29, 49, 50, 51, 52, 53, 54, 55, 56, 57].

Alkaline phosphatase and gamma-glutamyl transpeptidase.

An increase in the level of GGTP occurs as a protective mechanism in response to the development of IR due to the antioxidant activity of this enzyme [58, 59]. Also, these enzymes are markers of intrahepatic cholestasis, that is, pathological accumulation of bile acids in the liver and their toxic effect on hepatocytes [60]. According to the literature, an increase in the pool of hydrophobic toxic bile acids is observed in NAFLD [11, 61, 62, 63]. With an increase in the concentration of fatty acids in hepatocytes, the autoregulation of the farnesoid X receptor is disrupted, and there is no activation of protective mechanisms to suppress the resynthesis of hydrophobic bile acids, which mediates the hepatocyte mechanism of biliotoxicity in cholestasis [64]. In addition, in the absence of farnesoid X receptor activation, insulin sensitivity worsens due to a decrease in the production of type 1 glucagon-like peptide by enteroendocrine cells [65, 66]. In patients with NAFLD, bile acids, alkaline phosphatase, and GGTP were found to be closely correlated with insulin, glucose, and HOMA-IR levels [67, 69, 69, 70], fibrosis levels [39, 64, 67, 70, 71], and hepatic cell apoptosis. [29, 72, 73].

Insulin resistance and the HOMA-IR index. In obesity, visceral adipose tissue is infiltrated by macrophages that secrete TNF-α [74]. It is this cytokine and an excess of FFA that lead to disruption of the insulin receptor and the development of IR [75, 76, 77]. FFA metabolites, diacylglycerols (DAG) also negatively affect the insulin signaling pathway, disrupting the type 2 insulin receptor, resulting in increased gluconeogenesis, decreased glycogen synthesis in the liver, increased insulin production by the pancreas in response to hyperglycemia, and hyperinsulinemia stimulates lipogenesis [78, 79, 80, 81, 82]. The HOMA index reflects the presence of systemic IR. A close association of the IR index in NAFLD with fibrosis, degree of obesity, IL-6, TNF-α, cytolysis, cholestasis, and dyslipidemia has been revealed [46, 77, 83, 84, 85, 86, 87, 88, 89, 90].

Approbation of the developed method on NAFLD patients and healthy volunteers.

The described method was tested on 244 patients of the therapeutic department and polyclinic of the ChUZ KB RZD-Medicine at st. Petrozavodsk JSC Russian Railways.

Body mass index (BMI), waist circumference (WC), clinical blood count, standard liver function tests - ALAT, ASAT, bilirubin, alkaline phosphatase (APF), cholesterol, high density lipoproteins (HDL) and low density lipoproteins (LDL), triglycerides (TG), glucose, albumin using an automatic analyzer "Random Access F-15" ("BioSystems", Spain). All patients underwent abdominal ultrasonography (Vivid Pro-7, General Electric, USA). To assess systemic inflammation, tumor necrosis factor-alpha (TNF-α) was determined by enzyme immunoassay using commercial Human TNFα Platinum ELISA test systems (eBioscience, Austria). Fragments of CK-18 (FCK-18) were evaluated by enzyme immunoassay using the TPS ELISA test system (Biotech, Sweden). Blind percutaneous and laparoscopic targeted liver biopsy was performed in 19 (7.8%) patients. Histological parameters were assessed according to the Nonalcoholic fatty liver disease activity score (NAS) [91]. Fibrosis index NAFLD fibrosis score (NFS) was calculated: (age, BMI, presence of disorders of carbohydrate metabolism, ALT, ACT, albumin and platelet levels): < -1.455: predictor of the absence of significant fibrosis (F0-F2 fibrosis), ≤ -1.455 to ≤ 0.675: intermediate index, > 0.675: predictor of significant fibrosis (F3-F4 fibrosis).

Table 1 shows the main indicators of anthropometric data, laboratory parameters reflecting the state of the liver, lipid and carbohydrate metabolism, and liver fibrosis index depending on the normal or elevated level of cytokeratin-18 (CK-18) in patients with early forms of NAFLD.

As can be seen from Table 1, in patients with early forms of NAFLD in combination with an elevated level of CK-18, the levels of leukocytes, ESR, ALT, ACT, alkaline phosphatase, GGTP, TG, TNF-α, HOMA index and fibrosis index were significantly higher and the level of platelets was lower . Also, the level of CK-18 in these patients showed significant correlations with ESR - r = 0.54 (p = 0.015), ALT - r = 0.26 (p = 0.007), ACT - r = 0.32 (p = 0.006 ), TNF-α - r = 0.62 (p=0.016), HOMA-IR - r = 0.30 (p=0.004), NAFLD FS - r = 0.60 (p=0.0001) and was negatively correlated with the level of platelets - r = -0.33 (p = 0.001). These data prove the direct effect of hepatocyte apoptosis on the processes of inflammation, hepatocellular damage, insulin resistance and fibrosis in patients with early forms of NAFLD, which actualizes the need to determine their level of CK-18.

Table 2 shows the main indicators of anthropometric data, laboratory parameters reflecting the state of the liver, lipid and carbohydrate metabolism, and liver fibrosis index depending on the normal or elevated level of tumor necrosis factor-alpha (TNF-α) in patients with early forms of NAFLD.

Table 2 shows that in patients with early forms of NAFLD in combination with an elevated level of TNF-α, the levels of OT, leukocytes, CRP, ALT, GGTP, cholesterol, TG, CK-18, HOMA and fibrosis indices were significantly higher. The levels of albumin and HDL were lower. Also, the level of TNF-α in these patients showed significant correlations with OT - r = 0.33 (p=0.04), TG - r = 0.68 (p=0.031), CK-18 - r = 0.66 (p=0.008), HOMA-IR - r=0.67 (p=0.004), NAFLD FS - r=0.84 (p=0.035). These data prove the direct effect of this marker on the processes of inflammation, hepatic cell damage, cholestasis, dyslipidemia, insulin resistance and fibrosis in patients with early forms of NAFLD, which actualizes the need to determine their TNF-α level.

Table 3 shows the main indicators of anthropometric data, laboratory parameters reflecting the state of the liver, lipid and carbohydrate metabolism, and liver fibrosis index depending on normal or elevated levels of interleukin-6 (IL-6) in patients with early forms of NAFLD.

As can be seen from Table 3, in patients with early forms of NAFLD in combination with an elevated level of IL-6, the levels of OT, leukocytes, lymphocytes, ESR, CRP, alkaline phosphatase, GGTP, cholesterol, TG, glucose, TNF-α, and fibrosis index were significantly higher. . The level of HDL was lower. Also, the level of IL-6 in these patients showed significant correlations with leukocytes - r = 0.49 (p = 0.04), lymphocytes - r = 0.62 (p = 0.013), ESR - r = 0.51 (p =0.04), TG - r=0.73 (p=0.016), ALP - r=0.63 (p=0.04), HDL - r=-0.52 (p=0.04), NAFLD FS - r = 0.70 (p = 0.02). These data prove the direct effect of this marker on the processes of inflammation, cholestasis, dyslipidemia, insulin resistance and fibrosis in patients with early forms of NAFLD, which actualizes the need to determine their IL-6 level.

Table 4 shows the main indicators of anthropometric data, laboratory parameters reflecting the state of the liver, lipid and carbohydrate metabolism, and liver fibrosis index, depending on the presence or absence of intrahepatic cholestasis (ITC) syndrome in patients with early forms of NAFLD.

Table 4 shows that in patients with early forms of NAFLD in combination with the syndrome of intrahepatic cholestasis, the levels of leukocytes, CRP, ALT, alkaline phosphatase, GGTP, TG, IL-6, CK-18, and indices of IR and fibrosis were significantly higher. The level of HDL was lower. ALP showed significant correlations with HDL in these patients - r = -0.57 (p=0.002), ACT - r = 0.21 (p=0.04), TG - r = 0.58 (p=0.003) , HOMA - r = 0.61 (p=0.04), NAFLD FS - r = 0.48 (p=0.04); GGTP significantly correlated with the level of ALT - r = 0.49 (p = 0.044) and with the level of leukocytes - r = 0.50 (p = 0.04). These data prove the direct effect of intrahepatic cholestasis on the processes of inflammation, hepatocellular damage, dyslipidemia, apoptosis, IR and fibrosis in patients with early forms of NAFLD, which actualizes the need to determine their levels of alkaline phosphatase and GGTP.

Table 5 shows the main indicators of anthropometric data, laboratory parameters reflecting the state of the liver, lipid and carbohydrate metabolism, and liver fibrosis index depending on the presence or absence of insulin resistance syndrome (IR) in patients with early forms of NAFLD.

As can be seen from Table 5, in patients with early forms of NAFLD in combination with insulin resistance syndrome, the levels of WC, BMI, ALT, AST, alkaline phosphatase, GGTP, cholesterol, TG, glucose, TNF-α, IL-6, CK-18 were significantly higher, and fibrosis index. The platelet count was lower. The HOMA index showed significant correlations with ACT in these patients - r = 0.26 (p=0.032), TG - r = 0.49 (p=0.021), CK-18 - r = 0.51 (p=0.016) , NAFLD FS - r = 0.66 (p = 0.038). These data prove a direct relationship between insulin resistance and the processes of inflammation, hepatocellular damage, dyslipidemia, apoptosis, intrahepatic cholestasis and fibrosis in patients with early forms of NAFLD, which actualizes the need to determine their glucose and fasting insulin levels with the calculation of the IR index.

Thus, the proposed technical solution makes it possible to simplify and reduce the economic costs of assessing the prognosis of the course of NAFLD, improve the quality of diagnosis, reduce the number of personnel involved in the examination, justify the use of more active tactics in patients with early forms of NAFLD with a high risk of progression. The technical result is achieved by simultaneously assessing all the main links in the pathogenesis of the progression of non-alcoholic fatty liver disease by one widely used laboratory method (ELISA) without the involvement of narrow specialists, additional examination methods. The proposed method can be used in any medical institution where there are conditions for conducting ELISA, the conclusion can be given by any doctor of a therapeutic profile, the method is not invasive and can be performed repeatedly to assess changes in risk over time in the same patient, which makes it simple , unified and public.

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Claims (1)

A method for predicting the progressive course of early forms of non-alcoholic fatty liver disease, including determining the levels of TNF-α, interleukin-6, cytokeratin-18, alkaline phosphatase, gamma-glutamyl transpeptidase, glucose and insulin on an empty stomach in peripheral blood in vitro, characterized in that patients are assessed for the presence of the following syndromes: systemic inflammation, hepatocyte apoptosis, intrahepatic cholestasis and insulin resistance; while systemic inflammation is diagnosed by an increase in the following parameters above the upper limit of normal: TNF-α more than 5 pg/ml and interleukin-6 more than 3.5 pg/ml; apoptosis of hepatocytes is detected with an increase in the level of cytokeratin-18 more than 100 U/l; intrahepatic cholestasis is determined by an increase in the following parameters above the upper limit of normal: alkaline phosphatase more than 270 U / l and / or gamma-glutamyl transpeptidase more than 32 U / l in women and 50 U / l in men; insulin resistance - with an increase in the HOMA-IR index value of more than 2.6, when this index is calculated using the level of fasting insulin; and in the presence of any two or more of the evaluated syndromes, the risk of progression of early forms of non-alcoholic fatty liver disease is predicted, in the presence of only one of the studied syndromes or in the absence of each of them, the absence of the risk of progression of non-alcoholic fatty liver disease is determined.