RU2128835C1 - Method for diagnosing hepatic cholestasis by means of blood serum lipoprotein electrophoresis - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves applying gel electrophoresis on agarous plate to detect basic classes of lipoproteins and determine percent contents of every class. Then the values of ratios between alpha-lipoproteins and beta-lipoproteins are calculated. The values are to be compared to a reference one to determine the marked degree of the cholestatic liver injury. EFFECT: enhanced accuracy of diagnosis; accelerated laboratory treatment. 2 dwg

Description

 The invention relates to medicine, in particular to clinical gastroenterology and can be used for laboratory diagnosis of chronic liver diseases, namely cholestatic lesions, based on an objective assessment of the quantitative content of the main classes of serum cholesterol-transport lipoproteins determined at different stages of liver cholestasis gel electrophoresis .

 Known methods for the clinical diagnosis of chronic liver lesions by morphological analysis of liver biopsy (Loginov AS, Aruin LI "Clinical morphology of the liver." M., Medicine, 1985), are performed using percutaneous puncture biopsy material, which is associated with surgical (laparoscopic) intervention in the patient’s body, as well as low predictability of the diagnosis, for example, due to insufficient information content of the obtained biopsy.

 There is also a method using biochemical enzyme diagnostics of chronic liver lesions, including primary biliary cirrhosis (PBC) (Loginov A.S., Matyushin B.N. et al., Copyright certificate N1809384 from 1993; Loginov A.S., Matyushin B . N. et al. "A new approach to the diagnosis of cholestasis by the activity of copper-containing enzymes" Ter. Archive. 1993, N 2, p. 34-36), which consists in determining the activity of a number of antioxidant enzymes in the liver biopsy. However, the existing method does not allow for differential diagnosis of the early stages of PBC from the terminal stage, when it is necessary to decide on the continuation of conservative treatment of PBC or on the most favorable terms for liver translation. In addition, with severe cholestasis, especially at the terminal stage of PBC, liver biopsy laparoscopy is contraindicated for the patient.

 The inventive method, in contrast to the known methods for the diagnosis of liver cholestasis, eliminates invasive intervention, simplifies the diagnosis and improves the reliability of diagnosis and, most importantly, provides an opportunity to distinguish between early stages favorable for the provision of liver transplantation.

The purpose of the proposed method is to improve the accuracy of diagnosis of chronic liver diseases by minimizing interference in the patient’s body based on electrophoretic isolation and quantification of the main classes of lipoproteins (LP) in serum: chylomicrons (chilo-LP), very low density lipoproteins (prebeta-LP), low density lipoproteins (beta-LR), high density lipoproteins (alpha-LP) (see table)
This goal is achieved through the use for a new purpose of the known method for the isolation of transport forms of cholesterol in serum and plasma, used for the diagnosis of hyperlipoproteinemia in atherosclerotic lesions of the cardiovascular system (New Engl. J. Med., 1967, v. 276, p. 34 and 147).

 Currently, the use of an electrophoretic method for isolating transport forms of cholesterol for the diagnosis of chronic liver lesions is unknown.

 The inventive method is as follows: samples of serum or plasma obtained on an empty stomach are applied in a volume of 5 μl to an agarose plate and electrophoresis is carried out for 30 min at a voltage of 100 volts, the plate is fixed, dried, stained with lipids in sudan black and discolor the background, after additional drying the plate is analyzed using a densitometer at a wavelength of 600 nm and the percentage of each class of lipoproteins is determined for a group of healthy individuals (normal) and for groups with different pathologies, including chno-stone disease (GSD), chronic pancreatitis (Chr. n), primary biliary cirrhosis (EVP). Comparative data on the class of LP in groups of healthy and sick people are summarized in the table.

 Based on the quantitative data, an indicator of the relationship between beta-drug and alpha-drug is calculated (see drawing).

 The table shows that there were no significant differences between the parameters of the corresponding classes of medications in individuals of the control group (normal) and in the group of individuals with PBC at stages 2–3 of the disease, although there is a slight tendency to increase beta-levels and decrease alpha level LP.

 A comparative analysis of the relations between the individual classes of lipoproteins in the studied groups (see the drawing) made it possible to establish significant differences in the ratios of beta-LP to alpha-LP in normal and in patients with PBC (p <0.024), normal and in patients with chronic pancreatitis (p <0.001), and also make sure that there are no significant differences between the norm and patients with cholelithiasis (p> 0.27).

 The results were carefully compared with the data of clinical, biochemical, instrumental and morphological studies. A total of 137 people were examined.

Examples of specific implementation of the method
Example 1

 Patient D., 53 years old.

 In the blood serum obtained for diagnostic purposes and subjected to electrophoresis by the present method, the percentage of the main classes of lipoproteins (chylomicrons, beta-LP, prebeta-LP, alpha-LP) is determined using a densitometer and the ratio of beta-LP / alpha-LP is calculated , in this patient the content of beta-LP is 56.1%, and the level of alpha-LP is 18.7%. Accordingly, the ratio of relations between these classes of lipoproteins is 3.0, which is within the corresponding values of the indicator for 2 - 3 stages of PBC (see drawing).

 Thus, it is possible to diagnose primary biliary cirrhosis of the liver, 2 - 3 stages. This coincides with the final clinical diagnosis: primary biliary cirrhosis, stage 3, portal hypertension, varicose veins of the esophagus, common osteochondrosis. At the same time, it should be noted that a conclusion about the diagnosis by the proposed diagnostic method was made within 45 minutes, which reduces the time spent by more than 2 times in comparison with enzyme diagnostics (2 hours).

 Example 2

 Patient M., 52 years old.

 After electrophoresis of blood serum according to the claimed method and densitometry of the gel plate, the values of beta-PL = 73.2 and alpha-LP of 15.8% were obtained. The ratio of the relations between these classes of lipoproteins is 4.6, which exceeds the corresponding average values for 2 - 3 stages of PBC (see graph in the drawing). In this regard, a diagnosis is possible: primary biliary cirrhosis, stage 3 with the possibility of transition to stage 4. Final clinical diagnosis: primary biliary cirrhosis.

 After 12.5 months, a second blood serum was taken from this patient and a new analysis was performed by the claimed method. Values for beta-LP = 77.2 and for alpha-LP = 11.0 were obtained. The corresponding indicator of relations between these classes is 7.2, which corresponds to the 4th stage of PBC. The final clinical diagnosis states: biliary cirrhosis, stage 3–4, active, portal hypertension, varicose veins of the esophagus.

 This example shows that the proposed method allows you to more accurately identify the stage of cholestatic liver damage and predict the dynamics of the disease.

 Example 3

 Patient S., 49 years old.

 After electrophoresis of blood serum according to the proposed method and densitometry, values of beta-LP = 81.9% and alpha-LP = 7.8% were found. The corresponding indicator between these classes of lipoproteins is 10.5 i.e. is within the confidence interval of similar indicators for the terminal stage of PBC. Based on comparative indicators, it can be concluded that there is primary biliary cirrhosis of the liver of the 4th (terminal) stage. The patient probably needs orthotopic liver transplantation. At the same time, on the basis of traditional methods of clinical, biochemical and instrumental research, the following final clinical diagnosis was made: primary biliary cirrhosis, stage 3, active, ovarian dysfunction, psoriasis.

 After 4 months, the patient died.

 This example shows that the inventive method more accurately reflects the severity of cholestatic liver damage and, therefore, can be used to identify the terminal stage of a chronic disease, when a liver biopsy is contraindicated. The proposed method may be crucial in deciding on the indications and contraindications for liver transplantation in patients with PBC at the terminal stage of the disease.

Using the proposed method for the diagnosis of liver cholestasis using electrophoresis of serum lipoproteins provides the following advantages compared with existing:
the possibility of non-invasive diagnosis of patients with cholestatic lesions of the liver,
the ability to quantify the pathological process,
the ability to control at each stage of the disease and, most importantly, at the terminal stage of the disease, when a puncture biopsy for a morphological or enzyme-diagnostic analysis is contraindicated,
It has a high prognostic value, and therefore can be used as the main biochemical test in deciding whether to continue conservative treatment or to clarify the optimal indications for surgical intervention.

 reduces more than half the time spent on laboratory research in comparison with the prototype.

 The use of this method will significantly improve the quality of diagnosis, and, accordingly, the prevention and treatment of cholestatic lesions of the liver and will help to understand the pathogenetic mechanisms and epidemiological patterns of diseases associated with cholestasis.

Claims (1)

 A method for the diagnosis of cholestatic lesions of the liver by means of a clinical and biochemical study of the blood serum of patients, characterized in that electrophoretic quantitative determination of the main classes of serum lipoproteins is carried out, an indicator of the relationship between beta-lipoproteins and alpha-lipoproteins is calculated, and on the basis of indicators of the magnitude of the ratio between the indicated classes of lipoproteins the severity of cholestatic liver damage in the early and terminal stages of primary biliary cirrhosis.

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