RU2234088C2 - Method for assay of stability of erythrocyte cellular membrane - Google Patents

Abstract

FIELD: medicine, laboratory diagnosis.

SUBSTANCE: invention can be used for diagnosis and prognosis of human health retention and their adaptation to extreme ecological conditions. Method involves biochemical blood analysis and comparison of calculated index with the norm and amount of easily oxidizable phospholipids is determined. The stability is estimated as deviation of tocopherol-index from norm that represents the ratio of tocopherol amount to amount of easily oxidizable phospholipids. Invention provides enhancing reliability and expanded diagnostic capacity of method.

EFFECT: improved assay method.

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Description

The invention relates to medicine, namely to laboratory diagnostics of clinical medicine. It can be used to diagnose and predict ways to maintain people's health and their adaptation to extreme environmental conditions.

A known method of studying the activity of metabolic processes in a cell, including biochemical blood testing, determining metabolic parameters, calculating an informative parameter and comparing it with the norm [patent 2050001 (RF), IPC ⁶ G 01 N 33/48. A method for studying the activity of metabolic processes in a cell // Bankova V.V. (RF), Banks V.N. (RF), I. Kislova (RF). - Application 92013706, claimed 22.12.1992, publ. 12/10/1995].

The known method is a simulation of artificial hemolysis of red blood cells. This helps to reduce the reliability of the results. Determination of the resistance of red blood cells only to those substances that are used in the known method, helps to reduce diagnostic capabilities.

The objective of the invention is to increase the reliability and expansion of the diagnostic capabilities of the method.

The problem is solved in that in the method for determining the stability of erythrocyte cell membranes, including a biochemical blood test and comparing the calculated indicator with the norm, according to the invention, the amount of readily oxidizable phospholipids is determined, and stability is judged by the deviation from the norm of the tocopherol indicator, which is the ratio of the amount of tocopherol to the amount easily oxidizable phospholipids. The deviation of the tocopherol indicator from the value of 0.013-0.017 rel. indicates a violation of the stability of the membrane.

The tocopherol indicator is calculated by the formula:

where TP is a tocopherol indicator;

TKF - the amount of tocopherol in the membranes of red blood cells, µmol / l;

LOFL - total amount of readily oxidizable phospholipids (phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol) in the structure of erythrocyte cell membranes, µmol / L.

Cell membranes form the boundaries of the cell, separating the cytoplasm from the environment. The membrane performs a variety of transport and biochemical functions, and it is also able to form a mechanical membrane of the cell.

The mechanical properties of membranes are manifested, first of all, in the maintenance by the cells of a certain shape in the resistance to deformation under the influence of external factors.

Cell membranes are active biochemical systems responsible for the selective transport of cells and subcellular counterparts into and out, binding of hormones and other regulatory molecules, enzyme-catalyzed reactions, transmission of electrical impulses, ATP synthesis. Cell membranes are composed of lipid and protein molecules. The lipid layer of cell membranes is a double layer of phospholipids with integrated proteins embedded in them. The bimolecular layer of lipids in the membranes is predominantly represented by fractions of phospholipids and cholesterol. At the same time, the fractions of phospholipids are oriented in such a way that their hydrocarbon chains of fatty acids are directed inward and retained by hydrophobic bonds, and the polar groups are facing the protein layer.

As structural components of cell membranes, phospholipids determine their viscosity (fluidity), the ability of cells to migrate, phagocytosis, pinocytosis and clumping. Depending on the structure of the polar "head groups" of glycerophospholipids, their structural and functional purpose changes.

In addition to phospholipids and cholesterol, cell membranes include tocopherols, which have the ability to stabilize lipids by improving the structure of the lipid bilayer of membranes. Tocopherol, which has hydrophobic properties, is completely immersed in the lipid bilayer in such a way that its OH group forms a hydrogen bond with the CO group of acyls associated with glycerol residues in the phospholipid molecules. As a result of this physicochemical interaction between the phytol side chain of the tocopherol and the hydrocarbon chain of the arachidonic acid residue, which is part of the fatty acids of readily oxidizable phospholipids, tocopherol orders the liquid crystal structure of the membrane lipid matrix, preventing the oxidation of phospholipids with unsaturated bonds. This leads to stabilization of their hydrophobic zone. It is this connection of tocopherol with fatty acid residues of arachidonic acid that helps stabilize the structure of biological membranes.

Tocopherol, an indicator representing the ratio of the amount of tocopherol to the amount of readily oxidizable phospholipids, allows us to determine if there is enough tocopherol for the stable state of erythrocyte membranes with a certain amount of readily oxidizable phospholipids.

The method is as follows by a standard method. Determine the amount of phospholipids FS + PHI and PEA by thin layer chromatography: the amount of tocopherol is determined using an MPF - Hitachi spectrofluorimeter. From venous blood a suspension of red blood cells in the amount of 0.04 ml + 0.2 water is prepared. This mixture is shaken for 30 minutes. Then, 0.2 ml of ethanol is added to the resulting liquid and again shaken for 30 minutes. After adding 1.0 ml of hexane, shake for another 40 minutes. Finally, this mixture is centrifuged at 1500 rpm for 10 minutes. Measure the fluorescence intensity of a suspension of erythrocyte membranes and standard alpha-tocopherol acetate (20 μmol / L) at a wavelength of 320 nm and a fluorescence excitation wavelength of 295 nm. The calculation is made according to the formula

With the sample is the concentration of the sample, µmol / l,

Fl.sample - an indicator of the fluorescence of the sample, fl.ed.,

Fl.st. - indicator of fluorescence standard, fl.ed.,

From Art. - an indicator of concentration of a standard sample, micromol / l.

The tocopherol indicator is calculated by the formula

and compare it with a normal value, previously determined by the results of a healthy examination, equal to 0.013-0.017 rel. Deviation from the norm indicates that tocopherol is not enough for the normal functioning of the cell membranes of red blood cells. This can lead to a decrease in cell resistance to external influences and contribute to a decrease in immunity.

Example 1. The red blood cell membrane was studied in patient No. 1

Conclusion: the deviation from the norm is 0.009 rel. To improve your health, you need to eat foods rich in fat-soluble vitamins.

Example 2. Investigated the erythrocyte membrane of patient No. 2

Conclusion: the red blood cell membrane is normal. You can think about the normal functioning of the body and a sufficient level of tocopherols in food intake.

The proposed method helps to increase the reliability and expansion of diagnostic capabilities for determining the stability of cell membranes. The optimal value of the tocopherol indicator indicates sufficient biological activity of tocopherols to preserve the easily oxidized phospholipids from the molecular structure from oxidation. Preserved easily oxidized phospholipids provide the enzymatic activity of transport proteins of erythrocyte membranes, generally increasing their stability and resistance to external influences.

Claims (1)

A method for determining the stability of erythrocyte membranes, including a biochemical blood test and comparing the calculated indicator with the norm, characterized in that the amount of easily oxidized phospholipids is determined, and stability is judged by the deviation from the norm of the tocopherol indicator, which is the ratio of the amount of tocopherol to the number of easily oxidized phospholipids.