Method for detection of alpha-2-macroglobulin molecule pathology in patients with cranial trauma

FIELD: medicine, neurosurgery, neuropathology, neurochemistry, physiology.

SUBSTANCE: method for detection of pathology of alpha-2-macroglobulin pathology in patients with cranial trauma involves simultaneous quantitative determination of alpha-2-macroglobulin content in cerebrospinal fluid of patient by electophoretic study and investigation of inhibitory enzymatic activity of alpha-2-macroglobulin with low-molecular synthetic N-benzoyl-DL-arginine-p-nitroanilide as a substrate. Pathology in alpha-2-macroglobulin in the patient with cranial trauma is detected in the case for presence of protein by electrophoretic method and by absence the inhibitory enzymatic activity of this protein. Using this method provides enhancing the precision in detection of alpha-2-macroglobulin pathology that promotes to well-timed prescribing the adequate therapy for patients with cranial trauma.

EFFECT: improved detecting method.

2 ex

 

The invention relates to medicine, namely to neurosurgery and neuropathology, neurochemistry laboratory, physiology and can be used to assess the pathology of protein molecules with inhibitory enzymatic activity in biological fluids, in particular in the cerebrospinal fluid (CSF).

One of the major proteins of the organism possessing inhibitory enzymatic activity, is a high-molecular inhibitor of proteolytic enzymes all four known classes of proteases, alpha-2-macroglobulin (α2-MG). It is characterized by a special mechanism of interaction with proteolytic enzymes and therefore it belongs to the most important regulators of their activity. Protein molecules α2MG exist in the form of macromolecular complexes, which interact with the receptor apparatus of the cell surface, which leads to changes in the functional activity of cells. All of the above makes you think about the feasibility study of this protein in biological fluids of a person, including CSF, and to assume its unconditional meaningfulness in different pathologies.

There is a method of assessing the pathology of protein molecules with inhibitory enzymatic activity, including radial immunodiffusion in the gel by Mancini (Manchini, G.,Carbonara, A.O. and heremans kept carrying out renovation, J.F. Immunochemical quantitation of atigens by single radial immunodiffusion. Int. J.Immunochem. 2 235-254 (1965) using specific antibodies. The disadvantage of this method is that it assesses only a quantitative parameter molecules α2MG and attests to a greater extent on the structure of the protein molecule, but not its biological activity.

Closest to the claimed method is a method of assessing the pathology of protein molecules with inhibitory enzymatic activity, by holding the disk-polyacrylamide gel electrophoresis (SDS page) (Ornstein L(1964)Disc electrophoresis I. Background and theory. Ann. No. 4. Acad. Sci. l21,321)adopted for the prototype. The method is based on the fractionation of protein mixtures depending on the electric charge and molecular weight members of the protein molecules. The method allows you to see the overall changes in protein fractions of the spectrum, including changing proteinase inhibitor α2-MG. When evaluating the results obtained by this method, evaluate quantitative differences in the protein fractions of the spectrum of the investigated biological fluid compared to the norm. However, the prototype is not sufficiently precise, as only estimates of the quantitative parameter of protein molecules on the electrophoretic mobility and molecular weight, but does not allow to evaluate their biological activity.

The invention is directed to a method of assessing pathology Belko what's molecules with inhibitory enzymatic activity, for enhancing the accuracy of the method due to the additional research inhibitor of the enzymatic activity of protein molecules.

This object is achieved in that in the known method of assessing the pathology of protein molecules with inhibitory enzymatic activity, including electrophoretic study of the protein, the peculiarity lies in the fact that at the same time conduct the study of the inhibition of the enzymatic activity of protein molecules with various low molecular weight synthetic substrates and identifying the presence of protein, but in the absence of the inhibitor of the enzymatic activity, ascertain the presence of pathology in the protein molecule.

Thanks to the simultaneous study of electrophoretic protein molecules and to determine their inhibitory enzymatic activity using a synthetic low-molecular-weight substrates assess the biological activity of α2MG (presence or absence), which indicates pathology in protein molecules.

The inventive method is carried out as follows. Conducting a study of protein molecules α2MG in the sample of biological fluid, such as CSF, obtained by lumbar puncture, define it the total protein content of any quantitative method, the number of cellular elements of the blood and, charge them by centrifugation at 1500-2000 rpm for 20 minutes. In the resulting supernatant determine inhibiting the enzymatic activity of the complex protein molecule inhibitor α2-MG trypsin in relation to synthetic low-molecular substrate N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) according to the modified method (Veremeenko PHD, Vasiliev YEAR, Dobrogowska L.N., Its juridical L.N., Goncharova V.P. α2-Macroglobulin in the cerebrospinal fluid during neurosurgical diseases. Questions of medical chemistry, 1989, No. 6, p.48-51): 0.8 ml CSFadd0.05 ml of trypsin solution containing 60 μg of enzyme solution and 0.05 inhibitor from soybean seeds containing 100 μg last, to neutralize the free trypsin. Bring a mixture of phosphate buffer to 1 ml, thermostatic in a water bath at a temperature of 30°C, add 3.2 ml of substrate solution BAPNA, which formed as a result of para-nitroaniline, the optical density of which is measured spectrophotometrically at a wavelength of 383 nm. The number of the resulting para-nitroaniline is a measure of the activity of the molecule α2MG or lack of it. At the same time conducting a study of the same sample of CSF method modified classic disc electrophoresis in SDS page (Alekseeva L.A., Valberg, A. Quality and quantity is the distribution of proteins in the cerebrospinal fluid of man according to the disc electrophoresis in polyacrylamide gel. Laboratory work, 1985, No. 4, S. 204-207). Electrophoresis is carried out in a 6.2% SDS page in Tris-glycine buffer at pH 8.3, at a current of 50 mA and a voltage of 400 V on any of the devices vertical electrophoresis in glass tubes. Each tube make a 0.1 ml sample of CSF (concentration of sample in the sample should not exceed 100 µg). After electrophoresis gels stained with a specific dye to protein. Colored bars gel densitometric at a wavelength of 620 nm. The height of each protein peak at proteinogram is directly proportional to its quantitative content. When determining the quantitative content of protein molecules α2MG in the sample of biological fluid, the absence of inhibition of the enzymatic activity indicates pathology of protein molecules α2MG.

The inventive method developed in the Polenov research neurosurgical Institute them. Professor and Operated and passed clinical trials in the study of 42 samples of CSF. While 11.9% of cases detected pathology of protein molecules with inhibitory enzymatic activity.

Examples of extracts from the log of biochemical studies No. 4.

Example 1.

Observation # 31. Sick And 46 years. Diagnosis: closed craniocerebral injury, brain contusion severe lesions crush zone parietotemporal area to the right, subdue the other hematoma, subarachnoid hemorrhage.

For the study was taken to 2.8 ml CSF, obtained by lumbar puncture in the first days after injury. To determine total protein and lymphocytosis in the obtained sample of CSF was taken to 1.6 ml Total protein amounted to 1.15 g/l, lymphocytosis - 6×106CL/l (neutrophils - 1×106CL/l, lymphocytes - 5×106CL/l), erythrocyte - 11930×106/l 1.2 ml of CSF was centrifuged at 2000 rpm for 15 minutes to precipitate the cellular elements of blood and the resulting supernatant was taken for further biochemical studies. 0.1 ml of CSF was taken for electrophoretic studies α2-MG. 1 ml was taken for determination triesysweeway activity α2MG using low molecular weight synthetic substrate BAPNA.

The study found that the absolute content of α2MG was 0,093 g/l (increased compared with the normal value), and triesysweeway activity - 0,087 g/l (increased compared with the normal value). Thus, this observation of the patient And the pathology of protein molecules is not revealed. On the 37th day after the injury the patient was transferred to the hospital gastromark in the rehabilitation Department for further treatment.

Example 2.

Observation No. 98. Patient B 32 years. Diagnosis: closed cranio-mo is gova injury, brain contusion severe lesions crush zone of the frontal-parietal-temporal region to the right, intracerebral hematoma of the right hemisphere, subarachnoid hemorrhage.

For study was taken 3.0 ml CSF, obtained by lumbar puncture in the first days after injury. To determine total protein and lymphocytosis in the obtained sample of CSF was taken to 1.8 ml Total protein was 1.25 g/l, lymphocytosis 42×106CL/l (neutrophils - 8×106CL/l, lymphocytes - 33×106CL/l, macrophages - 1×106CL/l), erythrocyte - 93900×106/l 1.2 ml of CSF was centrifuged at 2000 rpm for 15 minutes to precipitate the cellular elements of blood and the resulting supernatant was taken for further biochemical studies. 0.1 ml of CSF was used for electrophoretic studies α2MG, 1 ml to determine its triesysweeway activity using a synthetic low-molecular-weight substrate BAPNA.

The study found that the absolute content of α2MG was 0,098 g/l (increased compared with the normal value). The same definition triesysweeway activity of this protein did not reveal the presence of inhibitor (biological) activity. Thus, the patient B was observed phenomenon pathology of protein molecules α2MG, features resouses the presence of synthesized molecules but missing them inhibitor of the enzymatic activity. Death in the patient came on day 7 after injury.

Using the proposed method improves the accuracy of detecting pathology molecules alpha-2-macroglobulin. In patients with traumatic brain injury to identify disease molecules alpha-2-macroglobulin indicates a severe condition of the patient and a high probability of adverse outcome. This state of the body requires not only the use of inhibitors to compensate for the missing inhibitory capacity of alpha-2-macroglobulin, but additional detoxification activities such as hemosorption and liquorice. The detection of pathology of the molecule alpha-2-macroglobulin in patients with traumatic brain injury allows to assign adequate therapy.

Method of detecting pathology molecules alpha-2-macroglobulin in patients with traumatic brain injury, including electrophoretic study of the cerebral spinal fluid of a patient, characterized in that it further survey inhibitor of the enzymatic activity of alpha-2-macroglobulin with low molecular weight synthetic substrate N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and, upon detection of protein electrophoretic method, but otsutstvie he inhibiting enzymatic activity, identify pathology molecules alpha-2-macroglobulin in a patient with traumatic brain injury.



 

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