SU1686359A1 - Method for detection of serine proteases in lacrimal fluid in cornea eye chronic ulcers - Google Patents

Abstract

The invention relates to medicine, in particular to ophthalmology, and can be used to determine serine proteases in the tear fluid in chronic cases of the cornea of the eye. The purpose of the invention is to accelerate and improve the accuracy of the determination of proteases in the tear fluid. The essence of the invention is that the proteolytic activity of the material under study is determined spectrophotometrically by the rate of cleavage of the synthetic substrate of the N---benzoyl-arginine ester, and the residual activity is measured in the presence of specific inhibitors of serine proteases: c-aminocaproic acid, inhibitors of trypsin from soybean or horodox beans, and the relative content of plasmin and its activators, as well as tissue and plasma callecreins, is determined by the level of residual activity. The method accelerates the measurement of proteolytic activity from 24-48 hours to 9-15 minutes and allows quantitative determination of the activities of plasmin and its activators, as well as tissue and plasma callecreins. 1 tabl (L S

Description

The invention relates to the field of medicine, in particular to ophthalmology, and in particular to methods for the diagnosis of enzyme factors contributing to the development of chronic ulcerative cornea.

The aim of the invention is to improve the accuracy and acceleration of the method.

The method is carried out as follows.

Tearing fluid samples collected from patients (50 to 70 µl) are placed in plastic tubes with hermetically sealed caps and stored at -20 ° C until examination. One-time freezing and thawing of samples is acceptable.

Then, 5-10 µl of tear fluid determine the total activity of enzymes,

M-a-benzoyl-arginine ethyl ester cleaving (BAEE-esterase activity) For this, the sample is placed in a quartz spectrophotometric cuvette, buffered with 0.1 ml of 0.5 M Tris-HCl buffer, pH 8.0 and brought to volume 2 ml of buffer heated to 37 ° С 0.05M Tris-HCl, pH 8.0. After that, 1 ml of 1.5 x 10 M solution of N-a-benzoyl-arginine ethyl ester (BAEE) was added to the cuvette and the increase in optical density at 253 nm at 37 ° C was started. In the absence of activity, the sample volume should be increased. Optical control is a sample containing 2 ml of 0.05 M Tris-HCl buffer, pH 8.0 and 1 ml of substrate.

The activity calculation is carried out according to the formula

about with about

jCJ

ate about

YES 2.73

tv

where YES is the increase in optical density during the registration,

2.73 is a conversion factor taking into account the difference in molar extinctions of the substrate and the reaction product; t is the time of registration of optical density; v is the volume of the sample, ml. The analysis of the effect of inhibitors on the BAEE-esterase activity of the tear fluid is performed as follows.

Inhibitor solutions were prepared: trypsin inhibitors from soy bean -ITS (2 mg / ml), α-aminocaproic acid - e-AKK (20 mg / ml). As a tissue inhibitor of proteolytic enzymes use commercial preparations Gordoks (Gideon Richter, Vengry).

0.1 ml of 0.5 M Tris-HCl buffer, pH 8.0, and 0.1 ml of one of the inhibitors are added to 5-10 µl of the tear fluid sample. After 15 min of incubation, the sample volume was adjusted to 2 ml with 0.05 M Tris-HCI buffer warmed to 37 ° C and after adding 1 ml of 1.5 x 10 3 M BAEE solution, the BAEE esterase activity was measured. The optical density of the sample is recorded every 3 minutes for 9-15 minutes. The activity is calculated using the same formula, after which the percent inhibition is calculated.

Repeat the measurement with other inhibitors as shown.

Based on the suppression of the total BAEE-esterase activity by inhibitors, the ratio between plasmin, tissue and plasma kallikrein and other proteinases is established.

The data on the effect of the inhibitors used are given in the table.

Example 1. A total of 21 patients were examined with a diagnosis of chronic cornea of the eye, among them 12 women and 9 men aged 45 to 68 years

The total BAEE-esterase activity ranges significantly from 0.012 to 3.03 µmol / ml ae. G. The percentage of activity, sensitive to the action of g-ACC, varies in individual samples from 9 to 90%. indicating a different plasmin activity in the samples. The proportion of activity that is sensitive to the effect of ITS varies in the studied samples from 43 to 74%, which proves the fact that the tissue calicrerein and plasmin activators are not exactly the same in the tear fluid of the drug Trasilol, an analogue of Gordox, suppresses activity by 80–100%, which also testifies to the different content in the tear fluid of patients plasmin activators.

Example 2. A survey of the patient with a diagnosis; herpetic keratitis, secondary of the cornea. 0.1 ml of 0.5 M Tris-HCl buffer solution, pH 8.0 was added to 5 µl of the lacrimal fluid of the patient, the volume was adjusted to 2 ml with 0.05 M Tris-HCl buffer heated to 37 ° C, pH 8.0, 1 ml of the substrate (BAEE) was added and the increase in the density of the sample was measured on a Hitachi Y-3200 spectrometer at a wavelength of 253 nm. Absorbance readings were taken automatically every 3 minutes for 9 minutes. Optical density increased over 9 minutes to 0.025, which corresponded to an activity of 1.57 µmol BAEE per minute per 1 ml of sample.

0.1 ml of e-ACC solution was added to 5 µl of tear fluid, buffered with 0.1 ml of 0.5 M Tris-HCl buffer was incubated for 15 min at 37 ° C, after which the remaining BAEE-esterase activity was measured. The activity after e-ACC action is 1.43 µmol / min per ml (8.9% inhibited), the activity after ITS is -0.41 µmol / min (73.9% inhibited), pride completely suppresses the activity of the sample. Consequently, the plasmin activity in the tear fluid sample is about 10%. and tissue kallikrein activity is more than 25%. Conclusions for treatment can be recommended for instillation of gordox and chelating agents.

Example 3 A patient was diagnosed with a chronic corneal ulcer.

The total activity of the tear fluid — 3.03 µmol / min per ml of f-AKK suppressed activity by 78%, and ITS - by 73%. Conclusion: for treatment, a solution of β-AQA solution and chelating agents can be recommended.

The method is a quantitative method for measuring enzyme activities, in contrast to the prototype method, which limits the semi-quantitative method (measuring the spot diameter and recalculating the activity on a calibration curve), and also measures the activity of tissue and plasma callecreins, which increases the accuracy of the method.

The proposed method significantly accelerates the measurement of the proteolytic activity of the tear fluid from 24–48 hours to 9–15 minutes.

Claims (1)

DETAILED DESCRIPTION OF THE INVENTION A method for detecting serine proteases in the tear fluid in chronic occlusions of the cornea, including determining the proteolytic activity of plasmin and its activators by splitting the substrate, characterized in that, to accelerate and improve the accuracy of the method, the proteolytic activity is determined spectrophotometrically synthetic fission rate ethyl ether-N-a-benzoyl-arginine substrate, after which the residual activity is measured in the presence of e-aminocaproic acid, trypsin inhibitors from soybeans or gordox, and the relative content of plasmin, its activators, tissue and plasma cell lekreinov. The sensitivity of individual enzymes to the action of inhibitors