RU2280873C2 - Method for determining protein-bound iodine in saliva - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves determining protein-bound iodine content in saliva using modified method from optical density value at wavelength of 440 nm.

EFFECT: simplified method of high accuracy.

Description

The invention relates to medicine, in particular to laboratory diagnostics.

The synthesis of thyroid hormones occurs in the thyroid gland by iodination of tyrosine residues of thyroglobulin.

Under the influence of thyroxine, the activity of about 100 different enzymes in the body increases, affecting many physiological functions that regulate the exchange rate of proteins, carbohydrates, lipids, water, electrolytes, tissue growth and differentiation, electron transport in mitochondria and other processes. Therefore, both hypo- and hyperfunction of the thyroid gland causes deep disorders of the physiological status of the body.

Determining the concentration of thyroid hormones is used to diagnose thyroid dysfunction.

To assess thyroid function, it is necessary to determine the concentration of thyroxine in the blood. Quantitative determination of protein-bound iodine is an adequate test, since 99.95% of thyroxin is found in the blood in combination with thyroxin-binding globulin.

The closest in technical essence and the achieved result is a method for determining protein-bound iodine in blood plasma (Eremin Yu.N., Proskuryakova G.F., Tocharina M.G. Determination of iodine bound to protein in small amounts of blood serum (or plasma) // Laboratory, 1980, No. 7, p. 428-430).

This method is based on the fact that plasma proteins are precipitated and then burned when heated in a muffle furnace. This is necessary so that all iodine passes into J ^- ions. Further, for the quantitative determination of iodine ions, the property of these ions is used to accelerate the decomposition of iron rhodanide in the presence of nitrates and nitrites in an acidic medium. The magnitude of the acceleration of the named reaction is proportional to the iodine concentration. The amount of iron thiocyanate having an orange color is measured colorimetrically. The iodine concentration is determined by a certain optical density and a calibration curve, an increase in the concentration of which indicates an increase in the function of the thyroid gland, and a decrease in its concentration indicates a decrease in the function of the thyroid gland.

The disadvantage of this method is that as the biological material that is taken from people during the examination, use blood, a fence that is traumatic for humans and requires specially prepared premises, trained medical personnel, sterile instruments. In mass surveys at enterprises, many people refuse this service. Moreover, the known method for determining protein-bound iodine in human blood is not sensitive enough to determine this indicator in saliva.

The basis of the invention is the task of simplifying and cheapening the method for determining protein-bound iodine, reducing the morbidity of the study during the intake of biological fluid, increasing the efficiency of diagnosis.

The technical result is achieved by the fact that in the method for determining protein-bound iodine in the patient’s saliva, before the sampling, the subject rinses the oral cavity with boiled water, drying the cavity with a napkin, saliva is collected in chemically clean tubes in an amount of 0.5-1 ml, for analysis in centrifuge tubes pour 4 ml of water, add 0.2 ml of test saliva, 0.4 ml of a 10% solution of zinc sulfate, 0.2 ml of 1 N. sodium hydroxide solution, centrifuged for 10 minutes at 1500 rpm and the supernatant is removed, the precipitate is poured 5 ml of water, stirred, centrifuged for 10 minutes at 1500 rpm and the supernatant is removed, 0.1 ml 1 is added to the precipitate n sodium hydroxide solution, 0.2 ml of a 20% solution of potassium nitrate, place the tubes in a thermostat with a temperature of 70-75 ° C for 2-3 hours until completely dry, heat the tube in a flame of an alcohol lamp until the contents are completely burned, as evidenced by the light color of the contents without black spots, pour 10 ml of water into test tubes, mix and centrifuge for 15 minutes at 3000 rpm, transfer 2 ml of the supernatant to clean tubes, add 0.3 ml of 0.006 M potassium rhodanide, 0.5 ml of 2% sodium nitrite, 0.8 ml of a 10% solution of iron ammonium alum in 2n. nitric acid and placed in a water bath with a temperature of 37 ° C for 30 minutes, then the tubes are placed in ice water for 1-2 minutes, then the optical density of the samples is measured on a spectrophotometer at a wavelength of 440 nm and the concentration of protein-bound iodine is determined by a calibration curve .

The method for determining protein-bound iodine in the patient's saliva is as follows.

As a biological fluid, human saliva is used, before the sampling of which the subject is washing the oral cavity with boiled water, drying the cavity with a napkin.

Saliva is collected in chemically pure tubes in an amount of 0.5-1 ml.

For analysis, 4 ml of water is poured into centrifuge tubes, 0.2 ml of test saliva, 0.4 ml of 10% zinc sulfate solution, 0.2 ml of 1N are added. sodium hydroxide solution. Then centrifuged for 10 minutes at 1500 rpm and remove the supernatant.

5 ml of water are added to the precipitate, stirred, centrifuged for 10 minutes at 1500 rpm, and the supernatant is removed.

Then, 0.1 ml of 1N was added to the precipitate. sodium hydroxide solution, 0.2 ml of a 20% potassium nitrate solution, place the tubes in a thermostat with a temperature of 70-75 ° C for 2-3 hours until completely dry.

After that, the test tube is heated in the flame of an alcohol lamp until the contents are completely burned, as evidenced by the light color of the contents without black spots. Then 10 ml of water is poured into test tubes, mixed and centrifuged for 15 minutes at 3000 rpm.

Then 2 ml of the supernatant is transferred to clean tubes, 0.3 ml of 0.006 M potassium thiocyanate, 0.5 ml of 2% sodium nitrite, 0.8 ml of 10% solution of iron-ammonium alum in 2N are added. nitric acid and placed in a water bath with a temperature of 37 ° C for 30 minutes. After this, the tubes are placed in ice water for 1-2 minutes. Then, the optical density of the samples is measured on a spectrophotometer at a wavelength of 440 nm.

The specific optical density and calibration curve determine the concentration of protein-bound iodine.

An increased concentration of protein-bound iodine indicates an increase in the concentration of thyroid hormones, and a reduced concentration of it indicates a decrease in the concentration of thyroid hormones. With an increase in the concentration of thyroid hormones in the studied saliva, a conclusion is made about an increase in the function of the thyroid gland, and with a decrease in the concentration of hormones in the saliva, a conclusion is drawn about a decrease in the function of the thyroid gland.

As a result of experimental studies conducted by the applicant, previously unknown properties of human saliva were identified, namely, there is a positive correlation between the concentrations of protein-bound iodine in blood and saliva, determined by the ability of iodine ions formed during the mineralization of protein-bound iodine to accelerate the decomposition of iron rhodanide in the presence of nitrates and nitrite in an acidic environment. The correlation coefficient was 0.94 in healthy individuals and 0.76 in patients with thyrotoxicosis. It was found that in practically healthy people, the concentration of protein-bound iodine in saliva, determined by the claimed method, ranged from 1.1 to 2.2 μg%, in patients with thyrotoxicosis - from 2.3 to 6.2 μg%.

In this regard, the possibility and expediency of using saliva as a biological fluid to assess thyroid function by determining the concentration of protein-bound iodine in saliva has been established.

Due to the fact that the concentration of protein-bound iodine in saliva is lower than in blood, the applicant introduced changes aimed at increasing the sensitivity of the method for determining protein-bound iodine in saliva, for which the ratio of the concentration of nitrates and nitrites in the reaction mixture was changed and their optimal ratio was selected to determine protein-bound iodine in saliva. The burning of the test material, the applicants produced instead of a muffle furnace in a test tube on an alcohol lamp. This leads to a reduction in the cost of the proposed method for laboratories that do not have a muffle furnace.

It was found that the rate of decomposition of iron rhodanide in the presence of nitrates, nitrites and iodine ions substantially depends on the temperature of the reaction mixture, therefore, the reaction is carried out in a thermostat at a temperature of 37 ° C. The known method does not provide for stopping the reaction, which can lead to errors with a large number of samples. The placement of samples in ice water in the inventive method allows to reduce the reaction rate so that it makes it possible to measure several samples without significant changes in their optical density. This allows you to increase the efficiency of the analysis, because it makes it possible to study several samples simultaneously.

The analysis of the prior art, including a search by patent and scientific and technical sources of information, and the identification of sources containing information about analogues of the claimed invention, allowed to establish that the applicant did not find an analogue characterized by features identical (identical) to all essential features of the claimed invention.

The definition from the list of identified analogues of the prototype allowed us to identify a set of essential in relation to the perceived technical result of the distinguishing features in the claimed method for evaluating the function of the thyroid gland set forth in the claims. Therefore, the claimed invention meets the criterion of "Novelty."

To verify the compliance of the claimed invention with the condition "Inventive step", the applicant conducted an additional search for known solutions in order to identify signs that match the distinctive features of the claimed invention from the prototype. The search results showed that the claimed invention does not follow explicitly from the prior art as determined by the applicant. Therefore, the claimed invention "Method for the determination of protein-bound iodine in saliva" meets the criterion of "Inventive step".

The criterion of "Industrial applicability" is confirmed by the fact that the invention can be successfully used in mass screening of the population for diagnostic purposes to assess the state of the thyroid gland and is effectively implemented in the healthcare system of Russia and the CIS.

Claims (1)

A method for determining protein-bound iodine in the patient’s saliva, characterized in that before the sampling of which the subject rinses the oral cavity with boiled water, drying the cavity with a napkin, saliva is collected in chemically clean tubes in an amount of 0.5-1 ml, for analysis 4 are poured into centrifuge tubes ml of water, add 0.2 ml of the investigated saliva, 0.4 ml of a 10% solution of zinc sulfate, 0.2 ml of 1 N. sodium hydroxide solution, centrifuged for 10 min at 1500 rpm, and the supernatant was removed, 5 ml of water was added to the precipitate, stirred, centrifuged for 10 min at 1500 rpm, the supernatant was removed, 0 was added to the precipitate, 1 ml 1 n sodium hydroxide solution, 0.2 ml of a 20% potassium nitrate solution, place the tubes in a thermostat with a temperature of 70-75 ° C for 2-3 hours until completely dry, heat the tube in a flame of an alcohol lamp until the contents are completely burned, as evidenced by light coloring the contents without black spots, pour 10 ml of water into test tubes, mix and centrifuge for 15 min at 3000 rpm, transfer 2 ml of the supernatant to clean tubes, add 0.3 ml of 0.006 M potassium rhodanide, 0.5 ml 2% sodium nitrite, 0.8 ml of a 10% solution of iron ammonium alum 2N. nitric acid and placed in a water bath with a temperature of 37 ° C for 30 minutes, then the tubes are placed in ice water for 1-2 minutes, then the optical density of the samples is measured on a spectrophotometer at a wavelength of 440 nm and the concentration of protein-bound iodine is determined by a calibration curve .