RU2688922C1 - Method of producing mixtures which imitate normal and pathological human urine used to teach students glucose-oxidase method of determining glucose in biological fluids - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, namely to laboratory research methods, and aims at teaching students to the glucose-oxidase method of quantitative determination of urine glucose using mixtures which imitate normal and pathological human urine. For this purpose, urine-simulating sodium phosphate buffer (pH 6.0–7.0), which is tinted with 1 % titanium yellow to a color similar to that of normal urine, without or with the addition of glucose powder (dextrose) to 0.5–2.0 % (28–112 mmol/l) of the volume of the urine-simulating mixture, respectively. Results of the glucose oxidase method are used to establish the presence or absence of glucosuria in the potential patient.EFFECT: use of invention provides 100 % accuracy of determining the glucose level in the urine simulator, enables to repeatedly and independently execute the training techniques by their safety, simplicity, reproducibility, uniqueness of interpretation, low cost for mastering and fixing abilities and skills for quantitative determination of glucose in urine by glucose oxidase method.1 cl, 1 tbl, 1 ex

Description

The invention relates to biomedicine, namely to medical and biological education, and can be used as a component of educational simulation technologies for the acquisition and consolidation of skills and abilities to master the glucose oxidase method of quantitative determination of glucose in biological fluids (urine) in laboratory classes for students of universities and colleges .

The curriculum of medical and biological specialties of universities and colleges, approved by the Ministry of Health of the Russian Federation and the Ministry of Science and Higher Education of the Russian Federation, includes sections on working with human body fluids. Currently, the epidemiological situation in our country and other countries of the world, due to the sharp increase in the number of carriers and patients with hepatitis, AIDS, other viral and bacterial infections, requires an age limit and licensing of medical personnel groups allowed to work with human biological fluids ( for example, the Decree of the Government of the Russian Federation No. 29 dated January 26, 2010 (as amended on September 4, 2012 No. 882) “On the approval of technical regulations on safety requirements for blood, blood products replacing blood creates and equipment used in transfusion, infusion therapy "[1]). In addition, ensuring safe working conditions and conducting training sessions in chemical laboratories and when working with chemicals in higher and secondary specialized educational institutions requires strict compliance with the established standards of sanitary and epidemiological control (for example, GOST 12.4.113-82. "Safety Standards System labor. Laboratory educational work. General safety requirements "(approved by the USSR State Standard of June 25, 1982, No. 2525 [2] or the current Order of the State Education of the USSR of 13.10.1989, No. 789" On the introduction into effect of the Rules for the safe handling of chemicals in higher and secondary specialized educational institutions, at enterprises and in institutions of the USSR State Education System ”[3]).

In this regard, the use of samples of human biological fluids, including urine, in the educational process is not feasible, which significantly limits the acquisition by students of medical and biological specialties of universities and colleges of practical skills, in particular, to determine the quantitative level of glucose, urea, uric acid, creatinine and other components of normal and pathological urine of patients. Such an approach also limits the pedagogical perspective of the formation of professional competence of a future doctor and a biologist.

The standard glucose oxidase method for the determination of glucose in biological fluids, which uses real blood, serum or human urine to determine the glucose level [4-6], was used as the prototype and also the method closest in technical essence to the method proposed by us. Human urine is an aqueous solution of salts containing chlorides, phosphates, sulfates, bicarbonates, as well as other more complex organic compounds (urea, uric acid, creatinine, certain hormones, etc.). Of the salts in the highest concentrations in the urine are NaCl, KCl, Na ₂ HPO ₄ , KH ₂ PO ₄ , that is, the components of the standard narcotic-phosphate buffer, the osmolarity and concentration of ions in solution of which correspond to concentrations in the human body, the optimum pH value is 6.0 -7.0. The principle of the glucose oxidase method is that glucose oxidase catalyzes the oxidation of glucose with air oxygen to glucuronic acid with the formation of an equimolar amount of hydrogen peroxide, which, under the action of peroxidase, promotes the oxidative combination of 4-aminoantipyrine and phenol (or its derivatives) to a quinone-colored, dyed in a pink-and-white mali . The intensity of the color is proportional to the concentration of glucose in the sample. The method is highly specific for glucose and gives reliable quantitative results for its content in the blood, urine and other biological fluids.

The disadvantage of this method, which is important when using standard diagnostic reactions in the educational process, are expensive and unsafe samples of real human biological fluids. In addition, it is not always necessary biological sample is available, especially for samples of patients with hyperglucosemia and glucosuria. However, the use of this method in relation to urine with an acidic pH value of 4.5-5.5 underestimates the concentration of glucose by inhibiting the activity of enzymes, which leads to a distortion of the results. The use of an artificially created urine simulator will completely eliminate the listed disadvantages.

The technical result of the proposed method is its repeated and independent reproduction by students due to safety, simplicity, reproducibility, uniqueness of interpretation, low cost, which will allow to develop confidence in the formation of professional competence of the future physician and biologist. The accuracy of reproducing the results of the proposed simulation method does not differ from the standard method using real biological fluids and is 100%.

The technical result of this method is achieved by obtaining mixtures that mimic human urine, used to teach students the glucose oxidase method for the quantitative determination of glucose in biological fluids, including the steps of preparing simulators of normal and pathological human urine, where sodium phosphate buffer (pH 6.0 -7.0), in both mixtures of simulators of normal and pathological urine add 1% solution of titanium yellow so that the color of simulators was similar to the color of holes In addition, glucose powder (dextrose) is added to the simulator of pathological urine to a concentration of 0.5-2.0% (28-112 mmol / l) of the volume of the mixture simulating urine.

The algorithm of the steps of the proposed method is similar to the algorithm of standard tests [4-6], as well as the visually diagnosed evaluation result:

Stage 1 - preparation of a simulator of normal human urine. A simulator of a sample of normal human urine is sodium phosphate buffer (pH 6.0-7.0), tinted with a 1% solution of titanium yellow to a color similar to normal urine, without the addition of glucose powder (dextrose). Sodium phosphate buffer is prepared according to the standard procedure: 8.00 g of NaCl, 0.20 g of KCl, 1.44 g of Na ₂ HPO ₄ and 0.24 g of KH ₂ PO _{4 are} dissolved in 800 ml of distilled water, the pH is adjusted to 6, 0-7.0 35% HC1 or 40% NaOH, add distilled water to 1 liter [7] or with the help of commercially available tablet (or powder) preparations of the companies Applichem (Germany) or Amresco (USA). Tablets (or powder) diluted with distilled water to a predetermined volume and get a solution of a given concentration. The acidity (pH) of the urine simulator is adjusted with a mandatory registration on the pH meter.

Stage 2 - preparation of a simulator of pathological human urine. A simulator of a sample of pathological human urine is sodium phosphate buffer (pH 6.0-7.0), tinted with a 1% solution of titanium yellow to a color similar to normal urine, with the addition of glucose powder (dextrose) to 0.5-2.0% (28-112 mmol / l) of the volume of the mixture simulating urine. Sodium phosphate buffer is prepared according to the standard procedure: 8.00 g of NaCl, 0.20 g of KCl, 1.44 g of Na ₂ HPO ₄ and 0.24 g of KH ₂ PO _{4 are} dissolved in 800 ml of distilled water, the pH is adjusted to 6, 0-7.0 35% HCl or 40% NaOH, add distilled water to 1 liter [7] or using commercially available tablet (or powder) preparations from the companies Applichem (Germany) or Amresco (USA). Tablets (or powder) diluted with distilled water to a predetermined volume and get a solution of a given concentration. The acidity (pH) of the urine simulator is adjusted with a mandatory registration on the pH meter.

Stage 3 - preparation of a set of reagents for determining the level of glucose in the urine by the glucose oxidase method. The simplest method is to use commercially available reagent kits for the implementation of the glucose oxidase method for the quantitative determination of glucose in biological fluids. The composition of the reagent kit to reproduce this method should be as follows:

1) Working reagent (phosphate buffer (pH 7.5) 100 mmol / l; phenol - 5 mmol / l; 4-aminoantipyrine - 0.5 mmol / l, glucose oxidase> 10,000 IU / l, peroxidase> 2000 IU / l ;

2) Calibrator (aqueous glucose solution) (glucose - 5.55 mmol / l).

The working reagent can be stored in a dark place in a tightly closed container at a temperature of + 2-8 ° C for 1 month. Standard glucose solution should be prepared before each use. All reagents at the concentrations used are non-toxic.

To engage with students the preparation of the steps of this method is carried out by a laboratory assistant.

An example of a method for the quantitative determination of glucose in biological fluids by the glucose oxidase method using human urine simulators (the actions presented in the example are performed by students).

The student is given an individual tripod, equipped with simulators of normal and pathological human urine, as well as a working reagent, a glucose calibrator and distilled water. All reagents on a tripod should be labeled. Together with the tripod, the student should be given: automatic dispensers, allowing you to select a volume of liquid 10 and 1000 μl, with appropriate tips; test tubes with a capacity of 5-10 ml; rubber or plastic gloves.

The successful implementation of the method also provides for the presence in the laboratory of a spectrophotometer (wavelength 510 nm) or a photoelectric colorimeter (wavelength 480-520 nm); a ditch with a thickness of the light absorbing layer of 10, 5 or 3 mm; the thermostat providing temperature + 37 ° С; stopwatch or hours. This method can be implemented using automatic or semi-automatic biochemical analyzers.

Conduct analysis.

The components of the reaction mixture using automatic dispensers contribute to the test tube in the quantities indicated in the table.

Samples are thoroughly mixed and incubated at + 37 ° C for 10 minutes or at room temperature (+ 18 ... + 25 ° C) for 20 minutes. Next, measure the optical density of the experimental samples and calibration samples against the control sample at a wavelength of 500 (480-520) nm in cuvettes per 5 mm (3 mm). When using a ditch of a different volume, the consumption of reagents can be proportionally changed while maintaining the ratio of the volume of the analyzed samples to the volume of the working reagent 1: 100. The color is stable for 60 minutes after the end of incubation, provided that there is no exposure to direct sunlight.

The concentration of glucose in experimental samples containing simulators of normal and pathological urine, calculated by the formula:

C = Eo / Ekal × 5.55, where

C is the concentration of glucose in the experimental samples, (mmol / l);

Eo is the optical density of the test sample, units wholesale pl .;

Ekal - optical density of the calibration sample, units wholesale pl .;

5.55 - glucose concentration in the calibration sample, mmol / l.

The normal glucose content in the native urine of a healthy person, as determined by this method, is <0.5 mmol / l. With the use of urine simulators, the results will be as follows: in the case of a simulator of normal human urine, the optical density of the test sample does not change, the glucose level is not determined, the result is considered negative; in the case of a simulator of pathological human urine, the glucose concentration in the sample will be 28-112 mmol / l.

Thus, according to the results of the analysis performed in the case of using a simulator of pathological human urine, a student diagnoses a patient's glucosuria, which is similar to the evaluation of the results of a standard test-prototype for determining the level of glucose in the urine using the glucose oxidase method.

The proposed method has passed the test in laboratory classes in biological chemistry for students of the 2nd course of the specialties "General Medicine", "Pediatrics", "Pharmacy" and "Biology" in the FSBEI HE "Petrozavodsk State University"; implemented and successfully used for 3 years at the Department of Biomedical Chemistry, Immunology and Laboratory Diagnostics of the Medical Institute when teaching students of medical and biological specialties.

The accuracy of reproducing the results of the simulation method is 100%. The algorithm of all stages is similar to the algorithm of the standard method for quantitative determination of glucose in the urine by the glucose oxidase method, as well as the diagnosed estimated result. Each student can repeatedly and independently reproduce this teaching method using textbooks and guidelines for the course of biological chemistry. This will strengthen the knowledge and skills of students, develop independence and confidence, which is important for the formation of professional competence of the future doctor and a specialist biologist.

This training method replaces expensive and unsafe samples of real human biological fluids (urine) with their simulators. The chemical and biological reagents used in it are available, affordable, safe in recommended concentrations. The teaching method may well be introduced into the educational process, used in laboratory studies of students of medical and biological specialties of universities and colleges in the development of professional competencies. The method can also be successfully used in clinical diagnostic rapid tests (with test strips) for determining the content of glucose in the urine.

BIBLIOGRAPHY

1. Decree of the Government of the Russian Federation dated January 26, 2010 No. 29 (as amended on September 4, 2012 No. 882) “On the approval of technical regulations on the safety requirements of blood, its products, blood-replacement solutions and technical means used in transfusion-infusion therapy " // Russian newspaper. - 2012.

2. GOST 12.4.113-82. Occupational safety standards system. Works educational laboratory. General safety requirements (approved by the Decree of the USSR State Standard of 25.06.1982, No. 2525). - M .: Publishing house of standards, 1982.

3. Order of the State Education of the USSR of 13.10.1989, No. 789 “On the implementation of the Rules for the safe handling of chemicals in higher and secondary specialized educational institutions, enterprises and institutions of the State Education System of the USSR”. - M .: USSR State Education, 1989.

4. Laboratory research methods in the clinic / Ed. V.V. Menshikov. - M .: Medicine, 1987. - 504 p.

5. Aleinikova T.L., Rubtsova G.V. Guide to practical classes in biological chemistry. - M .: Higher School, 1988. - 239 p.

6. Biochemistry. A guide to practical exercises: a manual. Chernov N.N., Berezov T.T., Burobina S.S. et al. / ed. N.N. Chernov. - M .: GEOTAR-Media, 2009. - 240 p.

7. Dawson R., Elliot D. Handbook of biochemistry / Translation from English. -M .: Mir, 1991. -544 p.

Claims (1)

The method of obtaining mixtures that mimic human urine, used to teach students the glucose oxidase method for quantitative determination of glucose in biological fluids, including the steps of preparing simulators of normal and pathological human urine, where sodium phosphate buffer (pH 6.0) is used as the basis for mixtures simulating urine. -7,0), characterized in that in both mixtures of simulators of normal and pathological urine add 1% solution of titanium yellow in such a way that the color of simulators was similar to the color of normal oh urine, glucose powder (dextrose) is additionally added to the pathological urine simulator to a concentration of 0.5-2.0% (28-112 mmol / l) of the volume of the mixture simulating urine.