Causes of increased protein in the urine. Daily urinalysis for proteinuria. Protein in urine - what does it mean? Treatment of proteinuria

Proteins are high molecular weight compounds that normally cannot pass through the filtering system of the kidneys (the system in which reabsorption takes place). However, a small amount of protein can pass into the urine. The norm of protein content in the urine should not exceed 0.033 g / l. During the day in the urine, the amount of protein can reach 50-100 mg. The permissible concentration of proteins in the urine test is called traces (up to 0.1 g / l). Part of the protein in the urine does not come from the blood, but from the bladder or urethra. During the day, the amount of protein in the urine can change. If the amount of protein in the urine exceeds the norm, this condition is called proteinuria. It may be physiological healthy people). Increased protein in the urine can be if a person consumed a large amount of protein food before the test, as well as after stress, nervous tension, heavy physical exertion or prolonged exposure to the cold. Such proteinuria disappears within a short period of time.

Proteinuria is determined after the collection of daily urine by qualitative, quantitative and semi-quantitative methods. Proteins in the human body tend to denature. It is on this ability that the principle of operation of qualitative methods for determining proteinuria is based. This is the first step in determining the amount of protein in the urine. After it, quantitative methods are used to exclude a false positive result. Quality methods include:

1. Heller ring test. The coagulation reaction is the basis of this study. In order for the result to be as accurate as possible, it is necessary to create an acidic reaction of urine (if it is alkaline), and also the urine itself must be transparent without the content of pathological impurities. The disadvantage of the method is its cost, duration and the likelihood of a false positive result.
2. Sample with a solution of sulfosalicylic acid. It is also based on the coagulation reaction and the urine must meet the same requirements as for the Heller ring test. However, this test is more preferable for detecting pathological proteinuria.
3. A method by which urine is boiled.

Express diagnostics can be carried out using test strips. This method is referred to as semi-quantitative methods for determining proteinuria. During the analysis, the test strip becomes a greenish tint, which becomes saturated if there is a large amount of protein in the urine. This method allows you to determine glomerular proteinuria.

The disadvantage of this method is that it does not make it possible to monitor the change in the amount of protein in the urine for a long time. There is a possibility of a false positive result if mucus is present in the urine. Quantitative methods are:

• Turbidimetric;
• Colorimetric.

In addition to measuring the amount of protein in a daily portion of urine, a number of other studies are used:

1. Urinalysis according to Zimnitsky;
2. Urinalysis according to Nechiporenko;
3. Urinalysis for microalbuminuria;
4. Urinalysis for sugar.

If you suspect proteinuria for self-diagnosis, you can use test strips that are sold in pharmacies. If deviations from the norm were noticed after using the strips, you should immediately consult a doctor.

Causes

There are three groups of elevated protein in the urine, the causes of which are:

• Burns, malignant processes in the body or hemolysis of red blood cells. This proteinuria is called prerenal.
• Kidney (tubular and glomerular) disease - renal proteinuria;
• Infectious and inflammatory processes of the genitourinary system - postrenal proteinuria. These include cystitis, urethritis, or orchiepididymitis.

Renal proteinuria is associated with damage to the renal filter, which increases the permeability of the glomerular epithelium and high-molecular compounds (proteins) enter the urine. It is also possible damage to the tubules of the kidneys. IN this case the protein is not reabsorbed and passes into the urine. Diseases that lead to renal proteinuria are:

1. Pyelonephritis;
2. Congenital anomalies of the kidneys;
3. Glomerulonephritis;
4. Amyloidosis of the kidneys;
5. Autoimmune pathologies in the body.

If protein is found in the urine, the causes are also physiological (stress, hypothermia) or pathological ( common diseases or pathology of the urinary system). Separately, among the reasons for the increased content of protein in the urine, pregnancy can be distinguished.

Proteinuria during pregnancy

If traces of protein appear in the urine during pregnancy, the cause is an increase in the load on the kidneys. The normal amount of protein in pregnant woman in the urine can reach 0.14 g/l. This condition is physiological. However, with late gestosis, an increase in the amount of protein and given state will be considered pathological. Proteinuria in pregnancy occurs due to impaired blood circulation in the juxtaglomerular apparatus of the kidney. Ischemia develops, dystrophic changes and a large amount of protein enters the urine. After childbirth, changes in the kidneys disappear and proteinuria disappears. An increase in protein in pregnant women is associated with toxic damage to the kidneys, which requires the prompt appointment of appropriate treatment. The first thing doctors can suggest is an abortion. In case of refusal of the mother, therapy is carried out, which is aimed at stabilizing the indicators and maintaining them throughout the pregnancy. A diet with increased protein in the urine in pregnant women is mandatory. It is necessary to reduce the consumption of foods rich in proteins. If proteinuria is detected in the urine test, a consultation with a nephrologist is mandatory. This condition usually occurs in the third trimester of pregnancy. Along with an increase in the protein content in the urine, there is an increase in pressure and swelling.

Symptoms

Symptoms for which proteinuria can be suspected are varied and depend on the cause of the pathology, as well as the degree of pathology. Depending on the amount of protein isolated, proteinuria is divided into:

• Weakly expressed (150-500 mg / day);
• Moderately expressed (500-2000 mg/day);
• Expressed (more than 2000 mg / day).

With mild proteinuria, accompanying symptoms may be absent or manifest slightly. Foamy urine appears and a slight deterioration in general well-being. Moderate proteinuria manifests itself:

1. Clouding of consciousness, headache, dizziness;
2. Weakness, fatigue, difficulty climbing stairs, drowsiness;
3. Decreased appetite, nausea, vomiting;
4. Edema on the lower extremities, face;
5. Increase in body temperature;
6. Increased pressure, tachycardia;
7. Changes skeletal system, bone deformities and pain in them;
8. Change in color of urine (appearance of red or white urine).

With severe proteinuria, these symptoms become more pronounced, a person periodically loses consciousness, is unable to walk for a long time, he has disturbances in all body systems.

Treatment

Therapy for proteinuria should be carried out under the supervision of a urologist and only after complete examination patient and determine the cause of the pathology. Treatment of protein in the urine with folk remedies is strictly prohibited, as there is a risk of developing severe complications up to death. After detecting a protein in the urine, it is necessary to take the tests again, since there is a possibility of a false positive result and the treatment in this case will have a negative effect on the body.

The answer to the question of how to reduce protein in the urine, first of all, is to follow a diet that excludes the intake of protein foods, and also requires the intake of large amounts of fluid. If the cause of proteinuria is an infectious and inflammatory disease of the kidneys or genitourinary system, antibiotics are prescribed or antiviral drugs along with non-steroidal anti-inflammatory drugs. The rest of the treatment is symptomatic and requires the elimination of the underlying disease. With proteinuria, the following groups of drugs can be used:

• Diuretics;
• Steroids;
• Immunosuppressants;
• antihypertensive drugs;
• Insulin.

Also, in parallel with drug therapy, decoctions of herbs are used that have diuretic properties (thyme, chamomile, horsetail, birch buds, lingonberry leaves), and also eat a large amount of fruits, vegetables and fish.

Prevention

In order to notice an increase in protein in the urine in time, each person, with a preventive purpose and with the appearance of changes in the genitourinary system, should regularly take general analysis urine and consult a urologist. The question of how to reduce protein in the urine should be dealt with exclusively by a doctor. Self-diagnosis and self-treatment in case of proteinuria are contraindicated due to the risk of complications. In order to avoid an increase in the amount of protein in the urine, it is necessary to follow some recommendations:

1. Eliminate the use of alcohol;
2. drink clean spring water drink during the day at least 1.5 liters water;
3. Do not abuse protein foods;
4. Avoid hypothermia and nervous overexertion;
5. Timely treat diseases of the genitourinary system, endocrine disorders and cardiovascular pathologies, since proteinuria is a complication of these diseases.

It is also worth avoiding infection in the genitourinary system and at the first symptoms of any disease in the body, seek help from a doctor.

The composition of the workplace for the determination of protein in urine includes the following elements:

1. Chemical test tubes, agglutination.
2. A set of graduated pipettes.
3. Pipettes with a narrow drawn end.
4. Alcohol lamps or gas burner.
5. Black paper.
6. Glacial acetic acid.
7. sulfosalicylic acid.
8. Concentrated nitric acid.
9. Distilled water.

Methods for determining protein in urine

All methods used for the qualitative determination of protein in urine are based on protein coagulation. Protein coagulation is manifested by turbidity expressed to varying degrees (from opalescence to high turbidity) or flaking.

Qualitative determination of protein in urine can be carried out in one of the following ways:

1. boiling with 10% acetic acid solution;
2. reaction with a 20% solution of sulfosalicylic acid;
3. reaction with 50% nitric acid solution (Geller test);
4. reaction with a 1% solution of nitric acid in a saturated solution of common salt (modified Geller test according to Larionova).

Before the qualitative determination of protein in the urine, the following preparatory work is carried out:
1. Turbid urine is filtered through a paper filter. If it is not possible to obtain a transparent filtrate, re-filtration through the same filter is performed, or urine is mixed with a small amount of diatomaceous earth or talc, after which it is filtered.
2. If urine has an alkaline reaction, it is acidified with a 10% solution of acetic acid to a slightly acidic reaction under the control of litmus or universal indicator paper.
3. With a low salt content (light yellow or pale yellow urine with a low specific gravity) to each
a few drops of a saturated sodium chloride solution are added to the sample, since the lack of salts causes the protein to coagulate.
4. The degree of turbidity is observed using a black background. The background is black cardboard or black paper used in photography. Accounting for the reaction on a black background allows you to identify the slightest degree of turbidity.

Numbered test tubes are placed in a separate rack. They produce one of the following reactions.

1. Boiling test with 10% acetic acid solution. This test requires a 10% solution of acetic acid, which is prepared as follows: 10 ml of glacial acetic acid is placed in a cylinder and topped up with distilled water to the 100 ml mark.

Protein determination technique. 10-12 ml of filtered urine of slightly acidic reaction is placed in a chemical test tube. Then the upper part of the test tube with urine is gently heated to a boil and 8-10 drops of a 10% solution of acetic acid are added to it. A test tube with urine is viewed against a black background in transmitted light. In the presence of protein in the urine, turbidity of varying degrees appears (from opalescence to great turbidity) or flakes fall out. The control is Bottom part test tubes that have not been heated. This test detects the amount of protein, starting from 0.015% o (% o - promille).

2. Reaction with 20% sulfosalicylic acid solution. A 20% solution of sulfosalicylic acid is prepared as follows: 20 g of sulfosalicylic acid is dissolved in 70-80 ml of distilled water, transferred to a 100 ml cylinder and topped up with distilled water to the mark. The prepared reagent is stored in a dark glass container.

Protein determination technique. In two tubes of the same diameter, 2-3 ml of filtered urine of a weakly acid reaction are placed, 3-4 drops of a 20% solution of sulfosalicylic acid are added to one of the tubes, the other tube serves as a control. If protein is present in the reagent tube, turbidity or flakes of coagulated protein will appear. In the control tube, the liquid remains clear. Sulfosalicylic acid, along with serum protein, precipitates albumoses (peptides), which are a product of protein breakdown. In order to clarify the cause of cloudy urine, the test tube with urine is heated. The turbidity caused by the serum proteins is enhanced, while the turbidity due to the presence of albumose disappears. This test has the same sensitivity as the previous one.

3. Reaction with 50% nitric acid solution (Geller test). A 50% solution of nitric acid is prepared as follows: 50 ml of distilled water (1:1 dilution) is added to 50 ml of nitric acid with a specific gravity of 1.2-1.4.

Protein determination technique. Pour 1 ml of 50% nitric acid into a narrow small test tube (agglutination ooze). 1 ml of filtered test urine is collected into a pipette with a narrow drawn end, layered on the reagent, and the tube is transferred to a vertical position. In the presence of protein, a white ring appears at the interface of the liquids. The time of the appearance of the ring, its properties depend on the amount of protein: if the protein is low, then the ring does not appear immediately, therefore, its appearance is monitored for 2.5-3 minutes. The minimum amount of protein determined by this method is 0.033°/oo. With a lower protein content in the urine, the ring does not form. Accounting for the results of the reaction produced on a black background in transmitted light.

4. The reaction with a 1% solution of nitric acid in a saturated solution of common salt is a modified Geller test (according to Larionova). For the test, a 1% solution of nitric acid is used, prepared in a saturated solution of common salt (Larionova's reagent). 35 g of common salt is dissolved in 100 ml of distilled water, the solution is filtered, 99 ml of the prepared saturated sodium chloride solution is added to 1 ml of concentrated nitric acid with a specific gravity of 1.2-1.4.

Protein Determination Technique the same as in the reaction with a 50% solution of nitric acid (Geller's test), but instead of 1 ml of a 50% solution of nitric acid, 1 ml of Larionova's reagent is poured into a test tube and 1 ml of urine is layered on it. The appearance of a white ring at the boundary of the liquids indicates the presence of protein in the test urine. The Larionova test is as sensitive as the Heller test.

5. Colorimetric (dry) test for the qualitative determination of protein. The colorimetric (dry) test for qualitative determination of protein in urine is based on the effect that protein has on the color of the indicator in a buffer solution.

Protein determination technique. A piece of indicator paper designed to determine the protein is immersed in urine for a short time. The sample is considered positive if the paper turns blue-green.

Quantification of protein in urine

The quantitative determination of protein in the urine is based on the fact that when layering urine containing protein on a 50% solution of nitric acid or Larionova's reagent, a white ring forms at the border of two liquids, and if a clear white ring appears by 3 minutes, then the protein content is 0.033% o or 33 mg per 1000 ml of urine. The appearance of the ring earlier than 3 minutes indicates a higher protein content in the urine.
When quantifying protein in urine, the following rules are followed:

1. Quantitative determination of protein is carried out only in those portions of urine where it was detected qualitatively.
2. The determination is made with carefully filtered urine.
3. Accurately follow the technique of layering the test urine on a 50% solution of nitric acid or Larionova's reagent in the ratio of the reagent to urine (1:1).
4. The time of appearance of the ring is determined by a stopwatch: in the final calculation of the amount of protein, the time of layering of urine on nitric acid is taken into account, which is 15 seconds.
5. Urine is diluted based on the properties of the ring. In this case, each subsequent dilution of urine is prepared from the previous one.
6. Rings are identified on a black background.

The most common are two methods for the quantitative determination of protein in urine: the Roberts-Stolnikov-Brandberg method and the method of S. L. Erlich and A. Ya. Althausen.

1. Roberts-Stolnikov-Brandberg method. According to this method, the amount of protein in the urine is determined by diluting it until the next layering of urine on a 50% solution of nitric acid or Larionov's ring reagent appears exactly by 3 minutes. The amount of protein is calculated by multiplying 0.033% by the degree of urine dilution. The result obtained expresses the amount of protein in milligrams per 1000 ml of urine, i.e. in ppm (% o).
2. The method of S. L. Erlich and A. Ya. Althausen. A number of agglutination tubes are placed in a rack, into which 1 ml of a 50% solution of nitric acid or Larionova's reagent is first poured. The test urine is taken with a separate clean, dry pipette with a narrow drawn-out end and layered on the reagent, after which the stopwatch is turned on. The time of appearance of the ring is monitored by placing the test tube on a black background. When the ring appears, the stopwatch is turned off.

When layering urine, depending on the amount of protein, a compact, wide or threadlike ring may appear. A compact, wide ring appears immediately after layering urine on the reagent. The thread-like ring may appear immediately, before the expiration of one minute, or in the interval from one to 4 minutes.

When a filiform ring appears within one to 4 minutes, it is not necessary to dilute the urine!
To calculate the amount of protein in this case, it is sufficient to use the table-plan proposed by the authors (Table 1).

Example 1 When layering urine on the reagent, a filiform ring formed after 2 minutes. If the ring had formed by 3 minutes, then the amount of protein would be 0.033%.

In this case, the ring formed earlier. The corresponding correction, according to the plan table, for a time of 2 minutes is 1 + 1/8. This means that the protein in this portion of urine will be 1 + 1/8 times more than 0.033 ° / oo, i.e. 0.033% o X (1 + 1/8) \u003d 0.037 ° / oo.

When a filiform ring appears up to 1 minute, i.e., after 40-60 seconds, one dilution of urine is made by 1.5 times (2 parts of urine + 1 part of water), and then the diluted urine is again layered on the reagent and the appearance of the ring is recorded. When calculating the results, it is taken into account that the urine was diluted 1.5 times.

Example 2 After layering urine diluted 1.5 times, a filiform ring appeared by 2 minutes. If the ring appeared by 3 minutes, then the protein would be 0.033%. The corresponding amendment according to the table-plan, for a time of 2 minutes is 1 + 1/8. Protein in the urine contains 0.033% oX1.5X (1 + 1/8) \u003d 0.056% o.

If the filiform ring appears immediately, the urine is diluted 2 times (1 part urine + 1 part water). The diluted urine is again layered on the reagent and the appearance of the ring is noted after 1 minute.

Example 3 When layering urine diluted 2 times on the reagent, a filiform ring appeared after 1 minute 15 seconds. Then the amount of protein in the test urine, by analogy with the previous calculations, will be equal to
0.033% oX2X (1 + 3/8) \u003d 0.091%.
If a wide ring appears, urine is diluted 4 times (1 part urine + 3 parts water).
With subsequent layering of diluted urine, a filiform ring can form both before and after one minute. In such cases, the calculation of the amount of protein is carried out by analogy with the previous examples, i.e., 0.033% o is multiplied by the degree of dilution and by the corresponding correction.

Example 1 The ring after dilution of urine 4 times appeared immediately. Urine is diluted 2 times. After layering urine diluted 8 times (4X2), a filiform ring formed after 1.5 minutes. In this case, the amount of protein is 0.033% oX8X1.25 \u003d 0.33% o, etc.
When a compact ring appears, urine is diluted 8 times (1 part urine + 7 parts water). Upon subsequent layering of diluted urine on the reagent, either a compact, or wide, or filiform ring may form.

Example 2 When urine was layered on nitric acid, a compact ring immediately formed. Urine is diluted 8 times (1 part of urine + 7 parts of water) and again it is layered. This again resulted in a compact ring. Then the urine is diluted another 8 times (for this, 1 part of the diluted urine is taken into a cylinder or into a test tube and 7 parts of water are added to it). After another layering of diluted urine, a filiform ring formed immediately. Urine is diluted 2 times (1 part urine + 1 part water). After the next layering of diluted urine, a filiform ring formed by 2 minutes. The calculation of the amount of protein in a given portion of urine is carried out as follows: 0.033,% oX8X8X2X (1 + 1/8) = 4.8% o.

In addition to the plan table, there is a table with calculated protein numbers (Table 2). If the urine is not diluted, then the amount of protein is found in the column "Whole undiluted urine". When diluting urine by an integer number of times (8,4,2), Table 1 is used. 1. When diluting urine by 1.5 times, use the table. 2.

Technique for using a table to determine the protein content in urine

In the corresponding columns of the table, the time of the appearance of the ring and the degree of urine dilution are indicated.
The number located at the point of intersection of the horizontal and vertical lines drawn from these two indicators indicates the amount of protein in the test urine (% o).

It is possible that with a positive qualitative test for protein, the ring does not form when layered on a 50% solution of nitric acid. This means that the amount of protein in the urine is less than 0.033%. In such cases, the amount of protein in the analysis form is referred to as "traces".

If the protein is quantified, the protein content in ppromille is noted in the urinalysis form, for example, “protein - 0.66% o”.

In addition to the quantitative determination of protein in a separate portion of urine, its daily amount in grams is calculated. For this purpose, daily urine is collected, its quantity is measured and the protein content in the promille is determined. Then a calculation is made. For example, the daily amount of urine is 1800 ml, protein - 7 ° / oo. This means that the protein in the daily amount of urine contains: 1.8X7 \u003d 12.6 g.

For the clinic, both qualitative and quantitative determination of protein in the urine is important.

Qualitative tests for the determination of protein in the urine
More than 100 reactions for the qualitative determination of protein in urine have been proposed. Most of them are based on the precipitation of protein by physical (heating) or chemicals. The presence of protein is proved by the appearance of turbidity.

Colorimetric dry samples are also of interest.

Only the most important samples for practice will be described below.

Sample with sulfosalicylic acid. To several milliliters of urine, add 2-4 drops of a 20% solution of sulfosalicylic acid. With a positive reaction, turbidity appears. The result is denoted by the terms: opalescence, weakly positive, positive or strongly positive reaction. The sulfosalicylic acid test is one of the most sensitive tests for determining protein in the urine. It detects even the most insignificant pathological increases in protein in the urine. Thanks to a simple technique, this test has found wide application.

Aseptol test. Aseptol is a substitute for sulfosalicylic acid. It can be prepared from materials available in any laboratory (phenol and sulfuric acid). A 20% solution of aseptol is used as a reagent. The test is carried out as follows: in a test tube containing 2-3 ml of urine, add 0.5-1 ml of aseptol solution to the bottom. If a white ring of coagulated protein forms at the interface between the two fluids, the test is positive.

Geller test. Under a few milliliters of urine, add 1-2 ml of 30% nitric acid (sp. weight 1.20). If a white ring appears at the interface of both liquids, the sample is positive. The reaction becomes positive if the protein is more than 3.3 mg%. Sometimes a white ring is obtained in the presence of large amounts of urates. Unlike the protein ring, the urate ring does not appear at the border between both fluids, but a little higher. Larionova suggests that instead of 30% nitric acid, use as a reagent a 1% solution of nitric acid in a saturated solution of common salt; this gives a great saving of nitric acid.

Test with ferruginous potassium and acetic acid. This reaction makes it possible to distinguish serum proteins from nucleoalbumins.

Equal amounts of urine are poured into two test tubes. A few drops of a 30% acetic acid solution are added to one of them. If there is haze compared to the control tube, the urine contains nucleoalbumin. If turbidity does not appear, the contents of both tubes are mixed and again divided into two parts. To one of the two test tubes, add a few drops (an excess can turn a positive sample into a negative one) of a 10% solution of yellow blood salt (potassium ferricyanide). In the presence of whey proteins, turbidity is obtained.

With concentrated urine containing large amounts of uric acid and urates, a test with potassium ferricyanide and acetic acid should be performed after preliminary dilution (2-3 times) of urine with water. Otherwise, cloudiness caused by settled uric acid may occur.

It has especially importance when examining the urine of infants containing a lot of uric acid and urates.

Of the other qualitative tests for protein in the urine, based on the precipitation of proteins, the following have been used: boiling test, Esbach, Purdy, Roberts, Almen, Balloni, Buro, Claudius, Corso, Dome, Goodmann-Suzanne, Jollet, Exton, Camlet, Kobuladze, Liliendal-Petersen, Polacci, Pons, Spiegler, Tanre, Thiele, Brown tests , Tsushiya and others.

When producing quality urine protein samples based on protein precipitation, the following should be observed: general rules, violation of which leads to significant errors in the study.

1. The urine to be tested must be acidic. In an alkaline reaction, the urine is slightly acidified with acetic acid. Producing an alkaline urine sample when acid is used as a reagent may result in neutralization of the acid and negative result with a positive reaction. This is especially true for the sulfosalicylic acid sample, since the acid is added in very small amounts and can be easily neutralized.

2. The urine to be tested must be clear.

3. Samples for the determination of protein in the urine should always be made in two test tubes, one of which serves as a control. Without a control tube, you may not notice slight turbidity in the reactions.

4. The amount of added acid in the samples should not be too large. A large amount of acid can lead to the formation of soluble acidalbumins and to the transformation of a positive sample into a negative one.

deserve great attention, thanks to its simple technique, colorimetric dry samples. These tests use the influence that a protein has on the color of an indicator in a buffer solution (the so-called indicator protein error). A filter paper strip impregnated with acidic citrate buffer and bromophenol blue as an indicator is immersed for a short time in the urine. The test is positive if a blue-green color is obtained. By comparing the color intensity with color paper standards, tentative and quantitative conclusions can also be drawn. Indicator papers are sold in reams of appropriate color standards, similar to universal indicator papers.

Methods for quantitative determination of protein in urine
Many methods have been proposed for the quantitative determination of protein in urine. Precise quantitative methods for the determination of proteins in biological material have not found wide application in the determination of protein in urine, due to complex and time-consuming techniques. Volumetric methods, especially the Esbach method, are widely used. They are very simple, but, unfortunately, they are not very accurate. The methods of the Brandberg-Stolnikov group are also convenient for the clinic, giving more accurate results than volumetric methods, with a relatively simple technique. In the presence of a photometer or nephelometer, nephelometric methods are also convenient.

Esbach method. It was proposed by the Parisian doctor Esbach in 1874. Urine and a reagent are poured into a special test tube (Esbach's albuminometer). The test tube is corked with a rubber stopper, thoroughly stirred (without beating!) and left in vertical position until the next day. They report the division, to which the column of protein sediment reaches. The number found shows the protein content. It is very important with the Esbach method that the urine is acidic. Alkaline urine can neutralize the acidic constituents of the reagent and prevent the precipitation of proteins.

Advantages of the method: it is simple and convenient in practice.

Disadvantages: the method is inaccurate, the result is obtained after 24 - 48 hours.

Brandberg-Stolnikov method. It is based on a qualitative Geller test. The Heller test can be used for quantitation as it gives a positive result above 3.3 mg% protein. This is the limiting protein concentration below which the sample becomes negative.

Erlich and Althausen modification. Soviet scientists S. L. Erlikh and A. Ya. Althausen modified the Brandberg-Stolnikov method, indicating the possibility of simplifying the study and saving time in its production.

The first simplification is related to the time of the appearance of the ring. The exact time of its appearance is determined, without necessarily adhering to the 2nd and 3rd minutes.

The second simplification makes it possible to establish what dilution should be made. The authors proved that the required dilution can be approximately determined by the type of the resulting ring. They distinguish filiform, wide
and a compact ring.

Of the nephelometric methods, it deserves to be noted Kingsberry and Clark method. 2.5 ml of filtered urine is poured into a small graduated cylinder, replenished with 3% aqueous solution sulfosalicylic acid up to 10 ml. Stir thoroughly and after 5 minutes photometer in a 1 cm cuvette, with a yellow filter, using water as a compensation liquid. With a Pulfrich photometer, the extinction found, multiplied by 2.5, gives the amount of protein in %o. In the case when the extinction index is higher than 1.0, the urine is pre-diluted 2 times, 4 times or even more.

In order to have a clear idea of ​​the amount of proteins excreted in the urine, it is necessary to determine not only their concentration in a separate portion of urine, but also their total daily amount. To do this, collect the patient's urine for 24 hours, measure its volume in milliliters and determine the protein concentration in a portion of daily urine in g%. The amount of proteins excreted in the urine in 24 hours is determined depending on the daily amount of urine in grams.

Clinical significance of protein in urine

Human urine normally contains minimum quantities protein, which cannot be established by ordinary qualitative samples of the study of protein in the urine. The excretion of large amounts of protein, in which ordinary qualitative tests for protein in the urine become positive, is an abnormal phenomenon called proteinuria. Proteinuria is physiological only in a newborn, in the first 4-10 days after birth. The commonly used name albuminuria is incorrect, because not only albumins, but also other types of proteins (globulins, etc.) are excreted in the urine.

Proteinuria, as a diagnostic symptom, was discovered in 1770 by Cotugno.

The most important functional renal proteinuria in children are as follows:

1. Physiological proteinuria newborn. It occurs in most newborns and has no adverse significance. It is explained by a weak kidney filter, damage at birth, or loss of fluids in the first days of life. Physiological proteinuria disappears on the 4-10th day after birth (later in premature babies). The amount of protein is small. It is nucleoalbumin.

Long-term neonatal albuminuria may be a symptom of congenital lues.

2. Stroke albuminuria. They are caused by exceeding the threshold of normal irritability of the renal filter by significant mechanical, thermal, chemical, mental and other irritations - loss of fluid in infants (dehydrational proteinuria), cold bathing, abundant, protein-rich food (alimentary proteinuria), palpation of the kidney (palpatory albuminuria), physical overwork, fear, etc.

Stroke albuminuria appears more easily in children at an early age than in older children and adults, since the kidneys of the chest and small child more easily irritated. Dehydration albuminuria (malnutrition, hydrolability, toxicosis, diarrhea, vomiting) is especially often observed in infants.

Stroke albuminuria is benign. They disappear immediately after the elimination of the causes that cause them. In a deposit sometimes there are single leukocytes, cylinders and erythrocytes. The protein is most often nucleoalbumin.

3. Orthostatic proteinuria. This condition is typical for preschool children and school age. It occurs on the basis of vasomotor disorders of the blood supply to the kidney. Typical for orthostatic albuminuria (hence its name) is that it appears only when the child is standing, when the spine is in a lordotic position. IN lying position she disappears. Nucleoalbumin is released. In doubtful cases, you can resort to orthostatic experience, which is as follows: in the evening, an hour before going to bed, the child empties the bladder; in the morning, getting out of bed, he urinates again. This urine does not contain protein. Then the child is put on his knees for 15-30 minutes with a stick behind his back, between the bent elbows of both hands. A position of lordosis is created, which leads to the release of protein, without changes in the sediment.

With orthostatic albuminuria, 8-10 g of protein can be secreted per day.

The most important clinical significance among all proteinuria are organic renal proteinuria. They are caused by organic diseases of the kidneys (nephritis, nephrosis, nephrosclerosis). Proteinuria is one of the most important and best known symptoms of organic kidney disease.

1. In acute and chronic glomerulonephritis, proteinuria occurs regularly. The amount of protein is moderate, and there is no parallel between the degree of proteinuria and the severity of the disease. In contrast, chronic and more severe nephritis often occurs with less protein than acute ones. After acute nephritis, sometimes for a long time (years), small amounts of protein in the urine are established, which do not have pathological significance ("residual albuminuria"). We should not forget that "nephritis without proteinuria" can also occur. Sometimes the protein is found in one portion of the urine, but not in the other. The ratio of albumins to globulins in acute nephritis is low, and in chronic nephritis it is higher.

2. With nephrosclerosis, the amount of protein in the urine is very small, often there are forms of the disease without protein in the urine.

3. Of all kidney disease nephrosis occur with the most pronounced proteinuria.

4. In infectious and toxic conditions, so-called febrile and toxic proteinuria occur. These are acute nephroses, in which the amount of protein is small. This group also includes proteinuria in convulsive conditions (convulsions), hyperthyroidism, jaundice, intussusception, enterocolitis, burns, severe anemia, etc. These albuminuria are benign and pass quickly (transient albuminuria).

5. With stagnation of blood in the kidneys, the so-called congestive albuminuria occurs, which is characteristic of heart patients in the stage of decompensation. It is also found in ascites and tumors of the abdomen.

With febrile, toxic and congestive albuminuria, the increased permeability of the renal filter is especially pronounced. According to some authors, many of these proteinuria occur without organic damage to the kidney parenchyma.

Extrarenal albuminuria are usually caused by protein impurities (secretions, decayed cells), which are excreted by diseased urinary tract and genital organs. Extrarenal albuminuria is more common due to cystopyelitis (pyuria), less often due to vulvovaginitis, calculi and tumors of the urinary tract.

With extrarenal albuminuria, a large number of leukocytes and bacteria are found in the sediment. Renal elements are almost never found. The amount of protein is small. Filtered or centrifuged urine usually does not test positive for protein.

In those recovering from pyelitis, albuminuria disappears after bacteriuria and pyuria.

It should be emphasized as a characteristic phenomenon that in the early childhood organic kidney disease is extremely rare, so organic proteinuria is also rare. Of these, there are mainly febrile and toxic. In contrast to organic proteinuria, stroke albuminuria is very common in children at an early age.

In older children, organic proteinuria is more often functional. In general, with age, functional proteinuria is less common, and organic more often.

Electrophoretic studies of proteins in urine

A number of authors use the electrophoretic method to study proteins in the urine (uroproteins). It can be seen from the obtained electrophoregrams that they have the same qualitative composition as plasma proteins. This indicates that the proteins in the urine are derived from plasma proteins.

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in the urine healthy child protein is contained in a small amount and is not detected by conventional qualitative samples. The amount of protein in the urine (proteinuria) increases with kidney disease, toxicosis, leukemia, pernicious anemia, congestion in the kidneys, after palpation of the kidneys (palpation albuminuria) and physical overwork, with emotional overload, fever, hypothermia and other conditions.
Qualitative determination of protein in urine. For the qualitative determination of protein in urine, the test with sulfosalicylic acid, the Heller test with nitric acid, the boiling test, etc. are most widely used. When using samples based on protein precipitation, it is important to follow some general rules in order to avoid errors.

1. Urine should be acidic. If the test urine is alkaline, it is slightly acidified by adding acetic acid. However, the amount of acid should not be large so as not to cause dissolution of the albumin.
2. Urine should be clear. If turbidity is present, it should be removed without causing protein precipitation.
3. The sample must be made in two test tubes - experimental and control. In the absence of control, you may not notice a slight turbidity of urine in an experimental test tube.

The sulfosalicylic acid test is one of the most sensitive to the presence of protein in the urine. 3-5 ml of urine is poured into a test tube and a 20% solution of sulfosalicylic acid is added at the rate of 2 drops per 1 ml of urine.
In another modification, for setting the sample, 1 ml of urine is poured into a test tube and 3 ml of a 1% solution of sulfosalicylic acid is added to it.

In the first and second cases, in the presence of protein in the urine, turbidity appears after the addition of sulfosalicylic acid. The result is taken into account by the intensity of turbidity: a weakly positive reaction (opalescence) is indicated by (short +), positive (+), sharply positive.
The Heller test is carried out as follows: several milliliters of urine are carefully (without mixing) layered on 1-2 ml of a 30% solution of nitric acid with a relative density of 1.20. If there is 0.033 g/l of protein or more in the urine, a white ring is formed at the border of both liquids, which is evaluated as a positive test. With a large amount of urate in the urine, a white ring can also form, but it is located just above the border between the liquids. With slight heating, the urate ring disappears.
The boil test gives reliable results, but only if the urine has a pH of 5.6. This test is best carried out in Ruppert's modification with Bange's acetate buffer, which includes 56.5 ml of glacial acetic acid, 118 g of crystalline sodium acetate, dissolved in 1 liter of water. Add 5 ml of urine to 1-2 ml of Bange's buffer and boil for 1/2 min. In the presence of protein in the urine, turbidity is formed.
Quantitative determination of protein in urine. For the quantitative determination of protein in urine, the Esbach method, the Brandberg-Roberts-Stolnikov method, the Sols biuret method, etc. are most often used.
The Brandberg - Roberts - Stolnikov method is based on a qualitative Geller test and allows you to determine the presence of protein in the urine from 0.033 g / l and above. A number of test tubes are being prepared. Urine containing protein is diluted with saline or water until a white ring ceases to form at the boundary of the liquids (Roberts-Stolnikov reagent and urine). A ring at the boundary of two liquids between the second and third minutes appears at a protein content of 0.033 g/L. If the ring appears earlier than after a few minutes, the urine is diluted with water. The highest dilution of urine, in which the ring is formed, contains 0.033 g/l of protein. The degree of dilution of the urine in the last tube is multiplied by 0.033 g/l and the concentration of protein in whole urine is obtained.

Proteinuria. In kidney disease, it is due to increased permeability of the renal filter. Protein can get into the urine in another way (from the mucous membranes of the urinary tract, vagina, prostate, etc.) - extrarenal proteinuria. Renal proteinuria is divided into organic and functional. Organic proteinuria is associated with damage to the kidney parenchyma, functional - with vasomotor disorders. One of the types of functional proteinuria is orthostatic (lordotic, intermittent, postural, cyclic) albuminuria. It is assumed that with a pronounced lordosis, a position is created in which the inferior vena cava is pressed against the spine by the liver, and this leads to stagnation in the renal veins and congestive albuminuria. Electron microscopic studies have established that orthostatic albuminuria has morphological signs of an inflammatory process in the renal glomeruli.

Our long-term observations show that orthostatic albuminuria is often caused by anomalies of the kidneys or their abnormal location. In this regard, we recommend using the orthostatic test as a screening test. After identifying orthostatic albuminuria with this test, we usually perform excretory urography to rule out abnormalities in the development of the urinary system.
Orthostatic test. On the eve of the test, in the evening, about an hour before bedtime, the child must empty the bladder. In the morning, getting out of bed, he immediately urinates, and this urine is collected in a separate bowl, marking it as a portion before the load. Then the child is offered to kneel on a semi-soft chair with a stick behind his back, clasping it with his elbows. In this position, the child should be 15-20 minutes, after which he empties the bladder and collected urine noted as a post-load portion. Protein is examined in portions of urine obtained before and after exercise. The detection or increase by 2-3 or more times in the protein content in the second portion (obtained after exercise) compared to the first portion is evaluated as a positive orthostatic test.
Determination of protein fractions in urine. Drawn elements are carried out. With glomerulonephritis, tuberculosis, polycystic kidney disease, kidney tumors, hemorrhagic vasculitis, collagenoses, inflammation of the bladder and other diseases of red blood cells in the urine, there may be a significant amount. There are macro- and microhematuria. With gross hematuria, it can already be macroscopically noted that the color of the urine has changed. Due to the presence of a large number of red blood cells in the urine, it becomes red or "the color of meat slops." With microhematuria, erythrocytes are detected only by microscopy of the sediment.
The penetration of erythrocytes into the urine during glomerulonephritis, intoxication is due to the increased permeability of the glomerular capillaries and their ruptures. In inflammatory diseases of the urinary tract, stones of the pelvis, ureters, bladder, erythrocytes enter the urine from damaged mucous membranes. When collecting urine in portions (two- and three-glass samples) during one urination, it is possible with a high probability to find out from which segment of the urinary system hematuria comes. So, with hematuria from the urethra, there may be blood clots in the first portion of urine. If hematuria is due to acute mucosal inflammation, a stone, or other bladder disease, more blood will be shed in the last urine stream. With hematuria associated with damage to the ureter, fibrin casts are sometimes found that correspond in shape to the lumen of the ureter. In diffuse kidney disease, hematuria stains the excreted urine evenly.
Leukocytes. In the urine of a healthy child, they may be single in the field of view. The detection of 5-7 leukocytes in each field of view indicates an inflammatory process in the urinary tract. However, it should always be excluded that leukocytes enter the urine from the external genitalia, which happens with phimosis, balanitis and balanoposthitis in boys and vulvovaginitis in girls. Two- and three-glass samples are widely used for leukocyturia.
cylinders. In urine, they can be in the form of hyaline, granular, epithelial and waxy casts. All of them can be formed in pathological conditions in the kidneys. Cylinders in the urine of healthy children are rare. Often they are found in quantitative methods of studying the urinary sediment. As a rule, these are hyaline cylinders, which are protein coagulated in the lumen of the tubules. Epithelial casts are indicative of damage to the renal parenchyma and consist of adherent epithelial cells of the renal tubules. With a more pronounced dystrophic process, granular and waxy cylinders appear in the kidneys. These are casts of torn cells of the tubular epithelium, which has undergone fatty degeneration. In addition, in the urine sediment, you can find cylinders formed from formed elements, hemoglobin, blood methemoglobin. The basis of such cylinders is usually protein, on which other elements are superimposed.
Cylindroids are formations similar to hyaline cylinders, consisting of crystals of ammonium urate salts, mucus, leukocytes, bacteria. Cylinderoids are found in the recovery phase in acute glomerulonephritis. They differ from hyaline cylinders in the heterogeneity of their structure.
inorganic residue. In the inorganic sediment in children, urates, oxalates, phosphates, and uric acid crystals are more common. Excessive excretion of them in the urine can lead to the formation of stones in the urinary tract.
Uraturia - increased excretion of uric acid salts in the urine. It is observed in the first days of life of newborns. Due to the significant amount of urate, the urine of newborns may have a brick red color. A large breakdown of cellular elements in newborns often leads to the formation of a uric acid infarction, which disappears by the end of the first week of life, since urate salts are removed with increasing diuresis. Uraturia in older children may be associated with eating large amounts of meat, with muscle fatigue, feverish conditions. Hyperuraturia may be due to hereditary hyperuricemia, which is especially pronounced in Lesch-Nyhan syndrome.
Oxalaturia - increased urinary excretion of calcium oxalate, may be associated with eating foods rich in oxalic acid. Products of this kind include sorrel, spinach, tomatoes, green pea, beans, radishes, tea, coffee, etc. The cause of oxalaturia is also a pathological process in the child's body, accompanied by tissue breakdown (dystrophy, tuberculosis, diabetes, bronchiectasis, leukemia, etc.). Oxalaturia is also known as a hereditary disease, often complicated by nephrolithiasis and chronic pyelonephritis. With severe oxalaturia, the content of oxalates in daily urine is 3-4 times or more higher than the permissible value (the norm is 8-10 mg%).
Phosphaturia - increased urinary excretion of phosphate salts that precipitate in alkaline urine. It is observed when eating plant products (vegetables, fruits, etc.), as well as during the inflammatory process in the urinary tract mucosa, when bacterial fermentation and alkalinization of urine occur. Phosphaturia can cause the formation of bladder stones.

Small amounts of protein are found in the daily urine of healthy individuals. However, such small concentrations cannot be detected using conventional research methods. The excretion of larger amounts of protein, at which the usual qualitative tests for protein in the urine become positive, is called proteinuria. There are renal (true) and extrarenal (false) proteinuria. In renal proteinuria, protein enters the urine directly from the blood due to an increase in its filtration by the glomeruli of the kidney or a decrease in tubular reabsorption.

Renal (true) proteinuria

Renal (true) proteinuria is functional and organic. Among functional renal proteinuria, the following types are most often observed:

Physiological proteinuria of newborns, which disappears on the 4th - 10th day after birth, and in premature babies a little later;
- orthostatic albuminuria, which is typical for children aged 7-18 years and appears only in the upright position of the body;
- transient (stroke) albuminuria, which can be caused by various diseases of the digestive system, severe anemia, burns, injuries or physiological factors: severe exercise stress, hypothermia, strong emotions, abundant, protein-rich food, etc.

Organic (renal) proteinuria is observed due to the passage of protein from the blood through damaged areas of the endothelium of the renal glomeruli in diseases of the kidneys (glomerulonephritis, nephrosis, nephrosclerosis, amyloidosis, nephropathy in pregnancy), disorders of renal hemodynamics (renal venous hypertension, hypoxia), trophic and toxic (including medicinal) effects on the walls of the glomerular capillaries.

Extrarenal (false) proteinuria

Extrarenal (false) proteinuria, in which the source of protein in the urine is an admixture of leukocytes, erythrocytes, bacteria, urothelial cells. observed in urological diseases ( urolithiasis disease, tuberculosis of the kidneys, tumors of the kidney and urinary tract, etc.).

Determination of protein in urine

Most qualitative and quantitative methods for determining protein in urine are based on its coagulation in the volume of urine or at the interface of media (urine and acid).

Among the qualitative methods for determining bedka in urine, the unified test with sulfosalicylic acid and the Heller ring test are most widely used.

A standardized sample with sulfasalicylic acid is carried out as follows. 3 ml of filtered urine are poured into 2 tubes. In one of them add 6-8 drops of a 20% solution of sulfasalicylic acid. Both tubes are compared against a dark background. Turbidity of urine in a test tube with sulfasalicylic acid indicates the presence of protein. Before the study, it is necessary to determine the reaction of urine, and if it is alkaline, then acidify with 2-3 drops of a 10% solution of acetic acid.

The Geller test is based on the fact that in the presence of protein in the urine at the border of nitric acid and urine, it coagulates and a white ring appears. 1-2 ml of a 30% solution of nitric acid is poured into a test tube and exactly the same amount of filtered urine is carefully layered along the wall of the test tube. The appearance of a white ring at the interface between two fluids indicates the presence of protein in the urine. It should be remembered that sometimes a white ring is formed in the presence of a large amount of urates, but unlike the protein ring, it appears slightly above the boundary between two liquids and dissolves when heated [Pletneva N.G., 1987].

The most commonly used quantitative methods are:

1) the unified Brandberg-Roberts-Stolnikov method, which is based on the Heller ring test;
2) photoelectrocolorimetric method for the quantitative determination of protein in the urine by the turbidity formed by the addition of sulfasalicylic acid;
3) biuret method.

The detection of protein in the urine by a simplified accelerated method is carried out by a colorimetric method using indicator paper manufactured by Lachema (Slovakia), Albuphan, Ames (England), Albustix, Boehringer (Germany), Comburtest, etc. blue, depending on the protein content in the urine. Tentatively, the concentration of protein in the test urine is determined using a standard scale. To obtain correct results, the following conditions must be met. urine pH should be in the range of 3.0-3.5; with too alkaline urine (pH 6.5) will be obtained false positive result, and at too acid urine(pH 3.0) - false negative.

The paper should not be in contact with the test urine for longer than indicated in the instructions, otherwise the test will give a false positive reaction. The latter is also observed when there is a large amount of mucus in the urine. Sensitivity various kinds and series of paper may be different, so the quantitative assessment of protein in the urine by this method should be treated with caution. Determining its amount in daily urine using indicator paper is impossible [Pletneva N.G., 1987]

Definition of daily proteinuria

There are several ways to determine the amount of protein excreted in the urine per day. The simplest is the Brandberg-Roberts-Stolnikov method.

Methodology. 5-10 ml of thoroughly mixed daily urine is poured into a test tube and a 30% solution of nitric acid is carefully added along its walls. In the presence of protein in the urine in an amount of 0.033% (i.e. 33 mg per 1 liter of urine), a thin, but clearly visible white ring appears after 2-3 minutes. At a lower concentration, the test is negative. With a higher protein content in the urine, its amount is determined by repeated dilutions of urine with distilled water until the ring ceases to form. In the last test tube in which the ring is still visible, the protein concentration will be 0.033%. Multiplying 0.033 by the degree of urine dilution, determine the protein content in 1 liter of undiluted urine in grams. Then the protein content in the daily urine is calculated by the formula:

K \u003d (x V) / 1000

Where K is the amount of protein in daily urine (g); x is the amount of protein in 1 liter of urine (g); V is the amount of urine excreted per day (ml).

Normally, from 27 to 150 mg (average 40-80 mg) of protein is excreted in the urine during the day.

This test allows you to determine in the urine only fine proteins (albumin). More precise quantitative methods (colorimetric Kjeldahl method, etc.) are quite complex and require special equipment.

In renal proteinuria, not only albumins are excreted in the urine, but also other types of protein. A normal proteinogram (according to Zeitz et al., 1953) has the following percentage: albumins - 20%, α 1 -globulins - 12%, α 2 -globulins - 17%, γ-globulins - 43% and β-globulins - 8%. The ratio of albumins to globulins changes with various kidney diseases, i.e. the quantitative ratio between protein fractions is broken.

The most common methods for fractionating uroproteins are the following: salting out with neutral salts, electrophoretic fractionation, immunological methods (radial immunodiffusion reaction according to Mancini, immunoelectrophoretic analysis, precipitation immunoelectrophoresis), chromatography, gel filtration, and ultracentrifugation.

In connection with the introduction of methods for fractionating uroproteins based on the study of electrophoretic mobility, variability in molecular weight, size and shape of uroprotein molecules, it became possible to isolate the types of proteinuria characteristic of a particular disease, to study the clearances of individual plasma proteins. To date, more than 40 plasma proteins have been identified in urine, including 31 plasma proteins in normal urine.

Selective proteinuria

IN last years the concept of proteinuria selectivity appeared. In 1955, Hardwicke and Squire formulated the concept of "selective" and "non-selective" proteinuria, determining that the filtration of plasma proteins into the urine follows a certain pattern: the greater the molecular weight of the protein excreted in the urine, the lower its clearance and the lower its concentration in the final urine. Proteinuria, corresponding to this pattern, is selective, in contrast to non-selective, for which the perversion of the derived pattern is characteristic.

The detection of proteins with a relatively large molecular weight in the urine indicates the absence of selectivity of the renal filter and its pronounced damage. In these cases, one speaks of a low selectivity of proteinuria. Therefore, at present, the determination of protein fractions of urine using the methods of electrophoresis in starch and polyacrylamide gels has become widespread. Based on the results of these research methods, one can judge the selectivity of proteinuria.

According to V.S. Makhlina (1975), the most justified is the determination of the selectivity of proteinuria by comparing the clearances of 6-7 individual blood plasma proteins (albumin, traneferrin, α 2 - macroglobulin, IgA, IgG, IgM) using accurate and specific quantitative immunological methods of the reaction of radial immunodiffusion according to Mancini, immunoelectrophoretic analysis and precipitation immunoelectrophoresis. The degree of proteinuria selectivity is determined by the selectivity index, which is the ratio of the compared and reference proteins (albumin).

The study of the clearances of individual plasma proteins allows obtaining reliable information about the state of the filtration basement membranes of the glomeruli of the kidney. The relationship between the nature of proteins excreted into the urine and changes in the basement membranes of the glomeruli is so pronounced and constant that the uroproteinogram can indirectly judge pathophysiological changes in the glomeruli of the kidneys. Fine the average size the pores of the glomerular basement membrane is 2.9-4 A ° NM, which can pass proteins having a molecular weight of up to 10 4 (myoglobulin, acid α 1 - glycoprotein, immunoglobulin light chains, Fc and Fab - IgG fragments, albumin and transferrin).

With glomerulonephritis, nephrotic syndrome, the pore sizes in the basement membranes of the glomeruli increase, and therefore the basement membrane becomes permeable to protein molecules. big size and masses (ceruloplasmin, haptoglobin, IgG, IgA, etc.). With an extreme degree of damage to the glomeruli of the kidneys, giant molecules of blood plasma proteins (α 2 -macroglobulin, IgM and β 2 -lipoprotein) appear in the urine.

Determining the protein spectrum of urine, one can conclude that certain parts of the nephron are predominantly affected. For glomerulonephritis with a predominant lesion of glomerular basement membranes, the presence of large and medium molecular weight proteins in the urine is characteristic. For pyelonephritis with a predominant lesion of the basement membranes of the tubules, the absence of macromolecular and the presence of increased amounts medium and low molecular weight proteins.

β 2 -Microglobulin

In addition to well-known proteins such as albumin, immunoglobulins, lipoproteins. fibrinogen, transferrin, urine contains plasma microprotein proteins, among which β 2 -microglobulin, discovered by Berggard and Bearn in 1968, is of clinical interest. Having a low molecular weight (relative molecular weight of 1800), it freely passes through the glomeruli of the kidney and is almost completely reabsorbed in the proximal tubules. This makes it possible to use the quantitative determination of β 2 -microglobulin in blood and urine to determine glomerular filtration and the ability of the kidneys to resorb proteins in the proximal tubules.

The concentration of this protein in blood plasma and urine is determined by radioimmunoassay using standard set"Phade-bas β 2 -mikroiest" (Pharmacia, Sweden). The blood serum of healthy people contains an average of 1.7 mg / l (range from 0.6 to 3 mg / l), in the urine - an average of 81 μg / l (maximum 250 μg / l) β 2 -microglobulin. Its excess in urine over 1000 mcg/l is a pathological phenomenon. The content of β 2 -microglobulin in the blood increases in diseases accompanied by impaired glomerular filtration, in particular in acute and chronic glomerulonephritis, polycystic kidney disease, nephrosclerosis, diabetic nephropathy, acute renal failure.

The concentration of β 2 -microglobulin in the urine increases in diseases accompanied by a violation of the reabsorption function of the tubules, which leads to an increase in its excretion in the urine by 10-50 times, in particular, with pyelonephritis, chronic renal failure, purulent intoxication, etc. It is characteristic that with cystitis, unlike pyelonephritis, there is no increase in the concentration of β 2 -microglobulin in the urine, which can be used for differential diagnosis these diseases. However, when interpreting the results of the study, it must be taken into account that any increase in temperature is always accompanied by an increase in the excretion of β 2 -microglobulin in the urine.

Average blood and urine molecules

Medium molecules (SM), otherwise called protein toxins, are substances with a molecular weight of 500-5000 daltons. Their physical structure is unknown. The composition of SM includes at least 30 peptides: oxytocin, vasopressin, angiotensin, glucagon, adrenocorticotropic hormone (ACTH), etc. Excessive accumulation of SM is observed with a decrease in kidney function and a large amount of deformed proteins and their metabolites in the blood. They have a variety of biological effects and are neurotoxic, cause secondary immunosuppression, secondary anemia, inhibit protein biosynthesis and erythropoiesis, inhibit the activity of many enzymes, and disrupt the phases of the inflammatory process.

The level of SM in blood and urine is determined by a screening test, as well as by spectrophotometry in the ultraviolet zone at a wavelength of 254 and 280 mm on a DI-8B spectrophotometer, as well as dynamic spectrophotometry with computer processing in the wavelength range of 220-335 nm on the same Beckman spectrometer. The content of SM in the blood is taken as the norm, equal to 0.24 ± 0.02 arb. units, and in urine - 0.312 ± 0.09 arb. units
Being normal products the vital activity of the body, they are normally removed from it at night by glomerular filtration by 0.5%; 5% of them are disposed of in another way. All SM fractions undergo tubular reabsorption.

Non-plasma (tissue) uroproteins

In addition to blood plasma proteins, there may be non-plasma (tissue) proteins in the urine. According to Buxbaum and Franklin (1970), non-plasma proteins account for approximately 2/3 of all urinary biocolloids and a significant proportion of uroproteins in pathological proteinuria. Tissue proteins enter the urine directly from the kidneys or organs anatomically associated with the urinary tract, or enter the blood from other organs and tissues, and from it through the basement membranes of the glomeruli of the kidney into the urine. In the latter case, the excretion of tissue proteins into the urine occurs similarly to the excretion of plasma proteins of various molecular weights. The composition of non-plasma uroproteins is extremely diverse. Among them are glycoproteins, hormones, antigens, enzymes (enzymes).

Tissue proteins in urine are detected using conventional methods of protein chemistry (ultracentrifugation, gel chromatography, various types of electrophoresis), specific reactions to enzymes and hormones, and immunological methods. The latter also make it possible to determine the concentration of non-plasma uroprotein in the urine and, in some cases, to determine the tissue structures that have become the source of its appearance. The main method for detecting non-plasma protein in urine is immunodiffusion analysis with antiserum obtained by immunization of experimental animals with human urine and subsequently depleted (adsorbed) by blood plasma proteins.

Examination of enzymes in blood and urine

In the pathological process, profound disturbances in the vital activity of cells are observed, accompanied by the release of intracellular enzymes into the liquid media of the body. Enzymodiagnostics is based on the determination of a number of enzymes released from the cells of the affected organs and not characteristic of blood serum.
Studies of the human and animal nephron have shown that in its individual parts there is a high enzymatic differentiation, closely related to the functions that each department performs. The glomeruli of the kidney contain relatively small amounts of various enzymes.

The cells of the renal tubules, especially the proximal ones, contain the maximum amount of enzymes. Their high activity is observed in the loop of Henle, direct tubules and collecting ducts. Changes in the activity of individual enzymes in various kidney diseases depend on the nature, severity and localization of the process. They are observed before the appearance of morphological changes in the kidneys. Since the content of various enzymes is clearly localized in the nephron, the determination of one or another enzyme in the urine can contribute to the topical diagnosis of the pathological process in the kidneys (glomeruli, tubules, cortex or medulla), the differential diagnosis of renal diseases and the determination of the dynamics (attenuation and exacerbation) of the process in the renal parenchyma.

For the differential diagnosis of diseases of the genitourinary system, the determination of the activity in the blood and urine of the following enzymes is used: lactate dehydrogenase (LDH), leucine aminopeptidase (LAP), acid phosphatase (AP), alkaline phosphatase (AP), β-glucuronidase, glutamine-oxaloacetic transaminase (GST), aldolase, transamidinase, etc. The activity of enzymes in blood serum and urine is determined using biochemical, spectrophotometric, chromatographic, fluorimetric and chemiluminescent methods.

Enzymuria in kidney disease is more pronounced and regular than enzymemia. It is especially pronounced in the acute stage of the disease (acute pyelonephritis, trauma, tumor decay, kidney infarction, etc.). These diseases are found high activity transamidinase, LDH, alkaline phosphatase and CP, hyaluronidase, LAP, as well as such non-specific enzymes as GST, catalase [Polyantseva LR, 1972].

Selective localization of enzymes in the nephron upon detection of LAP and ALP in the urine allows us to speak with confidence about acute and chronic diseases kidneys (acute renal failure, renal tubular necrosis, chronic glomerulonephritis) [Shemetov VD, 1968]. According to A.A. Karelin and L.R. Polyantseva (1965), transamidinase is found only in two organs - the kidney and the pancreas. It is a mitochondrial enzyme of the kidneys and is normally absent in the blood and urine. With various diseases of the kidneys, transamidinase appears in the blood and urine, and with damage to the pancreas - only in the blood.

The differential test in the diagnosis of glomerulonephritis and pyelonephritis Krotkiewski (1963) considers the activity of alkaline phosphatase in the urine, the increase of which is more typical for pyelonephritis and diabetic glomerulosclerosis than for acute and chronic nephritis. Increasing in dynamics amylasemia with a simultaneous decrease in amylasuria may indicate nephrosclerosis and wrinkling of the kidney, LAP is most important for pathological changes in the glomeruli and convoluted tubules of the kidney, since its content in these parts of the nephron is higher [Shepotinovsky V.P. et al., 1980]. For the diagnosis of lupus nephritis, the determination of β-glucuronidase and CF is recommended [Privalenko M.N. et al., 1974].

When evaluating the role of enzymuria in the diagnosis of kidney disease, the following provisions should be taken into account. Enzymes, being by their nature proteins, with a small molecular weight can pass through intact glomeruli, determining the so-called physiological enzyme. Among these enzymes, α-amylase (relative molecular weight 45,000) and uropepsin (relative molecular weight 38,000) are constantly detected in the urine.

Along with low-molecular enzymes in the urine of healthy individuals, other enzymes can also be found in small concentrations: LDH, aspartate and alanine aminotransferases, alkaline phosphatase and CP, maltase, aldolase, lipase, various proteases and peptidases, sulfatase, catalase, ribonuclease, peroxidase.

High-molecular enzymes with a relative molecular weight of more than 70,000-100,000, according to Richterich (1958) and Hess (1962), can enter the urine only if the permeability of the glomerular filter is impaired. The normal content of enzymes in the urine does not allow to exclude pathological process in the kidney with ureteral occlusion. With epimuria, the release of enzymes is possible not only from the kidneys themselves, but also from other parenchymal organs, cells of the mucous membranes of the urinary tract, prostate gland, as well as formed elements of urine with hematuria or leukocyturia.

Most enzymes are nonspecific to the kidney, so it is difficult to determine where the enzymes found in the urine of healthy and sick people come from. However, the degree of enzymuria, even for non-specific enzymes in kidney damage, is higher than normal or that observed in diseases of other organs. More valuable information can be provided by a comprehensive study of the dynamics of a number of enzymes, especially organ-specific ones, such as transaminase.

In resolving the issue of the renal origin of the enzyme in the urine, the study of isoenzymes helps to identify fractions typical of the organ under study. Isoenzymes are enzymes that are isogenic in action (catalyze the same reaction), but heterogeneous in chemical structure and other properties. Each tissue has its own isoenzyme spectrum. Valuable methods for the separation of isoenzymes are electrophoresis in starch and polyacrylamide gels, as well as ion-exchange chromatography.

Bence Jones protein

With multiple myeloma and Waldenström's macroglobulinemia, Bence-Jones protein is found in the urine. The method for detecting this protein in urine is based on the thermoprecipitation reaction. Previously used methods that evaluate the dissolution of this protein at a temperature of 100 ° C and re-precipitation upon subsequent cooling are unreliable, since not all Bence-Jones protein bodies have the appropriate properties.

More reliable detection of this paraprotein by its precipitation at a temperature of 40-60 °C. However, even under these conditions, precipitation may not occur in too acidic (pH< 3,0—3,5) или слишком щелочной (рН >6,5) urine, with low OPM and low Bence-Jones protein concentration. The most favorable conditions for its precipitation are provided by the method proposed by Patnem: 4 ml of filtered urine is mixed with 1 ml of 2 M acetate buffer pH 4.9 and heated for 15 minutes in a water bath at a temperature of 56 °C. In the presence of Bence-Jones protein, a pronounced precipitate appears during the first 2 minutes.

At a Bence-Jones protein concentration of less than 3 g / l, the test may be negative, but in practice this is extremely rare, since its concentration in the urine is usually more significant. Boil samples cannot be fully relied upon. With complete certainty, it can be detected in the urine by immuno-electrophoretic method using specific sera against heavy and light chains of immunoglobulins.