How urine is collected to determine daily protein loss. Urine protein (proteinuria). Determination of protein in urine

Protein in urine: methods of determination

Pathological proteinuria is one of the most important and constant signs of diseases of the kidneys and urinary tract. Determining the concentration of protein in the urine is a mandatory and important element of the study of urine. The detection and quantification of proteinuria is important not only in the diagnosis of many primary and secondary kidney diseases, but the assessment of changes in the severity of proteinuria in dynamics carries information about the course of the pathological process and the effectiveness of the treatment. The detection of protein in the urine, even in trace amounts, should be alarming in relation to possible disease kidney or urinary tract and requires re-analysis. Of particular note is the senselessness of the study of urine and, in particular, the determination of urine protein without observing all collection rules.

All methods for determining protein in urine can be divided into:

quality,

semi-quantitative,

Quantitative.

Qualitative Methods

All qualitative tests for protein in the urine based on the ability of proteins to denature under the influence of various physical and chemical factors. In the presence of protein in the test urine sample, either turbidity or flocculation appears.

Conditions for determining protein in urine based on the coagulation reaction:

Urine should be acidic. Alkaline urine is acidified with a few (2 - 3) drops of acetic acid (5 - 10%).

Urine should be clear. Turbidity is removed through a paper filter. If the haze persists, add talc or burnt magnesia (about 1 teaspoon per 100 ml of urine), shake and filter.

A qualitative sample should be carried out in two test tubes, one of them is a control one.

Look for turbidity should be on a black background in transmitted light.

Qualitative methods for determining protein in urine include:

Heller ring test,

sample with 15 - 20% sulfosalicylic acid,

boiling test, and others.

Numerous studies show that none of the a large number known methods for the qualitative determination of protein in urine does not allow obtaining reliable and reproducible results. Despite this, in most DLTs in Russia, these methods are widely used as screening - in the urine with a positive qualitative reaction, further quantitative determination of the protein is carried out. Of the qualitative reactions, the Heller test and the sulfosalicylic acid test are more commonly used, but the sulfosalicylic acid test is generally considered the most suitable for detecting pathological proteinuria. The boiling test is currently practically not used due to its complexity and duration.

Semi-quantitative methods

TO semi-quantitative methods relate:

Brandberg-Roberts-Stolnikov method,

determination of protein in urine using diagnostic test strips.

The Brandberg-Roberts-Stolnikov method is based on the Geller ring test, therefore, with this method, the same errors are observed as with the Geller test.

Currently, diagnostic strips are increasingly used to determine the protein in the urine. Bromophenol blue dye in citrate buffer is most often used as an indicator for the semi-quantitative determination of protein in urine on a strip. The protein content in the urine is judged by the intensity of the blue-green color that develops after contact of the reaction zone with urine. The result is evaluated visually or using urine analyzers. Despite the great popularity and obvious advantages of dry chemistry methods (simplicity, speed of analysis), these methods of urinalysis in general and protein determination in particular are not without serious shortcomings. One of them, leading to a distortion of diagnostic information, is the greater sensitivity of the indicator of bromophenol blue to albumin compared to other proteins. In this regard, the test strips are mainly adapted to the detection of selective glomerular proteinuria, when almost all of the urine protein is represented by albumin. With the progression of changes and the transition of selective glomerular proteinuria to non-selective (the appearance of globulins in the urine), the results of protein determination are underestimated compared to the true values. This fact makes it impossible to use this method determination of protein in the urine to assess the condition of the kidneys (glomerular filter) in dynamics. With tubular proteinuria, the results of protein determination are also underestimated. Protein testing with test strips is not a reliable indicator of low levels of proteinuria (most test strips currently available are not capable of detecting protein in urine at concentrations lower than 0.15 g/L). Negative results of protein determination on the strips do not exclude the presence of globulins, hemoglobin, uromucoid, Bence-Jones protein and other paraproteins in the urine.

Flakes of mucus with a high content of glycoproteins (for example, with inflammatory processes in the urinary tract, pyuria, bacteriuria) can settle on the indicator zone of the strip and lead to false positive results. False positive results may also be associated with high concentrations urea. Poor lighting and poor color perception can cause inaccurate results.

In this regard, the use of diagnostic strips should be limited to screening procedures, and the results obtained with their help should be considered only as indicative.

Quantitative Methods

correct quantitative determination of protein in urine in some cases it turns out to be a difficult task. The difficulties of its solution are determined by the following number of factors:

the presence in the urine of many compounds that can interfere with the course of chemical reactions;

significant fluctuations in the content and composition of urine proteins with various diseases, making it difficult to choose an adequate calibration material.

In clinical laboratories, the so-called "routine" methods for determining protein in urine are predominantly used, but they do not always provide satisfactory results.

From the point of view of an analyst working in a laboratory, a method designed to quantify protein in urine should meet the following requirements:

have a linear relationship between the absorption of the complex formed during the chemical reaction and the protein content in the sample in a wide range of concentrations, which will avoid additional operations when preparing the sample for research;

should be simple, do not require high qualification of the performer, be performed with a small number of operations;

have high sensitivity, analytical reliability when using small volumes of the test material;

be resistant to various factors (fluctuations in the composition of the sample, the presence of medicines and etc.);

have an acceptable cost;

be easily adaptable to autoanalyzers;

the result of the determination should not depend on the protein composition of the urine sample under study.

None of the currently known methods for the quantitative determination of protein in urine can fully claim to be the "gold standard".

Quantitative methods for determining protein in urine can be divided into turbidimetric and colorimetric.

Turbidimetric methods

Turbidimetric methods include:

determination of protein with sulfosalicylic acid (SSK),

determination of protein trichloroacetic acid(THU),

determination of protein with benzethonium chloride.

Turbidimetric methods are based on a decrease in the solubility of urine proteins due to the formation of a suspension of suspended particles under the influence of precipitating agents. The protein content in the test sample is judged either by the intensity of light scattering, determined by the number of light-scattering particles (nephelometric method of analysis), or by the weakening of the light flux by the resulting suspension (turbidimetric method of analysis).

The amount of light scattering in precipitation methods for detecting protein in urine depends on many factors: the speed of mixing the reagents, the temperature of the reaction mixture, the pH value of the medium, the presence of foreign compounds, photometric methods. Careful observation of the reaction conditions contributes to the formation of a stable suspension with a constant particle size and obtaining relatively reproducible results.

Some drugs interfere with the results of turbidimetric methods for determining protein in the urine, leading to the so-called "false positive" or "false negative" results. These include some antibiotics (benzylpenicillin, cloxacillin, etc.), radiopaque iodine-containing substances, sulfanilamide preparations.

Turbidimetric methods are difficult to standardize and often lead to erroneous results, but despite this, they are currently widely used in laboratories due to the low cost and availability of reagents. The most widely used method in Russia is the determination of protein with sulfosalicylic acid.

Colorimetric Methods

The most sensitive and accurate are colorimetric methods for determining total urine protein, based on specific color reactions of proteins.

These include:

biuret reaction,

lowry method,

methods based on the ability of various dyes to form complexes with proteins:

Ponceau S (Ponceau S),

Coomassie Brilliant Blue (Coomassie Brilliant Blue)

pyrogallol red (Pyrogallol Red).

From the performer's point of view, in the daily work of the laboratory with a large flow of research, the biuret method is inconvenient due to the large number of operations. At the same time, the method is characterized by high analytical reliability, allows the determination of protein in a wide range of concentrations and the detection of albumin, globulins and paraproteins with comparable sensitivity, as a result of which the biuret method is considered as a reference and is recommended for comparison of other analytical methods for detecting protein in urine. The biuret method for determining protein in urine is preferably performed in laboratories serving nephrological departments and used in cases where the results of determination using other methods are doubtful, as well as to determine the amount of daily protein loss in nephrological patients.

The Lowry method, which has a higher sensitivity than the biuret method, combines the biuret reaction and the Folin reaction for the amino acids tyrosine and tryptophan in the protein molecule. Despite its high sensitivity, this method does not always provide reliable results in the determination of protein content in urine. The reason for this is the non-specific interaction of Folin's reagent with non-protein components of urine (most often amino acids, uric acid, carbohydrates). The separation of these and other urinary components by dialysis or protein precipitation allows this method to be successfully used for the quantitative determination of protein in the urine. Some drugs - salicylates, chlorpromazine, tetracyclines can affect this method and distort the results of the study.

Sufficient sensitivity, good reproducibility, and ease of protein determination by dye binding make these methods promising, however high price reagents hinders their wider use in laboratories. At present, the method with pyrogallol red is becoming more widespread in Russia.

When examining the level of proteinuria, it must be borne in mind that different methods for determining proteinuria have different sensitivity and specificity for numerous urine proteins.

Based on empirical data, it is recommended to determine the protein by two different methods and calculate the true value using one of the following formulas: proteinuria = 0.4799 B + 0.5230 L; proteinuria = 1.5484 B - 0.4825 S; proteinuria = 0.2167 S + 0.7579 L; proteinuria = 1.0748 P - 0.0986 B; proteinuria = 1.0104 P - 0.0289 S; proteinuria = 0.8959 P + 0.0845 L; where B is the result of measurement with Coomassie G-250; L is the result of measurement with Lowry's reagent; P is the measurement result with pyrogallol molybdate; S is the result of the measurement with sulfosalicylic acid.

Given the pronounced fluctuations in the level of proteinuria in different times days, as well as the dependence of the protein concentration in the urine on diuresis, its different content in individual portions of the urine, it is now customary to assess the severity of proteinuria in the pathology of the kidneys by the daily loss of protein in the urine, that is, to determine the so-called daily proteinuria. It is expressed in g/day.

If it is impossible to collect daily urine, it is recommended to determine the concentration of protein and creatinine in a single portion of urine. Since the rate of creatinine release during the day is quite constant and does not depend on changes in the rate of urination, the ratio of protein concentration to creatinine concentration is constant. This ratio correlates well with daily protein excretion and, therefore, can be used to assess the severity of proteinuria. Normally, the protein/creatinine ratio should be less than 0.2. Protein and creatinine are measured in g/l. An important advantage of the method for assessing the severity of proteinuria by the protein-creatinine ratio is the complete elimination of errors associated with the impossibility or incomplete collection of daily urine.

Literature:

O. V. Novoselova, M. B. Pyatigorskaya, Yu. E. Mikhailov, "Clinical aspects of the detection and assessment of proteinuria", Handbook of the head of the CDL, No. 1, January 2007

A. V. Kozlov, "Proteinuria: methods for its detection", lecture, St. Petersburg, SPbMAPO, 2000

V. L. Emanuel, “ Laboratory diagnostics kidney diseases. Urinary Syndrome”, - Handbook of the head of the CDL, No. 12, December 2006.

IN AND. Pupkova, L.M. Prasolova - Methods for determining protein in urine (review of literature data)

Handbook of Clinical Laboratory Research Methods. Ed. E. A. Kost. Moscow, "Medicine", 197

Majority Development renal pathologies differs in that they can have a rather long latent (hidden) period. Therefore, such an analysis as a definition is undoubtedly the most important for doctors of many specialties.

Function of nephrons and proteinuria

During ultrafiltration, a large number of different protein molecules and amino acids enter the composition of the primary urine. It is not surprising, for most proteins the average size exceeds the threshold for passage through membranes. And amino acid molecules are polarly charged. This also plays a role in their "retention" on the surface of the membranes. However, during the passage of primary urine through the tubules of the nephron, almost all proteins and amino acids are reabsorbed (subjected to reabsorption) into the blood. Because of what, secondary urine can contain no more than hundredths of a gram of proteins.

Normal figures, when determining protein in urine by standard methods, should not exceed 0.033 grams per liter. For a day, an adult healthy person can excrete up to 0.15 grams. Moreover, about 1-2% of them are albumins. The remaining 90% are accounted for (there are about 30 types of them) and glycoproteins - the main proteins of membranes. The latter appear in the urine due to the activity of podocytes. Since, during the operation of any cell, permanent damage to the membrane is inevitable. But these damages are, firstly, not significant, and secondly, they are quickly “dragged out” due to the constant processes of its regeneration. Also, these 90% include mucoproteins - protein molecules that enter the lumen of the tubules during the work of the epithelium of the loop of Henle.

There are several types of proteinuria. They are built only on the basis of a urine test for daily protein.

• Normal proteinuria. The release of protein does not exceed the already known 0.15 grams per day.
• moderate proteinuria. It is set when more than 0.15, but less than 3.5 grams per day is allocated.
• Massive proteinuria means that more than 3.5 grams of protein molecules are excreted by the kidneys in 24 hours.

Causes of proteinuria and mechanisms of its development

According to the underlying mechanisms of ultrafiltration and reabsorption, excess protein excreted in the urine can result from damage to the tubules, glomeruli, and urinary tract.

• Glomerular injury. Leads to the so-called glomerular proteinuria. At normal conditions proteins are retained by the membrane. Only a small amount can pass through its pores. Therefore, the tubules do not provide for the presence of special mechanisms for the reabsorption of proteins, which practically do not get there. Here we are talking about albumins. Although they are retained by the membranes of podocytes, no more than 2% enter the composition of the primary urine, since due to their negative charge they are retained at the basement membranes and do not enter the cavity of the nephron capsule. However, when the membrane is destroyed, albumins rush into the cavity of the capsule and are no longer reabsorbed from the tubules. That is why a small degree of proteinuria is noted in glomerular pathology: the specific gravity of albumin does not allow the indicators that are used to determine the protein in the urine to respond more strongly.
• tubular pathology. Here, ultrafiltration is not disturbed. Since reabsorption suffers, the absolute degree of proteinuria will be significant due to the fact that a large amount of coarse proteins is included in the secondary urine. But even here it is necessary to make a reservation that more sensitive indicators are needed to determine it. Since the action of most of them is aimed at determining the number of charged protein particles. Which are primarily albumins and which in these situations do not increase significantly, or in all are within the normal range. As a result, urinalysis for total protein may give false results about the actual size of proteinuria.

Determination of protein in urine

It is important! The main way to record proteinuria is by testing for protein in the urine. What are indicators used for? The minimum threshold for the sensitivity of most of these indicator sticks is a value of 30 milligrams per liter.

However, here it must be remembered that the test strip is sensitive to protein concentration within hundredths of a millimeter from its surface. Therefore, with very dilute urine, the strip will show negative result, even if proteinuria is more than 50 or even 70 mg.

In order to avoid such "shortcomings" of laboratory diagnostic methods, it is best to conduct a urine test in the morning, when its concentration is highest. If at general analysis revealed proteinuria more than 0.033 g/l, to confirm it, a study is carried out on the daily protein content.

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. Minimal amount protein, determined by this method, 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 urine containing protein is layered 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% about or 33 mg in 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 urine under study, by analogy with 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.

Small amounts of protein are found in the daily urine of healthy individuals. However, such low concentrations cannot be detected using conventional methods research. 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 vertical position body;
- transient (stroke) albuminuria, which can be caused by various diseases of the digestive system, severe anemia, burns, injuries or physiological factors: heavy physical exertion, 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 kidney diseases (glomerulonephritis, nephrosis, nephrosclerosis, amyloidosis, nephropathy of pregnancy), disorders of renal hemodynamics (renal venous hypertension, hypoxia), trophic and toxic (including including medicinal) effects on the walls of 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.

Protein detection in urine by a simplified accelerated method is carried out by a colorimetric method using indicator paper, which is produced by Lachema (Slovakia), Albuphan, Ames (England), Albustix, Boehringer (Germany), Comburtest and others. The method consists in immersion in the urine of a special paper strip, impregnated with tetrabromophenol blue and citrate buffer, which changes its color from yellow to 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; when too alkaline urine(pH 6.5) a false positive result will be obtained, and if 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.

With renal proteinuria, not only albumins, but also other types of protein are excreted in the urine. A normal proteinogram (according to Seitz 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, having determined 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 urine. 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 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. Normally, the average pore size of the glomerular basement membrane is 2.9-4 A ° NM, which can pass proteins with 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 basal membranes of the tubules, the absence of large molecular proteins and the presence of increased amounts of medium and low molecular weight proteins are characteristic.

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 kidney glomeruli and reabsorbed in the proximal tubules. This allows 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). In the blood serum healthy people contains an average of 1.7 mg / l (fluctuations 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 with 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 in unlike pyelonephritis, there is no increase in the concentration of β 2 -microglobulin in the urine, which can be used to 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 the urine are detected using conventional methods of protein chemistry (ultracentrifugation, gel chromatography, various options 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, cortical or medulla), differential diagnosis kidney disease and determining the dynamics (attenuation and exacerbation) of the process in the renal parenchyma.

For 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.). In these diseases, a high activity of transamidinase, LDH, alkaline phosphatase and CP, hyaluronidase, LAP, as well as such non-specific enzymes as GST, catalase is found [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 kidney (acute kidney 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 the dynamics of amylazemia with a simultaneous decrease in amylasuria may indicate nephrosclerosis and wrinkling of the kidney, LAP has highest value at 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.

Instruction

Qualitative methods for the determination of protein in urine: Heller method, 20% sulfosalicylic acid test, boiling test, etc. Semi-quantitative methods: use of diagnostic test strips to determine protein in urine, Brandberg-Roberts-Stolnikov method. Quantitative methods: turbidimetric and colorimetric.

Determination of protein in the daily urine at a concentration of 0.033 g/liter or more is a pathology. As a rule, in the morning portion of urine, the protein concentration does not exceed 0.002 g / l, and in the daily urine protein concentration is not more than 50-150 mg of protein.

Sources:

• determination of protein in urine

Urine is a product of human metabolism. It is formed during the filtration of blood in the kidneys, which is why the composition of urine gives a clear description of the state of the human body.

Urine is a complex solution of more than 150 compounds. Some specific substances, for example, acetone, bile acids, protein, glucose, may be present only in certain diseases.

To control human health, first of all, it is necessary to determine the amount of urine. The norm is the formation of 1-1.8 liters of urine per day. When more than 2 liters of urine is excreted, this is a sign possible violation in the work of the kidneys, diabetes and a number of other diseases. If less than 0.5 liters of urine is formed per day, there is a blockage of the ureter or bladder.

urine color

The color of urine excreted depends on many factors, so it can vary, ranging from light yellow to orange. The presence of certain shades can be affected by certain foods, as well as medications taken by a person.

After taking medications, urine may become stained and acquire a reddish tint. If a person is actively moving, while he releases a large amount of sweat, urine will have an intense yellow, as well as when taking funds such as Nitroxoline or Biomycin.

If a person has not taken any coloring foods and medicines, but the color of his urine is different from usual, one can suspect the presence of a disease in the body. For example, in diseases of the liver, urine will have a dark yellow or greenish color.

The presence of blood in the excreted urine clearly indicates the presence of a stone in or renal bleeding, if pain is also observed.

If urination is difficult, this may indicate an inflammatory process caused by an infection in the bladder. But dirty and cloudy urine indicates serious illnesses kidneys.

Protein in the urine

There is no protein in the blood of a person or its amount is so small that it cannot be determined using laboratory tests. If a protein is detected in the urine, it is necessary to conduct repeated tests, since it may be present when a person wakes up in the morning, as well as after hard physical work or exercise in athletes.

To determine visually whether protein is present in the urine or not is 100% impossible. One can only guess when there is a large amount of whitish flakes in the urine.

If the protein in the urine is repeatedly detected, this indicates the presence of some kind of kidney disease. The inflammatory processes occurring in them provoke a slight increase in the amount of protein. If more than 2 grams are excreted in the urine, this is an alarm signal.

Pyelonephritis is an inflammatory disease that affects the renal pelvis, calyces, and parenchyma. In most cases, inflammation is caused by bacterial infection. Full recovery is possible only with timely diagnosis therefore, when symptoms appear, a thorough examination is necessary.