Proteinuria (proteinuria) - the appearance of protein in the urine in concentrations that make it possible to identify it with qualitative methods.
Distinguish
- Renal proteinuria and
- Extrarenal (postrenal) proteinuria
Renal proteinuria
Renal proteinuria is caused by damage to the glomerular filter or dysfunction of the epithelium of the convoluted tubules.
Distinguish between selective and non-selective proteinuria depending on the ratio of certain plasma and urinary proteins, their molecular weight and charge.
Selective proteinuria
Selective proteinuria occurs with a minimal (often reversible) violation of the glomerular filter, represented by low molecular weight proteins (molecular weight not exceeding 68,000) - albumin, ceruloplasmin, transferrin.
Non-selective proteinuria
Non-selective proteinuria is more common with more severe filter damage, when large molecular proteins begin to be lost. The selectivity of proteinuria is an important diagnostic and prognostic feature.
Renal proteinuria can be:
- organic and
- functional (physiological).
Organic renal proteinuria
Organic renal proteinuria occurs with organic damage to the nephron. Depending on the predominant mechanism of occurrence, certain types of organic proteinuria can be distinguished.
Glomerular proteinuria
Glomerular proteinuria - due to damage to the glomerular filter, occurs with glomerulonephritis and nephropathy associated with metabolic or vascular diseases. (glomerulonephritis, hypertension, infectious and allergic factors, cardiac decompensation)
tubular proteinuria
Tubular proteinuria - associated with the inability of the tubules to reabsorb plasma low molecular weight proteins that have passed through an unchanged glomerular filter. (amyloidosis, acute tubular necrosis, interstitial nephritis, Fanconi syndrome)
Prerenal proteinuria
Prerenal proteinuria (excessive) - develops in the presence of an unusually high plasma concentration of a low molecular weight protein, which is filtered by normal glomeruli in an amount exceeding the physiological ability of the tubules to reabsorb. (multiple myeloma, muscle tissue necrosis, erythrocyte hemolysis)
Functional renal proteinuria
Functional renal proteinuria is not associated with kidney disease and does not require treatment.
Functional proteinuria include:
- marching,
- emotional
- cold,
- intoxication,
- orthostatic (only in children and only in a standing position).
Extrarenal (postrenal) proteinuria
With extrarenal (postrenal) proteinuria, protein can enter the urine from the urinary and genital tract (with colpitis and vaginitis - with improperly collected urine). AT this case it is nothing more than an admixture of inflammatory exudate.
Extrarenal proteinuria usually does not exceed 1 g/day and is often transient.
Diagnosis of extrarenal proteinuria is helped by a three-cup test and urological examination.
Postrenal proteinuria occurs with cystitis, urethritis.
Methods for determining protein in urine
Necessary condition when conducting research on the presence of protein is the absolute transparency of urine.
Quality samples
Sample with sulfosalicylic acid
3–4 ml of filtered urine is poured into two test tubes. Add 6–8 drops of a 20% solution of sulfosalicylic acid to an experimental test tube. The second tube is the control. On a dark background, compare the control tube with the experimental one. In the presence of protein in urine samples, an opalescent turbidity appears.
The result is indicated as follows:
- the reaction is weakly positive (+),
- positive (++),
- sharply positive (+++).
The sample is highly sensitive.
You can also use a dry test, when a few crystals of sulfosalicylic acid or a filter paper pre-soaked with a solution of this acid are added to several milliliters of urine.
False positive results may be due to the intake of iodine preparations, sulfa drugs, large doses of penicillin and the presence of high concentrations of uric acid in the urine.
Nitric acid test (Geller test)
1–2 ml of a 50% solution of nitric acid is poured into a test tube, then an equal amount of urine is layered on the acid. In the presence of protein, a white ring appears at the interface between two liquids. Sometimes a reddish ring is formed slightly above the boundary between the liquids. purple from the presence of urates. The urate ring, unlike the protein ring, dissolves with slight heating.
Bright sample
The Bright boil test and proteinuria screening tests (dry colorimetric samples) require virtually no reagents.
When urine containing protein is boiled, it denatures, forming a cloudy precipitate or flakes that do not dissolve in 6% acetic acid, unlike phosphate salts. Screening tests are based on the ability of a protein (albumin) to change the color of paper coated with an indicator (usually bromophenol blue) and a buffer. A direct relationship between the color intensity of the indicator paper (Albufan, Albutest - Czech Republic; Labstix, Multistix - USA; Comburtest - Germany) and the amount of protein makes it possible to roughly estimate the amount of proteinuria. However, currently used screening tests are not without drawbacks. In particular, bromophenol blue does not detect Bence-Jones protein.
Quantitative Methods
Brandberg-Roberts-Stolnikov method
The method is based on a qualitative sample with nitric acid. The course of the test is described above. The appearance of a thin ring at the border of two liquids between the 2nd and 3rd minutes after layering indicates the presence of 0.033 g / l of protein in the urine (protein concentration in urine is usually expressed in ppm, i.e., in grams per liter). If the ring appears earlier than after 2 minutes, the urine should be diluted with water. Such a dilution of urine is selected so that when it is layered on nitric acid, the ring appears at the 2-3rd minute. The degree of dilution depends on the width and compactness of the ring and the time of its appearance.
The protein concentration is calculated by multiplying 0.033 g/l by the degree of urine dilution (Table 8).
The Roberts-Stolnikov dilution method has a number of disadvantages: it is subjective, time-consuming, the accuracy of determining the protein concentration decreases as the urine is diluted.
The most convenient and accurate are the nephelometric and biuret methods.
Nephelometric method
It is based on the property of the protein to give turbidity with sulfosalicylic acid, the intensity of which is proportional to the concentration of the protein. 1.25 ml of filtered urine is poured into a graduated test tube and a 3% solution of sulfosalicylic acid is added to a volume of 5 ml, thoroughly mixed. After 5 minutes, the extinction is measured on the FEK-M (or any other photometer) at a wavelength of 590–650 nm (orange or red light filter) against the control in a cuvette with a layer thickness of 0.5 cm. For control, 1.25 ml of filtered urine is used ( the same), to which an isotonic sodium chloride solution is added to a volume of 5 ml.
A calibration curve is preliminarily built depending on the extinction value on the protein concentration. Albumin standard solution (from human or bovine serum) is used to prepare various protein concentrations. Complete the worksheet.
Biuret method
It is based on the ability of the protein to give with copper sulfate and caustic alkali a violet biuret complex, the color intensity of which is directly proportional to the amount of protein. To 2 ml of urine, add 2 ml of trichloroacetic acid solution to precipitate the protein and centrifuge. The supernatant is discarded. To the sediment (protein) add 4 ml of 3% NaOH solution and 0.1 ml of 20% copper sulfate solution, stir and centrifuge. The violet supernatant is photometered at a wavelength of 540 nm (green light filter) against distilled water in a cuvette with a layer thickness of 1.0 cm. The protein concentration is determined from a table obtained empirically (the calibration curve is built as in the previous method).
Orthostatic test
Indicated for suspected orthostatic proteinuria and nephroptosis. After complete emptying Bladder the subject maintains a horizontal position for 2 hours. Then, without getting up, he passes one (control) portion of urine. Over the next 2 hours, the subject walks continuously, maintaining the position of maximum lumbar lordosis (holds a stick behind the lower back), after which he passes the second portion of urine. In both portions of urine, the protein concentration and protein content in grams are determined, and in case of nephroptosis, the number of red blood cells in 1 ml. With orthostatic proteinuria, proteinuria or a 2–3-fold increase in the initial protein content in grams is detected in the second serving. The appearance of hematuria, often in combination with trace proteinuria in the second portion, is characteristic of nephroptosis.
Determination of Bence-Jones uroproteins
Bence-Jones proteins are thermolabile low molecular weight paraproteins (relative molecular weight 20,000–45,000) found mainly in multiple myeloma and Waldenström's macroglobulinemia. They are light L-chains of immunoglobulins. Due to their small molecular weight, L-chains easily pass from the blood through an intact renal filter into the urine and can be determined there using a thermoprecipitation reaction. The study is advisable to conduct only with a positive test with sulfosalicylic acid. The definition is carried out as follows. To 10 ml of urine, add 3–4 drops of a 10% solution of acetic acid and 2 ml of a saturated sodium chloride solution, gently heat in a water bath, gradually increasing the temperature. If there are Bence-Jones proteins in the urine, then at a temperature of 45–60 ° C diffuse turbidity appears or a dense white precipitate forms. On further heating to boiling, the precipitate dissolves, and on cooling it reappears. This sample is not sensitive enough and must be checked by electrophoresis and immunoelectrophoresis.
Many diseases occur without pronounced clinical manifestations, therefore, the determination of protein in the urine for the purpose of timely detection and treatment pathological condition is important point for practical medicine.
Protein in urine can be determined by qualitative and quantitative methods.
Qualitative Methods
Currently, there are about 100 known qualitative reactions for protein. They consist in the precipitation of protein by the method of physical or chemical influences. With a positive reaction, turbidity occurs.
The most informative are the samples:
- With sulfosalicylic acid. It is considered the most sensitive and with its help it is possible to determine even the smallest amounts of protein bodies in the urine. The description of the result with a trace presence of protein is indicated by the term "opalescence", and with a larger amount - "weakly positive", "positive" and with a large loss of protein in the urine - "strongly positive reaction".
- With an acid substituent - aseptol. A solution of the substance is added to the urine, and when a ring is formed at the boundary of the solutions, it is said that the sample is positive.
- Geller. Produced using a solution of nitric acid. The result of the conduct is interpreted similarly to the one with aseptol. Sometimes the ring may be present during the presence of urates in the test fluid.
- With acetic acid with the addition of selezistosinerodisty potassium. With a high concentration of urine during such a test, it is diluted, otherwise a false positive result may be obtained, since the reaction will be to urates and uric acid.
Incorrectly conducting such a test can often give an incorrect result in newborns, since they produce urine with a high content of uric acid.
The basic rules for sampling are as follows - it is necessary that the urine being examined be transparent, have a slightly acidic environment (for this, a small amount of acetic acid is sometimes added to it), there should be two test tubes for control.
quantitation
When a urine test is performed, total protein is also determined by quantitative methods. There are a lot of them, but the following are most often used:
- Esbach method. Used since the 19th century. To do this, urine and a reagent are poured into a certain test tube. Then the mixture is shaken a little, and left closed for 24-48 hours. The resulting precipitate is counted according to the division in the test tube. The correct conclusion can be drawn only with acidic urine. This technique is quite simple, but it does not have high accuracy and is time consuming.
- Brandberg-Stolnikov method. Based on the Heller test, which allows you to get a result at a protein concentration of more than 3.3 mg%. Later, this method was modified and simplified.
- Nephelometric methods for determining the amount of protein are widely used.
- For correct result the first morning portion is poured, the collection begins with the second portion in one container, which is recommended to be kept in the refrigerator.
Application of test strips
The urine protein test works on the principle of indicators. Special strips can change their color depending on the protein concentration. They are convenient for determining changes that occur at different times, and are used both at home and in any medical and preventive institutions. Test strips
Test urine strips are used when needed early definition and monitoring the results of treatment for genitourinary pathologies. This diagnostic technique is sensitive, and reacts to albumin in its concentration from 0.1 g / l., and allows you to determine the qualitative and semi-quantitative changes in the protein content in the urine.
Based on the results of this diagnosis, it is possible to monitor the effectiveness of therapy, amend it, and prescribe the necessary diet.
All methods for determining protein in urine are based on protein coagulation under the influence of chemical or thermal agents. In the presence of protein in the urine, turbidity appears, the degree of which depends on the amount of protein.
A) quality tests determination of protein in the urine - are mandatory.
1. Sample with nitric acid- in a test tube with 1-2 ml of 50% nitric acid solution, carefully layer an equal amount of urine, trying not to shake the liquid. In the case of the presence of protein in the urine, a white ring appears at the border of the two liquids, better seen against a black background.
2. Sample with sulfasalicylic acid- 4-5 ml of urine is poured into a test tube and 8-10 drops of the reagent are added. In the presence of protein in the urine, depending on its amount, turbidity or flocculation may occur.
3. Express test (dry diagnostic test)- a strip of indicator paper "Albufan" is immersed in the test urine so that both indicator zones are moistened simultaneously (the upper zone is for pH determination, the lower one is for protein determination). After 2-3 seconds, the strip is placed on a white glass plate. The assessment is carried out 60 seconds after wetting the strip with urine, using a color scale printed on the case with indicator strips.
B) quantitative samples- carried out in those portions of urine where the protein was found during its qualitative determination; the determination is carried out in the supernatant after centrifugation
Brandberg-Roberts-Stolnikov method- 1-3 ml of 50% nitric acid solution is poured into a test tube and the same amount of urine is carefully layered along the wall. Time is recorded on a stopwatch. If the ring at the interface of the liquids is formed immediately or earlier than 2 minutes after layering, the urine must be diluted with water. After that, a repeated determination of protein in diluted urine is performed. Dilution is carried out until a white ring appears between the 2nd and 3rd minutes when the diluted urine is added to nitric acid. The amount of protein is determined by multiplying 0.033 ppm by the degree of dilution.
18. Technique for taking smears for flora, gonococci, Trichomonas, cytological examination, KPI.
Flora smear technique: the material is taken from the cervical canal and from the urethra with a special brush under visual control. The resulting sample is immediately placed on a glass slide and triturated.
Technique for taking smears for Trichomonas: first, the material is taken by scraping from the mucous membrane of the urethra (after its preliminary massage for 1 min on the pubic symphysis) and the posterior fornix of the vagina, then the vaginal part of the cervix is wiped with a sterile swab moistened with saline, the mucous plug is removed, in cervical canal carefully, a probe is inserted to a depth of no more than 1.0-1.5 cm and a scraping is taken from the mucous membrane of the cervix.
Smear technique for gonococci: the material is taken from the urethra, Bartholin glands and paraurethral passages after wiping them with a cotton swab moistened with saline, vaginal tweezers or a special probe. From the rectum, the material is taken with a blunt spoon. In chronic and torpid gonorrhea, a provocation is performed before the study to increase the likelihood of identifying the pathogen.
You can not take the material with a cotton swab and during menstruation.
Technique for taking smears for cytological examination: a smear is taken from the surface of the exocervix, vagina and vulva with a spatula, from the endocervix - with an endo brush. The material is applied in a thin layer on a specially treated skimmed glass and treated with a special compound to prevent the cells from drying out. Preparations are stained according to the Papanicolaou method (the so-called Pap smear) and microscoped.
Karyopyknotic index- percentage of surface cells with pycnotic nuclei to cells with vesicular (non-pycnotic) nuclei. CRPD at the beginning of the follicular phase menstrual cycle 25-30%, by the time of ovulation 60-70%, in the luteal phase it decreases to 25%.
Urine is an important biological fluid in the human body. Most of the products of exchange are removed with it. In a healthy person, its composition has relatively fixed values. When a disease occurs, some indicators change, which makes it possible for the attending physicians to clarify the diagnosis. An analysis of daily urine for protein will help identify problems in the functioning of the kidneys.
Analysis of daily urine or daily diuresis
This is a laboratory study that determines the amount of fluid released per day. This method is used to assess the state of functioning of the kidneys. Composition of urine:
Each component in the norm should contain a certain amount, the deviation from which is considered a pathology. In the study of daily protein intake, the norm for adult men and women is the same. During normal kidney function, this substance should not enter the urine or it may contain a small amount of 40-80 mg. Isolation of 150 mg or more is considered a pathology. However, with age, especially after 60 years, a slight excess is allowed. allowable indicator, and this is related to age-related changes in the body. In some cases, protein detection is due to improper preparation before collecting biomaterial or nutritional errors. In children, the norm depends on age, weight and body surface area.
general information
According to the results of laboratory studies of daily urine, the following information is obtained:
- The volume of fluid that is excreted during the day from the body. On average, it is 1750 ml and can fluctuate both up and down depending on the amount of fluid you drink.
- Sugar. This indicator is especially important for patients with diabetes mellitus.
- Oxalates. Exceeding the permissible limits is fraught with the formation of sand and stones in the kidneys.
- Metanephrine. This substance is formed after the breakdown of hormones. Deviations from the norm in the direction of increase are a sign of kidney pathology, of cardio-vascular system and etc.
- Protein. In healthy people, this indicator should not be observed in urine. This is one of the important parameters that is detected in the daily analysis of urine. An overestimated level indicates signs of kidney pathology, diseases of the central nervous system. In addition to the total amount of protein, a laboratory study of the biological fluid also allows you to detect protein compounds, which are also important for the correct diagnosis.
Indications for the appointment of a daily urine test for protein
Due to the fact that changes in the qualitative and quantitative composition of urine begin earlier than the symptoms of the disease, the analysis allows timely detection of pathology. Indications for the appointment of this type of research in adults and children is the presence of the following diseases:
- amyloidosis (violation of protein metabolism);
- diabetes;
- nephropathy, which is manifested by swelling of the legs;
- ischemia of the heart;
- kidney failure.
In addition, it is prescribed when the patient takes certain drugs: aminoglycosides, ACE inhibitors, thiazide diuretics, and some other drugs.
Preparatory stage
For greater information content and obtaining the most objective information based on the results of the analysis of daily urine for protein, certain rules must be observed, which are mandatory and are as follows:
- For a day or more, exclude the use of vitamin complexes, anticoagulants and diuretics.
- Change your diet in a day. Refuse spicy, fatty and sweet foods, as well as alcoholic and coffee drinks.
- Do not donate biomaterial during menstruation.
- To collect urine, purchase a special container at a pharmacy or you can use glass jar, with a volume of at least three liters.
for protein analysis?
The rules for collecting biomaterial are common for both adult men and women and children who have reached a certain age and can use the potty on their own.
- Immediately before collecting urine, it is necessary to perform a toilet of the external genital organs.
- The first portion of the biomaterial is not taken into account. However, the time of morning urination is fixed.
- Collect all excreted fluid within 24 hours. At night, urine collection also continues.
- It is recommended to store containers with biomaterial at a temperature not higher than 8 degrees and not lower than 5.
- Immediately after taking the last portion, the collected urine should be thoroughly mixed.
- Beforehand, the doctor should clarify how to take a daily urine test for protein in the laboratory. There are two ways. About 100 ml of biomaterial is poured into a container small size and take it to the laboratory or you can deliver the entire daily volume in a large container.
- In the case when the change in diuresis per day matters, the doctor recommends fixing the volume of any fluid consumed within 24 hours.
How to collect a daily urine test for protein in children under one year old? This question is of interest to young mothers. When prescribing such a study for babies, pediatricians are advised to purchase a special device for collecting biomaterial, which is freely sold in pharmacies. Do not squeeze liquid out of diapers or catch the moment when the baby wants to urinate.
Proteinuria or protein in daily urine
This term refers to the high levels of protein substances in urine. It monitors the functioning of the kidneys. There are the following types of proteinuria. Light - is not a harbinger of disease and occurs for natural reasons. Moderate and severe - these types indicate problems in the work of the kidneys. In these cases, in addition to protein in the daily urine test, there are symptoms such as:
- bone pain;
- dizziness;
- loss of appetite;
- drowsiness;
- nausea;
- vomit;
- severe fatigue.
Causes of protein in urine:
- heart failure;
- diseases of the central nervous system;
- hyperthyroidism;
- infective endocarditis.
A change in the composition of urine is indicated by its shade, the appearance of a whitish color is a sign of the presence of albumin, a substance of a protein nature.
Types of proteinuria
The main factor influencing the appearance of protein compounds in urine is a malfunction in the absorption of this substance. Distinguish proteinuria:
- tubular. In some diseases, protein absorption is impossible or difficult.
- Glomerular. In this case, protein molecules do not linger and are excreted along with the liquid. This phenomenon is typical for the following pathologies: pyelonephritis, kidney damage by toxins, glomerulonephritis.
- Extrarenal. Damage present urinary tract characteristic of urethritis, colpitis and cystitis.
To determine the type of proteinuria, its microscopic examination is used. Further, if necessary, the doctor prescribes other types of examinations, including ultrasound, magnetic resonance imaging and other hardware methods.
Protein in the urine during pregnancy. The reasons
Protein is a building material that takes part in various processes in the body of an individual. Its detection in urine is considered a sign of the development of pathological processes. The renal corpuscles lose their integrity and protein enters the urine along with harmful substances, and the cause of this failure is the inflammatory process in the kidneys. Urine analysis allows you to recognize the disease in a timely manner, make sure correct work kidneys in the most responsible and important period in a woman's life. Any, even the most insignificant detection of protein in pregnant women in a daily urine test is a sign of the onset of the development of pathology. The causes of its appearance are the following diseases:
- glomerulonephritis;
- lupus erythematosus;
- nephritis;
- hypertension;
- diabetes diabetes.
As well as infectious and inflammatory processes in the kidneys, their injuries of a mechanical nature, burns, hypothermia, poisoning with toxic substances or the treatment of malignant neoplasms with chemotherapeutic drugs before pregnancy.
Safe pregnancy
In order to diagnose kidney diseases, a simple method is used - this is a daily urine test for protein. Compliance with the conditions for proper collection urine is a must for any woman in position.
- The external genitalia must be clean. You can wash with regular soap. The use of antiseptics or herbal preparations is prohibited, as they will distort the reliability of the results.
- Collect urine in a clean, dry container with a wide neck.
If a protein is found in the urine, the doctor prescribes a treatment that will protect the fetus from the negative effects of the disease that caused the appearance of protein substances in urine. Next, you should find out the reason for this phenomenon. In most cases, it lies in the violation of the kidneys. The appearance of protein in the biomaterial in the second half of pregnancy is one of the symptoms of preeclampsia. Then edema, drops in blood pressure join.
- daily urine test for protein;
- regular visits to the doctor;
- pressure control;
- nutrition, enhanced with vitamin complexes;
- moderate fluid intake;
- refusal of spices and, if possible, salt or reducing its amount to a minimum.
With a preventive purpose, as well as to reduce protein compounds in the urine, the doctor may prescribe herbal medicines or decoctions of herbs that have a diuretic effect.
Treatment and prevention
The interpretation of the analyzes should be entrusted to your doctor. In case of detection pathological causes the appearance of protein substances in urine, the necessary therapy is prescribed only medical worker. It is not recommended to self-treat and get involved non-traditional methods. When identifying causes that are not related to pathological processes, they give Special attention diet. It is important to remember that the effectiveness of therapy depends on timely diagnosis.
26.02.2009
Kurilyak O.A., Ph.D.
Normally, protein is excreted in the urine in a relatively small amount, usually no more than 100-150 mg / day.
Daily diuresis in a healthy person is 1000-1500 ml / day; thus, the protein concentration under physiological conditions is 8-10 mg/dL (0.08-0.1 g/L).
The total protein of urine is represented by three main fractions - albumins, mucoproteins and globulins.
Urine albumin is that portion of serum albumin that has been filtered in the glomeruli and has not been reabsorbed in the renal tubules; normal urinary albumin excretion is less than 30 mg/day. Another main source of protein in the urine is the renal tubules, especially the distal part of the tubules. These tubules secrete two-thirds of the total urinary protein; of this amount, approximately 50% is represented by the Tamm-Horsfall glycoprotein, which is secreted by the epithelium of the distal tubules and plays an important role in the formation of urinary stones. Other proteins are present in the urine in small amount and originate from kidney filtered low molecular weight plasma proteins that are not reabsorbed in the renal tubules, renal tubular epithelium (RTE) microglobulins, and prostatic and vaginal secretions.
Proteinuria, that is, an increase in the protein content in the urine, is one of the most significant symptoms reflecting kidney damage. However, a number of other conditions can also be accompanied by proteinuria. Therefore, there are two main groups of proteinuria: renal (true) and extrarenal (false) proteinuria.
In renal proteinuria, the protein enters the urine directly from the blood due to an increase in the permeability of the glomerular filter. Renal proteinuria is often found in glomerulonephritis, nephrosis, pyelonephritis, nephrosclerosis, renal amyloidosis, various forms nephropathies, such as nephropathies of pregnant women, febrile conditions, hypertension, etc. Proteinuria can also be found in healthy people after heavy physical exertion, hypothermia, psychological stress. In newborns, physiological proteinuria is observed in the first weeks of life, and with asthenia in children and adolescents, combined with rapid growth at the age of 7-18 years, orthostatic proteinuria (in the upright position of the body) is possible.
With false (extrarenal) proteinuria, the source of protein in the urine is an admixture of leukocytes, erythrocytes, epithelial cells urinary tract urothelium. The breakdown of these elements, especially pronounced in the alkaline reaction of urine, leads to the ingress of protein into the urine, which has already passed the renal filter. Especially a high degree false proteinuria gives an admixture of blood in the urine, with profuse hematuria it can reach 30 g / l or more. Diseases that may be associated extrarenal proteinuria- urolithiasis, kidney tuberculosis, tumors of the kidney or urinary tract, cystitis, pyelitis, prostatitis, urethritis, vulvovaginitis.
Clinical classification includes mild proteinuria (less than 0.5 g/day), moderate (0.5 to 4 g/day), or severe (greater than 4 g/day).
Most patients with kidney disease such as acute glomerulonephritis or pyelonephritis have mild proteinuria, but patients with nephrotic syndrome typically excrete more than 4 g of protein daily in the urine.
A wide range of methods are used for the quantitative determination of protein, in particular, the unified Brandberg-Roberts-Stolnikov method, the biuret method, the method using sulfosalicylic acid, methods using Coomassie blue dye, pyrogallol red dye, etc.
The use of various methods for determining protein in the urine has led to serious confusion in the interpretation of the limits of the normal content of protein in the urine. Since 2 methods are most often used in laboratories - with sulfosalicylic acid and pyrogallol red dye, we will consider the problem of the correctness of the limits of the norms for them. From the position of the sulfosalicylic method in normal urine, the protein content should not exceed 0.03 g / l, from the position of the pyrogallol method - 0.1 g / l! Differences triple!
The low values of the norm of the concentration of proteins in the urine when using sulfosalicylic acid are due to the following points:
- the calibration curve is built on an aqueous solution of albumin. Urine in its composition is very different from water: pH, salts, low molecular weight compounds (creatinine, urea, etc.). As a result, according to Altshuler, Rakov and Tkachev, the error in determining protein in urine can be 3-fold or more! Those. correct determination results can only be obtained in cases where the urine has a very low specific gravity and in its composition and pH approaches water;
- higher sensitivity of the sulfosalicylic method to albumin in comparison with other proteins (while, as mentioned above, albumin in normal urine samples does not exceed 30% of the total urine protein);
- if the pH of urine is shifted to the alkaline side, sulfosalicylic acid is neutralized, which is also the reason for underestimating the results of protein determination;
- the sedimentation rate of precipitates is subject to significant variation - at low protein concentrations, precipitation is slowed down, and an early stop of the reaction leads to an underestimation of the result;
- the rate of the precipitation reaction essentially depends on the mixing of the reaction mixture. At high protein concentrations, vigorous shaking of the tube can lead to the formation of large flakes and their rapid settling.
All of the above features of the method lead to a significant underestimation of the protein concentration determined in the urine. The degree of underestimation is highly dependent on the composition of a particular urine sample. Since the sulfosalicylic acid method gives underestimated protein concentrations, the normal limit for this method of 0.03 g / l is also underestimated by about three times in comparison with the data given in foreign reference books on clinical laboratory diagnostics.
The vast majority of laboratories in Western countries have abandoned the use of the sulfosalicylic method for determining the concentration of protein in the urine and are actively using the pyrogallol method for this purpose. The pyrogallol method for determining the concentration of protein in urine and other biological fluids is based on the photometric principle of measuring the optical density of a colored complex formed by the interaction of protein molecules with molecules of the pyrogallol red dye complex and sodium molybdate complex (Pyrogallol Red-Molybdate complex).
Why does the pyrogallol method provide more accurate urine protein measurements? Firstly, due to the greater dilution of the urine sample in the reaction mixture. If in the sulfosalicylic method the ratio of urine sample / reagent is 1/3, then in the pyrogallol method it can range from 1/12.5 to 1/60, depending on the method variant, which significantly reduces the effect of urine composition on the measurement result. Secondly, the reaction proceeds in a succinate buffer, that is, at a stable pH. And, finally, the very principle of the method, one might say, is more “transparent”. Sodium molybdate and pyrogallol red dye form a complex with a protein molecule. This leads to the fact that the dye molecules in the free state, which do not absorb light at a wavelength of 600 nm, absorb light in combination with the protein. Thus, we label each protein molecule with a dye, and as a result, we find that the change in the optical density of the reaction mixture at a wavelength of 600 nm clearly correlates with the protein concentration in the urine. Moreover, since the affinity of pyrogallol red for different protein fractions is almost the same, the method allows you to determine the total urine protein. Therefore, the limit of normal values of protein concentration in the urine is 0.1 g/l (it is indicated in all modern Western guidelines for clinical and laboratory diagnostics, including the Clinical Manual for Laboratory Tests, edited by N. Titz) . Comparative characteristics pyrogallol and sulfosalicylic methods for determining protein in urine are presented in Table 1.
In conclusion, I would like to once again focus on the fact that when the laboratory switches from the sulfosalicylic method for determining protein in urine to the pyrogallol method, the limit of normal values increases significantly (from 0.03 g/l to 0.1 g/l!). The laboratory staff must certainly inform the clinicians about this, because. in this situation, the diagnosis of proteinuria can only be made when the protein content in the urine exceeds 0.1 g / l.
Bibliography.
- Altshuler B.Yu., Rakov S.S., Tkachev G.A. // Question. honey. chemistry. - 2001. - No. 4. - C.426-438.
- Kim Yu.V., Potekhin O.E., Tokar M.I., Shibanov A.N. // Lab. honey. - 2003. - No. 6. - C.94-98.
- Clinical Guide to Laboratory Tests, ed. N. Titsa.- M.- Unimed-press.-2003.- 942 p.
- Kozlov A.V., Slepysheva V.V. Methods for determining protein in urine: opportunities and prospects // Collection of works of the VII annual. SPb nephrol. seminar. - St. Petersburg: TNA. - 1999. - C.17-28.
- Pupkova V.I., Pikalov I.V., Khrykina E.N., Kharkovskiy A.V. // News "Vector-Best". - 2003. - No. 4 (30).
- Chambers R.E., Bullock D.G., Whicher J.T. // Ann. Clin. Biochem. - 1991. - Vol. 28 (Pt 5). - P.467-473.
- Clinical Laboratories Medicine. Ed. by Kenneth D. McClatchey. - 2nd ed.-2001.- 1993p.
- Eppel G.A., Nagy S., Jenkins M.A., Tudball R.N., Daskalakis M., Balazs N.D.H., Comper W.D. // clinic. Biochem. - 2000. - Vol. 33.-P.487-494.
- Franke G., Salvati M., Sommer R.G. Composition and device for urinary protein assay and method of using the same // US Patent No. 5326707. - 1994.
- Kaplan I.V., Levinson S.S. // clinic. Chem. - 1999. - Vol. 45.-P.417-419.
- Kashif W., Siddiqi N., Dincer H.E., Dincer A.P., Hirsch S. // Cleveland Clin. J. of Med. - 2003. - Vol. 70(6). - P.535-547.
- Koerbin G, Taylor L, Dutton J, Marshall K, Low P, Potter JM. // clinic. Chem. - 2001. - Vol. 47.-P.2183-2184.
- Le Bricon T., Erlich D., Dussaucy M., Garnier J.P., Bousquet B. // Article in French. - Ann. Biol. Clin. (Paris). - 1998. - Vol. 56(6). - P.719-723.
- Marshall T., Williams K.M. // clinic. Chem. - 2003. - Vol. 49 (12). - P.2111-2112.
- Pugia M., Newman D.J., Lott J.A., D'Mello L., Clark L., Profitt J.A., Cast T. // Clin. Chim. acta. - 2002. - Vol. 326(1-2). - P.177-183.
- Ringsrud K.M., Linne J.J. Urinalysis and body fluids: A ColorText and Atlas // Mosby. - 1995. - P.52-54.
- Shepard M.D., Penberthy L.A. // clinic. Chem. - 1987. - Vol. 33.-P.792-795.
- Williams K.M., Marshall T. // J. Biochem. Biophys. methods. - 2001. - Vol. 47.-P.197-207.
- Williams K.M., Arthur S.J., Burrell G., Kelly F., Phillips D.W., Marshall T. // J. Biochem. Biophys. methods. - 2003. - Vol. 57(1). - P.45-55.
