Methods of Examination 2 страница. Chemical analysis of urine.Reaction of the urine

Chemical analysis of urine. Reaction of the urine. The kidneys are im­portant for maintaining acid-base equilibrium in the body. The kidneys are capable of removing the ions of hydrogen and hydrocarbonate from the blood and this is a mechanism by which pH of blood is maintained cons­tant. The concentration of the hydrogen ions is the true reaction of urine (active acidity or pH of the medium). The sum of dissociated and un-dissociated hydrogen ions is the titration (analytical) acidity. The true reac­tion of urine may vary from pH 4.5 to 8.4. The pH of urine can be deter­mined colorimetrically and electrometrically. Colorimetry includes methods employing litmus paper, bromthymol blue, and other indicators, by which the pH is determined only tentatively. More accurate determina­tion of pH is done by comparing colour intensity of test solutions with standard solutions (the Michaelis method).

Special indicator papers can also be used for sufficiently accurate deter­mination of the pH of urine in the range from 5.0 to 9.0. The mean pH value of the urine in healthy subjects (with normal nutrition) is about 6.0. The value of pH is affected by the use of medicinal preparations (diuretics, corticosteroids). Acidity of urine can increase in diabetes mellitus, renal in­sufficiency, tuberculosis of the kidneys, acidosis, and hypokaliaemic alkalosis. Urine reacts alkaline in vomiting and chronic infections of the urinary tracts due to bacterial-ammoniacal fermentation.

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/ Determination of protein in urine. Normal urine does not practically contain protein. The small quantity of plasma proteins (to 150 mg/day), that is present in the urine, cannot be determined by qualitative tests used in practical medicine. The appearance of protein in the urine in concentra-* tions determinable by qualitative methods is called proteinuria. It can be of renal and extrarenal origin. Organic renal proteinuria occurs in kidney af­fections due to increased permeability of glomeruli which is underlain by vascular inflammation or structural disorganization of the basal mem­brane. Glomerular permeability is upset by the " molecular sieve" mechanism, i.e. low-molecular proteins are lost in the first instance. This proteinuria is called selective. As the process progresses, high-molecular proteins are also lost (non-selective proteinuria). Selectivity of proteinuria is an important diagnostic and prognostic sign.

Functional renal proteinuria is connected with the permeability of membranes in the renal filter in the presence of strong stimulation, slowing of the blood flow in the glomeruli, etc. Functional proteinurias include emotional, athletic (effort), cold, and orthostatic (a condition characteriz­ed by the appearance of protein in the urine when the patient is in the erect posture; hence the name). In cases with extrarenal proteinuria, proteins enter the urine from the urinary and sex ducts (admixtures of inflammatory exudate); extrarenal proteinuria does not exceed 1 g/1. Tests intended to reveal protein in the urine are based on its thermal or acid coagulation (the urine sample should first be filtered).

Acetic-acid test. The test gives reliable results provided the pH of the medium is 5.6. If the urine contains much phosphates, a few drops of acetic acid, which is usually added in this test, do not decrease the pH of the medium significantly and the proteins remain dissolved as alkalalbumins. In other cases it is enough to add a few drops of acetic acid to decrease the pH much below 5.6, and the proteins form acid albumins without giving cloudiness. The test should be better carried out with the Bang buffer (56.5 ml glacial acetic acid and 118 g of sodium acetate dissolved in 1 1 of water). To a 5-ml sample of urine added are 1-2 ml of the Bang buffer and the mixture is boiled for 30 s. The solution turns cloudly in the presence of even insignificant amount of protein.

Sulphosalicylic acid test. This is one of the most sensitive and popular tests. To 3-4 ml of filtered urine added are 6-8 drops of a 20 per cent solution of sulphosalicylic acid. Cloudiness develops if the test is positive.

Quantitative determination of albumin. A modified Heller's test is now popular: a white ring appears at the interface between the test liquid con­taining albumin and nitric acid. A thin but distinct ring appears by the end of the third minute to indicate the presence of 0.033 g/1 of albumin in the test urine. Filtered urine is layered on 1—2 ml of a 50 per cent nitric acid and the time is marked. If the white ring forms earlier than in 2 minutes,

the urine sample should be diluted with water so that the white ring should be formed during the course of the second or third minute. The amount of albumin contained in the urine is determined by multiplying 0.033 g/1 by the dilution degree.

Turbidimetric tests are widely used for determining protein in the urine. The sulphosalicylic acid is used for the purpose. Since turbidity is propor­tional to protein concentration in the urine, protein can be determined from the calibration curve after determining extinction (optical density) of the solution.

Rapid-diagnosis methods are very popular now. They are used for pro­phylactic large-scale examination of the population (with special paper in­dicators). Protein error of some acid-base indicators is used as the working principle. The paper is impregnated in bromphenol blue and citrate buffer solution. As the paper is wetted, the buffer dissolves to ensure the required pH of the medium for the indicator reaction. Amino groups of protein react with the indicator at pH 3.0-3.5 to alter its initial yellow colour to greenish-blue. By comparing the new colour with a special scale of stan­dards, it is possible to assess tentatively protein concentration in the test urine.

Protein concentration in urine expressed in grammes per litre does not express the absolute amount of protein lost. It is therefore recommended to express it in grammes per day. The protein concentration in the urine col­lected during 24 hours should first be determined, diuresis measured, and the amount of protein lost per day finally calculated. ^v Determining Bence-Jones proteins. Bence-Jones proteins occur in myeloma and Waldenstrom's macroglobulinaemia. These are light (L) polypeptide chains, which pass an intact renal filter because of their relatively small molecular weight, and are determined by thermal precipita­tion and electrophoretic study of urine.

^v Determining glucose in urine. The urine of a healthy person contains very small quantity of glucose (0.03-0.15 g/1) which cannot be detected by common qualitative tests. Glucose in the urine (gfycosuria) can be both physiological and pathological. In the presence of normal renal function, glycosuria occurs only in increased concentration of sugar in the blood (normal sugar content of blood is 4.6-6.6 mmol/1 or 0.8-1.2 g/1), i.e. in the presence of hyperglycaemia. The so-called renal glucose threshold (sugar concentration in the blood) does not usually exceed 9.9 mmol/1 (1.8 g/1); higher concentration of sugar indicates glycosuria.

Physiological glycosuria can be observed in persons whose diet is rich in carbohydrates (alimentary glycosuria), following emotional stress, and ad­ministration of some medicines (caffeine, corticosteroids). Less frequent is renal glycosuria associated with disturbed resorption of glucose in the

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tubules: glycosuria develops in the presence of normal amount of sugar in the blood. As a primary disease, glycosuria occurs in the form of renal diabetes. Secondary renal glycosuria occurs in chronic nephritis, nephrotic syndrome, and in glycogen-storage disease. Pathological glycosuria occurs most frequently in diabetes mellitus, less frequently in thyrotoxicosis, in pituitary insufficiency (Itsenko-Cushing syndrome), and in liver cirrhosis.

In order to assess correctly glycosuria (especially in patients with diabetes mellitus), it is necessary to calculate the daily loss of sugar with urine. Most qualitative tests used to detect glucose in urine are based on the reducing power of glucose.

Haines' test for sugar in the urine is based on the property of glucose to reduce copper hydroxide in an alkaline medium to yellow cuprous hydrox­ide or red cuprous oxide.

Nylander's test. The reaction is based on reduction of bismuth nitrate by glucose to bismuth metal. In the presence of sugar, the colour of solu­tion changes from brown to black. The test urine should be free from pro­tein. Extraneous reducing substances (antipyrin, benzoic acid, etc.) giving a false reaction should be removed by adding 1 ml of 95 per cent alcohol and a small amount of animal carbon to 9 ml of urine.

Glucose oxidase (notatin) test. This is a highly specific and very simple enzyme test. Glucose oxidase (notatin) is (3-d-glucose dehydrogenase. At its first stage, the enzyme acts on glucose to liberate hydrogen peroxide. At the second stage, the presence of hydrogen peroxide is established by a redox indicator (like in the benzidine test).

The principle of the glucose oxidase test is used in the indicator paper method. A paper strip impregnated with glucose oxidase, peroxidase and a benzidine derivative is dipped in urine: if the urine contains glucose the paper turns blue in 30-60 seconds.

Quantitative determination of glucose in urine. The amount of glucose contained in a given sample of urine can be determined by the angle of rotation of a polarized beam of light: glucose rotates the polarized light to the right.

Althausen colorimetric method. The method is based on the colour reaction occurring during heating a glucose solution with alkali. To 4 ml of urine added is 1 ml of 10 per cent sodium hydroxide (or potassium hydrox­ide) solution and the mixture is boiled for a minute. The solution is allowed to stand for ten minutes and its colour compared with this of colour stan­dards (either visually or photometrically).

Determining ketone (acetone) bodies. The presence of ketone bodies (acetone, acetoacetic and |3-oxybutyric acid) in the urine is called ketonuria. Ketonuria is usually observed in severe diabetes mellitus but it can also develop due to carbohydrate deficit (in grave toxicosis, long-

standing gastro-intestinal disorders, etc.); it may develop postoperatively. Ketone bodies in the urine occur simultaneously and their separate deter­mination is therefore clinically impracticable. The Lange test is most com­monly used for the detection of ketone bodies in the urine. The test urine sample is mixed with acetic acid and nitroprusside, and then ammonia is layered: a violet ring is formed at the interface of the liquids if the test is positive.

Determination of bilirubin. Normal urine is practically free from bilirubin. Increased amounts of bilirubin in the urine at which common qualitative bilirubin tests become positive (bilirubinuria) occur in hepatic and subhepatic jaundice at which the concentration of bound bilirubin (bilirubin glucuronide) in the blood increases. Most qualitative tests for bilirubin are based on its conversion into green biliverdin under the action of oxidizers.

Rosin's test. Lugol (1 per cent iodine solution) is layered upon 4-5 ml of urine: a green ring appears at the interface between the liquids if the test is positive.

Fouchet's test. To 10-12 ml of urine added are 5-6 ml of a 15 per cent barium chloride solution; the mixture is stirred and filtered. Barium chloride precipitates bilirubin. The precipitate is separated on a filter and 2-3 drops of Fouchet's reagent (100 ml of a 25 per cent trichloroacetic acid solution mixed with 10 ml of a 10 per cent ferric chloride solution) are added: green-bluish or light-blue spots appear on the filter if the test is positive. The Fouchet test is more sensitive.

Determining urobilinoids. Urobilinoids are urobilin (urobilinogens, urobilins) and stercobilin (stercobilinogens, stercobilins). Urobilin and stercobilin bodies are not determined separately. Excretion of large amounts of urobilinoids in the urine is called urobilinuria which occurs in diseases of the liver (hepatitis, cirrhosis), haemolytic anaemia, and in in­testinal diseases (enterites, etc.).

Neubauer's test. The test is based on the reaction between urobilin bodies and the Ehrlich reagent (2 g of p-dimethylaminobenzaldehyde + + 100 ml of a 20 per cent hydrochloric acid solution). To a few millilitres of urine (freshly taken and cooled to room temperature) added are a few drops of the Ehrlich reagent: colouration of the liquid during the first 30 seconds indicates increased content of urobilin bodies (positive test), while development of colour at later period indicates either their absence or the presence of their normal quantity.

Florence' test. Urobilinoids are extracted from the urine acidified with sulphuric acid by ether (8-10 ml of urine and 3 ml of ether). The ether ex­tract is then layered upon 2-3 ml of concentrated hydrochloric acid. The advantage of this test is that it is also positive in the presence of normal

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quantity of urobilinoids and can therefore be used to establish their com­plete absence.

Bogomolov's test. To 10 ml of urine added are 2-3 ml of a saturated copper sulphate solution. Next, a few drops of hydrochloric acid are added to clarify the solution. The mixture is allowed to stand for 5 minutes, 2—3 ml of chloroform are added, and the mixture is shaken: chloroform turns pink in the presence of urobilin bodies.

Quantitative determination of urobilinoids is based on their colour reaction (pink) with p-dimethylaminobenzaldehyde or with hydrochloric acid.

Rapid diagnosis (by indicator paper) of ketonuria, bilirubinuria, urobilinuria is based on the employment of the same chemical reactions with subsequent colorimetry.

Microscopy of urine sediment. A urine specimen is stirred thoroughly and its 10 ml are transferred into a centrifugal test tube. After centrifug-ing, the supernatant is decanted while the precipitate transferred onto an object glass for microscopy. The precipitate is first examined at small and then at large magnification to study the formed elements, cylinders, and salts.

Erythrocytes (red blood cells) can be altered and unaltered. Unaltered erythrocytes contain haemoglobin and appear as greenish-yellow discs. Altered erythrocytes are free from haemoglobin and are colourless one- or two-contour rings (Plate 21). These erythrocytes occur in the urine of low specific gravity; erythrocytes shrink in the urine of specific gravity. The urine of a healthy person can have single erythrocytes.

Erythrocytes may be liberated either from the kidneys or from the urinary tract. The presence of erythrocytes in the urine is called haematuria. Haematuria that can only be established by microscopy is call­ed microhaematuria, while haematuria revealed by macroscopy is called macrohaematuria. It is important practically to decide whether haematuria is of glomerular or non-glomerular origin. In the latter case blood is liberated into the urine from the urinary tract due to the presence of stones in the pelves, urinary bladder or ureters, and because of tuberculosis or malignant newgrowths of the urinary bladder. In the presence of glomerular haematuria, the urine usually contains much protein. Pro-teinoerythrocytic dissociation (i.e. haematuria with insignificant pro-teinuria) usually suggests haematuria associated with pathology of the urinary tract. An intermittent character of haematuria (with strongly vary­ing intensity) is another evidence of non-glomerular haematuria.

A three-glass test is used for differential diagnosis of haematuria. The patient urinates into three vessels. If the blood originates ki the urinary tract (urethra), the highest amount of blood is present in the first portion

of the urine; if bleeding occurs in the urinary bladder, haematuria is the highest in the last portion. If the source of haemorrhage is located in other parts of the urinary system, all three portions of the urine contain equal quantity of erythrocytes.

Leucocytes are found in the urine as small granular rounded cells. They swell in the urine of low specific gravity. Leucocytes in the urine of a healthy person are usually neutrophils and their amount is insignificant (to 1-2 in the microscope's vision field). Increased quantity of leucocytes in the urine (leucocyturia) indicates inflammation in the kidneys or urinary tract (urethritis, prostatitis, cystitis, pyelonephritis). Thompson's test is used for differential diagnosis of leucocyturia. The firts portion of an early morning urine specimen is collected in the first glass, the main bulk of the urine in the second glass, and only the residue in the third glass. If prevail­ing quantity of leucocytes is found in the first portion, it indicates the presence of urethritis and prostatitis. If the main quantity of leucocytes is found in the third portion, this suggests the disease of the urinary bladder. Uniform distribution of leucocytes in all portions of the urine may suggest affection of the kidneys. Cell structures are quickly destroyed in alkaline urine; it is therefore difficult to judge about the degree of leucocyturia. Eosinophils are sometimes found in the urine; they differ from other leucocytes by ample uniform refracting granularity. The presence of eosinophils suggests an allergic character of the disease.

The degree of leucocyturia does not always correspond to the gravity of affection in chronic pyelonephritis. In the absence of active inflammatory process, the quantity of leucocytes in the urine may remain normal. The method of supravital staining is widely used now. It was proposed in 1949 by Sternheimer and Malbin. Depending on their morphological properties, leucocytes (Plate 22) are coloured either red or pale-blue by a special stain (water-alcohol mixture of 3 parts of Gentian violet and 97 parts of safranine). Leucocytes that are coloured blue in the urine of low specific gravity are greater in size and contain vacuolized cytoplasm with granules that are set in Brownian movement. They are found in patients with pyelonephritis. Leucocyte cells (Sternheimer-Malbin cells) can be found in the urine of patients with iso- or hyposthenuria with any location of the source of inflammation in the urinary tract. These cells are more often call­ed " active leucocytes". They are determined by adding distilled water to urine precipitate to create low osmotic pressure.

Increased number of " active leucocytes" suggest activation of inflam­mation in the urinary tract or exacerbation of pyelonephritis.

Microscopy can reveal cells of squamous, transitional, and renal epithelium (Plate 23). Squamous epithelium cells are rounded or polygonal; they are large, colourless, and contain a small nucleus; they

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enter the urine from the external genitalia and the urethra; their diagnostic importance is low. Cells of transitional epithelium line the mucosa of the urinary tract; their configuration is quite varied; they are smaller than squamous epithelium cells; the nucleus is rounded. The presence of large amount of transitional epithelium in the urine indicates inflammatory pro­cess in the pelves or the bladder. Cells of renal (cuboidal) epithelium of tubules are rounded or polyhedral; they are small (slightly larger than leucocytes) and have a large, eccentrically located nucleus; their granularity is coarse. They are often found in hyaline cylinders. The presence of renal epithelium in the urine is a specific sign of acute and chronic affections of the kidneys, and also of fever, toxicosis, and infectious diseases.

Casts are proteinous or cell formations of tubular origin; they have cylindrical configurtion and variable size (Plate 24). gyalinecasts are pro­teinous formations of indistinct contour with smooth and slightly granular surface; they are found in acute and chronic nephritis, nephrotic syn­drome, and also in physiological transient albuminuria. Hyaline casts can be found in the urine of practically healthy people when the pH of the urine decreases sharply along with increasing specific gravity of the urine, which is characteristic of dehydration. It is believed that hyaline casts are formed by glycoprotein secreted in the tubules; but there are no reliable data that would confirm this conjecture. Qranul^casts^ have distinct contours; they consist of dense granular mass formed by degraded cells of renal epithelium. Their presence indicates dystrophic processes in the tubules. Wgxgjxists have distinct contours and a homogeneous yellow structure. Their presence is characteristic of chronic diseases of the kidneys. The urine can also contain epithelial, erythrocytary, haemoglobin and leucocyte casts, and cylindrical formations of amorphous salts, which are diagnostically unimportant.

" Non-organized sediment" of the urine consists of salts that precipitate as crystals and amorphous substances. Their character depends on the col­loidal composition of the urine, its pH, and other properties. Acid urine contains uric acid (yellow rhomboid-type crystals), urates (yellowish-brown amorphous salt), oxalic lime, or oxalates (colourless octahedral crystals that may also occur in alkaline urine) (Plate 25). Alkaline urine contains ammonium urate, calcium carbonate, triple phosphates, amor­phous phosphates, and neutral calcium phosphate (Plate 26). The sediment is diagnostically insignificant but pathological urine can contain crystals of cystine, thyrosine, and leucine. The presence of thyrosine and leucine is especially characteristic of subacute dystrophy of the liver and of phosphorus poisoning. The presence of lipoids in the urine is characteristic of nephrotic syndrome. In a polarizing microscope, lipoids give a dual reflection and appear as lustrous crosses.

Addis-Kakovsky test. The test is used for quantitative determination of the formed elements in the urinary sediment. Urine collected during ten hours is stirred thoroughly, its amount is measured and a 12-minute aliquot (l/5Oth of the full volume) is placed in a graduated centrifugal test tube. After centrifuging for 5 minutes at 2000 rpm the supernatant is removed by a pipette, while the remaining 0.5 ml sediment is stirred and transferred in­to a cell for counting blood formed elements. Leucocytes, erythrocytes, and casts are counted separately. The quantity of cells counted in one microlitre is multiplied by 60 000 to find the quantity of the formed cells of the urine excreted during the day. The normal counts are 1 000 000 for erythrocytes, 2 000 000 for leucocytes, and 20 000 for casts.

Nechiporenko's method is now widely used to count erythrocytes and leucocytes in 1 ml of urine. The main advantage of this method is that an average sample of urine is taken for analysis and the presence of pus from the sex organs is thus excluded. A disadvantage of the method is that it does not account for diuresis. The normal counts are 1000 erythrocytes, 4000 leucocytes, and 220 hyaline casts.

Bacterioscopic and bacteriological study of urine. Urine cultures are used to establish the infectious nature of a disease of the urinary system. Sterile glassware should be used for the purpose. Whenever necessary, the urine is studied bacterioscopically for the presence of tuberculosis mycobacteria. A smear is prepared from the urinary sediment with Ziehl-Nielsen staining. The urine is studied bacteriologically to determine qualitative and quantitative composition of its microbial flora. In the presence of bacteriuria, it is very important to determine its degree and microorganism sensitivity to various antibiotics.

FUNCTIONAL TESTS FOR KIDNEYS

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Assessing the renal function by specific gravity and amount of the urine excreted. In conditions of water deficit, a normal person excretes a small amount of the urine with high specific gravity; and vice versa: if excess li­quid is taken, the amount of the urine excreted increases while its specific gravity decreases. The kidneys thus maintain equilibrium in the bodily fluids, i.e. they maintain constancy of osmotic concentration and volumes of the fluids. If the body is dehydrated, osmotic concentration of ex­tracellular fluid increases and the amount of released antidiuretic hormone (ADH) increases to increase tubular resorption of water. If the amount of taken liquid increases, osmotic concentration of extracellular fluid decreases; this decreases secretion of ADH and water resorption to increase diuresis. In pathology, the kidneys are incapable of ensuring the required osmotic gradient in the medulla and the concentrating power of the kidneys

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is thus upset. The impaired power of the kidneys to resorb the osmoticafly active substances without water disturbs their diluting capacity.

* Zimnitsky's test. The main advantage of this method is that the renal function is tested without interfering with the normal life of the patient. The patient collects his urine at 3-hour intervals (8 portions during 24 hours). The volume of each portion and specific gravity of the urine are determined. The volumes of daily and night urine are compared and a con­clusion is derived concerning daily and nocturnal diuresis. Fluctuations in specific gravity of the urine during the course of the day and its maximum value are thus determined. Normally the daily diuresis exceeds the noctur­nal one; volumes of urine portions can vary from 50 to 250 ml, and their specific gravity from 1.005 to 1.028. Nocturnal diuresis (nycturia) prevails in renal insufficiency to indicate longer work of the kidneys because of their impaired functional capacity. If renal insufficiency is pronounced, decreased specific gravity becomes permanent (hyposthenuria). Combina­tion of polyuria with low specific gravity of the urine and nycturia is a specific sign of renal dysfunction.

Dilution test. The patient is given to drink 1-1.5 1 of water or thin tea within 30—45 minutes and then the urine is collected at 30-minute intervals during 4 hours. The portions are measured and their specific gravity deter­mined. A normal individual would eliminate about 75 per cent of the taken liquid during four hours, while the specific gravity of the urine decreases to 1.003-1.001. The first portions will be larger and their specific gravity lower. A more accurate method includes also calculations where the amount of liquid taken is referred to the body weight: 22 ml of liquid is given per kg body weight.

If the excretory function of the kidneys is decreased, the amount of urine excreted during 4 hours is markedly less than that of liquid taken; the specific gravity in all portions is about the same, but not below 1.006-1.007. If the renal function is upset significantly, the specific gravity of the urine in all portions is 1.009—1.011, which corresponds to the specific gravity of the primary urine. The dilution test is contraindicated in oedema and hypertension.

Urine concentration test. The patient receives no fluids for 36 hours (nor food containing much liquid). Urine is collected at 3-hour intervals during 24 hours (8 specimens). The volume and specific gravity of each specimen is determined. The specific gravity of the urine of a healthy in­dividual will in these conditions be not lower than 1.028. If the specific gravity of thus obtained urine does not rise over 1.022, this indicates im­paired renal function.

The urine concentration test is valid when applied to cases where the daily diuresis does not exceed 400 ml. The test is contraindicated in acute

inflammatory processes in the kidneys, in cardiovascular and renal insuffi­ciency, and in essential hypertension.

The renal function can be assessed by studying glomerular filtration, renal plasma flow, tubular transport of certain substances (e.g. glucose reabsorption), secretion of extraneous substances, urea and electrolyte ex­cretion in the urine. It is possible to reveal and assess the degree of renal in­sufficiency by studying concentration of urea, indican, residual nitrogen, creatinine, potassium, sodium, calcium, magnesium and phosphates in the blood (see Tables 7 and 8 of the " Appendix").