URINALYSIS

Urine is body fluid produced and excreted by kidney. The kidney plays an important role in acid-base balance as well as in maintaining the levels of electrolytes and water. A functional unite of the kidney is a nephron. The kidney contains 6 approximately 1,3 x 10 nephrons. Each nephron consists of a glomerulus, which is supplied with blood via an arteriolar capillary system. There exists a filtration pressure on a glomerular membrane to effect ultrafiltration of the lower molecular weight materials in the plasma. The glomerular filtrate collects in Bowmans capsule, which leads to the system of tubules. The osmolarity of glomerular filtrate is about 300 mmol.l-1, similarly to the blood plasma from which it originates, and contains principally Na+, K+, Cl-, HCO3-, HCO32-, SO42-, glucose, urea, creatinine, and proteins with small molecular weight. As the filtrate flows through the tubules, definitive composition of the urine is produced. Examination of urine consists of: 1. Description of physical properties, 2. Analysis of chemical composition, 3. Examination of urinary deposits (urine sediment - Addis count, Hamburger count), . 1. PHYSICAL EXAMINATION OF THE URINE involves subjective data (appearance - color, transparency, lather, and odor) and objective data (volume excreted during 24 h, pH, specific gravity, and osmolality). The normal urine is yellow, pellucid liquid, with white lather disappearing immediately. COLOR: The yellow color of normal urine is attributed to urochrome, small amounts of uroerythrin, urobilin, uroporphyrin and coproporphyrin may also contribute to the color of normal urine. The traces of uroporphyrin and coproporphyrin may be present but they do not normally occur in sufficient quantity to be detected by ordinary tests. The colorless urobilinogen in urine is oxidized on standing to urobilin, but the small amount normally present contributes little to the color. The main determinant of the color intensity of normal urine is the volume excreted daily. Very concentrated urines, recognized by their very high osmolality, may be so deeply colored as to suggest the presence of abnormal pigments. Other pigments may also be increased in disease. A marked increase in urobilin excretion is seen in cirrhosis and in hemolytic anaemia to give the urine a characteristic brickred color. Of the pathological pigments, the greenish-yellow or brown color of bilirubin is well known. Blood pigments which may be found in the urine range from the red color of oxyhemoglobin to the brown of methemoglobin. The presence of abnormal amounts of porphyrins gives a brick-red color. Of the other pathological pigments, melanin and homogentisic acid only occur rarely. Melanin occurs in patients with a malignant melanoma. Freshly passed urines contain a colorless precursor, melanogen, which is oxidized to melanin when the urine is allowed to stand in contact with air. As melanin is dark brown or black, the urine slowly darkens. Homogentisic acid in patients with alkaptonuria causes, that the urine darks by standing. Homogentisic acid has reducing properties and can be

estimated with Fehlings reagent giving the brown-black color product. Abnormally colored urine may be also due to ingested substances (drugs, dyes ...). Riboflavin taken in multiple vitamin preparations gives the urine a deeper amber color with a greenish fluorescence. ODOR: Odor of normal urine is specific and can be influenced by food intake.. In some pathological conditions we can find out characteristic acetone odor (in diabetics) or mouse-like odor (in patients with phenylketonuria). In patients with urinary infection a typical ammonia-like odor may be noted. VOLUME: Volume of urine excreted per 24 h is called diuresis, it depends on the kidney function and the fluid intake and loss. In general, 1.0 1.5 l of urine are excreted per day. The diuresis is necessary information for an estimation of a daily excretion of some minerals or for an evaluation of a daily loss of some compounds that are not present in urine of a healthy person (i.e. glucose, albumin etc.). More then 2 l urine per day is called polyuria, less than 500 ml oliguria and anuria is diuresis less than 200 ml. SPECIFIC GRAVITY: Urine has a specific gravity of 1,003 to 1,030 g/ml, depending on the homeostatic control of total body fluid and solids. Measurement of urine concentration is best done using a urometer. Put some urine in cylinder and float the urometer. Before taking the reading off the scale, make sure the urometer is free of the sides of the vessel. OSMOLALITY: Osmolality ranges commonly within 500 - 850 mmolkg-1, border values are 40 1 450 mmolkg-1. We can measure the osmolality using a osmometer. pH: Measurement of pH is useable for diagnosis of disturbance of tubular cells leading in renal tubular acidosis. Urine pH also changes in cases of urinary infections with a bacteriuria. 2. CHEMICAL ANALYSIS OF URINE Reagents: Sulkowitchs reagent: oxalic acid 25 g, ammonium oxalate 25 g, glacial acetic acid 50 ml, water up to 1 500 ml Sulfosalicylic acid (200 gl-1) Concentrated nitric acid Fehlings solution I: copper (II) sulphate cryst. 70 gl-1 Fehlings solution II: sodium hydroxide 250 g, sodium-potassium tartarate cryst. 350 g diluted in 1 l of distilled water Benedicts reagent: a) copper (II) sulphate cryst. 17.3 g diluted in 100 ml of distilled water b) sodium carbonate 100 g, sodium citrate cryst. 173 g diluted in 700 ml ad distilled water. Mix a) and b), add water up to 1l

Nylanders reagent: alkaline bismuth (III) nitrate 2 g, sodium-potassium tartarate cryst. 4 g, sodium hydroxide 10 g diluted in distilled water, added up to 100 ml Resorcinol Bials reagent: orcinol 0.3 g diluted in 100 ml of hydrochloric acid 300 gkg-1, added 5 drops of iron(III)chloride 100 gl-1 Concentrated hydrochloric acid Glacial (concentrated) acetic acid Hydrogen peroxide 30 gl-1 O-tolidine Alkoholic solution of amidopyrine ("pyramidone"): aminophenasone 2.5 g in 500 ml of ethanol 900 gkg-1 Heitz-Boyers reagent (red. phenolphthalein in alkaline medium): 50 g KOH and 5 g phenolphthalein diluted in 800 ml of distilled water, added 25 g zinc dust, heated for 2 hours till the color faded away (reduction), filtrated an added by water up to 1 l Ethanol Sodium nitropruside cryst., dissolve several grains in distilled water in a test tube Sodium hydroxide 100 gl-1 Lestradets reagent: ammonium sulphate 20 g, sodium carbonate 20 g, sodium nitropruside cryst. 0.2 1 g Sodium nitrite 5 gl-1 Lugols solution: potassium iodide 2 g, iodine 1 g, distilled water 300 ml Iodine tincture: solution of iodine in ethanol (10 gl-1) Iron(III)chloride 100 g.l-1 Ehrlichs aldehyde reagent: 1 g of 4-dimethylaminobenzaldehyde dissolved in 100 ml of hydrochloric acid (200 gkg-1) Chloroform (trichloromethane) PHYSIOLOGIC PARTS: A qualitative detection of physiologic parts of urine is of little value in the diagnosis. But quantitative monitoring of some physiologic parts of urine is necessary to judge the kidney functions, acid-base equation and some metabolic disorders. Methods for evaluation of the physiological parts of urine are based on the same principles as in blood. Semiquantitative evaluation of calcium according to Sulkowitch: Take about 1 ml of urine. Measure pH of urine and make it slightly acid with diluted acetic acid (500 g/kg), if it is not acidic. Add 1 ml of Sulkowitchs reagent. Mix and observe a precipitate after 2 minutes. A slight opacity and minimum precipitates is a physiological finding. PATHOLOGIC COMPONENTS: Proteins Normally only a small amount of protein is present in urine (up to about 150 mg/24 h). Tests for qualitative detection of protein in urine are based on denaturing of protein. Sulfosalicylic acid test: To 1 ml of urine in a test tube add several drops of sulfosalicylic acid (200 g/l). A white precipitate shows the presence of protein.

Heat coagulation test: Heat 1 ml of clear urine to boiling in a test tube. The formation of a precipitate at this point is due to protein or to phosphates. Add 2 drops of diluted acetic acid. An excess of acid is to be avoided, for it may dissolve the proteins. Does the precipitate disappear or became heavier? The phosphates dissolve readily in the acidified solution, whereas the proteins remain precipitated. Hellers test: Put 1 ml of urine in a test tube. Incline the test tube and pour gradually down its side 1 ml of concentrated nitric acid, so as to form a layer between urine and nitric acid. Wait about 1 minute. A white precipitate or zone indicates presence of protein. The use of a black or dark background is helpful. Carbohydrates If the estimation of protein is positive, deproteination of the sample is necessary before detection of carbohydrates. Do it in this way: Mix a sample of urine with several drops of diluted acetic acid in a test tube. An excess of acid is to be avoided. Heat the sample to boiling. Than filter the sample through a paper filter. Glucose The detection is possible either using non-specific tests based on reducing properties of glucose or by specific glucose oxidase reaction. Fehlings test: Prepare Fehlings reagent before the test - mix Fehlings solutions I and II (1:1). Shake the reagent to get blue solution. Exclude presence of reducing agents, which would give false positive reaction: Heat 1 ml of the solution to boiling it must not change color. Than mix 1 ml of protein-free urine with 1 ml of Fehlings reagent in a test tube. Boil the sample in water bath. If glucose is present it develops green-yellow, yellow, to brick red precipitate. The color depends on the glucose concentration of the sample. Benedicts test: To 1 ml of Benedicts reagent in a test tube, add several drops of protein-free urine. Boil in water bath. The amount of reduction varies from a greenish tinge to a red precipitate, depending on the amount of sugar present. Nylanders test: Mix 1 ml of urine and 0.5 ml of Nylanders reagent. Boil for a few minutes. If the glucose is present yellow-brown, brown, to black color (depending on the glucose concentration) develops. Test strip test: The detection of glucose using the test strip is based on the glucose oxidase - peroxidase chromogen reaction. The oxidation of glucose is catalyzed by glucose oxidase to form gluconic acid lactone and hydrogen peroxide. Peroxidase catalyzes the reaction of hydrogen peroxide with a chromogen. If oxidized the chromogen turns to a color compound (green-blue or red). An intensity of the color corresponds to concentration of glucose. Pathological glucose concentration is indicated by a color change of a test field. Semiquantitative estimation of glucose concentration is possible using a color scale marked on a box with test strips. Each color from the scale corresponds to appropriate glucose concentration. . Ascorbic acid Urine containing ascorbic acid reduces Fehlings reagent while cold.

Fructose Selivans reaction: Into a test tube with a grain of resorcinol add 1 ml of concentrated hydrochloric acid and 2 ml of urine. Shake the test tube and heat it to boil. Red color of the solution indicates presence of fructose. Pentoses Bials test: Add several drops of urine to 2 ml of Bials reagent (orcinol) in a test tube. Heat the test tube to boiling and than cool it. A green colored solution or precipitate denotes a positive reaction. Blood, hemoglobin, myoglobin The detection is based on the pseudoperoxidative activity of hemoglobin (myoglobin). Iron involved in these compounds catalyze the oxidation of an indicator by hydrogen peroxide, producing a colored compound. Avoid presence of drinking water which contains ferric ions and than it may give false positive reaction. Orthotolidine test: Dissolve several grains of o-tolidine in 1 ml of ethanol in a test tube. Add several drops of glacial acetic acid, 1 ml of hydrogen peroxide, and 1 ml of urine. Shake the test tube and watch color changing of the solution. A bluish (to bluegreen) color will develop in the presence of blood. "Pyramidon" test: Mix approximately 1 ml of urine and 1 ml of alcoholic solution of pyramidon (amidopyrine, aminophenasone). Add 2 drops of diluted acetic acid and 3 drops of hydrogen peroxide. Shake well the solution. If the test is positive pink-violet to blue-violet color develops. Heitz-Boyers test (or reduced phenolphthalein test): Mix 1 ml of urine and 1 ml of Heitz-Boyers reagent in a clean test tube. Decline the test tube and layer some hydrogen peroxide onto the mixture along a wall of the test tube. Work carefully to prevent mixing of reagents. A pink to red-violet colored ring is positive for the blood. The principle described above is also useful for test strips. Ketone bodies Proof of diacetic acid and acetone is based on reaction with sodium nitropruside in alkaline medium. A product of the reaction is a violet complex. Legals nitroprusside test: To 1 ml of urine, add 2 drops of freshly prepared water solution of sodium nitroprusside. Make alkaline with 1 ml of sodium hydroxide (2mol.l-1). A violet color may be due to acetone, diacetic acid, or creatinine. Divide the sample into two parts. Acidify one part of the sample with a few drops of glacial acetic acid. In the presence of acetone or diacetic acid, the color remains or is intensified (compare with the non-acidified part of the sample). If the violet color turns to the yellow color, it were caused by creatinine (the creatinine is a physiological part of urine). In the presence of indol and its derivatives, for example indol melanogenes, the violet color turns to the blue (or blue-black) color. This way of estimation of indol melanogenes is called Thrmahlens test.

Lestradets nitroprusside test: Put a small amount of Lestradets reagent on a filter paper. Moisten the reagent by 1 drop of urine. If purple color develops within 1 minute the test is positive. Test strips: The test is based on the principle of Legals test. Acetotoacetic acid and acetone form a violet colored complex with sodium nitroprusside in alkaline medium. D-Hydroxybutyric acid is not detected using this reaction. Conjugated bilirubin Principle of the detection usually is oxidation of bilirubin glucuronide on biliverdin glucuronide (green) or bilicyanin (blue). The reaction is not specific. Gmelins test: Put 1 ml of urine in a test tube. Incline the test tube and pour gradually down its side 1 ml of concentrated nitric acid, so as to form a layer between urine and nitric acid. A green ring between urine and nitric acid indicates presence of conjugated bilirubin. Note: Compare with Hellers test for detection of protein. Rosins test: Put 1 ml of urine in a test tube. Incline the test tube and pour gradually down its side 1 ml of Lugols solution (or iodine tincture), so as to form a layer between urine and iodine tincture. A green ring between urine and iodine tincture indicates presence of conjugated bilirubin. Test with sodium nitrite: Put 1 ml of urine into a test tube. Add 1 drop of sodium nitrite and 1 drop of concentrated hydrochloric acid. The solution will be green if bilirubin is present. Test strips: The test is based on specific reaction. A reacting field of the test strip is impregnated by diazonium salt, which reacts with bilirubin and gives red azo dye. Semiquantitative estimation using a colored scale on the box is possible. Urobilinogen Ehrlichs reaction: Mix 1 ml of urine with a small amount of Ehrlichs aldehyde reagent. Wait for 5 minutes. If the compounds mentioned above are present the pinkred color develops. The reaction with Ehrlichs aldehyde reagent is not specific. It is positive in presence of urobilinogen, stercobilinogen, indol and its derivates (indolic melanogenes), porphobilinogen, and some drugs (e.g. sulphonamides). There rises pink-red condensing products. To differentiate indol, indolic melanogenes: perform Thormahlens test (see the Legals test for ketone bodies). To differentiate urobilinogen and porphobilinogen: If the colored condensing compound is conditioned on urobilinogen a batch extraction is possible. Procedure: Mix 1 ml of urine with a small amount of Ehrlichs aldehyde reagent. Wait for 5 minutes. If the compounds mentioned above are present the pink-red color develops. Add 0.5 ml of chloroform to the test tube with the examined pink-red condensing product. Shake it well. The chloroform separates and forms a lower layer of the mixture. The urobilinogen conditioned pink condensing products get to the chloroform and thus the color limits only to the lower layer. The porphobilinogen conditioned pink condensing products does not do it.

Test strips for urobilinogen are usually based on specific reaction. The urobilinogen reacts with diazonium salt contained in a reagent field of the test strip. A product of the reaction is red azo dye. Phenylpyruvic acid (Phenylketonuria) Add several drops of iron (III) chloride to 1 ml of urine. Wait for several minutes. The test is positive if dark green color appears. Homogentisic acid (2,5-dihydroxyphenylacetate) (Alkaptonuria) Urine containing homogentisate reduces Fehlings reagent while cold (see the procedure for proof of glucose described above). But color rises in this case is brownblack (or yellow-black), produced by phenolic melanin, which superimposes the yellow-red color reduced copper. 3. SEDIMENT ANALYSIS Centrifugation of urine produces sediment which may contain a variable number of leucocytes, erythrocytes, epithelial cells, hyaline casts and crystals. The elements like bacteria, red and white blood cells and casts are typical for urogenital disorders. The casts are formed in the tubules; the epithelial cells originate in the bladder and the urethra. The crystals present in sediment may contain uric acid, calcium oxalate, phosphates, tyrosine, cystine, leucine.