Chemical Examination of Urine PDF

Email: [email protected] 1 CHAPTER FOUR Chemical Analysis of Urine  Email: [email protected] 2 Chapter Outline Objectives Determination of Urinary sugar Determination of Urinary ketone bodies Determination of Urinary protein Deter...

Email: [email protected] 1 CHAPTER FOUR Chemical Analysis of Urine  Email: [email protected] 2 Chapter Outline Objectives Determination of Urinary sugar Determination of Urinary ketone bodies Determination of Urinary protein Determination of Urinary hemoglobin Determination of Urinary Bilirubin Determination of Urinary urobilinogen Determination of Urinary urobilin Determination of Urinary Porphybilinogen Determination of Urinary lecuocyte estrase  Email: [email protected] 3 Chapter Outline… Determination of Urinary Nitrite Determination of Urinary Calcium Determination of Urinary indicant Determination of Urinary Melanin Determination of Urinary Ascorbic Acid Determination of Urinary phenyl pyruvic Acid Determination of Urinary Mucopolysacchride Determination of Urinary Hemosiderine  Email: [email protected] 4 Learning objectives ❖At the end of this chapter the students will able to:- to carry out how to perform chemical tests and show the various types of methods used to determine the chemical constituent:- – Know the interfering factors during their work – Expected to differentiate the principles of each test – Explain the test procedures – Know the clinical significance of the tests  Email: [email protected] 5 Introduction Chemical analysis of urine deals with those procedures or examinations which are accomplished by means of chemical reactions. Chemical test are done for a quick screening of urine for certain substances which have a diagnostic significance. Abnormal or pathological dissolved constituents of urine (chemicals) performed in routine urinalysis. Chemical analysis of urine is important in the detection of many diseases. In abnormal (pathological) conditions its composition varies in kind and quantities.  Email: [email protected] 6 Introduction… So the chemical changes of urine can indicate disease at the early stage. Urine composition depends greatly on how much and what specific waste material is to be excreted. Urea, creatinine, uric acid, ammonium salts, chlorides, sulphates and phosphates of sodium, potassium, calcium and magnesium are the normal composition of urine. Glucose, protein, ketone bodies, bilirubin, bile salts etc. are the abnormal constituents of urine. Normally these substances do not appear in the urine in detectable amount. So their appearance in the urine shows the pathological condition.  Email: [email protected] 7 Chemical analysis Chemical analysis of urine can be classified as:- ✓Screening test ✓Qualitative test ✓Quantitative test Screening test:- tells the presence or absence of the abnormal components of urine. e.g., dipstick tests Qualitative test:- tells semi-quantitatively the amount of solutes that exist at the detectable level in pathological or physiological conditions. e.g., bilirubin, protein Quantitative test:- tells the exact amount of abnormal constituents if they are detectable. e.g., 24-hour urine protein  Email: [email protected] 8 Chemical Reaction Chart  Email: [email protected] 9 General Precaution for reagent strips Store strips in a cool, dry place to prevent deterioration. Do not use strips if they are discolored. Do not touch the test areas. Keep the test areas away from detergents or other contaminating substances. Dip the test areas into the urine specimen completely, but briefly to avoid dissolving the reagents out of the test area. Remove only the number of reagent strips required at a particular time, and keep the container tightly covered at all times to prevent deterioration. Read the test results carefully, at the time specified by the manufacturer, in a good light and with the test area held near the appropriate color chart on the bottle label. Email: [email protected] 10  Email: [email protected] 11 Determination of Glucose Glucose is the sugar most commonly found in the urine, although other sugars, such as lactose, fructose, galactose, and pentose, may be found under certain condition. The presence of detectable amount of glucose in the urine is known as glycosuria. Normally almost all the glucose, which passes from the blood into the glomerular filtrate, is reabsorbed back into the circulation by the kidney tubules (proximal convoluted tubules). Usually less than 15 - 20 mg/dl (0.8 mmol) is excreted in the urine. But this amount cannot be detected by the routine laboratory tests.  Email: [email protected] 12 Glucose cont’d… The blood glucose concentration normally lies between 70 and 120 mg/dl. After a meal it may increase to 120 - 160 mg/dl. If the blood glucose concentration becomes too high (usually greater than 170 - 180 mg/dl), the excess glucose will not be reabsorbed into the blood and glucose start appearing in urine. The lowest blood glucose concentration that will result in glycosuria is termed as the renal threshold. The renal threshold refers to the concentration of a substance in the blood at which the kidneys begin to excrete that substance into the urine. The most common condition in which the renal threshold for glucose exceeds is diabetes mellitus.  Email: [email protected] 13 Causes of Glycosuria Physiological Pathological Physiological a. After large ingestion of carbohydrates b. Anything that stimulates sympathetic nervous system c. 15 to 20% cases of pregnancy d. Renal Glycosuria: in some persons, glycosuria is found when blood glucose is in normal range.  Email: [email protected] 14 Causes of Glycosuria Pathological Glycosuria A. Diabetes Mellitus:- a chronic metabolic disorder characterized by high blood sugar levels due to either:- insufficient insulin production or the body's cells not responding effectively to insulin. ✓ Glucose is not properly metabolized and blood glucose concentration rises, and ✓ When it is in range of 170 - 180 mg/dl, glucose starts appearing in urine. B. Glycosuria due to other endocrine disorders:- A number of endocrine disorders can cause hyperglycemia, and this may result in glycosuria, - Hyperthyroidism - Hyperadrenalism - Hyperpitutarism - Some diseases of pancreas  Email: [email protected] 15 Types of Urinary Sugar (Glucose) Test 1. Non-specific reduction tests based on the reduction of certain metal ion by glucose. 2. Specific (Enzymatic) tests based on the action of glucose oxidase on glucose.  Email: [email protected] 16 Non-Specific Test for Glucose Glucose is acting as a reducing agent and any compound with a free aldehyde or ketone group will give the same reaction. Hence glucose is not the only reducing substance that may be found in urine. Urine contains non-glucose reducing substances (NGRS) such as: uric acid, creatinine, galactose, fructose, lactose, pentose, homogenestic acid, ascorbic acid chloroform, and formaldehyde. ❖ Commonly used non-specific tests for urinary sugar are:- ✓ Benedict’s Qualitative test ✓ Fehling test ✓ Clinitest tablet test  Email: [email protected] 17 Benedict’s Qualitative Test Principle When urine is boiled in an alkaline copper sulphate solution, glucose and other reducing substances reduce (convert) the blue copper (II) in Benedict's qualitative reagent to copper (I) oxide (Cu2O), which is orange to red in color. A positive reaction is graded as a change in color ranging from blue to green, yellow, orange and finally red.  Email: [email protected] 18 The overall reaction is The copper (II) ions are supplied in Benedict's qualitative reagent in the form of copper sulphate (CuS04). In the presence of a strong alkali this is converted to copper ( I) oxide (Cu2O ). The heat is supplied by means of a boiling water (1000C) bath. The tubes are brought back to room temperature, and the results are read when convenient.  Email: [email protected] 19 Procedure 1. Arrange test tubes (12ml) in a row rack. 2. Take two extra tubes for control runs:- ✓ negative with water and ✓ positive with 1.0 g/dl of glucose solution. 3. Add eight drops of well-mixed urine. 4. Add 5ml of Benedict’s reagent. 5. Mix well and place in a boiling water bath for 5 min. 6. Remove and cool to room temperature. 7. A positive: presence of a fine yellow to brick red 8. Graded on the basis of the color of solution.  Email: [email protected] 20 Grade results according to the following criteria: 1. Negative: No change in the blue color of the reagent or the occurrence of a white or green precipitate from phosphates. 2. Trace: Slight amount of yellow precipitate with a greenish blue to bluish green mixed solution less than 500mg/dl of sugar). 3. +: Moderate amount of yellow precipitate with green, often referred to as apple green, mixed solution. (Approximately 500mg/dl of sugar). 4. ++: Large amount of yellow precipitate with a yellowish green, often called muddy green mixed solution. (Appr.750mg/dl of sugar). 5. +++: Large amount of yellow precipitate with green yellow, or muddy orange, mixed solution. Some blue color remains in supernatant. (Appr.1000mg/dl of sugar). 6. ++++: Large amount of yellow to red precipitate with reddish yellow to red mixed solution. No blue remains in the supernatant (Appr.2000mg/dl).  Email: [email protected] 21 Fehling’s Test Principle: The same as benedict test Fehling’s reagent Solution A Solution B Copper sulphate 34.65 g Sodium hydroxide 125 gm Distilled water 500 ml Sodium potassium tartarate173 gm Distilled water 500 ml  Email: [email protected] 22 Procedure of reagent preparation 1. Take equal volume of Fehling’s solution A and solution B. 2. Mix and boil. 3. Add a few drops of urine and boil again. 4. If sugar is present, the same type of change occur as in the Benedict’s test and are reported similarly.  Email: [email protected] 23 Advantage of Bendict’s Test over Fehling’s test It uses a single solution already prepared It uses a more stable reagent It is about ten times more sensitive and traces of glucose will not be caramelized. If the test is carried out using the proper proportions of the reagent and urine, it is more specific, as uric acid and creatinine will not ordinarily reduce Benedict’s solution, under these conditions.  Email: [email protected] 24 Clinitest Tablet Tests Principle: The same as that of Benedict's Qualitative Test. Contains anhydrous sodium hydroxide, which results in moderate boiling when added to dilute urine gives heat in its reaction with citric acid. Results are graded as negative, trace, 1+, 2+, 3+, or 4+ by comparison on color chart. Sensitivity ✓ Clinitest reagent tablets will detect as little as 250mg of sugar in 100ml of urine.  Email: [email protected] 25 Procedure Five drop method Follow the direction supplied with the clintest tablets 1. Place 5 drops of urine in a test tube and add 10 drops of distilled water. 2. Add one clintest tablet by easing it in to the tube with out touching it. It contains strong alkali. 3. Watch while boiling takes place, but don’t shake or touch the bottom of tube. 4. Wait 15 seconds after boiling stops, then shake the tube gently and compare the color of the solution with the color scale.  Email: [email protected] 26 Cont… Grade the results as negative, trace, 1+, 2+, 3+, 4+ The results correspond to the following concentrations (mg/dl): trace = 250mg/dl;1+ = 500mg/dl; 2+ = 750mg/dl: 3+ = 1000mg/dl: 4+ = 2000mg/dl. Watch the solution carefully while it is boiling. If it passes through orange to dark shade of greenish brown, the sugar concentration is more than 2000mg/dl and the results should be recorded as 4+ without reference to the color scale.  Email: [email protected] 27 Specific (enzymatic) test for glucose These tests are based on the use of glucose oxidase The glucose oxidase in the presence of atmospheric oxygen oxidize glucose to gluconic acid and hydrogen peroxide. The principle of all enzymatic test is the same They differ only on the uses of different type of chromogen (a color indicative).  Email: [email protected] 28 Cont… Specific test for urinary glucose that will be discussed here are: ✓Clinistix reagent strip ✓Test tape ✓Diastistix Which are available in the reagent strip – Separately or in combination with other urine test  Email: [email protected] 29 Principle Clinstix It is a double sequential enzyme reactions:- Glucose oxidase will oxidize glucose to gluconic acid and at the same time reduce atmospheric oxygen to hydrogen peroxide. The hydrogen peroxide formed will, in the presence of the enzyme peroxidase, oxidize the reduced form a dye to the oxidized form, which is indicated by the color change of an oxidation - reduction indicator.  Email: [email protected] 30 Clinistix Reagent Strip Test  Email: [email protected] 31 Chemical Exam When the test strip is dipped in urine the reagents are activated and a chemical reaction occurs. The chemical reaction results in a specific color change.  Email: [email protected] 32 Positive Glucose (Normal for comparison)  Email: [email protected] 33 Procedure Follow the directions supplied with the strips. 1. Rapidly dip the test end of the strip in the urine. 2. Read the results after exactly 10 seconds 3. Record the results as positive or negative. If the test area remains green, the result is negative. A positive result is indicated by the appearance of a purple color in the test area.  Email: [email protected] 34 Test-Tape for Glucose Principle Test-Tape is a screening test Specific for glucose The principle is the same to clinistix Differ in the oxidation - reduction indicator employed, and the material the reagents are impregnated on. In Test-Tape the reagents are impregnated on a tear strip of special paper, and the indicator is yellow in its reduced form and green to blue in its oxidized form.  Email: [email protected] 35 Procedure Follow the manufacture direction: 1. Tear off approximately 1 and 1/2 inch. 2. Dip part of the tape into the urine specimen; remove it immediately. 3. Wait for 30 seconds; then observe the appearance of any green color. 4. Record the result as positive or negative. – If the test area remains yellow after 30 seconds, the result is negative. – If any green color is present at this time, the result is positive.  Email: [email protected] 36 Diastix Reagent Strip for Glucose Diastix is a specific test for glucose based on the use of glucose oxidase. The chemical reaction is the same as clinistix The difference being the chromogen system used to indicate the presence of glucose. The reagent area contains glucose oxidase, peroxidase, a blue background dye, and potassium iodide as the chromogen.  Email: [email protected] 37 Procedure ❖ Follow the directions supplied with the reagent strip. 1. Dip the reagent area of the strip briefly into the specimen. 2. Compare the test area with the color chart after 10 seconds to see whether the reaction is positive or negative for glucose. 3. Compare the test area with the color chart at 30 seconds, for a semi-quantitative result, and report the results as indicated on the chart.  Email: [email protected] 38 Rubner’s Test To 10 ml of undiluted urine add 3 gms (excess) lead acetate, Shake well, Filter Boil the filtrate and add 1 ml concentrated ammonium hydroxide and boil. The precipitate formed is the criterion of the test. Lactose: Red solution + Red precipitate Glucose: Red solution + Yellow precipitate  Email: [email protected] 39 Fructose Fructose may be found in the urine mostly after fruits, honey, syrup and jams food. It may be found in liver disease and along with glucose, in the urine of diabetics.  Email: [email protected] 40 Seliwanoff’s Test for Fructose Hydrochloric acid acts on fructose to form a derivative of furfuraldehyde, which gives a red-colored compound when linked with resorcinol. Procedure 1.Add a few drops of urine (about 0.5 ml) to about 5 ml of the reagent in a test tube 2. Boil. Interpretation: Fructose gives a red color within half a minute  Email: [email protected] 41 Clinical Significance Normally, urine doesn't contain a sufficient amount of sugar to react with any of the popular enzyme or reducing tests. When sugar appears in the urine, it shows the abnormality caused by disease diabetes mellitus. Test for urine sugar is used in screening to detect diabetic mellitus, confirming diagnosis of diabetes mellitus, and monitoring the effectiveness of diabetic therapy.  Email: [email protected] 42 Ketones Ketones are also called Ketone bodies. are a group of three related substances:- 1. Acetone 2. Acetoacetate (acetoacetic acid) and 3. -hydroxybutyrate (-hydroxybutyric acid). Formed in liver from acetoacetate (oxidation product of amino acid, fatty acid) Used as energy source for extrahepatic tissue like brain and muscle cells.  Email: [email protected] 43 Ketone bodies an alternative fuel for cells Ketone bodies are normal products of fat metabolism. Normally they are not detectable in the blood or urine of health individuals. In normal metabolism, fat is broken down in the tissues to glycerol and fatty acids. The free fatty acids are transported by the plasma albumin to the liver where they are broken down to acetyl coenzyme A (acetyl Co-A) molecules. Liver mitochondria have the capacity to divert any excess acetyl CoA derived from fatty acid or pyruvate oxidation into ketone bodies. The compounds categorized as ketone bodies are acetoacetate, 3- hydroxybutyrate and acetone (a volatile non-metabolizable side product). They are transported in the blood to peripheral tissues, where they can be reconverted to acetyl CoA and oxidized by the TCA cycle.  Email: [email protected] 44 Cont… The liver actively produces ketone bodies, but it cannot itself use them as fuels. Ketone bodies are used in extrahepatic tissues such as the skeletal and cardiac muscle and the renal cortex, in proportion to their concentration in blood. Even the brain can utilize ketone bodies for fuel if the level rises sufficiently, this is important during prolonged periods of fasting. Extrahepatic tissues efficiently oxidize acetoacetate and 3- hydroxybutyrate. The liver lacks succinyl CoA: acetoacetate CoA transferase, and therefore is unable to use acetoacetate as a fuel.!! Red blood cells cannot efficiently oxidize acetoacetate and 3- hydroxybutyrate because they lack mitochondria. Email: [email protected] 45  Email: [email protected] 46 Tests for ketone bodies in Urine:- Rothera’s test Lang’s test Acetest tablet test Acetone powder test Reagent strip tests (eg. ketostix)  Email: [email protected] 47 Rothera’s test Principle Both acetone and acetoacetic acid give a purple color with alkaline sodium nitroprusside. This is the general principle for the tests mentioned above. Results:- Report the test as positive or negative  Email: [email protected] 48 Procedure 1. To 5 ml of fresh urine, add ammonium sulphate crystals until saturated (about 1 g.). 2. Add 2 drops of sodium nitroprusside reagent and mix 3. Overlay with ammonium hydroxide solution (28%). 4. If acetone or acetoacetate is present, a red to purple color will develop at the line of contact. ✓ The color may not appear for 10-15 minutes. 5. Report the test as positive or negative. Note: Urine collected after a big meal may give a purplish color within 30 seconds but it fades within 3-4 minutes. This is not a positive test.  Email: [email protected] 49 Ketostix Reagent Strip Test Principle:- is based on Rothera’s test, Acetoacetic acid will react with sodium nitroprusside in a alkaline medium to form a purple color. If glycine is added, the test is slightly sensitive to acetone. N.B Multistix and ketostix have been formulated to react only with acetoacetic acid. They don’t react with acetone.  Email: [email protected] 50 Ketostix Reagent Strip Test Procedure Dip test-end of the strip in urine At 15 seconds compare the color of dipped-end with the color chart. Report as indicated by the color chart. Content of Reagent Strip Sodium Nitroprusside, Glycine, and a strongly Alkaline buffer.  Email: [email protected] 51 Positive urine Ketones (Normal for comparison)  Email: [email protected] 52 Gerhardt’s Test for acetoacetate It is specific for acetoacetate; However, it is capable of detecting only large amounts of acetoacetate. The test has been used as a means of determining the severity of ketosis. A positive result indicates severe ketosis, and treatment must be started immediately. Principle of the Test When acetoacetate reacts with a ferric chloride (FeC13) solution, Bordeaux red color is formed. Reagent = 10% Ferric chloride reagent Weigh 10 g of ferric chloride and transfer to a 100 ml volumetric flask. Dissolve and dilute to 100 ml volume with water and mix.  Email: [email protected] 53 Clinical Significance When the rate of formation of ketone bodies is greater than the rate of their use, their levels begin to rise in the blood, which is called ketonemia, and eventually in the urine, which is known as ketonuria. Both ketonemia and ketonuria are seen most often in cases of:- Starvation and diabetes mellitus Also in the urine during prolonged vomiting, severe diarrhea, anesthesia, high fat intake and low carbohydrate diet.  Email: [email protected] 54 Clinical Significance… The excessive production and accumulation of ketone bodies may lead to ketosis. Its physiological effect is serious because acetoacetic acid and -hydroxybutyric acid contribute excess hydrogen ions to the blood, ✓resulting in acidosis:- a condition that tends to lower the blood pH. ✓If not corrected in time this may result in death. Another physiological effect found to be toxic to brain tissue when present in increased amounts in the blood. So this condition can result in permanent brain damage. For diagnosis of the severity of diabetes.  Email: [email protected] 55 Determination of Urinary Protein Introduction ❖ Test for urinary protein is one of the most important and valuable parts of the routine urinalysis. ✓ Albumin is one of the important proteins, which appears in urine during a pathological condition. ✓ It often occurs as a symptom of renal disease. ✓ Globulins are excreted less frequently. ✓ Bence Jones protein is a specific type of globulin excreted in multiple myeloma.  Email: [email protected] 56 Causes of Proteinuria 1. Increased permeability of the glomerulus:- Normally, the glomerular membrane in the formation of urine, is not permeable for protein molecules. If the glomerular membrane is damaged these large protein molecules can pass through it. 2. A decrease in normal reabsorption in the tubules:- disease condition which affect reabsorption.  Email: [email protected] 57 Types of proteinurea Classified according to the relationship of its etiology to the kidney and also the mechanism involved:- A. Functional Proteinuria B. Systemic disease or renal pathology Proteinuria A. Functional Proteinuria: not associated with renal damage. 1. Accidental or false proteinurea:- Occurs when mixing of urine with a proteinous fluid such as pus, blood or vaginal discharge.  Email: [email protected] 58 Functional Proteinuria cont’d… 2. Physiological proteinurea:- It is usually less than 0.5 gm/24 hrs. which is associated with fever, exposure to heat or cold, emotional stress, and later stage of pregnancy. 3. Postural (orthostatic) proteinurea:- ✓ Associated only with the upright position or sitting for a long time. ✓ The proteinurea is intermittent and disappears when the individual lies down.  Email: [email protected] 59 B. Systemic disease or renal pathology Proteinurea 1. Pre-renal proteinuria:- not due to primary renal disease Fever and a variety of toxic condition Venous congestion Renal hypoxia Hypertension Myxedema(hypothyroidism) Bence Jones protein  Email: [email protected] 60 Cont… 2. Renal proteinuria:- primarily kidney disease Gomerulonephritis Nephrotic syndrome, primary and secondary Destructive parenchymal lesions (tumour, infection) 3. Post renal proteinuria:- protein added to the urine at some point farther down the urinary tract from the renal parenchyma. Infection of the renal pelvis or ureter Cystitis Urethritis or prostatitis Contamination with vaginal secretion  Email: [email protected] 61 Test for urinary protein 1. Precipitation or turbidimetric test Principle: Protein is either precipitated out of the urine specimen by means of a chemical, or it is coagulated out of solution with heat. These tests include:- a) Robert’s Test ring tests b) Heller’s Test c) Sulphosalicylic Acid Test non-ring tests d) Heat and Acetic Acid Test  Email: [email protected] 62 Robert's Test Principle:- The principle of this test is based on the precipitation of protein and formation of white compact ring using concentrated Nitric acid (HNO3).  Email: [email protected] 63 Procedure of Robert's Test Place 3-5 ml urine in a test tube. Place the Robert's Reagent to the bottom of the tube and allow 3 ml of the reagent to lay beneath the urine. A white ring at the zone of contact indicates a positive test. The ring must be read within 3 minutes Report the result according to the type of the ring formed  Email: [email protected] 64 Heller's Test Principle:- The same as Robert`s Test Heller's Reagent: It is concentrated nitric acid. Procedure the same as roberts test  Email: [email protected] 65 Sulphosalicylic Acid Test Principle Sulphosalicylic acid solution (20%) precipitates any protein in the urine specimen. It is an anion precipitant that works by the neutralization of the protein cation. This method is more sensitive and reliable than the heat method. It can detect 5 to 10 mg/dl of protein in urine specimen  Email: [email protected] 66 Procedure of Sulphosalicylic Acid Test Take 2 ml of a centrifuge (clear) urine specimen in a test tube. Add equal amount of Sulphosalicylic acid reagent. Shake the test tube gently and let stand for 10 minutes Note the degree of turbidity by looking at the illuminated tube against a dark background Grade and report the result as non ring test Interpretation:- report the result based on the turbidity formed  Email: [email protected] 67 Heat and Acetic Acid Test Principle: The test is based on the precipitation of protein by heat. Procedure Fill a test tube three-fourth full of clear urine, and gently heat the upper portion of urine for 2 minutes to boil, being careful not to shake the tube more than necessary. The lower portion of urine is not heated so that it can be used as a control for comparing. If turbidity (a white cloud) can arise due either of phosphates, carbonates, or protein.  Email: [email protected] 68 Procedure Cont’d… Add 3 drops of 10% acetic acid drop by drop, boiling between each drop. A white cloud that disappeared is due to phosphates or carbonates. Persistence or development of turbidity implies proteinuria. Read the test and record results according to the chart for non-ring precipition test. Sensitivity This method is the most sensitive for small amount of protein and can reliably detect protein concentrations of 2 to 3 mg/dl  Email: [email protected] 69 Colorimetric Reagent Strip (Dipstick) Principle The Colorimetric reagent strip test is based on the ability of protein to alter the color of some acid-base indicators without altering the pH. When an indicator, such as tetrabromophenol blue is buffered at pH 3, it is yellow in solutions without protein. Tests are more specific. They require only a drop of urine enough to moisten the reagent area.  Email: [email protected] 70 Procedure of Reagent Strip Observe the precautions and follow the instructions supplied by the manufacturer. 1. Dip the reagent area of the strip briefly into the specimen. 2. Remove excess urine by tapping or drawing the edge of the strip along the rim of the urine container. 3. Compare the color that develops with the color chart supplied by the manufacturer and report as indicated on the chart.  Email: [email protected] 71 Chemical Exam When the test strip is dipped in urine the reagents are activated and a chemical reaction occurs. The chemical reaction results in a specific color change.  Email: [email protected] 72 Quantitative 24 hour protein Determinations ✓ Estimates the protein content of urine. ✓ Performed by quantitating the amount of precipitation formed following the addition of a specific chemical to the urine. ✓ The precipitate is measured either by comparison with known standards (sulphosalicylic acid turbidity test) or ✓ By recording the height of the column of precipitate in a specially-designed tube (Esbach's test).  Email: [email protected] 73 Quantitative 24 hour protein Determinations A. Sulphosalicylic Acid Turbidity Test 1. Pipette 2.5 ml of urine into a test tube. 2. Add 7.5 ml of 3% sulphosalicylic acid. 3. Invert to mix 4. Let stand 30 minutes. Compare the turbidity with known standards prepared from solutions containing 10, 20, 30, 40, 75 and 100mg albumin/dl, and estimate the concentration of the unknown (urine protein). If the unknown urine contains more than 100mg/dl protein, dilute the urine and repeat the test.  Email: [email protected] 74 Quantitative 24 hour protein Determinations B. Esbach’s Test 1. measure the total volume; then filter some of the urine. 2. Do qualitative protein test, Robert’s or strip test. if the urine is +3, made 1:5 dilution. if the urine is +4 make 1:10 dilution. if the urine is trace, +1 or +2 non dilution is needed. if the urine is negative, a quantitative test is not done. N.B: measure the pH of the urine. It should be acidic. If not, add 10 % acetic acid  Email: [email protected] 75 Esbach’s Test Cont’d… 4. Add pumice powder to the 0.5 mark of the Esbach’s tube. 5. Add urine to the “U” mark. 6. Add Esbach’s reagent to the “R” mark. 7. Mix slowly by inversion, 10 times. 8. Wait for 30 minutes. Read the highest of the column. Do not subtract the amount of the pumice. 9. The result is now in gram per liter of protein in the urine. If the urine has been diluted, multiply by the dilution factor, calculate, and record the g % and the g / 24 hrs.  Email: [email protected] 76 Clinical Significance Proteinuria: >6-8gm/l. renal damage cause proteinuria. A positive protein test for urine may be correlated with findings of casts. In cases of renal disease, it is essential that the diagnosis be made and treatment started as soon as possible to prevent extensive and permanent renal damage. Proteinuria with the presence of leukocytes and bacteria in the urine will be suggestive of urinary tract infection.  Email: [email protected] 77 Bence - Jones Protein It is an abnormal low molecular weight globulin consisting of light chains of immunoglobulin, which contains the amino acid found in other proteins except methionine. It may be found in the urine of patients with multiple myeloma, which is a malignant disease of the plasma cells, mainly affecting bone. The incidence of Bence-Jones proteinuria in multiple myeloma has been estimated as 50% to 80%.  Email: [email protected] 78 Heat test to screen Bence -Jones Protein Principle: Bence Jones protein coagulates when heated to 40 to 600C and re-dissolves partially or wholly on boiling. Albumin coagulates above 600C and does not re-dissolve on boiling.  Email: [email protected] 79 Heat test to screen Bence -Jones Protein Procedure 1. Take 5ml of clear fresh urine into each of three test tubes. 2. Acidify the urine by adding 1 drop of glacial acetic acid to the first tube, 2 drops to the second tube, and 3 drops to the third tube. 3. Place the three tubes in a beaker of tap water and insert a 0- 100 OC thermometer in one of the tube. 4. Using a gentle flame slowly heat the beaker of water. 5. When the temperature reaches 40OC look for cloudiness and precipitate in the tubes. Continue to observe until the temperature reaches 60OC.  Email: [email protected] 80 Clinical significance Suggests the diagnosis of multiple myeloma, leukemia or waldenstorms macroglobulinemia. Interfering factor: False positive result may occur in connective tissue disease, renal insufficiency, contamination of the specimen with menstrual blood and prostatic secretion or semen. coagulable protein denatures or decompose at room temperature. Email: [email protected] 81