TITLE The analysis of normal and abnormal urinary components AIMS AND OBJECTIVES 1. 2. 3.

To know the various urinary components To understand the significance of these components To get a brief (hands-on) introduction to clinical biochemistry

INTRODUCTION The quantity and composition of urine reflects various biochemical processes that occur in the body. Thus the composition of the urine of an individual may change when a person has a disease. In a diseased condition, it is quite common to observe the presence of abnormal components (Compounds which are not present in the urine of a normal, healthy individual) or normal components in abnormal quantities in the urine. THE FOLLOWING TABLE SHOWS THE NORMAL COMPOSITION OF THE URINE. Types VOLUME SPECIFIC GRAVITY PH UREA AMMONIA URATE CREATININE CREATINE Indicator 600 - 200 ml / 24 hour 1.003 – 1.030 4.6 - 8.0 15 – 35 g (250 – 580 mmol) / 24 Hour 0.5- 1.0 g (29 – 58 mmol ) / 24 Hour 0.5 – 2.0 g (3 – 12 mmol) / 24 Hour 1.0 – 2.0 g (9 – 17 mmol) / 24 Hour 0.1g / 24 Hour

THE ABNORMAL URINARY COMPONENTS ARE AS FOLLOWS Glucose, ketone bodies, indole and related compounds, porphyrine and related compounds, protein ( albumin and Bence-Jones protein), bile pigments, blood, melanin and so on. EXPERIMENT 1 Tests for normal urinary components The urine sample is necessary free from any contamination and using own urine samples. PROCEDURE • AMMONIA 1. 2. • pH of 2-3 ml urine is adjust with sodium carbonate to a pH of 9.0. The solution is boiling and tested for the presence of ammonia.

SULPHATE 1. 2. 3. 4. Mix 2 ml dilutes HI with 10 ml urine and then adds 2 ml chloride solution. Filter the barium sulphate precipitate that is formed. Use half the filtrate as blank and boil the other half. Notice the change that takes place in the boiled filtrate. (It should become cloudy).


INDICAN 1. 2. 3. 4. 5. 6. Mix 3ml of urine with 3 ml of concentrated HC1 and 1 ml of chloroform. Add a few drops of sodium hypochlorite (NaOCl) and shake thoroughly. Observe the colour of the chloroform phase. Do not add too much of NaOCl. What is the source of indican in the urine? This test should be negative in normal urine.

URIC ACID 1. 2. 3. 4. Add 2 drops of Folin-fosfotungstic acid reagent to 2 ml of urine which has been alkalinized with NaOH. In another tube add 2 drops of Folin-fosfotungstic acid to 2 ml sodium urate. Compare the colors formed. For a more specific reaction, carry out the above reaction with 20 ml of urine that has been saturated with ammonium chloride and alkalinized with concentrated ammonium hydroxide. (Note: Ammonium urate is not soluble in ammonium chloride).

CREATININE (WELY TEST) 1. 2. 3. Add a few drops of sodium nitropruside into 2 ml of urine and 2 ml of creatinine solution in a separate tube. Add a few drops of dilute NaOH and observe the colour formation. Add a few drops of glacial acetic acid and notice the colour change. (Note: Nitropruside is also used to test for the presence of acetone in urine. However the effect of glacial acetic acid is different.)

UROBILIN 1. 2. 3. 4. 5. 6. 7. 8. 9. This compound is formed by the reduction of bile pigment in the intestine and the oxidation of urobilinogen. The amount of urobilin present in the urine increases certain types of liver diseases. The most common test used is the Schlesinger test. Add 2 drops of 2.5 % alcoholic iodine solution into a test tube that contains 5 ml urine. (This is to oxidize urobilinogen in the urine to urobilin.) Then, add 5 ml solution of 10% zinc acetate in alcohol. Mix thoroughly by inverting the tube several times. Filter the mixture and ensure that the filtrate has an acidic pH. If the filtrate is found to be neutral or basic, acidify the filtrate with acetic acid. Ensure that the colour of the filtrate is pendafluor green. If this colour does not disappear even after 20x dilution, it indicates a pathological condition.

UROBILINOGEN 1. 2. 3. 4. 5. 6. Urobilinogen is not easily detectable because it is easily oxidized. Thus it is crucial that only freshly obtained urine sample is used. To test for urobilinogen. Add 1ml of 2% p-dimethylamino-benzaldehyde in20 % HCl. The formation of red colour complex indicates the presence of urobilinogen. The test shows positive results even in normal urine that has been diluted up to 20 X. However, pathological condition is indicated if the red colour complex is observed in urine that has 2

been diluted greater than 20 X. EXPERIMENT 2 Tests for abnormal urinary components. Detection of sugar in the urine. The most common type of sugar that is found in the urine is glucose (in diabetes and in kidney failure) and lactose (sometimes during pregnancy or lactation). Medically, the presence of glucose or lactose in the urine is significant. It is also important to differentiate between glucose and lactose. The presence of lactose is not indicative of a pathological condition but the presence of glucose at above a certain concentration indicates diabetes mellitus. PROCEDURE • BENEDICT TEST FOR REDUCING SUGARS 1. 2. 3. 4. • Add 8 drops of urine to 5 ml of Benedict qualitative reagent in a test tube. Mix thoroughly and place the tube in a boiling water-bath for 5 min. The formation of orange-brown complex confirms the presence of reducing sugars. This reagent specifically shows positive result with reducing carbohydrates and does not react with aldehyde or any other forms of reducing agents. FEARON TEST FOR GLUCOSE, LACTOSE AND MALTOSE 1. 2. 3. 4. 5. Boil 5 ml of glucose, lactose and maltose (in separate test tubes) with 0.5 ml of 5% methylamineHCl. Then add 2 ml of hot, dilute NaOH. Cool to room temperature. Lactose and maltose produce red colour complex. Repeat the test using the urine sample.

Test for ketone bodies The ketone bodies in the urine include acetoacetic acid, b-hydroxybutyric acid and also acetone. These compounds are excreted in the urine when fats are metabolized excessively. Acetoacetic acid may easily undergo decarboxylation to form acetone. PROCEDURE • ROTHERA TEST ( TO DETECT ACETON AND ACETOACETA TE) 1. 2. 3. 4. 5. 6. Test for proteins In a healthy individual, very little protein (30 -200 mg/24 hr) is excreted in the urine. The amount of protein that is excreted increases in pathological conditions such as heart disease, toxemia, pregnancy, kidney failure and liver 3 Add ammonium sulphate crystals into 3 ml of urine and shake until it becomes saturated. Add a few drops of sodium nitropruside (5%, w/v). Gently add 2 ml of concentrated ammonia to the surface of the solution. The addition of ammonia causes the formation of a purple ring at the interface (between the urine and the ammonia) in the presence of ketone bodies. Shake the tube gently to ensure that the colour is homogeneously distributed. Note: the formation of the colour complex may take up to 5 - 10 minutes if the ketone concentration is very low. This test is sensitive up to 100 ppm (parts per million) acetone or 8 ppm acetoacetic acid in urine.


PROCEDURE • HEAT TEST 1. 2. 3. • Acidify 10 ml of urine with dilute acetic acid in a test tube. Heat it over a boiling water-bath. The presence of coagulated precipitate indicates the presence of protein.

SULPHOSALISILIC ACID TEST 1. 2. 3. 4. 5. 6. Add a few drops of 20% sulphosalisilic acid to 10 ml of urine in a test tube. The formation of precipitate indicates the presence of protein. In some cases the precipitate may be caused by Bence-Jones proteins that are usually present in the urine samples of patients with multiple myeloma. The following method can be used to differentiate the Bence-Jones proteins from other types of proteins: Heat the urine sample (with sulphosalisilic acid) to 50-60°C . A precipitate would form. Heat it further to 100°C. The precipitate should become soluble and form again when cooled.

HELLER TEST 1. 2. Add 5 ml of concentrated nitric acid to 3 ml of urine very carefully. In the presence of protein, a whitish layer would form between the to solutions.

EXPERIMENT 3 Analysis of urinary components using the dipstick method. The routine urine analysis is currently carried out using dipsticks (strips that have been precoated with the appropriate reagents). This strip is dipped into the urine sample and the change in colour in the test region within a specified time indicates the presence of the compound tested. The colour that is formed is compared with a suitable colour chart (for the particular test) that is provided, for the rough estimation of concentration. This method is certainly much simpler and more rapid than the conventional chemical method! One of the latest in the series of dipsticks is the ' multistix' which contains at least 10 different stripes (streaked with different reagents for the corresponding tests ) to analyse the various urinary components such as glucose, bilirubin, ketone bodies, specific gravity, blood, pH, protein, urobilinogen, nitrite and leukocyte . PROCEDURE • • • • • • Place the fresh urine specimen in a clean container. Dip a strip of the 'multistix' into the urine sample. Ensure that the strip is properly dipped. Remove the strip immediately to avoid the solubilization of the regents. Remove any excess urine that may be present and hold the strip horizontally for the appropriate length of period (as indicated in the chart). Compare the colours for the various test compounds as indicated in the chart. 4

RESULT EXPERIMENT 1 TEST Ammonia Sulphate Indican • • • • • • • • OBSERVATION Stingy smell produced pH paper turned to blue (pH 9.0) Solution in the test tube turn to cloudy Colorless solution at the bottom of the test tube 1st tube (2 ml Urine) : Formed purple solution 2nd tube (2 ml Sodium urate) : Formed dark green solution. 3rd tube (2 ml Urine) : Formed purple solution Urine Sample : o Adding Sodium Nitropruside :  o Urine color change from yellow to light orange. Adding NaOH :  o Creatinine • Urine color change from light orange to orange red. Adding Glacial acetic acid :  Urine color change from orange red to light orange. Creatinine solutions : o Adding Sodium Nitropruside :  o Change from colorless to light orange.

Uric Acid

Adding NAOH :  Change from light orange to dark yellow.

o Urobilin Urobilinogen • • •

Adding Glacial acetic acid :

 Change from dark yellow to light yellow. Filtrate shows pale yellow Formed rd color complex On 20 times Diluted o Solution change to colorless.


Test for ketone Detection of sugar in the urine bodies


OBSERVATION Formed orange brown color.

• •

INTERPRETATION Presence of reducing sugar Presence of maltose, lactose, and glucose on urine sample.


Produces red color formation. •

Presence high of ketone bodies.


• • •

Formed dark purple ring. Formed coagulated precipitate. At 50 C – 60 C : o Formed orange precipitate • • Presence of protein substances Presence of protein substances.


Test for Proteins


• •

At 100 C : o Precipitate dissolve

Cooling down : o Formed orange • Presence of protein substances.


precipitate again Formed whitish layer at top of solution.

EXPERIMENT 3 INDICATOR Glucose Bilirubin Ketone Blood pH Protein Urobilinogen Nitrite Leukocyte RESULT PATHOLOGICAL URINE SAMPLE STUDENT URINE SAMPLE 4+ Negative 1+ Negative 3+ Negative 4+ Negative 6 6 3+ Negative 1+ Normal Negative Negative Negative Negative


DISCUSSIONS EXPERIMENT 1 Urine is a fluid that is composed of waste products from the blood. It is formed in the kidney and is eliminated from the body by the urinary system. Urinalysis is performed to aid in screening, diagnosis, and prognosis and to monitor the treatment of disease. In this experiment, the random normal sample urine is obtained from the student, where the urine is collected by using a urine container that clean, dry and sterile. Sterility is important because to avoid any contamination of microorganisms. The Urine is analyzed immediately as to maintain in normal physiological condition within 30 minutes. In the ammonia test, the urine had shown positive of ammonia substances. It shown by ungenerous smell of that urine and when tested with pH paper, the resulted is blue color. When the urine tested with sulphate, the solution showed turn to cloudy means that a positive result of presence sulphate. Unfortunately, in the Indican test, the urine had shown negative, which formed a colorless solution at the bottom of the test. In the creatinine test, the urine gives a color orange solution, when added the sodium nitropruside. It also occurs when adding the glacial acetic acid, but when added NaOH solution, the urine turned to the color light orange. So, as a result, the urine sample is presence of creatinine. To detecting the urobilin, Selesinger test is used for this experiment. The urine sample showed a negative of urobilin by seeing the filtrate product that not formed a pale yellow. For the urobilinogen test, the urine sample also gave a negative result, where if positive the urine formed the red complex substances. EXPERIMENT 2 Urine reflects various biochemical reactions that took place in the body. In fact, the composition of urine in an individual may be changed when a person is infected with a disease. Most frequently, in a disease condition, there will be present of abnormal compounds or present of normal compound in abnormal quantities. In order to detect the presence of sugar in urine, two methods are used which are the benedict test and the Fearon test. In benedict test, the formation of orange brown color indicates presence of reducing sugars. In Fearon test, if there is presence of maltose and lactose, a red color complex will be formed. Meanwhile, if glucose is absence, the solution will change to yellowish color. But, on this experiment we proved that the urine pathological sample shown stronger positive of glucose. The false positive for these tests, occur when it mixes up with bleach or others strong oxidizing reagents. While for the false negative is when the urine contain ascorbic acid, high amount of ketone, on medication such as aspirin, prolonged sample which causes the microorganisms metabolized quickly. The important clinical implication for this protein test is diabetes diseases. Rothera test is carrying out for detecting the presence of ketone bodies. In our experiment, the pathological formed a purple ring clearly; this indicates that ketone bodies are presence with high concentration. In fact that, the ketone bodies in human urine is abnormal. False positive reactions are not common but could happen if high pigmented urine or levodopa and false negative due to the ascorbic acid or volatile of ketone bodies. The clinical significance on this test is formed during incomplete fat metabolism. Clinical implications of ketone bodies may include decreased intake of carbohydrates, starvation, prolonged vomiting, dehydration, fever, and diabetes insipidus. For detection of protein, three methods are used which are the Heat test, sulphosalisic acid test and Heller test. All these tests gave positive result of protein. In the Heat test, coagulated precipitate is formed. While, in the Sulphosalisilic acid test the orange precipitate is formed in temperature of 50’C and forming again when is cooled down after boiling in 100’C. For the Heller test, the presence of protein is by formed whitish layer between to solutions. False positive could occur when the urine sample in high alkaline pH and false negative, when the urine in high salt concentration. The clinical significance for the protein tests is a proteinuria, that first indication of renal disease. The clinical implications of this test are renal abnormality, glomerular, tubular damage or excess overflow. 7

Moreover, in the cases of Non disease, the protein resulted are strenuous exercise, cold exposures and fever. EXPERIMENT 3 The routine urinalysis currently in laboratory is carried out by using the reagent strips, which performed by read as color changes. A urinalysis is an array of tests performed on urine and one of the most common methods of medical diagnosis. A typical medical urinalysis usually includes ph, specific gravity, glucose, protein, RBC, leucocytes, ketone, urobilinogen, bilirubin, and nitrite. This reagent strips have been proven their sensitivity and specificity. There are lots of product of reagent strip such as ‘Clinitest’, ‘Acetest’, ‘Isototest’ and others products. The method of using the strip is by dipped into urine, leave it for few second, then reads the colors strip by compared it onto their colors indicator bottles. From this experiment, the pathological sample has shown many changes for their color reagent strip, if compared to the normal urine from student and the both results as shown on the table above. As additional, it is important to be the urine sample tested with microscopy. CONCLUSIONS EXPERIMENT 1 The normal urine composed of ammonia, sulphate, uric acid, creatinine, urobilin and urobilinogen. This method can be detected by using specific method on each of the compound. From this experiment, the sample have shown a normal urine. EXPERIMENT 2 From the experiments, we can conclude that the urine sample from the pathological sample have shown an abnormal urine, which gave a positive results in protein and glucose. EXPERIMENT 3 The reagent strips is a rapid test for detecting the some compounds. The pathological had shown many changes of colors reagent strip from normal, where indicates having a protein, glucose, ketone, urobilinogen, bilirubin, and blood. The urine from student has shown a negative of all reagent strips.




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