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The analysis of normal and abnormal urinary components

The analysis of normal and abnormal urinary components

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the quantity and composition of urine reflects various biochemical processes that occur in the body.
the quantity and composition of urine reflects various biochemical processes that occur in the body.

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Published by: kiedd_04 on Feb 01, 2008
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05/20/2013

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TITLE
The analysis of normal and abnormal urinary components
AIMS AND OBJECTIVES
1.To know the various urinary components2.To understand the significance of these components3.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. Thusthe composition of the urine of an individual may change when a person has a disease. In a diseased condition, it isquite common to observe the presence of abnormal components (Compounds which are not present in the urine of anormal, healthy individual) or normal components in abnormal quantities in the urine.
THE FOLLOWING TABLE SHOWS THE NORMAL COMPOSITION OF THE URINE.TypesIndicator
VOLUME
600 - 200 ml / 24 hour 
SPECIFIC GRAVITY
1.003 – 1.030
PH
4.6 - 8.0
UREA
15 – 35 g (250 – 580 mmol) / 24 Hour 
AMMONIA
0.5- 1.0 g (29 – 58 mmol ) / 24 Hour 
URATE
0.5 – 2.0 g (3 – 12 mmol) / 24 Hour 
CREATININE
1.0 – 2.0 g (9 – 17 mmol) / 24 Hour 
CREATINE
0.1g / 24 Hour 
THE ABNORMAL URINARY COMPONENTS ARE AS FOLLOWS
Glucose, ketone bodies, indole and related compounds, porphyrine and related compounds, protein ( albumin andBence-Jones protein), bile pigments, blood, melanin and so on.
EXPERIMENT 1Tests for normal urinary components
The urine sample is necessary free from any contamination and using own urine samples.
PROCEDURE
 AMMONIA
1.pH of 2-3 ml urine is adjust with sodium carbonate to a pH of 9.0.2.The solution is boiling and tested for the presence of ammonia.
SULPHATE 
1.Mix 2 ml dilutes HI with 10 ml urine and then adds 2 ml chloride solution.2.Filter the barium sulphate precipitate that is formed.3.Use half the filtrate as blank and boil the other half.4.Notice the change that takes place in the boiled filtrate. (It should become cloudy).1
 
 INDICAN 
1.Mix 3ml of urine with 3 ml of concentrated HC1 and 1 ml of chloroform.2.Add a few drops of sodium hypochlorite (NaOCl) and shake thoroughly.3.Observe the colour of the chloroform phase.4.Do not add too much of NaOCl.5.What is the source of indican in the urine?6.This test should be negative in normal urine.
URIC ACID
1.Add 2 drops of Folin-fosfotungstic acid reagent to 2 ml of urine which has been alkalinized with NaOH.2.In another tube add 2 drops of Folin-fosfotungstic acid to 2 ml sodium urate.3.Compare the colors formed.4.For a more specific reaction, carry out the above reaction with 20 ml of urine that has been saturatedwith ammonium chloride and alkalinized with concentrated ammonium hydroxide. (Note:Ammonium urate is not soluble in ammonium chloride).
CREATININE (WELY TEST)
1.Add a few drops of sodium nitropruside into 2 ml of urine and 2 ml of creatinine solution in aseparate tube.2.Add a few drops of dilute NaOH and observe the colour formation.3.Add a few drops of glacial acetic acid and notice the colour change. (Note: Nitropruside is also usedto test for the presence of acetone in urine. However the effect of glacial acetic acid is different.)
UROBILIN 
1.This compound is formed by the reduction of bile pigment in the intestine and the oxidation of urobilinogen.2.The amount of urobilin present in the urine increases certain types of liver diseases. The mostcommon test used is the
Schlesinger test.
3.Add 2 drops of 2.5 % alcoholic iodine solution into a test tube that contains 5 ml urine. (This is tooxidize urobilinogen in the urine to urobilin.)4.Then, add 5 ml solution of 10% zinc acetate in alcohol.5.Mix thoroughly by inverting the tube several times.6.Filter the mixture and ensure that the filtrate has an acidic pH.7.If the filtrate is found to be neutral or basic, acidify the filtrate with acetic acid.8.Ensure that the colour of the filtrate is pendafluor green.9.If this colour does not disappear even after 20x dilution, it indicates a pathological condition.
UROBILINOGEN 
1.Urobilinogen is not easily detectable because it is easily oxidized.2.Thus it is crucial that only freshly obtained urine sample is used.3.To test for urobilinogen. Add 1ml of 2% p-dimethylamino-benzaldehyde in20 % HCl.4.The formation of red colour complex indicates the presence of urobilinogen.5.The test shows positive results even in normal urine that has been diluted up to 20 X.6.However, pathological condition is indicated if the red colour complex is observed in urine that has2
 
 been diluted greater than 20 X.
EXPERIMENT 2Tests 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. Itis 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.Add 8 drops of urine to 5 ml of Benedict qualitative reagent in a test tube.2.Mix thoroughly and place the tube in a boiling water-bath for 5 min.3.The formation of orange-brown complex confirms the presence of reducing sugars.4.This reagent specifically shows positive result with reducing carbohydrates and does not react withaldehyde or any other forms of reducing agents.
 FEARON TEST FOR GLUCOSE, LACTOSE AND MALTOSE 
1.Boil 5 ml of glucose, lactose and maltose (in separate test tubes) with 0.5 ml of 5% methylamine-HCl.2.Then add 2 ml of hot, dilute NaOH.3.Cool to room temperature.4.Lactose and maltose produce red colour complex.5.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 areexcreted in the urine when fats are metabolized excessively. Acetoacetic acid may easily undergo decarboxylation toform acetone.
PROCEDURE
 ROTHERA TEST ( TO DETECT ACETON AND ACETOACETA TE)
1.Add ammonium sulphate crystals into 3 ml of urine and shake until it becomes saturated.2.Add a few drops of sodium nitropruside (5%, w/v).3.Gently add 2 ml of concentrated ammonia to the surface of the solution.4.The addition of ammonia causes the formation of a purple ring at the interface (between the urineand the ammonia) in the presence of ketone bodies.5.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.6.This test is sensitive up to 100 ppm (parts per million) acetone or 8 ppm acetoacetic acid in urine.
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 isexcreted increases in pathological conditions such as heart disease, toxemia, pregnancy, kidney failure and liver 3

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