Qualitative* Analysis of Urine for Abnormal Constituents (Albumin Protein, Ketone Bodies, Sugar)

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KETONE BODIES DETERMINATION
It is seen in conditions in which the body produces excess ketones as an indication that it is using an alternative source of energy. It is seen during starvation or more commonly in type I diabetes mellitus. Production of ketone bodies is a normal response to a shortage of glucose, meant to provide an alternate source of fuel from fatty acids.

Rothera’s Test Principle
Acetoacetic acid and acetone react with an alkaline solution of sodium nitroprusside to form a purple-colored complex. This method can detect above 1-5 mg/dl of acetoacetic acid and 10-20 mg/dl of acetone. Beta-hydroxybutyrate is not detected.

Rothera’s Test Procedure
1. Take a clean test tube and add 5 ml of urine to it.
2. Transfers 1 gm of Rothera’s powder mixture within the test tube and mix well.
3. Add 1-2 ml of concentrated ammonium hydroxide to the urine sample within the test tube. It will create a thin layer over the urine sample.
4. Observe the pink-purple ring at the interface.

Results
Positive Test: If a purple permanganate-colored ring is immediately formed at the interface, means Ketone bodies are present.
Negative Test: If no permanganate colored ring is formed at the interface, means ketone bodies are absent within the test sample.

Principle of Benedicts test
Benedict’s test is performed by heating the reducing sugar solution with Benedict‘s reagent. The presence of the alkaline sodium carbonate converts the sugar into a strong reducing agent called enediols. During the reaction, enediols decrease the cupric particles (Cu2+) present in Benedict’s reagent to cuprous particles (Cu+) which appear as red copper oxide (Cu2O) which is insoluble in water. When Benedict’s reagent solution and reducing sugars are heated together, the solution changes its color to orange-red/ brick red precipitate. The red-colored cuprous oxide is insoluble in water and hence, separate out from the solution. When the concentration of the reducing sugar is high in the solution, then the color becomes more intense (reddish) and the volume of the precipitate increases in the test tube making it clearly visible.

The procedure of Benedict’s Test
1. Pipette out 2 ml (10 drops) of Benedict’s reagent and placed it in the clean test tube
2. Approximately 1 ml of sample (urine) is added to Benedict’s reagent.
3. The test tube is placed over the boiling water bath for 3-5 minutes (can be heated directly over flame).
4. Observe for color change in the solution of test tubes or precipitate formation.

Result Interpretation of Benedict’s Test
1. If the color upon boiling is changed into green, then there would be 0.1 to 0.5 percent sugar in solution.
2. If it changes color to yellow, then 0.5 to 1 percent sugar is present.
3. If it changes to orange, then it means that 1 to 1.5 percent sugar is present.
4. If color changes to red,then 1.5 to 2.0 percent sugar is present.
5. And if color changes to brick red,it means that more than 2 percent sugar is present in solution.

ALBUMIN TEST
Proteins circulate throughout your blood to help your body maintain fluid balance. Albumin is a type of protein the liver makes. It’s one of the most abundant proteins in your blood. Albumin is the protein which is found in blood, if the kidney is healthy it does not allow albumin to pass out through urine, and if kidney Is not working a properly small amount of albumin get excreted by the urine, so this test plays an important role in the determination of albumin content in urine.

There are two types of test for identifying albumin
1 Sulfosalicylic acid test- if you add sulfosalicylic acid in urine sample it will appear as turbid means albumin is present, and if it remains as earlier means there is no evidence of albumin in the urine.
2 Nitric Acid- if we add in dropwise in the test tube containing nitric acid it will create a white ring at the contact point, which means the presence of albumin.

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