1.Enumerate the tests for detection of Glucose and reducing substances in urine
Copper reduction test – Fehling test, Benedicts test
Enzymatic test – Glucose oxidase reagent strip test
Osazone test
2. What is the principle of Benedicts test ?
When reducing sugar is subjected to heat in the presence of an alkali, it gets converted to an enediol which is powerful reducing agent. This reduces cupric ions in Benedicts reagent to cuprous ions. These cuprous ions form copper oxide with the reaction mixture and precipitate out as brick colored compound
3. What is the composition Benedicts reagent ?
Sodium carbonate (anhydrous) – 100g
Sodium citrate – 173g
Cupric sulfate – 17.3g
Distilled water to – 1000ml
In this copper sulfate act as source of cuprous ions. sodium carbonate provides an alkaline medium and sodium citrate forms complexes with cuprous ions
4. How do we test for purity of Benedicts test ?
Heat the benedicts reagent prior to the test. No color change in benedicts reagent indicates that it is pure
5. Why only reducing sugars give positive result with Benedicts test ?
All reducing sugars have free aldehyde group or free ketone group that can reduce the cupric sulfate. Hence they give positive result with Benedicts test
6. What is the procedure of Benedicts test
Take 5 ml of Benedicts reagent in a test tube and heat it to check for purity of reagent
If there is no color change then add 8 drops or 0.5 ml of urine and boil for 3-5 minutes
7. How do you interpret the Benedicts test
No color change – Negative
Pale green with slight cloudiness – Trace
Green deposit – 1 + (0.5 to 1 g%)
Yellow deposit – 2+ ( 1 to 1.5 g%)
Orange to red precipitate – 3+ ( 1.5 to 2.5 g%)
Brick red precipitate – 4 + (> 2.5g%)
8. How does the quantity of urine in Benedicts test affects results ?
If the amount of urine is less there will be false negative result and if more there will be false positive result
9. What are reducing sugars ?
Fructose
Lactose
Galactose
Xylose
Pentose
Glucose
10. What are the other reducing substances other than sugars which give positive result with Benedicts test ?
Uric acid
Creatinine
Salicylic acid
Homogentisic acid
Ascorbic acid
Morphine
Paraldehyde
INH
11. What are the causes of glycosuria ?
Diabetes mellitus
Hyperthyroidism
Cushing syndrome
Renal glycosuria
Hypopituitarism
Alimentary Glycosuria
Increased intracranial pressure
Fanconi syndrome
12. What is renal glycosuria ?
Renal glycosuria is the excretion of glucose in urine in detectable amounts at normal blood glucose concentrations in the absence of renal tubular dysfunction
Example – Familial renal glucosuria with mutations in sodium- glucose cotransporter 2 gene
13. What is alimentary glycosuria ?
It is a condition when high carbohydrate diet are taken and it is rapidly absorbed in conditions where part of stomach is surgically removed. Excess of glucose absorbed appears in urine producing glycosuria.
14. What are the conditions of lactosuria?
Lactating mothers, late pregnancy
15. What is Fehling test and who introduced it?
Hermann Von Fehling, a German chemist introduced Fehling test to differentiate between reducing sugars and non reducing sugars
16. What is the composition of Fehling solution?
Fehling solution is prepared by combining equal amount of two solutions i,e. Fehling A and Fehling B
Fehling A solution
Copper sulfate – 34.65g
Distilled water – 500ml
Fehling B solution
Sodium hydroxide – 125g
Sodium potassium tartrate – 173g
Distilled water – 500ml
17. What is the procedure of Fehling test?
Take 2.5 ml of Fehling A solution and Fehling B solution each in a test tube
Mix them and boil
Then add 0.5ml (8 drops) of urine and boil again for 3 to 5 minutes
If reducing sugars or reducing substances are present, color changes will be similar to benedicts test
18. What are the uses of Fehling reagent
It distinguishes aldehyde from ketone groups
Aldehydes are oxidized and give positive results where as ketones does not give positive results
19. What is the principle of Fehling test
The Bistartrato cuprate complex oxidizes the aldehyde to carboxylate anion and in this process the copper ions of the complex are reduced to copper ions and later red copper oxide precipitates which indicates positive result.
20. What is the principle and mechanism of Glucose oxidase reagent strips
When blood is placed onto the test strip, it reacts with a chemical called glucose oxidase producing gluconic acid from glucose
At the other end of the test strip, the meter transfers a current to the test strip
Test strip has electric terminals which allow the meter to measure the current between the terminals
The current between the terminal changes depending on the level of gluconic acid that has been produced
Blood glucose meter then uses an algorithm to work out the blood glucose level based upon the differences in current
21. What are the other tests for Glucose detection in urine
Enzymatic test (Glucostix or Clinistix)
It is a glucose oxidase reagent strip test
The test end of the strip is dipped into urine
The color of the reagent area on the strip is compared with color chart supplied by manufacturer
Osazone test
This test is also termed as Phenyl hydrazine test
Principle – Carbohydrates with free carbonyl groups react with phenylhydrazine to form osazone. Condensation-oxidation-condensation reaction between 3 molecules of phenylhydrazine and carbon one and two of aldoses and ketoses yield 1,2-diphenylhydrazone, known as Osazone.
procedure –
Take 5ml of test solution in a clean, dry test tube
Add 0.3g of osazone mixture (0.5g of phenylhydrazine hydrochloride and 0.1g of sodium acetate) and 5 drops of glacial acetic acid to the test tube
mix well (warm the test tube in water bath if required to dissolve the contents)
Keep the test tube in boiling water and observe the formations of crystals
Examine the crystals under low magnification in microscope
Osazone appears as yellow colored crystals and are different for different sugars
By
Dr.Shanthi Vissa, Associate professor, Pathology, Sri Venkateswara Institute Of Medical Sciences, Tirupathi.
Dr. Byna Shyam Sundar Rao, Professor, Pathology, Narayana Medical College, Nellore