1. COPPER REDUCTION METHODS
A. Benedict’s qualitative test:
When urine is boiled in Benedict’s qualitative solution, blue alkaline copper sulphate is reduced to red-brown cuprous oxide if a reducing agent is present (Figure 820.1). The extent of reduction depends on the concentration of the reducing substance. This test, however, is not specific for glucose.
Figure 820.1 Principle of Benedict’s qualitative test for sugar in urine. Sensitivity is 200 mg of glucose/dl
(like lactose, fructose, galactose, pentoses), certain metabolites
, homogentisic acid, uric acid, creatinine), and drugs (ascorbic acid, salicylates, cephalosporins, penicillins, streptomycin, isoniazid, para-aminosalicylic acid, nalidixic acid, etc.) also reduce alkaline copper sulphate solution.
- Take 5 ml of Benedict’s qualitative reagent in a test tube (composition of Benedict’s qualitative reagent: copper sulphate 17.3 gram, sodium carbonate 100 gram, sodium citrate 173 gram, distilled water 1000 ml).
- Add 0.5 ml (or 8 drops) of urine. Mix well.
- Boil over a flame for 2 minutes.
- Allow to cool at room temperature.
- Note the color change, if any.
Sensitivity of the test is about 200 mg reducing substance per dl of urine.
Since Benedict’s test gives positive reaction with carbohydrates other than glucose, it is also used as a screening test (for detection of galactose, lactose, fructose, maltose, and pentoses in urine) for inborn errors of carbohydrate metabolism
in infants and children. For testing urine only for glucose, reagent strips are preferred (see below).
The result is reported in grades as follows (Figure 820.2):
- Nil: no change from blue color
- Trace: Green without precipitate
- 1+ (approx. 0.5 grams/dl): Green with precipitate
- 2+ (approx. 1.0 grams/dl): Brown precipitate
- 3+ (approx. 1.5 grams/dl: Yellow-orange precipitate
- 4+ (> 2.0 grams/dl): Brick- red precipitate.
Figure 820.2 Grading of Benedict’s test (above) and reagent strip test (below) for glucose
B. Clinitest tablet method (Copper reduction tablet test): This is a modified form of Benedict’s test in which the reagents are present in a tablet form (copper sulphate, citric acid, sodium carbonate, and anhydrous sodium hydroxide). Sensitivity is 200 mgs/dl of glucose.
2. REAGENT STRIP METHOD
This test is specific for glucose and is therefore preferred over Benedict’s and Clinitest methods. It is based on glucose oxidase-peroxidase reaction. Reagent area of the strips is impregnated with two enzymes
(glucose oxidase and peroxidase) and a chromogen. Glucose is oxidized by glucose oxidase with the resultant formation of hydrogen peroxide and gluconic acid. Oxidation of chromogen occurs in the presence of hydrogen peroxide and the enzyme peroxidase with resultant color change (Figure 820.3). Nature of chromogen and buffer system differ in different strips. Also read: URINE STRIP TEST — UNDERSTANDING ITS LIMITATIONS.
The strip is dipped into the urine sample and color is observed after a specified time and compared with the color chart provided (Figure 820.2).
Figure 820.3 Principle of reagent strip test for glucose in urine. Each mole of glucose produces one mole of peroxide, and each mole of peroxide reduces one mole of oxygen. Sensitivity is 100 mg glucose/100 ml
This test is more sensitive than Benedict’s qualitative test and specific only for glucose. Other reducing agents give negative reaction.
Sensitivity of the test is about 100 mg glucose/dl of urine.
test occurs in the presence of oxidizing agent (bleach or hypochlorite used to clean urine containers), which oxidizes the chromogen directly.
test occurs in the presence of large amounts of ketones
, salicylates, ascorbic acid, and severe Escherichia coli infection
(catalase produced by organisms in urine inactivates hydrogen peroxide).