Diabetes Mellitus:- Part 5 – Glucose in Urine (Glycosuria), Benedict’s solution

Glucose in Urine (Glycosuria)

Sample of Glucose in Urine

1. The test sample is urine.
2. The best sample after 2 to 3 hours of the meal.

Indications for Glucose in Urine

1. To diagnose diabetes mellitus.
2. To monitor diabetes mellitus.
3. To evaluate the effectiveness of the therapy.
4. To diagnose gestational diabetes.
5. It is part of a routine urine examination.

Precautions for Glucose in Urine

1. Can see false-positive tests when any substance present in the urine can reduce the copper in the clinitest strips.
2. Can see the false positive tests in the presence of other sugars like galactose, fructose, and lactose.
3. Drugs giving false positive tests are acetylsalicylic acid, Ascorbic acid, cephalothin, chloral hydrate, streptomycin, sulphonamides, and aminosalicylic acid.
   1. Contamination from the oxidizing agents or bleach.
   2. Improper storage of the urine strips.
4. Drugs giving false-negative results are levodopa, phenazopyridine, and ascorbic acid with clinitest strips.
   1. Multistix detects ≥50 mg/dL; if the amount is less, it will be negative.
   2. Sodium fluoride causes enzyme inhibition.
   3. Refrigerated specimens give decreased enzyme activity.
5. Some drugs increase glucose in the urine, like diuretics (thiazide), estrogens, isoniazid, lithium, nalidixic acid, nicotinic acid, chloramphenicol, chloral hydrate, cephalosporin, and aminosalicylic acid.

Glycosuria definition

Glycosuria is the presence of reducing substances (glucose, galactose, lactose, and fructose) in the urine.

Glucosuria is the presence of glucose in the urine. This is specific to the diagnosis of diabetes mellitus. Glucosuria may be with hyperglycemia and, in some cases, maybe without hyperglycemia.

Pathophysiology of Glucose in Urine:

1. Examination of urine for glucose is rapid, noninvasive, and inexpensive for screening urine.
   1. A large number of urine samples can be tested.
2. Glucose is filtered by the glomeruli and reabsorbed by proximal convoluted tubules.
3. When glucose exceeds the renal threshold level(>180 mg/dL) of tubules, glucose appears in the urine, called glycosuria. https://labpedia.net/gestational-diabetes-mellitus-oral-gtt/.
4. Tubular absorption is an active process to maintain the body’s glucose level.
   1. Renal threshold for glucose = 160 to 180 mg/dL.
5. After the renal threshold values, glucose appears in the urine.
6. This is not sensitive nor specific for the control of diabetes because we don’t know about the glucose level below 180 mg/dL.

The reducing substances in the urine are:

1. Lactose.
2. Fructose.
3. Galactose.
4. Maltose.
5. Arabinose.
6. Xylose.
7. Ribose.

Other substances found in the urine are:

1. Uric acid.
   1. Creatinine.
   2. Cysteine.
2. Ketone bodies.
   1. Oxalic acid.
   2. Glucuronic acid.
   3. Hippuric acid.
   4. Homogentisic acid.
3. Drugs are:
   1. Ascorbic acid.
   2. Isoniazid.
   3. Salicylates.
   4. Formaldehyde.

The normal value of glucose in the urine:

Source 2

1. Normally sugar (glucose ) is absent in the urine.
   1. Random specimen = negative
2. 24 hours specimen = < 0.5 g/day (<2.78 mmol/day).
3. Glucose appears in the urine when a blood glucose level of 180 mg/dL or more (crosses the renal threshold).
4. Its concentration in the urine correlates with the blood glucose level.

Procedure for reducing substances in the urine:

Benedict’s solution method:

Benedict’s reagents:

1. CuSO4 (cupric sulphate) = 17.3 grams
2.  Sodium citrate (Na₃C₆H₅O₇-2H₂O) = 173 grams
3. Sodium carbonate (Na₂CO₃) = 100 grams
4. Distle water = 1000 mL

Procedure to prepare the benedict solution:

1. 1. Dissolve cupric sulfate in hot water of 100 mL.
   2. Now dissolve sodium citrate and sodium carbonate with heating water of 800 mL separately. Let it cool.
   3. Mix solutions 1 and 2 and make up to 1000 ml of water.
   4. Benedict’s reagent is ready and stable.

The principle of Benedict’s reaction:

1. It is a reaction that is a qualitative method and is very common.
2. It contains cupric ions complex to citrate in an alkaline medium.
3. Reducing substances convert cupric to cuprous ions.
   1. It forms yellow cuprous hydroxide or red cuprous oxide.

Glucose in Urine (Glycosuria): The principle of Benedict’s reaction

Procedure for Benedict’s solution method:

1. This can be done on the solution of benedicts as well.
2. Take 2.5 mL of Benedict’s solution.
3. Add 0.2 ml of urine.
4. Place the tubes in a heat block or heat them directly to bring them to 100 °C.
5. Examine each tube’s color and the precipitate.
6. Different colors develop according to the quantity of sugar in the urine.
7. Greenish brown when there is a large quantity >2 g/dL.

Another way to do Benedict’s reaction:

1. Benedict’s solution is 5 mL.
2. Urine  0.4 mL (8 drops) .
3. Mix and keep in a boiling water bath for 3 minutes.

Tablet method for benedict’s reaction:

1. 5 drops of urine are mixed with 10 drops of water.
   1. Then add the tablet as known clinitest.
2. The procedure for Benedict’s reaction with the tablet (Clinitest) is shown in the following diagram.
   1. Also, run the negative control as well.

Benedict reaction

Reporting the result:

1. It can be reported as plus + signs, from 1+ to 4+.
2. It can report a percentage of 1% to 2 %. This reporting is more accurate.

A semiquantitative:

1. This method with different strips is available, like Clinistix, Diastix, and Chemstrip.
2. In all the above strips, the glucose-specific enzyme glucose oxidase is used.
3. This is more specific for glucose than Benedict’s method.
   1. This test is positive when glucose concentration is 100 mg/dL or more.

The sensitivity of the commercially available Discs:

1. Multistix = 75 to 125 mg/dL
2. Diastix  =  75 to 125 mg/dL
3. Chemstrip = 40 mg/dL in 90% of the specimen.

The drawbacks of the strips:

1. Urine strips detect mainly glucose, so there are chances for false-negative results due to interfering chemicals in the urine.
2. False-positive results are seen in the following:
   1. If the detergents contaminate the container.
   2. In very dilute urine, traces may be seen due to sensitivity at low specific gravity.
   3. If the strips are exposed to air due to improper storage.
3. False-negative results are seen in the following:
   1. It is seen in the intake of vitamin C (ascorbic acid) ad tetracyclines.
   2. In case there are high ketone bodies (≥40 mg/dL)  and low glucose levels (75 to 125 mg/dL).
   3. Sodium fluoride inhibits the enzyme reaction, so it should not be used in the urine as a preservative.
   4. If urine is refrigerated, it will give false-negative results because of decreased enzyme activity, bringing the urine to room temperature.

The quantitative method for Glucose in Urine (Glycosuria):

• This method uses hexokinase or glucose dehydrogenase procedures.

Increased glucose in urine seen in:

1. Diabetes mellitus.
2. Renal glycosuria.
3. Hereditary defects in the metabolism of other reducing substances like galactose, pentose, and fructose.
4. Pregnancy.
5. Liver diseases.
6. Pancreatic diseases.
7. Thyrotoxicosis.
8. Cushing’s syndrome.
9. Acromegaly.
10. Brain injuries.
11. Shock.
12. Fanconi’s syndrome (Tubular defect).
13. Advanced renal tubular diseases.
14. Nephrotoxic chemicals like carbon monoxide, lead, and mercury.

The false-negative result is seen in the following:

1. Mostly seen due to drugs.
   1. Ascorbic acid.
   2. Levodopa.
   3. Phenothiazine.

Comparison of Benedict reaction and Oxidase method:

Questions and answers:

• Please see more details on Fasting Blood Glucose.