Stool Examination:- Part 4 – Reducing substances, Breath Test, and Lactose Intolerance
- This is done on the stool of infants or adults.
- A small amount of stool is needed; just 5 grams are enough.
- Try to collect the fresh stool.
- The stool should be delivered to the laboratory as soon as possible, preferably within 1 hour.
- Because of lactose (or other sugars), the stool will normally be broken down by chemical processes within 2 to 4 hours.
- Avoid contamination with urine or other material like water or toilet paper.
- Bacterial fermentation may give a falsely low result if the stool is not tested immediately.
- To diagnose the intolerance to disaccharides.
- To diagnose reducing substances in the stool.
Definition of reducing substances:
Reducing substances can reduce cupric (Cu++) to cuprous (Cu+); the best example is Benedict’s solution. These may be present in the urine and the stool.
Reducing substances are:
|Carbohydrate group||Noncarbohydrate group|
|Pentose (Xylulose, arabinose)||Phenol|
|Lactose||Uric acid and urates|
|Sucrose (not reducing sugar)||Salicylate|
- Table sugar sucrose is not reducing sugar.
- Normally sugars are rapidly absorbed in the upper small intestine.
- Lactose break into glucose and galactose by the lactase enzyme.
- In such a case on the ingestion of sugars (lactose), blood glucose level does not increase.
- Lactose floods the small intestine and then by the bacterial catabolism produces Methane and H+.
- This is the basis for the breath test where Hydrogen (H+) gas is tested to find the lactase enzyme deficiency.
- If sugars are not absorbed, then these produce diarrhea due to the osmotic pressure produced by these unabsorbed sugars.
- These unabsorbed sugars draw fluid and electrolytes into the intestine.
- These unabsorbed sugars are measured as reducing substances.
- These Sugars are characterized as reducing substances based on their ability to reduce cupric ions to cuprous ions.
- Cu++ in hot alkaline solution to Cu+.
- But Galactose is an exception, cannot reduce copper.
- Cu++ in hot alkaline solution to Cu+.
- Reducing substances are:
- Xylulose and Arabinose.
- Other substances include ascorbic acid.
- Sucrose is not reducing sugar (nonreducing sugar).
- Sucrosuria when sucrose appears in the urine.
- Urine specific gravity is high (up to 1.070)
- Urine and stool test for reducing substances are negative.
- Carbohydrate malabsorption is a major cause of:
- Watery diarrhea:
- Osmotic diarrhea is due to the osmotic pressure of the unabsorbed sugars in the intestine.
- This will draw the fluid and electrolytes into the gut.
- Electrolyte imbalance.
- Watery diarrhea:
- Idiopathic lactase deficiency.
- This is seen in 70 to 75% of southern European, Greek, and Indian.
- Blacks have 70%.
- Asian adults have >90 %.
- Caucasian American adults have 5 to 20%.
- This is a familial disease of infancy with diarrhea, vomiting, failure to thrive, and malabsorption.
- Patients becoming asymptomatic when lactose is removed from the diet.
- Galactose appears in infants’ urine with galactosemia; this is characterized by the inability to metabolize galactose.
- These infants fail to thrive on milk, which contains mainly galactose.
- Failure to diagnose this condition will lead to:
- Liver disease.
- Mental retardation.
- Primary glucose-galactose malabsorption:
- This is a rare hereditary disorder of active absorption of glucose and galactose from the small intestine.
- It is inherited as an autosomal recessive trait.
- S/S is like other disaccharides malabsorption.
- Diarrhea is the main complaint.
- Stools are watery and contain several grams of glucose and galactose/100 mL.
- Diagnosis is to find the glucose and galactose in the stool by various methods like:
- Glucose oxidase method.
- Galactose oxidase method.
- Oral glucose and galactose tolerance test, where the flat curve is expected.
- Flat glucose tolerance curves are normal in newborn babies.
Laboratory diagnosis of reducing substances:
Various options to detect reducing substances are:
- Mostly there are commercial devices available, e.g, Clinitest (Benedict’s solution), etc.
- A yellow-brown color indicates reducing substances.
- This color indicates ++ sugars (lactose).
- Benedict’s reaction principle is as follows:
- The clinitest tablet contains anhydrous cupric sulfate, sodium hydroxide, citric acid, and sodium bicarbonate.
- Clinitest method:
- Procedure for reducing substances in the stool:
- Add one volume of stool and two volumes of water.
- Mix thoroughly.
- Transfer 15 drops of this suspension to a clean test tube.
- Add the Clinitest tablet.
- Interpretation of the result:
- The finding of these reducing substances is abnormal.
- Normal when reducing substances is 0.25 g/dL or less in the stool.
- Suspicious when the reducing substances are 0.25 to 0.5 g/dL in the stool.
- Positive when > 0.5 g/dL are reducing substances in the stool.
- Glucose oxidase reagent strip:
- This is specific for the detection of glucose.
- The glucose oxidase enzyme catalyzes the oxidation of the glucose to gluconic acid and the production of H2O2.
- The next step is the addition of the enzyme peroxidase and a chromogen Oxygen (O2) acceptor (O-tolidine or O-dianisidine), result in colored oxidized chromogen, which is measured and is proportional to the production of H2O2 by the oxidase enzyme.
- Thin-layer chromatography: Separation of the sugars can be done by thin-layer chromatography.
- pH: Also, check the pH of the stool.
- Normal stool pH is 7.0 to 8.0.
- Stool pH <6.0 is suspected of lactase enzyme deficiency.
- Another method is:
- Give a load of lactose.
- In the case of deficiency of lactase, there will be no increase in the glucose level.
- Keep in mind when there is sucrose intolerance, in these patients, instead of a small amount of sucrose, there is a large amount of glucose and galactose found in the stool, which is due to the hydrolysis of sucrose by the intestinal bacteria so that the test may be positive.
- Give a load of lactose.
Comparison of Glucose oxidase and copper reduction method:
|Chemical substance||Copper reduction tablets||Oxidase reagent strip|
|Hydrogen peroxide||May inhibit positive test||False-positive|
|Sodium fluoride||No effect||False-negative|
- Tests for other sugars:
- Galactose is diagnosed by thin-layer chromatography.
- The resorcinol test diagnoses fructose.
- Fructose also reduces copper at low temperatures by Benedict’s reaction.
- Thin-layer chromatography can also identify fructose.
- Pentose concentration of 250 to 300 mg/dL will reduce Benedict’s reagent at 50 °C within 10 minutes or at room temperature for several hours.
- OR can diagnose by thin-layer chromatography.
- Lactose: Keep in mind that a glass of milk contains approximately 11 grams of lactose.
- Lactose was diagnosed by thin-layer chromatography.
- OR qualitative lactose test.
- Lactose tolerance test when 50 grams of the lactose given orally.
- This is followed by the measurement of blood glucose.
- An oral lactose tolerance test will show a rise in blood sugar less than 20 mg/dL.
- A dose of 50 grams of glucose and galactose produces a rise in blood sugar of more than 25 mg/dL.
- Normal blood value (adult/elders) = There is rise in plasma glucose level >20 mg/dL (no abdominal cramps or diarrhea).
- Sucrose ferment yeast and can be separated chromatography and needs stains.
Normal Stool findings
|Stool parameter||Normal findings|
|Normal diet||Neutral to alkaline = 7 to 7.5|
|Infants on breastfeeding||Slightly acidic|
|Infants on formula milk||Neutral to alkaline|
|Normal||<250 mg/dL (<13.9 mmol/L)|
|Borderline cases||250 to 500 mg/dL (13.9 to 27.8 mmol/L)|
|Positive cases||>500 mg/dL (>27.8 mmol/L)|
This is advised to find the lactase enzyme deficiency.
Advised to the patient:
- Patients can have 12 hours fast.
- Ask the patient to blow out and measure the H+ in the analyzer.
- Give some sugar (lactose, sucrose, sorbitol, fructose, and lactulose) to the patient.
- Now check every 15 minutes H+ in the breath for 1 to 5 hours.
- Normal breath test = <50 ppm Hydrogen (H+) increase over the baseline.
Increased reducing substances are seen in:
- Disaccharidase enzyme deficiency in the intestine.
- Short bowel syndrome.
- Idiopathic lactase deficiency leads to lactose intolerance.
- Carbohydrate malabsorption is seen in :
- viral gastroenteritis.
- Celiac disease.
- False-positive result:
- It is seen due to the presence of ascorbic acid (>200 mg/dL).
- This will show a low-level positive result trace or 1+.
- Urine with low specific gravity contains glucose that may give a slightly raised level result.
- Antibiotics with large quantities like nalidixic acid, cephalosporin, and probenecid.
- Radiographic material gives black color, and this may be considered positive.
- False-negative result:
- Mixing the test tube before the 15 seconds wait after boiling stops; due to reoxidation of the cuprous ions to cupric ions by atmospheric oxygen.