Stool Examination:- Part 4 – Reducing substances, Breath Test, and Lactose Intolerance

Sample

1. This is done on the stool of infants or adults.
2. A small amount of stool is needed; just 5 grams are enough.
3. Try to collect the fresh stool.

Precaution

1. The stool should be delivered to the laboratory as soon as possible, preferably within 1 hour.
   1. Because of lactose (or other sugars), the stool will normally be broken down by chemical processes within 2 to 4 hours.
2. Avoid contamination with urine or other material like water or toilet paper.
3. Bacterial fermentation may give a falsely low result if the stool is not tested immediately.

Indication

1. To diagnose the intolerance to disaccharides.
2. To diagnose reducing substances in the stool.

Pathophysiology

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
Glucose	Ascorbic acid
Galactose	Salicylic acid
Fructose	Homogentisic acid
Pentose (Xylulose, arabinose)	Phenol
Lactose	Uric acid and urates
Sucrose (not reducing sugar)	Salicylate

1. Table sugar sucrose is not reducing sugar.
2. Normally sugars are rapidly absorbed in the upper small intestine.
3. Lactose break into glucose and galactose by the lactase enzyme.
   1. In such a case on the ingestion of sugars (lactose), blood glucose level does not increase.
   2. Lactose floods the small intestine and then by the bacterial catabolism produces Methane and H⁺.
   3. This is the basis for the breath test where Hydrogen (H⁺) gas is tested to find the lactase enzyme deficiency.
      

      lactose deficiency sign and symptoms and diagnosis by a breath test

4. If sugars are not absorbed, then these produce diarrhea due to the osmotic pressure produced by these unabsorbed sugars.
5. These unabsorbed sugars draw fluid and electrolytes into the intestine.
6. These unabsorbed sugars are measured as reducing substances.
7. These Sugars are characterized as reducing substances based on their ability to reduce cupric ions to cuprous ions.
   1.  Cu⁺⁺  in hot alkaline solution to Cu⁺.
      1. But Galactose is an exception, cannot reduce copper.
         

         Stool: Effect of reducing substances

8. Reducing substances are:
   1. Glucose.
   2. Fructose.
   3. Lactose.
   4. Galactose.
   5. Pentose.
      1. Xylulose and Arabinose.
   6. Other substances include ascorbic acid.
9. Sucrose is not reducing sugar (nonreducing sugar).
   1. Sucrosuria when sucrose appears in the urine.
   2. Urine specific gravity is high (up to 1.070)
   3. Urine and stool test for reducing substances are negative.

Clinical effects:

1. Carbohydrate malabsorption is a major cause of:
   1. Watery diarrhea:
      1. Osmotic diarrhea is due to the osmotic pressure of the unabsorbed sugars in the intestine.
      2. This will draw the fluid and electrolytes into the gut.
   2. Electrolyte imbalance.
2. Idiopathic lactase deficiency.
   1. This is seen in 70 to 75% of southern European, Greek, and Indian.
   2. Blacks have 70%.
   3. Asian adults have >90 %.
   4. Caucasian American adults have 5 to 20%.
   5. This is a familial disease of infancy with diarrhea, vomiting, failure to thrive, and malabsorption.
   6. Patients becoming asymptomatic when lactose is removed from the diet.
      

      Summary of Lactase enzyme deficiency

3. Galactose appears in infants’ urine with galactosemia; this is characterized by the inability to metabolize galactose.
   1. These infants fail to thrive on milk, which contains mainly galactose.
   2. Failure to diagnose this condition will lead to:
      1. Liver disease.
      2. Mental retardation.
      3. Cataract.
4. Primary glucose-galactose malabsorption:
   1. This is a rare hereditary disorder of active absorption of glucose and galactose from the small intestine.
   2. It is inherited as an autosomal recessive trait.
   3. S/S is like other disaccharides malabsorption.
      1. Diarrhea is the main complaint.
      2. Stools are watery and contain several grams of glucose and galactose/100 mL.
   4. Diagnosis is to find the glucose and galactose in the stool by various methods like:
      1. Glucose oxidase method.
      2. Galactose oxidase method.
      3. Oral glucose and galactose tolerance test, where the flat curve is expected.
         1. Flat glucose tolerance curves are normal in newborn babies.
      4. Chromatography.

Laboratory diagnosis of  reducing substances:

Various options to detect reducing substances are:

1. Mostly there are commercial devices available, e.g, Clinitest (Benedict’s solution), etc.
   1. A yellow-brown color indicates reducing substances.
   2. This color indicates ++ sugars (lactose).
2. Benedict’s reaction principle is as follows:
   1. The clinitest tablet contains anhydrous cupric sulfate, sodium hydroxide, citric acid, and sodium bicarbonate.
   2. Clinitest method:
   3. Procedure for reducing substances in the stool:
      1. Add one volume of stool and two volumes of water.
      2. Mix thoroughly.
      3. Transfer 15 drops of this suspension to a clean test tube.
      4. Add the Clinitest tablet.
      5. Interpretation of the result:
      6. Result
         1. The finding of these reducing substances is abnormal.
         2. Normal when reducing substances is 0.25 g/dL or less in the stool.
         3. Suspicious when the reducing substances are  0.25 to 0.5 g/dL in the stool.
         4. Positive when > 0.5 g/dL are reducing substances in the stool.
            

            Benedict reaction interpretation in the stool or urine

3. Glucose oxidase reagent strip:
   1. This is specific for the detection of glucose.
   2. The glucose oxidase enzyme catalyzes the oxidation of the glucose to gluconic acid and the production of H2O2.
   3. 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.
      

      Glucose oxidase method explanation

4. Thin-layer chromatography: Separation of the sugars can be done by thin-layer chromatography.
5. pH: Also, check the pH of the stool.
   1. Normal stool pH is 7.0 to 8.0.
   2. Stool pH <6.0 is suspected of lactase enzyme deficiency.
6. Another method is:
   1. Give a load of lactose.
      1. In the case of deficiency of lactase, there will be no increase in the glucose level.
   2. 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.

Comparison of Glucose oxidase and copper reduction method:

Chemical substance	Copper reduction tablets	Oxidase reagent strip
Glucose	Positive	Positive
Fructose	Positive	Negative
Galactose	Positive	Negative
lactose	Positive	Negative
Sucrose	Positive	Negative
Maltose	Positive	Negative
Pentose	Positive	Negative
Homogentisic acid	Positive	Negative
Creatine	False-positive	Negative
Hydrogen peroxide	May inhibit positive test	False-positive
Sodium fluoride	No effect	False-negative

1. Tests for other sugars:
   1. Galactose is diagnosed by thin-layer chromatography.
   2. The resorcinol test diagnoses fructose.
      1. Fructose also reduces copper at low temperatures by Benedict’s reaction.
      2. Thin-layer chromatography can also identify fructose.
   3. 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.
      1. OR can diagnose by thin-layer chromatography.
   4. Lactose: Keep in mind that a glass of milk contains approximately 11 grams of lactose.
      1. Lactose was diagnosed by thin-layer chromatography.
      2. OR qualitative lactose test.
      3. Lactose tolerance test when 50 grams of the lactose given orally.
         1. This is followed by the measurement of blood glucose.
         2. An oral lactose tolerance test will show a rise in blood sugar less than 20 mg/dL.
         3. A dose of 50 grams of glucose and galactose produces a rise in blood sugar of more than 25 mg/dL.
         4. Normal blood value (adult/elders) = There is rise in plasma glucose level >20 mg/dL (no abdominal cramps or diarrhea).
   5. Sucrose ferment yeast and can be separated chromatography and needs stains.

Normal Stool findings

Stool parameter	Normal findings
pH
Normal diet	Neutral to alkaline = 7 to 7.5
Infants on breastfeeding	Slightly acidic
Infants on formula milk	Neutral to alkaline
Reducing substances
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)
Glucose	Negative

Breath test:

This is advised to find the lactase enzyme deficiency.

Advised to the patient:

1. Patients can have 12 hours fast.
2. Ask the patient to blow out and measure the H⁺ in the analyzer.
3. Give some sugar (lactose, sucrose, sorbitol, fructose, and lactulose) to the patient.
4. Now check every 15 minutes H⁺ in the breath for 1 to 5 hours.
   1. Normal breath test = <50 ppm Hydrogen (H+) increase over the baseline.

Increased reducing substances are seen in:

1. Disaccharidase enzyme deficiency in the intestine.
2. Short bowel syndrome.
3. Idiopathic lactase deficiency leads to lactose intolerance.
4. Carbohydrate malabsorption is seen in :
   1. Sprue.
   2. viral gastroenteritis.
   3. Celiac disease.
5. False-positive result:
   1. It is seen due to the presence of ascorbic acid (>200 mg/dL).
   2. This will show a low-level positive result trace or 1+.
   3. Urine with low specific gravity contains glucose that may give a slightly raised level result.
   4. Antibiotics with large quantities like nalidixic acid, cephalosporin, and probenecid.
   5. Radiographic material gives black color, and this may be considered positive.
6. False-negative result:
   1. 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.

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