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 lactose (or other sugars) in 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 stool not tested immediately.

Indication

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

Pathophysiology

1. Definition of reducing substances:
   1. Reducing substances are those substances that 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	Phenol
      Lactose	Uric acid and urates
      Sucrose	Salicylate

2. Table sugar sucrose is not reducing sugar.
3. Normally sugars are rapidly absorbed in the upper small intestine.
4. 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 flood the small intestine and then by the bacterial catabolism produces Methane and H⁺.
   3. This is the bases for the breath test where Hydrogen (H⁺) gas is tested to find the lactase enzyme deficiency.
      

      lactose deficiency and its complications

5. If sugars are not absorbed then these produce diarrhea due to the osmotic pressure produced by these unabsorbed sugars.
6. These unabsorbed sugars draw fluid and electrolytes into the intestine.
7. These unabsorbed sugars are measured as reducing substances.
8. 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.
         

         Reducing substances and their effects

9. Reducing substances are:
   1. Glucose.
   2. Fructose.
   3. Lactose.
   4. Galactose.
   5. Pentose.
      1. Xylulose and Arabinose.
   6. Other substances include ascorbic acid.
   7. Sucrose is not reducing sugar (nonreducing sugar).
10. Clinical effects:
    1. Carbohydrate malabsorption is a major cause of:
    2. Watery diarrhea.
    3. Electrolyte imbalance.
11. There may be 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%.
12. Galactose appears in the urine of the infants with galactosemia, this is a condition 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.

13. 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.

How to test reducing substances:

1. Various options to detect reducing substances are:
2. Mostly there are commercial devices available e.g Clinitest (Benedict’s solution) and etc.
   1. A yellow-brown color indicates reducing substances.
   2. This color indicates ++ sugars (lactose).
3. Benedict’s reaction principle is as follows:
   

   Principle of Benedict’s reaction

4. Glucose oxidase reagent strip principle as follows:
   

   Principle of Glucose oxidase method

5. Separation of the sugars by thin-layer chromatography.
6. Also, check the pH of the stool.
7. Another method is:
   1. Give a load of lactose.
   2. In the case of deficiency of lactase, there will be no increase in the glucose level.
8. Procedure for reducing substances in the stool:
   1. 1. Add one volume of stool and two volumes of stool.
      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:
9. Result
   1. The finding of these reducing substance 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.
      1. Keep in mind when there is sucrose intolerance, in these patients instead of a small amount of sucrose, there is a large amount of the glucose and galactose found in the stool, which is due to the hydrolysis of sucrose by the intestinal bacteria, so 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

10. Tests for other sugars:
    1. Galactose is diagnosed by a thin layer chromatography.
    2. Fructose is diagnosed by the resorcinol test.
       1. Fructose also reduces copper at low temperatures by Benedict’s reaction.
       2. Thin-layer chromatography can also identify the 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 diagnosed by thin-layer chromatography.
       1. OR qualitative lactose test.
    5. Sucrose ferment yeast and this 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.

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 saw 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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