OCCULT BLOOD & REDUCING SUBSTANCES

REDUCING SUBSTANCES DEFINITION
Substances that donate electrons are called reducing agents. Some carbohydrates can reduce other compounds. To be reducing substances, they must contain free aldehyde or ketone group that reduces the blue cupric ions to red cuprous oxide.

Types of reducing substances in urine
CARBOHYDRATE TYPE
GLUCOSE FRUCTOSE PENTOSE LACTOSE GALACTOSE

NON CARBOHYDRATE TYPE
CREATININE URIC ACID ASCORPIC ACID HOMOGENTISIC ACID

DRUGS false +ve reaction
SALICYCLATE ASPIRINE PENICILIN STREPTOMYCIN ISONIAZID AMINOSALICYCLIC ACID

URINE SUGAR:
When reducing sugars are excreted in the urine the condition is called glycosuria. One of the earliest tests for the urine sugar of diabetic was a test for reducing sugar. This non specific test could be positive for other reducing hexoses or pentoses.

Glucosuria
The term glycosuria refers to the presence of more of the usual amount of glucose in urine. Usually < 15 mg/dl (0.8 mmol/l) is excreted in the urine.

Causes of glucosuria
Glucosuria with hyperglycemia (DM). A reduced rate of renal reabsorption of glucose (renal glucosuria). An increase in the rate of glomerular filtration as during pregnancy. Fanconi's syndrome.

REDUCING SUBSTANCES OF THE STOOL LACTOSE INTOLERANCE
Intolerance to lactose caused due to lactase deficiency ( -D- galactosidase). The syndrome should not be confused with intolerance of milk (result due to sensitivity to protein in milk, usually to lacto globulin).

LACTOSE INTOLERANCE
Symptoms:
Abnormal cramps. Diarrhea. Flatulence.

LACTOSE

GALACTOSIDASE (LACTASE)

GLUCOSE + GALACTOSE

PORTAL CIRCULATION

NORMAL LACTOSE METABOLISM

SMALL INTESTINE LACTOSE
-GALACTOSIDASE DEFICIENCY

LARGE INTESTINE LACTOSE 2-carbon metabolites BACTERIA H2, Can be measured in breath CO2 Bloating diarrhea (dehydration)
ABNORMAL LACTOSE METABOLISM

3-carbon metabolites

TYPES OF LACTASE DEFICIENCY SYNDROM

1. 2.

3.

INHERITED. SECONDARY LOW LACTASE ACTIVITY. PRIMARY LOW LACTASE ACTIVITY.

INHERITED LACTASE DEFICIENCY
Develops soon after birth. Feeding of lactose free diet result in absence of the symptoms. Can be treated by:  Active -galactosidase.  Calcium and energy to replace milk.

SECONDARY LOW LACTASE ACTIVITY
Because of intestinal diseases: Celian sprue. Kwashiorkor. Colitis. Gastroenteritis. After surgery of peptic ulcer.

PRIMARY LOW LACTASE ACTIVITY

Mechanism by which enzyme is lost is not clear.

REDUCING SUBSTANCES Laboratory Diagnosis

REDUCING SUBSTANCES Specimen
Freshly passed urine/stool is the specimen of choice. The specimen should be examined immediately. A diarrheal stool usually give good results. Collect urine/stool in a dry, clean container. The specimen should be uncontaminated with other body secretions.

QUALITATIVE TESTS for reducing sugars
1. 2. 3. 4. 5. 6. 7.

BENEDICT¶S TEST CLINITEST FEHLING¶S SOLUTION GLUCOSE STRIPS TEST SELIWANOFF¶S TEST METHYLAMINE TEST MUCIC ACID TEST

1-BENEDICT¶S TEST
Principle:
When boiled in alkaline copper sulphate, glucose and other sugars, reduce bluish cupric ions to redbrownish cuprous oxide, the degree of reduction corresponding to the concentration of reducing substance present.

Solution Benedict¶s Reagent Reagent preparation:
Dissolve 17.3 gm of copper sulphate in 800 ml DW. Add 100 gm of sodium carbonate. Mix and add 175 gm of trisodium citrate. Mix and make up to one liter with D.W. Stable for one year.

Solution Benedict¶s Reagent Procedure:
Dispense 2.5 ml of Benedict¶s reagent into test tube. Add 0.2 ml of urine to the tube. Mix and boil for 5 minutes. Cool and observe for any colour change.

Solution Benedict¶s Reagent Results
NO CHANGE IN COLOUR PALE GREEN GREEN WITH PRECIPITATE YELLOW ORANGE BRIOR RED

NILL

TRACE

+ 0.5 g/dl

++ 1 g/dl

+++ 2 g/dl

++++ >2 g/dl

Dry Benedict¶ reagent Reagent preparation
Copper-II sulphate 5hydrate(CuSO4.5H2O): 10 gm. Citric acid: 150 gm. Sodium hydroxide or potassium hydroxide pellets.

Dry Benedict¶ reagent Procedure
Place a pea-size amount of mixed dry reagent to a test tube. Add three drops of urine to the tube and shake. The mixture will begin to boil, continue shaking until the boiling stop. Observe for change of colour or precipitate.

Dry Benedict¶ reagent Result
appearance Blue Slight orange precipitate with blue supernatant Persistent orange precipitate Orange precipitate turning brown during boiling Green-brown precipitate rapidly turning dark brown Sugar concentration Nil or <0.2g% 0.2 g/dl (+) 0.5 g/dl (++) 1 g/dl (+++) >2 g/dl (++++)

2. CLINITEST
It is a modification of the Benedict¶s dry reagent in tablet form. Each tablet contains: copper sulphate, sodium carbonate, sodium hydroxide, citric acid.

2. CLINITEST
The citric acid is neutralized by sodium carbonate and sodium hydroxide with production of intense heat and release of carbon dioxide. The heat brings the mixture to boil and copper ions are reduced by the sugar.

2. CLINITEST
Quick and without boiling. In case of alkaptonuria, homogentisic acid as reducing substance react with Clinitest tablets.

2. CLINITEST Disadvantages
In humid climates tables are not stable and become unfit for use. Unable to detect concentrations < 250 mg/dl (low sensitivity).

Note that:
The faeces specimen is tested in the same way as described above for urine, except that 8 drops of fluid faeces are used instead of urine.

3. FEHLING¶S TEST
In this test the presence of aldehydes but not ketones is detected by reduction of the deep blue solution of copper(II) to a red precipitate of insoluble copper oxide.

3. FEHLING¶S TEST
Fehling's A:copper sulphate (CuSO4.5H2O):7 g dissolved in DW containing 2 drops of dilute sulfuric acid. Fehling's B potassium tartrate35g ,and NaOH: 12g in 100 ml DW. These two solutions should be stoppered and stored until needed.

3. FEHLING¶S TEST
Add 2 ml of Fehling's mixture to a test tube. Add 3 drops of urine to the tube. Place the tube in a water-bath at 60° C. A positive test is indicated by a green suspension and a red precipitate. The test is sensitive (1 g/dl of glucose will produce the red colour). More specific.

4. GLUCOSE STRIPS TEST
Principle:
Glucose is oxidized to gluconolactone by glucose oxidase with the release of hydrogen peroxide. A peroxidase and hydrogen peroxide convert the a chromogen from a reduced colorless state to a colored oxidized state.

4. GLUCOSE STRIPS TEST
Procedure:
The strip is dipped in urine and the colour is examined after 20-30 seconds and compared with colour present in kit.

4. GLUCOSE STRIPS TEST
It is specific for glucose. More sensitive (100 mg/dl or less). Quick and easy to perform.

4. GLUCOSE STRIPS TEST False strips test reactions: Oxygen receptors in urine:
ascorbic acid, drugs, large amount of acetoacetate( in urine of out of control diabetics).

4. GLUCOSE STRIPS TEST False strips test reactions:

Catalase enzyme:
severe E.coli infection (can destroy hydrogen peroxide).

4. GLUCOSE STRIPS TEST False strips test reactions: Disinfectants:
oxidize the chromogen directly causing false positive reaction.

5. SELIWANOFF¶S TEST (for detection of fructose)
. This test is used to differentiate between ketoses and aldoses. The acid when heated along with a sugar will produce furfural or hydroxymethylfurfural, which further reacts to give a red color.

5. SELIWANOFF¶S TEST
Ketoses react more quickly than aldoses and thus the reaction time is a means of separation or detection. Disaccharides containing fructose should react intermediately between that of fructose alone and one of the aldoses.

5. SELIWANOFF¶S TEST The reagent
Dissolve 1 g resorcinol in 330 mL concentrated HCl, dilute to one liter . This reagent is stable for a year.

5. SELIWANOFF¶S TEST PROCEDURE:
Add 0.5 ml of urine to 3 ml of Selivanoffís reagent. Boil for 30 seconds. Begin your observations immediately. Red colour appears if fructose is present.

6. FEARON¶S METHYLAMINE TEST (for detection of lactose)
a specific test for the reducing disaccharides When lactose are heated with methylamine, in alkaline conditions, a red coloration is produced.

6. FEARON¶S METHYLAMINE TEST
5% aqueous methylamine hydrochloride, 20% NaOH.

6. METHYLAMINE TEST
Add 1 ml of ethylamine hydrochloride and 1 ml of sodium hydroxide To 5 ml of urine. Keep at 56C for 30 minutes. Appearance of red colour indicates the presence of lactose.

7. MUCIC ACID TEST
The mucic acid test distinguishes galactose and lactose from other reducing sugars. The nitric acid oxidizes galactose to tetrahydroxyadipic acid(mucic acid) that crystallizes out. Other sugars, give similar acids, but they are water-soluble.

7. MUCIC ACID TEST
When urine is boiled with nitric acid, mucic acid crystals are formed indicating the presence of lactose or galactose.

Testing faeces for lactase deficiency
If using Benedict¶s reagent the specimen is tested in the same way for urine except that8 drops of freshly passed fluid feces are used instead of urine.

Testing faeces for lactase deficiency
If clinitest is used, add 5 drops of freshly passed fluid specimen to 10 drops of clean water. After boiling has stopped mix the contents of the tube and note the colour of the fluid. Yellowbrown colour indicates the presence of lactose (++).

Testing faeces for lactase deficiency
Testing the PH of the faeces PH meter: calibrate the PH meter using STD buffers, one having an acidic PH and the other an alkaline PH. Pour the specimen in a beaker and calibrate the PH meter again using the buffer of PH 7. And then measure the PH of the specimen.

Testing faeces for lactase deficiency
Testing the PH of the faeces
Narrow range PH papers: put a drop of freshly passed fluid specimen on a portion of PH paper. The colour obtained is compared with standard chart.

Testing faeces for lactase deficiency Interpretation of the results:
Lactase deficiency is indicated if a faecal specimen: Contain (++) or more of lactose sugar. Has PH of 6 or below.

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