‫بسم اهلل الرمحن الرحيم‬

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 NON DRUGS

TYPE CARBOHYDRATE false +ve reaction
TYPE
GLUCOSE CREATININE SALICYCLATE
FRUCTOSE URIC ACID ASPIRINE
PENTOSE ASCORPIC ACID PENICILIN
LACTOSE HOMOGENTISIC ACID STREPTOMYCIN
GALACTOSE 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 (β-D- galactosidase).
 The syndrome should not be confused with

intolerance of milk (result due to sensitivity

to protein in milk, usually to β- lacto
globulin).
LACTOSE INTOLERANCE

Symptoms:
 Abnormal cramps.
 Diarrhea.

 Flatulence.
 Β-
GALACTOSIDASE
(LACTASE)
LACTOSE GLUCOSE PORTAL
+ CIRCULATION
GALACTOSE

NORMAL LACTOSE
METABOLISM
 SMALL INTESTINE LACTOSE

Β-GALACTOSIDASE
DEFICIENCY

LARGE INTESTINE LACTOSE

2-carbon
metabolites BACTERIA H2, Can be
measured in
breath
3-carbon CO2
metabolites
Bloating diarrhea
(dehydration)

ABNORMAL LACTOSE METABOLISM

 TYPES OF LACTASE DEFICIENCY SYNDROM

1. INHERITED.
2. SECONDARY LOW LACTASE
ACTIVITY.
3. 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 β-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. BENEDICT’S TEST
2. CLINITEST
3. FEHLING’S SOLUTION
4. GLUCOSE STRIPS TEST
5. SELIWANOFF’S TEST
6. METHYLAMINE TEST
7. MUCIC ACID TEST
1-BENEDICT’S TEST

Principle:
When boiled in alkaline copper sulphate,
glucose and other sugars, reduce bluish
cupric ions to red-brownish 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 PALE GREEN WITH YELLOW ORANGE BRIOR

IN COLOUR GREEN PRECIPITATE RED

NILL TRACE + ++ +++ ++++

0.5 g/dl 1 g/dl 2 g/dl >2 g/dl
 Dry Benedict’ reagent
Reagent preparation
 Copper-II sulphate 5-
hydrate(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 Sugar
concentration
Blue Nil or <0.2g%
Slight orange precipitate with blue 0.2 g/dl (+)
supernatant

Persistent orange precipitate 0.5 g/dl (++)

Orange precipitate turning brown during 1 g/dl (+++)
boiling

Green-brown precipitate rapidly turning dark >2 g/dl (++++)

brown
 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 56C⁰ 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. Yellow-brown 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.