Carbohydrates

Laboratory Applications
Objective
s
• Know the specimen handling and storage of samples for Glucose testing.

• Discuss the different glucose methodologies .

• Describe the different laboratory testing for glucose

analysis.
SPECIMEN
CONSIDERATIONS
SPECIMEN CONSIDERATIONS

Possible samples:

- Whole blood 15% lower than serum or

plasma
-Plasma
Unhemolyzed
-Serum Venous plasma/serum
-CSF
-Pleural Fluid
-Urine
SPECIMEN CONSIDERATIONS

•Glycolysis decreases serum glucose by

approximately 5% to 7% in 1 hour (5 to 10 mg/L) in

normal uncentrifuged coagulated blood at room

temperature.

•In separated , nonhemolyzed sterile serum,

stable as long as 8 hours at 25°C an up to

SPECIMEN CONSIDERATIONS

Storage of samples
•Refrigerated (2-8ºC)

–serum or plasma: stable up to 48 hrs

–Whole blood: 2mg Na fluoride per mL of

whole blood (48 hrs)

 • CHEMICAL
Method

• ENZYMATI
C Method

GLUCOSE METHODOLOGIES
 CHEMICAL METHOD
A. OXIDATION REDUCTION METHOD

1. Alkaline Copper Reduction Method

a. Folin Wu Method
b. Nelson Somogyi Method
c. Neocuproine Method
d. Benedict’s Method
(Modification of Folin Wu)

2. Alkaline Ferric Reduction Method

Hagedorn Jensen

B. CONDENSATION METHOD
Dubowski
Method
 CHEMICAL METHOD

A. OXIDATION REDUCTION
METHOD
Principle:

Reduction of
1. Alkaline Copper Reduction cupric ions to
cuprous ions
Method forming cuprous
Glucose oxide in hot
Alkaline Copper Tartrate Cuprous Ions
alkaline solution
Heat
by glucose.
 CHEMICAL METHOD

1. Alkaline Copper Reduction

Method
a. Folin Wu
Method
Cuprous Ions +
Phosphomolybdate

Phosphomolybdic Acid or
Phosphomolybdenum
Blue
 CHEMICAL METHOD

1. Alkaline Copper Reduction

Method

b. Nelson Somogyi
Method
Cuprous Ions +
Arsenomolybdate

Arsenomolybdic Acid or
Arsenomolybdenum Blue
 CHEMICAL METHOD

1. Alkaline Copper Reduction

Method

c. Neocuproine (2,9 Dimethyl 1,10 Phenantroline Hydrochloride)

Method
Cuprous Ions +
Neocuproine

Cuprous-Neocuproine Complex
(Yellow or Yellow Orange)
 CHEMICAL METHOD

1. Alkaline Copper Reduction

Method

d. Benedict’s Method (Modification of

Folin-Wu)
• used for detection and quantitation of reducing substances
in body fluids like blood and urine.
• used citrate or tartrate as stabilizing agent
 CHEMICAL METHOD

Benedict’s Method (Modification of

Folin-Wu)
Fehling’s Test
 CHEMICAL METHOD

A. OXIDATION REDUCTION
METHOD

2. Alkaline Ferric Reduction Method (Hagedorn Jensen)

It involves reduction of a yellow ferricyanide to a colorless ferrocyanide

by glucose
(Inverse Colorimetry)
 CHEMICAL METHOD

B. Condensation Method
Ortho-toluidine
(Dubowski Method)
•condensation of glucose with a primary
aromatic amine in glacial acetic acid,
forming an equilibrium mixture of a
glycosylamine and the corresponding
Schiff base

Glacial HAC
Glucose + Aromatic glycosylamine + schff’s
heat
amines base
 CHEMICAL METHOD

B. Condensation Method
Ortho-toluidine
(Dubowski Method)
•Procedure:
–Glucose in a PFF (3% TCA)
reacts to O-Toluidine in hot
acidic solution will yield a
GREEN colored compound
with maximum absorbance at
630 nm.
ENZYMATIC METHODS
1. GLUCOSE OXIDASE METHOD
a. Colorimetric Glucose Oxidase
Method
(Saifer Gerstenfield Method)
b. Polarographic Glucose Oxidase

2. HEXOKINASE METHOD

3. GLUCOSE DEHYDROGENASE
METHOD

4. DEXTROSTICS (cellular strip)

5. INTERSTITIAL GLUCOSE MEASURING

DEVICE
 ENZYMATIC METHODS
1. GLUCOSE OXIDASE METHOD
a. Colorimetric Glucose Oxidase Method
(Saifer Gernstenfield Method)

B-D-glucose

Trinder Reaction

• 3-methyl-2-benzothiazolinone hydrazone

• N,N-dimethylaniline

1. Increased levels of uric acid, bilirubin, and ascorbic acid can cause
falsely decreased values as a result of these substances being oxidized by
peroxidase, which then prevents the oxidation and detection of the
chromogen.

2. Strong oxidizing substances, such as bleach, can cause falsely increased

values
 ENZYMATIC METHODS
1. GLUCOSE OXIDASE METHOD

b. Polarographic Glucose Oxidase

• Measures rate of oxygen consumption which is proportional to glucose concentration.

• Glucose oxidase in the reagent catalyzes the oxidation of glucose by oxygen under
first order conditions, forming hydrogen peroxide.

• Quantitated by the consumption of oxygen on an oxygen-sensing electrode.

• Hydrogen peroxide is prevented from re-forming oxygen by adding molybdate, iodide,

catalase and ethanol.
 ENZYMATIC METHODS
1. GLUCOSE OXIDASE METHOD

b. Polarographic Glucose Oxidase

catalase
H2O2 + C2H5OH CH3CHO + 2H2O

OR

molybdate
H2O2 + 2H + 2 I I2 + 2H2O
 ENZYMATIC METHODS
2. HEXOKINASE METHOD

• MOST SPECIFIC GLUCOSE METHOD; REFERENCE METHOD

• Less interference

• Plasma collected using heparin, EDTA, flouride, oxalate or citrate may be

used for this test.

• Other samples; Urine, CSF and serous fluids

 ENZYMATIC METHODS
2. HEXOKINASE METHOD

Gross hemolysis and

extremely elevated bilirubin
may cause a false decrease in
results.
 ENZYMATIC METHODS
3. GLUCOSE DEHYDROGENASE METHOD

• Glucose is reduced to produce a chromophore that is measured

spectrophotometrically or an electrical current
• NADH = glucose concentration
• Close agreement with hexokinase procedures
• Mutarotose is added to shorten the time
 ENZYMATIC METHODS

4. DEXTROSTICS (cellular strip)

An enzyme-impregnated strip used with a

small portable electronic colour-measuring
device for convenient estimation of the blood
sugar levels by diabetics.

It should not be used to

diagnose diabetes or
hypoglycemic disorders.
 ENZYMATIC METHODS
5. INTERSTITIAL GLUCOSE
MEASURING DEVICE

-used for cobtinuous monitoring of glucose

levels in people with diabetes

-uses electrochemical methods to automatically

and frequently measure glucose levels in the
interstitial fluid of dermis or subcutaneous fat
tissue.
 Laboratory
Testing
for GLUCOSE
Laboratory Test for Glucose

1.Random plasma glucose

2.Fasting plasma glucose
3. Tolerance test
4. HbA1c

5. Fructosamine
6. Urine Microalbumin
7. Ketone testing
“Dont fret or worry. Instead of
worrying, pray. Let petitions
and praises shape your worries
into prayer, letting God know
your concerns. Before you
know it, a sense of God’s
wholeness, everything coming
together for good, will come
and settle you down. It’s
wonderful what happens when
Christ displaces worry at the
center of your life.”
–Philippians 4: 6-7
Thank
you