Clinical Chemistry

Manual Glucose Determination

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Presentation Outline
01 Glucose Level

The Need for Glucose Measurement

02
Glucose Metabolism and the Fate of
03 Glucose

04 Manual Methods of Glucose

Determination
 Recall
Carbohydrates, Amino Acids, and Lipids
3 General Types of
Organic Compounds

Carbohydrates
Primary Energy
Source

The General Formula of

  𝐶 𝑥 ¿
Carbohydrates

Can only be use by the Glucose

body in the form of
Glucose Level
 Glucose Level

The blood sugar level,

blood sugar
concentration, or
blood glucose level is
the amount of glucose
present in the blood.

unit: mmol/L
 Why do we need to measure our
Glucose levels?
• High Blood Sugar
Abnormalities may • Low Blood Sugar
result if the glucose
levels becomes too
high or too low.
Glucose levels are
kept a narrow
range to maintain
the body’s
homeostasis.
 Glucose Metabolism
Insert the title of your subtitle Here

Glucose is a primary
source of energy for
It is stored in the body
humans. The nervous as glycogen. The liver
system, including the is an important
brain depends on the storage site for
glucose from the glycogen.
surrounding ECF for
energy.

The endocrine
pancreas secretes the When the concentration
hormones responsible falls below a certain
for the regulation of level, the nervous tissue
blood glucose levels. loses the primary source
These are glucagon of energy.
and proinsulin.
 The Fate of Glucose
Pathways in Glucose Metabolism

Glycogenesis
Glycolysis
Conversion of glucose to glycogen
Metabolism of glucose molecule to for storage
pyruvate or lactate for the
production of energy

Lipogenesis
Gluconeogeneis
Conversion of carbohydrates to
Formation of glucose-6-phosphate fatty acids
from non-carbohydrate sources

Glycogenolysis Lipolysis
Breakdown of glycogen to glucose Decomposition of fat
for use as energy
 Methods of Glucose Measurement

• Glucose can be measured from serum, plasma, or whole blood.

• Most glucose measurements are performed on serum or plasma.
• Gray tops are typically used as anticoagulant and preservative of whole blood,
particularly if analysis is delayed.
• FBG should be obtained in the morning after an approximately 8-10 hr. fast.
 Test Analyte Tested Reagent Methodologies and Principle Normal Value

ENZYMATIC METHOD

 Glucose oxidase converts β-D-glucose to

gluconic acid. Normal:
Colorimetric Glucose venous plasma -Glucose oxidase/  Mutarotase may be added to the reaction to <100 mg/dL
Oxidase Method glucose kinase facilitate the conversion of α-D-glucose to β-D-glucose.
β-D-glucose 
Pre-diabetic:
-Mutarotase Oxygen is consumed and hydrogen peroxide
(H2O2) is produced. 100–125 mg/dL
 The rate of disappearance of oxygen is Diabetic:
measured by using an oxygen electrode or by ≥126
consuming H2O2 in a side reaction.

 Considered more accurate than the glucose

Polarographic venous plasma oxidase methods because the coupling reaction using
glucose Horseradish
Glucose Oxidase glucose-6-phosphate dehydrogenase is highly specific; Normal:
β-D-glucose peroxidase therefore, it has less interference than the coupled.
method   <100 mg/dL
 An oxygen consumption electrode can be used to
perform the direct measurement of oxygen Pre-diabetic:
 Enzymatic conversion of glucose is quantitated by 100–125 mg/dL
the consumption of oxygen on an oxygen-sensing Diabetic:
electrode hydrogen peroxide is prevented from ≥126
re-forming oxygen by adding molybdate, iodide,
catalase and ethanol.
  May be performed on serum or plasma
collected using heparin,
ethylenediaminetetraacetic acid (EDTA), fluoride,
oxalate, or citrate.

-Triethanolamine  can also be used for urine, Normal:

-ATP cerebrospinal fluid, and serous fluids. <100 mg/dL
-NAD Pre-diabetic:
-Hexokinase  Hexokinase in the presence of ATP 100–125 mg/dL
venous plasma converts glucose to glucose-6-phosphate.
Hexokinase glucose (Recombinant Diabetic:
β-D-glucose Yeast) ≥126
-G-6-PDH  Glucose-6-phosphate and the cofactor NADP+
 
(Recombinant are converted to 6 phosphogluconate and
Leuconostoc) NADPH by glucose-6-phosphate dehydrogenase.

 NADPH has a strong absorbance maxima at

340 nm, and the rate of appearance of NADPH can
be monitored spectrophotometrically and is
proportional to the amount of glucose present in
the sample.

Glucose -Hexokinase  
dehydrogenase venous plasma -Glucose 6  glucose is reduced to produce a chromophore
method glucose Phosphate that is measured spectrophotometrically or an
    dehydrogenase electrical current
β-D-glucose  
 CHEMICAL METHODS

Normal:
 When glucose or other reducing agents are 8 to 120 mg / 100ml
-Alkaline copper treated with alkaline copper solution they
venous solution
reduce the copper with the result insoluble Mild diabetic
plasma -Phosphomolybdic
glucose acid
cuprous oxide is formed. conditions
Folin-Wu Method   -10% Sodium  The reaction depends on temperature, 140. -300 / 100ml
β-D-glucose tungstate duration of heating, degree of alkalinity.
 The ratio of glucose to cuprous oxide form Severe diabetic
-2/3 N H2SO4 may be varied after heating far a period. conditions
 The cuprous oxide form is allowed to react 1200mg / 100ml
with phospharomolybdate to form  
molybdenum blue colored complex which
can be read colorimetrically using red filter
on at 680nm.

 
 The reducing sugars when heated with alkaline
copper tartrate reduce the copper from the
-Alkaline Copper – cupric to cuprous state and thus cuprous oxide is
-Tartrate formed.
-Arsenomolybdate  When cuprous oxide is treated with
Nelson-Somogyi β-D-glucose reagent arsenomolybdic acid, the reduction of molybdic acid
Method to molybdenum blue takes place.
 The blue colour developed is compared with a
set of standards in a colorimeter at 620 nm.
  Reducing sugars under alkaline condition
tautomerise and form enediols, which are powerful
reducing agents.
-Copper(II) sulphate
β-D-glucose -Sodium carbonate
Benedict’s Method -Sodium citrate  They can reduce cupric ions (Cu2+) to cuprous
(modification of Folin- form (Cu+), which is responsible for the change in
Wu) color of the reaction mixture. This is the basis of
Benedict’s test.

 When the conditions are carefully controlled, the

colouration developed and the amount of
precipitate formed (Cuprous oxide) depends upon the
amount of reducing sugars present.

 o-Toluidine, 6% (v/v) in glacial acetic acid, is

used to determine glucose in biologic material after
Ortho-toluidine deproteinization with 3% (w/v) trichloracetic acid.
(Dubowski Method) β-D-glucose -Glacial HAC
   A stable green color develops after heating at
1000 for 10 mm., and the absorbance is determined
at 630 or 635 mp.
  
 A solution containing 75 g of glucose  
is administered, and a specimen for plasma glucose Normal:
measurement is drawn 2 hours later. The patient <140 mg/dL
glucose solution/
should be ambulatory and on a normal-to high  
load:
Glucose Tolerance Adult : 75 g carbohydrate intake for 3 days before the test. Pre-diabetic:
and 2-Hour β-ᴅ-glucose   140–199 mg/dL
Postprandial Tests Children: 1.75 g/kg,  If that level is ≥ 200 mg/dL and is confirmed on a  
maximum subsequent day by either an increased random Diabetic:
dose of 75 g. or fasting glucose level, the patient is diagnosed with ≥200 mg/dL
diabetes

 The patient should be fasting for at least 10

hours and not longer than 16 hours, and the test
should be performed in the morning because of the
hormonal diurnal effect on glucose.

 The glucose molecule attaches non-enzymatically to

the hemoglobin molecule to form a ketoamine.
Pre-diabetes:
Hemoglobin A1c  The rate of formation is directly proportional to the 5.7–6.4%
Glycosylated plasma glucose concentrations  
from EDTA
Hemoglobin / whole blood hemolysate Diabetes mellitus:
Hemoglobin A1c  glycosylated hemoglobin level at any one time ≥6.5%
sample. reflects the average blood glucose level over the previous
2 to 3 months.

 hemolysate affinity chromatography is the preferred

method of measurement

 glycosylated hemoglobin attaches to the boronate

  
 measures blood glucose levels after a Normal:
period of fasting, usually at least eight hours <100 mg/dL
FASTING Fasting sample without food or liquid (except water). Pre-diabetic:
PLASMA of venous 100–125 mg/dL
GLUCOSE (FPG) plasma glucose  This test is more definitive than a random Diabetic:
  test, because there is no chance that it has ≥126
been influenced by recent food intake.
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