CLINICALCHEMITRY LABORATORY NOTES

CARBOHYDRATES – the major component of the human diet, an important source of energy. The
medically important carbohydrates that contain six carbons ( HEXOSES ) are glucose, fructose, and
galactose. Important disaccharides are lactose ( glucose and galactose ) and sucrose ( glucose and
fructose ).

GLUCOSE MEASUREMENT
- Can be measured from serum, plasma or whole blood.
- The glucose concentration in whole blood 11% lower than the concentration in serum or
plasma
- Fasting flucose in whole blood is 15% lower than serum or plasma
- Venous blood glucose 7 mg/dL lower than capillary blood due to tissue metabolism, while
capillary blood glucose is the same with arterial blood glucose.
- Plasma glucose levels increase with age:
a. Fasting – 2 mg/d: per decade
b. Postprandial – 4 mg/dL per decade
c. Glucose Challenge – 8-13 mg/dL per decade

Specimen considerations
- Serum or plasma must be refrigerated and separated from the cells within one hour
( preferably within 30 min. ) to prevent substanstial loss of glucose by the cellular fraction,
particularly if the white blood cell count is elevated.
- At room temperature ( 20 – 25oC), glycolysis decreases glucose by 5-7% per hour ( 5-10
mg/dL) in normal uncentrifuged coagulated blood.
- At refrigerated temperature ( 4oC), glucose is metabolized at a rate of about 1-2mg/dL per
hour
- WBC and RBC metabolize glucose, resulting to decrease value in clotted, uncentrifuged
blood ( leukocytes can lead to excessive glycolysis )
- The rate of metabolism is higher with bacterial contamination or leukocytosis
- Sodium fluoride ions ( gray-tip tubes ) are often used as an anticoagulant and preservative
of whole blood, particularly if analysis is delayed.
- Fluoride inhibits glycolytic enzymes
- Fasting Blood Sugar (FBS) should be obtained in the morning after an approximately 8-10 hr.
fast ( not longer than 16 hours )

GLUCOSE METHODS:
1. Chemical Methods
A. Reduction – Oxidation methods
1. Alkaline Copper Reduction Methods –
Principle : Reduction of cupric ions to cuprous ions, forming cuprous oxide in hot alkaline
solution by glucose.
glucose
Alkaline Copper Tartrate ------------- Cuprous ions
heat
 a. Folin Wu Method :
Principle: Glucose reduces the cupric ions present in the alkaline copper reagent to
cuprous ions or the cupric sulphate is converted into cuprious oxide, which reduces the
phosphomolybdic acid to phosphomolybdous acid, which is blue when optical density is measured at
420 nm.
b. Nelson Somogyi Method

Principle: The reducing sugars when heated with alkaline copper tartrate reduce the
copper from the cupric to cuprous state and thus cuprous oxide is formed. When cuprous oxide is
treated with arsenomolybdic acid, the reduction of molybdic acid to molybdenum blue takes place. The
blue colour developed is compared with a set of standards in a colorimeter at 620 nm.
c. Neocuproine Method ( 2,9 Dimethyl 1,10 Phenantroline HCl)

Cuprous ions + Neocuproine ------Cuprous-Neocuproine complex ( yellow or yellow orange )

d. Benedict’s Method ( Modification of Folin Wu )

- Used for the detection and quantitation of reducing substances in body fluids like
blood and urine.
- Use citrate or tartrate as stabilizing agent.

2. Alkaline Ferric Reduction / Ferricyanide Methods ( Hagedorn Jensen )

Principle: Reduction of a yellow ferricyanide to a colorless ferrocyanide by glucose
( inverse colorimetry )
* Most chemical mesuarements of glucose depend on its redusing properties; mpost are no
longer used because of lack of specificity.

B. Condensation Methods
1. Condensation with aromatic amines
*Ortho- Toluidine ( Dubowski Method )
- Ortho-toluidine is the only chemical method still widely used and is based on
the condensation of aldosaccharide, such as glucose, with an aromatic amine and glacial acetic acid. The
stable green color that develops then is measured spectrophotometrically. This method can be used for
plasma, urine and CSF without protein precipitation. Galactose and mannose react as well as glucose,
lactose, maltose, sucrose, and fructose also react but to a much lesser extent. Hence, values for this
Method are slightly higher than for more specific enzymatic methods.
Major disadvantage:
1. Corrosiveness of the reagent to laboratory equipment
2. Toxicity

II. Enzymatic Methods

- Acts on glucose but not on other sugars and other reducing substances
- Yield maximum specificity for glucose measurements.
1. Glucose oxidase Method ( GOD)
- measures the β-D-glucose
- Also measures CSF glucose
 a. Colorimetric Glucose Oxidase Method ( Saifer Gernstenfield Method )
- Glucose can be measured by its reaction with glucose oxidase, in which gluconic acid and
hydrogen peroxide are generated.
- Hydrogen peroxide then reacts woth an O2 accepteor, such as ortho-dianisidine phenylamine-
phenazone ( Trinder’s reagent), 0-toluidine, or other chromogenic acceptors in a reaction
catalyzed by peroxidise to form a color.
- Glucose in solution exists in 2 forms ( approximately 35% alpha and65% beta )
- Glucose oxidase is highly specific for the β-isomer ( β –D-glucose ), and any glucose present in
the α form must be converted to the β form before reacting.
- Some preparations contain the enzyme Mutarotase which accelerated this process.
- The second step involving peroxide is less specific than the first, and numerous reducing
substances inhibit oxidation of the chromogens used in the peroxidise reaction.
- Although uric acid and creatinine cause little interference in most of these methods, ascorbic
acid can lead to spuriously decreased values.
- One of the chief advantages: low cost

b. Polarographic Glucose Oxidase

- Enzymatic conversion of glucose is quantitated by the consumption of oxygen on an oxygen
sensing electrode
-measures the rate of oxygen consumption which is proportional to glucose concentration
- GOD in reagent catalyzes the oxidation of glucose by oxygen under first order conditions,
forming hydrogen peroxide.
- Hydrogen peroxide is prevented from re-forming oxygen by adding molybdate, iodide, catalase
and ethanol.
- an oxygen consumption electrode can be used to perform the direct measurement of oxygen
by the polarographic technique.
- oxygen depletion is measured and is proportional to the amount of glucose present

2. Hexokinase Method
- most specific glucose method ( reference method )
- Plasma collected using heparin, EDTA, fluoride, oxalate or citrate may be used for this test.
- other samples: Urine, CSF, and serous fluids.
- Provodes a high degree of specificity for estimating glucose ( accepted as a reference method )
- Main disadvantage : cost
3. Glucose Dehydroginase Method
- The amount of NADH generated is proportional to the glucose concentration
- provides results in close agreement with hexokinase procedures
-Mutarotase is also added to shorten the time necessary to reach equilibrium.
4. Dextrostics ( cellular strip )
- important in establishing correct insulin amount for the next dose.
- effective in reducing the rate of development of diabetic complication.
5. Glycosylated haemoglobin / Glycohemoglobin/ Glycaated haemoglobin / HbA1C
- Term used to describe the formation of a hemoglobin compound produced when glucose ( a
reducing sugar ) reacts with the amino group of hemoglobin ( a protein )
- Reliable method in monitoring long term glucose.
- Aim of diabetic management is to maintain the blood glucose concentration within or near the
nondiabetic range with a minimal number of fluctuations.
- A glucose/ haemoglobin derivative that increases as blood glucose level increases.
 - Provide an indication of glucose control over the last three months or so
- used to monitor how well the patient has maintained dietary and medication regimens over
the last week or month.
- For every 1% change inHbA1C value, 35 mg/dL is added to plasma glucose
- 2 factors that determine HbA1C :
-Average glucose concentration
- Red blood cell life span
- elevated values suggest that the therapy is not working or that the patient is not complying
with the prescribed program.
- Methods of measurement are grouped into two major categories:
A. Based on charge differences between glucosylated and non-clycosylated hemoglobin
a. cat-ion exchange chromatography
b. electrophoresis
c. Isoelectric focusing
d. HPLC
B. Structural characteristics of glyco groups on haemoglobin
a. Affinity chromatography
b. Immunoassay

6. Fructosamine
- also called glycosylated or glycated albunin/ plasma protein ketoamine
- reflection of short term glucose control ( 2-3 weeks )
- May be useful for monitoring diabetic individuals with chrhonic haemolytic anemias and
haemoglobin variants ( HbS or HbC) – decreased RBC life span
- Should not be measured in cases of low plasma albumin ( < 30 g/L ) – low fructosamine
- Reference values : 205 – 285 umol/L
- Methods: Affinity chromatography, HPLC, and Photometric

Test of Glucose Determination:

1. Fasting blood Sugar (FBS) – NPO ( Non Per Orem ) at least 8 hours before the test.
2. Random Blood Sugar ( RBS) – requested during insulin shock, hyperglycaemic ketonic coma.
3. 2 hr. Post Prandial Blood Sugar ( 2 hr. PPBS)
4. Glycosylated Hemoglobin ( HbA1C)
5. Glucose Tolerance Tests ( GTT) – multiple blood sugar test; to determine how well the body
metabolizes glucose over a given time required.

Kinds of Glucose Tolerance Test :

* Oral Glucose Tolerance Test ( OGTT )
- A solution containing 75 g. of glucose ( glucola ) is administered and a specimen for plasma
glucose measurement is drawn 2 hrs. later
- The adult dose of glucose solution is 75 g. children receive 1.75 g/kg of glucose to a maximum
dose of 75 g.
- If the level is >200 mg/dL and is confirmed on a subsequent day by either an increased RBS or
FBS, the patient is diagnosed with diabetes.
- Janney- Isaacson Method ( Single Dose Method ) – most common} Types of IVGTT
- Exton Rose Method ( Divided Oral Dose or Double Dose Method ) }
Added Plasma glucose after OGTT:
30 min= 30-60 mg /dL above fasting
1-hour= 20-50 mg/dL above fasting
2-hour= 5-15 mg/dL above fasting
3-hour= fasting level or below
Requirement of OGTT:
- The patient should be ambulatory and on a normal to high carbohydrate intake for 3 days
before the test.
- The patient should be fasting for at least 10 hours and not more 16 hours
- The test should be performed in the morning because of the hormonal diurnal effect on
glucose
- Just before tolerance and while the test is in progress, patients should refrain from exercise,
eating, drinking ( except that the patient may drink water ), and smoking

Factors that affect the tolerance results include:

- Medications
Salicylates, diuretics, anticonvulsants, contraceptives, corticosteroid
- Gastrointestinal problems
Malabsorption problems, gastrointestinal surgery, vomiting
- Endocrine dysfunction
Intravenous Glucose Tolerance Test
- Used for diabetes patients with gastrointestinal disorders
- 0.5 g. of glucose/kg body weight ( given within 3 min ) administered intravenously
- Fasting sample is also required
- The first blood collection is after 5 min. of IV glucose

Indications of IVGTT:
- Those who are unable to tolerate a large carbohydrate load
- Those with altered gastric physiology
- Those who had undergone previous operation or surgery
- Those with chronic malabsorption syndrome