March 15 (LAB) Flashcards
Condensation method
Ortho-toluidine (dubowski method)
Condesation of glucose with witha primary aromatic amine in glacial acetic acid, forming an equilipibrium mixture of a glycosylamine and the corresponding schiff base
Ortho-toluidine
glycosylamine + schiff’s base
Ortho-toluidine
Colormetric glucose oxidase method (saifer gerstenfield method)
Glucose oxidase method
Polarographic glucose oxidase
Glucose oxidase method
Whole blood is __ lower than serum or plasma
11%
leukocytosis (increased WBCs) is up to __ mins
90 mins
is present in the gray tube
which is used for glucose determination
Sodium fluoride
inhibits the glycolytic enzyme
enolase which acts as an anticoagulant
Sodium fluoride
There is also __ which inhibits the
glycolytic enzyme, glyceraldehyde-3-phosphate
dehydrogenase
acetate
Glycolysis decreases serum glucose by approximately
__ (5 to 10 mg/L) in normal
uncentrifuged coagulated blood at room temperature
5% to 7% in 1 hour
Refrigerated (___C)
○ Serum or Plasma: stable up to ___
○ Whole blood: 2mg Na fluoride per mL of whole
blood (48 hrs)
2-8*C, 48 hrs
Refrigerated (___C)
○ Serum or Plasma: stable up to ___
○ Whole blood: 2mg Na fluoride per mL of whole
blood (48 hrs)
2-8*C, 48 hrs
Different lab methods to determine glucose levels in the
sample
○ Chemical method
○ Enzymatic 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
- Alkaline Ferric Reduction Method
a. Hagedorn Jensen
B. Condensation Method
a. Dubowski Method
Principle: reduction of cupric ions to cuprous
ions forming cuprous oxide in hot alkaline
solution by glucose
Alkaline Copper Reduction Method
The glucose reduces cupric ions present in the
alkaline copper reagent to cuprous ions or the
cupric sulfate is converted to cuprous oxide
which reduces the phosphomolybdic acid to
phosphomolybdous acid
Folin Wu Method
Sugars with reducing properties are rising out
of the presence of the potential aldehyde or
keto group called the reducing sugars
● Ex of reducing sugars
Nelson Somogyi Method
The reducing sugars when heated with the
alkaline copper tartrate reduce copper from the
cupric state and cuprous oxide is formed
Nelson Somogyi Method
When cuprous oxide is heated with
arsenomolybdate, there will be reduction of ___ to ___
which has the color ___
molybdic acid to molybdenum, blue
420 nm
Folin Wu method
The copper in oxidation state reacts with
neocuproine forming a complex, this complex is
extracted into a chloroform methanol mixture
giving a yellow or yellow-orange solution
Neocuproine method
Used for detection and quantitation of reducing
substances in body fluids like blood and urine
Benedict’s method
Can be used for identification of glucose in the
urine
Benedict’s method
It has a principle that when the reducing sugars
are mixed with the Benedict’s reagent and
heated, a reduction reaction causes the
benedict’s reagent to change color
Benedic’ts method
Color varies from green to brick red,
rusty brown = depending on the
amount of sugar
Benedic’ts test
Aldolases are easily
oxidized to yield carboxylic acid
Fehling’s test
■ Benedict’s solution contains
the Cu 2+ ___
■ Fehling’s solution contains
the Cu 2+ ___
citrate, tartrate
It involves reduction of yellow ferricyanide to a
colorless ferrocyanide by glucose (inverse
colorimetry)
Alkaline Ferric Reduction Method (Hagedorn Jensen)
Condensation of glucose with primary aromatic
amine in glacial acetic acid, forming an
equilibrium mixture of a glycosylamine and the
corresponding Schiff base
Ortho-toluidine (Dubowski Method)
Acts on glucose but not on other sugars and reducing
substances.
ENZYMATIC METHODS
is used to oxidize a dye compound to commonly
chromogens are used in this type of method.
H2O2
is used to catalyze the second
reaction.
Horseradish peroxidase
Increased levels of __,___,___
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.
uric acid, bilirubin, and ascorbic acid
Measures rate of oxygen consumption which is
proportional to glucose concentration.
Polarographic Glucose Oxidase
Glucose oxidase in the 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 ___,___,___, and ____.
molybdate, iodide, catalase and ethanol
___ can be used to perform the direct
measurement of oxygen by polarographic technique.
Oxygen consumption
More accurate than glucose oxidase method
Hexokinase Method
Hexokinase in the presence of ATP converts glucose to
___
glucose 6 phosphate
___ is added to shorten the time
Mutarotose
___ is touched to a drop of blood and inserted
into the meter which gives a digital reading of the blood
sugar; gives the unit of mmol/L
Dextrostics
An enzyme-impregnated strip used with a small portable
electronic colour-measuring device for convenient
estimation of the blood sugar levels by diabetics.
Dextrostics
used for continuous monitoring of glucose levels in
people with diabetes
Interstitial glucose monitoring device
Laboratory Testing for GLUCOSE
- Random plasma glucose (random blood sugar)
- Fasting plasma glucose (FBS)
- Tolerance test
- HbA1c (glycosylated hemoglobin test)
- Fructosamine
- Urine Microalbumin
- Ketone testing
Random Plasma Glucose
● Formerly random blood sugar (RBS)
● Specimens collected anytime of the day
● Usually done in a glucometer
● NO NORMAL VALUES
○ Bcs the glucose is measured randomly
First step to measure the glucose in the blood is to
convert glucose concentration into ___ or amount or
the current signal
voltage
●___ To determine how well your body metabolizes glucose
over a required period of time
● ___ samples in total
Oral glucose tolerance test, 3
Hemoglobin A is irreversibly glycosylated at
one or both N-terminal valines of the B-chains
of the tetrameric hemoglobin molecule
(International Federation of Chemistry Working
Group on HBA1c)
HbA1c
Is the largest subfraction of normal HBA
involved in diabetic and nondiabetic individuals
HbA1c
It will cause the decreased value of your HBA1c
Hemoglibonpathy
A genetic defect that results in abnormal
structure of one of the globin chains of the
hemoglobin molecule
Hemoglibonpathy
Also known as the glycosylated albumin
Fructosamine
Most useful if the HBA1c is unreliable due to
hemoglobinopathy or hemolysis
Fructosamine
Most widely used to assess short-term (__to__ week)
glycemic control
Fructosamine, 3-6 weeks
Test to detect very small levels of protein (albumin) in
urine
Urine Microalbumin
beta-HBA, acetoacetic acid, and acetone
Ketone
greatly increased in the Keto acidosis
due to the altered rate and elevated levels of
___ in hepatic mitochondria
NADH
In serum acetone, it is indicative of defect in
carbohydrate metabolism when the same
acetone is increased
beta-HBA, acetoacetic acid, and acetone
Used of ferric chloride reacted with
acetoacetic acid to produce a ___ color
Gerhatt’s, Red color
■ Reacts with acetoacetic acid in an
alkaline pH to form a ___ color
■ Urine reagent strip test and Acetest
tablets
Sodium Nitroprusside, Purple Color
To detect either 3-b-hydroxybutyric
acid or acetoacetic acid
3-hydroxybutyrate dehydrogenase
