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Clinical diagnostic biochemistry - 3
Dr. Maha Al-Sedik2015
CLS 334
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Glycation: is the non-enzymatic addition of a sugar residue to amino
groups of proteins.
Glycated Hemoglobin
Formation of GHb ( glycated hemoglobin ) is essentially
irreversible, and the concentration in the blood depends on:
1. Lifespan of the red blood cell (average 120 days).
2. The blood glucose concentration.
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GLYCATED PROTEINS
Measurement of glycated proteins, primarily GHb, is effective
in monitoring long term glucose control in people with
diabetes mellitus.
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Patients with hemolytic disease or other conditions with
shortened red blood cell survival exhibit a substantial reduction in
GHb. Similarly, individuals with recent significant blood loss
have falsely low values owing to a higher fraction of young
erythrocytes.
Normally , Glycated Hb (HbA1C) = 4.5 – 8 % of total Hb.
In D.M. may reach 30 %
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When is Glycated Albmin measured?
Glycated albumin is measured when diabetes therapy is initiated to
determine medication regimens and doses and to assess overall
therapy efficacy.
What's Glycated Albumin (GA)?
Glycation is the bonding of a sugar molecule, such as glucose, to a
protein molecule, such as albumin OR non enzymatic addition of
sugar residue to albumin.
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DETERMINATION OF GLUCOSE IN BODY
FLUIDS
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A number of methods are used to measure glucose in blood, serum, plasma, and urine
Spacemen collection and storage:
• In individuals with a normal hematocrit, fasting whole-blood
glucose concentration is approximately 10% to 12% lower than
plasma glucose.
• In most clinical laboratories, plasma or serum is used for most
glucose determinations.
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• During fasting, capillary blood glucose concentration is only 2
to 5 mg/dL higher than that of venous blood.
• After eating, however, capillary blood glucose concentrations
are 20% to 25% higher than the concentrations in
concurrently drawn venous blood samples.'‘
• Glycolysis decreases serum glucose by approximately 5% to 7%
in 1 hour (5 to 10 mg/dL) in normal un-centrifuged coagulated
blood at room temperature. The rate of in vitro glycolysis is
higher in the presence of Leukocytosis or bacterial
contamination.
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• In separated, non hemolyzed sterile serum, the glucose
concentration is generally stable as long as 8 hours at 25 oC and
up to 72 hours at 4 "C.
• Glycolysis is inhibited and glucose stabilized for as long as
3 days at room temperature by adding sodium fluoride (NaF).
Why we use sodium floride tube in blood
sample used for glucose?
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• Fluoride is also a weak anticoagulant because it binds calcium;
however, clotting may occur after several hours, and it is therefore
advisable to use a combined flouride-oxalate mixture
( antiglycolysis and anti coagulant).
• Fluoride ions in high concentration inhibit the activity of urease
and certain other enzymes; consequently the specimens may be
unsuitable for determination of urea in procedures that require
urease and for direct assay of some serum enzymes.
• No need for flouride tubes , if glucose is measured within 60
minutes of blood collection.
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• CSF may be contaminated with bacteria or other cells and should
be analyzed for glucose immediately.
• If a delay in measurement is unavoidable, the sample should be
centrifuged and stored at 4 "C or -20°C.
CSF
In 24-hour collections of urine, glucose may be preserved by adding
5 mL of glacial acetic acid to the container before starting the
collection.
With this approach, the final pH of the urine is usually between 4
and 5, which inhibits bacterial activity.
24 – hour urine collection
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1-Hexokinase method:
Measurement of Glucose in Blood
NADPH is measured at
340
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2- Glucose Oxidase Method : Glucose oxidase catalyses the oxidation of glucose to gluconic acid
and hydrogen peroxide.
This H2O2 is broken down to water and oxygen by a peroxidase in
the presence of an oxygen acceptor which itself is converted to a
coloured compound, the amount of which can be measured
colorimetrically. This method is used in various autoanalyzers.
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Gluconic acid
500 nm
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Glucose oxidase is highly specific for B -D-glucose. Because 36%
and 64% of glucose in solution are in the a- and B -forms,
respectively, complete reaction requires mutarotation of the a-
to B –form.
Some commercial preparations of glucose oxidase contain an
enzyme, mutarotase, that accelerates this reaction.
Otherwise, extended incubation time allows spontaneous
conversion.
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Various substances, such as uric acid, ascorbic acid, bilirubin
and hemoglobin inhibit the reaction (presumably by
competing with the chromogen for H202), producing lower
values.
Calibrators and unknowns should be simultaneously analyzed
under conditions in which the rate of oxidation is proportional
to the glucose concentration.
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Glucose oxidase methods are suitable for measurement of
glucose in CSF.
Urine contains high concentrations of substances that interfere
with the peroxidase reaction (such as uric acid), producing
falsely low results.
The glucose oxidase method therefore should not be used for
urine.
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3- Glucose dehydrogenase methods:
GLUCOSE + NAD
Gluconolactone + NADH2
Glucose dehydrogenase
THE BEST METHOD IN URINE
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MEASUREMENT OF GLUCOSE IN URINE :
Qualitative method:
By Bendect reagent ( cupric complexed to citrate in alkaline solution
) reducing substances convert cupric ion complexed to cuprous ions
forming yellow cuprous hydroxide or red cuprous oxide).
Quantitative method: Using urine test strips.
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GLUCOSE
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MEASUREMENT OF KETON BODIES
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Urine
Rotheras test:For both acetone and acetoacetic acid in which sodium
nitroprusside in alkaline solution decompose into strong oxidising
agents and in presence of acetone or acetoacetic acid , It gives a
rose purple color.
Gerhardts test : for acetoacetic acid , careful addition of ferric
chloride solution 10 % drop by drop , It leads to percipitation of
phosphate and formation of red color .
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Harts test: for B hydroxy buteric acid , performed by boiling urine
with water then by addition of H2O2. B hydroxy buteric acid is
transformed to acetone to be completed as Rothera test.
Detection of keton bodies by ketostix.
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Q. Explain patient with sever ketoacidosis but
when you perform urine tests for keton
bodies , it gives negative results?
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Because most common methods used in keton body
detection in urine do not detect B hydroxy buterate .
Although it is the most common type in urine, in sever
diabetes ratio between B- hydroxy buterate :
acetoacetate in blood is 6 : 1 .
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Reference: Burtis and Ashwood Saunders, Teitz fundamentals of Clinical Chemistry, 4th edition, 2000.
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THANK YOU