Clinical diagnostic biochemistry - 3

Dr. Maha Al-Sedik2015

CLS 334

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.

GLYCATED PROTEINS

Measurement of glycated proteins, primarily GHb, is effective

in monitoring long term glucose control in people with

diabetes mellitus.

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 %

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.

DETERMINATION OF GLUCOSE IN BODY

FLUIDS

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.

• 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.

• 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?

• 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.

• 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

1-Hexokinase method:

Measurement of Glucose in Blood

NADPH is measured at

340

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.

Gluconic acid

500 nm

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.

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.

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.

3- Glucose dehydrogenase methods:

GLUCOSE + NAD

Gluconolactone + NADH2

Glucose dehydrogenase

THE BEST METHOD IN URINE

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.

GLUCOSE

MEASUREMENT OF KETON BODIES

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 .

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.

Q. Explain patient with sever ketoacidosis but

when you perform urine tests for keton

bodies , it gives negative results?

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 .

Reference: Burtis and Ashwood Saunders, Teitz fundamentals of Clinical Chemistry, 4th edition, 2000.

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