Clinical diagnostic biochemistry - 3 Dr. Maha Al-Sedik 2015 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 o C 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.