Biochemistry 2: Glycated Haemoglobin Flashcards
Glucose intolerance
- Impaired glucose tolerance
- Impaired fasting glucose
Measuring glycated proteins
∵ Single fasting blood glucose measurement only indicate patient’s immediate past condition (within hours)
—> not represent true status of blood glucose regulation over an extended period
—> measure glycated protein to establish an accurate index of mean blood glucose concentration
—> glycated haemoglobin = mean glycaemia over 2 months (reflected by t1/2 of RBC)
Glycation = Non-enzymatic addition of sugar residue to amino groups of proteins
Human adult haemoglobin
HbA: α2β2
- HbA0
—> those HbA NOT migrate quickly
—> glycation occurs at site other than the end of β-chain
—> cannot be separated from non-glycated HbA by chromatography based on charge
—> measured by ***affinity chromatography
- Minor haemoglobins (HbA1a, HbA1b, **HbA1c)
—> collectively referred to as **HbA1
—> Fast haemoglobin (migrate more rapidly than HbA in electric field) / Glycohaemoglobin / Glycated haemoglobin: 4-7% of HbA
HbA1 has **carbohydrate attached to **N-terminal amino acid Valine of ***β-chain
2 subtypes of HbA1a:
- HbA1a1: Fructose-1,6-diphosphate
- HbA1a2: Glucose-6-phosphate
HbA1b:
- Pyruvate attached
- probably via ketamine / enamine bond
- **HbA1c (80% of HbA1):
- formed in 2 steps of Non-enzymatic glycation of HbA:
1. Reversible reaction (condensation) between Carbonyl group of **glucose and N-terminal Valine of β-chain —> **Unstable aldimine (Schiff base, pre-HbA1c): directly proportional to blood glucose concentration
2. some Schiff base converted (slow Amadori rearrangement) to HbA1c (Stable ketoamine) during RBC circulation
Methods for determination of HbA1c / HbA1
Separation based on:
- Charge differences
- Ion-exchange chromatography —> Cation exchange chromatography
- High performance liquid chromatography
—> high pressure speeds up analysis compared to by gravity
—> can use smaller samples
—> HbA0 (4 mins), HbA1c (2.7 mins), HbA1b (1.6 mins), HbA1a (1.1 mins) - Electrophoresis
- Isoelectric focusing
- Chemical analysis
- Colorimetry
- Spectrophotometry - Structural differences
- Affinity chromatography
- Immunoassay
Regardless of method, result is expressed as a percentage of total Hb
Many of these assays have acceptable precision when they are properly performed
Ion-exchange chromatography
e.g. ***Cation exchange chromatography:
—> Ions electrostatically bound to the insoluble / chemically inert matrix
—> reversibly replaced by charged solutes in solution
—> pH and [salt] of buffer solution in which target protein is dissolved is chosen
—> the desired protein is preferably bound to the selected ion exchanger
—> e.g. Na displaces HbA1C, only A0 binds
—> ↑ [NaCl] in continuous gradient
—> competes with binding
—> exchanges with the ones bound increasingly strong to resin
Hb has a net +ve charge —> ***weakened by glycation depending on sugar type:
1. HbA0: 4+
- bind most tightly to resin (polymer beads with -ve charged functional group as stationary phase)
—> hardest to be exchanged
—> eluded the last
2. HbA1c: 3+
3. HbA1b: 2+
4. HbA1a: 1+ (less net +ve charged —> weakly bound —> move faster, elute earlier)
All absorb at 415nm
Formation of glycated haemoglobin
Irreversible
Blood levels depends on:
- Life span of RBC (120 days)
- amount of HbA1c / HbA1 represents the integrated values for glucose over the preceding 6-8 weeks —> able to assess glucose control - Blood glucose concentration
- values are free of day-to-day glucose fluctuations, unaffected by exercise / recent food ingestion
—> measurement of HbA1c has been accepted for clinical management of diabetes
***Clinical usefulness of measurement of HbA1c level
False positive:
- HbF > normal / Persistence of HbF
- some ion-exchange based separation cannot separate HbF and HbA1c because they co-migrate and co-elute out of column
- HbF usually elevated on genetic basis, i.e., hereditary persistence of HbF (HbFH)
- Beta-thalassaemia have increased HbF production - Fe deficiency anaemia
- high proportion of old RBC - Polycythaemia / Post-splenectomy
- longer lifespan of RBC - Non-diabetic hyperglycaemia e.g. Cushing’s syndrome
- Increased serum TAG / Bilirubin, Uaemia (Renal failure), Alcoholism, Lead poisoning, Chronic high dose salicylate intake
False negative:
- Haemolytic anaemia / Blood loss
- shortened RBC lifespan - Pregnancy (2nd half)
- fetomaternal transfusion
- increased metabolic demand in 3rd trimester —> high proportion of young red cells in circulation (less exposure to glucose) - Chronic renal failure
- lack of erythropoietin —> lower RBC count - Ingestion of large amount of Vit C/E
- Vit C/ E inhibit Hb glycation by competing with glucose for binding to RBC - Haemoglobinopathy
- HbC/D/S (decreased); HbF/H (increased)
- combination of abnormal haemoglobin + excessive intramedullary haemolysis
***Calculation
Standard (已經知有10% HbA1):
HbA1 absorbance: 0.480
Haemolysate (contain all types of Hb): 0.575
—> R: 0.480/0.575 = 0.835
Patient
HbA1 absorbance: 0.746
Haemolysate (contain all types of Hb): 0.962
—> R: 0.746/0.962 = 1.290
Calculation: 如果0.835即係10%, 咁1.290就係(1.290/0.835)x10% = 15.4%
—> 15.4%係HbA1既percentage!!!
要計算HbA1c:
(0. 838 x 15.4) - 0.732 = 12.2% HbA1c
(0. 838, 0.732係depend on kit, will vary between kits, just a arbitrary number)
Reference values of HbA1c
Normal: 4.2-6.2
Good control: 5.5-6.8
Fair control: 6.8-7.6
Poor control: above 7.6
Alternative to cation-exchange chromatography
Glycation of proteins - Fructosamine:
- Turnover rate of protein: past 15-20 days
- show changed glucose levels earlier than HbA1c
- monitor degree of hyperglycaemia during the previous 2-3 weeks when glycated Hb cannot be used reliably as glycaemic control index
Limitations:
Changes in protein level have effects on proportion of protein that is glycated
- Shortened albumin half-life
- Increased loss of protein
