Title: Total Glycated Haemoglobin
Aim: To determine the total concentration of glycated haemoglobin within an individual in
order to identify the health status of an individual; whether they are healthy, diabetic or
prediabetic, via glycohemoglobin separation and colorimetry assay.
Introduction: Round, biconcave and flattened cells that are responsible for transporting oxygen
throughout the body are identified as red blood cells. These cells lack a nucleus, which provides
the cell with a large surface area to accommodate maximum space for haemoglobin.
Haemoglobin is a red protein that is found within the red blood cells and is the molecule which
allows for the red blood cells to transport oxygen to all cells within the body. It can bind to
oxygen and carbon dioxide molecules hence it is required for the functioning of the red blood
cells. Haemoglobin consists of four protein molecules called globins (α, β, δ chains) that are
associated with four haem molecules. In adults, the normal Hb is made up of 97 - 98%Hb-A,
which has 4 polypeptide chains, 2 alpha (α) globin chains and two beta(β) chains (α2 β2), and 2 -
3% Hb-A2 (α2 δ2). There are four haem groups present on each haemoglobin molecule, where
each haem can bind one oxygen molecule, thus the four haem groups together binds four oxygen
molecules. Red blood cells have a life span of 120 days and during this time, some of the
haemoglobin covalently bonds to glucose forming glycosylated haemoglobin or
glycohaemoglobin (GlyHb) (HbA1 c). Due to the difference in electrophoretic mobilities from
haemoglobin of the complexes, the complexes are referred to as HbA1. Usually, 5-10% of
circulating haemoglobin is HbA1, however, for red blood cells that are 120 days old they contain
10-15%. Glycosylation has very little effect on the oxygen-carrying properties.
Glycosylated haemoglobin is a product from the irreversible reaction of spontaneous
combination of the ammonia (NH2) terminal group of the haemoglobin β chain and the glucose
molecule. There are several glycosylated derivatives, such as A1a1, A1a2, A1b and A1c that
contribute to HbA1. HbA1c is the principal complex that contributes to this and is about 5% of the
circulating Hb. There are methods that are used for determining the total HbA1 and HbA1c.
Glycosylated haemoglobin can be used to determine the average glucose concentration in the
blood. The A1c test is one method that can be used to determine the total Glycosylated
haemoglobin. This test shows the average glucose present in the blood for the past three months
by measuring the amount of haemoglobin with attached glucose and is used to test for diabetes
type 2 and prediabetes. It is expressed in percentage, where the higher the percentage, the higher
the glucose level. Generally, for a normal non-diabetic person, the normal range for haemoglobin
A1c level in the blood is 4% to 5.7%. Levels between 5.7% to 6.4% wold indicate that an
individual is prediabetic, while levels above 6.4% would indicate that an individual has diabetes.
It should be noted that in healthy person 5% of the circulating haemoglobin is glycosylated
whereas in diabetic persons, approximately 30% of the circulating haemoglobin is glycosylated
because of greater blood glucose concentrations. The complete reaction with glucose is non-
enzymatic and irreversible and the GlyHb therefore reflects the average blood glucose
concentration. Thus, the level of GlyHb increases proportionally in patients with uncontrolled
diabetes. The glycosylated glucose levels in the blood is relatively constant thus provides a more
accurate means of testing and monitoring glucose levels in diabetic patients than the glucose
tolerance test. It may also provide a simpler index of control of diabetic persons since it avoids
having to do glucose loading tests. A diabetic has haemoglobin A1c levels of 6.5% and higher. A
diabetic is expected to be unable to lower blood glucose levels due to either poor insulin
response or the inability to produce any insulin. Therefore, since insulin production is not
working appropriately, the more glucose in the blood would result in more glycohaemoglobin
formed. There would be a high level of HbA1c which would lead to the determination of the
diabetic status of a person. In other words, the HbA1c values of persons can be measured and
compared to pre-existing literature values to determine whether they have diabetes, prediabetes
or normal/healthy.
One of the most common method to separate the haemoglobin molecules from the red
blood cells. It is where whole blood is hemolyzed and mixed with a weak binding cation-
exchange resin. The resin binds to all the non-glycosylated haemoglobin molecules present
following the use of a resin separator and the supernatant is isolated with the glycosylated
haemoglobin molecules. This supernatant can be assayed to determine the number of
glycohaemoglobin molecules present, where it can then be calculated as a percentage of the total
haemoglobin which is known as the glycohaemoglobin percentage.
