ABSTRACT
The influence of age and gender on the levels of glycosylated haemoglobin among non- diabetic Nigerians were investigated in this study. Seventy nine non-diabetic individuals volunteered for the study and were grouped into male and female and then into four groups according to age: ≤ 20 years, 21 – 40 years, 41 – 60 years and ≥ 61 years. Fasting blood glucose, 2-hour post-load glucose, packed cell volume and genotype analyses of subjects
were initially determined to ensure that subjects were really non-diabetic and had no glucose metabolic impairment. Subsequently, glycosylated haemoglobin and body mass index were measured. Student’s t-test, Pearson correlation and one-way analysis of variance were used to compare the data which were presented as mean ± standard deviation for continuous variables and statistical significance accepted at p ˂ 0.05. The results obtained showed that glycosylated haemoglobin (HbA1c) increased across the age groups (4.27 ± 0.64, 4.97 ±
0.61, 5.13 ± 0.71, and 5.26 ± 0.49 for age groups ≤ 20, 21 – 40, 41 – 60 and above 60 years respectively).  These increase were found to be significant  (p < 0.05) from each  other. A positive  correlation  was  also  observed  between  age  and  HbA1c  levels.  There  was  no significant (p > 0.05) association between HbA1c and gender. The results did not show any correlation  between  HbA1c  and  gender.  The  study  showed  that  HbA1c  is  positively correlated (p = 0.01) with body mass index (BMI). The mean BMI for men increased with age up to the 41 – 60 year-old age group and then declined, while the average values for women increased across the age groups. Men had the highest mean BMI in the 41 – 60 year- old  age-group,  while  women  had  the  highest  mean  BMI  in  the  age  group  above  60. Generally, women had higher mean BMI than men. The mean values obtained at the different age groups were significant (p < 0.05) compared with each other within each gender except at age group 21- to 40-year old in women. Thus this study showed that age and BMI positively correlated with the levels of HbA1c in normal glucose tolerant individuals whereas gender did not. Consequently,  it may be necessary that their contributions are taking into account when making diagnostic and therapeutic decisions with regard to diabetes care using HbA1c. From this study, an HbA1c range of (4.0 – 5.2) % could be considered as the normal range for individuals below sixty one years while HbA1c level of ≤ 5.27% is suggested for individuals above sixty years. However,  further studies  is required  especially to  investigate  the  non- glycaemic  factors affecting HbA1c levels in normal glucose  tolerant populations so as to really understand the actual role glycosylated haemoglobin play in diabetes management and diagnosis.
CHAPTER ONE
INTRODUCTION
Studies on chronic complications of diabetes established the role of glycosylated haemoglobin, HbA1c, as a marker of evaluation of long-term glycaemic control, glycaemic risk and prediction of diabetic complications, and as a screening tool for the diagnosis of diabetes (Nathan et al., 1984; Manjunathaet al., 2011). It is considered as one of the best achievements in the history ofdiabetes mellitus.HbA1c is a specific haemoglobin produced by a two-stage non-enzymatic attachment of glucose molecule to the N-terminal valine of the ß-chains of the haemoglobin molecule. Once formed, the HbA1c remains throughout the life span of the erythrocyte. Hence, it is primarily measured to identify the average plasma glucose concentration over the previous 2-3months. If other factors that may affect the HbA1c such as haemoglobinopathies, anaemia, etc, are kept constant, normal levels of plasma glucose will produce a normal amount of HbA1c. However, as the average plasma glucose increases (as in diabetes), the fraction of HbA1c increases in a predictable way. Hence a marker of glycaemic control. HbA1c measurement is the most preferred test by clinicians and patients for monitoring glycaemia. This is because, its measurement has substantially less biologic variability, needing no fasting or timed samples and it is a better index of overall glycaemic exposure and risk for long-term complications (Kim et al., 2010). However, certain studies have shown that the HbA1c levels are altered by various other coexisting factors, along with diabetes. Such factors are age, gender and body mass index, among others. According to the available research reports in this regard, there seems to be no agreement on whether age and gender have significant effect on HbA1c values. Studies conducted by Arnetzet al. (1982), Kilpatrick et al. (1996) and Yates and Laing. (2002), indicated that there is positive correlation between age and HbA1c whereas those by Kabadi (1998), Wiener and Roberts (1999) and Valleeet al. (2004), showed no significant relationship between age and HbA1c. There are also very scanty reports on the influence of gender on HbA1c values (Yanget al., 1997). To these effect, it is pertinent to ensure that these factors are well accounted for, as to what degree they affect HbA1c values. This is aimed at ensuring that clinical decisions using HbA1c are accurate.
1.1 Glycosylated Haemoglobin
1.1.1 Historical Perspective of Glycosylated Haemoglobin
It was Maillard(1912) who described the reaction of reducing sugars with amino acids which resulted in a stable ketoamine adduct formation.Allen et al. (1958) demonstrated that normal adult haemoglobin could be separated chromatographically on a cation exchange resin into a main component, accounting for more than 90% of the haemoglobin and three negatively charged minor components, which they designated as HbA1a, HbA1b and HbA1c, and collectively known as HbA1. However, they did not demonstrate the nature of these fractions. Later, Huisman and Dozy (1962) observed a two- to three-fold rise in the HbA1 fraction in four diabetic patients who were being treated with tolbutamide. After five years of documenting this unusual finding, HbA1c was described as “an abnormal haemoglobin in diabetes” byRahbar (1968). In the process of screening for the presence of abnormal haemoglobins in persons with diabetes mellitus, Rahbar (1968)demonstrated that a normal fraction of haemoglobin A1 was found in slightly higher amounts in their blood. Further investigations in patients with poorly controlleddiabetes mellitus resulted in the finding of a “diabetic haemoglobin component” that was reported in
1968. Later on, several structural studies were carried out in patients with diabetes mellitus and that so called “abnormal haemoglobin” was found to be identical to HbA1c fraction. Trivelliet al (1971) found a two-fold increase of HbA1c over values observed in non-diabetic subjects. Thus, by the mid-1970s, it was clear that HbA1 and HbA1c are elevated in humans with diabetes mellitus. Furthermore, this “abnormal haemoglobin” was found to be increased in direct proportion to the degree of hyperglycaemia (Koeniget al., 1976; Gabbayet al, 1977; Gonenet al.,
1977).Significance of HbA1c in monitoring blood glucose control in patients with diabetes mellitus was then proposed by Koenig in 1976. By the 1980s, HbA1c evolved as a better index of glycaemic control in clinical trials. (Kahnand Fonesca,2008; Gallagheret al., 2009) This, along with the other method that emerged by that time, namely, self-monitoring of blood glucose (SMBG),greatly enhanced the achievement of glycaemic control. Regular SMBG had a positive effect on improving glycaemia especially in individuals treated with insulin. SMBG reflects the immediate
plasma glucose levels, whereas HbA1c measures long-term glycaemic control (Saudeket al., 2006). After the data from The Diabetes Control and Complications Trial (DCCT) and UK Prospective Diabetes Study (UKPDS) became available, HbA1c has become an integral part of monitoring the glycaemic control indiabetes mellitus. The American Diabetic Association (ADA) recommendation of the goal of achieving aHbA1c level of less than 7 as evidence of satisfactory glycaemic control in patients treated for diabetes mellitus revolutionized the significance of HbA1c as a diagnostic test for assessing the adequacy of glycaemic control (Reddyet al., 2012).
1.1.2 Formation of Glycosylated Haemoglobin
The normal lifespan of an erythrocyte is 120 days. In the presence of hyperglycaemia, as the erythrocyte circulates, the N-terminal valine residues of β chains of haemoglobin gradually undergoes non-enzymaticglycosylation (Dods, 2010). The HbA1c thus formed, constitutes about 60% to 80% of the total glycosylated haemoglobin. The number “1c” represents the order of haemoglobin detection on chromatography. The glycosylation of haemoglobin occurs over the entire 120-day life span of erythrocyte (Bunnet al., 1976). The glycosylation process follows a peculiar pattern. The initial 25% of glycosylation occurs in the first 1 to 2 months of the life span of the erythrocyte, another 25% occurs in the next month. The remaining HbA1c is formed during the senescence of the erythrocyte corresponding to the period of 1-2 months prior to measurement. Consequently, older, senescent erythrocytes have more HbA1c than the reticulocytes. Furthermore, this forms the rationale behind the assumption that HbA1c represents average glycaemia over the last 6 to 8 weeks (Bunnet al., 1976; Goldsteinet al., 1986; Taharaand Shima, 1995).
Due  to  the  post-translational,  post-secretory  glycosylation,  an  unstable  aldimine- Schiff  base  is  formed,  which  is  a  reversible  process.  This  slowly  undergoes  an Amadori rearrangement  to form a stable irreversible  ketoamine  linkage(see  fig.1), which  is  an  advanced  glycosylation  end-product.    The  reaction  is  essentially irreversible,  meaning that once the haemoglobin  molecule  becomes glycosylated  it remains so until the end of its lifespan. While  the senescent erythrocytes lose their ability  to  metabolize  glucose,they  remain  permeable  to  glucose.     Thus,  the intracellular  glucose  concentrations  reflect the extracellular  glucose concentrations. The clinical  assay  of HbA1c  measures  total glycosylation  of haemoglobin:  i.e., it measures glycosylation of haemoglobin in both less glycosylated young erythrocytes as well as more glycosylated senescent erythrocytes (Reddyet al., 2012).
Fig.1: Reactions leading to the formation of haemoglobin HbA1c (Peacock, 1984).
Glycosylated HaemoglobinFormation is Non-Enzymatic
The formation of HbA1c is actually different from other forms of protein glycosylation in that it is non-enzymatic. This unique property is part of what confirms its clinical use in the monitor of glycaemic control. The non-enzymatic formation nature of HbA1c was concretized by the discovery that it could be formed by incubating either whole blood or purified haemoglobin in the presence of glucose
at 37˚C (Flückiger andWinterhalter, 1976). The rate of absorption of (14C) labelled
glucose into haemoglobin to form HbA1c was the same whether purified or crude haemoglobin was used, suggesting that the action was not mediated by a red cell enzyme. Studies of the kinetics of conversion of HbA to HbA1c in vivo have lent further weight to the theory that the process is non-enzymatic (Bunn et al., 1976). Here, injections of 59Fe-bound transferrin into a normal volunteer enabled specific radioactivity in the haemoglobin to be measured over a period of 100 days. The activity in the major part of adult haemoglobin HbA, designated as HbA0, reached a peak at 15 days and was then relatively constant for the next 80 days, consistent with normal erythropoiesis and a cell viability of approximately 100 days. The specific activities of HbA1a, HbA1b and HbA1c increased only gradually however, and continued to rise throughout the 100 days, exceeding the activity of HbA0 from approximately 60 days onwards (Bunn et al., 1976). Several other observations were consistent with HbA1c being formed throughout the life span of the red cell as a post-
synthetic modification of HbA. When human reticulocytes or marrow were incubated with radioactively labelled amino acids, the specific activity of HbA1c was much lower than that of HbA0. Young red cells, isolated by density gradient, have lower levels of HbA1c than older red cells(Fitzgibbonset al., 1976). The amino terminus of the beta chain is not the only site of formation of glucose adducts with haemoglobin. The amino terminus of the alpha chain is similarly modified, although at an eight- to ten-fold lower rate, both in vivo and in vitro (Shapiroet al., 1980). Moreover, the modification at that site has an insufficient effect on the charge of the protein to permit separation by ion exchange chromatography in the same way as with HbA1c. There are a number of epsilon amino groups of lysine throughout the alpha and beta chains, but due to the conformational structure of the haemoglobin molecule, more reactive amino groups may be less accessible to free glucose (Kennedy, 1992).
1.1.3 Factors Affecting Glycosylated Haemoglobin Levels
Some factors are known to affect HbA1c levels. These factors are grouped into those leading to falsely elevated HbA1c levels and those causing falsely low HbA1c levels.
1.1.3.1 Factors leading to falsely low Glycosylated Haemoglobin levels
Falsely low HbA1c is seen mainly in conditions of high red cell turnover, such as haemoglobinopathies (including variant haemoglobins, thalassaemias, and sickle cell disease), glucose-6-phosphate dehydrogenase deficiency, treatment of anaemia with iron or erythropoietin and autoimmune haemolyticanaemia. Recent blood loss and blood transfusion result in greater proportion of reticulocytes or transfused red cells in blood stream thereby reducing the average age of red cells (Horton and Huisman,
1965; Bernstein, 1980; Starkmanet al., 1983). Patients of chronic kidney disease on
dialysis and chronic liver failure may also have less than expected level of HbA1c.In chronic renal failure patients, haemolysis and sometimes the gastrointestinal loss of blood lowers the HbA1c levels. Hence, the effect of urea on HbA1c levels varies (Nitin, 2010). All these conditions result in shortened average age of erythrocytes, resulting in decreased exposure time of haemoglobin to glucose and therefore less percentage of HbA1c. Falsely low levels of HbA1c are also observed because glycosylated haemoglobin variant is separated from HbA1c so it is excluded from calculation (Syed and Khan, 2011).
1.1.3.2 Factors Leading to Elevated Levels of Glycosylated Haemoglobin
Anaemia of iron, folic acid, and vitamin B12 deficiency could result in falsely high levels of HbA1c. Haemoglobin variants eg, Hb Raleigh, Hb Graz and persistence of foetal haemoglobin or rise in HbF during pregnancy may also yield falsely high HbA1c levels (Paiseyet al., 1984)). The possible explanation is that HbF is separated from haemoglobin A and therefore, the proportion of HbA1c increases. Urea reacts with haemoglobin molecules at the same site as does glucose therefore HbA1c and carbamylatedHb have the same isoelectric point and assayed together and results in falsely high percentage of HbA1c in uraemic patients. Alcoholism and hyperbilirubinaemia are also said to falsely increase HbA1c (Flückigeret al.,1981). Iron Deficiency Anaemia
Iron deficiency anaemia is associated with higher HbA1c and higher fructosamine in
both diabetic and non-diabetic individuals (Sundaramet al.,2007). Consistent with these observations, iron replacement therapy lowers both HbA1c and fructosamine concentration in diabetic and non-diabetic individuals, (Tarimet al., 1999; Cobanet al.,2004; Sundaramet al.,2007). HbA1c, but not glycosylated albumin, is increased in late pregnancy in non-diabetic individuals owing to iron deficiency, (Hashimoto et al.,1995). Insight into the mechanism was recently obtained by observation that malondialdehyde, (a marker of oxidative stress), which is increased in patients with iron deficiency anaemia (Sundaramet al.,2007), enhances the glycosylation of haemoglobin (Selvarajet al.,2006). Thus, alternative measure of glycaemic assessment (e.g. glucose monitoring) is advised pending the successful treatment of the anaemia.
1.1.4Labile Glycosylated Haemoglobin
Svendsenet al. (1979), demonstrated that the short- term (6–12 hours) exposure of red blood cells to high glucose concentrations, both in vivo and in vitro, led to significant increases in the glycosylated haemoglobin, as measured by ion exchange chromatography. At the same time, it was demonstrated by Goldstein et al (1980) that HbA1c measured by high performance liquid chromatography (HPLC) increased two hours after a standard breakfast, that the increase in HbA1c correlated closely with the
plasma glucose increase, and that incubating the red cell in 0.9% saline for five hours at 37ËšC before HbA1c assay eliminated the post-prandial increment. This phenomenon is due to the unstable Schiff base or aldimine (also known as labile HbA1c) formed as an intermediate step in the glycosylation reaction, and it may be a potential source of error in any assay that relies on the effect of glycosylation on the charge of the molecule. In order for the original concept of glycosylated haemoglobin as an index of long-term integrated glycaemia to hold well, the labile fraction should be removed before such an assay is performed, and this can be achieved by saline incubation, dialysis or other chemical methods (Nathanet al., 1982). It is clear that in some patients, the perception of glycaemic control, as reflected by glycosylated haemoglobin, could be altered (Kennedy, 1985). To eliminate this fraction, the chromatographic kits now contain an additive in their haemolytic reagent that eliminates the labile fraction.
1.1.5 Methods of Measuring Glycosylated Haemoglobin
The methods of HbA1c measurements can be divided into two groups: (i) those that depend on the effect of glycosylation on the charge of the molecule and, (ii) those that depend on identifying a specific property of the ketoamine linkage in glycosylated haemoglobin. Methods that depend on the effect of glycosylation on the charge of the molecule, measuresHbA1c or HbA1, but not the ‘total’ glycosylated haemoglobin, i.e. HbA1, plus haemoglobin glycosylated at sites other than the amino terminal valine of the beta chain.
1.1.5.1 Methods that are Dependent on the Charge of the Molecule
Ion Exchange Chromatography
This has been the most widely used technique. The results are expressed as the percentage of total haemoglobin. The original method utilized macrocolumns and enabled the separation of the individual fractions HbA1a, HbA1b and HbA1c, which being more negatively charged than HbA0, elute before HbA0.The first eluted glycosylated haemoglobin and the second unmodified haemoglobin, the concentrations of each being measured by spectrophotometry (Trivelliet al., 1971). However, this is a tedious process that requires large quantities of buffer and cyanides. The pH is critical, with small changes affecting the degree of separation of the minor haemoglobins. The minicolumn system, which has now largely replaced the above system, (Kynoch and Lehmann, 1977; Welch and Boucher, 1978) has the
advantage of speed and ease of handling, and is available in the form of a kit, as repacked columns with prepared buffers, standards and additives to eliminate labile adducts. However, with the minicolumn technique, the glycosylated haemoglobin cannot be measured as separate fractions but as HbA1. This separation is influenced by temperature; for each 1˚C rise in temperature, HbA1 increases by 0.25% (Kortlandtet al., 1985). Therefore, a constant temperature has to be maintained. Another problem is the presence of variant haemoglobins like foetal haemoglobin (HbF), which co-elutes with HbA1 and gives falsely high readings, whereas haemoglobin C (HbC) and sickle cell haemoglobin (HbS) co-elute with HbA and lead to the underestimation of HbA1 (Eberentz-Lhommeet al., 1984). Hence, where there is a prevalence of variant haemoglobins, this technique should be used with caution. Labile haemoglobin can also lead to high HbA1 and should be eliminated before assay. In most assays, the range of HbA1 in non-diabetic subjects is 5%–9%, with the levels in diabetic patients ranging up to approximately 20%. The coefficient of variation is usually 2%–3% for the same day analysis, while the inter-assay variation is 4%–5% (Welch and Boucher, 1978; Kortlandtet al., 1985).
High Performance Liquid Chromatography
For HPLC, the principles of measurement of HbA1c and HbA1 are the same as for ion exchange chromatography, but the use of high flow pressures and finely divided resins results in a more constant flow rate, as well as a faster and more accurate separation (Goldsteinet al., 1980). However, this is an expensive method.
Isoelectric Focusing
In this technique, haemolysate is applied to a thin-layer polyacrylamide gel containing an ampholyte with a pH level of 6–8, followed by the application of a suitable voltage to separate the haemoglobin fractions, and finally quantification by high resolution microdensitometer (Spiceret al., 1978; Simon and Cuan, 1982). Despite the fact that the difference in isoelectric points between HbA1c and HbA0 is only 0.02 pH units, accurate separation can be achieved. This method has the advantage of having the HbF, HbC and HbS migrate separately. The inter-assay variation is 6.9%–12.6%, which is higher than that of other techniques.Because of the high resolution which can be achieved, isoelectric focusing is valuable as a research method. Commercial equipment is available and is particularly useful for identifying varianthaemoglobins which may be eluted together with HbAl inmicrocolumn methods. Another advantage
is that stable HbA1c is readily separated from the intermediate Schiff base
(Sticklandet al., 1982).
Agar Gel Electrophoresis
In this technique, haemolysate is applied to the agar gel at the anodic site, and after electrophoresis with a citrate buffer at 60V for 40 minutes, HbA1 is located cathodic to HbA0 and is then quantified by scanning densitometry at 420 nm after the gel has been fixed by heat drying for 20 minutes (Menardet al., 1980; Thorntonet al., 1981). It is also essential to eliminate the labile component here. HbC and HbS migrate to points anodic to HbA and do not interfere with its estimation, but HbF migrates to the same point as HbA1. The intra-assay variation is 1.6%–7.3%, while the inter-assay variation is 2.6%–7.3%.
1.1.5.2 Methods Dependent on Detecting aKetoamine Linkage
WeakAcid Hydrolysis
This is one of the oldest methods, where glycosylated haemoglobin is hydrolyzed by a weak acid and the amount of 5-hydroxymethyl furfural (5-HMF) released is quantified colourimetrically after reaction with thiobarbituric acid. This is an inexpensive method, but has some disadvantages. 5-HMF is destroyed as it is being released and its production is non-stoichiometric. Glucose itself interferes with the colour formation in proportion to its concentration, and the hydrolysis step lasts for several hours. To overcome these disadvantages, scrupulous adherence to the rigid assay conditions is required. By performing the hydrolysis in an autoclave at increased temperatures and pressures, the yield of 5-HMF is enhanced and is more constant over a much shorter period of time. The use of fructose or glycosylated haemoglobin standards helps to correct the variation in hydrolysis between assays. These precautions and modifications lead to an intra- and inter-assay coefficient of variation of less than 2% and 3%, respectively, as well as shorten the procedure to less than two hours. The advantages of this method are its ability to detect glycosylation at all sites and non- interference from the labile fraction or haemoglobin variants.
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INFLUENCE OF AGE AND GENDER ON THE LEVELS OF GLYCOSYLATED HAEMOGLOBIN AMONG NON- DIABETIC NIGERIAN SUBJECTS>
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