INFLUENCE OF AGE AND GENDER ON THE LEVELS OF GLYCOSYLATED HAEMOGLOBIN AMONG NON- DIABETIC NIGERIAN SUBJECTS

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   37˚C   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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