UNIVERSITY OF BUEA

FACULTY OF HEALTH DEPARTMENT OF MEDICAL

SCIENCES LABORATORY SCIENCES

THE RELATIOINSHIP BETWEEN SERUM FERRITIN LEVELS AND

GLYCEMIC CONTROL IN TYPE 2 DIABETIC PATIENTS
ATTENDING THE BUEA REGIONAL HOSPITAL

BY:
BERINYUY BEDE
(HS20P168)

A Thesis Submitted to the Department of Medical laboratory science,

Faculty of Health Sciences of the University of Buea in
Partial Fulfillment for the Requirements for
the Award of Master of Science
(M.Sc) Degree in Chemical
Pathology.

SUPERVISOR CO-SUPERVISOR

Prof. ASSOB CLEMENT Dr. ACHIDI ADUNI UFUAN

JUNE, 2022

i
 CERTIFICATION

This is to certify that the thesis entitled ‘THE RELATIOINSHIP BETWEEN FERRITIN
LEVELS AND GLYCEMIC CONTROL IN TYPE 2 DIABETIC PATIENTS
ATTENDING THE BUEA REGIONAL HOSPITALis the original work of BERINYUY
BEDE (HS20P168) submitted to the Department of Medical Laboratory Sciences, Faculty of
Health Science, University of Buea in partial fulfillment of the requirements for the award of
a Master of Science Degree in Chemical Pathology under the supervision of:

Supervisor

ASSOB JULES CLEMENT

Professor of Chemical Pathology

SIGN……….………………..

DATE………………………..

Co-Supervisor

ACHIDI ADUNI UFUAN

Associate Professor of Biochemistry

SIGN……….…………………….DATE…………………………….

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 ACKNOWLEDGEMENTS

My profound gratitude goes to my supervisor Prof Assob Jules Clement, co-supervisor Dr.

AchidiAduniUfuan for their guidance and supervision which led to the successful completion

of this work. I also wish to appreciate the staff of the Department of Medical Laboratory

Sciences for the knowledge imparted in me throughout this program. Special thanks to my

family for their constant support especially my grandmother Ma Emily. I am thankful to my

brothers Tem Luiz and Bwemba Paul, my classmates and friends for their encouragement and

support in all aspects.

I will also like to say a big thank you to the entire staff of the diabetic unit at Buea Regional

Hospital for their warm reception and assistance in recruiting study participants. Special

thanks to Mr. Richard for his assistance in carrying out all the laboratory procedures.

Above all, I give all the glory to God Almighty for making this work a success.

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 TABLE OF CONTENTS
CERTIFICATION.......................................................................................................................................i
ACKNOWLEDGEMENTS..........................................................................................................................ii
LIST OF ABBREVIATIONS.......................................................................................................................vi
LIST OF FIGURES...................................................................................................................................vii
LIST OF TABLES....................................................................................................................................viii
ABSTRACT.............................................................................................................................................ix
CHAPTER ONE: INTRODUCTION.............................................................................................................1
1.1 Background......................................................................................................................................1
1.2) Rationale........................................................................................................................................3
1.3) Hypothesis......................................................................................................................................4
1.4) Research Question..........................................................................................................................4
1.5) Main objective................................................................................................................................4
1.6) Specific Objectives..........................................................................................................................4
CHAPTER TWO: LITERATURE REVIEW....................................................................................................5
2.1 Iron Overview..................................................................................................................................5
2.2 Iron Homeostasis.............................................................................................................................6
2.2.1) Iron Metabolism..........................................................................................................................7
2.2.2) Metabolic Functions of Iron........................................................................................................9
2.3) Etiology of Diabetes......................................................................................................................11
2.4) Etiological Classification of Diabetes Mellitus...............................................................................11
2.5) Epidemiology of Diabetes.............................................................................................................12
2.6) Pathophysiology of Diabetes........................................................................................................13
2.7) Diabetic Complications and Management Paradigm for Diabetes...............................................15
2.7.1 Diabetic Complications...............................................................................................................15
2.7.1.1 Acute Diabetic Complications..................................................................................................15
2.7.1.2 Chronic Diabetic Complications...............................................................................................16
2.7.2 Management Paradigm for Diabetes..........................................................................................19
2.8) Diagnosis and Monitoring of Type 2 Diabetes..............................................................................20
I. Diagnosis and of Type 2 Diabetes Mellitus.......................................................................................20
II. Monitoring of Type 2 Diabetes Mellitus..........................................................................................23
CHAPTER THREE: MATERIALS AND METHODS.....................................................................................26
3.1 Study Design.................................................................................................................................26
3.2 Study Area.....................................................................................................................................26

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3.3 Inclusion and Exclusion Criteria....................................................................................................26
3.4 Ethical considerations....................................................................................................................27
3.5 Sample Size Calculation................................................................................................................27
3.6 Sampling Method..........................................................................................................................28
3.7 Sample Collection..........................................................................................................................28
3.8 Laboratory Procedures..................................................................................................................28
3.9 Study variables and Data Management.........................................................................................32
3.9.1 Study variables............................................................................................................................32
3.9.2 Data Management......................................................................................................................32
CHAPTER FOUR: RESULTS....................................................................................................................32
4.1 General characteristics of the study population............................................................................33
Table 1: Socio-demographic characteristics of study population........................................................33
4.2 Age Distribution of Diabetic patients in the Study.........................................................................34
4.3 BMI Distribution in the Study Population......................................................................................35
4.4 Correlation between Ferritin and Markers of Glycemic control....................................................36
4.4.1 Correlation between Ferritin and FBG........................................................................................36
4.4.2 Correlation between Ferritin and HbA1C....................................................................................37
4.5.1 Association between ferritin and age.........................................................................................37
4.5.2 Association between Ferritin and Gender..................................................................................38
4.6 Associations between glycemic control and Socio- Demographic Factors.....................................39
4.6.1 Association between HbA1C and Age.........................................................................................39
4.6.2 Association between HbA1C and Gender...................................................................................39
4.6.3 Association between HbA1C and BMI........................................................................................40
4.7 Indices of Glycemic Control and Ferritin in Study population........................................................40
CHAPTER FIVE: DISCUSSION, CONCLUSION AND RECOMMENDATION...............................................41
5.1 DISCUSSION...................................................................................................................................41
5.1.1 Findings and Comparison with other studies..............................................................................41
5.1.2 Influence of ferritin imbalance on FBG and HbA1C....................................................................43
5.1.3 Limitations of this study..............................................................................................................43
5.2 Conclusion.....................................................................................................................................44
5.3 Recommendation and projection for further studies....................................................................44
REFERENCES........................................................................................................................................45
APPENDIX............................................................................................................................................51
CONSENT FORM..................................................................................................................................51

iv
QUESTIONNAIRE..................................................................................................................................52
Authorisations to carryout research....................................................................................................54

v
 LIST OF ABBREVIATIONS

CVD Cardiovascular Disease

DNA Deoxyribonucleic Acid

DM Diabetes Mellitus

Dcytb Duodenal Ferric Reductase

DMT1 Divalent Metal Transporter

DPP-4 Dipeptidyl Peptidase-4

Fe Iron

GLP-1 Glucagon Like Peptide

GSIS Glucose Stimulated Insulin Secretion

HbA1c Glycated Hemoglobin

ID Iron Deficiency

IDA Iron Deficiency Anemia

MHC-1 Major Histocompatibility Complex

WHO World Health Organisation

SLGT2 Sodium-Glucose Cotransporter 2

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 LIST OF FIGURES

Figure 1: Illustration of iron homeostasis ...............................................................................13

Figure 2: Fe-S clusters in the electron transport chain helps transport electron to molecular

O2 enabling proteins to carry out their function in energy production ...................................16

Figure 3: Worldwide Prevalence of Diabetes ........................................................................20

Figure 4: Pathophysiology of type 2 diabetes .........................................................................22

Figure 5:  Major Microvascular and macrovascular complications associated with diabetes

mellitus…………………………………………………………………………………...….26

Figure 6: Management paradigm for type 2 diabetes ..............................................................28

Figure 7: Age distribution of study participants………………………………………..........34

Figure 8: BMI distributions of study participants....................................................................35

Figure 9: Correlation between serum ferritin and FBG...........................................................36

Figure 10: Correlation between serum ferritin and HbA1C4.5 Associations between Ferritin

and Socio- Demographic Factors.............................................................................................37

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 LIST OF TABLES

Table 1: Characteristics of study population............................................................................33

Table 2: Biochemical and Anthropometric Measurements of Study Population.....................35

Table 3 : Association between Ferritin and Age......................................................................38

Table 4: Association between Ferritin and Gender..................................................................38

Table 5: Association between HbA1C and Age......................................................................40

Table 6: Association between HbA1C and Gender.................................................................40

Table 7: Association between HbA1C and BMI......................................................................41

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 ABSTRACT

Background: Diabetes mellitus is a metabolic disorder that impairs the body's glucose
utilization. It's one of the most common chronic diseases, defined by high blood sugar levels that
gradually affectbody systems. Diabetes affects life expectancy by a decade on the average,
which is mostly due to macrovascular disease. Type 1 diabetes is caused by lack of insulin,
whereas Type 2 diabetes is due to lack of insulin, insulin resistance or a combination of both.
Glycemic control is essential in avoiding diabetic complications. Diabetics must invest in
appropriate blood glucose control to avoid diabetic emergencies including ketoacidosis and non-
kinetic hyperosmolar coma. Diabetes is becoming more common among people in Cameroon's
cities. Ferritin is the major storage form of iron and iron deficiency anemia is associated with
greater HbA1C levels in diabetics, while iron surplus is associated with lower HbA1C levels.
Thus while interpreting HbAlC values in diabetic patients; the iron statuses should be taken into
account. Iron imbalance is common in type 2 diabetesdue to inadequate absorption of iron in
diabetics, Multiple factors appear to alter assessment of glycemic control one of which may be
iron overload or iron deficiency reflected by high and low ferritin levels respectively.
Objectives:The main objective of this study was to determine the relationship between ferritin
and glycemic control in type 2 diabetic patients attending the Buea Regional Hospital.
Methods: This study was hospital based cross sectional study at the Buea Regional Hospital.
Data was collected using a structured questionnaire and blood samples were collected and
analyzed. Data was keyed into SPSS and statistical analysis done using person’s correlation test
to determine correlation between ferritin and HbA1C, ferritin and FBG. Chi – square test was
done to determine the association between ferritin, markers of glycemic control and socio-
demographic factors of the study population.
Results:In this study ferritin levels were found to be high in diabetics. For ferritin, 50(59.5%)
patients had high ferritin levels of which 41(82%) were females (ferritin >200ng/ml) and
9(18%) were males, (ferritin >350ng/ml) while 13(15.5%) patients had low ferritin levels, of
which 11(84.6%) were females (ferritin <10ng/ml) and 2(15.4%) were males (ferritin
<20ng/ml). For indices of glycemic control, 54(64.3%) patients had poor control of which
47(87.04%) were females and 7(12.96%) were males, (HbA1C >8%) while 30(35.7%) patients
had good control, (HbA1C <7%) of which 20(66.7%) were females and 10(33.3%) were males.
A positive correlation (R=0.348) with P-value 0.001 was found between serum ferritin levels
and FBG thus ferritin levels increase as FBG increase. Also, a positive correlation (R=0.147)
with P-value 0.08 was found between serum ferritin levels and HbA1C.Ferritin was also found
to be associated with age (odds ratio= 5.33) and P-value (0.001) and HbA1C also had positive
associations with age, gender and BMI (Odds Ratio>1) and P-value (<0.05.)
Conclusion:In this study, serum ferritin levels were high in diabetics attending the Buea
Regional Hospital for glycemic control and positively correlated with FBG and HbA1C levels.
Therefore, ferritin levels should be routinely monitored in diabetic patients when assessing
glycemic control as it influences their glycemic status.

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 CHAPTER ONE: INTRODUCTION

1.1Background

Ferritin is the major storage form of iron and it is a mineral found in every cell of the body

[1]. It is considered an essential mineral because it is needed by several molecules such as

myoglobin found predominantly in muscles and hemoglobin found predominantly in red

blood cells (RBCs) to carry out their oxygen-carrying capacity. Iron is needed to maintain the

normal structure and functioning of these molecules and also for cells to live, grow and

proliferate [1]. A study by Kim C and Bullard KM showed that reduced iron stores have a

link with increased glycation of hemoglobin A1C (HbAlc), leading to false-high values of

HbAlc [2]. Some studies suggest that among diabetic individuals iron deficiency anemia is

associated with higher concentrations of HbA1c and iron in excess is associated with lower

levels of HbA1c [2].This implies that the iron states should be considered during the

interpretation of HbAlc concentrations in diabetic patients. Iron deficiency anemia appears to

be more common in diabetic patients due to poor absorption of iron and can impair glucose

hoemeostasis and also may negatively affect glycemic control. Iron deficiency anemia

predisposes a person to more complications [3].

Iron can be toxic in excessive amounts especially inhemosiderosis (deposition of hemosiderin

in tissues), and certain genetic disorders like hemochromatosis and also poses problems when

deficient as such it is tightly regulated by different proteins [1]. The human homeostatic iron

regulator gene provides information for producing a protein that is located on the surface of

cells primarily the liver and intestinal cells [1]. This protein interacts with other proteins on

the cell surface to detect the amount of iron in the body and regulates iron levels in liver cells

by preventing transferrin from binding to transferrin receptor 1. Also, the Human

Homeostatic Gene (HFE protein) regulates the synthesis of hepcidin from the liver which

1
determines the amount of iron that is absorbed from the diet and released from storage sites in

the body [4]. Iron overload and deficiency is reflective of ferritin levels as it is the major

storage form of iron. Iron absorption is tightly regulated when the proteins involved in iron

sensing and absorption are functioning properly [4].Iron toxicity is a factor for diabetes

particularly in iron-overload states such as hemochromatosis and recurrent transfusions in

thalassemia conditions [5].

Evidence that iron imbalance could contribute to abnormal glucose metabolism was derived

from the observation that the frequency of diabetes is increased in classic hereditary

hemochromatosis (HH) [5]. However, with the discovery of novel genetic disorders of iron

metabolism, it is said that iron overload irrespective of the cause or gene involved results in

an increased incidence of type 2 diabetes [5].Certain factors such as age, alcohol

consumption, obesity, gender, multiple blood transfusions, leukemia, liver disease,

hyperthyroidism, inflammatory diseases and infectious diseases alter ferritin levels [6].

Diabetes mellitus is a collective term referring to a group of metabolic disorders which affect

how the body uses glucose [7]. It is one of the most widely prevalent chronic condition with

about 422 million people affected, characterized by increased levels of blood sugar, which

progressively leads to serious damage to the cardiovascular system, circulatory system, renal

system, neurological system and the sensory system [8]. On the average, diabetes reduces life

expectancy by a decade, an effect largely attributable to macrovascular disease [9]. The most

common isType 1which is as a result of insulin deficiency while type 2 diabetes mellitus is a

metabolic disorder characterized by hyperglycemia resulting from insulin defect, insulin

resistance or a combination of both [7]. Multiple factors appear to be involved in the

pathogenesis of type 2 diabetes one of which may be iron overload [5]. Type 2 diabetes has

mostly been implicated in people with obesity and inactive lifestyles. However, genetic

susceptibility also plays a role in its etiology [7].

2
Glycemic control is critical to the prevention of diabetic complications. To prevent diabetic

emergencies such as ketoacidosis and non-kinetic hyperosmolar coma, diabetics must invest

in proper control of blood glucose levels.In Cameroon, the prevalence of diabetes in adults in

urban areas is currently on the rise [10].

1.2) Rationale

In Cameroon, there is an increase of diabetes in adults in urban areas and the prevalence is

currently estimated to be 6-8% the main drivers include increasing age, overweight, sedentary

lifestyle, and obesity with as many as 80% of people living with diabetes who are currently

undiagnosed in the population [10]. Iron imbalance is common in patients with type 2

diabetes and may vary in patients with different levels of glycemic control and severe forms

may lead to fatal complications. Ferritin, the storage form of iron has been reported to be

inconsistently correlated with markers of glycemic control. In a study carried out by Shubam

and Aparnaet al., (2018) in India to determine the correlation of glycated hemoglobin

(HBA1C) and fasting blood glucose(FBS) with serum ferritin concluded that there was a

positive correlation [11]. This result correlated with findings from Rhaul and Seloemet al.,

(2018) in Egypt in which it also concluded that there was a positive correlation between

serum ferritin and FBS [12]. Despite these reports, other controversial studies by Todd et

al ,and Basantaet al.,(2021) in India and Spain respectively have reported a negative

correlation between FBS, glycatedhaemogloblinand serum ferritin [13]. These controversies

could be due to difference in environmental factors, genetic factors, sex, ethnicity and certain

undefined confounders in the population which alter ferritin levels. It is therefore essential

that more studies be carried out in different geographical locations to determine the

association of ferritin with glycemic control in diabetic patients, such as diabetics attending

the Buea Regional Hospital.

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1.3) Hypothesis

Serum ferritin is associated with glycemic control

1.4) Research Question

Is there a relationship between serum ferritin and glycemic control?

1.5) Main objective

The main objective of this study is to determine the relationship between ferritin and

glycemic control in type 2 diabetic patients.

1.6) Specific Objectives

1) To measure ferritin, glycatedhaemoglobin and fasting blood glucose levels in diabetic

patients.

2) To determine the correlation between ferritin and glycatedhaemoglobin, ferritin and

fasting blood glucose level in diabetic patients.

3) To determine the association between ferritin, glycemic control and socio-

demographic factors of diabetic patients.

4
 CHAPTER TWO: LITERATURE REVIEW

2.1 Iron Overview

Iron is one of the most essential elements present in almost all cells [14]. It is the fourth most

abundant element in the Earth’s crust right in front of oxygen (32.1% and 30.1%

respectively) with an atomic number of 26 [14]. Chemically, iron is found in group 8 of the

periodic table and exists in a wide range of oxidation states (-2 to +7). The biological

oxidation states of iron are +2 (ferrous iron) and +3 (ferric iron). These two ionic forms are

interchangeable via oxidation/ reduction and are important for the function of many proteins

[14].Iron is a mineral found in every cell of the body, it is considered an essential mineral

because it is needed by several molecules such as myoglobin found predominantly in muscles

and hemoglobin found predominantly in red blood cells (RBCs) to carry out their oxygen-

carrying capacity. Hence, iron is needed to maintain the normal structure and functioning of

these molecules and also for cells to live, grow and proliferate [1].

There are two forms of dietary iron: heme and non-heme[15]. The RDA for non-vegetarians

is 1.8 times lower than that of vegetarians because heme iron from meat is more bioavailable

than non-heme iron from plant foods.The total body content of iron in an adult human is

about 3 to 5 grams stored in the form of ferritin or hemosiderin [15]. Serum ferritin

concentration is currently the most efficient and cost-effective test for diagnosing iron

deficiency and iron overload [15].

5
2.2 Iron Homeostasis

Intracellular levels of iron are tightly regulated because disorders of iron can be detrimental

to the health of the cell leading to the development of several pathological conditions like

diabetes, complications of diabetes and CVD. [4, 5]

Iron homeostasis is of great importance because iron is required for several diverse cellular

functions[16]. The regulation of iron in mammals is at the level of intestinal absorption, as

the body lacks a defined mechanism to excrete iron. Figure 1 shows the regulation of iron in

the cell.

Figure 1: Illustration of iron homeostasis [16].

In the duodenal enterocyte, dietary iron is reduced to the ferrous state by duodenal ferric

reductase (Dcytb), transported into the cell by divalent metal transporter 1 (DMT1), and

released by way of ferroportin into the circulation [4, 16]. Hephaestin (a transmembrane

copper-dependent ferroxidase necessary for effective iron transport) facilitates enterocyte

iron release into the circulation. Hepatocytes take up iron from the circulation either as free

6
iron or as transferrin-bound iron (through transferrin receptors 1 and 2) [16]. Transferrin

receptor 2 may serve as a sensor of circulating transferrin-bound iron, thereby influencing the

expression of the iron regulatory hormone hepcidin [4, 16]. The hepcidin response is also

modulated by HFE and hemojuvelin. Hepcidin is secreted into the circulation, where it

downregulates the ferroportin-mediated release of iron from enterocytes, macrophages, and

hepatocytes [16].

2.2.1) Iron Metabolism

 Absorption and transport

Iron absorption involves the entry, movement through, and release of iron from the intestinal

cell to the circulation. The fraction of iron absorbed from ingested food is typically low and

depends heavily on the physical state of the iron atom [17].

There are two types of absorbable dietary iron: heme and non-heme iron.

1. Heme iron, derived from hemoglobin and myoglobin of animal food sources (meat,

seafood, and poultry), is the most easily absorbable form (15% to 35%) and contributes

10% or more of the total absorbed iron [17].

2. Non-heme iron is derived from plants and iron-fortified foods and is less well

absorbed [17].

There are two main forms of iron in the body: the ferrous (Fe 2+) and oxidized ferric (Fe 3+)

state. To be absorbed, iron must be in the ferrous state or bound by a protein. At

physiological pH, iron exists in the oxidized ferric state [18]. Iron travels through the stomach

into the lumen of the small intestine in the ferric iron (Fe 3+) form [4]. The absorption of most

dietary iron occurs in the duodenum and proximal jejunum. At the proximal duodenum, the

low pH of gastric acid allows a ferric reductase enzyme, duodenal cytochrome B (Dcytb) on

the brush border of the enterocytes to convert the insoluble ferric (Fe 3+
) iron to absorbable

7
ferrous iron (Fe2+) [16, 17]. Ferrous iron gets absorbed by the enterocytes through a divalent

metal transporter 1 (DMT1), a solute carrier group of membrane transport protein found on

the apical side of the duodenum [4, 16]. In the enterocytes, the ferrous iron (Fe2+) can be

oxidized back to ferric iron to be stored as ferritin. Iron not stored as ferritin (ferrous Fe 2+)

leaves the cell through the basal surface through a transporter called ferroportin. This process

is coupled by the re-oxidation of ferrous iron (Fe 2+) to the ferric form (Fe ) by
3+

ceruloplasmin–ferroxidase activity [17]. Ferric iron binds with specific iron-binding protein

transferrin, the transport form of iron synthesized in the liver. The normal plasma level of

transferrin is 250 mg/dl and a molecule of transferrin can transport 2 ferric atoms. The total

iron-binding capacity (TIBC) of transferrin is 250 µg/dl. It is then transported through the

duodenal mucosa into the blood [4, 16]. The major transport protein is transferrin, a protein

capable of binding two atoms of ferric iron. It accounts for about 0.1% of the total body iron.

It transports iron from the reticuloendothelial system and the intestine to the bone marrow for

the synthesis of hemoglobin in developing red blood cells [16].

 Iron storage

The human body stores iron in the form of hemosiderin and ferritin. Iron is stored in an

insoluble form (Fe3+) in the liver, spleen, duodenum, skeletal muscle, and bone marrow.

Hemosiderin less readily releases iron for body needs while the majority of iron is highly

conserved in ferritin[16].

The serum ferritin concentrations correlate well with the total body iron stores under steady-

state conditions [4]. Serum ferritin is the most convenient laboratory test to estimate iron

stores. Ferritin is an acute-phase reactant protein elevated in inflammatory conditions.Human

ferritin is a large molecule consisting of a protein shell around an iron core. High

8
concentration of ferritin is found in the cytoplasm of the reticuloendothelial system, the liver,

spleen and bone marrow [4]. The measurement of ferritin in serum is useful in determining

changes in body iron stores. Serum ferritin levels can be measured routinely and are

particularly useful in the early detection of iron deficiency anemia in apparently healthy

people [16]. Ferritin levels are clinically significant in the monitoring of iron status of

pregnant women, blood donors, renal dialysis patients and also evaluating clinical conditions

not related to iron storage such as malignancy, inflammation, diabetes [16].

The amount of ferritin iron is slightly greater than that of hemosiderin iron in the range of

normal to iron deficiency. Hemosiderin iron becomes larger than ferritin in the iron overload

range. Unless the stores are exhausted, their amount has no discernible influence on any

physiological or biochemical function other than iron absorption [16, 17].

 Excretion

Iron is heavily conserved and is not readily lost from the body [4]. It is lost due to

menstruation, blood donations, and pregnancy. Excessive menstrual blood loss is the most

common cause of iron deficiency in women. Iron is not excreted, but in nephrotic syndrome

loss of transferrin may lead to increased loss of iron in urine [4, 16].

2.2.2) Metabolic Functions of Iron

i. Mitochondrial proteins contain iron within the cell and these are heme proteins and

nonheme containing iron-sulphur (Fe-S) complexes [15].Fe-S clusters in the electron

transport chain helps transport electron to molecular O2 enabling proteins to carry out

their function in energy production [15] as shown in figure 2.

9
 Figure 2: Electron transport chain showing Fe-S complexes [15]

ii. Iron is an important constituent of hemoglobin and myoglobin needed to maintain their

structure and carry out their function of transporting oxygen to tissues and muscles [1].

iii. Iron helps immune cells to proliferate and maturate and also acts as a constituent of

peroxidase, the lysosomal enzyme required for phagocytosis and killing of bacteria. Iron

overload suppresses host immunity and favors pathogen growth [15].

iv. Iron in the form of Fe-cluster aids in DNA replication by acting as a prosthetic group for

polymerase alpha, delta, and epsilon which are very essential for the DNA replication

process [15].

v. Iron is important for pigment and porphyrin metabolism. In porphyrin metabolism,

enzymes such as haem synthase and uroporphyrinogen decarboxylase are under the

feedback control of iron.

vi. Iron is required by phenylalanine hydroxylase for the formation of homogentisic and

melanin quinones.

vii. Iron acts as a component of enzymes involved in the synthesis of some

neurotransmitters. Iron is an important component of the neuronal monoamine oxidase

catecholamine involved in adrenergic neurotransmission. Dopamine receptors are

downregulated and alter gamma-aminobutyric acid (GABA) metabolism during iron

deficiency [15].

10
viii. Iron is a major component of catalase and peroxidases such as glutathione peroxidase

for the detoxification of hydrogen peroxide and free radicals.

ix. Iron is needed for collagen synthesis

2.3) Etiology of Diabetes

Diabetes is a chronic disease that occurs either when the pancreas does not produce enough

insulin due to the destruction of β-cells or when the body cannot effectively use the insulin

produced (insulin resistance) leading to a state of hyperglycemia [7]. According to the World

Health Organisation (WHO), 422 million people worldwide have diabetes, the majority living

in low and middle-income countries [8]. In 2014, 8.5% of adults aged 18years and older had

diabetes and by 2019 diabetes was the direct cause of about 1.5 million deaths and 48% of all

deaths due to diabetes occurred before the age of 70 years [8]. The primary category of

diabetes is presently differentiated into type 1 and type 2 depending on whether it emerges

from the destruction of β-cells of the pancreas with predominant absolute insulin deficiency

(Type 1) or evolves out of a complex interaction between impairment of insulin action and

relative inadequacy of insulin secretion (Type 2) [20].

2.4) Etiological Classification of Diabetes Mellitus

I. Type 1 diabetes (β-cell destruction, usually leading to absolute insulin deficiency) [20].

A. Immune-mediated

B. Idiopathic

II. Type 2 diabetes (may range from predominantly insulin resistance with relative insulin

deficiency to a predominantly insulin secretory defect with insulin resistance)

III. Other specific types of diabetes [20].

A. Genetic defects of β-cell function characterized by mutations in:

1. Hepatocyte nuclear transcription factor (HNF) 4α (MODY 1)

2. Glucokinase (MODY 2)

11
B. Drug- or chemical-induced; pentamidine, nicotinic acid, glucocorticoids, thyroid hormone,

diazoxide, β-adrenergic agonists, thiazides, phenytoin, α-interferon, protease inhibitors,

clozapine, beta-blockers [8].

C. Gestational diabetes: A form of hyperglycemia in pregnant women. Those who develop

gestational diabetes are at high risk of having type 2 diabetes later in life.

2.5) Epidemiology of Diabetes

In Cameroon, the prevalence of diabetes in adults in urban areas is currently estimated to be

6-8%, the main drivers include increasing age, overweight, sedentary lifestyle, and obesity

with as manyas 80% of people living with diabetes who are currently undiagnosed in the

population [10]. Wide variations are present between neighboring areas in Europe, Africa,and

North America [10]. Studies show that the incidence of type 1 diabetes is on a steady rise in

several countries and the number is going to double over in the next decade [20]. There is no

underlying mechanism to explain the geographical incidence and increased rates of type 1

diabetes but can be considered as attributes of environmental influences because genetic

alterations or improvement in the delivery rate of offspring’s of type 1 diabetes mothers could

alone not explain the rates of increase [21]. Moreover, genetic predisposition contributes less

now than in the past as a factor prerequisite for developing type 1 diabetes [22]. It should be

noted that the rise in incidence has not been equal across all age groups. A significant

increase in incidence is observed in individuals of age group below 35 years [23] as well as in

young children (age group 5–7 years) from high incidence countries. A wide range of

environmental influences has been shown to affect the epidemiology of diabetes. Developing

models to study the influence of the environment on diabetes including hygiene are being

proposed [24]. Figure 3 shows an overview of the world wide prevalence of diabetes [20, 21]

12
Figure3: Worldwide Prevalence of Diabetes [20, 21].

The world prevalence of diabetes among adults aged 29 to 79 years in 2010 was about 6.5%

affecting 285 million adults and is expected to rise about 7.7% and 500million adults by 2030

[10]. Epidemiological studies had predicted that between 2010 and 2030 there will be an

increase in the numbers of adults with diabetes in developing countries and about a 20%

increase in developed countries [8]. These predictions based on a large number of studies

indicate a growing burden of diabetes particularly in developing countries [10].

2.6) Pathophysiology of Diabetes

The pathogenesis of type 1 diabetes is a result of the autoimmune destruction of insulin-

secreting pancreatic islet β-cells. The basis of this observation is the presence of chronic

inflammatory infiltrates, which affect the pancreatic cells at the symptomatic onset of the

disorder [24]. The pancreas in patients with long-standing type 1 diabetes is devoid of

13
insulin-producing cells and the remaining β-cells are incapable of regeneration. However,

reports show that, despite type 1 diabetic patients having a small number of residual β-cells,

the evidence of regeneration potential of β-cells is only present in infants and very young

children [24] but the regeneration potential is not seen in adolescents and adults [24, 25].

Most findings on the pathogenesis of type 1 diabetes are derived from analysis of pancreatic

samples, serum, and peripheral blood lymphocytes obtained from the patients. Hence,

functional defects in the bone marrow, thymus, immune system, and β-cells collectively

conribute towards type 1 diabetes [24]. In mid-1980’s Eisenbarth developed a model

combining the genetic, autoantibody, and metabolic markers of type 1 diabetes to better

understand the disease [26]. According to the model, individuals with high genetic

predisposition having a fixed number of β-cells when exposed to environmental triggers

induce β-cell autoimmunity. This develops islet reactive autoantibodies, which signals the

development of activated auto-reactive T-cell capable of destroying β-cells resulting in the

progressive loss of insulin secretory function [26]. The symptoms associated with the disease

are classically polyuria, polydipsia, and polyphagia.

Type 2 diabetes is caused by a combination of genetic factors related to impaired insulin

secretion, insulin resistance and environmental factors such as obesity, lack of exercise, and

stress [7, 19]. It is typically a multifactorial disease involving multiple genes and

environmental factors that affect the β-cell function and insulin sensitivity. The

pathophysiology of type 2 diabetes is characterized by peripheral insulin resistance, impaired

regulation of hepatic glucose production, and β-cell dysfunction [19]. Figure 4 shows the

pathophysiology of type 2 diabetes.

Genetic predisposition Environmental factors

14
 Decrease insulin release Insulin resistance Obesity

Increased hepatic glucose outputDecrease glucose uptakein muscle

Hyperglycemia

Type 2 diabetes

Figure4: Pathophysiology of type 2 diabetes [19].

2.7) Diabetic Complications and Management Paradigm for Diabetes

2.7.1 Diabetic Complications

Over time, diabetes can damage the heart, blood vessels, eyes, kidneys, and nerves.

2.7.1.1 Acute Diabetic Complications

1) Diabetic Ketoacidosis (DKA)

It occurs when the body produces high levels of blood acids called ketones. This condition

develops when the body cannot produce insulin to help glucose enter the cells [27]. Without

enough insulin, the body begins to break down fat as fuel leading to the buildup of keto-acids

in the bloodstream eventually leading to diabetic ketoacidosis [19]. DKA is caused by low

levels of effective circulating insulin and a rise in counter regulatory hormones such glucagon

and catecholamine, cortisol, as well as growth hormone. This combination leads to catabolic

changes in the metabolism of carbohydrates, fat, and protein [28]. Impaired glucose

utilization and increased glucose production by the liver and kidneys result in hyperglycemia.

Lipolysis leads to increased production of ketones, especially beta-hydroxybutyrate (β-OHB),

ketonemia, and metabolic acidosis, which is exaggerated by ongoing fluid and electrolyte

15
losses [29].Diabetic ketoacidosis (DKA) is a common and life-threatening complication of

type 1 diabetes, particularly at the time of diagnosis.

2) Hypoglycaemia

Diabetic hypoglycemia is a low blood glucose level occurring in a person with diabetes

mellitus. The result of unregulated delivery of endogenous or exogenous insulin into the

circulation initiate the sequence that may or may not culminate in an episode of

hypoglycaemian [30]. Absolute therapeutic insulin excess of sufficient magnitude can cause

isolated episodes of hypoglycemia.It is distinguished by several risk factors and a

complicated pathogenesis [30]. The brain requires a constant supply of glucose for energy,

while it can also use ketone bodies. The spectrum of outcomes ranges from mild cognitive

impairment to coma, seizure, and sudden death [31].

3) Hyperglycemic Hyperosmolar State (HHS)

The fundamental mechanism for HHS is a decrease in the effective action of circulating

insulin combined with an increase in counter-regulatory hormones [32]. These changes result

in increased hepatic and renal glucose production and decreased glucose utilization in

peripheral tissues, resulting in hyperglycemia and changes in extracellular osmolality. [27,

33]. HHS is associated with glycosuria, leading to osmotic diuresis, with loss of water,

sodium, potassium, and other electrolytes. In HHS, insulin levels are inadequate for glucose

utilization by insulin sensitive tissues but sufficient as determined by residual C-peptide to

prevent lipolysis and ketogenesis [34].

2.7.1.2 Chronic Diabetic Complications

Chronic complications develop overtime and affect the tiny blood vessels of the retina, the

kidney, the nerves, and the heart.

A. Micro vascular Complications

1)Diabetic Retinopathy

16
It involves the growth of abnormal blood vessels in the retina. Over time, too much sugar in

the blood can lead to the blockage of tiny blood vessels that nourish the retina cutting off its

blood supply [8, 19]. There are 2 types of diabetic retinopathy; non-proliferative diabetic

retinopathy whereby new blood vessels are unable to grow making the walls of the retina

weak and proliferative diabetic retinopathy where damaged blood vessels close off causing

the growth of new abnormal blood vessels in the retina and leak into the vitreous body.

According to the WHO, about 1 million people are blind due to diabetes [8].

2) Diabetic Nephropathy

It results when blood vessels and other cells in the kidney are damaged due to the

accumulation of glucose. The kidney contains millions of tiny clusters (glomeruli) that filter

waste from blood [8]. Severe damage to these blood vessels due to poorly controlled diabetes

leads to kidney damage. Diabetic nephropathy is one of the leading causes of kidney failure

[8].

3) Diabetic Neuropathy

It occurs when uncontrolled high blood sugar damages nerves interfere with their ability to

send signals and also weakens the walls of the capillaries that supply the nerves with oxygen

and nutrients [8]. Combined with reduced blood flow, neuropathy in the feet increases the

chance of foot ulcers, infection, and the eventual need for limb amputation [8].

B. Macro vascular Complications

1) Cardiovascular Disease (CVD)

Diabetes increases the risk that an individual will develop cardiovascular disease.

Although the precise mechanisms through which diabetes increases the likelihood of

atherosclerotic plaque formation are not completely defined [35]. The association

17
between the two is profound. CVD is the primary cause of death in people with diabetes.

Diabetes is also a strong independent predictor of stroke and cerebrovascular disease [8].

2) Stroke

There are several possible mechanisms wherein diabetes leads to stroke. These include

vascular endothelial dysfunction, increased early-age arterial stiffness, systemic

inflammation and thickening of the capillary basal membrane. Abnormalities in early left

ventricular diastolic filling are commonly seen in type II diabetes [35, 36]. Uncontrolled

diabetes puts subjects at risk for both ischemic and hemorrhagic strokes. There are

specific clinical patterns of ischemic stroke in individuals with diabetes. For example,

individuals with diabetes are more likely to have limb weakness and dysarthria as signs of

lacunar cerebral infarction when compared with those without diabetes [8].

3) Dementia

Studies have shown that type 2 diabetes can be a risk factor for vascular dementia and

alzheimer’s disease [36]. This is because the same cardiovascular problems that increase

the risk of type 2 diabetes also increase the risk dementia. These include:Obesity,Heart

disease or family history of heart disease, impaired blood vessels, high cholesterol,

andigh blood pressure [8, 35]. Glucose is not used properly in the brains of people with

diabetes leading to Alzheimer’s disease. This may be caused by nerve cell death, which

reduces the brain’s ability to interpret messages. In the case of vascular dementia, brain

cells die due to lack of oxygen, preventing brain cells from communicating with each

other [36].

4) Diabetic macroangiopathy

A specific form of accelerated atherosclerosis due to hyperglycemia characterized by

intra-plaque new vessel formation due to abnormal angiogenesis, increased vascular

18
 permeability of the capillary vessels, and tissue edema, resulting in frequent

atherosclerotic plaque [36].  Figure 5 shows the major macrovascular and microvascular

complications of diabetes.

Figure5: Major microvascular and macrovascular complications associated with

diabetes mellitus. [35, 36]

2.7.2 Management Paradigm for Diabetes

Recent advances in anti-diabetic medications and glucose monitoring have led to a paradigm

shift in diabetes care [26]. The previous care strategy was the use of sulfonylureas in the

1950s, sulfonylureas were developed and used as a treatment for diabetes by enhancing

insulin activity [26]. These sulfonylureas were later reported to cause significant

hypoglycemia, weight gain, and burn out the pancreas. High mortality and morbidity has

being recorded due to the adverse effects of the sulfonylureas leading to a paradigm shift in

diabetes care [26]. New anti-diabetic medications such as dipeptidyl peptidase-4 (DPP-4)

inhibitors, glucagon-like peptide-1 (GLP-1) receptor agonists, and sodium-glucose

cotransporter 2 (SGLT2) inhibitors have enabled optimal glycemic control to be achieved

19
without increasing the risk of hypoglycemia and weight gain thereby making this new shift

better [26,37]. Treatment with GLP-1 receptor agonists and SGLT2 inhibitors have been

demonstrated to improve cardiorenal outcomes thereby making these agents as the mainstay

of treatment of patients with type 2 diabetes [26]. Lifestyle improvement includes dietary

habits and physical activity have been reported to be the best way to attain optimal glycemic

control [37]. Figure 6 shows the management paradigm for type 2 diabetes.

Figure 6: Management paradigm for type 2 diabetes [37].

2.8) Diagnosis and Monitoring of Type 2 Diabetes

I. Diagnosis of Type 2 Diabetes Mellitus

Both Laboratory Investigations and clinical features are employed in the diagnosis of diabetes

mellitus. A preliminary screening test may identify the presence of urinary glucose, although

this is not diagnostic of diabetes mellitus [8]. Four diagnostic tests for diabetes are currently

recommended. They are: fasting blood glucose; 2-hour post-load plasma glucose after a 75 g

20
oral glucose tolerance test (OGTT); HbA1c; and arandom blood glucose in the presence of

signs and symptoms of diabetes [38].

i. Fasting blood glucose

Fasting blood glucose test is a test of moderate sensitivity which assesses the body’s glucose

concentration after at least 8 hours of fasting. People with fasting blood glucose values ≥ 7.0

mmol/L (126 mg/dl; or a random blood glucose ≥ 11.1 mmol/L (200 mg/ dl) [8] in the

presence of signs and symptoms are considered diabetic. If elevated values are detected in

asymptomatic people, testing is repeated preferably with the same test as soon as practicable

on a subsequent day to confirm the diagnosis. It is relatively inexpensive and simple and a

single blood glucose level measurement usually suffices [38]. It is also highly correlated with

presence of complications. It however will require the patient fasts overnight (at least 8

hours) if not there is a potential of processing error. Point measurements of fasting blood

glucose can as well be affected by short-term lifestyle changes and there are risks of wrong

phlebotomy.[38]

ii. Oral glucose Tolerance Test

OGGT is the current gold standard for the diagnosis of diabetes mellitus [38]. It is used in

assessing the body’s response to glucose. During the OGTT, the patient is kept on a normal

diet for three days prior to the test and then fasts overnight prior to the test. A basal venous

blood sample is taken for glucose determination before the patient drinks 75 g of anhydrous

glucose dissolved in about 50µl of water [8]. 2 hours after the patient drinks the glucose,

another blood sample is taken and measured for blood glucose. Plasma glucose values greater

than or equal to 5.5 mmol/L for the basal sample or 7.8 mmol/L for the 2 h samples are

diagnostic of diabetes mellitus [8]. Individuals with plasma glucose concentrations less than

6.9 mmol/L for the basal sample or between 7.8 and 11.1 mmol/L for the 2h samples are

categorized as having impaired glucose tolerance (IGT). This group has an increased risk of

21
developing cardiovascular disease [8]. It is the most sensitive test for impaired glucose

tolerance but is disadvantageous in that, it requires 8-hour fasting, it is lengthy and the overall

test-retest reproducibility is lower than with other tests [38].

iii. Glycated Hemoglobin

HbA1c is used as diagnostic test for diabetes at a threshold of ≥ 6.5%. People with HbA1c≥

6.5% (48 mmol/mol) are considered diabetic [8]. It is also used as a stable marker of long-

term glycemic level. No fasting is required and it can be completed at any time. This test is

not affected by short-term lifestyle changes and it requires only venous blood or a point-of-

care testing capillary sample. Disadvantages of this test include; value may vary with assay

method used; possible non-glycemic causes of error such as hemoglobinopathies and anemia;

may be insensitive for detection of impaired fasting glucose or impaired glucose tolerance;

high cost (more expensive than routine glucose testing) and there is limited availability in

some areas of the world [38]

iv. Random blood glucose

Random blood glucose test is used as a screening tool but it is somewhat limited by its low

screening performance [8]. People with a random blood glucose ≥ 11.1 mmol/L (200 mg/ dl)

in the presence of signs and symptoms are considered diabetic [8]. If the individual under

investigation lacks the typical symptoms of diabetes then diagnosis cannot be confirmed

unless tests are repeated as soon as practicable on a subsequent day for confirmation. This

test is relatively inexpensive and easy to obtain and the patients are not required to fast prior

to the test. Disadvantages of using a random blood sample for glucose measurement is that, it

requires prompt processing (<two hours) thus the potential for error and point measurement

can be affected by several factors such as short-term lifestyle changes as well as timeof

sample collection.[38].

22
 II. Monitoring of Type 2 Diabetes Mellitus

Maintaining tight glycemic control is essential for the prevention of both microvascular and

macrovascular complications. It is essential to monitor glucose levels in order to achieve the

therapeutic targets. Blood glucose monitoring can be done by different methods: glycated

hemoglobin, self-monitoring of blood glucose (before and after meals) with a glucometer and

continuous glucose monitoring with a system that measures interstitial glucose concentrations

[39]. Even though glycated hemoglobin is considered the gold standard of diabetes care, it

does not provide complete information about the magnitude of the glycemic disequilibrium

[8]. Therefore, the self-monitoring and continuous monitoring of blood glucose are

considered an important adjunct for achieving and maintaining optimal glycemic control.

A. Home Monitoring

To achieve and sustain glycaemic control goals, home monitoring, which is a type of self-

monitoring blood glucose, is essential. It adds to the information provided by HbA1c to a

certain extent. It's done with the use of a glucometer and is based on glucose dosage in

capillary blood [41]. Glucose monitoring is done in real time and provides information on

suspected hyperglycemia or hypoglycemia. In general, glucometer values are less precise

than those acquired using laboratory methods, although they are more precise than the earlier

color-reaction methods. The patient can make instantaneous therapeutic decisions, such as

pharmaceutical and dietary alterations, as well as changes in physical activity, resulting in

moreefficient illness management [41, 42]. The ability of glucometers is always being

improved.

B. Continuous Glucose Monitoring

Continuous glucose monitoring from the interstitial fluid is a new technical technology that

has only recently been developed and used. It gives a complete picture of the glucose profile,

23
with or without the ability tosee real-time glucose values [39]. It allows assess to medication,

nutritional therapy, physical activity, education, and therapy adherence. Invasive

measurements, minimally invasive approaches, and non-invasive techniques are all options

for these systems [41]. The level of glycemia in constant glycemic circumstances is similar to

the glucose from interstitial fluid. However, with rapid glycemic value exchange, changes in

the interstitial fluid level occur with a delay compared to venous blood, known as the lag

period, which can last up to an hour. As a result, while this sort of glucose monitoring does

not exactly match the glycemic level, the information it provides is incredibly useful. This is

because it is able to recognize the glycemic propensity and behave accordingly [42]. It also

provides a more complete picture of the disease's progression and therapy. The least invasive

systems are the most commonly utilized. These devices require a sensor to be implanted in

subcutaneous tissue, most commonly in the abdomen area. [39, 42]

A. Glycated Hemoglobin

HbA1C is the standard and preferred test for measuring long-term glycemic control in

persons with diabetes. HbA1C has been approved by the World Health Organization for use

as a screening test for people at high risk of diabetes, and more critically, as a test for

predicting the risk of microvascular complications [8]. Glycated hemoglobin is created by the

irreversible glycation of adult hemoglobin, which occurs due to non-enzymatic bonding of

adult hemoglobin with glucose in the blood. If hyperglycemia persists for a long time, a high

proportion of HbA molecules get glycated. This glycatedHbA circulates for roughly 120

days, which is the same as a red blood cell's lifespan. As a result, its value reflects long-term

glycemic exposure, and it represents average glucose concentration over the last 8–12 weeks

[8].Although it shows the average of the exposure over the previous 2–3 months, allowing for

a long-term assessment of control, it does not provide information on daily glycaemic levels,

and hence provides no instant feedback to the patient on treatment or nutritional decisions.

24
HbA1c also does not provide information on the amplitude of glycaemic fluctuation to which

the patient is subjected on a daily basis [40]. Several medical disorders, such as liver illness,

kidney disease, hemoglobin abnormalities, blood transfusions, pregnancy, hemolytic anemia,

iron deficiency anemia, hypertriglyceridemia, hyperbilirubinemia, uremia, certain medicines,

and alcohol misuse, can all affect the value of HbA1c. This was the method used to assess the

glycemic control of the study participants.

25
 CHAPTER THREE: MATERIALS AND METHODS

3.1 Study Design

This was a cross-sectional study based on laboratory investigations evaluating glycemic

control parameters (FBG and HBA1C) and ferritin levels in diabetic patients attending the

Buea Regional Hospital. The samples analyzed were obtained from randomly collected

specimens from patients visiting the Buea Regional Hospital for glycemic control between

the periods of March to May 2022. Participants who met the study inclusion criteria were

assisted in filling the research questionnaires after proper explanation of the study through

informed consent.

3.2 Study Area

This research was carried out in Buea regional hospital, South West region of Cameroon

located on the eastern slope of Mount Cameroon having a population of about 300,000

people. The climate is humid with cooler temperatures. The study population included males

and females of age greater than 30 years at the time of onset of diabetes. Samples were then

collected from confirmed diabetic patients attending the Buea Regional Hospital who fulfill

the inclusion criteria and voluntarily accepted to participate in the study.

3.3 Inclusion and Exclusion Criteria

I. Inclusion criteria

1) Patients with confirmed diabetes

2) Patients of both sexes greater than 30 years of age from time of onset.

II. Exclusion criteria

1) Patients suffering from iron deficiency anemia

2) Patients on iron supplements

26
3) Patients with chronic kidney disease

3.4 Ethical considerations

Ethical clearance for this study was obtained from the Institutional Review Board of the

Faculty of Health Sciences, University of Buea. Administrative authorization was obtained

from the South-West Regional Delegation of Public health. Also, written permission to carry

out this study was obtained from the director of the Buea Regional Hospital. Participants

were also given the free will to withdraw from this study without any sanctions either directly

or indirectly. The use of sterile material for sample collection and appropriate procedures to

reduce risk of injury was strictly implemented. Participant information was strictly kept

confidential.

3.5 Sample Size Calculation

The minimum sample size was obtained using the Lorentz formula as follows:

The prevalence of 5.8% was obtained from a study carried out by Bigna to determine the

Prevalence of pre-diabetes and diabetes mellitus among adults residing in Cameroon in 2018

[10].

N= Z2 PQ/D2

Where;

Z= 1.96 for 95% confidence interval

P= 5.8%, the prevalence of diabetes in Cameroon as estimated by (Bigna et al, 2018)

Q= 1-P

D= relative precision of 5% (0.05)

Therefore, sample size (N) = 83.7 approximated to 84 participants.

3.6 Sampling Method

27
 Participants were selected using the convenience sampling method.Participants were

recruited into the study as long as they agreed to take part in the study and met the inclusion

criteria. This was done until the required sample size was obtained.

3.7 Sample Collection

A structured questionnaire was used to collect demographic data and other relevant

information from the participantsbefore blood sample collection.

 Materials: Syringes, tourniquet, 75% alcohol, EDTA tubes, Plain tubes,Eppendorf

tubes,Spectrophotometer, centrifuge, Pipette, Glass tubes, HbA1C reagent kit,

Glucometer, Glucose test strips, disposable pipette tips, distilled water, Ferritin

ELISA kit, ELISA plate reader.

 Sample collection: Bloodsamples were collected by venipuncture using a syringe into

EDTA and plain tubes for HbA1C and ferritin measurement respectively.

 Sample preparation:Bloodsamples in plain tubes were centrifuged and serum

extracted into eppendorf tubes and stored at -70ºC while blood samples in EDTA

tubes were stored at 4ºC as whole blood was needed for analysis.

3.8Laboratory Procedures

i. Fasting blood glucose measurement (FBG)

FBG was measured with the aid of a glucometer using the glucose oxidase method. A finger

prick was done using a lancet and a drop of blood placed on a test strip inserted into the

glucometer which displays the glucose concentration automatically.

Principle: The enzyme glucose oxidase reacts with glucose, water, and oxygen to form

gluconic acid and hydrogen peroxide. The hydrogen peroxide can then be used to oxidize a

28
 chromogen or the consumption of oxygen measured to estimate the amount of glucose

present. The glucometer then gives a reading of the glucose concentration in the sample.

Reactions:

Glucose + H₂O + O₂ GOX Gluconic acid + H₂O₂

4 Amino Phenazone + Phenol + 2H₂O₂ PODQuinonimine + 4H2O

ii. Glycatedhaemoglobin measurement (HbA1C)

HbA1C was measured spectrophotometricallyusing the ion exchange resin method.

Principle: A weak binding cation-exchange resin is used for the rapid separation of

glycatedhaemoglobin from all the other hemoglobins. A hemolysed preparation of the whole

blood is mixed for 5minutes with a weak binding cation–exchange resin. During this time

HbA0 (non-glycosylated Hb) binds to the resin. HbA0 consist of all the other hemoglobins

except A1C which remains in solution. After the mixing period, a filter is used to separate the

supernatant containing the A1C from the resin. The per cent glycohemoglobin is determined

by measuring the absorbance at 415nm of the A1C fraction and the total haemoglobin

fraction. The ratio of the two absorbance gives the per cent of HbA1C.

Procedure

 The spectrophotometer was calibrated according to the commercial kit. Glass tubes

were labeled as standard, sample and resin tubes.

 500µL of the lysing reagent was added into the tubes.

 100µL of sample and standard were pipetted into the respective tubes, mixed and

allowed to stand for 5 minutes.

 70µL of the hemolysate was added into the resin tube and filter separators inserted

into the tube and mixed on a rocker for 5 minutes.

29
  The resin was firmly packed as the filter separators were pushed into the tubes and the

supernatant was collected in another tube and absorbance measured at 415nm. This

gives the absorbance of the glycohemoglobin.

 1ml of deionized water was added into the tubes (sample and standard)

 20µL of the hemolysate was added into the respective tubes and mixed. The

absorbance was measured at 415nm and this was the absorbance of Hb total.

|of|glycohemoglobin
 The results were then calculated as, %HbA1C= x standard conc
|of|Hb total

Reference range: 7 - 8%

iii. Ferritin measurement

Ferritin was assessed using the ERBAlisasandwich ELISA method as per the

manufacturers’procedure. The sandwich assay method uses two antibodies, a capture

antibody which is mobilized on a surface (biotin) and a detection antibody conjugated to an

enzyme (HRP).

Principle: TheERBAlisa ferritin ELISA kit is a solid phase sandwich assay method which

makes use of two highly specific monoclonal antibodies, based on a streptavidin-biotin

principle. The standards, samples and the biotinylated anti-ferritin antibody reagent are added

into designated wells, coated with streptavidin. Endogenous ferritin in the patients’ serum

binds to the antigenic site of the biotinylated anti-ferritin antibody. Simultaneously, the

biotinylated antibody is immobilized on to the wells through the high affinity streptavidin-

biotin interaction. Unbound protein and excess biotin conjugated antibody are washed off by

was buffer. Upon addition of the peroxidase (HRP) conjugated anti-ferritin antibody reagent,

a sandwich complex is formed, the analyte of interest (ferritin) being in between the two

30
highly specific antibodies, labeled with biotin and HRP. Upon addition of the substrate, the

intensity of color developed is directly proportional to the concentration of ferritin in the

samples. The enzymatic reaction is terminated by addition of the stopping solution. The

absorbance is measured on a micro titer plate reader. The intensity of the color formed by the

enzymatic reaction is directly proportional to the concentration of ferritin in the sample. A set

of standards is used to plot a standard curve from which the amount of ferritin in patient

samples and controls can be directly read.

Procedure

 The samples and reagent kit were brought to room temperature (25°C) before used for

analyses.

 25µL of ferritin standards, controls and samples were pipetted into the coated wells.

 100µL of the biotin reagent was pipetted into each well. The plate was rocked for

30seconds, covered and incubated for 30 minutes at room temperature.

 The liquid was removed from all wells and the wells washed three times with 300µL

of 1X wash buffer.

 100µL of the enzyme reagent was pipetted into each well, the plate covered and

incubated for 30 minutes at room temperature.

 The liquid was removed from all wells and the wells washed three times with 300µL

of 1X wash buffer.

 100µL of TMB substrate was pipetted into each well and incubated for 15 minutes at

room temperature

 50µL of stop solution was added to end the enzymatic reaction and the plate rocked

for 20 seconds to mix the solution.

 The absorbance was read on ELISA reader at 450nm within 15 minutes after adding

the stop solution.

31
  The assay was done in duplicate and the average absorbance was calculated and the

concentration of ferritin in each sample was recorded.

Reference range

Males: 20-350ng/ml

Females: 10-200ng/ml

3.9 Study variables and Data Management

3.9.1 Study variables

 Qualitative variables: gender , age, level of education, occupation, marital status

 Quantitative variables: FBG, Ferritin, HbA1C, Height, Weight, BMI, Blood

pressure.

3.9.2 Data Management

Codes were assigned to each participant and the results were kept confidential in a log book

to be inputted in to SPSS for statistical analysis.

 Frequency distribution tables and cross tabulations were used to describe data, paying

attention to how the variables relate to some demographic aspects of the population

like gender, age, marital status and level of education.

 Pearson’s correlation was done to determine the correlation between ferritin and

glycated hemoglobin, ferritin and FBG.

 Chi–square test was used to determine the association between ferritin, socio-

demographic data and markers of glycemic control.

32
 CHAPTER FOUR: RESULTS

4.1 General characteristics of the study population

The general characteristics of the study population were obtained using a questionnaire.

Table 1 shows the socio-demographic data of diabetics attending the Buea Regional Hospital.

Table 1: Socio-demographic characteristics of study population

Parameter Category n (%)

mean SD
Age
59.52 10.71

30-40 4(4.8)
41-50 15(17.8)
51-60 21(25)
61-70 32(38.1)
>70 12(14.3)
Total 84
Females 67(79.7)
Gender Males 17(20.3)

Total 84

Primary 40(47.7)
level of education Secondary 33(39.3)
Tertiary 11(13.1)
Total 84
Employed 26(31.9)
Occupation Self-employed 28(33.3)
Unemployed 30(35.7)

Total 84

Married 71(84.5)
Marital status Single 13(15.5)
Total 84

33
Eighty four (84) participants were recruited in this study of which 67(79.7%) were females

and 17(20.3%) were males. Their ages ranged from 30 – 78 years and most of the participants

where between 60-70 years with the mean age being 59.5 years and standard deviation of

10.7 years. Only 13.1% of the study population had attained tertiary education and majority

of the participants (47.7%) had just primary education and 39.3% had attained secondary

education. 35.7% of study participants were unemployed, 33.3% were self-employed and

31.9% were employed as shown in table 1.

4.2 Age Distribution of Diabetic patients in the Study.

Among the 84 participants, 38.1% of the population was between 61-70 years, and just 4.8%

of the study population was between 30-40 years as shown in figure 7.

Age distribution of participants

35

30

25

20
participants

32
15

10 19
16
14
5
5
0
31- 41-50 51-60 61-70 70-80
40

Age groups

Figure 7: Age distribution of study participants

34
4.3 BMI Distribution in the Study Population

In this study, 30(35.7%) of the total population were overweight (BMI>=25), 2(2.4%) of the

study population were class 3 obese (BMI >=40.0), 12(14.3%) were class 2 obese (BMI;

35.0-39.9), 23(27.4%) were class 1 obese (BMI; 30.0-34.9) and 17(20.2%) of the total

population had normal BMI values (BMI; 18.5-24.9) as shown in figure 8

BMI distribution
35

30

25
participants

20

15 30
23
10
17
5 12

0 2
normal class 1 class2 class3 overweight
BMI category

Figure 8: BMI distributions of study participants

Table 2: Biochemical and Anthropometric Measurements of Study Population

Parameter mean SD Reference range

Diastolic blood 80.22 10.78 75-85
pressure (mmHg)
Systolic blood 134.98 20.12 115-135
pressure (mmHg)
Height (m) 1.61 0.06 -

Weight (kg) 76.75 14.85 -

BMI (kg/m2) 29.05 5.20 18.5-24.9

HbA1C (%) 8.56 1.19 7-8%

Serum ferritin 354.44 285.94 Males: 20-350ng/ml

35
ng/ml Females: 10- 200ng/ml
FBG (mg/dl) 157.01 51.4 102-154mg/dl

4.4 Correlation between Ferritin and Markers of Glycemic control

Positive correlations were obtained between serum Ferritin and FBG, Ferritin and HbA1C.

4.4.1 Correlation between Ferritin and FBG

There was a positive correlation R= 0.348 (R> 0.1) between ferritin and FBG which was

statistically significant with p- value 0.0011 (P<0.05) with 95% confidence interval as shown

in figure 9. This implies that ferritin levels tend to increase as fasting blood glucose levels

increase in diabetics attending theBuea Regional Hospital for glycemic control.

Correlation between serum ferritin and FBG

1200

1000

800
ferritin

600

400

200

0
50 100 150 200 250 300 350 400
FBS

Pearson Correlation coefficient (R) = 0.3486777

P-value = 0.001152

Figure 9: Correlation between serum ferritin and FBG

36
4.4.2 Correlation between Ferritin and HbA1C

There was a positive correlation R= 0.147 (R> 0.1) between ferritin and HbA1C but it was

not statistically significant with p- value 0.0808 (P>0.05) with 95% confidence interval as

shown in figure 10. This implies that HbA1C levels increase as ferritin levels get higher but

these variations can be negligible due to the high p- value.

Correlation between Ferritin and HbA1C

1200
1000
800
ferritin

600
400
200
0
5 6 7 8 9 10 11 12
HbA1C

Pearson Correlation coefficient (R) = 0.1474351

P-value = 0.0808

Figure 10: Correlation between serum ferritin and HbA1C

4.5 Associations between Ferritin and Socio- Demographic Factors

4.5.1 Association between ferritin and age

There was a significant positive association between ferritin and age with P- value 0.001 (p-

value <0.05) and odds ratio 5.33 (OR>1). This implies that an increase in age brings about an

increase in ferritin levels.

37
Out of the total number of participants with high ferritin, 25(50%) with was from the 60-70

age group, 11(22%) was from the51-60 age group and 10(20%)was from the 41-50 age group

as shown in table 3.

Table 3: Association between Ferritin and Age

Serum ferritin levels P value Odds

ratio
High (%) Normal (%) Low (%) Total (%)
30-40 0 0 4(30.7) 4(4.8)
Age groups 41-50 10(20) 3(14.3) 2(15.4) 15(17.8)
51-60 11(22) 8(38.1) 2(15.4) 21(25) 0.001 5.328
61-70 25(50) 4(19.0) 3(23.1) 32(38.1)
>70 4(8) 6((28.6) 2(15.4) 12(14.3)
Total 50(59.5) 21(25) 13(15.5) 84

4.5.2 Association between Ferritin and Gender

In this study, there was an association between ferritin and gender with odds ratio 1.875

(OR>1). This association was not statistically significant as the P- value was 0.535 (p-

value>0.05) as shown on table 4. This implies that gender had negligible effect on ferritin

levels.

Table 4: Association between Ferritin and Gender

Serum ferritin levels P value Odds

ratio
High (%) Normal (%) Low (%) Total
(5)
Gender Female 41(82) 15(71.4) 11(84.6) 67(79.8) 0.535 1.875
Male 9(18) 6(28.6) 2(15.4) 17(20.2)
Total 50(59.5) 21(25) 13(15.5) 84

38
4.6 Associations between glycemic control and Socio- Demographic Factors

4.6.1 Association between HbA1C and Age

There was a significant positive association between HbA1C and age with P- value 0.038 (p-

value <0.05) and odds ratio 4.667 (OR>1). This implies that HbA1C levels increase with

increase in age. Participants within the 60-70 age groups had the highest HbA1C levels

21(38.8%) whereas just 2(3.7%) from the 30-40 age group had high HbA1C out of the 54

participants with high HbA1C as shown on table 5.

Table 5: Association between HbA1C and Age

HbA1C levels P value Odds

ratio
High (%) Normal (%) Total (%)
30-40 2(3.7) 2(6.6) 4(4.8)
Age groups 41-50 14(25.9) 1(3.3) 15(17.8)
51-60 9(16.6) 12(40) 21(25) 0.038 4.667
61-70 21(38.8) 11(36.6) 32(38)
>70 8(14.8) 4(13.3) 12(14.2)
Total 54(64.3) 30(35.7) 84

4.6.2 Association between HbA1C and Gender

In this study, there was an association between HbA1C and gender with odds ratio 3.375

(OR>1). This association was statistically significant as the P- value was 0.048 (p-

value<0.05) as shown on table 6. This implies that HbA1C varies with gender; HbA1C was

higher in females than in males.

Table 6: Association between HbA1C and Gender

HbA1C levels P value Odds

ratio

39
4.6.3 Association between HbA1C and BMI

In this study, there was an association between HbA1C and BMI with odds ratio

4.277(OR>1). This association was statistically significant as the P- value was 0.047 (p-

value<0.05) as such HbA1C increased with increasing BMI. Majority of the population

18(33.3%) was overweight and had the highest HbA1C levels, 12(22.2%) with high HbA1C

were class 1 obese and 10(18.5%) with high HbA1C were class 2 obese as shown on table 7.

Table 7: Association between HbA1C and BMI

HbA1C levels P value Odds

ratio
High(%) Normal(%) Total (%)
Class 1 12(22.2) 11(36.7) 23(27.4)
Class 2 10(18.5) 2(6.7) 12(14.3)
BMI levels Class 3 0(0) 2(6.7) 2(2.4) 0.047 4.277
Healthy 14(25.9) 3(10) 17(20.2)
Overweight 18(33.3) 12(40) 30(35.7)
Total 54(64.3) 30(35.7) 84

4.7 Indices of Glycemic Control and Ferritin in Study population

For indices of glycemic control, 54(64.3%) patients had poor control of which 47(87.04%)

were females and 7(12.96%) were males, (HbA1C >8%) while 30(35.7%) patients had good

control, (HbA1C <7%) of which 20(66.7%) were females and 10(33.3%) were males. For

ferritin, 50(59.5%) patients had high ferritin levels of which 41(82%) were females (ferritin

>200ng/ml) and 9(18%) were males, (ferritin>350ng/ml) while 13(15.5%) patients had low

ferritin levels, of which 11(84.6%) were females (ferritin <10ng/ml) and 2(15.4%) were

males (ferritin <20ng/ml).

40
 CHAPTER FIVE: DISCUSSION, CONCLUSION AND RECOMMENDATION

5.1 DISCUSSION

5.1.1 Findings and Comparison with other studies

In this study ferritin levels were found to be high in diabetics. For ferritin, 50(59.5%) patients

had high ferritin levels of which 41(82%) were females (ferritin >200ng/ml) and 9(18%)

were males, (ferritin >350ng/ml) while 13(15.5%) patients had low ferritin levels, of which

11(84.6%) were females (ferritin <10ng/ml) and 2(15.4%) were males (ferritin <20ng/ml).For

indices of glycemic control, 54(64.3%) patients had poor control of which 47(87.04%) were

females and 7(12.96%) were males, (HbA1C >8%) while 30(35.7%) patients had good

control, (HbA1C <7%) of which 20(66.7%) were females and 10(33.3%) were males. A

positive correlation (R=0.348)with P-value 0.001 was found between serum ferritin levels

and FBG (figure 9). Also, a positive correlation (R=0.147)with P-value 0.08 was found

between serum ferritin levels and HbA1C. This correlation was not statistically significant as

the P-value is >0.05.this implies that the effect of ferritin on HbA1C is negligible in this

population because the P-value is highly affected by the population size (figure 10). Ferritin

was also found to be associated with age (odds ratio= 5.33) and P-value (0.001), ferritin can

increase with age as a part of the low grade chronic inflammatory state which occurs with

aging(Table 3). Ferritin was also associated with gender (OR=1.875) and p value 0.5 which

is not statiscally significant.This could be due to the fact that gender was not evenly

distributed in the study population [67(79.7%) of the population were females and 17(20.3%)

were males] (Table 4).

41
HbA1C was found to be positively associated with age (OR=4.667) and p value (0.038). The

concentration of HbA1C changes as people age due to fluctuations in hemoglobin and

glucose levels(Table 5).

A positive association was found between HbA1C and gender OR=3.375 and p value 0.048.

HbA1C is affected by hemoglobin levels and males have higher hemoglobin levels than

females. In this study females had higher HbA1C levels due to the fact that 67 (79.7%) of the

population were females(Table 6).

HbA1C was associated with BMI (OR=4.227) and p value (0.0047). HbA1C increases with

increasing BMI. Over and obesity can cause systemic oxidative stress which enhances

glycation of hemoglobin leading to higher HbA1C (Table 7).

Many studies have been carried out worldwide evaluating the relationship between body iron

stores in diabetic patients. Some of these studies failed to identify a significant relationship

between serum ferritin levels and DM [43] A study conducted by Elimam et al. [44] observed

significant positive correlations between HbA1c and serum ferritin levels.

There have been various studies suggesting a relationship between elevated body iron stores

and serum insulin and blood glucose levels [45]. Ferritin, the storage form of iron has been

reported to be inconsistently correlated with markers of glycemic control. Shubam and

Aparnaet al.in 2018 in India reported positive correlation between glycated hemoglobin

(HBA1C) and fasting blood glucose (FBS) with serum ferritin [11]. This result correlated

with findings from Rhaul and Seloemet al. 2018 in Egypt who reported a positive correlation

between serum ferritin and FBS [12].Despite these reports, other controversial studies by

Todd et al , 2021 and Basantaet al, 2021 in India and Spain respectively have reported a

negative correlation between FBS, glycatedhaemogloblin and serum ferritin [13]. These

42
controversies could be due to difference in environmental factors, genetic factors, sex,

ethnicity and certain undefined confounders in the population which alter ferritin level

Although the results of these studies seem to be inconsistent, a review of literature reveals a

higher number of studies suggesting a positive correlation as the case in this study. In a study

by Ford et al. [46] Conducted in 1999, serum ferritin levels were found to be elevated in

9,486 diabetic patients in the United States and positively correlated with FBG and HbA1C.

Furthermore, a study conducted by Wolide et al. [47] in Ethiopia reported higher ferritin,

waist circumference, BMI, and blood pressure values in patients with type 2 DM and positive

correlations with FBG.

5.1.2 Influence of ferritin imbalance on FBG and HbA1C

The specific mechanisms that could lead to high ferritin in type 2 diabetic patients remain

unclear. In recent years, there has been a mounting body of evidence that suggests a major

role of inflammation in the development of type 2 diabetes. Inflammatory cytokines such as

tumor necrosis factor, interlukin- 6 (IL-6) and advanced glycated end products( AGEs)have

been detected in significant amounts in people with type 2 diabetes, due to insulin resistance.

Hepatic iron stores have also been found to increase in diabetic patients. Therefore, high

ferritin levels observed in this study could be due either an increase in extra-hepatic iron

stores or an inflammatory phenomenon as part of an acute phase reaction due to the

production of inflammatory cytokines and advanced glycated end products.

5.1.3 Limitations of this study

1.This study was a cross sectional study, hence causality could not be ascertained as lack of

control group limits to a certain degree the possible range of conclusions.

2. Potential of selection bias since recruitment was restricted only to patients attending the

diabetic clinic at the Buea Regional Hospital.

43
5.2 Conclusion

In this study, serum ferritin levels were high in diabetics attending the Buea Regional

Hospiatal for glycemic control and positively correlated with FBG and HbA1C levels.

Therefore, ferritin levels should be routinely monitored in diabetic patients when assessing

glycemic control as it influences their glycemic status.

5.3 Recommendation and projection for further studies

Findings from this study suggest that ferritin levels should be routinely monitored when

assessing glycemic control status as this could help prevent development of severe diabetic

complications due to either iron overload or iron deficiency as reflected by ferritin levels.

Further studies directly exploring the link between proinflammatory mediators are neccessry

to investigate the nature and significance of the relationship between serum ferritin, insulin

resistance in diabetics. Also, further large scale case-control studies should also be carried out

taking into consideration red cell indices such mean cell volume, hemoglobin content and

mean corpuscular hemoglobin.

44
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45.  Sheu WH, Chen YT, Lee WJ, Wang CW and Lin LY. A relationship between serum

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47.  Wolide AD, Zawdie B, Alemayehu T and Tadesse S. Evaluation of serum ferritin and

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2.3.1 Type 2 Diabetes Mellitus (Type 2 DM)

Ranging from predominantly insulin resistance with relative insulin deficiency to

predominantly an insulin secretory defect with insulin resistance; this form of diabetes,

which accounts for 90–95% of those with diabetes, previously referred to as non–

insulindependent diabetes, type 2 diabetes, or adult onset diabetes, encompasses individuals

who have insulin resistance and usually have relative (rather than absolute) insulin

deficiency. At least initially, and often throughout their lifetime, these individuals do not need

insulin treatment to survive (11). There are probably many different causes of this form of

diabetes. Although the specific etiologies are not known, autoimmune destruction of cells

50
does not occur, and patients do not have any of the other causes of diabetes listed above or

below.

Most patients with this form of diabetes are obese, and obesity itself causes some degree of

insulin resistance. Patients who are not obese by traditional weight criteria may have an

increased percentage of body fat distributed predominantly in the abdominal region.

Ketoacidosis seldom occurs spontaneously in this type of diabetes; when seen, it usually

arises in association with the stress of another illness such as infection. This form of diabetes

frequently goes undiagnosed for many years because the hyperglycemia develops gradually

and at earlier stages is often not severe enough for the patient to notice any of the classic

symptoms of diabetes.

Nevertheless, such patients are at increased risk of developing macrovascular and

microvascular complications. Whereas patients with this form of diabetes may have insulin

levels that appear normal or elevated, the higher blood glucose levels in these diabetic

patients would be expected to result in even higher insulin values had their cell function been

normal. Thus, insulin secretion is defective in these patients and insufficient to compensate

for insulin resistance. Insulin resistance may improve with weight reduction and/or

pharmacological treatment of hyperglycemia but is seldom restored to normal. The risk of

developing this form of diabetes increases with age, obesity, and lack of physical activity.

It occurs more frequently in women with prior GDM and in individuals with hypertension or

dyslipidemia, and its frequency varies in different racial/ ethnic subgroups. It is often

associated with a strong genetic predisposition, more so than is the autoimmune form of type

1 diabetes. However, the genetics of this form of diabetes are complex and not clearly

defined.

51
 APPENDIX

CONSENT FORM

INTRODUCTION: My name is BERINYUY BEDE a master's student in Chemical

Pathology in the Faculty of Health Sciences of the University of Buea carrying out research

entitled: THE RELATIONSHIP BETWEEN SERUM FERRITIN LEVELS AND

GLYCEMIC CONTROL IN TYPE 2 DIABETIC PATIENTS ATTENDING THE

BUEA REGIONAL HOSPITAL..Underthe supervision of Prof. Assob and Dr. Achidi.

“INVITATION TO STUDY”: You are kindly ENCOURAGED to enroll yourself in this

study which I will proceed to describe. This study is to enable us to evaluate the use of iron

parameters like ferritin for the early diagnosis of the onset of diabetes to avoid severe

complications

VOLUNTARY PARTICIPATION: If you decide to participate in this study, you will be

required to answer some questions, and your blood sample will be collected. If you decide to

quit at any time during the study, your decision will be respected without any further

52
 discussion. YOU ARE FREE TO DECIDE TO PARTICIPATE. IT IS NOT AN

OBLIGATION.

STUDY PROTOCOL: You will be asked some questions about your general information
RISKS: NO RISK IS INVOLVED.

CONFIDENTIALITY: All information obtained from you will be kept strictly confidential
and will be used solely for the intended purpose.

I (your name) ______________________________________________________ having

understood the study, after having the participant information sheet well explained
to me, having being given the opportunity to ask questions and time to consider my
participation in the study, do hereby agree to participate in this study.

Date/signature of participant’s guardian Date/signature of investigator

QUESTIONNAIRE
All information will be kept confidential

Name:…………………………………………………………………………………

Age: ………………………………….. Gender………………………………...

Address: ……………………………… Phone number…………………………

Marital status …………………............. Profession: ….…………………………

Weight ………………………………… Height…………………………………

Blood pressure………..………………………………………………………………..

1. Have you been diagnosed with anemia (low level of blood) before? Yes No

2. Have you had malaria within the last 3 months? Yes No

53
3. When where you first diagnosed with diabetes (year)?.............................................................

4. Do you have any other disease(s): Yes No

5. If (5) is No, skip to question (7). If (5) is yes, what other disease(s) do you

have?.....................................................................................................................................

6. Are you currently on any drug(s): Yes No

7. If (7) is no, skip to question (10). If (7) is yes, please indicate the drug(s) you are currently

using and how long you have been using each?..................................................................

…………………………………………………………………………………………

8. Are the drugs prescribed: Yes No

9. Are you using any supplements (If yes, please list them)?

…………………………………………………………………………………………

10. Do you monitor your blood sugar levels at home using a glucometer? (If yes, how often)

……………………………….…………………………………………………………

11. How do you control your blood sugar level at home? (traditional medicine, diet, exercise)

……………………………………………………………………………………………..

12. Do you consume alcohol? (If yes, please indicate the average number of bottles or cans per

week)……………………………………………………………………………

13. Do you exercise? (If yes, please indicate which type of exercise and how often you do it)

…………………………………………………………………………………………

14. How do you control your blood sugar level at home? (diet, exercise, herbs…)

…………………………………………………………………………………………

15. Which foods do you eat frequently (more than twice a week)?

…………………………………………………………………………………………

16. Do you have any complications of diabetes (high blood pressure, kidney problems, cataracts,

urinary tract infections, low blood sugar crisis, diabetic foot ulcer, eye problems etc)? If

54
 yes, list

them......................................................................................................................................

17. How frequently do you visit the hospital for follow up (please indicate the hospital you’ve

been visiting as well)?

……………………………………………………………………………………………

18. When last did you do a glycated haemoglobin test (and what was the result)?

……………………………………………………………………………………………

19. When last did you do a fasting blood sugar test (and what was the result)?........................

Authorisations to Carryout Research

55