This study was conducted at the Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC), Ile-Ife, Osun State. The hospital serves as a tertiary health institution, supporting the health needs of the people of Osun, Ondo and Ekiti States of Nigeria. From hospital records, the average weekly patient attendance at the Diabetes clinic is about 100.
3.2.1 STUDY POPULATION
The study population was made up of people with Type 2 Diabetes, diagnosed using the WHO criteria,39,40 aged between 30 and 64 years who presented to, or are being followed up at the diabetes medical outpatient clinic. The lower age limit of 30 years was chosen because the prevalence of Type 2 Diabetes increases with age greater than 30 years.41 The upper age limit of 64 years was chosen because atherosclerosis increases with age, hence including people who are above 64 years could be confounding.
3.2.2 SAMPLE SIZE DETERMINATION
A pilot study was done to determine factors that may influence the final protocol and also to estimate the sample size by determining the prevalence
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of PAD in PLWDM and controls. The pilot study included 50 people in each study group aged 31-64 years. 13 PLWDM and 4 controls were found to
have PAD, that is a prevalence of 26% and 8% for PLWDM and controls respectively.
The minimum sample size was determined by using the formula for calculating sample size for the comparison of two independent proportions.42 N per group = 2(Zα + Zβ)2 X p(1-p)
(p0 – p1)2
Where N per group = minimum sample size per group.
Zα = Standard normal deviate of α (where α is type 1 error probability) Zβ = Standard normal deviate of β (where ß is type 2 error probability) p = Arithmetic average of the two proportions (p0 & p1)
p0 – p1 = the difference between the prevalence of PAD in people with diabetes and people without diabetes.
The sample size was calculated as follows:
Zα – Standard normal deviate corresponding to the probability α, i.e the probability of making a type 1 error = 5% = 1.96.
Zβ – Standard normal deviate corresponding to the probability β, i.e the probability of making a type 2 error = 5%. Power(1- β) = 95% = 1.65 Thus p0 and p1 are 26% and 8% respectively (from pilot study)
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p = (26+ 8)/2 = 17.
Thus N per group = 2(1.96 + 1.65)2 x 0.17 (1 – 0.17) (0.18)2
N per group = 114.
Correction for non-response43 - Nc = N + 2/(p0-p1)
Where Nc is the sample size after correction for non-response.
Nc = 114 + 2/ (p0-p1) = 125
To enhance the statistical significance of the study, a sample size of 150 was used for each group.
3.2.3 Inclusion Criteria
Type 2 diabetic patients aged between 30 and 64 years ( either known or newly diagnosed) diagnosed using the WHO criteria40 were recruited after informed consent had been obtained in the best understood language of the patient.
The controls were apparently normal people, similar in age and sex with the subjects recruited from the PLWDM, who presented themselves following an advertisement which was pasted at conspicuous areas in the hospital. These controls were not previously diagnosed diabetic. Control subjects found to have impaired plasma glucose measurements during evaluation were also excluded from the study.
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3.2.4 Exclusion Criteria:
Patients who were unwilling to participate in the study.
Patients with bilateral amputation which would make measurement of ABI impossible.
Patients with casts, ulcers, dressings, or other conditions of the participant that may interfere with evaluation of ABI.
Patients with acute febrile illness within 1 month of study which may affect the result of the inflammatory markers that are to be measured.
Patients presenting with diabetic emergencies and other acute clinical conditions e.g myocardial infarction which may also interfere with the results of the inflammatory markers.
● Patients with Ankle-Brachial index (ABI) >1.3. This suggests poorly
compressible arteries at the ankle level due to the presence of medial arterial calcification,22 hence, denoting a falsely elevated reading.
3.2.5. Limitations of Study
1. There is paucity of data in Nigeria on PAD (especially in PLWDM), which precluded the use of local prevalence rates in calculating the sample size. It is hoped that determining the prevalence will add to the literature in this locality.
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2. Normal values and population distribution for the non-traditional risk factors in Nigeria are not known.
3. Facilities for the measurement of other inflammatory markers such as tumor necrosis factor , brain natriuretic peptide (BNP), interleukin-6, adhesion molecules etc, were not available.
4. High sensitivity C-reactive protein is an acute phase reactant and has low specificity,44 hence may be elevated in acute stressful states or inflammatory conditions.
3.3.0 ETHICAL CONSIDERATION
The research proposal was approved by the Research and Ethics Committee of the Obafemi Awolowo University Teaching Hospital, Ile-Ife (Appendix 1). Informed verbal consent was obtained from each subject.
3.4.0 MATERIALS, EQUIPMENT AND REAGENTS 3.4.1. Materials
(1) Biorad In2it glycated hemoglobin test cartridges (Deeside, UK)
(2) General laboratory materials e.g. EDTA bottles, Fluoride oxalate bottles, syringes, cotton wool etc.
(3) Measuring tape (4) Ultrasonic gel
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3.4.2. Equipment
(1) LifeDop Hand held Doppler with 8Hz probe. Model 150R, Madras Engineering, Chennai, India
(2) Hanson weighing scale H89 model, UK.
(3) Leaidal Stadiometer RGZ 160 (Leaidal Medical Ltd, UK) (4) Sphygmomanometer (Accoson Ltd, England).
(5) Stethoscope (Littmann Quality, TM).
(6) Bio-Rad in2it System (Deeside, UK) - for measuring glycated haemoglobin.
(7) Sysmex KX-21N Haematology Analyzer, (Illnois, USA) - for white blood cell count.
(8) Refrigerator.
(9) Bio-Rad PR 3100 TSC Microplate Reader (California, USA) - for reading hs-CRP.
(10) Unico PowerspinTM Fx C806 Centrifuge (New Jersey, USA).
3.4.3 Reagents
(1) High-sensitivity C-reactive protein immunoassay kit (Diagnostic Automation Inc, California, USA).
(2) Reagents for glucose (Fortress Diagnostics, UK).
(3) Reagents for lipids (Fortress Diagnostics, UK).
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3.5.0 STUDY DESIGN AND METHODS
To obtain the requisite number of diabetic subjects, all patients aged between 30-64 years presenting with Type 2 Diabetes who met the inclusion criteria were selected, until 150 patients were obtained. Using data collection sheets (appendix 2), the demographic data, history of cigarette smoking, alcohol consumption, duration of diabetes, duration of hypertension and
history of intermittent claudication were obtained. Other information obtained include medications used (anti-diabetic, anti-hypertensive and anti-lipid) and family history of diabetes. One hundred and fifty apparently healthy people who were matched for age and sex were recruited as control from hospital staff and relatives of patients who presented themselves following an
advertisement which was pasted in conspicuous places in the hospital.
3.5.1. Clinical assessment
(i) The weights and heights of the study subjects were measured using a stadiometer which is made up of a standard weight scale and a graduated height scale. The weight was measured in kilogram (kg)
nearest to 0.50kg (Appendix 3) The height was measured in meters (m) and recorded to the nearest 0.01m with the subjects bare-foot. The body mass index (BMI) was calculated using the formula:
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BMI= Weight (kg)/Height(m2).34,45
(ii) Blood pressure was recorded using mercury Sphygmomanometer with appropriate cuff (encircling at least 75-80% of the arm) after the patient had rested for at least five minutes in a sitting position.
The phase one and five of Korotkoff sounds were used to determine the systolic and diastolic blood pressure respectively.
Two measurements were taken and their average was taken as the individual’s blood pressure.46
(iii) Waist circumference measurements were taken in a standing position at the end of a normal expiration, with the arms at the side, using a non-elastic measuring tape placed in the middle point between the iliac crest and the costal border in mid-
axillary line.47 The hip circumference was measured at the maximum circumference around the hips.48 Waist-to-hip ratio (WHR) was calculated, and central obesity using WHR was defined as > 0.9 in male and > 0.85 in female.49
(iv) Palpation – palpation of the pedal pulses ( dorsalis pedis and posterior tibial artery) were done and graded as normal, reduced or absent.37
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3.5.2. Ankle Brachial Index
Peripheral arterial disease was assessed by determining the ankle- brachial index (ABI) using the hand-held Doppler which is reproducible, non- invasive and reasonably accurate.22 Right and left ABI were calculated by dividing the higher of the systolic pressure from dorsalis pedis or posterior tibial artery (on the right and left respectively) with the higher brachial systolic pressure (procedure in appendix 4).50 The lower of the two ABI values obtained for the right and the left foot was used for analysis.50 PAD was defined as an ABI < 0.9.22,50
3.5.3. Non-traditional markers for Peripheral Arterial Disease
The study also examined traditional risk factors such as age, gender, smoking, obesity, hypertension and dyslipidaemia in relation to the occurrence of PAD. Assessment of non-traditional risk was limited to hs-CRP and white blood cell count because the other markers of non-traditional risk factors are not readily available, and because of cost.
3.5.4 Laboratory Measurements
To assess some of the other associated risk factors for PAD, venous blood was drawn under sterile conditions for the following investigations:
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1. Fasting plasma glucose - measured using glucose oxidase method51 (Appendix 5).
2. Glycosylated haemoglobin- measured using the boronate affinity chromatography52 (Appendix 6).
3. C-reactive protein- measured by the ELISA method44 ( Appendix 7).
4. Fasting lipid profile – measured using enzymatic method53 (Appendix 8) 5. White blood cells count (total and differential) - was be measured using
the Sysmex KX-21N Haematology Analyzer, which utilizes the direct current detection method for white blood cell and differential counting.
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