2 'Class is a relationship and not a thing'
2.2 The struggle of classifications
Study participants were chosen from consenting Nigerian subjects with type 2 DM attending the Diabetes Clinic of LUTH and apparently healthy persons from among LUTH members of staff and General Outpatient Department (GOPD) Clinic.
3.4.1. SELECTION OF STUDY PARTICIPANTS
Type 2 DM participants who met the inclusion criteria were selected from the LUTH Diabetes Clinic register. The controls for the study who had similar demographics were apparently healthy persons. They were selected from LUTH staff and GOPD on satisfying the inclusion criteria.
3.4.2 SAMPLE SIZE DETERMINATION
The sample size was calculated using the Kish and Leslie87 formula.
N = Z2Pq d2 Where,
N = desired sample size.
53
Z = Standard deviation set at 1.96 corresponding to 95% confidence interval.
P = Population of T2DM patients estimated to have insulin resistance (Assuming an unknown prevalence rate of 50% of insulin resistance in type 2 DM subjects in Nigeria)
q = 1.0 – P
d = degree of accuracy required set at 0.10 (10%).
Therefore,
N = (1.96)2 x 0.5 x (1 – 0.5) (0.10)2
= 3.842 x 0.25 0.01
= 96 (nearest whole number) To allow for 20% attrition, 120 subjects with type 2 DM were in the study group while 60 participants without DM were in the control group. Hence, total sample size = 180.
3.4.3 Inclusion and Exclusion Criteria
a.
Inclusion and Exclusion Criteria for DM Subjects Inclusion criteria for DM Subjects included the following:– Type 2 DM
– Aged 30 – 60 years – Nigerian of either sex – Consent to participate.
Exclusion criteria for DM Subjects included the following:
- Type 1 DM
54
- Type 2 DM currently or previously on insulin therapy.
- Outside the age range of 30 and 60 years.
- Presence of liver disease.
- Kidney disease.
- Sickle cell disease
b. Inclusion and Exclusion Criteria for Control Participants Inclusion criteria for control participants included the following:
Being apparently a healthy person aged 30 – 60 years, Nigerian of either sex, or consent to participate.
Exclusion Criteria for Control Participants - Diabetes mellitus
- Impaired fasting glucose - Hypertension
- Outside the age range of 30 and 60 years.
- Liver disease - Kidney disease - Sickle cell disease
55 3.5 MATERIALS AND EQUIPMENT
3.5.1 MATERIALS AND SUPPLIES
- 20ml syringes and 21G needles, (GmbH, Germany)
- Fluoride oxalate, Lithium Heparin, EDTA and plain sample bottles - Methylated spirit and cotton wool
- Disposable gloves
- Flexible non stretch measuring Tapes
- Precision Pipettes (AccuTek Laboratory, San Diego California)
3.5.2 REAGENTS /KITS
- Plasma glucose assay kit(QCA, Spain)
- Glycosylated haemoglobin kits (QCA, Spain )
- Human Insulin assay ELISA kit (Cusabio Biotech Co LTD, USA.
- Serum Bilirubin assay kit (Cusabio Biotech Co LTD, USA.
- Total cholesterol assay kit (Mountain view, CA, USA)
- Triglyceride colorimetric assay kit (Cayman Chemical, Ann Arbor, MI, USA) - Human C-peptide ELISA kit (Webster, TX, USA)
3.5.3 EQUIPMENT
- Weighing Scale (Seca 770 Floor Digital Scale, Hamburg Germany) - Portable stadiometers (Seca 240 wall mounted, Hamburg Germany)
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- Mercury Sphygmomanometer (Accoson England) - Littman’s Stethoscope
- Centrifuge (Gallenkamp, England)
- Ultracentrifuge(Beckman Coulter XL-90, Brea, CA)
- Spectrophotometer (Spectronic ZOD, Milton Roy Company England).
- Refrigerator(Haier Thermocool, Thermocool Ltd,UK)
- Ultra-low temperature freezer (LABFREEZ Instruments Co., Ltd, China) 3.6 CLINICAL AND LABORATORY PROCEDURES
3.6.1 CLINICAL PROCEDURES
On the day of appointment, the following clinical procedures were carried out:
a. Completion of the focused history section of the study protocol. (see Appendix 2).
b. Physical measurements which included
Weight Measurement – Weight was measured with an electronic weighing scale without shoes and with the participants on light clothing to the nearest 0.1kg.
Height Measurement – Height was measured with a portable stadiometer to the nearest 0.1m.
Waist circumference – Using a non-stretch tape, the waist circumference was taken midway between the inferior margin of the last rib and the iliac crest in a horizontal plane to the nearest 0.1cm at the end of normal expiration. 88
Hip circumference – Using a non-stretch flexible tape, the hip circumference was taken around the maximum circumference of the buttocks in a horizontal plane to the nearest 0.1cm. 88
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Blood Pressure Measurement – This was done using a mercury sphygmomanometer in both the sitting and standing positions using the appropriate sized cuff, after the subjects are well relaxed for 5 minutes.
3.6.2 LABORATORY ANALYSIS
In a fasting state, between 08.00am and 10.00am, 14 ml of venous blood was collected from the antecubital fossa of each subject. The blood collected was shared into lithium heparin (5 ml), fluoride oxalate (3 ml), EDTA (3 ml) and plain sample bottles (3 ml) for the relevant tests.
The whole blood in lithium heparin bottles was centrifuged at a speed of 3000 r.p.m for 10 minutes. The plasma derived was stored at – 200C for bilirubin, lipids, creatinine, alanine transaminase, aspartate transaminase and alkaline phosphatase analysis. The whole blood in fluoride oxalate bottles was centrifuged at a speed of 3000 r.p.m for 10 minutes. The plasma derived was stored at – 200C for glucose analysis.
The whole blood in the plain bottle was kept at room temperature to clot before they were centrifuged at a speed of 3000 r.p.m for 10 minutes. The serum derived was stored at – 800C for insulin and c-peptide analysis.
Two ml of the whole blood sample was stored at a temperature of between 2oC and 8oC for glycated haemoglobin assay for no longer than a week.
3.6.2.1 Assay of Plasma Bilirubin.
58
Two hundred µl of plasma was used for plasma total and direct bilirubin assays. The vanadate oxidation method89 was employed for the assay. The intra-assay precision was assessed using duplicates per assay and inter-assay precision was assessed by performing the assays on different days. Details of the procedure are described in Appendix 9.
3.6.2.2 Plasma Glucose Assay
One millilitre of plasma was used for plasma using the Trinder glucose oxidase method.90 The procedure is described in Appendix 6.
3.6.2.3 Serum Insulin Assay
Serum insulin was estimated using a kit based on the ELISA technique with 25µL of serum. The intra-assay precision was assessed using duplicates per assay and inter-assay precision was assessed by performing the assays on different days. The procedure is described in Appendix 4.
3.6.2.4 Assay of Plasma Lipids
Plasma total cholesterol levels was estimated using 0.2 ml of plasma based on the modified method of Liebermann-Burchard.91 A kit employing enzymatic hydrolysis of triglycerides with lipases was used to estimate plasma triglyceride levels using 0.2 ml of plasma.92 HDL-Cholesterol was assayed by precipitation method using 50µl of plasma.93 The procedure is described in Appendix 8. LDL-C was measured using 10µl of plasma by precipitation technique.94 (see Appendix 8).
59 3.6.2.5 Assay of Glycated Haemoglobin
One ml of the whole blood sample which was stored at a temperature of between 2 and 8oC, for no longer than a week was used for glycated haemoglobin assay. Analysis was done using a kit based on the boronate affinity chromatography method using 0.1ml of whole blood.95 (See Appendix 7)
3.6.2.6 Assay of Serum Pancreatic C-Peptide
Serum C-Peptide levels was estimated using a kit based on the ELISA technique with 50µL of serum. The intra-assay and inter-assay precisions of the assays was determined using coefficients of variations. The procedure is described in Appendix 5.
3.7 DATA MANAGEMENT AND STATISTICAL ANALYSIS