Incremental Conductance +Integral Regulator

The sample size was calculated for the initial study proposal compring only two groups using the sample size equation for a study comparing two means as stated below.^{83, 84}

N= 4σ² (Zcrit + Zpwr)² D²

Where N = the total sample size (the sum of the sizes of both comparison groups) σ = The assumed SD of each group (assumed to be equal for both groups)

zcrit = The standard normal deviation value that is given for the desired significance criterion which is 1.960 for the selected significance criteria of 0.05 (95% confidence level) that was used for this study

zpwr = The standard normal deviation value that is given for the desired statistical powers which is 0.842 for the statistical power of 0.80 that was used for this study

D = The minimum expected difference between the two means

On the basis of the results of a previous study which showed the means and standard deviations of the serum leptin concentrations for obese women with and without type 2 diabetes,⁵⁸ the equation above gave the value of N to be 101.2.

This calculated total sample size was increased by 10% in order to accommodate for attrition.

Therefore, N became 111.32 which was rounded off to 112. However, the total sample size was increased to 180 which now covered 3 groups of subjects as follows:

A) 60 Obese type 2 diabetic females, B) 60 Obese non-diabetic female,

C) 60 Non-obese females with type 2 diabetes mellitus.

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3.9.0 MATERIALS: EQUIPMENTS AND REAGENTS 3.9.1 EQUIPMENTS:

1.Mercury Sphygmomanometer (Accoson brand, England) 2.Littman Classic II Stethoscope (Littman Quality TM, USA) 3.SECA weighing scale, England

4.SECA stadiometer, England

5.Flexible non stretch measuring tape

6.Accu-check active Glucometer and test strips.( Roche diagnostics, Germany) 7.Spectrophotometer ( Spectro SC2OD) Labomod Inc England.

8.Glycosylated Haemoglobin Auto-Analyzer (in2it^TM A1c Bio-Rad Laboratories) 9.URIT 8020 Chemistry Auto-Analyzer (URIT Medical Electronic Co,. Ltd, China)

10.ELISA Machine for Leptin and Insulin Assay: (Chemwell 2910 Auto-Analyser [Awareness Technology Incorporated, USA]).

General laboratory equipment including, centrifuge, test tubes, pipettes, sample trays, plain specimen bottles, fluoride oxalate specimen bottles, lithium heparin specimen bottles, EDTA specimen bottles,5ml and 10ml syringes with 21G needles.

3.9.2 REAGENTS:

1.RANDOX Glucose oxidase preparation from RANDOX Laboratories Ltd., UK.

2.RANDOX Lipid profile test kits from RANDOX Laboratories Ltd., UK.

3. Insulin ELISA test kits (Cusabio Biotech Co. Ltd, USA)

4. Human Serum Leptin Sandwich ELISA test kits (by Diagnostic Automation, Inc., USA)

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3.10.0 DATA COLLECTION METHOD: This was by quantitative research method using clinical records, clinical observation including physical examination and laboratory tests.

3.11.0 DATA COLLECTION AND COLLATION: Subjects that met the inclusion criteria were recruited into the study after written informed consent had been obtained from them. Each subject was interviewed using a data proforma (Appendix 1). Demographic data and clinical history were obtained by interviewing the study subjects and also from information in their hospital case folders. Physical examination was performed on each subject to exclude those with physical findings in keeping with the exclusion criteria. Body weight, height, body mass index (BMI)_, waist circumference (WC), hip circumference (HC), waist to hip ratio (WHR) and blood pressure were measured in all study subjects and recorded.

All non-diabetic obese subjects had initial fasting plasma glucose (FPG) screening test done with Accu-check active glucometer in order to aid in the selection of subjects with normoglycaemia alone. Laboratory assessment for all subjects included the collection of 20 mls of venous blood sample under aseptic procedure from the cubital fossa of each subject between 8.00 and 8.30 a.m after an overnight fast of at least 8 hours but not more than 12 hours. These samples were then divided in aliquot into the following specimen bottles consisting; fluoride oxalate bottle, ethylene-diamine-tetra-acetic acid (EDTA) bottle, lithium heparin bottle and a plain specimen bottle respectively and kept in aliquots till ready for analysis.

The samples in the fluoride oxalate bottles were used for analysis of plasma glucose, those samples in the EDTA bottles were used for glycosylated haemoglobin levels (HBA1c) while the samples in the lithium heparin bottles were used for analysis of lipid parameters (total cholesterol, high density cholesterol, low density cholesterol and triglyceride).The samples in the plain specimen bottle were allowed to clot and thereafter were centrifuged at 3000 revolution per

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minute for five minute to extract the serum into another set of plain specimen bottles which were then stored frozen at -20^oC till ready for analysis and measurement of fasting serum insulin and fasting serum leptin.

HbA1C was measured in subjects with type 2 diabetes as an index of glycaemic control. Based on HbA1C results, type 2 diabetic subjects were then categorized as controlled or non-controlled. All subjects were assessed for insulin resistance usingthe homeostasis model assessment of insulin resistance (HOMA-IR) as described by Matthews et al.^{85, 86}

3.11.1 MEASUREMENT OF CLINICAL AND ANTHROPOMETRIC PARAMETERS Weight: Body weight was measured with subjects in light clothing and shoes off to the nearest 0.1 kilogram (Kg) using a standardized weighing scale (Seca weighing scale) placed on an even horizontal hard surface.

Height: Height in metres (m) were measured in subjects without wearing head gear and shoes in erect position against a graduated height scale (Seca stadiometer) to the nearest 0.5 centimetres (cm). Subject stood with both feet flat on the platform at the base of the stadiometer with both heels together and touching the vertical and toes apart pointing outward at about angle 60 degree.

The arms by the side with shoulders relaxed, buttocks, upper back and back of the head touching the vertical.

Head was oriented in Frankfort horizontal plane with the subject looking straight at a distant object. The head is in the Frankfort horizontal plane when the horizontal line from the ear canal to the lower border of the orbit of the eye is parallel to the floor platform and perpendicular to the vertical backboard. Subject was then asked to take a deep breath in and hold his/her breath at maximum inspiration while the height was measured against the vertical at the level of vertex.

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Body Mass Index (BMI): Body mass index (kg/m²) was calculated as the weight of subject in kilogram divided by square height of the same subject in metres and recorded in kilogram per square meter (Kg/m²).⁶⁸

.Waist Circumference (WC): This was measured to the nearest 0.1 cm using a flexible non stretch measuring tape at a point half way between the lower margin of the lowest palpable rib and the top of iliac crest with the tape parallel to the floor. The subject stood in relaxed position with feet close together, arms at the side and body weight evenly distributed and the measurements were taken at the end of a normal expiration. Each measurement was repeated; if the measurements were within 1 cm of one another, the average was calculated.⁷²

Hip Circumference (HC): This was measured to the nearest 0.1 cm using a flexible non stretch measuring tape at a point around the widest portion of the buttocks ( point of maximum extension of the buttock) with the tape parallel to the floor. The subject stood in relaxed position with feet apposed together, arms at the side and body weight evenly distributed. Each measurement was repeated; if the measurements were within 1 cm of one another, the average was then calculated.⁷²

Waist to Hip Ratio (WHR): This was calculated by dividing the measured waist circumference of a subject by the measured hip circumference of the same subject.

Blood Pressure (BP): Brachial blood pressure measurements were taken by auscultatory method using standard mercury sphygmomanometer with appropriate cuff size (cuff bladder encircling at least 80 percent of the arm) for all subjects after five minutes rest in a sitting and a relaxed position. All measurements were taken to the nearest 2.0 millimeters of mercury from the right arm which was positioned at the level of the heart of the subject. Systolic BP was recorded at phase 1 Korotkoff sound while diastolic BP was recorded at phase 5 korotkoff sound. The

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average of two BP measurements taken at least one minute interval were calculated and recorded for subjects according to JNC VII classification⁸⁷ as shown below.

Normal Less than 120/80 mmHg

Pre hypertension 120-139/80-89mmHg

Stage1 hypertension 140-159/90-99mmHg

Stage 2 hypertension Greater or equal to 160/100mmHg

3.11.2 LABORATORY MEASUREMENTS

FASTING PLASM GLUCOSE: This was measured with the spectrophotometer with the aid of glucose oxidase preparation supplied by RANDOX Laboratory Ltd., United Kingdom using the principle of Trinder reaction as described below (Appendix 5).

Enzymatic indicator test based on the Trinder reaction quantified by the formation of a pink quinoneimmine dye. In this reaction plasma glucose is determined after enzymatic oxidation in presence of glucose oxidase (GOD). The hydrogen peroxide formed is catalyzed by peroxidase (POD) and reacts with phenol and 4-aminoantipyrine to form the dye indicator.⁸⁸ The absorbance of the dye indicator formed were the measured by the spectrophotometer to determine the plasma glucose levels.

Glucose + O2 + H2O ^GOD Gluconic acid + H2O2

H2O2 + 4-aminoantipyrine + phenol ^POD Quinoneimine + H2O2

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GLYCOSYLATED HAEMOGLOBIN: This was measured from the venous blood samples by a principle based on boronate affinity chromatography with the aid of the Biorad in-2-it glycosylated haemoglobin autoanalyser and test catridges after the initial standardization of the autoanylyser with a system check cartridge (Appendix 6).

FASTING LIPID PROFILE: Total Cholesterol, Low Density Lipoprotein-Cholesterol, High Density Lipoprotein-Cholesterol and Triglyceride were measured from the plasma samples by spectroscopy technique using URIT 8020 Chemistry Auto-Analyzer (URIT Medical Electronic Co., Ltd., China) with RANDOX Lipid test kits from RANDOX Laboratories Ltd., UK

(appendix 7).

FASTING SERUM LEPTIN: This was measured by double assay from the sera of subjects as total serum leptin. This quantitative estimation of human serum leptin assay was done using the Human leptin kit with batch number 1242-6 (supplied by Diagnostic Automation, Inc, Calabasas, CA 91302USA.) using a Chemwell 2910 miceowll ELISA immunoo-analyser (Appendix 8).

Assay sensitivity: 0.3ng/ml

Specificity of Antibodies (Cross Reactivity) for human insulin: 100%

Intra assay coefficient of variation (CV): 6.42%) Inter assay coefficient of variation (CV): 10.11%

The expected values for a normal weight male = 3.84 ± 1.79ng/ml and for a normal weight female = 7.36 ± 3.73ng/ml.

FASTING SERUM INSULIN: Double assay for serum insulin by a quantitative method with microwell enzyme linked immunosorbent assay (ELISA) human insulin test kits (supplied by Cusabio Biotech Co Ltd.,USA) were performed by Chemwell 2910 Auto-analyser (Appendix 9). The expected reference values for normal adults range from 0.7 to 9.0 µIU/ml and values for

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adults with type 2 diabetes mellitus range from 0.7 to 25 µIU/ml. The sensitivity of this assay was 0.75µIU/ml and the test has no cross reactivity with C-peptide, proinsulin and glucagon. The intra assay coefficient of variation (CV) was 6.0%, while the inter assay coefficient of variation (CV) was 7.4%

INSULIN RESISTANCE (HOMA-IR): The homeostasis model assessment of insulin resistance (HOMA-IR) was used to estimate insulin resistance as stated in the formula below.

HOMA-IR= Fasting Glucose (mmol/l) x Fasting Insulin (µIU/ml) / 22.5 as described by Matthews et al.^{85, 86}

3.11.3 DEFINITION OF TERMS

1. Obesity was defined as BMI ≥ 30 Kg/m ² while non-obese was defined as BMI < 30 Kg/m² and Central Obesity was defined as Waist Circumference (WC) ≥ 88cm or Waist to Hip Circumference Ratio (WHR) ≥ 0.85 for females.^{28, 68, 71}

2. Type 2 diabetic subjects were defined as all previously diagnosed diabetic subjects based on the WHO classification and diagnostic criteria⁷¹ who are currently on oral hypoglycaemic agents and or dietary therapy but not on insulin treatment.

3. Controlled diabetic subjects were taken as subjects with HbA_1C < 7.0% while the Non-controlled diabetic subjects were subjects with HbA1C ≥ 7.0%.

4. HOMA-IR cut off level of ≥ 2 was used to define individuals with insulin resistance as previously described by Oli et al.,⁸⁹ for Nigerians.

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3.11.4 DATA ANALYSIS: This was done using Statistical Package for Social Sciences (SPSS) version 17.0 (SPSS Inc. Chicago Illinois). Except where otherwise stated, results were expressed as mean ± standard deviation (SD) and number count (N) with proportions (%). Descriptive analyses were presented with frequency tables and charts as appropriate.

Continous variables were compared among the three groups with Analysis of Variance (ANOVA).Thereafter, post –hoc test were carried out on the result of ANOVA tables generated to identify the two groups with a statistically significant difference. Relationship between the serum leptin levels and diabetic control of obese and non-obese type 2 diabetic subjects was determined using Student T-Test while correlation using Spearman’s correlation coeficient was used on continuous variables. Classification of diabetes control between the obese and non-obese type 2 diabetic subjects groups and other categorical variables were also compared among the groups using Chi-square test with level of statistical significance set as p ≤ 0.05.

3.11.5 ETHICAL CONSIDERATION: Ethical approval was obtained from the Ethics and Research Committee of OAUTHC (appendix 4). In addition, signed informed consent form was obtained from each subject after a discussion session explaining the required procedure to each subject in her best understood language (Appendices 2 and 3).