De la encuesta a directivos

On the day of appointment, the clinical procedures were done as follows:

a. Completion of the focused history section of the study protocol (see Appendix 3).

b. Physical measurements which included:

 Weight Measurement – Weight was measured with an electronic weighing scale without shoes and with the subject on light clothing to the nearest 0.1kg⁷³ (Details in Appendix 4 )

 Height Measurement – Height was measured with a portable stadiometer to the nearest 0.1cm (Details in Appendix 4 ).⁷³

 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 (Details in Appendix 4 )⁷³

 Hip circumference – using a non-stretch tape, the hip circumference was taken around the widest portion of the buttocks, to the nearest 0.1cm (Details in Appendix 4)⁷³

 Blood Pressure Measurement was done using a mercury sphygmomanometer in both the sitting and standing positions using the appropriate sized cuff, after the subjects were well relaxed for 5min (see Appendix 4)⁷³

3.6.2. Laboratory Procedures

In a fasting state, between 08.00am and 10.00am, 10 ml of venous blood was collected from the antecubital fossa of each subject. Tourniquet was lightly applied about 2 to 4 cm above puncture site and left for less than 2 minutes. The blood sample collected was shared into appropriate sample bottles for the relevant tests as stated below.

3.6.2.1. Assay of serum vitamin D

Three milliliters of blood sample were collected into plain bottles. The samples were left for 15 to 20 minutes to clot at room temperature and then centrifuged at a speed of 3000 r.p.m for 10 minutes to obtain the serum which was stored at – 80⁰C (in ultralow freezer) until when used.

The Agilent series 1120 HPLC system with quaternary pump was employed for the assay. Prior deproteinization of the serum was done by liquid phase extraction using acetonitrile. The extracted sample was transferred to an auto sample vial and 20µL injected into the HPLC system. Separation was performed on a Zorbrax eclipe XDB-C8 column (4.6 x 150mm, 5µm size) maintained at 40 °C. The mobile phase was methanol/acetonitrile (15:85) and the flow rate was 1 ml/min. Detection was at 280 nm, the injection volume was 20 µL. The vitamin D3 peaks were resolved with retention time of 4.6 to 4.8 min.Details of the HPLC procedure is described in Appendix 5.

3.6.2.2. Plasma Glucose Assay: About 2ml of blood was transferred into fluoride oxalate bottles. The sample was then centrifuged at a speed of 3000 r.p.m for 10 min. The plasma was separated and stored at – 20⁰C until the time for analysis. The Trinder (Glucose-oxidase) method⁷⁴ was employed. The process involved the addition of 2ml of plasma into about 2ml of a glucose oxidase containing solution in a test-tube. The mixture was incubated at room

temperature for 15 minutes; it was then allowed to cool to room temperature during which time the colour changes from colourless to pink depending on glucose concentration. The absorbance was read in a spectrophotometer and the reading is compared with that of a standard glucose solution with a known glucose concentration which had been incubated with the glucose oxidase solution.

The procedure is described in Appendix 6.

3.6.2.3. Glycated haemoglobin assay: About 1ml of blood was transferred into potassium EDTA bottles. The blood sample was stored at a temperature of between 2^oC and 8 ^oC, for not longer than four days. Analysis was done via the Boronate affinity chromatography technique, ⁷⁵ using the Bio-rad in2it automated Glycohemoglobin analyzer.

In this analytical technique, a boronate such as phenylboronic acid is bonded to the surface of the column support. When a solution of proteins (e.g. hemolysate) is passed through the column, the glycated component is retained by the complexing of its diol groups with the boronate. After the unretained non-glycated component elutes from the column and passes through the spectrophotometric detector, where it is detected at wavelength of 413 ±2 nm, the glycated component is eluted from the column with a reagent that displaces it from the boronate and then passes through the detector. Details of the Boronate affinity chromatography technique in Appendix 7.

3.6.2.4. Assay of serum insulin and other tests

About 4ml of blood was transferred into plain bottles for the assay of serum insulin and the other tests. The serum obtained following centrifugation of the clotted blood at a speed of 3000 r.p.m

for 10 min, was then stored at – 20°C to be used for serum insulin assay, serum creatinine, alanine transaminase, calcium, phosphate and albumin.

For the insulin assay, the Enzyme-linked immunosorbent (ELISA) method was used. The Insulin ELISA Kit is a solid phase ELISA based on the sandwich principle. The micro-titer wells are coated with a monoclonal antibody directed towards a unique antigenic site on the insulin molecule. An aliquot of patient sample containing endogenous insulin was incubated in the coated well with enzyme conjugate, which is an anti-insulin antibody conjugated with Biotin.

After incubation the unbound conjugate was washed off. During the second incubation step Streptavidin Peroxidase enzyme complex binds to the biotin-anti-Insulin antibody. The amount of bound hormone complex is proportional to the concentration of insulin in the sample. Having added the substrate solution, the intensity of colour developed is proportional to the

concentration of Insulin in the patient sample.

(See details of procedure in Appendix 8). Insulin resistance and beta cell function were estimated with the Homeostasis model assessment (HOMA). ⁷⁶

3.7 DATA MANAGEMENT AND STATISTICAL ANALYSIS