The making of valued selves

The World Health Organization criteria (1999) require presence of diabetes mellitus, impaired glucose tolerance, impaired fasting glucose or insulin resistance, AND two of the following:

- Blood pressure: ≥ 140/90 mmHg

- Dyslipidaemia: triglycerides (TG): ≥ 15039 mg/dl and/or high-density lipoprotein cholesterol (HDL-C) ≤ 35 mg/dl (male), ≤ 39 mg/dl (female)

- Central obesity: waist:hip ratio > 0.90 (male), > 0.85 (female), and/or body mass index >

30kg/m²

- Microalbuminuria: urinary albumin excretion ratio ≥ 20 mg/min or albumin:creatinine ratio ≥ 30mg/g

131 7.4 APPENDIX 4

SERUM INSULIN ASSAY TEST PRINCIPLE

Cusabio Biotech insulin ELISA is a solid phase two-site enzyme immunoassay based on the sandwich technology, in which two monoclonal antibodies are directed against separate antigenic determinants on the insulin molecule. Insulin in the sample reacts with anti-insulin antibodies bound to micro-tiration wells and peroxidase-conjugated and anti-insulin antibodies in the solution.

ASSAY PROCEDURE

1. Prepare sufficient microplate wells to accommodate calibrators, controls and all test samples in duplicate

2. Pipette 25µL each of Calibrators, controls and samples into appropriate wells 3. Add 100µL enzyme conjugate solution.

4. Incubate 1 hour on shaker at room temperature (18 to 25°C) 5. Wash plate six times with automatic plate washer.

6. Add 200µL substrate solution, tetramethyl benzydine (TMB – H2O2) 7. Incubate 15mins on shaker at room temperature

8. Add 50µL Stop solution. Shake for approximately 5 seconds on shaker 9. Measure absorbance at 450nm and calculate results. Read within 30mins.

132 7.5 APPENDIX 5

SERUM C-PEPTIDE ASSAY Test principle

The C-Peptide ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA), based on the principle of competitive binding. The microtiter wells are coated with anti-mouse antibodies, which bind a monoclonal antibody directed towards a unique antigenic site on the C-Peptide molecule. Endogenous C-C-Peptide of a patient sample competes with a C-C-Peptide horseradish peroxidase conjugate for binding to the coated antibody. After incubation the unbound conjugate is washed off. The amount of bound peroxidase conjugate is reverse proportional to the concentration of C-Peptide in the sample. After addition of the substrate solution, the intensity of colour developed is reverse proportional to the concentration of C-Peptide in the patient sample.

Assay procedure

All standards, samples, and controls should be run in duplicate concurrently so that all conditions of testing are the same.

1. Secure the desired number of microtiter wells in the holder.

2. Dispense 50 µl of each Standard, controls and samples with new disposable tips into appropriate wells.

3. Dispense 50 µl Antiserum into each well

4. Dispense 100 µl Enzyme Conjugate into each well.

133 5. Thoroughly mix for 10 seconds.

6. Incubate for 60 minutes at room temperature.

7. Briskly shake out the contents of the wells. Rinse the wells 3 times with diluted Wash Solution (400 µl per well). Strike the wells sharply on absorbent paper to remove residual droplets.

8. Add 100 µl of Enzyme Complex to each well.

9. Incubate for 30 minutes at room temperature.

10. Briskly shake out the contents of the wells.

11. Add 100 µl of Substrate Solution to each well.

12. Incubate for 20 minutes at room temperature.

13. Stop the enzymatic reaction by adding 100 µl of Stop Solution to each well.

14. Read the optical density at 450±10 nm with a microtitre plate reader within 10 minutes after adding the Stop Solution

134 7.6 APPENDIX 6

PLASMA GLUCOSE ASSAY

Trinder’s analytical method will be used. It uses glucose oxidase enzyme buffered in phenoxylate and dissolved in a coloured reagent. A solution constituted in 100mls containing:

Potassium dihydrogen sulphate 38.9mls, Disodium hydrogen phosphate 61.1mls, Glucose oxidase 1.0ml, Phenol 0.64 ml, 4-4aminophenaxone 20mg, sodium 100mg.The solution is stored at 4⁰C before use.

Glucose Estimation

1. One ml of glucose oxidase containing solution is placed inside a test-tube.

2. One ml of plasma is added and the mixture is incubated at room temperature for 15mins.

3. It is allowed to cool to room temperature during which time the colour changes from colourless to pink depending on glucose concentration.

4. The absorbance is read in a spectrophotometer at wave length of 540nm.

5. The reading is compared with the one for a standard glucose solution with a known glucose concentration which would have been incubated with the glucose oxidase solution.

135 7.7 APPENDIX 7

GLYCATED HAEMOGLOBIN ASSAY

Measurement will be done using the Boronate affinity chromatography technique.

Test Principle

Glycated proteins differ from non-glycated proteins by the attachment of a sugar moiety(s) at various binding sites by means of a ketoamine bond. Glycated haemoglobin, thus, contains 1,2-cis-diol groups not found in non-glycated proteins. These diol groups provide the basis for separation of glycated and non-glycated components by boronate affinity chromatography. In this analytical technique, a boronate such as phenyl boronic 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, the glycated component is eluted from the column with a reagent that displaces it from the boronate.

Test procedure

HbA1C SEPARATION

1. The resin suspension is mixed thoroughly and 3ml is dispensed into one plastic tube.

2. 0.1ml of the haemolysed sample is added and a separator is introduced into the tube at approximately I cm height over the liquid surface.

3. The resin suspension and the haemolysed sample are mixed for 5mins

4. The separator is pressed against the bottom of the tube until the resin is completely packed.

5. The supernatant is decanted and the absorbance (OD,) read.

136 TOTAL HAEMOGLOBIN

1. 20ul of the haemolysed sample is dispensed into a test tube.

2. 5ml of deionized H20 is added and mixed vigorously.

3. The absorbance is read (OD total) Reading Wave length: 415nm

Blank: water Stability: 1 hour

CALCULATIONS

The value of the ratio C = (OD/OD total) of the sample and corresponding to the standard are determined.

% HbAlC sample = (C sample / C std) x % HbAlC STD (Stated on the label of the vial)

137 7.8 APPENDIX 8

ASSAY FOR SERUM LIPIDS

A. SERUM TOTAL CHOLESTEROL ASSAY USING MODIFIED