PUBLICACIÓN DEL 18 DE MAYO  Identificación.

metres. (kg/m²).

4.9.4 Measurement of Waist Circumference (Abdominal Obesity)

Measurement of the waist circumference was taken using a stretch-resistant tape (HTS, China) which was standardised by measuring known lengths traceable to NIST. The tape measure was validated by taking each subject’s waist circumference three times and each waist circumference measurement was taken from the zero-reading of the tape rule. The tape rule was wrapped snugly, but not constrictingly, around the subjects at a level parallel to the floor, midpoint between the top of the iliac crest and the lower margin of the last palpable rib in the mid axillary line. It was read to the nearest centimetre, at the end of several consecutive natural breaths.¹¹⁹

4.9.5 Precision and Controls

Precision controls were run during the laboratory analyses. Precision studies were carried out for plasma glucose using bovine precision multi-sera (levels 1, 2 and 3) (RANDOX laboratories, UK). Human sera with protease inhibitors, (GenWay Biotech, USA) levels one and two (low and normal), representing clinically relevant pathologic and normal ranges respectively; were assayed for osteocalcin. Low and high controls (Fortress diagnostics, UK), were assayed for glycated haemoglobin. Two of each level of control for the analytes were included in each run. Between-run precision studies were carried out by determining the mean, standard deviation and coefficient of variation for the different control levels.

Within-run precision was performed by analysing twenty control specimens for each level of

38 control in one run and estimating the mean, standard deviation and coefficient of variation for each level.

4.9.6 Plasma Osteocalcin Assay¹²⁰

A solid phase sandwich enzyme linked immunosorbent assay (GenWay Biotech, USA), was used for the quantitative determination of intact human osteocalcin in plasma, and read out using Acurex plate read (Acurex Diagnostics, USA). Low and normal level controls were assayed in duplicate during each run.

Assay Principle

The assay uses monoclonal antibodies directed against distinct epitopes of the human osteocalcin. The calibrators with known concentration of target protein, controls and samples with unknown concentrations of osteocalcin, react with the capture monoclonal antibody (Mab1), coated on a microtitre plate, and with a monoclonal antibody (Mab2) labeled with an enzyme. After an incubation period to allow formation of a sandwich:

Mab1-human osteocalcin-Mab2-enzyme, the microtitreplate is washed to remove unbound enzyme-labeled antibody. Bound enzyme-labeled antibody is measured through a chromogenic reaction. Chromogenic solution is added and incubated. The reaction is stopped with the addition of a stop solution and the microtitreplate is then read at the appropriate wavelength. The amount of substrate turnover is determined spectrophotometrically by measuring the absorbance, which is proportional to the osteocalcin concentration.

Reagent Preparation

A. Calibrators: Each calibrator was reconstituted with 1.0 ml distilled water.

B. Controls: Each control was also reconstituted with 1.0 ml distilled water.

39 C. Working anti-osteocalcin-Horse Radish Peroxidase Conjugate: Extemporaneous preparation was carried out, 40μl of the concentrated anti-osteocalcin-Horse Radish Peroxidase conjugate was added to 2 ml of conjugate buffer. A vortex mixer was used to homogenize the solution.

D. Working Wash Solution: An adequate volume of working wash solution was prepared by adding 199 volumes of distilled water to 1 volume of Wash Solution (200x). Unused solution was discarded at the end of the day.

Procedure

i. The required number of wells for a run was selected. The unused wells were resealed in the bag with a desiccant and stored at 2-8°C.

ii. The wells for the assay are secured into the holding frame.

iii. Twenty five μl of each calibrator, control and sample were pipette into the appropriate wells.

iv. One hundred μl of working anti-OST-HRP conjugate was pipetted into all the wells.

v. The mixture was incubated for 2 hours at room temperature . vi. The liquid was aspirated from each well.

vii. The plate was washed 3 times by:

Dispensing 0.4 ml of Wash Solution into each well

Aspirating the content of each well

viii. One hundred μl of the chromogenic solution was pipetted into each well within 15 minutes following the washing step.

ix. The microtiterplate was incubated for 30 minutes at room temperature, horizontally and away from direct sunlight.

40 x. One hundred μl of Stop Solution was pipetted into each well.

xi. The absorbances and corresponding concentrations at 450 nm (reference filter 630 nm) were read within 1 hour.

Assay Characteristics

The within-run and between-run coefficient of variation (CV) values were 4.7% and 6.8%

respectively for the level 1 control and 3.6% and 5.5% respectively for the level 2 control.

The assay reference range is 5.0 – 25.0 ng/ml. Haemolysis causes a negative interference while lipaemia causes a falsely elevated result.

4.9.7 Glycated Haemoglobin (HbA1c) Assay¹²¹

An ion-exchange chromatographic-spectrophotometric method was used for the HbA1c assay with reagents from Fortress Diagnostics, UK and the absorbances were read using SM23A spectrophotometer (Microfield Instruments, England). A low and high level control were assayed in duplicate in each run.

Assay Principle

After preparing a haemolysate where the labile fraction is eliminated, haemoglobins are retained by a cationic exchange resin. HbA1c is specifically eluted after washing away the HbA1a+b fraction and is quantified by direct photometric reading at 415nm.

Assay Procedure

a) Haemolysate preparation and labile fraction elimination i. Columns and reagents were brought to room temperature.

41 ii. Fifty µl of sample and 200µl of reagent 1 (Potassium Pthalate solution) were pipetted into a test tube, shaken thoroughly and left for 10 – 15 minutes. This haemolysate was used in steps v and x.

b) Column Preparation

iii. The upper cap of the column was removed and the tip snapped at the bottom.

iv. The column was left to drain completely to waste.

c) HbA1c Separation

v. 50µl of the haemolysate was pipetted carefully onto the upper filter in the column and the column was left to drain to waste.

vi. 200µl of reagent 2 (Phosphate buffer- 30mmol/l) was pipetted onto the filter paper to drain any sample left on it.

vii. 2ml of reagent 2 was pipette into the column and the column was left to drain to waste.

viii. The column was placed over a new test tube and 4 ml of reagent 3 (Phosphate buffer- 72mmol/l) was added and the eluate collected (HbA1c fraction).

ix. The eluate was thoroughly mixed and the absorbance (A) of the HbA1c fraction read at 415nm against distilled water.

d) Reading of HbA total

x. Twelve ml of reagent 3 and 50µl of haemolysate were pipetted into a test tube.

xi. The solution was mixed thoroughly and its absorbance (A total), read against distilled water at 415nm.

e) Calculation

The percentage HbA1c in the sample was calculated using the expression.

42

% HbA1c = (Absorbance of HbA1c) / (Absorbance Hb total) x (volume of HbA1c {4ml}) / (volume of Hb total {12ml}) x 100%

For standardization of results the values obtained were converted to equivalent of the United States National Glycohaemoglobin Standardization Programme (NGSP), certified method using the formular:

% HbA1c (NGSP) = 0.86 x % HbA1c (Fortress) + 0.24¹²² Assay Characteristics

The within-run CV and between-run CV for the level 2 control was 4.7% and 5.0%

respectively, while the level 3 control had within-run CV of 3.2% and between run CV of 4.6%. The assay reference range is 4.0 -6.0% while the analytical measurement range is 4.0% -17.0%. Lipaemia may cause falsely elevated results. Interfering substances may also include haemoglobins S and C, however, this had been taken care of in the exclusion criteria.

4.9.8 Fasting Plasma Glucose Assay¹²³

A quantitative-enzymatic-spectrophotometric method, was used for the assay of glucose with reagents from Biolabo laboratories, France and SM 23A spectrophotometer (Microfield Instruments, England). Levels 1, 2 and 3 control sera were assayed in duplicate in each run.

Assay Principle

Glucose in the calibrator, controls and plasma is oxidised by glucose oxidase to gluconic acid and hydrogen peroxide, which in conjunction with peroxidase, reacts with chloro-4-phenol and 4-amino-antipyrine to form a red quinoimmine. The absorbance of the coloured