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4.2.1 The Whitehall II study

The Whitehall study began in 1967 and originally recruited 18,403 British civil servants, examining the relationship between cardiovascular risk factors and subsequent morbidity and mortality (Fuller et al, 1980). A steep inverse association between social class and mortality from a wide range of diseases was one o f the main findings (Marmot et al, 1984). The Whitehall II study was designed to investigate the causes of the social gradient in a new cohort o f 10,304 civil servants recruited inl985 (Marmot et al, 1991). Between November 1985 and March 1988 participants completed a detailed questionnaire and attended a screening examination (phase 1, baseline). The questionnaire included details on the prevalence of concurrent disease, socio-economic status and lifestyle. A range of anthropometric measurements were recorded and risk factors determined at the screening examination.

During 1991-1993, 8355 (83%) of participants took part in the third phase of data collection (phase 3) when additional risk factors including glucose tolerance and insulin were measured. Phase 5 of the Whitehall II study commenced in 1997, when all participants were invited back for a further detailed assessment.

4.2.2 Study design

Participants who attended the phase 5 follow-up examination (1997 - 1998) with no history of clinical cardiovascular disease who were non-smokers and not treated diabetics were identified. From this large group three separate study groups were selected.

1. Subjects with the metabolic syndrome (MS), defined as being in the adverse quintile for 3 out of 5 of waist-hip ratio, 2 hour post OGTT glucose concentration, systolic blood pressure, plasma triglyceride and HDL cholesterol level, were identified (Brunner et al, 1997).

2. A large unselected (other than no history of clinical cardiovascular disease, non- smokers and not treated diabetics) group individuals with a range of risk factors

but who did not satisfy the criteria for diagnosis of theh MS were recruited as a “control” (C) sample of the general study population.

3. To examine the effect of glucose tolerance and insulin resistance on vascular function. Individuals, with a phase 3 (1991-1993), 2 hour post OGTT glucose concentration in the lowest quintile or highest quintile, were defined as glucose tolerant (GT) and glucose intolerant (GI) respectively. Subjects whose HDL- cholesterol level, fasting triglyceride level, systolic blood pressure and waist-hip ratio were in the adverse quintile were excluded to avoid the confounding effects of these risk factors.

4.2.2.1 Blinding and selection

All subjects who attended the phase 5 examination and satisfied the criteria for one of the groups above were invited, by letter, to attend for further detailed assessment of vascular function. Selection of subjects was performed independently such that investigators having further contact with the subjects were blinded to the subjects group. The local research ethics committee approved this study and all subjects gave written informed consent.

4.2.3 Power calculations

Previous studies have demonstrated wide differences in endothelial function between risk factor groups and healthy controls. On the basis of these studies power calculations determined that 60 subjects in each group would be necessary to detect a half standard deviation difference between the GT and GI groups.

4.2.4 Data collection

Participants in the Whitehall II study, had a detailed assessment at each stage o f the study including a questionnaire and screening examination. The questionnaire included details on current civil service employment grade (divided into 6 levels) which was used as a measure of socio-economic status. Smoking was categorised (never, ex and current) and quantified by packyears.

4.2.4.1 Blood pressure and anthropometry

At each assessment blood pressure was measured twice, in the sitting position after 5 minutes rest, with the Hawksley random-zero sphygmomanometer. Hypertension was defined as systolic blood pressure > 160mmHg and / or diastolic blood pressure > 90mmHg or on antihypertensive medication. Height and weight were measured, and body mass index (BMI) was calculated as weight (Kg) / height (m)^. Waist circumference (smallest circumference at or below the costal margin) and hip circumference (at the level of the greater trochanter of the right femur) were recorded with subjects in the standing position, unclothed using a fiberglass tape measure at 600 g tension. Waist-hip ratio was calculated as the ratio of the two circumference measurements.

4.2.4.2 Assessment o f insulin resistance

Glucose tolerance was defined on the basis of a 75g oral glucose tolerance test (OGTT) (Brunner et al, 1997). Fasting and 2 hour post OGTT plasma glucose (electrochemical glucose oxidase) and insulin (radioimmunoassay using polyclonal guinea pig antiserum) levels were measured. Whole body insulin sensitivity was inferred by calculation of an insulin sensitivity index (ISI) as recently described (Matsuda and DeFronzo, 1999). ISI = 10,000/square root of ([fasting glucose x fasting insulin] x [mean glucose x mean insulin during OGTT])

A fasting venous sample of blood was drawn from each subject. Cholesterol concentration was determined by cholesterol oxidase-peroxidase colorimetric method (Boehringer Mannheim) and triglycerides by enzymatic colorimetry. High-density lipoprotein (HDL) cholesterol was determined after dextran sulphate-magnesium chloride precipitation of non-HDL cholesterol. LDL cholesterol was calculated using the Friedewald formula (Friedwald et al, 1972).

4.2.4.4 Inflammatory and coagulation factors

Levels of von Willebrand factor (vWF), plasminogen activator inhibitor type-1 (PAH) C-reactive protein (GRP), interleukin-6 (1L6) and serum amyloid A (SAA)

were measured using ELISA. Fibrinogen levels were measured using a modification of the clotting method of Clauss (Oosting and Hoffmann, 1997).

4.2.4.5 Risk factors and cardiovascular risk index (CVRI)

To analyse overall differences in risk factors and metabolic measurements between the groups, a ‘z-score’ was calculated for each variable and the risk burden for each subject, in relation to the population as a whole, determined as the mean z-score for the risk factors (age, total, HDL and LDL cholesterol level, triglyceride level and systolic and diastolic blood pressure) and insulin resistance (fasting and two-hour post OGTT glucose and insulin level and the ISI) calculated.

In addition, a composite cardiovascular risk index (CVRI) (including age, gender, systolic blood pressure, presence o f diabetes, and smoking status) was calculated for each subject, for phase 3 and phase 5 data, using an algorithm derived from the Framingham database (Anderson et al, 1991). Whilst this algorithm does not take into account many of the more recently recognised risk factors it has proven a reliable predictor of risk and risk reduction following intervention in previous studies (Haqe/ûf/., 1999).

4.2.5 Assessment of vascular function

Brachial artery flow-mediated dilatation (FMD) and carotid artery distensibility and intima media thickness were measured non-invasively using high-resolution external ultrasound as previously described. All studies were performed in a temperature controlled laboratory after subjects had been resting for at least 10 minutes.

4.2.5.1 Assessment o f brachial artery endothelial function

Brachial artery flow-mediated dilatation and response to glyceryl trinitrate were assessed in the right arm using the standard protocol. Brachial artery reactivity was measured over the whole time course of the experiment using the digital edge detection software. A 5 minute period of forearm ischemia was used to stimulate reactive hyperaemia and GTN (400 pg sublingually) used as the stimulus for endothelium-independent dilatation. Blood flow velocity was estimated using pulsed

wave Doppler at rest, prior to release of the cuff and at 15 second intervals following its release.

Baseline brachial diameter was determined as the average of all measurements made over the first minute of rest prior to cuff inflation or administration of GTN. Following release of the tourniquet images acquired over 12 second intervals (4 frames) were averaged whilst after administration of GTN the mean vessel diameter between 180 and 240 seconds was used as the measure of endothelium-independent dilatation. The maximal flow-mediated dilatation (FMD) and dilatation to GTN were expressed as a percentage in relation to their respective baselines. Peak reactive hyperaemia (VTIpeak) and the AUC of reactive hyperaemia (VTIauc) over the first 60 seconds were calculated as a measure of the stimulus to the endothelium.