CONCISE DESCRIPTION OF THE STUDY PROCEDURES
The Study Protocol was approved by local ethics committee and participants gave informed consent to participate
Population and field procedures
All examinations were performed in the morning, in a quite and comfortable environment, according to a carefully standardized protocol. The participants underwent physical examination, anthropometric measurements, blood pressure measurements and blood tests. A fixed sequence questionnaire was given, including demographic information, job history, medical history, food history, questionnaires on habitual physical activity.
Blood pressure measurement
Blood pressure measurements were performed by trained observers who had attended training sessions for standardization of the procedures. The operator code was recorded in order to check for possible measurement biases. Blood pressure was measured between 8.00 and 10.00 h, after the participants had been sitting upright for at least 10 min. Systolic and diastolic blood pressure were taken three times 2 min apart with a random zero sphygmomanometer (Gelman Hawksley Ltd. Sussex, UK). The first reading was discarded and the average of the second and third reading was recorded for systolic and diastolic blood pressure.
Anthropometric measurements were performed by trained observers who had attended training sessions for standardization of the procedures. The operator code was recorded in order to check for possible measurement biases. Body weight and height were measured on a standard beam balance scale with an attached ruler. Body weight was measured to the nearest 0.1 kg and height was measured to the nearest cm, with participants wearing only light indoor clothing without shoes. The body mass index was calculated as weight in kg divided by the square of the height in metres.
The umbilical circumference was measured at the umbilicus level with the subject standing erect with his abdomen relaxed, his arms at the sides and his feet together; the arm circumference was measured at the mid- point between the acromion and the olecranon with the arm relaxed and hanging just away from the side of the body, after marking the acromion with the arm lexed at a 90° angle. Measurements were performed to the nearest 0.1 cm using a flexible inextensible plastic tape. The sagittal (antero-posterior) abdominal diameter (SAD) was measured using the Holtain- Kahn abdominal caliper (Holtain Ltd. Cross- well, UK). The distance between the participants’ back and the front of the participants’ abdomen was read on the centimetre scale of the calliper to the nearest 0.1 cm. Since with the participants lying on his back in the supine position, the abdominal subcutaneous fat tends to slipper along the flanks, the sagittal abdominal diameter is taken as an indirect estimate of the amount of visceral fat. This index has been validated against direct measurements of visceral at by computerized tomography and nuclear magnetic resonance (Kvist et al, 1988; Van der Kooy et al, 1993). Subscapular and triceps skinfold thickness were measured using a Lange skinfold calliper (Beta Technology inc., Santa Cruz, California, USA). The subscapolar fold was picked- up just below the inferior angle of the scapula at 45° to the vertical. The tricipital fold was measured at the mid- point of the back of the upper arm between the tip of the olecranon and the acromion process of the scapula. The means of the repeat measurements at each site were used for analysis.
Blood sampling and biochemical assays
A fasting venous blood sample was taken in the seated position without stasis between 8.00 and 10.00 h, after the blood pressure measurements, for determination of serum lipids, insulin, glucose, creatinine, sodium, lithium and uric acid levels. The blood specimens were immediately centrifuged and stored at -70° until analysed. Serum cholesterol, triglyceride, glucose and uric acid levels were measured with automated methods (Cobas- Mira; Roche, Italy); creatinine by the picric acid colorimetric method, serum and urinary electrolytes by atomic absorption spectrophotometry, uric acid by an enzymatic colorimetric method.
Plasma insulin concentration was measured by radioimmunoassay (Insulin Lisophase, Technogenetics, Milan, Italy) and insulin resistance was estimated by the homeostasis model assessment (HOMA) using the formula: fasting plasma insulin (uU/ml) X fasting plasma glucose (mmol/l)/ 22.5, as described by Matthews et al.. Although this method does not give a direct measure of insulin- dependent glucose utilization, it has been validated against the euglycemic hyperinsulinemic clamp (Bonora et al, 1998) and has no practical alternatives in large- scale epidemiological investigations.
Plasma aldosterone was measured by radioimmunoassay (DRG Instruments, GmbH- Germany).
C.R.P. was measured by immunoturbidimetric assay (Roche Diagnostics GmbH).
Microalbuminuria was measured by immunoturbidimetric assay (ABX Diagnostics).
Leptin was measured by Elisa immunoassay (Biovendor Laboratory Medicine, Inc.).
Adiponectin was measured by Elisa immnuoassay (Biovendor Laboartory Medicine, Inc.)
Protocol for the study of renal sodium handling
The participants consumed their evening meal at no later than 19.00 hours and took a 300 mg lithium carbonate capsule (carbo- lithium, IFI, Milan, Italy) delivering 8.1 mmol elemental lithium at 22.00 hours with 400 ml tap water. On the morning of the Study, after voiding and discarding overnight urine and having drunk 400 ml tap water, they produced a fasting timed urine collection. The collection time and volume were recorded and a specimen was used for the analysis. At the midpoint of the urine collection a blood sample was obtained by venepuncture with the subject in the seated position and without stasis between 08:00 and 11:00 hours. Creatinine, sodium, lithium and uric acid on serum and urine samples were measured as described.
Standard formulae were used to calculate the clearance of creatinine, sodium, lithium and uric acid. Values are expressed as fractional excretion (%), dividing the respective clearance by the clearance of creatinine, to allow for the confounding effects of age, body mass and possibly incomplete urine collections. The creatinine clearance was taken as an index of the glomerular filtration rate and was corrected for body surface area.
This protocol has been extensively validated in our laboratory, as previously described (Strazzullo et al 1988).
Protocol for the Food- Frequency Questionnaire
To evaluate dietary habit and lifestyle of the Olivetti Prospective Heart Study’s population it has been used a food- frequency questionnaire, one year closed- ended food- frequency questionnaire with 120 items.
Conversion in nutrients has been performed by Naf programm (Nutritional Analysis of Food Frequency Questionnaire), version 5.3, used by Epic Italy.
The food frequency questionnaire has been validated with food diary of 24 h for intake of nutrients.
Statistical analyses are performed using updated versions of the Statistical Package for Social Sciences (SPSS- PC; SPSS Inc., Chicago, Illinois, USA).