This study is part of the “Invecchiare in Chianti” (Aging in the Chianti area, InChianti) study, a prospective population-based study of older people, designed by the Laboratory of Clinical Epidemiology of the Italian National Research Council of Aging (INRCA, Florence, Italy).
The study included 1156 older participants (age 65–102 years), randomly selected from residents in two towns of the Chianti area (Greve In Chianti and Bagno a Ripoli, Tuscany, Italy)[20
]. The collection of the data started in September 1998 and was completed in March 2000. A detailed description of the sampling procedure and data collection method has been previously published [20
]. The Italian National Research Council on Aging (INRCA) Ethical Committee ratified the entire study protocol.
In the present study 1044 individuals aged 65 years and older in which inflammatory markers and plasma lipids had been measured at baseline were included. One hundred and twelve participants could not be included into the present study as they had refused to provide blood sample for analysis at baseline. They were characterized by older age (mean 79.9, S.D. 8.6 years), while no differences in socio-demographic characteristics emerged.
2.1. Markers of inflammation
Blood was drown in the morning after a 12-h fasting, and after the patient has been sedentary in sitting or supine position for at least 15 min. Sampled blood was transferred, making it flowing down the side of the tube, never directly squirted into the center, in order to minimize mechanical disruption or turbulence that may result in haemolysis or activation. After having been aliquoted, serums was frozen and stored at −80 °C till the tests were performed.
IL-6, IL-1β, and TNF-α were quantified with immunoassay kits (BioSource Cytoscreen human IL-6 and human TNF-α UltraSensitive kits). The minimum detectable concentrations were 0.10, 8, and 0.09 pg/ml, respectively. The interassay coefficient of variation was 7% for all three kits.
Serum interleukin 10 (IL-10) was detected by Human IL10 CytoSETS TM ELISA kits (BIOSOURCE Internetional Inc., Camarillo, CA, USA). The minimum detectable concentration was 1.00 pg/ml; the inter-assay CV was 8.6%.
Serum interleukin 18 (IL-18) level was detected in duplicate using highly sensitive quantitative sandwich assays (ELISA) (Quantikine HS, R&D Systems, Minneapolis, MN). The lower limits of detection were 0.7 pg/mL; the coefficient of variation for IL-18 test was approximately 7%. Determination of CRP level was based on a high sensitivity enzyme-linked immunosorbent assay, a competitive immunoassay that uses purified protein and polyclonal anti-CRP antibodies. The interassay CV was 5.0%. The minimum detectable concentration was 0.03 mg/L.
All assays were done in duplicate (except IL-10) and were repeated if the second measure was >10% or <10% compared to the first. The average of the two measures was used in the analyses. The cut-off values for tertiles distribution (pg/ml) for each of the cytokines considered in the study was the following:
- - IL-6: I <1.06; II 1.06–1,91; III >1.91;
- - IL-10: I <1. 39; II 1.39–2.49; III >2.49;
- - IL-18: I <332; II 332–442; III >442;
- - IL-1β: I <0.12; II 0.12–0.16; III >0.16;
- - TNFα: I <3.51; II 3.51–5.74; III >6.74;
- - CRP: I <1.68; II 1.68–4.49; III >4.49.
2.2. Other clinical chemistry parameters
All parameters were measured on serum from fresh samples (not frozen) drawn after 12 h overnight fasting. Commercial enzymatic tests (Roche Diagnostics) were used for determining serum total cholesterol (TC), triglycerides (TG), and HDL-C concentrations. The interassay coefficient of variation was less than 3.8% for total cholesterol and less than 5.0% for HDL cholesterol.
For triglycerides, the analytical sensitivity (lower detection limit) was 4.0 mg/dL; the intra-assay CV was 3.1%, the inter-assay CV was 1.8%.
Low-density lipoprotein cholesterol (LDL-C) was calculated by the Friedewald's formula as follows: LDL-C: TC – (TG/5) – HDL-C. Low HDL-C was defined as ≤10th gender specific percentile (36 mg/dL for males, 41 mg/dL for females).
Fasting insulin was determined using a commercial double-antibody, solid-phase radioimmunoassay (Sorin Biomedica, Milan, Italy) with an intra-assay coefficient of 3.1%. Fasting blood glucose was determined by an enzymatic colorimetric assay using a modified glucose oxidase-peroxidase method (Roche Diagnostics, GmbH, Mannheim, Germany) and a Roche-Hitachi 917 analyzer.
2.3. Other covariates
Weight and height were measured by using standard techniques. Body mass index (BMI) was calculated as weight (kg) divided by the square of height (m). Waist circumference was measured to the nearest 0.5 cm by using a non-elastic plastic tape, at the midpoint between the lower rib margin and the iliac crest (normally umbilical level).
Daily alcohol intake (expressed in g/day) was calculated by using a specific food questionnaire validated in the Italian population (EPIC questionnaire) [21
Physical activity was assessed from self-report of recreational and work-related activities. For each activity, we estimated the metabolic equivalent tasks (METs), frequency (times per month), and time of exposure (years), and calculated the average METs consumed per year from the age of 60.
The presence of specific diseases was established by standardized criteria combining information obtained from self-reported history, medical records, and from clinical examination. The following diseases were assessed in this study: angina, acute myocardial infarction, peripheral arterial disease, stroke and transient ischemic attack, arterial hypertension, congestive heart failure, renal insufficiency, cancer, and dementia.
All participants were asked about smoking habits and pack-years (measure that combines intensity and duration) was calculated as packs smoked per day × years of smoking.
Drugs were coded according to the Anatomical Therapeutic and Chemical codes. The medications considered in the present study were: insulin, hypoglycemics, statins, fibrates, and beta-blockers.
Systolic and diastolic blood pressures were calculated from the mean of the three measures taken during physical examination. Hypertension was defined as the presence of at least one of the following conditions: systolic blood pressure >140 mmHg or diastolic blood pressure >90 mmHg; documented history of hypertension; current use of antihypertensive drugs. Diabetes mellitus was defined as the presence of at least one of the following conditions: previous physician's diagnosis, current treatment with insulin or oral hypoglycemics, self-report of diabetes mellitus, and fasting blood glucose ≥ 126 mg/dL.
2.4. Statistical analysis
Continuous variables were expressed as mean (S.D.). Due to the skewed distribution, cytokines, TG, and insulin values were log-transformed in order to approximate a normal distribution. Means were compared by the unpaired t-test, while prevalences were compared by the χ2-test.
The association (odds ratio) between low HDL-C levels and each inflammatory marker levels (expressed as tertiles) was evaluated by univariate logistic regression analysis.
In order to select the variables independently associated with low levels of HDL-C we calculated the odds ratio by using a multivariate logistic regression analysis (method: stepwise forward Wald). The independent variables included in the model were the following: age (years), smoking (three categories: current, former, never), alcohol intake (g/day), physical activity (METs per year), hypertension (yes/no), diabetes (yes/no), CHD (yes/no), stroke (yes/no), peripheral arterial disease (PAD: yes/no), plasma triglycerides, fasting insulin, oral hypoglycaemic drugs, BMI, waist circumference, IL-6, CRP, IL-18, and TNF-α levels.
Systat for Windows, version 5.0, and SPSS for Windows, version 7.0 (SPSS, Inc., Chicago, IL) statistical packages were used.