ABC | Volume 110, Nº4, April 2018

Original Article Cartolano et al LAP and lipoprotein size Arq Bras Cardiol. 2018; 110(4):339-347 Lipid Accumulation Product (LAP) was proposed as a simple, inexpensive and accurate surrogate index to estimate cardiovascular risk 8 and all-cause mortality. 9 This index combines anthropometric (waist circumference, WC) and biochemical (fasting triglycerides, TG) parameters, connecting anatomical to physiological changes associated with increased central accumulation of lipids in adults. Kahn 10 observed in the Third National Health and Nutrition Examination Survey (NHANES III) that LAP index evidenced the negative effect of large WC possibly related with small dense LDL, although direct measurement of LDL size has not been done. The validity and superiority of LAP to identify cardiovascular risk, metabolic syndrome, diabetes mellitus and insulin resistance have been compared with body mass index (BMI), WC and waist‑to‑hip ratio. 9-13 Despite the negative impact of LAP on glucose metabolism, monitored principally in postmenopausal 13,14 and polycystic ovary syndrome women, 15,16 its association with the size of lipoproteins has not been directly evaluated and reported yet. Previous studies based in LAP confirmed its association with classical risk factors for CVD. 17-20 Therefore, the aim of this study was to extend current knowledge of LAP, by evaluating the impact of this parameter on LDL and HDL size, considering the potential influence of confounders. Methods Subjects Three hundred fifty-one adults of both sexes and multiple cardiovascular risk factors were selected for this cross-sectional study after complete clinical evaluation and electrocardiogram (ECG). These subjects were recruited from the Research Center located at the University Hospital of the University of Sao Paulo. The non-probabilistic sampling was employed. According to inclusion criteria, the subjects included in the study were 30–74 years old and had at least one of the risk factors for CVD – dyslipidemia, diabetes mellitus , and/or hypertension. Pregnant or lactating women, individuals who participated in other studies, had severe hepatic or renal disease, type 1 diabetes mellitus, illicit drug users, alcoholics, and individuals under lipid-lowering drugs introduced or changed 30 days before blood collection were not enrolled in this protocol. This study was approved by the Research Ethics Committee of the University Hospital (n 1126/11) and the School of Public Health, University of Sao Paulo (n 2264) and all procedures followed the standards of the Declaration of Helsinki of 1975, revised in 2008. All subjects gave their written informed consent. Demographic and clinical profile Trained interviewers evaluated the demographic features of participants by a pre-structured questionnaire addressing sex, age, and ethnicity. The clinical evaluation consisted of current information on medical history, family history of chronic diseases (father and mother), and regular use of medication. Smoking was considered when the habit was reported by the subjects, regardless of the amount of cigarettes. Hypertension was confirmed by clinical history, use of antihypertensive medication and systolic (SBP) and diastolic (DBP) blood pressure monitored after at least five minutes at rest and mean of three measures was used for data analysis. Hypertension was defined as SBP ≥ 140 mmHg and/or DBP ≥ 90 mmHg. Type 2 diabetes mellitus was defined by previous diagnosis of diabetes, use of oral hypoglycemic agents and plasma glucose levels higher 100 mg/dl. The Framingham Risk Score (FRS) was calculated as previously described. 21,22 Anthropometric parameters Weight (Kg) and height (cm) were measured to the nearest 0.1 kg and 0.1 cm, respectively, with standard methods and equipment. BMI was calculated as weight (Kg) divided by the square of the standing height (m 2 ). The WC was measured using flexible inelastic tape with an accuracy of 1.0-mm (TBW ® ; Sao Paulo, SP, Brazil) without tightening it against the body. Body composition was assessed by bioelectrical impedance (BIA) (Analyzer®, model Quantum II; RJL Systems; Michigan, USA). Body fat percentage was calculated using the Cyprus (Body Composition Analysis System, v. 2.5; RJL Systems®; Detroit, MI, USA) program, which considered sex, age, weight, height, level of physical activity, resistance and reactance. All measurements were performed in duplicate by trained staff. Blood samples After fasting (12 h), blood samples (20 mL) were collected. For analyses using plasma, blood was collected in vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA; 1.0 µg/mL). The protease inhibitors aprotinin (10.0 µg/ml), benzamidine (10.0 µM), phenylmethylsulfonyl fluoride (PMSF; 5.0 µM) and the antioxidant butylated hydroxytoluene (BHT; 100.0 µM) were added to the samples. Plasma and serum were separated by centrifugation (3,000 rpm; 10 min; 4°C) and samples were kept frozen (−80 °C) until analysis. Biochemical Analysis Plasma TG, total cholesterol (TC), and HDL-C levels were measured using commercial kits (Labtest; Lagoa Santa, MG, Brazil). LDL-C levels were calculated using the Friedewald equation for subjects who had TG lower than 400 mg/dl. 23 Apolipoproteins B and AI (Apo B and Apo AI) were determined using standard methods (APOA1 and APOB Autokits, Randox; Kearneysville, WV, USA). Non-esterified fatty acids (NEFA) levels were determined using the Free Fatty Acid Quantification kit (Wako Chemicals – USA Inc.; Richmond, VA, USA). Glucose levels were determined using an enzymatic and colorimetric kit (Glucose PAP Liquiform; Labtest; Lagoa Santa, MG, Brazil). Plasma insulin was detected using the commercial Human Insulin Direct ELISA kit (Life Technologies; Grand Island, NY, USA). Insulin resistance was calculated using the homeostatic model assessment-insulin resistance (HOMA-IR) formula as follows: HOMA-IR = fasting insulin concentration (U/mL) x fasting glucose (mmol/L)/22.5. 24 These parameters were analyzed in duplicate in automatic Cobas system (Hitachi High Technology, Minato-ku, Tokyo, Japan). 340