Montserrat Cofán

Phytosterol plasma concentrations and coronary heart disease in the prospective Spanish EPIC cohort

Phytosterol intake with natural foods, a measure of healthy dietary choices, increases plasma levels, but increased plasma phytosterols are believed to be a coronary heart disease (CHD) risk factor. To address this paradox, we evaluated baseline risk factors, phytosterol intake, and plasma noncholesterol sterol levels in participants of a case control study nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) Spanish cohort who developed CHD (n = 299) and matched controls (n = 584) who remained free of CHD after a 10 year follow-up. Sitosterol-to-cholesterol ratios increased across tertiles of phytosterol intake (P = 0.026). HDL-cholesterol level increased, and adiposity measures, cholesterol/HDL ratios, and levels of glucose, triglycerides, and lathosterol, a cholesterol synthesis marker, decreased across plasma sitosterol tertiles (P < 0.02; all). Compared with controls, cases had nonsignificantly lower median levels of phytosterol intake and plasma sitosterol. The multivariable-adjusted odds ratio for CHD across the lowest to highest plasma sitosterol tertile was 0.59 (95% confidence interval, 0.36–0.97). Associations were weaker for plasma campesterol. The apolipoprotein E genotype was unrelated to CHD risk or plasma phytosterols. The data suggest that plasma sitosterol levels are associated with a lower CHD risk while being markers of a lower cardiometabolic risk in the EPIC-Spain cohort, a population with a high phytosterol intake.

Influence of HDL Cholesterol on Preclinical Carotid Atherosclerosis in Familial Hypercholesterolemia

Objective—The effect of risk factors on carotid atherosclerosis in heterozygous familial hypercholesterolemia (FH) is unclear. We evaluated carotid intima-media thickness (IMT) by sonography in relation to classical and emergent risk factors in a large FH cohort. Methods and Results—Risk factors and carotid IMT were assessed in 196 asymptomatic subjects aged ≥25 years fulfilling strict diagnostic criteria for clinical FH who were either undertreated or treatment-naive. Conventional risk factors, but not lipoprotein(a), homocysteine, or apolipoprotein E (apoE) genotypes were univariately related to IMT. Age-adjusted and gender-adjusted IMT increased with increasing low-density lipoprotein (LDL) cholesterol and decreased with increasing high-density lipoprotein (HDL) cholesterol. Compared with a total cholesterol/HDL ratio >5.0, a ratio ≤5.0 was associated with a lower adjusted IMT, with a mean difference of −0. 09 mm (95% confidence interval, −0.13 to −0.04). By multivariate analysis, age, HDL cholesterol (negatively), physical exercise, family history of early-onset coronary heart disease, LDL cholesterol, and leukocyte count, in this order, were independent associations of IMT (r2=0.429, P<0.001). Conclusions—Traditional risk factors account for a sizeable proportion of variation in carotid IMT in FH. Because the HDL cholesterol level and the total cholesterol/HDL ratio are strong predictors of preclinical carotid atherosclerosis, HDL cholesterol-raising strategies should have an important therapeutic role in FH.

Fatty acids in serum phospholipids and carotid intima-media thickness in Spanish subjects with primary dyslipidemia

BACKGROUND Low rates of incident ischemic heart disease (IHD) and cardiac death occur in Spain despite a high prevalence of cardiovascular risk factors. High consumption of unsaturated fatty acid-rich foods, such as olive oil, nuts, and seafood, might underlie this paradox. OBJECTIVE We investigated whether serum phosphatidylcholine enrichment in oleic, linoleic, alpha-linolenic, and n-3 (omega-3) long-chain polyunsaturated fatty acids (as biomarkers of olive oil, seed oil, walnut, and fish intake, respectively) relate to carotid atherosclerosis in Spanish subjects at risk of IHD. DESIGN In a cross-sectional study, we measured fatty acid concentrations in serum phosphatidylcholine and measured carotid intima-media thickness (IMT) by using ultrasound in 451 asymptomatic subjects (261 men, 190 women; mean age: 45 y) with primary dyslipidemia. Main and secondary outcomes were mean and maximum IMT in the common carotid artery (CCA) and other carotid segments, respectively. RESULTS Phosphatidylcholine fatty acid composition was similar to that reported for other Spanish populations. Multiple regression analyses showed that proportions of oleic and docosahexaenoic acids were inversely related to mean CCA IMT (P < 0.02, all) after adjustment for several confounders. In similar models, alpha-linolenic acid related inversely to mean and maximum internal carotid artery IMT (P < 0.05 for all). Linoleic and eicosapentaenoic acids were unrelated to IMT. CONCLUSIONS Higher phospholipid proportions of oleic, alpha-linolenic, and docosahexaenoic acids showed inverse associations with IMT at specific carotid segments in subjects with primary dyslipidemia. High intakes of healthy fats might explain, in part, the Spanish paradox of low IHD rates in the face of a high burden of cardiovascular risk factors.

Apolipoprotein E gene mutations in subjects with mixed hyperlipidemia and a clinical diagnosis of familial combined hyperlipidemia.

OBJECTIVE Rare mutations in the APOE gene, undetectable with the usual genotyping technique, are responsible for dominant familial dysbetalipoproteinemia (FD) and therefore could be easily misclassified as familial combined hyperlipidemia (FCHL). We aimed to identify APOE mutations associated with dominant combined hyperlipoproteinemia and to establish their frequency in subjects with a clinical diagnosis of FCHL. METHODS AND RESULTS In 279 unrelated subjects with FCHL in whom a functional LDLR mutation was excluded, sequencing of the entire APOE gene detected 9 carriers of a rare mutation: 5 subjects (1.8%) with the R136S mutation (arginine at residue 136 changed to serine) and 4 subjects (1.4%) with the p.Leu149del mutation, a 3-bp inframe deletion that results in the loss of leucine at position 149. Both genetic defects were detected with similar frequency (2.5% and 1.3%, respectively) in an independent group of 160 FCHL subjects from other locations in Spain. Family studies demonstrated cosegregation of these APOE mutations with hyperlipoproteinemia. R136S carriers showed dysbetalipoproteinemia, while the lipid phenotype of p.Leu149del carriers was IIa or IIb. CONCLUSIONS Rare APOE mutations are responsible for approximately 3.5% of FCHL cases in our population. APOE R136S and p.Leu149del induce autosomal dominant FD and a phenotype indistinguishable from FCHL, respectively.

Impact of low-density lipoprotein receptor mutational class on carotid atherosclerosis in patients with familial hypercholesterolemia

BACKGROUND AND OBJECTIVES Defects in the low-density lipoprotein receptor (LDLR) gene cause familial hypercholesterolemia (FH), a highly atherogenic condition. The effect of different LDLR mutations on coronary heart disease (CHD) risk is insufficiently defined. We assessed carotid intima-media thickness (IMT), a surrogate marker of CHD, in relation to LDLR mutational class in FH. METHODS In 436 Spanish FH patients (223 men and 213 women, age 44+/-14 years) with known LDLR mutations, alleles were classified by standard criteria as null (n=269), defective (n=162), or undetermined (n=5). LDLR defects were detected using a microarray (Lipochip) designed to uncover prevalent mutations in Spain and gene sequencing when no mutations were detected. Carotid IMT and plaque were assessed in FH patients and 268 healthy subjects. RESULTS All carotid measurements were increased in FH patients versus controls (p<0.05), irrespective of genotype. After adjustment for gender and age, patients with null alleles compared with defective alleles had similar mean and maximum common carotid artery (CCA) IMT, but higher maximum IMT at any carotid segment, with median values (95% confidence interval) of 1.25 mm (1.19-1.31) and 1.11 mm (1.05-1.18), respectively. Multivariate analysis showed that null alleles were independently associated with maximum CCA-IMT (beta=0.09, p=0.033) with an impact similar to that of gender (beta=0.10, p=0.035). CONCLUSIONS FH patients show advanced carotid atherosclerosis in relation to LDLR mutational class. The findings support the utility of genetic testing in FH beyond providing a secure diagnosis.

Femoral Atherosclerosis In Heterozygous Familial Hypercholesterolemia Influence Of The Genetic Defect

Objective—The purpose of this study was to assess femoral atherosclerosis by ultrasound in patients with molecularly defined heterozygous familial hypercholesterolemia (FH) in comparison with matched control subjects and in relation to mutational class in the LDL receptor and apolipoprotein B (APOB) genes. Methods and Results—Femoral intima-media thickness (IMT) and plaque were evaluated in 146 FH patients carrying null alleles (n=48), defective-receptor alleles (n=62), undetermined-function alleles (n=25), or APOB defects (n=11) and in 193 healthy subjects. Twenty-three patients had coronary heart disease (CHD). The frequency of both tendon xanthomas and CHD was ≈2-fold higher and average LDL cholesterol was 30 mg/dL higher in null-allele genotype compared with receptor-defective mutations. All femoral measurements were increased in FH patients versus controls (P<0.001), and null-allele mutations showed higher age-, sex-, and LDL cholesterol-adjusted maximum IMT than receptor-defective or APOB defects (P for trend, 0.001). By multivariate analysis, independent associations of mean IMT, a measure of early atherosclerosis, were age, LDL cholesterol, sex, and systolic blood pressure. Age, null-allele genotype, sex, and smoking explained 42% of the variability of maximum IMT, a measure of advanced atherosclerosis. Conclusions—FH patients have increased femoral IMT in relation to mutational class. The findings support the usefulness of genetic testing in FH beyond securing the diagnosis.

Simultaneous determination of oxysterols, phytosterols and cholesterol precursors by high performance liquid chromatography tandem mass spectrometry in human serum

A fast and sensitive high performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (HPLC-APCI-MS/MS) method to identify and quantify oxysterols, phytosterols and non-cholesterol sterols at the pico-molar concentration level in human serum in only one run was developed in this study. This method allows the simultaneous separation and quantitation of individual phytosterols, cholesterol precursors and oxidized derivatives of cholesterol without a derivatization step in a single run, thus providing a more confident quantitation of sterols in serum. After saponification, solid-phase extraction (SPE) used as a clean-up step and HPLC separation, detection by MS was developed using APCI and multiple ion monitoring modes. This method employs reversed-phase C18 SPE cartridges and serum calibrators, as well as isotopically labelled cholesterol as an internal standard added before sample processing. The time consumed for a single sample is reduced from the 4 hours of conventional sterol analysis to 1 hour including the chromatographic run time. The method has been evaluated by analyzing a certified cholesterol sample as well as by comparison to other two methods used as reference, based on GC and enzymatic reaction, respectively. Serum from 14 individuals was successfully analyzed. Detection limits for oxysterols, phytosterols and non-cholesterol sterols, all determined in a single run in small serum volumes, were between 0.47 and 1.69 pM. Intra-day precision was <7% for all sterols in in-house-made lipoprotein-deficient serum. The limits of detection showed high sensitivity with very good intraday- and interday-precision for all sterols. The recoveries of the phytosterols, cholesterol precursors and cholestanol ranged from 77% to 92%. These analytical parameters provide a reliable and reproducible method for the identification and quantitation of human sterols.

Basal plasma concentrations of plant sterols can predict LDL-C response to sitosterol in patients with familial hypercholesterolemia

Background:Familial hypercholesterolemia (FH) is associated with a high risk of coronary heart disease. Pharmacological treatment and diet are both essential for the management of FH. Foods rich in plant sterols (PS) may play an important role in the treatment of patients with these disorders.Objective:To test the effect of the intake of PS on low-density lipoprotein (LDL) concentration, endothelial function (EF) and LDL particle size in 30 patients with FH.Design:Randomized and crossover dietary intervention study.Setting:Tertiary outpatient care.Subjects:Thirty-eight were recruited, but only 30 were subjected to four low-fat dietary intervention periods, each of 4 weeks.Methods:Each intervention had a different content of cholesterol (<150 or 300 mg/day) and sitosterol (<1 or 2 g/day). Lipid response, EF and LDL particle size were analysed after the intervention.Results:Plasma sitosterol/cholesterol ratio was higher during both plant sterol-rich periods than during the low plant sterols periods. Basal sitosterol concentrations predicted the LDL-cholesterol response during the intake of plant sterol-enriched diets. The change in LDL-cholesterol was significantly greater in subjects in the upper and intermediate tertiles of basal plasma sitosterol concentrations (−21±8 mg/dl, P=0.03; −19±7 mg/dl, P=0.04, respectively) than in subjects in the lower tertile (8±5 mg/dl) when they changed from a low cholesterol diet to a low cholesterol plus plant sterol diet.Conclusion:Our study demonstrates that basal sitosterol values can predict hypolipidemic response in patients with FH.

White blood cell count is associated with carotid and femoral atherosclerosis

OBJECTIVE Chronic low-grade inflammation is associated with atherosclerosis. Ultrasound imaging allows measurement of intima-media thickness (IMT) and plaque. We investigated the association between inflammatory markers and carotid and femoral atherosclerosis. METHODS We studied 554 subjects with primary dyslipidemia (57% men, median age 49 years) and 246 age- and sex-matched normolipidemic subjects. Carotid and femoral arteries were imaged bilaterally with a standardized protocol. Mean and maximum common carotid IMT (CC-IMT and MaxCC-IMT) and common femoral IMT (F-IMT and MaxF-IMT), and carotid and femoral plaque were assessed. Carotid atherosclerosis was defined by CC-IMT and/or plaque height >75th percentile of a reference population. White blood cell count (WBCC) was measured in all subjects. High-sensitivity C-reactive protein (CRP) was measured in 330 dyslipidemic subjects. RESULTS The age- and sex-adjusted probability of carotid atherosclerosis and femoral plaque increased by 20% (odds ratio [OR] 1.20; 95% CI, 1.10-1.31) and 25% (1.25; 1.13-1.38), respectively, for each 1000/mm(3) WBCC increment. WBCC was associated with age- and sex-adjusted CC-IMT and MaxCC-IMT (p<0.05, both), and F-IMT and MaxF-IMT (p<0.001, both). Adjustment for cardiovascular risk factors did not influence these associations. CRP was associated with CC-IMT and MaxCC-IMT (p<0.05, both), but the associations disappeared after adjustment for body mass index. CRP was unrelated to carotid plaque or measures of femoral atherosclerosis. CONCLUSIONS WBCC, but not CRP, related to early and advanced measures of atherosclerosis independently of risk factors. Our findings support using the heretofore undervalued WBCC as an easy-to-measure, low-cost diagnostic marker of atherosclerosis.

FABP4 plasma levels are increased in familial combined hyperlipidemia

The lipid profile of familial combined hyperlipidemia (FCHL) shares some characteristics with atherogenic dyslipidemia seen in diabetes, metabolic syndrome, and obesity. Adipocyte fatty acid-binding protein 4 (FABP4) appears to be a determinant of atherogenic dyslipidemia. We examined relationships between FABP4 plasma concentrations, dyslipidemia, and metabolic variables in patients with FCHL. We studied 273 unrelated FCHL patients and 118 control subjects. FABP4 was higher in FCHL than controls, with mean levels of 21.8 (10.1) microg/l and 19.2 (9.2) microg/l, respectively (adjusted P= 0.012). In FCHL, FABP4 correlated to body mass index (BMI), waist circumference, insulin levels, and homeostasis model assessment (HOMA) index (all P< 0.05), but not to lipid levels, whereas in obese patients, FABP4 correlated to triglyceride levels (r = 0.303, P= 0.014) and very low density lipoprotein size (r = 0.502, P = 0.001), as determined by nuclear magnetic resonance. Associations of FABP4 with BMI and waist circumference, but not with insulin levels, persisted in this subgroup. Plasma FABP4 does not influence the lipid phenotype of FCHL. In a small subgroup of obese FCHL, FABP4 levels were associated with triglyceride-rich lipoproteins independent of insulin resistance. These results support a hyperlipidemic mechanism of FCHL different from similar metabolic conditions where fat mass is strongly related to FABP4 and hypertriglyceridemia.