Lucía Baila-Rueda

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.

Omega-3 Fatty Acids and HDL. How Do They Work in the Prevention of Cardiovascular Disease?

Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) from marine origin have been strongly associated with cardiovascular protection, even at low doses ( < 1g/d). Despite the research performed in this promising area, basic aspects, such as the ideal doses and the mechanisms by which ω-3 PUFAs act, are not precisely defined. The best known biological property of ω-3 PUFAs is their hypotriglyceridemic effect, but other cardioprotective actions, such as reduction of arrhythmia susceptibility, antithrombotic, antiinflammatory and antioxidant effects, improvement of endothelial function, and delayed atherosclerosis development have received an increased interest in recent years. Some of these actions are also ascribable to high-density lipoproteins (HDL). Abundant epidemiological evidence links increasing HDL-cholesterol concentrations to cardiovascular protection. Recently, the protein cargo (proteome) of HDL particles has been attributed a key role in their functionality. In this review, we summarize the main effects of ω-3 PUFAs on HDL-cholesterol, HDL subfractions, and its main proteins, apolipoproteins (apo) AI and AII. The shared cardioprotective actions of ω-3 PUFAs and HDL are reviewed as well.

Common genetic variants contribute to primary hypertriglyceridemia without differences between familial combined hyperlipidemia and isolated hypertriglyceridemia.

Background—The majority of hypertriglyceridemias are diagnosed as familial combined hyperlipidemia (FCHL) and primary isolated hypertriglyceridemias. The contribution of common genetic variants in primary hypertriglyceridemias and the genetic difference between FCHL and isolated hypertriglyceridemias have not been thoroughly examined. Methods and Results—This study involved 580 patients with hypertriglyceridemias and 403 controls. Of the 37 single nucleotide polymorphisms examined, 12 located in 10 genes showed allelic and genotype frequency differences between hypertriglyceridemias and controls. The minor alleles of APOE, APOA5, GALNTN2, and GCKR variants were positively correlated with plasma triglycerides, whereas minor alleles of ADIPOR2, ANGPTL3, LPL, and TRIB1 polymorphisms were inversely associated. Body mass index, glucose, sex, rs328 and rs7007797 in LPL, rs662799 and rs3135506 in APOA5, and rs1260326 in GCKR explained 36% of the variability in plasma triglycerides, 7.3% of which was attributable to the genetic variables. LPL, GCKR, and APOA5 polymorphisms fit dominant, recessive, and additive inheritance models, respectively. Variants more frequently identified in isolated hypertriglyceridemias were rs7412 in APOE and rs1800795 in IL6; rs2808607 in CYP7A1 and rs3812316 and rs17145738 in MLXIPL were more frequent in FCHL. The other 32 single nucleotide polymorphisms presented similar frequencies between isolated hypertriglyceridemias and FCHL. Conclusions—Common genetic variants found in LPL, APOA5, and GCKR are associated with triglycerides levels in patients with primary hypertriglyceridemias. FCHL and isolated hypertriglyceridemias are probably trace to an accumulation of genetic variants predisposing to familial and sporadic hypertriglyceridemias or to hypertriglyceridemias and hypercholesterolemia in case of FCHL.

The human HDL proteome displays high inter-individual variability and is altered dynamically in response to angioplasty-induced atheroma plaque rupture

Recent findings support potential roles for HDL in cardiovascular pathophysiology not related to lipid metabolism. We address whether HDL proteome is dynamically altered in atheroma plaque rupture. We used immunoaffinity purification of HDL samples from coronary artery disease patients before and after percutaneous transluminal coronary angioplasty (PTCA), a model of atheroma plaque disruption. Samples were analyzed by quantitative proteomics using stable isotope labeling and results were subjected to statistical analysis of protein variance using a novel algorithm. We observed high protein variability in HDL composition between individuals, indicating that HDL protein composition is highly patient-specific. However, intra-individual protein variances remained at low levels, confirming the reproducibility of the method used for HDL isolation and protein quantification. A systems biology analysis of HDL protein alterations induced by PTCA revealed an increase in two protein clusters that included several apolipoproteins, fibrinogen-like protein 1 and other intracellular proteins, and a decrease in antithrombin-III, annexin A1 and several immunoglobulins. Our results support the concept of HDL as dynamic platforms that donate and receive a variety of molecules and provide an improved methodology to use HDL proteome for the systematic analysis of differences among individuals and the search for cardiovascular biomarkers. Biological significance The HDL proteome is an interesting model of clinical relevance and has been previously described to be dynamically altered in response to pathophysiological conditions and cardiovascular diseases. Our study suggests that interindividual variability of HDL proteome is higher than previously thought and provided the detection of a set of proteins that changed their abundance in response to plaque rupture, supporting the concept of HDL as dynamic platforms that donate and receive a variety of molecules.

Serum Lipid Responses to Weight Loss Differ between Overweight Adults with Familial Hypercholesterolemia and Those with Familial Combined Hyperlipidemia

The effect of weight loss on lipids differs among individuals, although whether it can modify the management of hereditary hyperlipidemias has not yet been explored. The objective of this study was to examine the effect of weight loss on cholesterol metabolism, assessed by circulating noncholesterol sterols, in overweight adults with familial hypercholesterolemia (FH) and familial combined hyperlipidemia (FCHL). We conducted a 6-mo weight loss intervention in untreated individuals (FH: n = 28; FCHL: n = 50) with a body mass index of >25 kg/m(2) and mean age of 46.9 ± 11.3 y, of whom 53.8% were men. A hypocaloric diet was implemented and serum lipid analyses, including noncholesterol sterols, were assessed. Global significant mean weight losses of 5.7 kg (-6.6%) and 6.6 kg (-7.6%) were achieved after 3 and 6 mo, respectively. Mean non-HDL cholesterol and triglyceride (TG) changes at 3 and 6 mo compared with baseline were -5.8% (P = 0.004) and -7.1% (P = 0.014), and -30.1% (P < 0.001) and -31.4% (P < 0.001), respectively. Among participants who lost ≥5% body weight, only significant changes in TGs and non-HDL cholesterol were observed in FCHL participants. Sterol precursors of cholesterol synthesis decreased significantly by 10.4% at 6 mo in FCHL participants, mostly because of a 23.9% lathosterol reduction. Baseline synthesis precursors were associated with TG reduction in FCHL participants (P = 0.039; R(2) = 0.20), and intestinally derived sterols were inversely associated with non-HDL cholesterol changes in FH participants (P = 0.036; R(2) = 0.21). Thus, FCHL participants had a better lipid-lowering response to weight loss than did FH participants. This response was positively associated with baseline cholesterol synthesis, which was reduced by weight loss. Our results confirm the cholesterol overproduction mechanism of FCHL and its interaction with fat mass, while also supporting the differential management of familial hyperlipidemias if obesity coexists. This trial was registered at clinicaltrials.gov as NCT01995149.

Cosegregation of serum cholesterol with cholesterol intestinal absorption markers in families with primary hypercholesterolemia without mutations in LDLR, APOB, PCSK9 and APOE genes

BACKGROUND AND AIM The genetic cause and pathogenic mechanism of approximately 20-40% of autosomal dominant hypercholesterolemias (ADH) are unknown. Increased cholesterol intestinal absorption has been associated to ADH. If this variation contributes to their pathogenesis is unknown. METHODS AND RESULTS We studied cholesterol absorption (phytosterols and cholestanol serum concentrations) and cholesterol synthesis (desmosterol serum concentration) in 20 families with ADH without causal mutations in LDLR, APOB, PCSK9 or APOE genes (non-FH ADH) selected from 54 non-FH ADH probands with (non-cholesterol sterol concentrations above 75th percentile) and without (under 75th percentile) hyperabsorption. The concentrations of cholestanol, sitosterol, campesterol and stigmasterol were higher in affected than in non-affected subjects (p = 0.003, <0.001.<0.001, 0.002, respectively). There was a strong cosegregation of hyperabsorption with high LDL cholesterol within hyperabsorber families with odds ratio 6.80 (confidence interval 1.656-27.9), p = 0.008. In hyperabsorber families, 60.5% of subjects were hyperabsorbers and 76% of them had high LDL cholesterol versus 38.3% and 63% in non-hyperabsorber families, respectively. CONCLUSION Most hypercholesterolemic family members with a hyperabsorber proband are hyperabsorbers. These absorption markers are significantly and positively associated with LDL cholesterol, and predispose to high LDL cholesterol in family members. Our data suggest that complex interindividual variation in cholesterol absorption is involved in many non-FH ADH.

Severe hypercholesterolemia and phytosterolemia with extensive xanthomas in primary biliary cirrhosis: Role of biliary excretion on sterol homeostasis

Primary biliary cirrhosis (PBC) is an autoimmune, chronic, cholestatic liver disease that affects primarily women. PBC is commonly associated with hypercholesterolemia that has been associated with cholestasis. We report an exceptionally high blood cholesterol and phytosterols with just mild cholestasis indicating a selective defect in sterol biliary secretion in a patient with PBC.

Single Nucleotide Variants Associated With Polygenic Hypercholesterolemia in Families Diagnosed Clinically With Familial Hypercholesterolemia

INTRODUCTION AND OBJECTIVES Approximately 20% to 40% of clinically defined familial hypercholesterolemia cases do not show a causative mutation in candidate genes, and some of them may have a polygenic origin. A cholesterol gene risk score for the diagnosis of polygenic hypercholesterolemia has been demonstrated to be valuable to differentiate polygenic and monogenic hypercholesterolemia. The aim of this study was to determine the contribution to low-density lipoprotein cholesterol (LDL-C) of the single nucleotide variants associated with polygenic hypercholesterolemia in probands with genetic hypercholesterolemia without mutations in candidate genes (nonfamilial hypercholesterolemia genetic hypercholesterolemia) and the genetic score in cascade screening in their family members. METHODS We recruited 49 nonfamilial hypercholesterolemia genetic hypercholesterolemia families (294 participants) and calculated cholesterol gene scores, derived from single nucleotide variants in SORT1, APOB, ABCG8, APOE and LDLR and lipoprotein(a) plasma concentration. RESULTS Risk alleles in SORT1, ABCG8, APOE, and LDLR showed a statistically significantly higher frequency in blood relatives than in the 1000 Genomes Project. However, there were no differences between affected and nonaffected members. The contribution of the cholesterol gene score to LDL-C was significantly higher in affected than in nonaffected participants (P = .048). The percentage of the LDL-C variation explained by the score was 3.1%, and this percentage increased to 6.9% in those families with the highest genetic score in the proband. CONCLUSIONS Nonfamilial hypercholesterolemia genetic hypercholesterolemia families concentrate risk alleles for high LDL-C. Their contribution varies greatly among families, indicating the complexity and heterogeneity of these forms of hypercholesterolemias. The gene score explains a small percentage of LDL-C, which limits its use in diagnosis.

Effect of different fat-enriched meats on non-cholesterol sterols and oxysterols as markers of cholesterol metabolism: Results of a randomized and cross-over clinical trial

BACKGROUND AND AIM Different kinds of fatty acids can affect the synthesis, absorption, and elimination of cholesterol. This study was carried out to assess the associations of cholesterol metabolism with the intake of two meats with different fatty acid composition in healthy volunteers. METHODS AND RESULTS The study group was composed of 20 subjects (12 males and eight females; age, 34.4 ± 11.6 years; body mass index (BMI), 23.5 ± 2.3 kg/m(2); low-density lipoprotein (LDL) cholesterol, 2.97 ± 0.55 mmol/l; high-density lipoprotein (HDL) cholesterol, 1.61 ± 0.31 mmol/l; triglycerides (TG), 1.06 ± 0.41 mmol/l) who completed a 30-day randomized and cross-over study to compare the cholesterol metabolism effect of 250 g of low-fat lamb versus 250 g of high-fat lamb per day in their usual diet. Cholesterol absorption, synthesis, and elimination were estimated from the serum non-cholesterol sterol and oxysterol concentrations analyzed by a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). No changes in weight, plasma lipids, or physical activity were observed across the study. Cholesterol intestinal absorption was decreased with both diets. Cholesterol synthesis and elimination decreased during the low-fat lamb dietary intervention (ρ = 0.048 and ρ = 0.005, respectively). CONCLUSION Acute changes in the diet fat content modify the synthesis, absorption, and biliary elimination of cholesterol. These changes were observed even in the absence of total and LDL cholesterol changes in plasma. REGISTRATION NUMBER FOR CLINICAL TRIALS ClinicalTrials.gov PRS, NCT02259153.

Atherosclerosis progression in patients with autosomal dominant hypercholesterolemia in clinical practice

BACKGROUND Autosomal dominant hypercholesterolemias (ADH) are associated with high risk of premature cardiovascular disease (CVD). No data on progression of atherosclerosis in ADH population in clinical practice are available. OBJECTIVE To investigate atherosclerosis progression in ADH patients and its relationship with CVD risk factors. METHODS A total of 463 patients, 279 with familial hypercholesterolemia and 184 with familial combined hyperlipidemia, were prospectively followed during a median of 36.5 months. Carotid intima-media thickness (cIMT) was assessed at baseline and at the end of the follow-up by ultrasonography. RESULTS A total of 259 patients (55.9%) showed cIMT progression and 149 (32.2%) remained within normal age-adjusted cIMT. Baseline cIMT was the variable most inversely associated with cIMT progression (B = -0.313; P < .001). Hypertension, diabetes, and smoking during follow-up were variables positively associated with progression. Patients who began statin treatment during the study period had less progression than former statin users. The 83.7% of ADH with normal baseline cIMT, absence of major CVD risk factors and non-high-density lipoprotein (HDL) cholesterol <190 mg/dL at follow-up remained with normal cIMT at the end of the study. Non-HDL cholesterol concentration reached during the follow-up was associated with cIMT only in subjects with abnormal cIMT at baseline. In this subgroup, cIMT tended to avoid progression with non-HDL cholesterol <130 mg/dL. CONCLUSION Atherosclerosis progression varies greatly among ADH patients. cIMT progression was inversely related to baseline cIMT and previous use of statins, and positively with age and CVD risk factors during the follow-up. Patients previously treated with statins may not be the preferred candidates for atherosclerosis regression trials. Treatment recommendations in ADH should be based on baseline risk.