In vitro effects of sitosterol and sitostanol on mitochondrial respiration in human brown adipocytes, myotubes and hepatocytes.

PURPOSE: Lowering of LDL cholesterol levels by plant sterols and stanols is associated with decreased risk of cardiovascular disease in humans. Plant sterols and stanols also lower triacylglycerol (TG). However, it is not fully understood how reduction in TG is achieved and what the full potential of plant sterols and stanols is on whole-body metabolism. We here hypothesize that high levels of plant sterols and stanols stimulate whole-body energy expenditure, which can be attributed to changes in mitochondrial function of brown adipose tissue (BAT), skeletal muscle and liver. METHODS: Phytosterolemic mice were fed chow diets for 32 weeks to examine whole-body weight gain. In vitro, 24-h incubation were performed in adipocytes derived from human BAT, human myotubes or HepG2 human hepatocytes using sitosterol or sitostanol. Following mitochondrial function was assessed using seahorse bioanalyzer. RESULTS: Chow feeding in phytosterolemic mice resulted in diminished increase in body weight compared to control mice. In vitro, sitosterol or sitostanol did not change mitochondrial function in adipocytes derived from human BAT or in cultured human myotubes. Interestingly, maximal mitochondrial function in HepG2 human hepatocytes was decreased following sitosterol or sitostanol incubation, however, only when mitochondrial function was assessed in low glucose-containing medium. CONCLUSIONS: Beneficial in vivo effects of plant sterols and stanols on lipid and lipoprotein metabolism are well recognized. Our results indicate that alterations in human mitochondrial function are apparently not involved to explain these beneficial effects.

Thyroid Hormone Status in Sitosterolemia Is Modified by Ezetimibe.

OBJECTIVES: To assess the association between biomarkers of thyroid status and 5α-stanols in patients with sitosterolemia treated with ezetimibe (EZE). STUDY DESIGN: Eight patients with sitosterolemia (16-56 years of age) were studied during 14 weeks off EZE therapy and 14 weeks on EZE (10 mg/day). Serum thyroid biomarkers (free triiodothyronine [FT3], free thyroxine [FT4], FT3/FT4 ratio, thyroid-stimulating hormone), 5α-stanols (sitostanol and cholestanol), and cholestanol precursors (total cholesterol and its synthesis marker lathosterol, and 7α-hydroxy-4-cholesten-3-one cholestenol) were measured at baseline and during the 14 weeks off EZE and on EZE. RESULTS: EZE increased FT3/FT4 (10% ± 4%; P = .02). EZE reduced plasma and red blood cells sitostanol (-38% ± 6% and -20% ± 4%; all P < .05) and cholestanol (-18% ± 6% and -13% ± 3%; all P < .05). The change in plasma cholestanol level on EZE inversely correlated with the change in FT3/FT4 (r = -0.86; P = .01). EZE lowered total cholesterol (P < .0001) and did not affect 7α-hydroxy-4-cholesten-3-one cholestanol. EZE increased (P < .0001) lathosterol initially, but the level was not sustained, resulting in similar levels at week 14 off EZE and on EZE. CONCLUSION: In patients with STSL, 5α-stanols levels might be associated with thyroid function. EZE reduces circulating 5α-stanols while increasing FT3/FT4, implying increased conversion of T4 to T3, thus possibly improving thyroid hormone status. TRIAL REGISTRATION: ClinicalTrials.govNCT01584206.

Expression and properties of three novel fungal lipases/sterol esterases predicted in silico: comparison with other enzymes of the Candida rugosa-like family.

Lipases from the Candida rugosa-like family are enzymes with great biotechnological interest. In a previous work, several enzymes from this family were identified by in silico mining of fungal genomes. Here, we describe the cloning, expression, and characterization of putative lipases from the genomes of Nectria haematococca, Trichoderma reesei, and Aspergillus niger and compared their catalytic properties with those of OPE, a well-characterized sterol esterase/lipase from Ophiostoma piceae. All of them hydrolyzed p-nitrophenol esters and triglycerides with different efficiency, but their activity against sterol esters was dissimilar, and the enzyme from A. niger was unable of hydrolyzing these substrates while OPE showed the best k cat values, which in general leads to an improved catalytic efficiency. Similarly, OPE was the best catalyst in the synthesis of ß-sitostanyl oleate, followed by the commercial CRL from C. rugosa, while the A. niger enzyme was unable to produce this compound. When the enzymes were evaluated for caprolactone oligomerization, the A. niger enzyme gave similar results than CRL, being OPE slightly more efficient. The expression of the putative selected proteins allowed their functional validation, suggesting that the hydrophobicity of the lid region may be an important factor, although the enzymatic efficiency is also influenced by other parameters, as the aggregation state and the size and morphology of the tunnel, where substrate recognition and catalysis takes place.

Obesity Does Not Interfere with the Cholesterol-Lowering Effect of Plant Stanol Ester Consumption (as Part of a Heart-Healthy Diet).

Dietary modifications including plant stanol ester consumption are recommended measures to control serum and low-density lipoprotein (LDL)-cholesterol concentrations, but obesity can affect their responses. We investigated whether body mass index (BMI) affects serum cholesterol levels during plant stanol (mainly sitostanol) ester consumption. This ad hoc analysis was based on earlier results of a cross-over, randomized controlled trial of postmenopausal women consuming rapeseed oil-based margarine without or with plant stanol ester (3 g plant stanols/day) for seven weeks. We classified the subjects as normal-weight (BMI ≤ 25 kg/m2, n = 9, mean 22.6 kg/m2) or overweight/obese (BMI > 25 kg/m2, n = 11, mean 28.4 kg/m2), and recalculated the results, focusing on cholesterol absorption, cholesterol synthesis, and fecal steroid outputs. Serum cholesterol levels were similar in the groups during the control diet. Plant stanol ester reduced serum cholesterol by 0.63 ± 0.19 mmol/L (11%) in normal-weight and by 0.75 ± 0.13 mmol/L (12%) in overweight/obese subjects (p < 0.05 for both), and cholesterol absorption was reduced in both groups. However, relative and dietary cholesterol absorption were more effectively reduced in normal-weight subjects. In conclusion, overweight/obesity did not interfere with the serum cholesterol response to plant stanol ester consumption despite substantial differences in cholesterol metabolism between the groups.

Changes provoked by nixtamalization and tortilla making in the lipids of two corn varieties. A study by 1H NMR.

The aim of this study is to analyze in depth, by means of proton nuclear magnetic resonance, 1H NMR, the changes caused by nixtamalization and tortilla making in the lipid composition of two corn varieties. This technique permits the characterization of not only main but also minor lipid components of both corn and tortilla. Ferulates have been found for the first time among the minor components of these lipids. It has been proved that this processing affects the lipids of both corn varieties in a similar way. The total loss of fatty acids occurs as does partial loss of minor components. Furthermore, a slight oxidation is provoked during this processing as well as a small reduction in the unsaturation degree of the lipids. In spite of this a similar distribution of the different kinds of acyl groups has been found in corn and tortilla within each variety.

Methodological Aspects of Phytosterol Measurements in Biological Samples.

Phytosterol measurement has gained a lot of interest during the last two decades after foods and supplements with added 4-desmethyl phytosterols were recognized and used as effective and safe non-pharmacologic hypocholesterolemic agents, and also after the mechanisms of intestinal absorption and hepatic excretion of sterols were unraveled. In addition, the wide use of serum phytosterols as biomarkers of cholesterol absorption has increased the interest in their measurement. In this review, the basic methods are discussed without going into details of the practical operations. The analysis includes first lipid extraction and saponification from various biologic matrices such as serum/plasma, feces, or tissues, after which the individual sterols are separated by adsorption chromatography (gas-liquid or liquid or high performance liquid chromatography) based on the polarity of the various sterols. We also deal with some specific aspects of phytosterol measurements in biological samples such as the need of harmonization of their analysis in biological samples, the discrepancies in the results of sitosterol and campesterol concentrations between different studies, and what is known about their biological day-to-day fluctuation. Phytosterols have a remarkable role in human health, so that their complicated and time consuming measurements call attention to routine ways of standardization between the sterol research laboratories.

Serum, liver and bile sitosterol and sitostanol in obese patients with and without NAFLD.

Background and aims: Non-alcoholic fatty liver disease (NAFLD) associates with low levels of serum plant sterols in cross-sectional studies. In addition, it has been suggested that the hepatic sterol transport mechanisms are altered in NAFLD. Therefore, we investigated the association between serum, liver and bile plant sterols and sitostanol with NAFLD.Methods: Out of the 138 individuals (age: 46.3 ± 8.9, body mass index: 43.3 ± 6.9 kg/m², 28% men and 72% women), 44 could be histologically categorized to have normal liver, and 94 to have NAFLD. Within the NAFLD group, 28 had simple steatosis and 27 had non-alcoholic steatohepatitis. Plant sterols and sitostanol were measured from serum (n=138), liver (n=38), and bile (n=41). The mRNA expression of genes regulating liver sterol metabolism and inflammation was measured (n=102).Results: Liver and bile sitostanol ratios to cholesterol were higher in those with NAFLD compared to those with histologically normal liver (all P<0.022). Furthermore, liver sitostanol to cholesterol ratio correlated positively with histological steatosis and lobular inflammation (rs > 0.407, P<0.01 for both). In contrast, liver sitosterol to cholesterol ratio correlated negatively with steatosis (rs = -0.392, P=0.015) and lobular inflammation (rs = -0.395, P=0.014). Transcriptomics analysis revealed suggestive correlations between serum plant sterol levels and mRNA expression.Conclusion: Our study showed that liver and bile sitostanol ratios to cholesterol associated positively and liver sitosterol ratio to cholesterol associated negatively with liver steatosis and inflammation in obese individuals with NAFLD..

Optimization of lipase-catalyzed synthesis of ß-sitostanol esters by response surface methodology.

The esters of ß-sitostanol and fatty acids are known for their effect as cholesterol-lowering agents. In this work, the efficiency of three lipases as biocatalysts of the esterification of ß-sitostanol and C16 and C18 fatty acids was compared. The sterol esterase of Ophiostoma piceae (OPEr) yielded the highest esterification rates and was selected for further optimization of the reaction. The effects of four parameters (temperature, enzymatic dosage, acyl donor concentration, and reaction time) on ester synthesis were investigated and the process conditions were optimized using response surface methodology (RSM). The best conditions for esterification for each fatty acid were predicted using a second-order model, and experimentally validated. Very high esterification efficiencies (86-97%) were observed using the predicted values for the four variables. This approach was shown to be suitable for optimizing the enzymatic production of ß-sitostanol esters, which represents a green alternative to the chemical synthesis of these dietary complements.

Dietary plant stanols or sterols neither accumulate in stenotic aortic valves nor influence their structure or inflammatory status.

BACKGROUND & AIMS: Consumption of plant stanols and plant sterols decreases LDL cholesterol level and increases serum concentrations of plant stanols/sterols, but it is practically unexplored whether also their tissue concentrations increase. Thus, the aim of this study was to assess whether consuming plant stanols/sterols increases their concentrations in stenotic aortic valves and affect the valvular structure (collagen and elastin) or inflammation (macrophages and mast cells). METHODS: In a randomized, double-blind controlled intervention patients with severe aortic stenosis consumed margarine without (n = 11) or with 2 g of plant stanols (n = 12) or sterols (n = 13) until valve replacement surgery (2.6 months, on average). The effects of sitostanol and sitosterol on the expression and secretion of proinflammatory cytokines by cultured aortic valve myofibroblasts were also assessed. RESULTS: Control-related LDL-cholesterol was diminished by 16% (p < 0.05) by plant stanol and by 11% (NS) by plant sterol consumption, respectively. In the resected valves, cholesterol, plant stanol and sterol levels were similar in all groups. Consumed plant stanols or sterols had no effect on valvular structure or mast cell or macrophage numbers in valves. Incubation of cultured myofibroblasts derived from stenotic valves with sitostanol or sitosterol decreased mRNA expression of the monocyte chemotactic protein-1 (p < 0.05) and interleukin-1 beta (p < 0.05). CONCLUSIONS: In this study, plant stanol/sterol consumption did not affect cholesterol, plant stanol or sterol levels in stenotic aortic valves; neither did they influence the structure or the inflammatory status of the valves. However, these findings need to be confirmed in a larger-scale intervention. ClinicalTrials.govRegister #NCT00738933.

Identification of Trace Sterols in the Seeds of Foxtail Millet (Setaria italica Beauv.).

Trace sterols in the seeds of foxtail millet (Setaria italica Beauv.) were investigated by GC-MS. Eleven of these trace sterols, i.e., brassicasterol, episterol, 24-methyllathosterol, 24-ethyllathosterol, avenasterol, 24-methylenecholesterol, fucosterol, isofucosterol, 24-methyl-5α-cholest-24(28)-en-3ß-ol, 24-ethyl-5α-cholest-24(28)Z-en-3ß-ol, and 24-ethyldesmosterol, were identified, suggesting that the previously reported high content of sitostanol was possibly contaminated with a small amount of isofucosterol.