First international descriptive and interventional survey for cholesterol and non-cholesterol sterol determination by gas- and liquid-chromatography-Urgent need for harmonisation of analytical methods.

Serum concentrations of lathosterol, the plant sterols campesterol and sitosterol and the cholesterol metabolite 5α-cholestanol are widely used as surrogate markers of cholesterol synthesis and absorption, respectively. Increasing numbers of laboratories utilize a broad spectrum of well-established and recently developed methods for the determination of cholesterol and non-cholesterol sterols (NCS). In order to evaluate the quality of these measurements and to identify possible sources of analytical errors our group initiated the first international survey for cholesterol and NCS. The cholesterol and NCS survey was structured as a two-part survey which took place in the years 2013 and 2014. The first survey part was designed as descriptive, providing information about the variation of reported results from different laboratories. A set of two lyophilized pooled sera (A and B) was sent to twenty laboratories specialized in chromatographic lipid analysis. The different sterols were quantified either by gas chromatography-flame ionization detection, gas chromatography- or liquid chromatography-mass selective detection. The participants were requested to determine cholesterol and NCS concentrations in the provided samples as part of their normal laboratory routine. The second part was designed as interventional survey. Twenty-two laboratories agreed to participate and received again two different lyophilized pooled sera (C and D). In contrast to the first international survey, each participant received standard stock solutions with defined concentrations of cholesterol and NCS. The participants were requested to use diluted calibration solutions from the provided standard stock solutions for quantification of cholesterol and NCS. In both surveys, each laboratory used its own internal standard (5α-cholestane, epicoprostanol or deuterium labelled sterols). Main outcome of the survey was, that unacceptably high interlaboratory variations for cholesterol and NCS concentrations are reported, even when the individual laboratories used the same calibration material. We discuss different sources of errors and recommend all laboratories analysing cholesterol and NCS to participate in regular quality control programs.

Oxidation of sitosterol and campesterol in foods upon cooking with liquid margarines without and with added plant sterol esters.

Plant sterol (PS) oxidation products (POP) derived from sitosterol and campesterol were measured in 15 foods cooked with liquid margarine without (control) and with added 7.5% PS. POP were analyzed using a GC-MS method. PS liquid vs. control margarine resulted in a higher median POP content per food portion (1.35mg, range 0.08-13.20mg versus 0.23mg, 0.06-0.90mg), a lower PS oxidation rate (0.63 vs. 1.29%) and lower oxidation susceptibility of sitosterol vs. campesterol. POP formation was highest in shallow-fried potatoes with PS liquid margarine (64.44mg per portion food plus residual fat). Mean relative abundances of epoxy-, 7-keto-, 7-hydroxy- and triol-PS derived from sitosterol and campesterol were 40.0, 34.4, 21.5 and 4.0% with control vs. 44.1, 23.8, 29.6 and 2.4% with PS liquid margarine. In conclusion, PS liquid margarine increased POP content in foods with a POP profile characterized by a higher ratio of epoxy- to 7-keto-derivatives.

Development and validation of methodologies for the quantification of phytosterols and phytosterol oxidation products in cooked and baked food products.

Chromatography-mass spectrometry (GC-MS) methodologies for the analysis of the main phytosterols (PS) and phytosterol oxidation products (POPs) present in 19 different foodstuffs cooked or baked using margarines with or without added plant sterols are presented. Various methods for fat extraction were evaluated to allow the GC-MS analysis of large numbers of prepared vegetable, fish and meat products, egg and bakery items in a practically feasible manner. The optimized methods resulted in a good sensitivity and allowed the analysis of both PS and POPs in the broad selection of foods at a wide range of concentrations. Calibration curves for both PS and POPs showed correlation coefficients (R(2)) better than 0.99. Detection limits were below 0.24mgkg(-1) for PS and 0.02mgkg(-1) for POPs, respectively. Average recovery data were between 81% and 105.1% for PS and between 65.5 and 121.8% for POPs. Good results were obtained for within- and between-day repeatability, with most values being below 10%. Entire sample servings were analyzed, avoiding problems with inhomogeneity and making the method an exact representation of the typical use of the food by the consumer.

Formation of Plant Sterol Oxidation Products in Foods during Baking and Cooking Using Margarine without and with Added Plant Sterol Esters.

Plant sterols (PS) in foods are subject to thermal oxidation to form PS oxidation products (POP). This study measured POP contents of 19 foods prepared by typical household baking and cooking methods using margarines without (control) and with 7.5% added PS (as 12.5% PS-esters, PS-margarine). Median POP contents per portion size of cooked foods were 0.57 mg (range 0.05-1.11 mg) with control margarine versus 1.42 mg (range 0.08-20.5 mg) with PS-margarine. The oxidation rate of PS (ORP) was 0.50% (median) with the PS-margarine and 3.66% with the control margarine. Using the PS-margarine, microwave-cooked codfish had the lowest POP content, with 0.08 mg per portion, while shallow-fried potatoes had the highest POP content, 20.5 mg per portion. Median POP contents in cookies, muffins, banana bread, and sponge cake baked with the control or PS-margarine were 0.12 mg (range 0.11-0.21 mg) and 0.24 mg (range 0.19-0.60 mg) per portion, with a corresponding ORP of 1.38% and 0.06%, respectively. POP contents in all the cooked and baked foods did not exceed 20.5 mg per typical portion size. A wide variation in the distribution of individual POP among different foods existed, with 7-keto-PS and 5,6-epoxy-PS being the major oxidation products.

Sterols heating: degradation and formation of their ring-structure polar oxidation products.

Cholesterol and phytosterols can be oxidised under heating conditions to give sterol oxidation products (SOPs), known by their toxic effects. This paper studied the degradation of cholesterol and three plant sterols during a 360 min heating treatment (180 °C). The formation and further degradation of SOPs was also analysed by GC-MS. Results revealed a sterol susceptibility to degradation according to the following decreasing order: campesterol≈ß-sitosterol≥stigmasterol>cholesterol. The degradation curve fit (R(2)=0.907-0.979) a logarithmic model. SOPs increased their concentration during the first 5-10 min and thereafter, their degradation rate was higher than their formation rate, resulting in a decrease over time. Irrespective of the sterol from which they had derived, 7-keto derivatives presented the highest levels throughout the entire process, and also SOPs with the same type of oxidation followed a similar degradation pattern (R=0.90-0.99).

Development and validation of a comprehensive two-dimensional gas chromatography-mass spectrometry method for the analysis of phytosterol oxidation products in human plasma.

Phytosterol oxidation products (POPs) have been suggested to exert adverse biological effects similar to, although less severe than, their cholesterol counterparts. For that reason, their analysis in human plasma is highly relevant. Comprehensive two-dimensional gas chromatography (GC×GC) coupled with time-of-flight mass spectrometry (TOF-MS) has been proven to be an extremely powerful separation technique for the analysis of very low levels of target compounds in complex mixtures including human plasma. Thus, a GC×GC/TOF-MS method was developed and successfully validated for the simultaneous quantification of ten POPs in human plasma. The calibration curves for each compound showed correlation coefficients (R(2)) better than 0.99. The detection limits were below 0.1 ng mL(-1). The recovery data were between 71.0% and 98.6% (RSDs <10% for all compounds validated). Good results were obtained for within- and between-day repeatability, with most values being below 10%. In addition, non-targeted sterol metabolites were also identified with the method. The concentrations of POPs found in human plasma in the current study are between 0.3 and 4.5 ng mL(-1), i.e., 10-100 times lower than the typical values found for cholesterol oxidation products.

Stability of sterols in phytosterol-enriched milk under different heating conditions.

Commercially available phytosterol-enriched milk was subjected to usual and drastic heating conditions to evaluate the stability of the sterols at different treatments. Products showed 422.2 mg of phytosterols/100 g of milk and 132 microg of sterol oxidation products (SOPs)/g of fat (277 microg of SOPs/100 g of milk). Schaal oven conditions (24 h/65 degrees C, equivalent to 1 month of storage at room temperature) reduced the phytosterol content by only 4%. Drastic heating treatments (2 min of microwave heating at 900 W or 15 min of electrical heating at 90 degrees C) led to a 60% decrease of total phytosterol content, with a significant increase of TBARs. The oxysterol amount under those conditions (which was higher in microwave-treated samples) was lower than expected, probably because of the degradation of the oxidation products. Usual heating conditions (1.5 min of microwaves) maintained phytosterol content on physiologically active values (301 mg/100 g of milk) with oxidation percentages around 0.12-0.40% for phytosterols and 1.13% for cholesterol.

Validation of a gas chromatography-mass spectrometry method for the analysis of sterol oxidation products in serum.

A validated gas chromatography-mass spectrometry (GC-MS) detection method for the quantitative analysis of sterol oxidation products (SOPs) in serum is described. After a lipid extraction procedure with chloroform-methanol, a cold saponification and purification by solid phase extraction, oxysterols were derivatized to form trimethyl-sylil-ethers which were subjected to GC-MS analysis. Calibration curves for cholesterol oxidation products showed determination coefficient (R(2)) of 1.0, with low values for the coefficient of variation of the response factors (< 1%). Detection and quantification limits were below 5 ng/mL and 10 ng/mL, respectively. Recovery data were between 77.65% and 110.29% (CV < 10% for all compounds). Good results were obtained for within- and between-day repeatability, with values below 10%. In conclusion, the method performed is suitable for the determination and quantification of SOPs in serum.

Óxidos de colesterol en alimentos cocinados con presencia habitual en nuestra dieta / Cholesterol oxidation products in common cooked foods

Los productos de oxidación del colesterol (COPs) poseen demostrados efectostóxicos y están implicados en el desarrollo de aterosclerosis. Pueden estar presentesen organismos animales y por ende, en alimentos de origen animal, siendosusceptibles de ser absorbidos a través de la dieta. Su formación en los alimentosse favorecería, al tratarse de un proceso de oxidación química, por la elevación dela temperatura y la presencia de oxígeno. En este trabajo se presenta una estimaciónde la presencia de COPs en diferentes tipos de alimentos cocinados mediantediferentes tecnologías culinarias y almacenados mediante distintas modalidades deconservación. El análisis se llevó a cabo por cromatografía de gases-espectrometríade masas. Tanto pescados (salmón y langostinos) como carnes (hamburguesas,pechugas de pollo, lomo y salchichas tipo frankfurt) mostraron valores bajos deCOPs en crudo (0.003-0.552 mg/100 g alimento), incrementándose significativamentetras el cocinado (hasta 0.7 mg/100 g alimento). El cocinado con microondassupuso el mayor incremento de COPs en comparación con la fritura, plancha yasado. El almacenamiento a vacío disminuyó drásticamente la formación de COPs respecto al almacenamiento en aerobiosis. La congelación ralentizó más eficazmentela formación de COPs que la refrigeración

Cholesterol oxidation products (COPs) have shown different toxic effects andare involved in the development of atherosclerosis. These compounds can be foundin animal organisms, and inconsequence in animal origin foods, and they aresusceptible to be absorbed from the diet. Their formation in foods would be increasedby high temperatures and the presence of oxygen, as it is a chemicaloxidation process. In this paper, an estimation of the presence of COPs in differenttypes of foods treated by different cooking technologies are shown. Also differentstorage conditions are studied. The analysis was carried out by gas chromatography-mass spectrometry. Both fish (salmon and shrimps) and meat (hamburgers,breast chicken, pork loin and frankfurters) showed low COPs values in raw products(0.003-0.552 mg/100 g food), increasing significantly after the application ofcooking technologies (up to 0.7 mg/100 g food). Microwave treatment leaded to thehighest increase of COPs in comparison to frying, grilling and roasting. Vacuumstorage dramatically decreased COPs formation with regard to aerobic storage.Freezing minimized COPs formation more efficiently than refrigeration