Phytosterols determination

Mitei et ah (2009) determined the relative percentage composition of phytosterols in morama bean oil and foimd 4-desmethylsterols to be the most abimdant phytosterols in the oil (77% or 149.15 gg/g). The content of 4-desmethylsterols is comparable to the phytosterols content of olive oil... 

A capillary gas chromatographic method is described for determination of major phytosterols and cholesterol in edible oils and fats. To extract the unsaponifiable matter and for sample cleanup, solid-phase extraction with C18 absorbent was used. 

Feces collection return the mice to their respective cages and collect feces for 24 h (see Note 3). Longer collection times have the potential to underestimate absorption since some absorbed cholesterol will end up in HDL and subsequently be taken up by the liver, transported to the bile, and secreted into the intestine. However, semi-purified diets are usually very low in fiber, which may reduce intestinal motility. If necessary, sterol transit time is measured in a preliminary experiment where only radiolabeled phytosterol is given and total fecal output is collected on days 1, 2, and 3. Sterols are quantitatively extracted (see below Section 3.1.3) and the time for 90% excretion determined. 

Prepare GC vials to generate standard curves for 5a-cholestane, cholesterol, coprostanol, and lathosterol. Put appropriate amounts of standard from the ethanol stocks into GC vials, dry, and resuspend in 100 pi hexane. Sensitivity varies between instruments but 0.3-10 pg (in halflog steps) is a good initial range. Also prepare vials of the various phytosterols (see Section 2.8) to verify peak identification and sufficient separation between peaks to be quantitated (see Note 5). Determine area under the curve (AUC) for all peaks of interest. 

The oils present in the QF differ from those found in the germ (corn oil) and in particular are enriched for phytosterols, which are cholesterol-lowering agents (5-9). A prior study with corn fiber demonstrated that the oils withstood dilute-acid) pretreatment and became enriched in the pretreated solids (22). Therefore, it was of interest to determine whether dilute-acid-pretreated QF solids might also serve as a source for these valuable nutraceutical chemicals. Following SSF, the solids residue was recovered and analyzed for the presence of oils. It was determined that only 1.12% oils was present in the residual solids (post-SSF, Table 3), which is comparable with that found in untreated QF (1.24-3.49% oil as noted in ref. 2). 

Some of the tests involved relatively simple colour reactions such as the Baudouin reaction for sesame oil, and the Halphen test for cottonseed oil. In both cases a compound characteristic to an oil is used to determine the presence of the oil. Here again the test detected a component that today would be detected and quantified by gas chromatography (GC) or high performance liquid chromatography (HPLC). It was even possible to determine the presence of cholesterol or phytosterols, although, after separation, the identification as to which type was present depended on microscopic examination and fractional... 

Guardiola, F., Boatella, J., Codony, R. 2002. Determination of cholesterol oxidation products by gas chromatography. In Cholesterol and Phytosterol Oxidation Products Analysis, Occurrence, and Biological Effects (F. Guardiola, P.C. Dutta, R. Codony, G.P. Savage, eds.), pp. 50-65, AOCS Press, Champaign, IL. 

The fatty/waxy products contained the lipophilic substances, including fatty oils, waxes, resins and colorants. Valuable pharmacological effects were proved for some minor constituents of these products (e.g. triterpenes, diterpenes, sterols and carotenoids). Thin layer chromatography and on-line UV-VIS spectroscopy were used for the quick identification and quantity determination of these compounds using authentic samples as standards. The SFE method proved favorable in terras of both extraction yield and speed of carotenoids. The CO2 extracts of the lavandin, clary sage and thyme have been enriched in triterpenic compounds (a-es P-amyrin, oleanic acid, ursolic acid, etc.) and phytosterols. Both free and esterified triterpenoids were present in the extracts of the different samples. Furthermore camosol and other diterpenes were detected in the SFE extract of Lamiaceae plants. The fatty acid composition was only slightly different for extracts obtained by SFE and conventional hexane extraction. 

Lu, B. Zhang, Y. Wu, X. Shi, J. 2007. Separation and determination of diversiform phytosterols in food materials using supercritical carbon dioxide extraction and ultraperformance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry. Anal. Chim. Acta 588 50-63. 

A concern has been raised that phytosterol doses that are effective for cholesterol reduction may impair the absorption and lower blood concentrations of fat-soluble vitamins and antioxidants. A number of studies showed that phytosterols had no effect on plasma concentrations of vitamin D, retinol, or plasma-lipid-standardized alpha-tocopherol. Moreover, the reports of the effect of phytosterols on concentrations of blood carotenoids (lutein, lycopene, and alpha-carotene) are controversial. There seems to be general agreement that phytosterol doses >1 g/d significantly decrease LDL-C standardized beta-carotene concentrations however, it remains to be determined whether a reported 15-20% reduction in beta-carotene due to phytosterol supplementation is associated with adverse health effects. Noakes et al. found that consumption of one or more carotenoid-rich vegetable or fruit servings a day was sufficient to prevent lowering of plasma carotenoid concentrations in 46 subjects with hypercholesterolemia treated with 2.3 g of either sterol or stanol esters. 

Adulteration of fats and oils is an old problem. Many older tests involved determination of physical properties such as refractive index, melting point, and viscosity. However, color tests were later used for this purpose. Thus, Baudonin reaction for sesame oil and the Halpben test for cottonseed oil have been noted. In both cases, a compound characteristic to an oil determines the presence of the oil. However, today such detections and quantitations are carried out with GC and HPLC procedures. Thus, cholesterol and phytosterols may be determined by gas chromatography for fingerprinting purposes however, fatty acid analysis might also be used for higher levels of contamination (31). Detailed discussion of issues related to oil authentication and adulteration has taken place (11). 

Paprika Capsicum annuum) is a commonly used flavor enhancer, and following production, the seeds are treated as waste. Paprika seed oils have been evaluated for their physicochemical properties (22, 23, 32). Paprika seed oil contained more than 82% of total unsaturated fatty acids, with polyunsaturated fatty acids comprising 67.8% of total fatty acids (Table 4) (22, 23). Oleic acid was the second major fatty acid at approximately 15% of the total. This fatty acid profile was consistent with a previous observation by Domokos et al. (32) on the fatty acid profile of Hungarian paprika seed oils. Linoleic acid comprised 74.4% of the total fat, whereas oleic and palmitic acid made up 9.8% and 11.2% of total fat, respectively (32). The paprika seed oil was determined to contain 870 mg/kg oil total tocopherols, 380 mg/kg oil carotenoids, and 0.92% phytosterols (32). 

Schmeltz, L, K.G. Ching, and D. Hoffmann Formation and determination of ethyl carbamate in tobacco and tobacco smoke J. Anal. Toxicol. 2 (1978) 265-268. Schmeltz, I., A. de Paolis, and D. Hoffmann Quantitative analysis of major phytosterols in tobacco and smoke 28th Tobacco Chemists Research Conference, Program Booklet and Abstracts, Vol. 28, Paper No. 43, 1974, p. 29 Chemical studies on tobacco smoke. XXXI. Phytosterols in tobacco Quantitative analysis and fate in tobacco combustion Beitr. Tabakforsch. 8 (1975) 211-218. 

Coleoptera. The confused flour beetle, Tribollum confusum, was the first phytophagous insect we found that produces an appreciable amount of a sterol other than cholesterol from radiolabeled dietary C28 and C29 phytosterols. We found this insect produced large quantities of 7-dehydrocholesterol, equivalent to as much as 70% of the total tissue sterols isolated (12). It was further determined that cholesterol and 7-dehydrocTfolesterol were in equilibrium in this flour beetle. Another new intermediate, 5,7,24-cholestatrien-3B-ol was identified as an intermediate between desmosterol and 7-dehydrocholesterol (Figure 3). We found very similar pathways of sterol metabolism to exist in the closely related flour beetle, Tribolium castaneum (13). However, another flour beetle, Tenebrio moHtor, nad only about one-third or less of the levels of 7-dehydrocholesterol as the two Tribolium species, but still much higher levels of this sterol than has been found in most species. Fucosterol 24,28-epoxide was also implicated as an intermediate in the synthesis of cholesterol from sitosterol in T. mol i tor (14). 

Vegetable oil modification involves not only the FA qualitative and quantitative manipulations, but also the manipulation of components such as tocopherols and phytosterols. To fully characterize oils from genetically modified plants, these oils must be analyzed for their stereochemical stracture, which determines several oil properties and the suitability of the oil for various purposes. 

Phytosterols has been found to be beneficial for health due to its antioxidant, antiinflammatory, and antibacterial activities, as well as for its capacity in protection against cancers, such as breast, colon, and prostate [67-71], Furthermore, in 2000, the U.S. Food and Drug Administration officially recognized that products containing phytosterols decreased the risk of the cardiovascular diseases if they are associated to low saturated fat and low-cholesterol nutrition [72], Hence, there is a great interest in determining their content and characterizing their structure. 

It should be noted that the content of these compounds varies considerably, but always within the rank of composition fixed by the COI standard, according to several factors, being the most important ones the olive variety [76-78] and the ripening degree of olives [79,80]. The determination of these compounds is also of major interest because their composition can be used to detect adulteration and genuineness or to check geographical origin and varietal authenticity [81]. Thereby, the study of the VOO phytosterols composition usually represents a subject of interest and... 

Likewise, although the quantification of phytosterols in VOO analysis is usually performed by GC with different detectors [80,85,86], recently published research has proposed new analytical methods for the precise determination of phytosterols in olive oil using HPLC-MS [22,87], and more recently, highly sensitive methods based on UHPLC-MS were applied in the determination of VOO phytosterols [88,89]. 

Johnsson L, Dutta PC (2(X)6) Determination of phytosterol oxides in some food products by using an optimized transesterification method. Food Chem 97(4) 606-613... 

Plant lipids contain 0.15-0.9% sterols, with sitosterol as the main component (Table 3.52). In order to identify blends of fats (oils), the data on the predominant steroids are usually expressed as a quotient. For example, the ratio of stigmas-terol/campesterol is determined in order to detect adulteration of cocoa butter. As seen from Table 3.53, this ratio is significantly lower in a number of cocoa butter substitutes than in pure cocoa butter. The phytosterol fraction (e. g. sito- and campesterol) has to be determined in order to detect the presence of plant fats in animal fats. 

HE Indyk. Simultaneous hquid chromatographic determination of cholesterol, phytosterols and tocopherols in foods. Analyst 115 1525-1530, 1990. 

Indyk, H.E. Simultaneous liquid chromatographic determination of cholesterol, phytosterols and toco-pherols in foods, AreoZyst, 1990,115, 1525-1530. 

---