Analysis sterol

Based on the clinical suspicion of a cholesterol biosynthesis defect, the first line of diagnosis for these various defects often involves sterol analysis in patient material where detection of a specific sterol intermediate is indicative for the respective defect. Confirmative diagnostic procedures involve enzymatic assays and/or molecular testing. 

Sterol analysis and quantification can be performed using plasma, serum, and cerebrospinal fluid (CSF) samples. Analysis of sterols from tissues and cells requires a pre-extraction work-up. The requirements for the different specimens are as follows ... 

As internal control for sterol analysis in plasma, serum, and liquor, each series of samples includes a 150 pi aliquot of a large batch of control plasma samples in which cholestanol and cholesterol concentrations are determined and compared with previously determined concentrations. The values for these should fall within 2 standard deviations. The control plasma sample is composed of pooled plasma samples of different individuals stored as 150-pi aliquots at - 18°C. 

Fig. 5.1.3 Sterol analysis in patients with defective cholesterol biosynthesis. Gas chromatography-mass spectrometry analysis of trimethylsilyl derivatives of sterols extracted from primary skin fibroblasts of a control subject, a Smith-Lemli-Opitz syndrome (SLOS) patient, and a Con-radi-Hunermann syndrome (CDPX2) patient, and lymphoblasts of a desmosterolosis patient cultured in lipoprotein-deficient medium reveals the accumulation of sterol intermediates indicative of a defect in cholesterol biosynthesis. Similar spectra can be obtained by sterol analysis of the plasma of such patients...

As an alternative, primary skin fibroblasts or lymphoblasts of patients suspected to be affected with a cholesterol biosynthesis defect can be cultured for 3-7 days in medium supplemented with fetal calf serum depleted of lipoproteins to induce cholesterol biosynthesis, whereupon the specific defect can be determined by sterol analysis using GC-MS as described above. This procedure will readily identify patients affected with Smith-Lemli-Opitz syndrome, desmosterolosis, lathosterolosis, hydrops-ectopic calcification-motheaten (HEM) skeletal dysplasia and most patients with Conradi-Hunermann syndrome (CDPX2). Patients with congenital hemidys-plasia with ichthyosiform nevus and limb defects (CHILD) syndrome may not be identified with this assay, but they can be readily diagnosed on the basis of their typical clinical presentation. 

An example for a GC-analysis of isolated sterols from P. oryzae is shown in Figure 8. An elution diagram of an untreated control is compared to the sterol analysis after application of 10 ppm triadi-menol. It can easily be seen that in the region where the sterols are eluted (framed area), the pattern becomes totally different. The elution index is the first criterion for the chemical nature of an accumulating sterol. However, structure elucidation has been per-... 

The above shows that rapeseed oil can easily be detected, or eliminated, as a contaminant by sterol analysis. It is also, at least in Europe, the oil most likely to be used to dilute another oil. Although low levels (as a percentage of the total sterols) have been reported in some other oils (Desbordes etal., 1993), the presence of brassicasterol in an oil is good evidence of contamination in any oil from a non-Brassica species. It is likely that the traces reported as present in some other oils arise from contamination of the sample with rapeseed oil, or from some other Brassica species, or from traces of some similarly behaving non-sterol not fully separated from the sterol fraction during the work-up of the sample (Desbordes et al., 1983). 

Sterol analysis can be useful other than for detecting rapeseed oil. Accepted ranges for many oils and all major oils, given as a percentage of the total sterols, are available (Codex Alimentarius, 1997 FOSFA, 1994 AOCS, 1997). In all... 

Sterol analysis, fatty acid analysis of the whole fat and of the acids at the 2-position, and triglycerides by GC, can be very useful in determining adulteration of and by animal fats. In the future HPLC of triglycerides is likely to provide an even better method for some purposes, but much more data need to be collected before this can be evaluated. 

In some cases where extreme refining conditions have been used, complete removal of the sterols, both free and esterified, may occur (Grob et al., 1994). By removing the sterols from an oil in this way, it is possible to prepare an adulterant that is undetectable by sterol analysis. As with adulteration of virgin olive oil with refined olive oil, this type of adulteration may be detected by analysis of sterol degradation products. 

Analytical methods for plant sterol analysis are commonly based on procedures used for cholesterol analysis. However, a significant shortcoming of these methods is the fact that cholesterol occurs only as free cholesterol and fatty acid esters. Therefore, the analytical methods optimized for cholesterol analysis are not suitable, or only suitable with some restrictions, for the analysis of conjugates found only in plants (SFs, SGs, and ASGs). Further, the methods described below only give the total amount of plant sterols and no information of the different sterol species found in the samples. However, if detailed information about the sterol composition is not required, and the amount of sterols to be analyzed is sufficiently high for these less sensitive but simpler methods, they provide a less laborious alternative for the analysis. 

The columns used for the GC separation of phytosterols are currently almost exclusively capillary columns with 0.1-0.3 mm internal diameter, and fused-silica capillary columns with chemically bonded stationary phases are commonly used (Abidi, 2001). The best separation of structurally very similar sterols, such as sitosterol and its saturated counterpart sitostanol, is obtained with slightly polar stationary phases like 5% diphenyl-95% dimethylpolysiloxane, and they are currently the most used columns for the separation of phytosterols (Lagarda et al., 2006). For detailed lists of different columns used in sterol analysis, see the papers by Abidi (2001) and Lagarda (2006). 

The main shortcoming of the current analytical methods is the fact that none of them is streamlined to analyze all classes of phytosteryl conjugates separately in one analysis. The methods currently used focus on, and are highly optimized to analyze, one or two groups of the sterol classes but not all of the classes in the same analysis. This multiplies the number of samples to be analyzed and creates some overlaps in the data. On the other hand, analytical procedures for total sterol analysis (without information on the conjugation) are easily available. 

Sterol Composition. Sterol analysis involves preparation of the unsaponifiable fraction, fractionation by thin-layer chromatography (TLC), and gas chromatographic analysis of the TMS derivatives (66). The following limits apply to aU types of olive oil (12) ... 

Dihydroxyterpene Alcohol Content. Olive-pomace oil contains relatively high levels of erythrodiol, uvaol, and wax esters. Erythrodiol and uvaol (total diol) content is determined by the same procedure as that used for sterol analysis (80, 81). Limits for total diol content (as % of total sterols) are as follows ... 

Italian and Spanish ohve oil from the 1991-1992 crop year contained a very high level of 9,19-cyclolanosterol (>400 mg/kg), which was not found with the standard method for sterol analysis. Two isomers of this sterol were identified by GC/MS of the unsaponifiable fraction, and their levels were found to be inversely proportional to the levels of p-sitosterol in the oils. GC/MS of the unsaponifiable fraction with high-resolution GC capillary columns provides a relatively rapid means of checking product purity and the identity of individual components. Thus, triterpene diols were identifiable at m/z 203, ot-tocopherol at m/z 165, squalene at m/z 69, cholesterol at m/z 386, and brassicasterol, characteristic of canola oil and other Brassica oils, at m/z 398. 

Several studies have been aimed at the detection of a fraudulent addition of vegetable oils to olive oil. In particular, different analytical methods can be applied to determine blends of regular and high-oleic sunflower oil with olive oil. The minimum sunflower oil detection level depends on the analytical method used. For example, a minimum detectable level of 0.7% of regular or high-oleic sunflower oil may be achieved through methods of sterol analysis, and analysis of the fatty acids will not enable detection of additions below 20% (45). 

When sterol analysis is performed after saponification of ester bonds, a total value is obtained for the level of each sterol by GC analysis. Free sterols can be separated from sterol esters before saponification by means of thin-layer chromatography (326) or solid phase extraction (327-330) using hexane to elute the sterol esters and diethyl ether or a mixture of diethyl ether and hexane to elute the sterols. Once separated, sterol esters need be saponified to release sterols before gas... 

Jayasinghe, L. Y., Marriott, P. J., Carpener, P. D., and Nichols, P. D., SFE and GC electron capture detection method for sterol analysis of environmental water samples. Anal. Commun., 35, 265-268,... 

Olive oil is often illegally adulterated with other less expensive vegetable oils. Oils widely used for this purpose include olive pomace oil, corn oil, peanut oil, cottonseed oil, sunflower oil, soybean oil, and poppy seed oil. °° Among the varions chemical and physical methods employed toward the detection of the adulteration of olive oil by low-grade olive oils and seed oils are (a) Sterol analysis (presence of stigmasterol and 3-sitosterol), (b) alkane analysis (C27, C29, and C31), (c) wax and aliphatic alcohol analysis, (d) fatty acids/(with HPLC) trans fatty acid, and (e) Triacylglycerol. 

Homberg, E. and Bielefeld, B. Main components of 4-methyl-sterol and triterpene fraction of twelve vegetable fats and their influence on sterol analysis. Fat Science and Technology, 92(12), 478-480. 1990. 

Sterol analysis was also as described previously (Rossell, King and Downes, 1983) except that derivatization was by means of trimethylchloro-silane (Rossell, King and Downes, 1985). 

Copius Peereboom, J. W. Chromatographic Sterol Analysis, as applied to... 

Special laboratory cartilage Sterol analysis (P, tissue, lymphoblasts) 4-methylsterols 4,4 -dimethylsterols> 4-carboxysterols T T... 

Special laboratory larynx Sterol analysis (P, FB, LYM) (9)-cholestenol HT T-n... 

Special laboratory Sterol analysis (tissue, P) desmosterol tn T... 

Special laboratory Sterol analysis (P, FB LYM) 7 -dehy droch olesterol n (<1%) t TT-m... 

Sterol analysis None P. LYM, FB Keep frozen (-20 °C) None... 

Bergmann also recognized the importance of sterol analysis for the taxonomy and phylogeny of sponges, and attempted to correlate sterol distribution with sponge taxonomy (77). 

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