Cholesterol quantitative determination

Tea (Camellia sinensis) is one of the most frequently consumed beverages in the world and, consequently, an important agricultural product [168], It has been proved many times that tea may reduce cholesterol level, hypertension, and shows antioxidant and anti-microbial effects [169], Because of its importance, a considerable number of analytical methods have been developed for the separation and quantitative determination of the constituents of tea [170,171]. Thus, the application of high-speed counter-current chromatography [172,173], and HPLC-APCI-MS [174] have been reported. 

An evaporative light scattering detector was coupled with a UV spectrophotometer, and was applied to HPLC for the quantitative determination of cholesterol oxides in edible oils and fats. 

Shaler and Jerpe combined gas chromatography and infrared spectroscopy for the identification of heroin and commonly used diluents, such as quinine and procaine in illicit seizures. The quantitative determination of heroin was carried out gas chromatographically using cholesterol TMS as an internal standard. 

The quantitative determination of total cholesterol is the most widely requested lipid procedure in the clinical laboratory, since it is implicated in atherosclerotic heart disease. 

Suckling rats were given an oral dose of 3.3 mg [U- C]-a-linolenate at 10 d old and were killed 3 h to 30 d later. The C enrichment was quantitatively determined in palmitate, docosahexaenoate, and cholesterol of liver, gut, and brain (Menard et al., 1998). The tracer dose was higher than for a radiotracer but was within physiological limits, thereby avoiding the possibility of overloading any of the pathways under investigation. No [U- C]-a-linolenate itself was found in the brain at any time-point, but enrichment in several n-3 PUFA, especially docosahexaenoate, was detected. Many saturated fatty acids in the brain were also labeled as was brain cholesterol (Table 4). 

The clinical usefulness of the LP-X test is well established in the literature. The cumulative specificity is almost 100%. This means that persons without cholestasis can be classified very accurately. Consequently the positive predictive value is almost 100%. This means, the presence of LP-X invariably indicates cholestasis, i.e., the presence of biliary lipids in the blood. The quantitative determination of LP-X is useful in the differentiation of mechanical cholestasis and cholestatic hepatitis. In hepatitis the relative contribution of LP-X to total cholesterol is much higher than in mechanical cholestasis. A high percentage of cholesterol carried by LP-X in combination with a GPT activity below 100 U/1 is helpful in ruling out metastatic liver disease. 

Blank et al. [73] have found that the calibration curves of various lipids when using another charring procedure and expressing the applied amount as the equivalent amount of carbon, all had the same gradient (Fig. 74). Charring was carried out with a fine spray of saturated potassium dichromate solution in 70% sulphuric acid and heating 25 min at 100° C. Spots from carbonisation of cholesterol with perchloric acid have been evaluated similarly [526]. The quantitative determination of charred spots has found frequent use so far, though principally in lipid analysis... 

In Waldi s simple, semi-quantitative determination of progesterone in serum [179], it is separated from cholesterol by TLC after extraction from 3.5 ml of serum. Comparison with standards is the basis of the evaluation. Luisi et al. [101] have extracted from the serum, purified by liquid/liquid partition and TLC and determined the progesterone quantitatively by gas chromatography. The progesterone content of the serum of 10 normally menstruating women fluctuated during the menstrual cycle between 0.25 and 3.5 (xg/100 ml. 

A Chromatographic Method for the Quantitative Determination of Cholesterol 14-Methylhexadecanoate (Carcino-lipin) in Biological Material J. Chromatogr. 32(3) 511-518 (1968) ... 

Lipid extraction with chloroform-methanol, followed by quantitative thin layer chromatography yields the phospholipid pattern, while fatty acids are determined by gas chromatography (Borggreven et al., 1970). Protein and cholesterol are determined colorimetrically. 

Cholesterol occurs in most biological materials in both the free and esterified forms. Quantitative determination of cholesterol is based on color reactions, which are not absolutely specific but suffice since cholesterol is the predominating sterol in most materials. The color reactions include the Liebermann-Burchard reaction with sulfuric acid and acetic acid anhydride, the Tschugaeff reaction with zinc chloride and acetyl chloride in acetic acid, or the Lifschiitz reaction with ferric chloride in acetic acid and sulfuric acid. Both free and esterified cholesterol participate in these reactions. Methods for the determination of cholesterol which are commonly used are those of Schoenheimer and Sperry (1934), Sperry and Webb (1950), Zak et al. (1951), Zlatkis et al. (1953). They were recently reviewed by Vanzetti (1964). 

For the quantitative determination of Solarium alkaloids in plant materials some new methods have been developed for instance, gravimetric procedures using the afore-mentioned cholesterol precipitation (15) for tetraosides, especially with saturated aglycones volumetric methods with titration of the bases in water-free solvents using aromatic sulfonic acids (26c, 47, 48) a number of simplified spectrophotometric methods by applying the Clarke reaction (49), that is, the hlue color obtained with paraformaldehyde-phosphoric acid in the case of J -unsaturated steroid alkamines and their glycosides (12, 50-52a, 525), or with the aid of amphi-indicators of the tropaeolin type (52,53-57,57a). The well-known Liebermann-Burchard reaction is not applicable to nitrogenous 3 -hydroxy-J -steroids (55). ... 

Considerable use of a number of its chemical and physical properties has been made in the isolation and quantitative determination of cholesterol. Cholesterol forms a very stable and quite insoluble molecular compound with the saponin, digitonin. The majority of sterols with hydroxyl groups at Cs in the 3 orientation likewise form more or less sparingly soluble digitonides. When treated with strong mineral acid, cholesterol forms a variety of colored complexes useful for the qualitative and quantitative determination of the compound. The most widely recognized of these is the Liebermann-Burchard reaction, in which the sterol Windaus, A., Ber. deut. diem. Oee. 42, 238 (1909). 

The quantitative determination of phospholipid acyl chain conformational disorder in hydrated systems of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamlne (DPPE), mixtures containing DPPC and cholesterol, and mixtures of DPPC and Gramicidin D has been made using FT-IR. The... 

The polyene macrolide filipin was isolated in 1955 from the cell culture filtrates of Sterptomyces filipinensis, and was later shown to be a mixture of four components [36]. Although too toxic for therapeutic use, the filipin complex has found widespread use as a histochemical stain for cholesterol and has even been used to quantitate cholesterol in cell membranes [37]. The flat structure of filipin III, the major component of the filipin complex, was assigned from a series of degradation studies [38]. Rychnovsky completed the structure determination by elucidating the relative and absolute stereochemistry [39]. The total synthesis plan for filipin III relied heavily on the cyanohydrin acetonide methodology discussed above. 

Coupled enzyme assays have been developed for the determination of substances as diverse as glucose, uric acid, and cholesterol, the principal application being quantitation in biological fluids such as blood, plasma, and urine. Typical examples are illustrated by Eqs. (9)-(12). 

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. 

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). 

Allen MP, DeLizza A, Ramel U, Jeong H, Singh P. A non-instrumental quantitative test system and its application for determining cholesterol concentration in whole blood. Chn Chem 1990 36 1591-7. 

Preparation of Extracts. Leffler (L2) and others have previously used isopropanol as a lipid solvent for determining cholesterol. Connerty et al. (C6) and Lofland (L4) also showed that triglycerides and phospholipids are extracted quantitatively. Under our extraction conditions, complete recovery of these lipid fractions is obtained at a serum-to-solvent ratio of 1 10 through 1 25. We routinely use 1 20 dilution (0.5 ml of sera+ 9.5 ml of isopropanol). We find that the addition of Lloyd s reagent removes bilirubin and other chromogenic material present in serum, as well as approximately 80% of the phospholipids present. 

A semiautomated fluorometric procedure for quantitating triglycerides was presented by Kessler and Lederer in 1965 (K6). This is based on the Hantzsch condensation reaction between an amine, j8-diketone, and an aldehyde (Bl, Nl). Isopropanolic extracts of serum are prepared manually, and at this point can also be used for the determination of cholesterol and phospholipids. Triglycerides can be determined after treatment with Zeolite, as employed by Van Handel and Zilversmit... 

---