Journal of Chromatography B: Biomedical Sciences and Applications
A new method for determination of serum cholestanol by high-performance liquid chromatography with ultraviolet detection
Introduction
The elevation of cholestanol (5α-cholestan-3β-ol) in the serum is known to be one of the biochemical markers of cerebrotendinous xanthomatosis (CTX) [1], [2] and liver and biliary tract diseases [3], [4], [5], [6]. A definite diagnosis of CTX is based on the molecular identification of homozygosity to mutant CYP27 alleles [7]. The determination of serum cholestanol [3], [7] and bile alcohols together with cholestanol has been used for monitoring treatment efficacy in CTX [8], [9], [10], [11], [12], [13]. Cholestanol measurement was also used for the follow-up of patients with cirrhosis, primary biliary cirrhosis, or after liver transplantation [4], [5], [6].
Methods for the simultaneous analysis of cholestanol and cholesterol include gas chromatography (GC) [14], [15], capillary GC [3], [6], [16], gas chromatography–mass spectrometry (GC–MS) [17], high-performance liquid chromatography (HPLC) with fluorescence detection [18], [19], [20], and HPLC with UV detection [21], [22], [23], [24]. The simultaneous quantification of cholesterol (5-cholesten-3β-ol) and cholestanol in the serum, and comparison of the concentration ratio, provide useful information on the disease-state [3], [16], [19], [20], [21], [25]. However, setting up a method for determination of cholestanol and cholesterol in serum is difficult, due to similarity of these steroids and the large concentration differences [3], which in most cases exceeds 1:500 [2], [16], [18].
In the studies cited above, special derivatizations and/or chromatographic systems were used to separate the metabolites. Some methods included pretreatment of the samples to enhance separation of the metabolites by thin-layer chromatography (TLC) [14], or oxidation with m-chloroperbenzoic acid and estimation of the resulting 5,6-epoxide and cholestanol by GC [26], [27], [28] or HPLC [18], [27] directly or after separation [26]. The epoxidation prior to chromatography improved the separation of the metabolites but, the resulting chromatographic baselines were unstable [18], [28]. The fluorescence detection methods are very sensitive, however the derivatization reagents are not available commercially, and their preparation is involved with elaborated synthetic methods [18], [19], [20]. When GC, capillary GC or HPLC with UV detection were used the baseline separation was generally not perfect [3], [21], [22], [23], [24]. An undefinitive peak separation may considerably influence the results of the minor component (cholestanol), especially when very high cholesterol/cholestanol ratio samples are tested. Therefore, all of the existing methods do not have the necessary degree of accuracy under all conditions.
In this paper we offer to measure cholestanol alone. In this way we overcome the difficulty of separating the two metabolites. Since the cholesterol level is included in the lipoprotein profile, which is routinely analyzed by the enzymatic colorimetric method [25], making the chromatographic measurement of cholesterol less important for the calculation of the concentration ratio of the two metabolites.
The purpose of the present study was to develop a method for measuring microgram amounts of cholestanol in human serum by modifying Stoll reaction [29], [30] to eliminate cholesterol from the system, using commercially available reagents and HPLC with UV detection. This new method enables us to measure microgram amounts of cholestanol in an accurate way which is not affected by the cholesterol/cholestanol ratio.
Section snippets
Chemicals
All chemicals and solvents were of analytical reagent grade. Double distilled water was used. Cholesterol, cholestanol, 5β-cholestan-3α-ol, 5β-cholestan-3β-ol, lathosterol, stigmasterol (24-ethyl-5,22-cholestadien-3β-ol), stigmastanol (24α-ethyl-5α-cholestan-3β-ol) and campesterol (24α-methyl-5-cholesten-3β-ol), were purchased from Sigma (St. Louis, MO, USA) and 4-bromobenezenesulfonyl chloride from Aldrich (Milwaukee, WI, USA). A mixture of 3.7% 5α-cholestan-3α-ol in cholestanol was prepared
Subjects
Six molecularly diagnosed CTX Jewish patients of North African origin were examined. CTX 215-5 and CTX 224-1 are homozygous for a guanosine to adenosine substitution at the 3′ splice acceptor site intron 4 of CYP27. CTX 216-6 and 206-3 are homozygous for a frameshift mutation resulting from a deletion of thymidine in exon 4 of the gene. The siblings CTX 208-1 and CTX 208-5 are homozygous for a threonine to methionine substitution at residue 306. The three mutations previously described in CTX
Procedure
Venous blood was collected in the fasting state for lipoprotein typing [25]. Aliquots (0.3 ml) of serum were used for cholestanol analysis. I.S. (3–10 μg) was added and the samples were saponified with five volumes of 90% ethanolic 1 M sodium hydroxide at 60°C for 60 min. Water (one-fifth of the total volume) and light petroleum (3 ml) were added. The test tube was thoroughly shaken and the lower phase was removed. The upper phase was further washed with 80% aqueous methanol, and anhydrous
Sterol detection after derivatization with and without solvolysis
Derivatization and solvolysis of the sterols were performed with 4-bromobenzensulfonyl chloride and isopropanol as described in the Section 4. The results are given in Fig. 1.
4′-Bromobenzenesulfonyl esters of cholesterol and cholestanol were detected at 235 nm after 9 and 10 min, respectively. After solvolysis the cholestanol was not changed but cholesterol was converted to cholesteryl-3β-isopropyl ether.
The separation capacity of the 4′-bromobenzenesulfonyl derivatives performed with 10 μg of
Discussion
The elimination of cholesterol from cholestanol was based on the Stoll reaction [29], who esterified cholesterol with p-toluenesulfonyl chloride and solvolyzed the product to cholesteryl-3β-methyl ether by heating with methanol, but cholestanol like other C-5-saturated sterols, was stable under the same conditions [30]. In order to achieve a more rapid derivatization and solvolysis of the product in an open tube, 4-bromobenzenesulfonyl chloride and isopropanol, respectively, were used. The
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