Steroids 68 (2003) 733–738 Synthesis of deuterated isotopomers of 7- and (25R,S)-26-hydroxycholesterol, internal standards for in vivo determination of the two biosynthetic pathways of bile acids Pierangela Ciuffreda, Silvana Casati, Laura Alessandrini, Giancarlo Terraneo, Enzo Santaniello ∗ Dipartimento di Scienze Precliniche LITA Vialba, Universitá degli Studi di Milano, Via G.B. Grassi, 74-20157 Milan, Italy Received 29 April 2003; received in revised form 30 June 2003; accepted 14 July 2003 Abstract Deuterated isotopomers of 7- and (25R,S)-26-hydroxycholesterol, internal standards for in vivo determination of the two biosyn- thetic pathways of bile acids formation from cholesterol, were prepared from [2,2,3,4,4,6- 2 H 6 ]-cholesterol and (20S)-[7,7,21,21- 2 H 4 ]-3- (tert-butyldimethylsilyl)oxy-20-methylpregna-5-en-21-ol, respectively. © 2003 Elsevier Inc. All rights reserved. Keywords: Deuterated isotopomers; 7-hydroxycholesterol; (25R)-26-hydroxycholesterol; Bile acids; Cholesterol metabolism 1. Introduction It is now commonly accepted that two main metabolic pathways are involved in the formation of bile acids, the clas- sical microsomal pathway that starts from 7-hydroxylation of cholesterol [1] and an alternative 27-hydroxylation that may occur also in extrahepatic tissues [2,3]. The rate of the “in vivo” formation of bile acids via the “27-hydroxylation” has been determined measuring the natural/deuterated (25R)-26-hydroxycholesterol ratio in plasma by mass spectrometry analysis [4]. In connection with a program aimed to a simultaneous evaluation in human healthy subjects of 7- and 27-hydroxylation pathways by gas chromatographic/mass spectrometric analysis of deuter- ated isotopomer, it became necessary to prepare deuterated 7- and (25R)-26-hydroxycholesterol for intravenous in- fusions. For the synthesis of a deuterated isotopomer of 7-hydroxycholesterol (1a), we could rely on commer- cially available hexadeuterated cholesterol, that could also be prepared via a multiple exchange methodology [5]. (25R)-26-Hydroxycholesterol (2a) labeled with five to nine deuterium atoms has been prepared from kryptogenin [4] but this triterpene is no longer commercially available. Al- ternatively, the deuterated compound 2b (Fig. 1) can be prepared from diosgenin [6] but we preferred a more general ∗ Corresponding author. Tel.: +39-02-50319691; fax: +39-02-50319631. E-mail address: Enzo.Santaniello@unimi.it (E. Santaniello). and versatile approach that relies on the construction of the side chain and can therefore be extended to the preparation of deuterated 2b and any other labeled sterols as well. 2. Experimental 2.1. General Melting points were recorded on Stuart Scientific SMP3 instrument and are uncorrected. All reagents were purchased from Sigma (USA). [2,2,3,4,4,6- 2 H 6 ]-Cholesterol was ob- tained from CDN Isotopes (Canada) and (20S)-[7,7,21,21- 2 H 4 ]-3-(tert-butyldimethylsilyl)oxy-20-methylpregna-5-en- 21-ol (5) was prepared according to [7]. Solvents were purified and dried in the usual way; chromatography eluents were petroleum ether (40–60 ◦ C b.p.)/AcOEt. All reactions were monitored by TLC on Silica Gel 60 F 254 precoated plates with a fluorescent indicator (Merck). Flash chromatography [8] was performed using silica gel 60 (230–400 mesh, Merck). In the experimental protocol, unless differently specified, work-up consists in three ex- tractions with CH 2 Cl 2 , washing of the organic solution with water, drying with anhydrous Na 2 SO 4 and evaporation of the solvent at reduced pressure. Mass spectra were carried out using a Hewlett-Packard 5988A instrument that was set at 70 eV ion energy, 0.1 mA emission current, and 280 ◦ C transfer line temperature. Syn- thetic [2,2,3,4,4,6- 2 H 6 ]-cholest-5-en-3,7-diol (1b) and 0039-128X/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0039-128X(03)00116-8