Identification of Two Sulfated Cholesterol Metabolites Found in the Urine of a Patient with Niemann-Pick Disease Type C as Novel Candidate Diagnostic Markers.

In the urine of a Niemann-Pick disease type C (NPC) patient, we have identified three characteristic intense peaks that have not been observed in the urine of a 3ß-hydroxysteroid-Δ5-C27-steroid dehydrogenase deficiency patient or a healthy infant and adult. Based on accurate masses of the protonated molecules, we focused on two of them as candidate NPC diagnostic markers. Two synthesized authentic preparations agreed with the two compounds found in NPC patient urine in regard to both chromatographic behavior and accurate masses of the deprotonated molecules. Moreover, the isotopic patterns of the deprotonated molecules, twin peaks unique to the sulfur-containing compounds appearing in their second isotope positions, and accurate masses of product ions observed at m/z 97 also agreed between the target compounds and authentic preparations. We identified the two compounds as the sulfated cholesterol metabolites as 3ß-sulfooxy-7ß-hydroxy-5-cholen-24-oic acid and 3ß-sulfooxy-7-oxo-5-cholen-24-oic acid. These two compounds represent more promising candidate diagnostic markers for NPC diagnosis than three other candidates that are multiple conjugates of cholesterol metabolites, 3ß-sulfooxy-7ß-N-acetylglucosaminyl-5-cholen-24-oic acid and its glycine and taurine conjugates, although we have reported an analytical method for determining the urinary levels of these compounds using liquid chromatography/electrospray ionization tandem mass spectrometry, because of their lack of N-acetylglucosamine conjugation.

Novel, major 2α- and 2ß-hydroxy bile alcohols and bile acids in the bile of Arapaima gigas, a large South American river fish.

Bile alcohols and bile acids from gallbladder bile of the Arapaima gigas, a large South American freshwater fish, were isolated by reversed-phase high-performance liquid chromatography. The structures of the major isolated compounds were determined by electrospray-tandem mass spectrometry and nuclear magnetic resonance using (1)H- and (13)C-NMR spectra. The novel bile salts identified were six variants of 2-hydroxy bile acids and bile alcohols in the 5α- and 5ß-series, with 29% of all compounds having hydroxylation at C-2. Three C27 bile alcohols were present (as ester sulfates): (24ξ,25ξ)-5α-cholestan-2α,3α,7α,12α,24,26-hexol; (25ξ)-5ß-cholestan-2ß,3α,7α,12α,26,27-hexol, and (25ξ)-5α-cholestan-2α,3α,7α,12α,26,27-hexol. A single C27 bile acid was identified: (25ξ)-2α,3α,7α,12α-tetrahydroxy-5α-cholestan-26-oic acid, present as its taurine conjugate. Two novel C24 bile acids were identified: the 2α-hydroxy derivative of allochenodeoxycholic acid and the 2ß-hydroxy derivative of cholic acid, both occurring as taurine conjugates. These studies extend previous work in establishing the natural occurrence of novel 2α- and 2ß-hydroxy-C24 and C27 bile acids as well as C27 bile alcohols in both the normal (5ß) as well as the (5α) "allo" A/B-ring juncture. The bile salt profile of A. gigas appears to be unique among vertebrates.

Chemical Synthesis of the Epimeric (23R)- and (23S)-Fluoro Derivatives of Bile Acids via Horner-Wadsworth-Emmons Reaction.

A method for the synthesis of two (23R)- and (23S)-epimeric pairs of 23-fluoro-3α,7α,12α-trihydroxy-5ß-cholan-24-oic acid and 23-fluoro-3α,7α-dihydroxy-5ß-cholan-24-oic acid is described. The key intermediates, 23,24-dinor-22-aldehyde peracetates were prepared from cholic and chenodeoxycholic acids via the 24-nor-22-ene, 24-nor-22ξ,23-epoxy, and 23,24-dinor-22-aldehyde derivatives. The Horner-Wadsworth-Emmons reaction of the 23,24-dinor-22-aldehydes using triethyl 2-fluoro-2-phosphonoacetate in the presence of LiCl and 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), and subsequent hydrogenation of the resulting 23ξ-fluoro-22-ene ethyl esters, followed by hydrolysis, gave a mixture of the epimeric (23R)- and (23S)-fluorinated bile acids which were resolved efficiently by preparative RP-HPLC. The stereochemical configuration of the fluorine atom at C-23 in the newly synthesized compounds was confirmed directly by the X-ray crystallographic data. The (1)H and (13)C NMR spectral differences between the (23R)- and (23S)-epimers were also discussed.