Identification of 22‐, 24‐ and 26‐Hydroxycholesterol in the Steroid Sulphate Fraction of Faeces from Infants

Abstract

A fraction of solvolyzable sterol conjugates with the chromatographic properties of steryl sulphates was isolated from pooled faeces collected from infants 1-4 and 6-12 months old. The major sterols in this fraction were analyzed by gas chromatography-mass spectrometry. The pool from the younger infants was shown to contain cholesterol and 22a-, 24&, and 26-hydroxycholesterol and the pool from the older infants contained cholesterol, campesterol, /?-sitosterol, and 22a-, and 24~-hydroxycholesterol. The concentrations of cholesteryl and 24-hydroxycholesteryl sulphates were higher in faeces from the older than from the younger subjects.I n a series of papers we have described the identification of steroids in faeces from germfree and conventional rats (for references see [l]). These studies have shown a pronounced difference between the two types of animals in the composition and nature of the steroid metabolites excreted. Evidence has been presented for the appearance of bacterial metabolites in the urine [2]. The pronounced effect of the intestinal bacteria on the steroid excretion in rats suggests that the establisment of an intestinal flora could influence the nature of steroid metabolites and their route of excretion in the newborn infant. A striking change in the urinary steroid pattern during the first year is the decrease of 16a-hydroxysteroids [3] and one of the important bacterial reactions in the rat is the removal of the 16a-hydroxy group in C,,. It is therefore of interest to investigate the nature of steroids in faeces from infants during the first year of life. Since most of the steroids in infant urine occur as sulphates [5] the sulphate fraction in faeces has been investigated. As shown in the present paper a major group of compounds in this fraction consists of C,,O, steroids, the identification of which will be described. MATERIALS AND METHODS Collection of FaecesFaeces were collected in polythene vials or plastic bags from two groups of infants (both sexes): 1 to 4 months old (yielding faecal pool PI), and 6 to 12 months of age (yielding pool PII). Pool PI, obtained from 28 subjects, consisted of 285 g of faeces; poolPII (from 10 subjects) weighed 225g. The samples were stored a t -20" until analyzed. The infants were kept on different regular diets (see [S]).

Excretion of 3β‐hydroxy‐5‐cholenoic and 3a‐hydroxy‐5a‐cholanoic acids in urine of infants with biliary atresia

“…Normally, sulphates of these compounds are excreted in faeces [5] . The atresia also leads to excretion in urine of conjugated cholic and chenodeoxycholic acids which are the major bile acids in infant bile [6] .…”

Steroids in Germfree and Conventional Rats

“…In most of the spectra a molecular ion could be distinguished a t mle 728, indicating a pentahydroxypregnane structure. The loss of the fragment of mass 103 indicated a 21-trimethylsiloxy group [4]. Fig.4 also shows a mass spectrum of 5n-pregnane-3B,16a,20o,Zl-tetrol silyl ether.…”

“…31, Israel) with 3n-hydroxysteroid oxidoreductase as described previously [4]. A mixture of the 20/3 and 20n epimers of 5n-pregnane-3n,11/3,20,21-tetrol was prepared by sodium borohydride reduction of 3a,ll/3,21-trihydroxy-5a-pregnan-20-one.…”

“…3a, 11/3,21 -Trihydroxy-5n-pregnan-20-one was prepared by reduction of 1 I/3,2l -dihydroxy-5n-pregnane-3,20-dione (purchased from Ikapharm, Ramat-Gan, P.O.B. 31, Israel) with 3n-hydroxysteroid oxidoreductase as described previously [4]. A mixture of the 20/3 and 20n epimers of 5n-pregnane-3n,11/3,20,21-tetrol was prepared by sodium borohydride reduction of 3a,ll/3,21-trihydroxy-5a-pregnan-20-one.…”

“…3a,7a-Dihydroxy-5a-androstan-l7-one was obtained from a purified sample of the monosulphate fraction of faeces from germfree female rats [4].…”