Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS

Akira Honda,Kouwa Yamashita,Hiroshi Miyazaki,Mutsumi Shirai,Tadashi Ikegami,Guorong Xu,Mitsuteru Numazawa,Takashi Hara,Yasushi Matsuzaki

doi:10.1194/jlr.d800017-jlr200

Abstract

We have developed a highly sensitive and specific method for the analysis of serum sterol profiles. Sterols in 1 mul of dried serum were derivatized into picolinyl esters (3beta-picolinate) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the electrospray ionization (ESI) mode. In addition to cholesterol, 19 cholesterol precursors, cholestanol, campesterol, sitosterol, and sitostanol were identified simultaneously. Quantitative analyses for the picolinyl esters of 11 available sterols were performed, and detection limits were found to be less than 1 pg on-column. Reproducibilities and recoveries of 8 noncholesterol sterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.6% to 8.2% and 2.5% to 16.5%, respectively. The recovery experiments were performed using 1 mul aliquots of normal human serum spiked with 1 ng to 6 ng of sterols, and recoveries of the sterols ranged from 88.1% to 102.5% with a mean recovery of 98.1%. The present method provides reliable and reproducible results for the identification and quantification of neutral sterols, especially in small volumes of blood samples, which is useful for serological diagnosis of inherited disorders in cholesterol metabolism and for noninvasive evaluation of cholesterol biosynthesis and absorption in humans.

Highlights

We have developed a highly sensitive and specific method for the analysis of serum sterol profiles
Afterwards, the cholesterol biosynthetic pathway was investigated in other malformation syndromes and the following deficiencies were discovered, i.e., desmosterolosis (3b-hydroxysteroid D24-reductase deficiency) (2), AntleyBixler syndrome (3, 4), hydrops-ectopic calcification-“moth-eaten” (HEM)/Greenberg skeletal dysplasia (3b-hydroxysteroid D14reductase deficiency) (5), congenital hemidysplasia with ichthyosis and limb defects (CHILD) syndrome or NAD (P)H steroid dehydrogenase-like (NSDHL) deficiency (6, 7), CHILD syndrome, X-linked dominant chondrodysplasia punctata type 2 (CDPX2) or Conradi-Hünermann-Happle syndrome (3b-hydroxysteroid D8,D7-isomerase deficiency) (8–10), and lathosterolosis (3b-hydroxysteroid 5-desaturase deficiency) (11)
Thirteen sterols were converted into the corresponding picolinyl ester derivatives and positive electrospray ionization (ESI)-MS, MS/MS, Selected reaction monitoring (SRM), and HPLC data were obtained for each of them (Table 1)

Summary

Introduction

We have developed a highly sensitive and specific method for the analysis of serum sterol profiles. The present method provides reliable and reproducible results for the identification and quantification of neutral sterols, especially in small volumes of blood samples, which is useful for serological diagnosis of inherited disorders in cholesterol metabolism and for noninvasive evaluation of cholesterol biosynthesis and absorption in humans.— Honda, A., K. Sensitive analysis of sterol profiles in human serum by LC-ESIMS/MS. Afterwards, the cholesterol biosynthetic pathway was investigated in other malformation syndromes and the following deficiencies were discovered, i.e., desmosterolosis (3b-hydroxysteroid D24-reductase deficiency) (2), AntleyBixler syndrome (functional deficiency of lanosterol 14ademethylase due to cytochrome P450 oxidoreductase gene mutations) (3, 4), hydrops-ectopic calcification-“moth-eaten” (HEM)/Greenberg skeletal dysplasia (3b-hydroxysteroid D14reductase deficiency) (5), congenital hemidysplasia with ichthyosis and limb defects (CHILD) syndrome or NAD (P)H steroid dehydrogenase-like (NSDHL) deficiency (deficiency of 3b-hydroxysteroid dehydrogenase in 4amethylsterol-4-demethylase complex) (6, 7), CHILD syndrome, X-linked dominant chondrodysplasia punctata type 2 (CDPX2) or Conradi-Hünermann-Happle syndrome (3b-hydroxysteroid D8,D7-isomerase deficiency) (8–10), and lathosterolosis (3b-hydroxysteroid 5-desaturase deficiency) (11).

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