Determination of serum levels of unesterified lanosterol by isotope dilution-mass spectrometry

Abstract

The synthesis of ²H₃-labelled lanosterol is described. This compound was used for assay of unesterified lanosterol in serum by isotope dilution-mass spectrometry. After addition of a fixed amount of internal standard (150 ng) to a fixed amount of serum (250 u. 1) the steroids were extracted with chloroform and subjected to Lipidex 5000 chromatography. The fraction containing lanosterol was eluted with methanol. This fraction was converted into trimethylsilyl derivative and subjected to mass spectrometric analysis with selected monitoring of the ions at m/z 498 (molecular ion of unlabelled lanosterol) and m/z 501 (molecular ion of ²H₃-labelled lanosterol). A standard curve was used for quantification of lanosterol in serum. Under the conditions employed the coefficient of variation was less than 4%. In a recovery experiment the maximal difference between expected and found value was about 5%.

Sera from 10 healthy subjects were found to have a mean concentration of lanosterol of 225 ng/ml with a SD of 63 ng/ml.

Using a less accurate method for analysis of lanosterol we have shown previously that there is a high correlation between the hepatic HMG CoA reductase and the absolute and relative concentration of lanosterol in serum (concentration of lanosterol relative to cholesterol). When using the same (old) sera as in the previous investigation this could not be confirmed, most probably because of the degradation of lanosterol as a consequence of prolonged storage and/or freezing and thawing. It appears that serum lanosterol must be analysed as soon as possible after the collection of the blood sample in order to obtain accurate results. This restricts the use of lanosterol as a marker for cholesterol biosynthesis in vivo; thus, we recommend using the more stable lathosterol for this purpose.

Key Words:

• cholesterol precursor
• cholesterol synthesis
• HMG CoA reductase
• mass fragmentography