ESI-MS/MS quantification of 7alpha-hydroxy-4-cholesten-3-one facilitates rapid, convenient diagnostic testing for cerebrotendinous xanthomatosis.
BACKGROUND
The genetic disorder cerebrotendinous xanthomatosis (CTX) frequently remains undiagnosed for many years. Left untreated CTX is associated with the development of cataracts, xanthomas and severe neurological dysfunction. The method routinely used to screen for CTX is GC-based measurement of elevated 5alpha-cholestanol from hydrolyzed plasma. A plasma test for CTX utilizing ESI-MS/MS methodology would be beneficial.


METHODS
Development of rapid, simple LC-ESI-MS/MS methodology to test plasma for CTX is described. Two hour Girard derivatization allowed for 7alpha-hydroxy-4-cholesten-3-one quantification by isotope dilution LC-ESI-MS/MS within 12 min from un-hydrolyzed affected patient plasma (5 microl).


RESULTS
Adequate sensitivity and reproducibility were achieved for quantification of 7alpha-hydroxy-4-cholesten-3-one, which demonstrated improved utility as a diagnostic marker of disease and to monitor treatment compared to 5alpha-cholestanol. The mean plasma concentration of 7alpha-hydroxy-4-cholesten-3-one in untreated CTX-affected patients (n=6) was 107-fold that in unaffected subjects (n=9), with the lowest concentration in affected patients >10-fold the highest concentration in unaffected subjects.


CONCLUSIONS
Quantification of the bile acid precursor 7alpha-hydroxy-4-cholesten-3-one with LC-ESI-MS/MS is a novel approach to improved diagnostic testing of plasma for CTX, amenable to high-throughput analysis and automated sample handling. Development of ESI-MS/MS methodology should make CTX testing more widely available and facilitate easier diagnosis of CTX.
DeBarber
Andrea E
AE
Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, OR 97239, USA. debarber@ohsu.edu
Connor
William E
WE
Pappu
Anuradha S
AS
Merkens
Louise S
LS
Steiner
Robert D
RD
D002783:Cholestenones / Q000097:blood*; D002853:Chromatography, Liquid; D006801:Humans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D019294:Xanthomatosis, Cerebrotendinous / Q000097:blood / Q000175:diagnosis*
D002783:Cholestenones; C002656:7 alpha-hydroxy-4-cholesten-3-one