Oxysterol mass spectrometry

There are, as yet, no standard methods for determining oxysterols in food. As a conseqnence, considerable variations in cholesterol oxide levels in similar kinds of foods have been reported by varions laboratories (Dutta et al., 1999). For quantification of oxysterols in samples, the nse of an internal standard technique is recommended. For oxysterol separation, capillary columns with a nonpolar stationary phase are most often nsed. For confirmation of oxysterol structure, the use of gas chromatography/mass spectrometry (GC/MS) is highly recommended. During analytical procednres, serious attention must be paid to the possibility of artifacts of oxysterols from cholesterol. In 1992, Wasilchuk et al. developed a procedure for detecting artifactual generation of oxysterols using [ Hg] cholesterol as an internal standard. 

Although cholesterol is about 10-fold less reactive in autoxidation than PUFA there is still interest in the products that have been associated with oxidant stress. Cholesterol, free and bound to ester, is measured after separation into free and ester cholesterol by selective extraction and hydrolysis using gas chromatography-tandem mass spectrometry (GC/MS/MS) [61]. Oxidized cholesterol, oxysterols, can be analyzed using LC-MS/MS with a minimum of manipulation or by using GC/MS/MS after derivatization [62-66]. 

Meljon, A., Griffiths, W.X. (2010) Analysis of oxysterols in mature and developing brain, charge-tagging and LC-MS . Book of Abstracts, 31st Annual Meeting of the British Mass Spectrometry Society, Cardiff, September 5th-8th 2010. 

GC-MS analysis is used to confirm the identities of ions in the LSI-MS urine spectrum and show that the excretion of abnormal cholanoids is >20 times normal. In the case of 5 ff-reductase deficiency GC-MS analysis should show that S-oxo-A" bile acids account for >70% of the total urinary bile acid excretion. In the case of sterol 27-hydroxylase deficiency (CTX), GC-MS analysis should indicate that the major cholestane pentols in the urine are 3,7,12,22,25 and 3,7,12,23,25-pentols. (One patient has been described who had familial cholestatic liver disease associated with greatly increased urinary excretion of 5jff-cholestane-3a,7a,12a,24 S,25-pentol [see previous table]). Liquid secondary ion-tandem mass spectrometry (LSI-MS/ MS) is an alternative method to GC-MS and can rapidly confirm the identity of a number of diagnostic ions that are found in the LSI-MS spectrum of urine. These include sulphated and taurine-conjugated abnormal metabolites such as those observed in 3 ff-HSDH deficiency (32.1), 5)9-reductase deficiency (32.2), oxysterol 7a-hydroxylase deficiency (32.4) and peroxisomal disorders [13]. 

Diagnosis of the 4 inborn errors of bile acid synthesis discussed in this chapter is important because three are treatable by oral bile acid supplementation (such treatment was not successful in the single reported case of oxysterol 7a-hydroxylase deficiency). Liquid secondary ion mass spectrometry (LSI-MS) is a simple and rapid method which can be used to screen urine samples for abnormal cholanoids (bile acids and bile alchohols). It should be applied to neonates with unexplained cholestatic liver disease, particularly if familial and associated with steatorrhoea and fat-soluble vitamin malabsorption, to infants and children with developmental delay whether or not this is associated with specific features suggestive of a peroxisomal disorder (e.g. hypotonia, seizures, dysmorphic features, ocular and auditory abnormalities and hepatic dysfunction) or CTX (e.g. juvenile cataracts). 

Jiang, X., Ory, D.S. and Han, X. (2007) Characterization of oxysterols by electrospray ionization tandem mass spectrometry after one-step derivatization with dimethylglycine. Rapid Commun. Mass Spectrom. 21, 141-152. 

Karu, K., Turton, J., Wang, Y. and Griffiths, W.J. (2011) Nano-liquid chromatography-tandem mass spectrometry analysis of oxysterols in brain Monitoring of cholesterol autoxidation. Chem. Phys. Lipids 164,411-424. 

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