Purchase this article with an account.
Radhika Susarla, Angela E. Taylor, H. Susan Southworth, Sreekanth Sreekantam, Geraint P. Williams, Philip I. Murray, Graham R. Wallace, Saaeha Rauz; Analysis of Steroid Metabolites Using Liquid Chromatography with Tandem Mass Spectrometry in Biological Fluids of Healthy Eyes. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3145.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The corneal epithelium (CEC) and non-pigmented epithelium (NPE) of the ciliary body have the molecular components necessary for the autocrine production of cortisol. Using liquid chromatography with tandem mass spectrometry (LC/MS/MS), we examined various metabolites in the steroid hormone synthesis pathway, and their relationship in ocular biofluids.
Tears, aqueous humour (AqH) and serum samples were collected from 17 healthy subjects prior to cataract surgery (males=11, females=6, aged 21-85 years). Steroids were identified and quantified using LC/MS/MS which combines the physical separation capabilities of liquid chromatography with the sensitivity of mass spectrometry (Waters Xevo with Acquity uPLC). Each steroid was extracted by liquid/liquid extraction and quantified by comparison to a calibration series with respect an internal standard.
This study revealed that several steroid metabolites: pregnenolone, progesterone, 11-deoxycortisol, cortisol (F), cortisone (E), corticosterone, dehydroepiandrosterone (DHEA), androstenedione, and testosterone were detected in tears and AqH. For 9/10 analytes assayed, the relative expression of these hormones was in the proportion serum>tears>AqH. The exception was DHEA which was at a similar concentration in serum and AqH, but lower in tears. All biofluids showed positive correlation between cortisone and cortisol levels: serum (r=0.6929 p<0.0001), AqH (r=0.6946, p<0.0177) and tears (r=0.4730, p<0.0147). There was a positive correlation between cortisone (r=0.7857, p<0.0362) and cortisol (r=0.8960, p<0.0026) levels in AqH vs tears, whilst in serum it remained unchanged, suggesting local production of cortisol both within the eye and on the surface of the eye. The F:E ratios were similar in serum and AqH but reduced to half in tears of healthy subjects irrespective of the gender. 17-OHP was detected in serum and tears, but was below the level of detection in AqH. Whether this is due to rapid turnover or AqH kinetics is currently not known. No gender difference was seen in any of the analytes measured.
These data show that LC/MS/MS can detect metabolites of the corticosteroid pathway and that such metabolites are present in tears and AqH from healthy subjects. Steroid concentrations varied, but the pattern of expression was consistent. Interestingly there were no gender differences of these metabolites in healthy subjects. These data support the use of LC/MS/MS in the analysis of ocular biofluid steroid metabolites. Alterations of the steroid profile from healthy to diseased may be important for understanding disease progression and to aid future treatments.
This PDF is available to Subscribers Only