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Kenneth Gek-Jin Ooi, Mittra Rahimi-Oztan, Nicholas Proschogo, Pauline Khoo, Stephanie L Watson; Evaluation of Sex Hormone and Cholesterol Sterols in Human Tears by Mass Spectroscopy. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3276.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate for cholesterol (chol.) and sex hormones in human tears with High Performance Liquid Chromatography-Atmospheric Pressure Chemical Ionisation Mass Spectrometry (MS)
Healthy volunteer (n=10) tears were obtained with glass micropipettes and stored at -80oC. Male (M) and female (F) tears were pooled together in aliquots of 250uL, 100uL and 500uL, without meibomian gland expression (MGE). MGE was performed only on separate M and F samples: F tears were pooled in aliquots of 400uL, 400uL and 30uL and M tears pooled in aliquots of 80uL and 40uL. Pooled tear aliquots of < 100uL in volume were diluted with MilliQ water to make a total volume of 100uL. Aliquots with a volume of > 100uL were left undiluted.MS was performed according to published methodology with a variation in extraction procedure. Extraction involved a combined mixture of standards: estrone, estradiol, testosterone (test.) and chol. as positive controls. To controls and test samples, 5uL of 100mg/mL D6-chol. was added as an internal standard prior to 10:90 ethyl acetate: hexane extraction. MilliQ water was the negative control. Dried samples were dissolved in 20uL of methanol and injected into MS. To measure sterols against positive controls, extracted ion chromatographs were generated for molecular masses of chol. (369.3m/z), D6-chol. (375.3m/z), test. (289.2m/z), estradiol (255.2m/z), estrone (275.2m/z) with a window of 0.3 Dalton. Spectral background subtraction was used to determine sterol spectra in test samples and controls.
Mean F and M ages were 26.6 (range 22-32) and 38 (range 29-44) years, respectively. Strong peaks were detected from the combined mixture of standards at all concentrations. Chol. concentrations observed in non-MGE samples pooled from M and F tears combined were mean 40.6 ± 19.0 (range 21.7-75.3) ng/uL. Chol. concentrations in samples with MGE in F alone were mean 30.7 ± 11 (range 20.3-43.0 0) ng/uL and M alone were mean 36.7 ± 17.0 (range 24.7-48.7) ng/uL. In the largest non-MGE of pooled F and M tear samples, test. was observed (3pg/uL) but estrone and estradiol were not.
Chol. levels in pooled tear samples from normal volunteers were similar to published levels. A novel finding of test. was detected in the one largest pooled sample but not in the others. Larger sample sizes and more sensitive techniques may be needed to detect any trace sex hormones present in human tears.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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