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M.-J. Lu, J. S. Pulido, K. H. Baratz, H. Qian, J. C. Erie, S. A. Shippy; Amino Acid Analysis of Human Tears in Dry vs. Normal Eyes by Capillary Electrophoresis. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5307.
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© ARVO (1962-2015); The Authors (2016-present)
To explore the possibility of determining the content of amino acids, particularly glutamate, in human tears.
Schirmer strips were wetted with standard amino acid and protein solutions or, for human tear collection, Schirmer strips were placed in the unanesthetized cul-de-sac of the eyes of patients with keratoconjuntivitis sicca (KCS) or normal controls for 5 minutes. Physiological saline was applied to dried strips and collected by centrifugation to elute strip amino acids. In vitro studies of the volume recovered with centrifugation and the extent of amino acid recovery with multiple extractions were performed. Amino acid analysis of eluent was performed by capillary electrophoresis (CE) following fluorescent labeling.
Optimized elution conditons with dried strips that were wetted and centrifuged with 20 microL and then 10 microL of saline provided consistent eluent volumes. Strips wetted with 10 microL of 10 to 100 microM amino acid standards show that the optimized elution resulted in 90-100% of the mass of applied amino acids was recovered. Similar masses of amino acids loaded onto strips or volumes provide indistinguishable eluent concentrations determined by CE. Tear samples from control and KCS eyes show 5 of 17 tested amino acids were resolved by CE. Control eyes (n=8) show significantly lower amino acid levels (microM/mm of wetted strip) compared to levels seen from eyes with KCS (n= 21) for Glu (6.87 vs. 27.57, p=0.003), Asp (2.68 vs. 9.18, p=0.005), Tau (11.75 vs. 39.29, p=0.001) and phosphoethanolamine (2.75 vs. 22.24, p=0.01). While the levels of Arg (1.44 vs. 2.56, p=0.1) was statistically indistinguishable.
The elution of amino acids from Schirmer strips is dependent upon the mass of amino acids adsorbed to the strips and allows a quantitative measure of tear amino acid concentration when precise tear volume is not known. KCS patients have statistically higher amino acid levels than control subjects. The measurement of tear amino acids may be useful in evaluating corneal health.
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