Tear analysis of ascorbic acid, uric acid and malondialdehyde with capillary electrophoresis.
Tears have a significant role in antioxidant defense in ocular tissues and since their collection is quick and noninvasive, their analysis would facilitate monitoring of pathophysiological changes. However, their low volume and low content of antioxidants makes analysis difficult; methods of high sensitivity are needed. In this paper, we present a method for tear analysis of two antioxidant molecules (ascorbic and uric acid) and of a lipid peroxidation indicator (malondialdehyde) with capillary electrophoresis. Tears were collected with Schirmer strips, extracted with a low-pH phosphate buffer, centrifuged through membrane filters and an antioxidant was added. They were stable at -70 degrees C for 15 days. After pilot experiments, optimum electrophoretic separation was achieved in a 25 mM borate buffer, pH 10.0, containing 100 mM sodium dodecyl sulfate at 25 degrees C and 20 kV. The developed method has good repeatability (<5% RSD), precision (<15% relative error values) and high sensitivity (LLOQ values of 20, 2.3 and 2.5 microM for ascorbate, urate and malondialdehyde, respectively). It was applied to the analysis of tears from healthy individuals and the antioxidant levels are in agreement with those obtained with other techniques. This method might serve as a tool to clarify the role of endogenous antioxidants in the pathophysiology of ocular diseases.
Georgakopoulos
Constantine D
CD
Department of Ophthalmology, School of Medicine, University of Patras, 26504 Patras, Greece.
Lamari
Fotini N
FN
Karathanasopoulou
Ioanna N
IN
Gartaganis
Vassiliki S
VS
Pharmakakis
Nikolaos M
NM
Karamanos
Nikos K
NK
D000328:Adult; D000368:Aged; D000975:Antioxidants / Q000032:analysis; D001205:Ascorbic Acid / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D008315:Malondialdehyde / Q000032:analysis*; D008875:Middle Aged; D013666:Tears / Q000737:chemistry*; D014527:Uric Acid / Q000032:analysis*
D000975:Antioxidants; D014527:Uric Acid; D008315:Malondialdehyde; D001205:Ascorbic Acid