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Effect of storage on protein concentration of tear samples
T Sitaramamma1, S Shivaji, G N Rao
1Prof. Brien Holden Eye Research Centre, L. V. Prasad Eye Institute, Hyderabad, India. tsita@lvpeye.stph.net
Purpose:
To determine the effect of storage on protein concentration of tear samples stored at room temperature (RT), 4 degrees C, -20 degrees C and -70 degrees C.
Methods:
Total protein concentration of closed, open (basal tears) and reflex tears (stimulated tears) was measured by modified Bradford's method. SDS-PAGE gel electrophoresis was used to determine the intensity of protein bands. Quantity of various tear proteins was determined by HPLC analysis.
Results:
Compared to control samples (0 h) protein concentrations decreased significantly in tear samples stored beyond 4 h (for closed) and 8 h (for open and reflex) at RT. However, no significant change in protein concentrations was observed in closed and reflex tears when stored up to 1 week at 4 degrees C, up to 2 months at -20 degrees C; and up to 4 months at -70 degrees C. Multiple freeze-thaw procedures (6 x per day at 2 h intervals) resulted in 8% decrease (at -20 degrees C) and 10% decrease (at -70 degrees C) of protein concentrations in closed eye tears. SDS-PAGE analysis of tear samples stored at -20 degrees C and -70 degrees C for 4 months greatly affected the intensity of the protein bands. HPLC analysis of tear samples stored at these conditions showed the significant reduction in the peak corresponding to secretory IgA in closed eye tears and a split in the peak corresponding to lysozyme in case of reflex tears.
Conclusions:
Reduction in the total protein concentration, intensity of the protein bands as well as changes in the quantity of tear proteins were observed in the tear samples stored for longer duration of time at various temperatures.