Comparison of metabolic profile of normal and keratoconus corneas using HR MAS 1HNMR spectroscopy and HPLC

Abstract

Abstract Purpose The aim of this study is to examine possible differences in the metabolic profile between keratoconus and normal corneas. Methods Samples of corneas with keratoconus were obtained during the transplantation surgery from 3 patients (age: 22‐27 years). The control corneal tissues were excised during enucleating of eyes with malign melanoma in the retina from 4 patients (age: 65‐72 years). The samples were immediately frozen at ‐80 ºC. The metabolic profiles of the samples were investigated either with HR MAS (High Resolution Magic Angle Spinning) 1H NMR (Nuclear Magnetic Resonance) spectroscopy (14.1 T) operating at 600.132 MHz and HPLC (High Performance Liquid Chromatography). All data obtained with both methods were analysed using special software for: (i) analysis of complex mixtures, (ii) principal component analysis – PCA, (iii) detailed statistical analysis. Results In all samples, 9 amino acids were detected with HPLC, and 19 metabolites (including amino acids) with HR MAS 1H NMR spectroscopy, respectively. PCA analysis of NMR spectra showed no grouping pattern between the keratoconus and control samples. Detailed analysis of data obtained with NMR spectroscopy and with HPLC revealed no significant differences in amino acid profiles between keratoconus and control corneas. Conclusion The study presents metabolic profiles of human cornea examined with new approach combining NMR spectroscopy and HPLC. Significant differences in the metabolic profiles of corneas with keratoconus and healthy corneas examined with both methods were not revealed. Small number of samples analysed in this study represents a limitation of the results, and further investigation will be performed to focus on particular metabolites.