Characterization of Human Meibum Lipid using Raman Spectroscopy

Purpose: Raman spectroscopy could potentially be used as a diagnostic tool for the detection of meibomian gland dysfunction by measuring the composition, conformation, and amount of meibum lipid on the lid margin. Toward this goal and to test our studies of meibum lipid using infrared spectroscopy, we measured the Raman spectra of samples from human donors with and without meibomian gland dysfunction. Materials and Methods: Human meibum was expressed from the eyelids and collected on a spatula. Meibum was placed onto an AgCl window for analysis using a Raman spectrometer. Results: Raman spectra of human meibum closely resembled that of wax. The Raman band intensity ratio, 1303/1267 cm(-1), was used to calculate a saturation level CH2/=CH of 12 for meibum from normal donors and 11.8 for meibum from donors with meibomian gland dysfunction. Human meibum was found to fit well in the linear relationships between lipid saturation and lipid order at physiological temperature and between lipid saturation and the lipid phase transition temperature. The CH stretching band region predominates the Raman spectra of human meibum. If sample temperature could be controlled, the CH stretching bands could be used to calculate the lipid structural order in terms of trans and gauche rotomer levels. Raman spectra indicate that carotenoid-like bands decrease from the equivalent of about 90 mu g carotenoid per gram of meibum wax at 19 years of age to about 0 at 80 years of age in samples from patients with meibomian gland dysfunction. The carotenoids may serve as antioxidants. Conclusions: Principal component analysis shows that a component containing carotenoid-like bands may be a promising spectral feature that could be used to distinguish differences between the Raman spectra of donors from normal and those with meibomian gland dysfunction. This study demonstrates the wealth of structural and compositional information that Raman spectra of human meibum provide.