Abstract
Purpose: :
Quantitative and qualitative differences in meibum lipids have been suggested to play a role in the etiology of dry eye disease. Human meibum contains a complex mixture of lipids, including approximately 30% each wax esters (WE) and cholesterol esters (CE), with the remainder including (O-acyl)-omega-hydroxy fatty acids, di/tri acylglycerols and possibly free fatty acids and cholesterol. Variable quantities of protein and carbohydrates may also be present. Current quantitative analysis of meibum lipids is predominantly based on gravimetric analysis of total collected meibum samples. The goal of this study was to evaluate the utility of the sulfophosphovanillin assay (SVA) as a method to quantitate the total lipid content in meibum clinical samples.
Methods: :
Human meibum samples were collected from healthy donors and their dry weight recorded prior to dissolving in 500ul chloroform/methanol, 2:1 (v/v). Individual, and mixtures of, lipid standards were dissolved at 1mg/ml in chloroform-methanol, 3:1 (v/v). Defined volumes of meibum and standard lipids were assayed for total lipid using a microplate modified SVA. Chemical intermediates generated by incubation in sulphuric acid, the first step in the assay, were analyzed both spectrophotometrically and mass spectrometrically. After addition of vanillin in phosphoric acid in the second assay step all lipids generated a common product which was assayed colorimetrically at 535nm.
Results: :
A linear dose response, with varying sensitivity, was obtained for pure lipid standards representing lipid classes found in meibum. These included both saturated and unsaturated WE and CE and cholesterol. Saturated triglycerides were not reactive but surprisingly stearyl stearate was. The nature of the step one intermediate generated by the latter will be discussed. Spectrophotometric analysis revealed different step one intermediates for different reactive lipids though all generated a maxima at 535nm follow addition of vanillin. A model mix of lipid standards mimicking the lipid composition of meibum gave a linear assay response in the sub 100ug range. Assay values for human meibum samples fell within the linear range of this standard curve for volumes between 20-100ul.
Conclusions: :
A model mix of pure lipids representing the distribution of lipid species in human meibum gives a linear and reproducible response in the SVA. The assay has sufficient sensitivity to allow routine use, in conjunction with gravimetric analysis, to assay the lipid content of human meibum samples.
Keywords: lipids • eyelid