Date on Master's Thesis/Doctoral Dissertation
Yappert, Marta C.
Hyaluron; Lipid; Vitreous; Nuclear magnetic resonance; NMR; Mass spectrometry; MS
Hyaluronan (HA) is a major structural component of the ocular vitreous humor (VH) and lipids are essential building blocks of cell membranes. However, little is known about their possible interactions. The VH is known to undergo liquefaction throughout the lifetime that may result in complications such as posterior vitreous detachment. The possible roles of lipids in the age-dependent liquefaction of the VH are unknown. As a first step in this new area of research in our group, model studies of the repeating unit of polymeric HA were performed. This unit contains glucuronate (GlcU) linked to N-acetylglucosamine (G1cNAc) via a 1-3 linkage. The sample contained both a and ß anomers leading to significant NMR (nuclear magnetic resonance) spectral complexity. Through the powerful combination of inverse heteronuclear 2D experiments, the resolution and assignment of all resonances for both anomers were achieved. Temperature-dependent changes in the NH resonances revealed the formation of weak or water-mediated intramolecular H-bonds. The polymeric form of HA was then investigated in D2O and its interactions with various lipids were probed. The analysis of chemical shifts and bandwidths of 1H NMRresonances revealed the strongest interactions to be between HA and unsaturated oleic acid and lyso-oleoyl-phosphatidylcholine. Because matrix-assisted laser desorption/ionization mass spectrometry (MALDIMS) will be used to analyze the components of the VH in situ, we evaluated changes in relative ionization efficiencies (RIE) when the repeating unit of HA and lipids were present in the model samples. Smaller lipid metabolites caused the greatest reduction of the RIE of HA but phosphatidic acid had no effect. On the other hand, HA decreased the ionization of all lipid species, almost equally, by ~50%. These findings suggest that the interactions of these species in the gas phase lead to partial blockage of the ionization sites. Preliminary results have been obtained for the VH by both NMR and MALDI-MS and do show the presence of lipids within the VH. In addition, oxidation of the VH leads to the presence of oxidized compounds and liquefaction. Future studies will focus on the isolation and characterization of these products of oxidation.
Magness, Kristen Ann 1981-, "Biophysical and bioanalytical studies of hyaluronan and lipids." (2012). Electronic Theses and Dissertations. Paper 883.