Browsing by Author "Stillwell, William"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    DHA Alters Raft-like Membrane Domains as Revealed by Solid State 2H NMR Spectroscopy
    (Office of the Vice Chancellor for Research, 2015-04-17) Kinnun, Jacob J.; Williams, Justin A.; Stillwell, William; Bittman, Robert; Shaikh, Saame Raza; Wassall, Stephen R.
    Dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as docosahexaenoic acid (DHA, 22:6), are correlated with the prevention of neurological and autoimmune disorders in humans. These fatty acids must be obtained from the diet, such as oil fish or fish oil supplements, as they cannot be generated within the human body. The origin of the health benefits at the molecular level is still under question. A membrane-mediated mechanism in which n-3 PUFAs are incorporated into phospholipids and modulate molecular organization is one possibility. Cellular membranes are inhomogeneous where structurally diverse lipids can exist in separate domains. Regions rich in sphingomyelin (SM) and cholesterol, commonly called lipid rafts, contain important signaling proteins. In a recent solid-state 2H nuclear magnetic resonance (2H NMR) study of a model membrane composed of 1-[2H31] palmitoyl-2-docosahexaenoyl-phosphatidylcholine (PDPC-d31), a deuterated analog of a DHA-containing phospholipid, in mixtures with SM and cholesterol, we discovered that DHA could significantly enter raft-like domains. How DHA affects the molecular organization within the raft-like domains is addressed here by observing PSM-d31, an analog of SM with a perdeuterated N-palmitoyl chain. The 2H NMR spectra for PSM-d31, in mixtures with PDPC and cholesterol, exhibit two spectral components, a larger more ordered component that we attribute to raft-like domains and a smaller less ordered component that we attribute to non-raft-like domains. On average, the order of PSM-d31 is reduced and, thus, disordering of PSM-d31 by PDPC is indicated. Our observations confirm that DHA can infiltrate rafts and affect molecular organization, which has implications for the signaling of raft and non-raft proteins. Furthermore, these results are consistent with in vivo studies showing that DHA infiltrates rafts.
  • Thumbnail Image
    Item
    Raft Busters: A Molecular Role for DHA in Biological Membranes?
    (Office of the Vice Chancellor for Research, 2016-04-08) Kinnun, Jacob J.; Williams, Justin A.; Stillwell, William; Bittman, Robert; Shaikh, Saame Raza; Wassall, Stephen R.
    Dietary consumption of fish oils rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as docosahexaenoic acid (DHA, 22:6), has a wide variety of health benefits. However, a complete molecular mechanism is yet to be elucidated. One model that has emerged from biochemical and imaging studies of cells postulates that n-3 PUFAs are taken up into phospholipids in the plasma membrane of cells and, due to their high disorder and aversion for cholesterol, reorganize lipid rafts. Lipid rafts are ordered domains within biological membranes which contain high amounts of sphingomyelin (SM) and cholesterol. To investigate this model, we studied lipid bilayers composed of SM, PDPC (a DHA-containing phospholipid), and cholesterol (1:1:1 mol). The molecular organization of each lipid was investigated with solid-state 2H NMR using deuterated analogs of the lipids. Spectral components assigned to ordered raft-like domains and disordered non-raft domains were resolved, from which the composition of the domains and the order within them could be determined. Most of the SM (84%) and cholesterol (88%) was found in the raft-like domain, together with a substantial amount of PDPC (70%). Despite the infiltration of PDPC there appears to be minimal effect on the order of SM or cholesterol. We speculate that PDPC molecules sequester into small groups minimizing the contact of DHA chains with cholesterol, thereby interrupting the continuity of the raft-like environment.
  • Thumbnail Image
    Item
    SPIN TRAPPING THE OXIDIZED PRODUCTS OF PUFA IN MODEL MEMBRANES: THE PROTECTION CONFERRED BY VITAMIN E
    (Office of the Vice Chancellor for Research, 2011-04-08) Wassall, Cynthia D.; Stillwell, William; Wassall, Stephen R.; Kemple, Marvin D.
    Electron paramagnetic resonance (EPR) spectroscopy is recognized as the most sensitive and noninvasive means to quantify free radicals of biological relevance such as reactive oxygen species (ROS). In spin trapping a molecule (the spin trap) reacts with the free radical producing a spin adduct that is sufficiently stable to be detected by EPR. Here we apply a novel spin trapping technique to investigate the protection that α-tocopherol (vitamin E), the major lipid soluble antioxidant in membranes, confers on polyunsaturated lipids in model membranes. Polyunsaturated fatty acids (PUFA) readily oxidize because they have a cis,cis-1,4-pentadiene motif that renders the central methylene group vulnerable to attack by ROS. Our method quantifies the oxidized products of PUFA in lipid vesicles that have been exposed to a peroxyl radical generator 2,2'-azobis-(amidinopropane) dihydrochloride (AAPH) that initiates the free radical chain reaction. By measuring the reduction in lipid peroxidation due to the presence of αtocopherol, we test the hypothesis that the vitamin co-localizes with polyunsaturated lipids in membrane domains to ensure close proximity to the most vulnerable lipid species.