betadex and 2-2--azobis(2-amidinopropane)

The effects of (2-hydroxypropyl)-β-cyclodextrin (HPβCD), a cyclic oligomer, on membrane electroporation-induced inward current (I(MEP)) in pituitary tumor (GH(3)) cells were experimentally and analytically characterized. Depletion of membrane cholesterol by exposing cells to HPβCD (2 mM) increased the activation time constant of delayed rectifier K(+) current. Such maneuver resulted in a significant reduction of I(MEP) density. 2,2'-Azo-bis(2-amidinopropane) dihydrochloride (AAPH), an initiator of free radicals, increased the magnitude of I(MEP). AAPH-stimulated I(MEP) was not reversed by the blockers of Ca(2+)-activated K(+) channels, but by LaCl(3) or MnCl(2). However, in HPβCD-treated cells, the ability of AAPH to enhance I(MEP) was abolished. Under such maneuver, the gating charge of I(MEP) activation was increased by 2 fold, along with a hyperpolarized shift of the activation curve by 30 mV. No change in single-channel conductance of MEP-induced channels during cell exposure to HPβCD was demonstrated. The energy change of I(MEP) in untreated and HPβCD-treated cells was estimated to be -17.7 and -44.8 kJ/mol, respectively, and the perturbation of free energy following HPβCD treatment was -27.1 kJ/mol. Based on an MEP model, cell exposure to HPβCD increased the edge energy of the electropore size. By use of a two barrier-one site barrier model, HPβCD treatment can increase both the peak height and well depth of the barrier profile. Taken together, depletion of membrane cholesterol by HPβCD can elevate the edge energy of pore formation, thereby decreasing the I(MEP) magnitude. The channel-suppressing properties during membrane cholesterol depletion with HPβCD might thus contribute to the underlying mechanisms by which such maneuver alters neuronal or neuroendocrine function.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; Amidines; Animals; Anticholesteremic Agents; beta-Cyclodextrins; Cell Line, Tumor; Cell Membrane; Cholesterol; Electroporation; Ion Channel Gating; Membrane Potentials; Pituitary Neoplasms; Rats

We recently reported the improved oxygen radical absorbance capacity (ORAC) assay using fluorescein (FL) as the fluorescent probe. The current ORAC(FL) assay is limited in hydrophilic antioxidant due to the aqueous environment of the assay. Lipophilic antioxidants mainly include the vitamin E family and carotenoids, which play a critical role in biological defense systems. In this paper, we expanded the current ORAC(FL) assay to lipophilic antioxidants. Randomly methylated beta-cyclodextrin (RMCD) was introduced as the water solubility enhancer for lipophilic antioxidants. Seven percent RMCD (w/v) in a 50% acetone-H(2)O mixture was found to sufficiently solubilize vitamin E compounds and other lipophilic phenolic antioxidants in 75 mM phosphate buffer (pH 7.4). This newly developed ORAC assay (abbbreviated ORAC(FL-LIPO)) was validated through linearity, precision, accuracy, and ruggedness. The validation results demonstrate that the ORAC(FL-LIPO) assay is reliable and robust. For the first time, by using 6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid as a standard (1.0), the ORAC values of alpha-tocopherol, (+)-gamma-tocopherol, (+)-delta-tocopherol, alpha-tocopherol acetate, tocotrienols, 2,6-di-tert-butyl-4-methylphenol, and gamma-oryzanol were determined to be 0.5 +/- 0.02, 0.74 +/- 0.03, 1.36 +/- 0.14, 0.00, 0.91 +/- 0.04, 0.16 +/- 0.01, and 3.00 +/- 0.26, respectively. The structural information of oxidized alpha-tocopherol obtained by liquid chromatography/mass spectrometry reveals that the mechanism for the reaction between the vitamin E and the peroxyl radical follows the hydrogen atom transfer mechanism, which is in agreement with the notion that vitamin E is the chain-breaking antioxidant.

Topics: alpha-Tocopherol; Amidines; Antioxidants; beta-Cyclodextrins; Binding, Competitive; Chromans; Chromatography, Liquid; Cyclodextrins; Fluorescein; Fluorescent Dyes; Free Radicals; Indicators and Reagents; Lipid Metabolism; Mass Spectrometry; Methylation; Oxygen Compounds; Peroxides; Sensitivity and Specificity; Solubility