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Rate constants for autoxidation propagation of several unsaturated lipids in benzene solution at 37°C and in phosphatidylcholine liposomes were determined by a linoleate radical clock. This radical clock is based on competition between hydrogen atom abstraction by an intermediate peroxyl radical derived from linoleic acid that leads to a trans,cis-conjugated hydroxyoctadecadienoic product and β–fragmentation of the same peroxyl that gives the trans,trans-product hydroxyoctadecadienoic acid. Rate constants determined by this approach in solution relative to linoleic acid (kp = 62 M−1s−1) were: arachidonic acid (kp = 197 ± 13 M−1s−1), eicosapentaenoic acid (kp = 249 ± 16 M−1s−1), docosahexaenoic acid (kp = 334 ± 37 M−1s−1), cholesterol (kp = 11 ± 2 M−1s−1), and 7-dehydrocholesterol (kp = 2,260 ± 40 M−1s−1). Free radical oxidations of multilamellar and unilamellar liposomes of various mixtures of glycerophosphatidylcholine molecular species were also carried out. In some experiments, cholesterol or 7-dehydrocholesterol was incorporated into the lipid mixture undergoing oxidation. A phosphatidylcholine bearing a linoleate ester at sn-2 was a component of each liposome peroxidation reaction and the ratio of trans,cis/trans,trans (t,c/t,t)-conjugated diene oxidation products formed from this phospholipid was determined for each oxidation reaction. This t,c/t,t-product ratio from linoleate was used to “clock” liposome constituents as hydrogen atom donors in the lipid bilayer. Application of this lipid bilayer radical clock gives relative autoxidation propagation rate constants of arachidonate (20:4), eicosapentaenoate (20:5), docosahexaenoate (22:6), and 7-dehydrocholesterol to be 115 ± 7, 145 ± 8, 172 ± 13, and 832 ± 86, respectively, a reactivity trend that parallels the one in solution. We also conclude from the liposome oxidations that linoleate peroxyl radicals at different positions on the eighteen-carbon chain (at C-9 and C-13) have different kinetic properties. This is in contrast to the results of solution oxidations of linoleate in which the C-9 and C-13 peroxyl radicals have similar reactivities. We suggest that peroxyl radical β–scission depends on solvent polarity and the polarity of the local environment of peroxyl radicals in liposomal oxidations depends on the position of the peroxyl radical on the eighteen-carbon chain.

Collision cross section (CCS) measurement of lipids using traveling wave ion mobility-mass spectrometry (TWIM-MS) is of high interest to the lipidomics field. However, currently available calibrants for CCS measurement using TWIM are predominantly peptides that display quite different physical properties and gas-phase conformations from lipids, which could lead to large CCS calibration errors for lipids. Here we report the direct CCS measurement of a series of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in nitrogen using a drift tube ion mobility (DTIM) instrument and an evaluation of the accuracy and reproducibility of PCs and PEs as CCS calibrants for phospholipids against different classes of calibrants, including polyalanine (PolyAla), tetraalkylammonium salts (TAA), and hexakis(fluoroalkoxy)phosphazines (HFAP), in both positive and negative modes in TWIM-MS analysis. We demonstrate that structurally mismatched calibrants lead to larger errors in calibrated CCS values while the structurally matched calibrants, PCs and PEs, gave highly accurate and reproducible CCS values at different traveling wave parameters. Using the lipid calibrants, the majority of the CCS values of several classes of phospholipids measured by TWIM are within 2% error of the CCS values measured by DTIM. The development of phospholipid CCS calibrants will enable high-accuracy structural studies of lipids and add an additional level of validation in the assignment of identifications in untargeted lipidomics experiments.

Free radical chain oxidation of highly oxidizable 7-dehydrocholesterol (7-DHC) initiated by 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile) was carried out at 37°C in benzene for 24 hours. Fifteen oxysterols derived from 7-DHC were isolated and characterized with 1D- and 2D-NMR spectroscopy and mass spectrometry. A mechanism that involves abstraction of hydrogen atoms at C-9 and/or C-14 is proposed to account for the formation of all of the oxysterols and the reaction progress profile. In either the H-9 or H-14 mechanism, a pentadienyl radical intermediate is formed after abstraction of H-9 or H-14 by a peroxyl radical. This step is followed by the well-precedented transformations observed in peroxidation reactions of polyunsaturated fatty acids such as oxygen addition, peroxyl radical 5-exo cyclization, and SHi carbon radical attack on the peroxide bond. The mechanism for peroxidation of 7-DHC also accounts for the formation of numerous oxysterol natural products isolated from fungal species, marine sponges, and cactaceous species. In a cell viability test, the oxysterol mixture from 7-DHC peroxidation was found to be cytotoxic to Neuro2a neuroblastoma cells in the micromolar concentration range. We propose that the high reactivity of 7-DHC and the oxysterols generated from its peroxidation may play important roles in the pathogenesis of Smith-Lemli-Opitz syndrome (SLOS), X-linked dominant chondrodysplasia punctata (CDPX2), and cerebrotendinous xanthomatosis (CTX), all of these being metabolic disorders having an elevated level of 7-DHC.

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