Preparation of pure lipid hydroperoxides

Abstract

Increasing evidence of lipid peroxidation in food deterioration and pathophysiology of diseases have revealed the need for a pure lipid hydroperoxide (LOOH) reference as an authentic standard for quantification and as a compound for biological studies in this field. Generally, LOOH is prepared from photo-or enzymatically oxidized lipids; however, separating LOOH from other oxidation products and preparing pure LOOH is difficult. Early studies showed the usability of reaction between hydroperoxide and vinyl ether for preparation of pure LOOH. Because the reactivity of vinyl ether with LOOHs other than fatty acid hydroperoxides has never been reported, here, we employed the reaction for preparation of a wide variety of pure LOOHs. Phospholipid, cholesteryl ester, triacylglycerol, or fatty acid was photo-or enzymatically oxidized; the resultant crude sample containing hydroperoxide was allowed to react with a vinyl ether [2-methoxypropene (MxP)]. Liquid chromatography (LC) and mass spectrometry confirmed that MxP selectively reacts with LOOH, yielding a stable MxP adduct (perketal). The lipophilic perketal was eluted at a position away from that of intact LOOH and identified and isolated by LC. Upon treatment with acid, perketal released the original LOOH, which was finally purified by LC. Using our optimized purification procedures, for instance, we produced 75 mg of pure phosphatidylcholine hydroperoxide (.99%) from 100 mg of phosphatidylcholine. Our developed method expands the concept of the perketal method, which provides pure LOOH references. The LOOHs prepared by the perketal method would be used as "gold standards" in LOOH methodology. Because lipid peroxidation is involved in food deterioration (1) and pathophysiology of human diseases (2, 3), there has been a great interest in the accurate measurement of lipid hydroperoxide (LOOH) concentration. This can be performed by several quantitative methods (4-11), and the most sensitive and reliable one is chemiluminescence detection-liquid chromatography (CL-LC) (9-11). Since there is no approved LOOH calibrator ("gold standard") available, it is impossible to compare the LOOH levels from various laboratories around the world.Currently, researchers prepare their own in-house reference LOOH [i.e., lipids (e.g., phospholipids, cholesterols, triacylglycerols, and fatty acids)] are subjected to photo-(12), free radical (13), or enzymatic oxidation (14, 15). The resultant crude or partly purified LOOH is generally used as a calibrator. However, these references are neither officially approved nor do they correspond to each other, particularly with regard to their purity. As frequently mentioned by LOOH researchers, this problem is mainly caused by the difficulty in distinguishing and isolating LOOH from other oxidation products such as hydroxides (13). Therefore, efficient purification of a wide variety of LOOHs is the key to the development of the gold standard not only for the accurate quantification of LOOH but also for the evaluation of its biological fu...

Preparation of 13 or 9-Hydroperoxy-9Z,11E (9E,11E) or 10E,12Z (10E,12E)-Octadecadienoic Phosphatidylcholine Hydroperoxide

“…In the present study, we improved our previously reported PCOOH preparation conditions 16 e.g., reaction between PCOOH and MxP , and produced 30 mg of PCOOH a pure mixture of 13-and 9-hydroperoxyoctadecadienoyl PCOOH isomers from 100 mg of PC without the formation of unknown brown byproducts. The major sn-2 fatty acid of the resultant PCOOH was 13-hydroperoxyoctadecadienoate.…”

“…With regard to stability, PCOOMxP was found to be more stable than PCOOH reported in our previous study 16 . We therefore recommend the following: researchers should prepare and store perketal in chloroform at low temperatures e.g., 80 in advance, regenerate PCOOH from PCOOMxP before the experiment e.g., quantitative study , and use the PCOOH as the reference material.…”

“…Other PCOOH isomers bearing 10-or 12-hydroperoxyoctadecadienoate can be obtained if PC 16:0/18:2 PC is oxidized by singlet oxygen, instead of enzyme followed by MxP and LC treatments. In addition to PCOOH and fatty acid hydroperoxide, the MxP method is also effective for preparation of other lipid hydroperoxides such as cholesterol esters hydroperoxide, triacylglycerol hydroperoxide, and fatty acid methyl ester hydroperoxide 16 . Such lipid hydroperoxides may be useful as reliable standards, not only for the accurate quantification of lipid hydroperoxides, but also for the evaluation of their pathophysiological functions.…”

“…Upon treatment with acid i.e., PPTS , perketal releases the original hydroperoxide molecule 16 , followed by gradual decomposition of the resultant hydroperoxide under the acidic conditions. Hence, the regeneration reaction from PCOOMxP was investigated by focusing on reaction time, in an effort to avoid a gradual decomposition of the resultant PCOOH during the reaction.…”

“…To prepare perketal PCOOMxP in our previously-reported method 16 , crude PCOOH equivalent to about 0.4 mg total PC, in which 0.3 mg PCOOH was included was reacted with 150 μmol MxP in 100 μL of dichloromethane containing 0.07 μmol catalytic PPTS. Nevertheless, as mentioned above, the reaction unexpectedly yielded unknown brown byproducts at times.…”