Rate of acyl migration in lysophosphatidylcholine (LPC) is dependent upon the nature of the acyl group. Greater stability of sn-2 docosahexaenoyl LPC compared to the more saturated LPC species.

Several previous studies reported that sn-2 acyl lysophosphatidylcholines (LPCs) undergo rapid isomerization due to acyl migration, especially at physiological pH and temperature. However, these studies have been carried out using mostly sn-2 palmitoyl LPC, whereas the naturally occurring sn-2 LPCs are predominantly unsaturated. In this study, we investigated the acyl migration in four naturally occurring sn-2 acyl LPCs (sn-2 16:0, sn-2 18:1, sn-2 20:4, and sn-2 22:6) stored at various temperatures in aqueous or organic solvents, employing LC/MS to analyze the isomer composition. At 37°C and pH 7.4, the order of acyl migration rates (from sn-2 to sn-1) in aqueous buffer was 16:0 LPC> 18:1 LPC> 20:4 LPC> 22:6 LPC. The rate of isomerization of sn-2 16:0 LPC was 2-5 times greater than that of sn-2 22:6 under these conditions. Complexing the LPCs to serum albumin accelerated the acyl migration of all species, but sn-2 22:6 LPC was least affected by the presence of albumin. The migration rates were lower at lower temperatures (22°C, 4°C, and -20°C), but the differences between the LPC species persisted. All the sn-2 acyl LPCs were more stable in organic solvent (chloroform: methanol, 2:1 v/v), but the effect of the acyl groups on acyl migration was evident in the solvent also, at all temperatures. Storage of sn-2 22:6 LPC at -20°C for 4 weeks in the organic solvent resulted in about 10% isomerization, compared to 55% isomerization for sn-2 16:0. These results show that the sn-2 polyunsaturated LPCs can be stored at -20°C or below for several days without appreciable isomerization. Furthermore, they demonstrate that the sn-2 polyunsaturated LPCs generated in vivo are much more stable under physiological conditions than previously assumed

Efficient Enrichment of Retinal DHA with Dietary Lysophosphatidylcholine-DHA: Potential Application for Retinopathies

Although decreased retinal docosahexaenoic acid (DHA) is a known risk factor for retinopathy, currently available omega-3 fatty acid supplements, which are absorbed as triacylglycerol (TAG), do not significantly enrich retinal DHA. We tested the hypothesis that lysophospahtidylcholine (LPC)-DHA which is absorbed as phospholipid, would efficiently increase retinal DHA because of the presence of LPC-specific transporter at the blood–retina barrier. In normal rats, LPC-DHA and di-DHA phosphatidylcholine (PC), which generates LPC-DHA during digestion, increased retinal DHA by 101% and 45%, respectively, but TAG-DHA had no significant effect at the same dose (40 mg/kg, 30 days). In normal mice, both sn-1 DHA LPC and sn-2 DHA LPC increased retinal DHA by 80%, but free DHA had no effect. Lipase-treated krill oil (which contains LPC-DHA and LPC-EPA (eicosapentaenoic acid), but not normal krill oil (which has little LPC), increased both retinal DHA (+76%) and EPA (100-fold). Fish oil, however, had no effect, whether lipase-treated or not. These studies show that retinal DHA can be efficiently increased by dietary LPC-DHA, but not by TAG-DHA or free DHA. Since DHA is known to be protective against retinopathy and other eye diseases, this study provides a novel nutraceutical approach for the prevention/treatment of these diseases

Stability of sn-2 acyl LPCs in organic solvent.

<p>The sn-2 acyl LPCs were dissolved in chloroform: methanol (2:1 v/v) at a concentration of 1 mg/ml, and stored at the indicated temperature for various periods. Aliquots were taken out at the indicated time points, brought to room temperature (22°C), and subjected to LC/MS, as described in Methods. The percentage of the sn-2 acyl isomer remaining was calculated from the area counts of the two isomers. The values shown are mean ± SD of 3 separate experiments.</p

Identification of the regioisomers of LPC from the product ion spectra.

<p>The product ion spectra were obtained for the early eluting (peak 1) and late eluting (peak 2) isomers of each LPC as described in Methods. The intensity of the molecular ion as percentage of that of phosphocholine ion (m/z 184) differentiates the two regioisomers, with the sn-1 acyl isomer showing much higher percent of molecular ion [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187826#pone.0187826.ref017" target="_blank">17</a>]. The relative intensities of the molecular ion, expressed as percent of phosphoryl choline ion (m/z 184.1) for the various LPCs are as follows: 16:0 LPC (4.7% for peak 1 and 65.5% for peak 2); 18:1 LPC (3.7% for peak 1 and 44.8% for peak 2); .20:4 LPC (10.2% for peak 1 and 60.5% for peak 2); 22:6 LPC (15.3% for peak 1, and,172% for peak 2). In addition, the choline fragment ion (m/z 104.1) is completely absent from the sn-2 acyl isomers, but was prominently present in all the sn-1 acyl isomers.</p

Time course of isomerization of albumin-bound sn-2 acyl LPCs at 37°C.

<p>Freshly prepared sn-2 acyl LPCs were complexed with 0.1% BSA in PBS buffer at pH 7.4, and incubated at 37°C for the indicated periods of time. The percent of the sn-2 acyl isomer remaining after each time point was determined by LC/MS/MS. The values shown are mean ± SD of 3 separate analyses. Note that the error bars too small to be visible at some points.</p

MRM transitions used for LC-MS analysis of lysophospholipids.

<p>MRM transitions used for LC-MS analysis of lysophospholipids.</p

Selected LC/MS chromatograms of sn-2 acyl LPC species incubated at 37°C in Tris-HCl buffer, pH 7.4.

<p>The aqueous solution of each sn-2 acyl LPC (2 mg/ml) was incubated at 37°C, and aliquots were taken out at the indicated time. The samples were diluted with equal volume of methanol, and analyzed by LC/MS/MS in the MRM mode, as described in Methods.</p

Stability of aqueous dispersions of sn-2 acyl LPCs at various temperatures.

<p>The sn-2 acyl LPCs were dispersed in Tris-HCl buffer, pH 7.4 at a concentration of 2 mg/ml and incubated under nitrogen in the dark at the indicated temperature for 24 h. Aliquots were taken out at 0 h, 4 h, 8 h, 12 h, 16 h, and 24 h, brought to room temperature, diluted with equal volume of methanol and subjected to LC/MS/MS analysis as described in Methods. The percentage of the sn-2 acyl isomer remaining was calculated from the area counts of the two isomers. The values shown are mean ± SD of 3 separate experiments.</p

Effect of serum albumin on the isomerization of sn-2 acyl LPC at 37°C.

<p>Freshly prepared sn-2 acyl LPCs were complexed with BSA in PBS, pH 7.4, and incubated at 37°C for the indicated periods. The isomer composition was then determined by LC/MS/MS as described in Methods. Chromatograms of only selected time periods are shown. The time course of the isomerization of each LPC is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187826#pone.0187826.g006" target="_blank">Fig 6</a>.</p