Isolation and purity determination of LCBs
GlcCers and CAEPs were purified from konjac tubers, Tamogi mushrooms, and scallops to prepare LCBs for rat lymph cannulation experiments. The obtained GlcCers and CAEPs were heated in Ba(OH)2 aqueous solution/dioxane. Most GlcCers were hydrolyzed to lyso-GlcCers, which were subsequently treated with β-glucosidase and LCBs were liberated. In contrast, CAEPs were completely hydrolyzed to LCB in Ba(OH)2 aqueous solution/dioxane. Liberated LCBs were finally purified by ODS HPLC, and LCB 18:2(4E,8Z);2OH, LCB 18:2(4E,8E);2OH, LCB 18(9Me):2(4E,8Z);2OH, LCB 18:3(4E,8E,10E);2OH, and LCB 18(9Me):3(4E,8E,10E);2OH were successfully isolated. Purified LCBs were analyzed by ESI-MS, and their associated ions were observed at m/z 298.2719 (calculated for 298.2746, Δ 9.06 ppm) [M + H]+ [LCB 18:2(4E,8Z);2OH], 298.2704 (calculated for 298.2746, Δ 14.09 ppm) [M + H]+ [LCB 18:2(4E,8E);2OH], 296.2573 (calculated for 296.2573, Δ 5.58 ppm) [M + H]+[LCB 18:3(4E,8E,10E);2OH], 312.2872 (calculated for 312.2903, Δ 9.78 ppm) [M + H]+ [LCB 18(9Me):2(4E,8Z);2OH], and 310.2727 (calculated for 310.2746, Δ 6.14 ppm) [M + H]+ [LCB 18(9Me):3(4E,8E,10E);2OH] (Additional file 1: Fig. S1A-F). Isolated LCBs were derivatized with OPA and analyzed by HPLC with a fluorescence detector. The purities were > 99% for LCB 18:2(4E,8Z);2OH, 95% for LCB 18:2(4E,8E);2OH, > 99% for LCB 18:3(4E,8E,10E);2OH, > 99% for LCB 18(9Me):2(4E,8Z);2OH, and > 99% for LCB 18(9Me):3(4E,8E,10E);2OH (Additional file 1: Fig. S2A-L).
Recovery of LCBs from chyle
Emulsions containing 10 mg of LCB (in brief LCB 18:2(4E,8Z);2OH, 33.6 μmol; LCB 18:2(4E,8E);2OH, 33.6 μmol; LCB 18(9Me):2(4E,8Z);2OH, 31.2 μmol; LCB 18:3 (4E,8E,10E);2OH, 33.9 μmol; LCB 18(9Me):3(4E,8E,10E);2OH, 32.3 μmol) emulsified with triolein and taurocholic acid were administered to rats through the duodenum tubes, and chyle were collected from the thoracic duct lymph over time. There were no significant differences in the amount of lymph output among rats administrated with each LCB emulsion (Additional file 1: Fig. S3), indicating that surgery and animal maintenance were carried out appropriately. First, LCBs extracted from the collected chyle and treated with 0.4 M NaOH-methanol solution were analyzed via LC/MS/MS. HPLC retention times and exact masses of protonated ion signals [the [M + H]+ m/z 298.2746, 298.2746, 296.259, 312.2903, and 310.2746 for LCB 18:2(4E,8Z);2OH, LCB 18:2(4E,8E);2OH, LCB 18:3(4E,8E,10E);2OH, LCB 18(9Me):2(4E,8E);2OH, and LCB 18(9Me):3(4E,8E,10E);2OH, respectively] were used for the identification of each LCB species (Additional file 1: Fig. S4A-E). Peak areas were integrated at m/z ± 0.05, and LCB amounts were determined by comparing the [M + H]+ ion signals of LCB 18:2(4E,8Z);2OH, LCB 18:2(4E,8E);2OH, LCB 18:3(4E,8E,10E);2OH, LCB 18(9Me):2(4E,8Z);2OH, and LCB 18(9Me):3(4E,8E,10E);2OH with the peak area of the LCB 17:1(4E);2OH internal standard. The amounts of LCBs in chyle at each time point are shown in Fig. 2. The amounts of all LCBs [LCB 18:2(4E,8Z);2OH, LCB 18:2(4E,8E);2OH, LCB 18:3(4E,8E,10E);2OH, LCB 18(9Me):2(4E,8Z);2OH, and LCB 18(9Me):3(4E,8E,10E);2OH], were elevated in the chyle of rats infused with the corresponding LCBs. The amount of LCB 18:2(4E,8Z);2OH was highest at 3 h after administration (0.83 nmol); whereas, the amount of LCB 18:2(4E,8E);2OH, a geometrical isomer of LCB 18:2(4E,8Z);2OH, was highest at 1 h after administration (4.38 nmol). The levels of both LCB 18:2(4E,8Z);2OH and LCB 18:2(4E,8E);2OH at 8 h decreased. The total amount of LCB 18:2(4E,8Z);2OH, up to 8 h after LCB administration, was ~ 4.4-fold higher than that of LCB 18:2(4E,8E);2OH. In the case of other LCBs, the amount of LCB 18:3(4E,8E,10E);2OH in chyle increased after 1–3 h, LCB 18(9Me):2(4E,8Z);2OH increased after 2–4 h, and LCB 18(9Me):3(4E,8E,10E);2OH was detected at 4 h in some samples after administration. This result suggests that the absorption percentages of LCBs in chyle differ with their structure, even among geometrical isomers. Herein, the percentage of absorption of LCB 18:2(4E,8Z);2OH, LCB 18:2(4E,8E);2OH, LCB 18:3(4E,8E,10E);2OH, LCB 18(9Me):2(4E,8Z);2OH, and LCB 18(9Me):3(4E,8E,10E);2OH into chyle as an LCB was 0.011 ± 0.006%, 0.048 ± 0.016%, 0.043 ± 0.016%, 0.004 ± 0.0004%, and 0.014 ± 0.013%, respectively, when integrated up to 8 h after administration (Fig. 2a-d).
Quantification of ceramide and HexCer with atypical LCB moieties in chyle
As the LCBs absorbed from the digestive tract were expected to be metabolized to ceramides and complex sphingolipids in intestinal epithelial cells, these LCB metabolites in the chyle were also analyzed. As expected, a portion of each administered LCB was processed into ceramides (Fig. 3a-d), and more than 80% were linked to FA 16:0, followed by FA 24:0 and FA 23:0 (Additional file 1: Figs. S5 and 6); moreover, the FA species linked to LCB did not depend on the structure of the administrated LCB (Additional file 1: Fig. S6). Similar to the results of LCB absorption, the amount of ceramide with an LCB 18:2(4E,8E);2OH moiety in chyle peaked at 1 h after administration and then gradually decreased to only trace amounts at 8 h (Fig. 3a). The percentages of ceramides with an LCB 18:2(4E,8Z);2OH moiety or ceramides with an LCB 18:2(4E,8E);2OH moiety absorption into the chyle were compared among groups administrated LCB 18:2(4E,8Z);2OH and LCB 18:2(4E,8E);2OH. As in the case of LCBs, ceramides with an LCB 18:2(4E,8Z);2OH moiety were absorbed more slowly and to a lesser extent than ceramides with an LCB 18:2(4E,8E);2OH moiety (Fig. 3a). In the case of other LCBs, the amount of ceramides with administrated LCBs in the chyle increased at 2–4 h after administration of LCB 18:3(4E,8E,10E);2OH, 2–6 h after administration of LCB 18(9Me):2(4E,8Z);2OH and 3–7 h after administration of LCB 18(9Me):3(4E,8E,10E);2OH (Fig. 3b-d). The absorption of ceramides with an LCB 18(9Me):3(4E,8E,10E);2OH moiety was the slowest among the LCBs tested in this study, and the maximum amount of ceramides detected in chyle recovered 6 h after LCB administration (Fig. 3d). The total absorption percentage of LCB 18:2(4E,8Z);2OH, LCB 18:2(4E,8E);2OH, LCB 18:3(4E,8E,10E);2OH, LCB 18(9Me):2(4E,8Z);2OH, and LCB 18(9Me):3(4E,8E,10E);2OH into chyle as a ceramide was 0.095 ± 0.008%, 0.143 ± 0.023%, 0.030 ± 0.008%, 0.054 ± 0.019%, and 0.088 ± 0.023%, respectively.
HexCers metabolized from the administrated LCB in the collected chyle were identified. Interestingly, LCB 18:2(4E,8E);2OH and LCB 18(9Me):2(4E,8Z);2OH were the only LCBs detected in chyle that were converted to HexCers (Fig. 4a, c), which was unlike the observations in case of LCBs or ceramide. HexCers of other LCBs were present only at trace levels (Fig. 4b, d). Only HexCer 18:2(4E,8E);2OH/16:0 and HexCer 18(9Me):2(4E,8E);2OH/16:0 were detected, and no other HexCers linked to FA such as 24:0 and 23:0 were detected in chyle. Changes in the amount of HexCer in the chyle showed a similar trend. The amount of reconstructed HexCer in the chyle was highest at 3 h after administration of LCB 18:2(4E,8E);2OH and at 4 h after administration of LCB 18(9Me):2(4E,8E);2OH (Fig. 4a, c). Both HexCers with LCB 18:2(4E,8E);2OH and LCB 18(9Me):2(4E,8Z);2OH moieties were decreased to trace levels at 6 h after administration (Fig. 4a, c). In addition, the absorption percentages of HexCers as LCB were lower than that of ceramides (Figs. 3a-d and 4a, c). Additionally, it is possible that the transport of HexCer, from the small intestine to chyle, may be slower than that of ceramide. The absorption percentage of HexCers with LCB 18:2(4E,8E);2OH and LCB 18(9Me):2(4E,8Z);2OH into chyle as a HexCer was 0.011 ± 0.004% and 0.017 ± 0.005%, respectively.
Analysis of the LCB backbones of SM by in-source CID/PRM in ESI-negative mode
To identify and quantify SMs with atypical LCB moieties, lipids extracted from chyle, from rats administered LCBs, were analyzed. In the ESI-positive mode, fragmentation of protonated ions ([M + H]+) of SM yielded a major typical product ion (m/z 184.0712) derived from phosphocholine; hence, information for LCBs was obtained using this mode (Additional file 1: Fig. S7A). Therefore, the in-source CID/PRM method was established, a combination of in-source CID and post-source CID, in the ESI-negative mode for identification of the LCB backbones of SMs. In this analysis, methyl group liberated by in-source fragmentation of SM ([M-CH3]−) were observed on the TOF survey scan when the declustering potential was set to − 200 V; hence, [M-CH3]− was selected as the precursor ion. The post-source CID of [M-CH3]− ions produced the product ion [M-CH3-fatty acyl]− (Additional file 1: Fig. S7), and these pairs were selected for the PRM mode (typical TOF-MS spectrums of SM fraction and TOF-MS based XICs which speculated to be demethylated SMs and typical product ion spectrums and XICs of demethylated SMs with atypical LCBs are shown in Additional file 1: Figs. S8 and 9).
Using this in-source CID/ PRM method in the ESI-negative mode, the levels of SM in the chyle of rats administrated LCBs were measured (Fig. 5). Metabolized SMs from administrated LCBs were detected, especially in the group administered LCB 18:2(4E,8Z);2OH (Fig. 5a). The amount of SMs with an LCB 18:2(4E,8E);2OH moiety in chyle increased at 4–7 h after administration of LCB 18:2(4E,8E);2OH. FA 16:0 was the most common fatty acid in SMs with an LCB 18:2(4E,8E);2OH moiety, as well as ceramides and HexCers with an LCB 18:2(4E,8E);2OH moiety (Figs. 3a, 4a and 5a). Upon comparing the detected SMs with an LCB 18:2(4E,8E);2OH moiety, as in the case of HexCers, the absorption of SM into chyle was observed to occur later than ceramide (Figs. 3a and 5a). In the case of groups administered LCB 18:2(4E,8E);2OH, LCB 18:3(4E,8E,10E);2OH, LCB 18(9Me):2(4E,8Z);2OH, and LCB 18(9Me):3(4E,8E,10E);2OH, the amount of SM detected in chyle was much lower than that in the LCB 18:2(4E,8E);2OH-administered group (Fig. 5b-d). The amount of LCB 18:2(4E,8Z);2OH, LCB 18:2(4E,8E);2OH, LCB 18:3(4E,8E,10E);2OH, LCB 18(9Me):2(4E,8Z);2OH, and LCB 18(9Me):3(4E,8E,10E);2OH adsorbed into chyle as SM was 1.064 ± 0.149%, 0.047 ± 0.068%, 0.026 ± 0.004%, 0.277 ± 0.023%, and 0.131 ± 0.053%, respectively, when integrated up to 8 h after administration. Total absorption percentage of LCB 18:2(4E,8Z);2OH was significantly higher than other LCBs (Fig. 6).
Uptake and transport of LCB 18:2(4E,8Z);2OH and LCB 18:2(4E,8E);2OH in differentiated Caco-2 cells
In rat chyle, the levels of HexCers and SMs with an LCB 18:2(4E,8Z);2OH or an LCB 18:2(4E,8E);2OH moieties clearly varied with the geometric isomerism of the 8-position of LCB 18:2(4E,8Z);2OH and LCB 18:2(4E,8E);2OH (Figs. 4a and 5a). These LCBs, having the same mass, are considered useful for investigating lipid metabolism and transport. On the other hand, there was no significant difference between LCB 18(9Me):2(4E,8E);2OH and LCB18(9Me):3(4E,8E,10E), thus LCB 18:2(4E,8Z);2OH and LCB 18:2(4E,8E);2OH were used for caco-2 experiments. Thus, the absorption behaviors of LCB 18:2(4E,8Z);2OH and LCB 18:2(4E,8E);2OH were analyzed using differentiated Caco-2 cells, an intestinal epithelial transport system model. Caco-2 cells, which were cultured on a cell culture insert for 21 days and differentiated into intestinal epithelium-like cells, were used for the experiments. The day 22, LCBs dissolved in medium at final concentrations of 10 μM were added to the apical surface of differentiated cells and incubated for 24 h. Lipids were extracted from the medium on the basolateral side and from cells. The LCBs, metabolized to ceramides, HexCers, and SMs were analyzed via LC/MS/MS (Fig. 7, Additional file 1: Fig. S10). There were no significant differences in the amounts of LCBs and ceramides in the medium on the basolateral side, between treatments with LCB 18:2(4E,8Z);2OH or LCB 18:2(4E,8E);2OH (Fig. 7a, b). Levels of HexCers in the basal-side medium were higher in LCB 18:2(4E,8E);2OH-treated cells than LCB 18:2(4E,8Z);2OH-treated cells, and the amounts of SMs in LCB 18:2(4E,8Z);2OH-treated cells were larger than those in LCB 18:2(4E,8E);2OH-treated cells, which was consistent with the rat chyle results. Quantitative analysis revealed that the amount of intracellular LCBs in the LCB 18:2(4E,8Z);2OH-treated cells were significantly higher than those in the control cells, but there was no significant difference between the LCB 18:2(4E,8E);2OH-treated cells and control cells (Fig. 7e). The amount of intracellular ceramides significantly increased only in the LCB 18:2(4E,8E);2OH-treated cells compared with that in the control cells (Fig. 7f), but there was no significant difference between the LCB 18:2(4E,8Z);2OH-treated cells and control cells. The amount of intracellular HexCers significantly increased in the LCB 18:2(4E,8E);2OH-treated cells compared with that in control cells, but there was no change in the amount of HexCers in the LCB 18:2(4E,8Z);2OH-treated cells compared with that in the control cells (Fig. 7g). These results followed a trend similar to that of the experiment on LCB absorption in the chyle of rats. However, unlike the rat experiment, the amount of intracellular SMs of both LCB 18:2(4E,8Z);2OH- and LCB 18:2(4E,8E);2OH-treated cells significantly increased compared with that in the control cells, and there were no significant differences in the amount of intracellular SMs between LCB 18:2(4E,8Z);2OH- and LCB 18:2(4E,8E);2OH-treated cells (Fig. 7h).