Structure-dependent absorption of sphingolipid long-chain bases from the digestive tract into lymph

doi:10.21203/rs.3.rs-88680/v1

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Structure-dependent absorption of sphingolipid long-chain bases from the digestive tract into lymph

https://doi.org/10.21203/rs.3.rs-88680/v1

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Journal Publication

published 01 Mar, 2021

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Background:

Dietary sphingolipids have various biofunctions, including skin barrier improvement, anti-inflammatory and anti-carcinoma properties. Long-chain bases (LCBs), the essential backbones of sphingolipids, are responsible for these bioactivities, and they vary structurally between species. Given these findings, however, the absorption dynamics of each LCB remain unclear.

Methods:

In this study, five structurally different LCBs were isolated and their absorption ratios and levels of their metabolites were analyzed using a lymph-duct-cannulated rat model by liquid chromatography tandem mass spectrometry (LC/MS/MS) with a novel multistage fragmentation method.

Results:

The five orally administered LCBs were absorbed and detected in lymph as free LCBs and several metabolites including ceramides, glucosylceramides, and sphingomyelins. The absorption rates of LCBs were 0.10-1.17% depending on their structure. The absorption rate of 4-trans,8-cis-sphingadienine was highest (1.17%), whereas that of 4-trans,8-trans,10-trans-sphingatrienine was lowest (0.10%). The amount of 4-trans,8-cis-sphingadienine-bound sphingomyelin in lymph was particularly higher than other four LCB-bound sphingomyelins.

Conclusion:

Structural differences among LCBs, particularly the geometric isomerism at the C8–C9 position, significant affect the absorption rate and amounts of metabolites. This is the first report revealed that the absorption rate and metabolism of sphingolipids are dependent on their LCB structure.

Endocrinology & Metabolism

sphingolipids

long-chain base

lipid metabolism

lipidomics

lymphatic absorption

sphingomyelin

Sphingolipids have long-chain aliphatic amino alcohol moieties called long-chain bases (LCBs) as their backbones. LCBs are widely distributed in nature and perform important functions in eukaryotic cells [1, 2]. Ceramides consist of LCB and fatty acid components, and the basic structure of glycosphingolipids and sphingomyelins (SM) includes the polar head groups sugars and phosphocholine, respectively [3] (Fig. 1A). Sphingolipids are involved in apoptosis, differentiation, proliferation, and cell migration in mammalian cells [2–7]. Ingested sphingolipids can induce important physiological activities, such as improvement of the skin barrier function, anti-inflammatory action, and suppression of the onset of colorectal cancer [4, 8–11]. Therefore, sphingolipids are predicted to be useful functional food components.

The structures of LCBs vary between species [1] (Fig. 1B). Sphingosine (d18:1, 2-amino-4-trans-octadecene-1,3-diol) is the most abundant LCB in mammals [1]. Plants and fungi produce LCBs with two double bonds, such as sphingadienine (4-trans,8-cis-sphingadienine, d18:2^4t8c), 4-trans,8-trans-sphingadienine (d18:2^4t8t), and 9-methyl,4-trans,8-trans-sphingadienine (9Me-d18:2). On the other hands, 4-trans,8-trans,10-trans-sphingatrienine (d18:3) and 9-methyl,4-trans,8-trans,10-trans- sphingatrienine (9Me-d18:3), which have three double—bond LCBs, are found in marine organisms [1].

Orally ingested d18:1-bound glucosylceramide (GlcCer) and SM are degraded to ceramide and polar head groups by specific enzymes in the digestive tract of rats, followed by the breakdown of ceramide to LCB and fatty acids [12–17]. Previous studies showed that maize-derived GlcCer and squid-derived ceramide 2-amynoethylphosphonate (CAEP), which have nonmammalian-type LCBs such as d18:2 and 9Me-d18:3, are also hydrolyzed in the digestive tract, and liberated LCBs are transferred to lymph as free LCBs and ceramides [18–21]. These findings suggest that liberated LCBs are incorporated into small intestinal epithelial cells, reconstructed to sphingolipids from incorporated LCBs in the form of ceramide, and then absorbed into lymph in rats [18, 22, 23]. Since the structures of LCBs in plants, fungi, and marine organisms are different from those in mammals [1], the absorption rates of different LCBs into lymph are likely to differ depending on their structure.

Thus, to elucidate the bioactivities and mechanisms of each dietary sphingolipid, it is important to clarify the relationship between their individual biokinetics and structure. Using conventional MS analysis methods, one can analyze the LCB backbones of ceramides and GlcCer [24]. In addition, a novel analytical method for LCB backbone identification of SM was developed. To explore the relationship between LCB structures and absorption rate, as well as their metabolites, the amount of LCBs and their metabolites in lymph from rats administered structurally different pure LCBs were analyzed.

General reagents

N-heptadecanoyl-d-erythro-sphingosine (C17 ceramide; d18:1-C17:0), N-heptadecanoyl-d-erythro-sphingosylphosphorylcholine (C17 sphingomyelin; d18:1-C17:0), d-glucosyl-1,1’-N-lauroyl-d-erythro-sphingosine (C12 glucosylceramide; d18:1-C12:0), and 2-amino-4-trans-heptadecene-1,3-diol (d17:1) were obtained from Avanti Polar Lipids (Alabaster, Alabama, USA). Triolein and sodium taurocholate were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Caco-2 cells were obtained from RIKEN BioResource Center. FBS was purchased from Life Technologies, Inc. (Gaithersburg, Maryland, USA). DMEM, non-essential amino acids solution (NEAA), penicillin-streptomycin solution (P/S), trypsin-EDTA solution, and b-glucosidase from almonds were from Merck KGaA (Darmstadt, Germany). Falcon six-well cell culture insert plates (high pore density, pore size 0.4 mm) and 10 cm cell culture dishes were purchased from Corning (New York, USA).

Extraction and isolation of LCBs

GlcCer and CAEP were extracted from the konjac tuber (Amorphophallus konjac), Tamogi mushroom (Pleurotus cornucopiae var. citrinopileatus), and giant scallop (Mizuhopecten yessoensis), and fractionated by silica gel column chromatography as described previously [6,20]. Purified GlcCer and CAEP were hydrolyzed in 10% Ba(OH)₂/dioxane (1:1, v/v) at 110°C for 24 h [25]. In the case of GlcCer hydrolysis, the resulting lyso-GlcCer products were extracted from the reaction mixture using ethyl acetate and treated with b-glucosidase to liberate LCBs [26]. Liberated LCBs were fractionated on a silica gel and purified using an Inertsil ODS-3 column (diameter, 20 mm; length, 250 mm; particle size, 5 mm; GL Science, Tokyo, Japan) attached to a HPLC instrument, resulting in d18:2^4t8c, d18:2^4t8t, 9Me-d18:2, d18:3, and 9Me-d18:3. LCB purity was measured by HPLC after derivatization with ortho-phthalaldehyde (OPA) [27]. In brief, methanol solutions of LCBs (50 mL) were mixed with OPA solution (250 mL) and incubated for 15 min at room temperature. The OPA solution was consisted of OPA (5 mg), 2-mercaptoethanol (5 mL), ethanol (100 mL), and 100 mM boric acid-KOH buffer of pH 10.5 (10 mL). The generated OPA-derivatized LCBs were analyzed by HPLC with an ODS-3 column (diameter, 4.6 mm; length, 75 mm; particle size, 3 mm; GL Science). A mixture of acetonitrile and water was used as the eluent, and OPA-derivatized LCBs were eluted at 0.5 mL/min over 50 min across the following gradient: 0-5 min, 55% acetonitrile; 5-15 min, 55-80% acetonitrile; 15-40 min, 80-100% acetonitrile; 40-42 min, 100% acetonitrile, 42-45 min, 100-55% acetonitrile; 45-50 min, 55% acetonitrile. Eluted OPA-LCBs were detected with a fluorescence detector set at excitation and emission wavelengths of 340 and 455 nm, respectively.

Lymph collection from rats administrated LCBs

Male Wistar/ST rats (Japan SLC, Inc., Shizuoka, Japan) aged 9 weeks were housed in individual stainless-steel cages and given a semipurified diet (AIN 93G formula) for a 5 day acclimation period. After overnight fasting, a vinyl catheter and a silicone catheter were implanted into the thoracic lymph duct and the duodenum, respectively, as described previously [28]. Test lipids (100 g/L) prepared in 1 mL of emulsified solution contained 100 mg of triolein (control group) or 90 mg of triolein plus 10 mg of each LCB (LCB-administered groups). Test lipids were emulsified with 10 mg of sodium taurocholate using a sonicator. After lymph collection for 30 min (initial lymph) on day 1 postoperation, the rats were infused with 1 mL of an emulsified test solution for 1 min, and the infusion of glucose-NaCl solution through the duodenal tube was continued at 1.8 mL/h until the end of the experiment. Lymph was collected in a test tube at 1 h intervals for 8 h following the administration of the test solution. The collected lymph was immediately frozen and stored at −80°C until subsequent analyses. Animal experiments were conducted in accordance with the guidelines of the Animal Committee of Iwate University (Authorization No. A201450).

Lipid extraction

Lipids were extracted from recovered lymph samples as described previously [6] with slight modification. The collected lymph samples (0.5 mL) were transferred to glass-capped test tubes and mixed with chloroform (1 mL), methanol (2 mL), PBS (0.3 mL), and 1 nmol of internal standards (C17 ceramide, C12 glucosylceramide, d17:1, C17 sphingomyelin). After incubation at 37°C for 2 h, chloroform (1 mL) and water (1 mL) were added and the mixture was centrifuged at 1000´g for 5 min, and lower organic phases were collected into fresh test tubes. Chloroform (1 mL) was added to the upper phase and residual lipids were re-extracted. After centrifugation, the resulting lower organic phases were combined and dried with nitrogen gas. 0.4 M NaOH-methanol solution (1 mL) was added to the extracted lipids and incubated at 37°C for 2 h to saponify glycerolipids. Chloroform (1 mL) and water (1 mL) were added, and lipids were collected from lower organic phases and evaporated with nitrogen gas. Dried residues were treated with cold acetone (1 mL) and centrifuged at 1000´g at 4°C for 5 min. Supernatants were discarded, and the pellet was analyzed by LC-MS/MS.

Cultures of Caco-2 cells and LCB treatment

Caco-2 cells were cultured in DMEM supplemented with 10% FBS, NEAA, and P/S (100 U/mL penicillin, 0.1 mg/mL streptomycin) at 37°C in a humidified 5% CO₂ incubator. Caco-2 cells were seeded into six-well cell culture inserts at a density of 1.5×10⁵ cells/insert. To generate differentiated Caco-2 cells for use as a small intestinal epithelial cell model, the Caco-2 cells were cultured for another 21 days after reaching confluency. The medium was changed with a fresh one every other day. For LCB treatment, 10 mM d18:2^4t8c or d18:2^4t8t dissolved in serum-free DMEM was added to the cell culture insert and incubated for 24 h. The final DMSO concentration was adjusted to 0.1%. The basal-side medium and cells were collected, and lipids were extracted as described above.

LC-MS/MS analysis of lymph lipids

Lymph lipids were analyzed with a TripleTOF 5600 LC-MS/MS system (AB SCIEX, Foster City, California, USA) equipped with an InertSustain NH2 column (diameter, 2.1 mm; length, 100 mm; particle size, 5 mm; GL Science) in the ESI positive mode as described previously with slight modification [6,24]. Sphingolipids extracted from lymph were dissolved in 200 mL of mobile phase A, and a 10 mL sample solution was used for sphingolipid analysis. Mobile phase A was acetonitrile:methanol:formic acid:1 M ammonium formate (95:5:0.2:5, v:v:v:v), and mobile phase B was methanol:formic acid: 1 M ammonium formate (100:0.2:5, v:v:v). The gradient method was used for analysis as follows: 0-5 min, 0% B; 5-10 min, 0-20% B; 10-12 min, 20% B; 12–15 min, 20-50% B; 15-22 min, 50% B; 22-27 min, 50-80% B; 27-30 min, 80% B; 30-45 min, 80-0% B. The flow rate of the mobile phase was set at 0.13 mL/min. LCB species were identified on the basis of their retention times and high resolution m/z of protonated ions; [M+H]⁺ and other sphingolipid species were identified in the MRM mode using characteristic product ions (d18:1-bound ceramide and GlcCer for m/z 264.27; d18:2-bound ceramide and GlcCer for m/z 262.25; d18:3-bound ceramide and GlcCer for m/z 260.24; 9Me-d18:2-bound ceramide and GlcCer for m/z 276.27; 9Me-d18:3-bound ceramide and GlcCer for m/z 274.25; SM for m/z 184.07). MS conditions were as follows: ion spray voltage floating, 5500 V; temperature, 300°C; declustering potential, 80 V; collision energy, 10 V; ion source gas 1, 20 psi; ion source gas 2, 50 psi; curtain gas, 20 psi; TOF accumulation time, 0.2 s; product accumulation time, 0.05 s. To generate product ions, the collision energy and collision energy spread were set at 35 and 5 V, respectively. Data acquisition and analysis were respectively performed using Analyst TF 1.7.1 software and MultiQuant 3.0.1 software (AB SCIEX). The amounts of target analytes were calculated using individual internal standards.

SM analysis by in-source collision-induced dissociation/multiple reaction monitoring (CID/MRM)

To identify the LCB backbones of SM, the parameters for MS were as follows: negative ion mode for TOF scans; ion spray voltage floating, -4500 V; temperature, 300°C; declustering potential, -300 V; collision energy, -10 V; ion source gas 1, 20 psi; ion source gas 2, 50 psi; curtain gas, 20 psi; accumulation time, 0.2 s. The detected in-source fragmented [M-CH₃]^- SM ions serving as precursor ions and [M-CH₃-fatty acid]^- ions providing LCB backbone information were generated by collision-induced dissociation (CID). The collision energy and collision energy spread were set at -50 and 5 V, respectively. LC conditions were the same as those described above for the positive ion mode. LCB species for SM were identified from their typical product ions (d18:1-bound SM for m/z 449.31; d18:2-bound SM for m/z 447.30; d18:3-bound SM for m/z 445.28; 9Me-d18:2-bound SM for m/z 459.30; 9Me-d18:3-bound SM for m/z 457.28).

Statistical analysis

Statistical significance was determined using Tukey's method and p < 0.05 was considered to indicate significant difference. Data are presented as mean ± standard deviation.

Isolation and purity determination of LCBs

GlcCer and CAEP were purified from konjac, Tamogi mushroom, and scallop to prepare LCBs for rat lymph cannulation experiments. The obtained GlcCer and CAEP were hydrolyzed in Ba(OH)₂ aqueous solution/dioxane, during which GlcCers were hydrolyzed to LCBs, but most GlcCers were hydrolyzed to lyso-GlcCer, and which was subsequently treated with b-glucosidase. By contrast, CAEP was completely hydrolyzed to LCB in Ba(OH)₂ aqueous solution/dioxane. Liberated LCBs were finally purified by ODS HPLC, and d18:2^4t8c, d18:2^4t8t, d18:3, 9Me-d18:2, and 9Me-d18:3 were successfully isolated. Purified LCBs were analyzed by ESI-MS, and their associated ions were observed at m/z 298.3 [M+H]⁺ (d18:2^4t8c), 298.3 [M+H]⁺ (d18:2^4t8t), 296.3 [M+H]⁺ (d18:3), 312.4 [M+H]⁺ (9Me-d18:2), and 310.4 [M+H]⁺ (9Me-d18:3) (Fig. S1A-D). Isolated LCBs were derivatized with OPA and analyzed by HPLC with a fluorescence detector. The purities were >99% for d18:2^4t8c, 95% for d18:2^4t8t, >99% for d18:3, >99% for 9Me-d18:2, and >99% for 9Me-d18:3.

Recovery of free LCBs from lymph

To investigate the absorption of LCBs from the digestive tract to lymph, emulsions with 10 mg of purified LCBs were enterally administrated to a thoracic-duct-cannulated rat. Lymph was collected every hour up to 8 h after LCB administration. There were no significant differences in the amount of lymph output among rats administrated each LCB (Fig. S2), indicating that surgery and animal maintenance were appropriately carried out. First, free LCBs extracted from the collected lymph were analyzed by LC/MS/MS. HPLC retention times and exact masses of protonated ion signals ([M+H]⁺m/z 298.27, 298.27, 296.26, 312.29, and 310.27 for d18:2^4t8c, d18:2^4t8t, d18:3, 9Me-d18:2, and 9Me-d18:3, respectively) were used for the identification of each LCB species. Peak areas were integrated at m/z ± 0.05, and LCB amounts were determined by comparing [M+H]⁺ ion signals of d18:2^4t8c, d18:2^4t8t, d18:3, 9Me-d18:2, and 9Me-d18:3 with the peak area of the d17:1 internal standard. The amounts of free LCBs in lymph at each timepoint are shown in Fig. 2. The amount of all LCBs, d18:2^4t8c, d18:2^4t8t, d18:3, 9Me-d18:2, and 9Me-d18:3, were elevated in the lymph of rats infused with the corresponding LCBs. The amount of d18:2^4t8c was highest at 3 h after administration (0.83 nmol). On the other hand, the amount of d18:2^4t8t, a geometrical isomer of d18:2^4t8c, was highest at 1 h after administration (4.38 nmol). The levels of both d18:2^4t8c and d18:2^4t8t at 8 h decreased to levels similar to those before administration. The total amount of d18:2^4t8c up to 8 h after LCB administration was ~4.4-fold higher than that of d18:2^4t8t. In the case of other LCBs, the amount of d18:3 in lymph increased after 1-3 h, 9Me-d18:2 increased after 2-4 h, and 9Me-d18:3 peaked at 4 h after administration. This result suggests that the absorption rates of free LCBs into lymph differ with their structure, even among geometrical isomers. Herein, the absorption rates of d18:2^4t8c, d18:2^4t8t, d18:3, 9Me-d18:2, and 9Me-d18:3 into lymph as free LCBs were 0.011% ± 0.006%, 0.048% ± 0.016%, 0.043% ± 0.016%, 0.004% ± 0.000%, and 0.014% ± 0.013%, respectively, when integrated up to 8 h after administration (Fig. 2).

Quantification of reconstituted ceramide and GlcCer in lymph

Since the LCBs absorbed from the digestive tract were predicted to be metabolized to ceramide and complex sphingolipids in intestinal epithelial cells, these LCB metabolites in lymph were also analyzed. As expected, a portion of each administrated LCB was processed into ceramide (Fig. 3A-D), and more than 80% was bound to the fatty acid C16:0, followed by C24:0 and C23:0 (Fig. S3), and the bound fatty acid species did not depend on the structure of the administrated LCB (Fig. S3). Similar to the results of free LCB absorption, the amount of d18:2^4t8t ceramide in lymph peaked at 1 h after administration and then gradually decreased to only trace amounts at 8 h (Fig. 3A). The rates of d18:2-bound ceramide absorption into the lymph of groups administrated d18:2^4t8c and d18:2^4t8t were compared. As in the case of free LCBs, ceramides containing d18:2^4t8c were absorbed more slowly and to a lesser extent than d18:2^4t8t ceramides (Fig. 3A). In the case of other LCBs, the amount of administrated LCB-bound ceramides in the lymph increased at 2-4 h after administration of d18:3, at 2-6 h after administration of 9Me-d18:2, and at 3-7 h after administration of 9Me-d18:3 (Fig. 3B-D). The absorption of 9Me-d18:3-bound ceramides were the slowest among the LCBs tested in this study, and the maximum amount of ceramides detected in lymph recovered at 6 h after LCB administration (Fig. 3D). The total absorption rates of d18:2^4t8c, d18:2^4t8t, d18:3, 9Me-d18:2, and 9Me-d18:3 into lymph as ceramide were 0.095% ± 0.008%, 0.143% ± 0.023%, 0.030% ± 0.008%, 0.054% ± 0.019%, and 0.088% ± 0.023%, respectively (Fig. 6).

GlcCers metabolized from the administrated LCB in the collected lymph were identified. Interestingly, d18:2^4t8t and 9Me- d18:2^4t8t were the only LCBs detected in lymph that were converted to GlcCer (Fig. 4A, C), which was unlike in the case of free LCBs or ceramide. GlcCer of other LCBs was present only at trace levels (Fig. 4B, D). Only C16:0-bound GlcCers of d18:2^4t8t and 9Me- d18:2^4t8t were detected, and no other fatty acids such as C24:0 and C23:0 were detected in lymph. Changes in the amount of GlcCer in lymph showed a similar trend. The amount of reconstructed GlcCer in lymph was highest at 3 h after administration of d18:2^4t8t, and at 4 h after administration of 9Me-d18:2^4t8t (Fig. 4A, C). Both d18:2^4t8t and 9Me-d18:2^4t8t bound to GlcCer were decreased to trace levels at 6 h after administration (Fig. 4A, C). In addition, the absorption rates of GlcCers as the LCB were lower than that of ceramides (Fig. 3A-D, 4A, C). The biosynthesis rate of GlcCer is estimated to be lower than the ceramide synthesis rate because it requires an additional glycosylation step. Additionally, it is possible that the transport of reconstituted GlcCer from the small intestine to lymph may be slower than that of ceramide. The absorption rates of d18:2^4t8t and 9Me-d18:2 into lymph as GlcCer were 0.011% ± 0.004% and 0.017% ± 0.005%, respectively (Fig. 6).

Analysis of the LCB backbones of sphingomyelin by in-source CID/MRM in ESI-negative mode

To identify and quantify nonmammalian-type LCB-bound SMs, lipids extracted from lymph from rats administrated LCBs were analyzed. In the ESI-positive mode, fragmentation of protonated ions ([M+H]⁺) of SM yielded only one typical product ion (m/z 184.07) derived from phosphocholine; hence, information for LCBs was not obtained using this mode (Fig. S4A). Therefore, the in-source CID/MRM method was established, a combination of in-source CID and postsource CID, in the ESI-negative mode to identify the LCB backbones of SMs. In this analysis, methyl groups 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 postsource CID of [M-CH₃]^- ions produced the product ion [M-CH₃-fatty acid]^-, and these pairs were selected for the MRM mode (Fig. S4C, D).

By this in-source CID/MRM method in the ESI-negative mode, the levels of SM in the lymph of rats administrated LCBs were measured (Fig. 5). Metabolized SMs from administrated LCBs were detected, especially in the group administered d18:2^4t8c (Fig. 5A). The amount of d18:2^4t8c-bound SMs in lymph increased at 4-7 h after administration of d18:2^4t8c. C16:0 was the most common fatty acid in d18:2^4t8c-bound SM as well as d18:2^4t8c-bound ceramides and GlcCers (Fig. 3A, 4A, 5A). By comparing the detected d18:2^4t8c-bound SMs, as in the case of GlcCer, the absorption of SM into lymph occurred later than ceramide (Fig. 3A, 5A). In the case of groups administered d18:2^4t8t, d18:3, 9Me-d18:2, and 9Me-d18:3, the amount of SM detected in lymph was much lower than that in the d18:2^4t8c-administrated group (Fig. 5B-D). The amounts of d18:2^4t8c, d18:2^4t8t, d18:3, 9Me-d18:2, and 9Me-d18:3 adsorbed into lymph as SM were 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 (Fig. 6).

Uptake and transport of d18:2 geometrical isomers in differentiated Caco-2 cells

In rat lymph, the levels of reconstituted GlcCers and SMs clearly varied with the geometric isomerism of the 8—position of d18:2 (Fig. 4A, 5A). Thus, the absorption behaviors of d18:2^4t8c and d18:2^4t8t were analyzed using differentiated Caco-2 cells, an intestinal epithelial transport system model. LCBs (10 mM) were added to the apical side of Caco-2 cells, which were cultured on a cell culture insert for 21 days and differentiated into intestinal epithelium-like cells, and incubated for 24 h. Lipids were extracted from the medium on the basolateral side and from cells. Free LCBs, reconstituted ceramide, GlcCer, and SM were analyzed by LC/MS/MS (Fig. 7A-D). There were no significant differences in the amounts of free d18:2- and d18:2-bound ceramides in the medium on the basolateral side between treatments with d18:2^4t8c and d18:2^4t8t (Fig. 7A, B). Levels of d18:2-GlcCer were higher in d18:2^4t8t-treated cells, and the amounts of d18:2-SMs in d18:2^4t8c-treated cells were larger than that in d18:2^4t8t-treated cells, which consistent with the rat lymph results. Quantitative analysis revealed that the amount of intracellular free d18:2 in the d18:2^4t8c-treated cells were significantly higher than that in the control cells, but there was no significant difference between the d18:2^4t8t-treated cells and the control cells (Fig. 7E). The amount of intracellular d18:2-bound ceramides significantly increased only in the d18:2^4t8t-treated cells compared with that in the control cells (Fig. 7F), but there was no significant difference between the d18:2^4t8c-treated cells and the control cells. The amount of intracellular d18:2-bound GlcCers significantly increased in the d18:2^4t8t-treated cells compared with that n the control cells, but there was no change in the amount of d18:2-bound GlcCers in the d18:2^4t8c-treated cells compared with that in the control cells (Fig. 7G). These results followed a similar trend to those of the experiment on LCB absorption in rat lymph. However, unlike in the rat experiment, the amount of intracellular d18:2-bound SMs of both d18:2^4t8c- and d18:2^4t8t-treated cells significantly increased compared with that in the control cells, and there were no significant differences in the amount of intracellular d18:2-bound SMs between d18:2^4t8c- and d18:2^4t8t-treated cells (Fig. 7H).

To explore the mechanism underlying the biological effects of dietary sphingolipids, in this study, the absorption and metabolism of nonmammalian-type LCBs from the digestive tract to lymph in rats were evaluated. It has been reported that ~ 0.3% of nonmammalian-type sphingolipids are absorbed into the lymph in rats as free LCBs or ceramides, and the amounts of free LCBs in lymph are ~ 20 times smaller than those of other sphingolipid classes such as ceramide [19, 21]. In addition, the rate of absorption of sphingosine, a major mammalian LCB, into the lymph is estimated to be three times higher than that of d18:2, a plant-derived LCB [19]. In this study, to exclude the efficiency of the degradation of sphingolipids in the gastrointestinal tract, the absorption of purified free LCBs in rats cannulated with thoracic lymph was examined, and free LCBs and reconstituted sphingolipids in lymph were analyzed by LC/MS/MS. The amount of free d18:2^4t8t in lymph, which has a double bond at C8–C9 in this trans form of d18:2, clearly increased at 1 h after oral administration of d18:2^4t8t, and the level was higher than that for d18:2^4t8c, which has a double bond at the 8-position in this cis form (Fig. 2A). On the other hand, d18:3, 9Me-d18:2, and 9Me-d18:3, which have the same C8–C9 double bond as d18:2^4t8t in the trans form, appear to be absorbed more slowly in the lymph than d18:2^4t8t (Fig. 2). These findings suggest that the absorption rate of LCBs varied depending on the geometrical isomerism of C–C double bonds and the presence of the C9 methyl group. The uptake rate of LCBs into the epithelial cells may depend on the structure of the LCBs.

Previous studies showed that about 0.2−0.3% of GlcCers from maize, whose major LCB is d18:2^4t8c, was absorbed into the lymph of rats [19]. The present analysis showed that the rate of absorption of d18:2^4t8c from the gastrointestinal tract into lymph was ~ 1.2% (Fig. 6). Previous studies showed that it is also necessary to consider the efficiency of degradation by sphingolipid digestive enzymes in the intestine [18, 19]. The absorption rate calculated in the present work was higher than that in a previous report because purified LCBs were used to the experiments [19].

Orally administrated sphingosine is converted to ceramide and incorporated into lymph [22]. Similarly to sphingosine, d18:2, d18:3, 9Me-d18:2, and 9Me-d18:3 are believed to be mainly converted into ceramide after absorption from the gastrointestinal tract and then transferred to the lymph. d18:2-, d18:3-, 9Me-d18:2-, and 9Me-d18:3-bound ceramides were also analyzed by LC/MS/MS. As expected, all of the LCBs in this study were detected as ceramides in the rat lymph (Fig. 3). The relationship between the absorption rate and the LCB structure of ceramides displayed the same tendency as that of free LCBs. Since the absorption rates of free LCBs and ceramides were similar among LCBs, it is speculated that the conversion of incorporated LCBs to ceramides occurs rapidly, and all LCBs are likely to be substrates of ceramide synthase (CerS). In each LCB-administrated group, C16:0-bound ceramides were the predominant species in lymph, which are nonmammalian-type LCBs (Fig. S3). In addition, most GlcCer and SM molecular species in lymph were also C16:0. These findings suggest that following administration, LCBs were metabolized to ceramides, and these re-synthesized ceramides were used for GlcCer and SM synthesis in intestinal cells. CerS has six isozymes with different substrate specificities; CerS6 has high substrate specificity for C16:0-CoA and is the major CerS in the mouse intestine [29], and it is also believed to be the major CerS family member in the rat small intestine.

When SM was analyzed in the ESI positive mode, only one fragmentation product ion (m/z 184.07) derived from a phosphocholine moiety was detected (Fig. S4A). SM compounds with the same molecular weight, such as d18:1/C24:1 and d18:2/C24:0, were not separated by normal phase HPLC. To determine the LCB backbones of SMs, an ion trap mass spectrometer (IT-MS) in the ESI negative mode was employed [30]. Alternatively, the product ion of ceramide generated from SM by in-source fragmentation can be decomposed by post fragmentation using an atmospheric pressure chemical ionization (APCI) probe to obtain information on LCBs [31]. Addition of alkali metals to the HPLC mobile phase can also yield product ions derived from LCBs by CID of alkali metal adduct ions of SM (Fig. S4B) [32]. To analyze the LCB backbone of SM using a conventional ESI-LC/MS/MS system, a method using a combination of in-source fragmentation and post-source fragmentation in the ESI negative mode was established. Since [SM-CH₃]⁻ ions can be observed by IT-MS [30], these ions were selected as precursor ions. The CID fragment ion of [SM-CH₃]⁻ was [SM-CH₃-fatty acid]⁻, which provides information on the LCB structure of SM. This method (in-source CID/MRM) is useful for quantifying the molecular species of SM; hence, the SM species in lymph and cultured cells were analyzed by in-source CID/MRM (Fig. 5, 7, S5). Comparison of the absorption behavior of LCB metabolites revealed that reconstituted GlcCer and SM were absorbed slower than ceramide (Fig. 3−5). Ceramide is de novo synthesized in the endoplasmic reticulum (ER) and transported to the Golgi by the ceramide transport protein (CERT), and transported ceramide is further metabolized to SM in the Golgi by sphingomyelin synthase (SMS) [33]. GlcCer synthesis requires the transfer of glucose from UDP-glucose to ceramide [34]. Therefore, it was suggested that differences in the absorption time between GlcCer/SM and free LCB/ceramide into lymph could be caused by enzymatic reactions. However, it is also possible that differences in absorption time are due to different mechanisms of transport to lymph.

Since the amount of LCBs absorbed depends on their structure, especially the geometric isomerism of C8-C9, the absorption mechanisms of LCBs were also investigated using Caco-2 cells. In the case of GlcCer, d18:2^4t8t-treated Caco-2 cells released more d18:2-GlcCer than d18:2^4t8c-treated Caco-2 cells (Fig. 7C). Compared with the amount of d18:2-GlcCer in Caco-2 cells, the amount in d18:2^4t8t-treated cells was 1.37-fold higher than that in d18:2^4t8c-treated cells (Fig. 7G). Since d18:2^4t8t is more easily metabolized to d18:2-GlcCer than d18:2^4t8c, it is presumed that d18:2-GlcCer accumulated more readily in Caco-2 cells when d18:2^4t8t was added than when d18:2^4t8c was added. In the case of d18:2^4t8t treatment, which resulted in significantly higher accumulation of GlcCer in Caco-2 cells, a larger amount of GlcCer was released into the medium than in the case of d18:2^4t8c treatment. On the other hand, d18:2-SM levels were significantly higher only when d18:2^4t8c was added, than in the control group (Fig. 7D). Although the amount of d18:2-SM in Caco-2 cells was similar for cells treated with both d18:2^4t8c and d18:2^4t8t (Fig. 7H), the amount of d18:2-SMs released into the medium were greater for the d18:2^4t8c-treated group than for the d18:2^4t8t-treated group (Fig. 7D). There was no significant difference in the amount of d18:2-SM between the d18:2^4t8t-treated group and the control group (Fig. 7D). Therefore, it is assumed that d18:2^4t8c-SMs were more easily transferred to the medium than d18:2^4t8t-SMs. The migration of SMs from cells to the basolateral medium may be related to the structure of the LCB portion of SM. The amounts of intracellular d18:2-GlcCer and d18:2-SM were increased by d18:2^4t8c or d18:2^4t8t treatment, which indicated that the level of d18:2-SM was over 100 times greater than that of d18:2-GlcCer (Fig. 7G, H). The migration behaviors of d18:2-GlcCer and d18:2-SM from Caco-2 cells into the medium were similar to the incorporation behaviors of d18:2-GlcCer and d18:2-SM into the lymph (Fig. 4A, 5A). In rat lymphatic cannulation experiments, although d18:2 was more easily metabolized to d18:2-GlcCer in the 8-trans form than in the 8-cis form, d18:2 was predominantly metabolized to d18:2-SM. Additionally, d18:2-SM with the 8-cis form of d18:2 more easily migrated to the lymph than d18:2-SM with the 8-trans form of d18:2, resulting in a greater amount of d18:2^4t8c being absorbed into the lymph fluid than d18:2^4t8t. The double bond at C8–C9 of d18:2^4t8t is a trans form, which makes this LCB molecule linear, whereas the double bond at C8–C9 of d18:2^4t8c is a cis form, which gives this molecule a bent form. Therefore, it can be concluded that the geometric isomer form of LCBs is a critical factor for their absorption and metabolism.

This is the first study demonstrating the absorption of purified nonmammalian-type LCBs and the metabolism of d18:2^4t8c, d18:2^4t8t, d18:3, 9Me-d18:2, and 9Me-d18:3 to ceramide, GlcCer, and SM in rats. Interestingly, the amounts of metabolized sphingolipids clearly varied depending on their structure. In particular, geometric isomers at C8–C9 of d18:2, which are abundant in higher plants, affect the metabolism and absorption od LCBs. The total absorption rate of d18:2^4t8c was higher than for any other LCBs, including the geometrical isomer of d18:2^4t8t (Fig. 6). Approximately 90% of the d18:2^4t8c metabolite absorbed into the lymph was SM (Fig. 6). Dietary sphingolipids show several beneficial functions such as skin barrier improvement. The presented findings provide information that will contribute to the development of effective sphingolipid-containing functional foods.

CID, Collision-induced dissociation

GlcCer, Glucosylceramide

CAEP, Ceramide 2-aminoethylphosphonate

MRM, Multiple reaction monitoring

Long-chain base (LCB)

glucosylceramide (GlcCer)

ceramide 2-amynoethylphosphonate (CAEP)

non-essential amino acids solution (NEAA)

penicillin-streptomycin solution (P/S)

ortho-phthalaldehyde (OPA)

ceramide synthase (CerS)

ion trap mass spectrometer (IT-MS)

atmospheric pressure chemical ionization (APCI)

trans-Golgi network (TGN)

ceramide transport protein (CERT)

Ethics approval and consent to participate

Animal experiment was approved by the Animal Committee of Iwate University.

Consent for publication

Not applicable.

Availability of data and materials

The dataset supporting the conclusions of this article is available upon request for corresponding author after publication.

Competing interests

The authors declare no competing interests.

Funding

This work was supported by funding from the Advanced Research and Development Programs for Medical Innovation (AMED-CREST) (to S.S. and K.Y.) from the Japan Agency for Medical Research and Development (AMED) and by Industry Creation Departments Funding (8607011, PCS8617001) provided by Daicel Corporation to the Lipid Biofunction Section of Hokkaido University.

Authors contributions

DM prepare the lipids and carried out LC/MS/MS analysis, and wrote manuscript. SS conceive the study. MN carried out lymph collection. KY work with SS on edition of manuscript. KM participated in the design of the study. YI supervised study and edited manuscript. All authors have given approval to the final version of the manuscript.

Acknowledgments

We thank our laboratory staff for fruitful discussion and clerical support.

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Structure-dependent absorption of sphingolipid long-chain bases from the digestive tract into lymph

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