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,2
* Division of Food Science, Incorporated Administrative Agency, National Institute of Health and Nutrition, 1–23-1 Toyama, Shinjuku-ku, Tokyo 162-8636, Japan;
Japan Society for the Promotion of Science, Tokyo 102-8471, Japan; and
** Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan
2To whom correspondence should be addressed. E-mail: sugawara{at}nih.go.jp.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • Caco-2 cells • ceramide • cerebroside • sphingadienine • sphingolipids
Sphingolipids are a family of compounds that have a sphingoid base (long-chain base) with an amide-linked fatty acid and a polar head group such as phosphorylcholine (for sphingomyelin) or carbohydrates (for cerebrosides, gangliosides and other complex glycolipids), except in the case of free ceramide. The most common sphingoid base of mammalian sphingolipids is sphingosine (trans-4-sphingenine, d18:14t). Smaller amounts of others, such as sphinganine (dihydrosphingosine, d18:0) and phytosphingosine (4-hydroxysphinganine, t18:0) are frequently present. In higher plants, the structures of the sphingoid bases of sphingolipids are more complicated than in mammals (1,2) (Fig. 1) because the sphingoid bases can be desaturated at the C8-position by a stereo-unselective 
8-cis/trans-sphingolipid desaturase, yielding cis- and trans- isomers of 8-unsaturated sphingoid bases (3,4).
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Although the daily intake by humans of sphingolipids from plant sources has been estimated to be as little as 50 mg (18), it is not known whether sphingolipids of plant origin composed of sphingoid bases distinctive from those of mammals have any effects on human health. We previously found that sphingoid bases prepared from wheat flour consisted mainly of 8-sphingenine and induced apoptosis in DLD-1 colon cancer cells (19). Thus, dietary sphingolipids from plant sources seem to influence human health. However, the fate of dietary sphingolipids of plant origin is still not well understood.
We investigated the digestion in rats of dietary cerebroside prepared from maize, which is one of major dietary sources of plant sphingolipids and compared the uptake and metabolism of the isomers trans-4, cis-8 (4t, 8c) and trans-4, trans-8 (4t, 8c) sphingadienine prepared from maize cerebroside with those of sphingosine in differentiated cultures of Caco-2 cells derived from human colonic carcinoma as a model of the intestinal epithelium (20–23).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Essentially fatty acid–free bovine serum albumin (BSA), glucocerebrosides from human Gaucher’s spleen, N,N-dimethylsphingosine, sodium taurocholate, sodium taurodeoxycholate, sphinganine, sphingosine and verapamil were purchased from Sigma Chemical (St. Louis, MO). Ceramide from bovine brain tissue and o-phthalaldehyde (OPA) were purchased from Wako Pure Chemical Industries (Osaka, Japan). MK-571 was purchased from Cayman Chemical (Ann Arbor, MI). The maize cerebroside-rich preparation was kindly donated by Nippon Flour Mills (Atsugi, Japan). Other chemicals and solvents were of reagent grade.
Preparation of cerebroside, ceramide and sphingoid bases.
The maize cerebroside-rich preparation contained 480 g/kg cerebroside; the other compounds were mainly steryl glucoside and pigments. To isolate cerebroside, this preparation was dissolved in chloroform and applied to a silica gel column, as described previously (24,25). The composition of the sphingoid bases of the maize cerebroside recovered was determined by oxidation with sodium periodate and subsequent GLC of the resultant fatty aldehydes (26,27). Constituent sphingoid bases were 3.4% d18:14t, 53.5% 4t, 8c-sphingadienine (d18:24t,8c), 17.2% 4t, 8t-sphingadienine (d18:24t,8t), 1.6% t18:0, 21.9% 4-hydroxy-cis-8-sphingenine (t18:18c), 1.7% 4-hydroxy-trans-8-sphingenine (t18:18t) and others. The purity of the cerebroside prepared was 96% by HPLC equipped with an evaporative light-scattering detector, as described previously (18). Ceramide was prepared from maize cerebroside by the method of Carter et al. (28).
To isolate each isomer of 4t, 8c and 4t, 8t-sphingadienine, the maize cerebroside preparation was subjected to strong alkaline hydrolysis [100 g/L aqueous Ba(OH)2/dioxane, 1:1 (v/v), 24 h at 110°C] (29). The liberated sphingoid bases were then extracted with diethyl ether. The mixture of free sphingoid bases was purified by reverse-phase HPLC. The HPLC system consisted of an LC-10AD pump (Shimadzu, Kyoto, Japan) and an SPD-10A UV-VIS absorbance detector (Shimadzu). An ODS column (TSKgel-ODS 80Ts, 250 x 4.6 mm, Tosoh, Tokyo, Japan) was eluted with a methanol/5 mmol/L potassium phosphate buffer, pH 7.5 (82:18, v/v) at a flow rate of 0.7 mL/min. The eluent was monitored at 210 nm. The purities of 4t, 8c- and 4t, 8t-sphingadienine prepared by HPLC were 99.8 and 96.0%, respectively, by GLC as described above.
Assays of cerebrosidase and ceramidase activity in vitro.
The experimental procedures used in this study met the guidelines of the Animal Committee of the Incorporated Administrative Agency, National Institute of Health and Nutrition (Tokyo, Japan). Male Sprague-Dawley rats, 6 wk old, were obtained from Japan SLC (Shizuoka, Japan) and housed at 22 ± 1°C with a 12-h light:dark cycle and free access to a commercial diet (CE-2, Clea Japan, Tokyo) and tap water for 1 wk. The small intestine and cecum were then excised from rats under ether anesthesia. The entire small intestine between the pylorus and the cecum was removed and rinsed in cold saline. The intestinal contents were flushed with cold saline, and then the small intestine was slit open. The mucosa was scraped off with glass slides and homogenized with a Teflon-glass homogenizer in 9 volumes of ice-cold 10 mmol/L sodium phosphate buffer, pH 7.2, containing 50 mmol/L sodium chloride. The homogenate was centrifuged at 10,000 x g for 30 min at 4°C, and the supernatant was discarded. The pellet, resuspended in the same buffer, was used as a crude enzyme source. The cecal contents were washed out and collected in 20 mL cold saline, mixed thoroughly with a vortex mixer and centrifuged at 1000 x g for 20 min (30). The supernatants were used for the assay. Aliquots of mucosal homogenate and cecal content supernatant were assayed for protein using the method of Lowry et al. (31).
The system used to assay cerebrosidase activity was based on the method of Kobayashi and Suzuki (32,33). Briefly, the reaction mixture contained 100 µg of cerebroside, 2 mg of sodium taurodeoxycholate in 0.1 mL of 0.1/0.2 mol/L sodium citrate/phosphate buffer, pH5.2, and 0.1 mL of the enzyme source. After incubation of the mixture for 2 h at 37°C, the glucose released enzymatically was determined with a commercially available kit (Glucose CII test kit, Wako Pure Chemical Industries). To avoid further hydrolysis during the glucose assay reactions, the reaction mixtures were heated with boiling water for 5 min before analysis of the glucose released.
Ceramidase activity was determined by the procedure of Duan et al. (34), with slight modification. Briefly, the reaction mixture contained 100 µg of ceramide as substrate, 2 mg of sodium taurocholate in 0.1 mL of 0.1 mol/L sodium phosphate buffer, pH 7.0, and the enzyme source solution (containing 
100 µg of protein). After incubation for 2 h at 37°C, total lipids were extracted with chloroform/methanol (2:1, v/v) (35). The amount of liberated sphingoid bases in the lipid extract was measured by the fluorometric method using fluorescamine (36).
Animal experiments.
Male Sprague-Dawley rats (6 wk old) were housed with free access to semipurified AIN-93G diet (37) and tap water for 1 wk. The rats were then deprived of food and allowed free access to water only for 14–15 h before cerebroside administration. The rats (200–225 g) were fed, by gastrogavage, 4 mg maize cerebroside (containing 2.7 µmol of 4t, 8c-sphingadienine and 0.9 µmol of 4t, 8t-sphingadienine) dispersed in 2 mL PBS containing 12 mmol/L sodium taurocholate. After 1, 3 or 6 h, the entire small intestine between the pylorus and cecum was removed under ether anesthesia. After the intestinal contents were collected by flushing with 40 mL cold saline, the small intestine was slit open and washed with cold saline, and the mucosa was scraped off with glass slides. The mucosa was then homogenized with Teflon-glass homogenizer in 9 volumes of ice-cold saline.
Lipid extraction.
Lipids were extracted from aliquots of homogenates of intestinal contents and mucosa with chloroform/methanol (2:1, v/v). After evaporation of the collected chloroform phase, the residue was saponified with 0.4 mol/L KOH in methanol at 38°C for 2 h to remove glycerolipids, as described previously (24–27). The alkali-stable fraction was subjected to HPLC analysis, as described below.
To separate the cerebroside and ceramide, aliquots of the alkali-stable fraction from the lipid extracts were taken for TLC; the TLC plate was developed in a solvent system consisting of chloroform/methanol/formic acid/water (56:30:4:2, by vol) (15). The Rf values for ceramide and cerebroside were 0.8 and 0.6, respectively. The ceramide and cerebroside fractions were collected and extracted from the silica according to the method of Bligh and Dyer (38) and were subjected to strong alkaline hydrolysis, as described above. The free sphingoid bases liberated from the ceramide and cerebroside were subjected to HPLC analysis, as described below.
HPLC analyses.
The OPA derivatives of the free sphingoid bases were analyzed by a reverse-phase HPLC system equipped with a fluorescent detector (39). The HPLC system consisted of an LC-10AD pump and an RF-10AXL fluorescence detector (Shimadzu). Sphingoid bases were separated on a TSK gel ODS-80Ts (Tosoh), 4.6 x 250 mm. The solvent used was acetonitrile/water (80:20, v/v). Isocratic analyses were performed at 1.0 mL/min, and the OPA derivatives were detected with an excitation wavelength of 334 nm and an emission wavelength of 440 nm.
Cell cultures.
Caco-2 cells (RIKEN Gene Bank, Tsukuba, Japan) were maintained in 10-cm dishes containing DMEM supplemented with 100 mL/L fetal bovine serum, 4 mmol/L L-glutamine, 40 000 U/L penicillin, 40 mg/L streptomycin and 0.1 mmol/L nonessential amino acids as described previously (22,23). For experiments, cells at passages 40–60 were seeded in 12-well plates at 1.2 x 105 cells/well. The experiments were performed 20–22 d postseeding on 12-well plates.
Cellular uptake of sphingoid bases.
The roles of P-glycoprotein and multi-drug resistance associated proteins were examined by measuring the cellular accumulation of sphingoid bases in the presence of selective inhibitors.The differentiated monolayers of Caco-2 cells in a 12-well plate were washed twice with 0.5 mL of serum-free DMEM and then supplemented with 5 µmol/L sphingoid base in 1 mL of serum-free DMEM containing 5 g/L BSA and 5 mmol/L sodium taurocholate, with or without several inhibitors (N,N-dimethylsphingosine for sphingosine kinase, verapamil for P-glycoprotein, and MK-571 for multidrug resistance–associated proteins). After incubation for an indicated time, the cell culture plates were placed on ice, the media were removed and the monolayers were washed twice with 0.5 mL of PBS containing 10 mmol/L sodium taurocholate to remove surface-bonded sphingoid bases; this was followed by two additional washings with 0.5 mL of PBS. The washed cells were harvested in 1 mL of PBS and pelleted by centrifugation at 1000 x g for 5 min. The supernatants were discarded, and the cell pellets were homogenized with a sonicator in 0.7 mL of ice-cold PBS. An aliquot of each cell homogenate was taken to determine the protein content. Lipids were then extracted from aliquots of cell homogenate and subjected to HPLC analysis for sphingoid bases, as described above.
To estimate the nonspecific uptake of sphingoid bases, some cells were pretreated for 1 h with a solution containing 37% formalin and DMEM (1:1, v/v) (40). These cells were washed twice with DMEM to remove the formalin solution before we added 5 µmol/L of each sphingoid base in DMEM containing 5 g/L BSA and 5 mmol/L sodium taurocholate.
Statistical analysis.
Values presented are means ± SEM. Data were analyzed by Student’s t test or one- or two-way ANOVA. Dunnett’s or Scheffé’s F-test was used to identify means that differed, P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cerebrosidase activity in the small intestinal mucosa and cecal contents toward cerebroside of plant origin prepared from maize did not differ from that toward glucocerebroside of mammalian origin [264 ± 60 vs. 259 ± 41 nmol/(h · mg protein) for intestinal mucosa and 86 ± 8 vs. 93 ± 12 nmol/(h · mg protein) for cecal contents, respectively, n = 4]. Similarly, the ceramidase activity, which hydrolyzes the amide linkage of ceramide, toward ceramide derived from maize cerebroside was the same as that toward ceramide of mammalian origin in both the small intestinal mucosa and cecal contents [18.0 ± 5.0 vs. 18.5 ± 2.9 nmol/(h · mg protein) for intestinal mucosa and 5.3 ± 1.6 vs. 8.3 ± 2.6 nmol/(h · mg protein) for cecal contents, respectively, n = 4].
Hydrolysis of maize sphingolipids in the intestinal tract of rats.
The formation in the intestinal lumen of free sphingoid bases such as 4t, 8c- and 4t, 8t-sphingadienine was confirmed 1 h after a single oral dose of maize cerebroside (Fig. 2). Similarly, in the small intestinal mucosa, isomers of sphingadienine hydrolyzed from maize cerebroside were found (Fig. 2). In addition, peaks ascribed to the trihydroxy sphingoid bases, cis and trans isomers of 4-hydroxy-8-sphingenine, were detected in both the lumen and tissue.
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3% of the dose given.
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The accumulation of 4t, 8c- and 4t, 8t-sphingadienine in Caco-2 cells incubated with the isomers reached a maximum between 0.5 and 1 h of incubation and then declined gradually (Fig. 4). When the cells were incubated with sphingosine, the predominant sphingoid base in most mammals, cellular accumulation of sphingosine reached a maximum after 1 h of incubation, and had not declined after 6 h. Here, the cellular sphingosine level was significantly higher than cellular levels of the sphingadienines of plant origin at each time point (P < 0.05).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In early studies, Nilsson investigated the digestion and intestinal absorption of cerebroside and sphingomyelin (12–14). Several reports indicated that the cerebrosidase activity in the intestine is lactase-phlorizin hydrolase in mice and rats (33,41,42). Lactase-phlorizin hydrolase activity is generally very low in adult rodents, whereas it may be relatively well maintained in humans (43). In addition, Duan et al. reported that specific ceramidase is present in the rat and human small intestine (34). Our findings of hydrolytic activity in the small intestine toward cerebroside and ceramide prepared from maize are consistent with the findings of such activity toward sphingolipids of mammalian origin. However, what remains uncertain is whether cerebrosides of plant origin are hydrolyzed before or after their uptake by intestinal epithelial cells. Schmelz et al. (15) speculated that dietary sphingomyelin trapped in intestinal mucin was still bound to the outer surfaces of the epithelial cells, or was taken up with the head group intact. Sphingolipids of plant origin might be hydrolyzed on the surfaces of the intestinal villi, because free sphingoid bases and ceramide appeared in both the small intestinal lumen and the tissues after ingestion of maize cerebroside.
Our results strongly suggest that the isomers of 4,8-sphingadienine prepared from maize cerebroside are not substrates for sphingosine kinase. Phosphorylation of the primary hydroxy group of sphingoid bases is the penultimate step in the degradation of sphingolipid (44). The phosphate esters formed are subsequently cleaved into ethanolamine phosphate and the corresponding aldehyde by sphingosine phosphate lyase. Long-chain aldehyde is further converted to fatty acids or an effective donor of the alkenyl chain of plasmalogen (45). Nilsson reported that sphingoid bases were converted to palmitic acid in the intestinal mucosa and incorporated into chylomicron triacylglycerol (12,14). Indeed, the activities of sphingosine kinase and sphingosine phosphate lyase are higher in the small intestine than in other tissues (45,46). Interestingly, Mao et al. (47) surmised that phosphorylation followed by dephosphorylation was required for the incorporation of exogenous sphingoid bases into sphingolipids. Thus, 4,8-sphingadienines digested from plant cerebroside would be poorly incorporated and resynthesized to complex sphingolipids in tissues. Given our knowledge of the metabolism of dietary sphingolipids, it would be interesting to evaluate the substrate specificity of sphingosine kinase.
Our observations support an important role for P-glycoprotein in the efflux of 4,8-sphingadienine, but not sphingosine, across the apical membranes of enterocytes after absorption. In differentiated Caco-2 cells, the extent of the expression of genes responsible for the efflux system is in good agreement with that in the human jejunum (48). P-glycoprotein (encoded by the MDR1 gene), a member of the ATP-binding cassette transporter superfamily, transports a wide variety of hydrophobic compounds, including chemotherapeutic drugs, natural products, toxicants and peptides, and contributes to the barrier function of the gut (49). It is possible that P-glycoprotein could distinguish 8-desaturated sphingoid bases from sphingosine.
The amount of maize-origin sphingadienine recovered from the small intestine 1 h after a single oral dose of maize cerebroside was very low. Schmelz et al. (15) reported that in the large intestine of mice, dietary sphingomyelin appeared after 
30 min. Therefore, dietary maize cerebroside might reach the lower digestive tract (cecum and colon) 1 h after ingestion. We confirmed that the cecal contents had hydrolytic activity toward sphingolipids, possibly because of the presence of enterobacteria in human fecal flora (50), although glucose recovery in the present cerebrosidasse assay could have been underestimated due to bacterial glycolysis, for example. The preventive effect of dietary plant-origin sphingolipid on carcinogenesis should occur in the lower digestive tract because plant-origin sphingoid bases induce apoptosis in DLD-1 colon cancer cells (19). However, we have only indirect evidence of this effect; the presence of metabolites of sphingolipids in the lower digestive tract has yet to be demonstrated. Further studies of structure/function relationships and cancer models are required to clarify the effect of dietary plant sphingolipids on human health.
In conclusion, we demonstrated that the digestibility of maize cerebroside is similar to that of cerebrosides of mammalian origin, and that the metabolic fate of sphingoid bases of plant origin absorbed by enterocytes differs from that of sphingosine. Our results suggest that isomers of 4,8-sphingadienine degraded from dietary plant cerebroside are absorbed poorly from the digestive tract. Our findings offer new insight into the metabolism of dietary sphingolipids from plant sources.
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FOOTNOTES |
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Manuscript received 16 April 2003. Initial review completed 22 May 2003. Revision accepted 18 June 2003.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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