Dietary Sphingomyelin Research Articles

Relevance of interactions between sphingomyelin and cholesterol in biliary and intestinal tract

AbstractPhosphatidylcholine and sphingomyelin are the major phospholipids of the hepatocytic canalicular membrane outer leaflet. Sphingomyelin may preferentially reside with cholesterol in liquid‐ordered domains. In contrast, phosphatidylcholine is the exclusive phospholipid secreted in rat bile (enriched in hydrophilic species compared to the canalicular membrane), subsequently incorporated into bile salt‐cholesterol micelles. We determined the bile lipid composition in 95 vertebrate species (Moschetta et al., J Lipid Res. 2005, 46, 2221–2232). Phospholipid was often virtually absent in bile of cartilaginous fish and reptiles, occurred in low relative amounts (compared to bile salts) in bony fish or birds and in high relative amounts in most mammals. Biles with low relative amounts of phospholipid often contained high proportions of sphingomyelin. Phosphatidylcholine was the predominant phospholipid in biles with high phospholipid contents. We then compared, in CaCo2 cells (without appreciable phospholipase A2 activity), the effects of incorporating sphingomyelin, egg yolk phosphatidylcholine or lyso‐phosphatidylcholine in apical bile salt‐cholesterol micelles. Egg yolk phosphatidylcholine and (more pronounced) sphingomyelin inhibited cholesterol absorption with decreased ABC‐A1 and ‐G1 expression. Lyso‐phosphatidylcholine enhanced cholesterol absorption with increased basolateral HDL‐dependent cholesterol efflux and high expression of ABC‐A1 and ‐G1. In conclusion, sphingomyelin plays a pivotal role in protecting hepatocytes against detergent bile salts. Dietary sphingomyelin may inhibit intestinal cholesterol absorption.

Effects of bile diversion in rats on intestinal sphingomyelinases and ceramidase

Alkaline sphingomyelinase (Alk-SMase) and neutral ceramidase (N-CDase) in the intestinal microvillar membrane are responsible for dietary sphingomyelin digestion. The activities of the enzymes require the presence of bile salt, and the enzymes can be released into the gut lumen in active forms by bile salts and trypsin. It is unclear to what extent that the intestinal presence of bile salts is critical for the intraluminal activity of these enzymes. We compared the activities of Alk-SMase, N-CDase, and other types of SMases in control and permanently bile diverted rats. In the intestinal tract of control rats, the activity of Alk-SMase was profoundly higher than those of acid and neutral SMases. Bile diversion reduced Alk-SMase activity by 85% in the small intestinal content, and by 68% in the faeces, but did not significantly change the activity in the intestinal mucosa. Western blot showed a marked reduction of the enzyme in the intestinal lumen but not mucosa. N-CDase activities both in the intestinal mucosa and content were reduced by bile diversion. Bile diversion also decreased aminopeptidase N activity in the content and increased that in the mucosa, but had no effects on that of alkaline phosphatase. In conclusion, the presence of bile salts is important for maintaining high intraluminal levels of Alk-SMase and N-CDase, two key enzymes for hydrolysis of sphingomyelin in the gut. We speculate that the sphingomyelin hydrolysis in cholestatic conditions is impaired not only by reduced hydrolytic activity but also by deficient dissociation of the enzymes from the membrane.

Absorption and lipoprotein transport of sphingomyelin

Dietary sphingomyelin (SM) is hydrolyzed by intestinal alkaline sphingomyelinase and neutral ceramidase to sphingosine, which is absorbed and converted to palmitic acid and acylated into chylomicron triglycerides (TGs). SM digestion is slow and is affected by luminal factors such as bile salt, cholesterol, and other lipids. In the gut, SM and its metabolites may influence TG hydrolysis, cholesterol absorption, lipoprotein formation, and mucosal growth. SM accounts for approximately 20% of the phospholipids in human plasma lipoproteins, of which two-thirds are in LDL and VLDL. It is secreted in chylomicrons and VLDL and transferred into HDL via the ABCA1 transporter. Plasma SM increases after periods of large lipid loads, during suckling, and in type II hypercholesterolemia, cholesterol-fed animals, and apolipoprotein E-deficient mice. SM is thus an important amphiphilic component when plasma lipoprotein pools expand in response to large lipid loads or metabolic abnormalities. It inhibits lipoprotein lipase and LCAT as well as the interaction of lipoproteins with receptors and counteracts LDL oxidation. The turnover of plasma SM is greater than can be accounted for by the turnover of LDL and HDL particles. Some SM must be degraded via receptor-mediated catabolism of chylomicron and VLDL remnants and by scavenger receptor class B type I receptor-mediated transfer into cells.

Detection of Alkaline Sphingomyelinase Activity in Human Stool: Proposed Role as a New Diagnostic and Prognostic Marker of Colorectal Cancer

Intestinal alkaline sphingomyelinase, by exerting a major role in dietary sphingomyelin digestion, is responsible for the generation of messengers able to trigger the rapid turnover and apoptosis in intestinal epithelial cells. Markedly reduced mucosal alkaline sphingomyelinase activity has been associated with human colorectal neoplasms. The aim of this study was to analyze the alkaline sphingomyelinase activity in feces from healthy subjects and colorectal adenocarcinoma patients and to correlate it with the enzyme activity in intestinal tissues. The enzyme activity was measured both in the intestinal samples from 12 healthy controls and 51 patients with colorectal adenocarcinoma (tumoral and paratumoral tissue) and in the fecal samples of 34 healthy subjects and 29 patients with adenocarcinoma. The relation between sphingomyelinase activity and Dukes' stage, cell differentiation degree, age, and gender was also analyzed. Alkaline sphingomyelinase was significantly decreased (P < 0.001; mean reduction >90%) in tumoral intestinal mucosa of patients compared with controls independently of Dukes' stage and tumor differentiation grade. Interestingly, the enzyme activity in histologically normal paratumoral tissues was statistically lower than control samples (P < 0.001). As occurs in neoplastic tissues, a relevant mean reduction (P < 0.0001; almost 90%) of alkaline sphingomyelinase was revealed in stool samples from tumor patients when compared with controls. These findings may have implications for cancer biology and perhaps also for the design of clinical test, thus suggesting that the fecal sphingomyelinase activity could really reflect the human intestinal mucosa enzyme level and could represent a new marker for human colorectal adenocarcinoma, mainly taking into account its early appearance in intestinal neoplasms.

Sphingomyelin in the suppression of colon tumors: prevention versus intervention

Intestinal cells are regularly exposed to sphingolipid metabolites, i.e., ceramide and sphingoid bases, after hydrolysis of complex sphingolipids from the diet. These metabolites are known regulators of cell growth, differentiation, and death. Non-pharmacological amounts in the diet have been shown to inhibit early stages of chemically induced colon cancer in mice. To distinguish between chemopreventive and chemotherapeutic effects of sphingomyelin supplements, mice were fed sphingomyelin before and after tumor initiation. Both applications drastically reduced tumor formation, without a significant difference among the groups, indicating that sphingolipids are as effective in the chemoprevention of tumors as in early intervention. The normalization of cell proliferation and rate of apoptosis, but not the induction of differentiation, seem to be key players in the suppression of tumor formation by dietary sphingomyelin. This may have implications for the development of a cancer prevention or treatment strategy with sphingolipids as an alternative to conventional drugs.

Identification of Human Intestinal Alkaline Sphingomyelinase as a Novel Ecto-enzyme Related to the Nucleotide Phosphodiesterase Family

Alkaline sphingomyelinase (alk-SMase) hydrolyzes dietary sphingomyelin and generates sphingolipid messengers in the gut. In the present study, we purified the enzyme, identified a part of the amino acid sequence, and found a cDNA in the GenBank coding for the protein. The cDNA contains 1841 bp, and the open reading frame encodes 458 amino acids. Transient expression of the cDNA linked to a Myc tag in COS-7 cells increased alk-SMase activity in the cell extract by 689-fold and in the medium by 27-fold. High activity was also identified in the anti-Myc immunoprecipitated proteins and the proteins cross-reacted with anti-human alk-SMase. Northern blotting of human intestinal tissues found high levels of alk-SMase mRNA in the intestine and liver. The amino acid sequence shared no similarity with acid and neutral SMases but was related to the ecto-nucleotide phosphodiesterase (NPP) family with 30-36% identity to human NPPs. Alk-SMase has a predicted signal peptide domain at the N terminus and a signal anchor domain at the C terminus. The ion-binding sites and the catalytic residue of NPPs were conserved, but the substrate specificity domain was modified. Alk-SMase had no detectable nucleotidase activity, but its activity against sphingomyelin could be inhibited by orthovanadate, imidazole, and ATP. In contrast to NPPs, alk-SMase activity was not stimulated by divalent metal ions but inhibited by Zn2+. Differing from NPP2, the alk-SMase cleaved phosphocholine but not choline from lysophosphatidylcholine. Phylogenetic tree indicated that the enzyme is a new branch derived from the NPP family. Two cDNA sequences of mouse and rat that shared 83% identity to human alk-SMase were identified in the GenBank. In conclusion, we identified the amino acid and cDNA sequences of human intestinal alk-SMase, and found that it is a novel ecto-enzyme related to the NPP family with specific features essential for its SMase activity.

Development of intestinal alkaline sphingomyelinase in rat fetus and newborn rat.

Sphingomyelin metabolism is a novel signal transduction pathway related to cell differentiation, proliferation, and apoptosis. Alkaline sphingomyelinase (alk-SMase) is specifically present in the intestinal tract of many species. The enzyme is important in digestion of dietary sphingomyelin. Milk is the only exogenous source of sphingomyelin for an infant, and digestion of milk sphingomyelin may be important for development of intestinal mucosa. It is unknown whether alk-SMase is present before birth and whether it changes after birth and during the suckling period. We studied activities, expression, and distribution of alk-SMase in rat fetus and newborn. The changes of acid and neutral SMase as well as alkaline phosphatase were analyzed for comparison. Little activity of alk-SMase was identified up to gestation day 20, but increased 10 times during the following 2 d. After birth, the activity continued to increase during the following 4 wk. Western blot using IgY antibody against rat alk-SMase failed to identify the enzyme at gestation day 20 but clearly showed the protein at day 22. The distribution pattern of the enzyme along the intestinal tract in fetus was largely the same as in adult animals, but became more pronounced after birth. Short-term weaning had no effect on alk-SMase activity. The activities of acid and neutral SMase were high at gestation day 20 and decreased significantly before birth. The changes of alk-SMase also differed from those of alkaline phosphatase, another brush border enzyme. Thus, we conclude that alk-SMase is rapidly expressed during the last days of gestation and that the newborn rat acquires the ability to digest milk sphingomyelin early in life.

Effects of dietary sphingomyelin on central nervous system myelination in developing rats.

Human milk contains sphingomyelin (SM) as a major component of the phospholipid fraction. Galactosylceramide (cerebroside), a metabolite of sphingolipids, increases along with CNS myelination, and is generally considered a universal marker of myelination in all vertebrates. l-Cycloserine (LCS) is an inhibitor of serine palmitoyltransferase (SPT), a rate-limiting enzyme for sphingolipid biosynthesis that is reported to show increased activity with development of the rat CNS. The present study examined the effects of dietary SM on CNS myelination during development in LCS-treated rats. From 8 d after birth, Wistar rat pups received a daily s.c. injection (100 mg/kg) of LCS. From 17 d after birth, the animals were fed an 810 mg/100g of bovine SM-supplemented diet (SM-LCS group) or a nonsupplemented diet (LCS group). At 28 d after birth, the animals were killed and subjected to biochemical and morphometric analyses. The myelin dry weight, myelin total lipid content, and cerebroside content were significantly lower in the SM-LCS and LCS groups than in a group not treated with LCS (the non-LCS group). However, these levels were significantly higher in the SM-LCS group than in the LCS group. Morphometric analysis of the optic nerve revealed that the axon diameter, nerve fiber diameter, myelin thickness, and g value (used to compare the relative thickness of myelin sheaths around fibers of different diameter) were significantly lower in the LCS group than in the other groups, but were similar in the SM-LCS and non-LCS groups. These findings suggest that dietary SM contributes to CNS myelination in developing rats with experimental inhibition of SPT activity corrected].

Sphingomyelin protects against apoptosis and hyperproliferation induced by deoxycholate: potential implications for colon cancer.

High fecal deoxycholate levels may promote colonic cancer. Phospholipids protect against bile salt-induced cytotoxicity. We therefore aimed to examine whether the dietary phospholipid sphingomyelin could decrease hyperproliferation induced by deoxycholate. In CaCo2 cells, hyperproliferation (by bromodeoxyuridine assay), phosphorylation state of cellular proteins, and apoptosis with concomitant caspase-3 activity were evaluated after incubation with 50-500 microM deoxycholate, with or without sphingomyelin. At 2 and 4 hr of incubation, deoxycholate induced dose-dependent apoptosis, with concomitant caspase-3 activation. At 16 hr, apoptosis had decreased markedly, but there was dose-dependent hyperproliferation (with changed phosphorylation status of cellular proteins) at this time point. Sphingomyelin dose-dependently reduced deoxycholate-induced apoptosis and hyperproliferation. In conclusion, sphingomyelin reduces deoxycholate-induced hyperproliferation and apoptosis. These findings may have implications for colonic cancer prevention by dietary modification.

Incorporation of cholesterol in sphingomyelin- phosphatidylcholine vesicles has profound effects on detergent-induced phase transitions

Vesicle ↔ micelle transitions are important phenomena during bile formation and intestinal lipid processing. The hepatocyte canalicular membrane outer leaflet contains appreciable amounts of phosphatidylcholine (PC) and sphingomyelin (SM), and both phospholipids are found in the human diet. Dietary SM enrichment inhibits intestinal cholesterol absorption. We therefore studied detergent-induced vesicle → micelle transitions in SM-PC vesicles. Phase transitions were evaluated by spectrophotometry and cryotransmission electron microscopy (cryo-TEM) after addition of taurocholate (3–7 mM) to SM-PC vesicles (4 mM phospholipid, SM/PC 40%/60%, without or with 1.6 mM cholesterol). After addition of excess (5–7 mM) taurocholate, SM-PC vesicles were more sensitive to micellization than PC vesicles. As shown by sequential cryo-TEM, addition of equimolar (4 mM) taurocholate to SM-PC vesicles induced formation of open vesicles, then (at the absorbance peak) fusion of bilayer fragments into large open structures (around 200 nm diameter) coexisting with some multilamellar or fused vesicles and thread-like micelles and, finally, transformation into an uniform picture with long thread-like micelles. Incorporation of cholesterol in the SM/PC bilayer changed initial vesicular shape from spherical into ellipsoid and profoundly increased detergent resistance. Disk-like micelles and multilamellar vesicles, and then extremely large vesicular structures, were observed by sequential cryo-TEM under these circumstances, with persistently increased absorbance values by spectrophotometry.These findings may be relevant for bile formation and intestinal lipid processing. Inhibition of intestinal cholesterol absorption by dietary SM enrichment may relate to high resistance against bile salt-induced micellization of intestinal lipids in presence of the sphingolipid.

35 P Dietary sphingomyelin protects against apoptosis and hyperproliferation induced by the hydrophobic bile salt deoxycholate potential implications for colon cancer

35 P Dietary sphingomyelin protects against apoptosis and hyperproliferation induced by the hydrophobic bile salt deoxycholate potential implications for colon cancer

In vitro effects of fat, FA, and cholesterol on sphingomyelin hydrolysis induced by rat intestinal alkaline sphingomyelinase.

Dietary sphingomyelin (SM) may have regulatory effects on cell proliferation and tumorigenesis in the colon. Alkaline sphingomyelinase (SMase) is the major enzyme responsible for hydrolysis of SM in the gut. Previously we purified the enzyme and showed that the presence of glycerophospholipids inhibited SM hydrolysis induced by alkaline SMase in vitro. In the present work, we studied the effects of TG, DG, FA, ceramide, and cholesterol on SM hydrolysis catalyzed by purified alkaline SMase. The results showed that both TG (triolein and tristearin) and DG (1,2-dioleoyl-sn-glycerol and 1,2-distearoyl-rac-glycerol) inhibited the activity of alkaline SMase. 1-Monooleoyl-rac-glycerol, 1-monostearoyl-rac-glycerol, stearic acid, oleic acid, linoleic acid, linolenic acid, and arachidonic acid stimulated the activity of alkaline SMase at 0.4-0.8 mM concentrations but inhibited the enzyme at higher concentrations. There was no difference between the effects induced by saturated and unsaturated FA. A short-chain FA such as lauric acid had a stronger stimulatory effect at low concentrations and weaker inhibitory effect at high concentrations than long-chain FA. Choosing linoleic acid as an example, we found that FA had similar effects on both alkaline SMase and neutral SMase. Cholesterol and ceramide when mixed with FA to increase its solubility in bile salt micelles inhibited SMase activity. In conclusion, glycerides, FA, ceramide, and cholesterol influence SM hydrolysis catalyzed by intestinal alkaline SMase. The presence of lipids in the diet may thus influence the course of SM digestion in the gut and thereby the exposure of colon to SM metabolites.

Dietary sphingomyelin suppresses intestinal cholesterol absorption by decreasing thermodynamic activity of cholesterol monomers

In humans, cholesterol absorbed from the intestine contributes appreciably to serum cholesterol levels. We hypothesized that cholesterol thermodynamic activity (A(t)) would predict bioavailability of cholesterol monomers in intestinal content, and that natural dietary phospholipids exhibiting high affinity for cholesterol would reduce its absorption. Cholesterol A(t) was determined by measuring partitioning of monomeric cholesterol from aqueous solutions of taurocholate, cholesterol, and either milk sphingomyelin (MSM), dipalmitoyl phosphatidylcholine (DPPC), or egg yolk phosphatidylcholine (EYPC) into wafers of polymerized silicone. Cholesterol absorption from the same mixtures was tested with monolayers of Caco-2 cells. For in vivo absorption studies (employing male C57L/J mice), we used the fecal dual isotope method during dietary enrichment with MSM, DPPC, or EYPC at varying dose levels. Cholesterol A(t) values were reduced significantly in MSM- and DPPC-containing systems compared with EYPC and correlated positively with reduced uptake and esterification of cholesterol by Caco-2 cells. Mice fed chow absorbed 31.4% +/- 6.9% (mean +/- SEM) cholesterol, whereas enrichment with MSM or DPPC led to dose-dependent decreases in cholesterol absorption; even at 0.1% MSM, cholesterol absorption was reduced by 20.4% +/- 15.4% (P < 0.05, n = 6). Different phospholipids have distinct effects on micellar cholesterol A(t), which predicts cholesterol uptake by enterocytes in vitro as well as in vivo. Natural phospholipids with high affinity for cholesterol, as evidenced particularly by sphingomyelin, decrease A(t) and curtail intestinal cholesterol absorption.

Dietary sphingomyelin (SM) significantly inhibits intestinal cholesterol (Ch) absorption by lowering thermodynamic activity (TA) of Ch in bile salt (BS) mixed micellar solution

Dietary sphingomyelin (SM) significantly inhibits intestinal cholesterol (Ch) absorption by lowering thermodynamic activity (TA) of Ch in bile salt (BS) mixed micellar solution

Dietary sphingomyelin (SM) significantly inhibits intestinal cholesterol (Ch) absorption by lowering thermodynamic activity (TA) of Ch in bile salt (BS) mixed micellar solution

Dietary sphingomyelin (SM) significantly inhibits intestinal cholesterol (Ch) absorption by lowering thermodynamic activity (TA) of Ch in bile salt (BS) mixed micellar solution

Dietary sphingomyelin and other sphingolipids in health and disease

Summary Sphingolipids are found in all eukaryotic and some prokaryotic cells and therefore most foods contain varying amounts. They are digested in the intestinal tract, and the bioactive metabolites (i.e. ceramide, sphingosine) are released. In mouse models, the supplementation of the diet with complex sphingolipids significantly reduced early stages in colon carcinogenesis and tumour formation, providing an insight into the association between diet and cancer.

Effects of phospholipids on sphingomyelin hydrolysis induced by intestinal alkaline sphingomyelinase: an in vitro study

Digestion of dietary sphingomyelin (SM) is catalyzed by intestinal alkaline sphingomyelinase (SMase) and may have important implications in colonic tumorigenesis. Previous studies demonstrated that the digestion and absorption of dietary SM was slow and incomplete and that the colon was exposed to SM and its hydrolytic products including ceramide. In the present work, we studied the influences of glycerophospholipids and hydrolytic products of phosphatidylcholine (PC; i.e., lyso-PC, fatty acid, diacylglycerol, and phosphorylcholine) on SM hydrolysis induced by purified rat intestinal alkaline SMase in the presence of 10 mM taurocholate. It was found that various phospholipids including PC, phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and phosphatidic acid (PA) inhibit alkaline SMase activity in a dose-dependent manner, with the degree of inhibition being in the order PA > PS > PI > PC > PE. Similar inhibition was also seen in a buffer of pH 7.4, which is close to the physiologic pH in the middle of the small intestine. When the effects of hydrolytic products of PC were studied, lyso-PC, oleic acid, and 1,2-dioleoyl glycerol also inhibited alkaline SMase activity, whereas phosphorylcholine enhanced SMase activity. However, in the absence of bile salt, acid phospholipids including PA, PS, and PI mildly stimulated alkaline SMase activity whereas PC and PE had no effect. It is concluded that in the presence of bile salts, glycerophospholipids and their hydrolytic products inhibit intestinal alkaline SMase activity. This may contribute to the slow rate of SM digestion in the upper small intestine.

Colonic Cell Proliferation and Aberrant Crypt Foci Formation Are Inhibited by Dairy Glycosphingolipids in 1,2-Dimethylhydrazine-Treated CF1 Mice

Dietary sphingomyelin (SM) inhibits early stages of colon cancer (appearance of aberrant crypt foci, ACF) and decreases the proportion of adenocarcinomas vs. adenomas in 1,2-dimethylhydrazine (DMH)-treated CF1 mice. To elucidate the structural specificity of this inhibition, the effects of the other major sphingolipids in milk (glycosphingolipids) were determined. Glucosylceramide (GluCer), lactosylceramide (LacCer) and ganglioside G(D3) were fed individually to DMH-treated (six doses of 30 mg/kg body weight) female CF1 mice at 0.025 or 0.1 g/100 g of the diet for 4 wk. All reduced the number of ACF by > 40% (P < 0.001), which is comparable to the reduction by SM in earlier studies. Immunohistochemical analysis of the colons revealed that sphingolipid feeding also reduced proliferation, with the most profound effect (up to 80%; P < 0.001) in the upper half of the crypts. Since the bioactive backbones of the glycosphingolipids (i.e., ceramide and other metabolites) are the likely mediators of these effects, the susceptibility of these complex sphingolipids to digestion in the colon was examined by incubating 500 microgram of each sphingolipid with colonic segments from mice and analysis of substrate disappearance and product formation by tandem mass spectrometry. All of the sphingolipids (including SM) disappeared over time with a substantial portion appearing as ceramide. Partially hydrolyzed intermediates (such as GluCer from LacCer or G(D3)) were not detected, which suggests that the cleavage involves colonic (or microflora) endoglycosidases. In summary, consumption of dairy SM and glycosphingolipids suppresses colonic cell proliferation and ACF formation in DMH-treated mice; hence, many categories of sphingolipids affect these key events in colon carcinogenesis.

Conjugated linoleic acid and other anticarcinogenic agents of bovine milk fat.

Prevention is an important strategy for conquering cancer. Milk fat contains a number of components, such as conjugated linoleic acid, sphingomyelin, butyric acid, ether lipids, beta-carotene, and vitamins A and D that have anticancer potential. Conjugated linoleic acid inhibits the growth of a number of human cancer cell lines and suppresses chemically-induced tumor development at a number of sites in animal models. As little as 0.1% of dietary conjugated linoleic acid inhibits the development of rat mammary tumors, independent of the amount and type of fat in the diet. Sphingomyelin, through its metabolites ceramide and sphingosine, participates in multiple antiproliferative pathways associated with suppression of carcinogenesis. Dietary sphingomyelin inhibits murine colon tumor development. Butyric acid, uniquely present in ruminant milk, is a potent antineoplastic agent and may ameliorate its potency through synergy with other milk fat components. Dietary butyric acid inhibits mammary carcinoma development in rats. In humans, ether lipids, beta-carotene, and vitamins A and D are associated with anticancer effects. Cows have the ability to extract anticarcinogenic components from pasture and feed and transfer them to milk. Use of genetic engineering and other techniques to increase the range and level of anticarcinogens in pasture and supplements may increase the anticancer potential of milk.

Localization and capacity of sphingomyelin digestion in the rat intestinal tract

Dietary sphingomyelin (SM) undergoes sequential cleavage to ceramide and sphingosine in the intestine. A distinctive intestinal sphingomyelinase (SMase) with alkaline pH-optimum was earlier identified by us. The activity was highest in middle and lower small intestine, but its role in SM digestion has not been clarified. In this study we examined the extension and capacity of SM digestion in vivo. After feeding rats 0.2, 6.6, or 32 μmol SM containing 2 μCi 3H-sphingosine-labeled milk SM ( 3H-SM), radioactivity was analyzed in intestinal contents and tissues 2, 4, and 8 hr later. The proportion of radioactivity in the contents of small intestine increased with the dose of SM; 9% of given dose with 0.2 μmol, 34% with 6.6 μmol, and 71% with 32 μmol, respectively after 2 hr. Lowest tissue radioactivity was found in duodenum and proximal jejunum and highest in distal jejunum and proximal ileum. Three to twenty one percent of radioactivity in the intestinal tissue was in ceramide, the proportion varying with the dose given, region of the intestine, and time after administration. After administration of 6.6 or 32 μmol SM, significant amounts of intact SM and ceramide was found in intestinal contents, colon, and excreted faeces. Colon was exposed to ceramide derived from exogenous SM in amounts that were rather proportional to the dose of SM fed. SM digestion is thus a process extending over the whole intestine and occurring mainly in the middle and lower parts of the small intestine. The site of digestion coincides with the distribution of the alkaline SMase, indicating that this enzyme catalyzes the first step in the digestion. The extension and limited capacity of the SM digestion leads to an exposure of the lower small intestine and colon to SM and sphingolipid metabolites.