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Nutrient Metabolism

Milk Sphingomyelin Is More Effective than Egg Sphingomyelin in Inhibiting Intestinal Absorption of Cholesterol and Fat in Rats^1,2

Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06269

⁴To whom correspondence should be addressed. E-mail: skoo{at}canr.uconn.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
We reported previously that egg sphingomyelin (SM) inhibits the intestinal absorption of cholesterol and fat in rats. This study was conducted to compare the relative efficiencies of milk and egg SM in inhibiting intestinal absorption of cholesterol and other lipids. Adult male rats with lymph cannulae were infused at 3.0 mL/h for 8 h via a duodenal catheter with a lipid emulsion (451.7 µmol triolein, 20.7 µmol cholesterol, 33.3 kBq ¹⁴C-cholesterol, 3.1 µmol -tocopherol, and 396.0 µmol sodium taurocholate in 24 mL PBS, pH, 6.5), without SM (controls), or with 80.0 µmol egg SM or milk SM. The lymphatic absorptions of ¹⁴C-cholesterol were significantly lower in rats infused with milk SM (19.5 ± 1.4% dose) and egg SM (24.4 ± 1.9% dose) than in those infused with no SM (37.6 ± 1.8% dose). In addition, the lymphatic outputs of fatty acids and phospholipid were significantly lowered by milk and egg SM. Similarly, the absorption of -tocopherol also was decreased by milk SM (13.6 ± 1.7% dose) and egg SM (18.3 ± 2.4% dose) compared with controls (27.0 ± 1.8% dose). Total lymphatic SM output was not affected by egg SM, but markedly decreased by milk SM, relative to controls. The results indicate that both milk and egg SM markedly inhibit the absorption of cholesterol, fat, and other lipids. However, milk SM is a more potent inhibitor than egg SM. The strong inhibitory effect of milk SM may be associated with the higher degree of saturation and longer chain length of its fatty acyl groups, which may slow the rate of luminal lipolysis, micellar solubilization, and transfer of micellar lipids to the enterocyte.

KEY WORDS: • cholesterol • sphingomyelin • egg • milk • -tocopherol

Studies have shown that sphingomyelin (SM)⁵ profoundly influences cholesterol trafficking and homeostasis (1,2). Evidence indicates that SM and cholesterol are colocalized in cell membranes and that perturbation of SM content or SM/cholesterol ratio alters cholesterol synthesis, transport, and balance (1,2). Recent studies also showed that dietary SM significantly influences the plasma and tissue levels of other lipids (3,4) and inhibits the intestinal absorption of cholesterol (5–7) in animals, suggesting that dietary SM may play an important role in regulating cholesterol homeostasis and lipid metabolism.

In our recent study using rats with lymph cannulae, we provided evidence that egg SM dose dependently inhibited the intestinal absorption of cholesterol and other lipids, including fat and -tocopherol (7). Although the mechanism underlying such effects of SM is currently unknown, one potential mechanism may be associated with the fact that SM may decrease micellar solubilization (6) and transfer of cholesterol from the micellar matrix to the enterocyte (5,6), thus reducing the rate of cell uptake. In previous studies (8,9), we showed that egg phosphatidylcholine (PC), structurally similar to SM, markedly decreased the absorption of cholesterol and that the complete saturation of the acyl groups of the PC by hydrogenation further decreased the lymphatic absorption of both cholesterol and fatty acids. In contrast, soy PC, which contains predominantly unsaturated fatty acids, increased their absorptions. These observations suggest that the degree of saturation or unsaturation of the PC’s acyl moiety is an important determinant of micellar solubility and absorption of cholesterol.

At present, little information is available concerning the way in which dietary SM from different food sources affects the intestinal absorption of cholesterol and other lipids. Although the amounts of sphingolipids in food vary widely, milk and egg are among the rich dietary sources of SM (10). SM has sphingosine as the hydrocarbon backbone with an amide-linked acyl chain that varies in saturation and chain length. Although both milk and egg SM are nearly completely saturated, milk SM consists largely of longer-chain SFA than egg SM. The primary objective of this study was to compare the effects of SM from these common food sources on the intestinal absorption of cholesterol and other lipids in rats with lymph cannulae under in vivo conditions.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Animals and diet. Male Sprague-Dawley rats (Harlan Sprague Dawley) weighing 274 ± 8 g were housed individually in plastic cages and subjected to a 12-h light:dark cycle with the light period starting at 1530 h in a temperature-controlled (22–25°C) room throughout the study. Rats had free access to a diet (7) formulated by Dyets Inc. according to the AIN-93G recommendations (11,12) and deionized water via a stainless steel watering system. All rats were cared for in an animal care facility in the Department of Human Nutrition, Kansas State University, in accordance with a protocol approved by the Institutional Animal Care and Use Committee.

    Cannulation of the mesenteric lymph duct. At 7 wk of feeding, rats were food deprived overnight (16 h) but had free access to water. The mesenteric lymph duct was cannulated; an indwelling infusion catheter was introduced via the gastric fundus into the upper duodenum and secured by a purse-string suture under halothane anesthesia as described previously (7–9). The rats were placed in restraining cages in a recovery chamber (30°C) for postoperative recovery and were infused continuously for 22–24 h via the infusion catheter with PBS containing glucose (pH 6.5) at 3.0 mL/h by a syringe pump, as described previously (7–9).

    Measurement of lymphatic ¹⁴C-cholesterol absorption. After postoperative recovery, each rat was infused with a lipid emulsion at 3 mL/h for 8 h via the duodenal catheter in subdued light. The lipid emulsion consisted of 451.7 µmol triolein (95%, Sigma Chemical), 33.3 kBq [4-¹⁴C]-cholesterol (>97% purity, specific activity, 1.9 GBq/mmol, DuPont NEN), 20.7 µmol cholesterol, 3.1 µmol -tocopherol (all-rac-dl--tocopherol, 97%, Aldrich Chemical), and 396.0 µmol sodium taurocholate (Sigma Chemical) in 24 mL of PBS buffer without SM (controls), or with 80.0 µmol egg SM or milk SM. The amount of triolein provided for 8 h was 29% of the daily fat intake of a rat consuming 20.0 g/d of the AIN-93G diet that contains 7.0% fat (11), representing a moderate fat intake, whereas the amount of cholesterol represented a moderately high intake in proportion to the total amount of fat given. The amount of -tocopherol was set at 100% of the daily intake of the vitamin in the AIN-93G diet formulation (11,12). The fatty acid compositions of egg and milk SM are shown in Table 1.

For lymph esterified cholesterol, each free and total cholesterol fraction was analyzed by HPLC using the method of Duncan et al. (13) with a slight modification. For the total cholesterol fraction, 100-µL aliquots of hourly lymph samples were saponified with 300 µL of 33% KOH and 3 mL ethanol at 60°C for 15 min. After the addition of hexane and water, the upper hexane phase was taken, dried under N₂, and redissolved in 500 µL of chloroform:methanol mixture (1:5, v:v). For the free cholesterol fraction, 100-µL aliquots of hourly lymph samples were extracted without KOH saponification as above. Each total and free cholesterol fraction was determined by a Beckman HPLC with System Gold Nouveau software (Beckman Instruments) equipped with a C-18 reversed-phase column (Alltima C18, 5 µm, 4.6 x 150 mm, Alltech Associates) and 507e autosampler (Beckman Instruments). Detection was monitored at 200 nm (Module 168, Beckman Instruments). Isopropanol:acetonitrile:water mixture (60:30:10, by vol) was used as the mobile phase at 1 mL/min. Lymph esterified cholesterol was calculated by the difference between the total and free cholesterol fractions.

    Determination of phospholipid classes in lymph by HPLC. From 200-µL aliquots of lymph samples, phosphatidylserine (PS), phosphatidylethanolamine (PE), PC, lysophosphatidylcholine (lysoPC), and SM were determined simultaneously by a modification of the methods described by Kaduce et al. (14) and Patton et al. (15). Total lipids were extracted by the method of Folch et al. (16), dried under N₂, and redissolved in 500 µL chloroform. Phospholipid classes were separated by a Beckman HPLC with System Gold Nouveau software (Beckman Instruments) equipped with a 4.6 x 250 mm silica column (Adsorbosphere HS Silica, 5 µm, Alltech Associates) and 507e autosampler (Beckman Instruments). The mobile phase consisted of acetonitrile:methanol:phosphoric acid:sulfuric acid (100:4.8:0.864:0.026, by vol) and the flow rate was 0.7 mL/min. Detection was monitored at 202 nm (Module 168, Beckman Instruments). Under these conditions, typical retention times (in min) were: 8.7 for PS, 10.4 for PE, 13.6 for PC, 19.5 for lysoPC, and 22.3 for SM. The concentration of each phospholipid class was calculated from the peak area responses using standard curves established with pure PL classes ranging from 47.6 to 190.5 ng.

    Lymph -tocopherol and fatty acid analyses. Lymph -tocopherol was extracted by the method of Bieri et al. (17). A 100-µL lymph sample was mixed vigorously with 10 volumes of ethanol containing 1% pyrogallol (99%, Acros Organics) as an antioxidant and then with 20 volumes of hexane and 10 volumes of water. After a brief centrifugation at 1000 x g for 10 min, the upper phase was transferred into a vial, dried under N₂, and resuspended in chloroform:methanol (1:3, v:v). -Tocopherol was determined by an HPLC method as detailed previously (18,19). Tocol (Hoffmann-La Roche) was added as an internal standard to monitor extraction efficiency, which generally exceeded 95%.

For lymph fatty acid analysis, total lipids from 100-µL lymph samples were extracted (20) and hydrolyzed with 1 mL of 0.5 mol/L methanolic NaOH in boiling water for 15 min; 17:0 was added to each sample as an internal standard. Lymph fatty acids were saponified and methylated simultaneously with 2 mL of 14% methanolic BF₃, as detailed elsewhere (7,21).

    Statistics. Data are presented as means ± SD. All statistical analyses were performed using PC SAS (SAS Institute). Repeated-measures ANOVA and the least significance difference test were used to compare multiple group means and time-dependent changes within groups. The level of significance was determined at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Lymph flow. In response to lipid infusion, the rate of lymph flow increased significantly in all 3 groups. The hourly rates of lymph flow were 2.3 ± 0.3, 2.1 ± 0.7, and 1.9 ± 0.2 mL/h in rats infused with no SM (control), egg SM, and milk SM, respectively, with no significant differences among the groups. Cumulative lymph volume did not differ (Table 2).

View this table: [in this window] [in a new window]   TABLE 2 Cumulative lymphatic absorption of 14C-cholesterol (14C-CH) and -tocopherol (TP) and output of oleic acid, PC, and SM during duodenal infusion of a lipid emulsion containing no SM or egg SM or milk SM in rats1

View larger version (14K): [in this window] [in a new window]   FIGURE 1 The lymphatic absorption of 14C-cholesterol (14C-CH) (A) and -tocopherol (TP) (B), and the lymphatic output of PC (C) and oleic acid (OA) (D) at hourly intervals for 8 h during intraduodenal infusion of lipid emulsion without SM (No SM) or with either egg SM or milk SM in rats. All values are expressed as means ± SD, n = 5. Means at a time without a common letter differ, P < 0.05.

    Lymphatic absorption of -tocopherol.

    Lymphatic outputs of major phospholipid classes. The outputs of total phospholipid were significantly decreased in rats infused with egg SM and milk SM, compared with controls (Table 3). The lymphatic output of PC was significantly lower in rats infused with egg SM and milk SM than in controls. The rates of PC output were 4.3 ± 0.2, 2.7 ± 0.3, and 2.3 ± 0.2 µmol/h in rats infused with no SM, egg SM, and milk SM, respectively. Similar trends were observed for PI and PE (Fig. 1C). Infusion of egg SM did not lower the lymphatic output of SM, whereas milk SM significantly lowered the output of SM compared with the rats infused with egg SM and controls.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

It is well documented that SM has a high affinity for and preferentially interacts with cholesterol in cell and model membranes (2). The SM-cholesterol interaction is presumably due to the ability of SM to form a hydrogen bond between the amide group of SM and the hydroxyl group of cholesterol (1). The interaction of SM with cholesterol is favored by the saturation of the SM acyl chain, whereas the presence of an unsaturated acyl chain introduces kinks, weakening the interaction (1). Eckhardt et al. (6) showed that milk SM significantly decreased cholesterol uptake by Caco-2 cells and that dietary milk SM even at 0.1% also markedly lowered the absorption of cholesterol in mice. This effect was attributed to the fact that milk SM and other phospholipids with high affinity for cholesterol decrease its micellar solubilization, thereby decreasing the concentrations of cholesterol monomers for uptake by the enterocyte. Also, it is probable that the tighter molecular packing, as produced via interaction of SM with cholesterol, may result in slower desorption of cholesterol and other lipids from the micellar matrix to the enterocyte, as suggested by previous studies with SM vesicles or membranes (22–24). Whether SM inhibits the intestinal absorption of other lipids such as -tocopherol via such molecular interactions remains to be determined.

In a recent study (7), we presented evidence that egg SM lowers the intestinal absorption of cholesterol and -tocopherol in a dose-dependent manner. The present data not only confirm this observation, but also provide new evidence that the efficiency of SM in inhibiting intestinal lipid absorption can vary depending on the food source it is derived from. Although not conclusive, our current data suggest that the acyl chain length of SM is an important factor determining its effectiveness in lowering lipid absorption. The acyl groups of both milk and egg SM are nearly completely saturated, but differ markedly in their chain length. Milk SM contains significantly less 16:0 (23 mol%), but more longer-chain SFA such as 22:0, 23:0, and 24:0, which account for 66% of its total fatty acid content (Table 1). In contrast, egg SM consists mostly of 16:0 (87 mol%) and relatively small amounts of longer-chain fatty acids [18:0 (6%), 20:0 (1.5%), 22:0 (3.0%), and 24:0 (3.0%)]. It is of interest to note that the longer-chain fatty acids (22:0–24:0) in milk SM were not detectable in mesenteric lymph, whereas 16:0 was significantly higher in the lymph obtained during egg SM infusion. This suggests that egg SM is more readily hydrolyzable by sphingomyelinase (SMase) than milk SM.

Our findings here suggest that the degree of saturation and chain length of the SM acyl group can be an important determinant of the inhibitory effect of intestinal lipid absorption because they may influence the interactions of SM with other lipids in the intestinal lumen. Among dietary lipids, SM is unique in that it is not readily hydrolyzed in the intestinal lumen because the activity of alkaline SMase, which is largely responsible for SM hydrolysis, is nearly absent in pancreatic juice or bile in rats, but localized in the brush-border membranes of the distal jejunum and lower in the ileum and colon (25). Furthermore, the enzyme, shown to be optimally active at pH 9, remains inactive under the conditions of the gastric and duodenal lumen. Thus, the hydrolysis of SM in the upper small intestine is a slow, inefficient, and incomplete process (26–28). Consequently, this permits sustained interactions among intact SM, lipolytic enzymes, and lipids in the upper intestinal lumen. Growing evidence suggests that the inhibitory effect of SM on lipid absorption may be associated in part with its slow rate of hydrolysis in the upper small intestine, interfering with micellar formation, solubilization, and transfer (or desorption) of lipids to the enterocyte for uptake. Although little information exists concerning SM’s ability to significantly alter the luminal digestion of lipids, the possibility was suggested that SM interferes with hydrolysis of triacylglycerol (TG) in the intestinal lumen. An earlier study (29) showed that the presence of SM in mixed bile-salt micelles significantly inhibited pancreatic lipase-colipase. Also, SM may inhibit pancreatic (type I) phospholipase A₂ (PLA₂), as suggested by the finding that secretory (type II) PLA₂, which is structurally related to type I, is inhibited by SM (30,31). Such effects of SM on lipolytic enzymes may explain in part the marked lowering of lymphatic lipid absorption and phospholipid output, as observed in this study. Previous studies with intestinal cell lines showed that minimal hydrolysis of TG by pancreatic lipase-colipase was required for stimulation of the cell uptake of cholesterol (32) and that a limited initial hydrolysis of PC to lysoPC by pancreatic PLA₂ facilitates the binding of lipase-colipase to the substrate interface, resulting in a rapid hydrolysis of TG (29,32–34). Thus, the slow and inefficient hydrolysis of SM in the upper small intestine may interfere with the activities of pancreatic PLA₂ and lipase/colipase, which are critical for luminal lipolysis and subsequent formation and transfer of mixed micelles to the enterocyte. Furthermore, the lack of lysoPC resulting from SM-mediated inhibition of PLA₂ may limit the availability of PC necessary for the construction of the chylomicron surface coat and adversely affect the intracellular processing of lipids within the enterocyte. Ample evidence indicates that once taken up by the enterocyte, lysoPC is used for synthesis of PC during chylomicron assembly and facilitates the intracellular reacylation and packaging of lipids and the formation and secretion of chylomicrons (35,36).

At present, it is not known whether dietary SM directly influences the intracellular events leading to chylomicron assembly and secretion. A study with Caco-2 cells in vitro (37) showed that treatment of the cells with SMase inhibited the secretion of large chylomicron-like lipoproteins from Caco-2 cells, without affecting the total amount of cholesterol secreted and that the addition of carboxyl ester lipase, which hydrolyzes ceramide, alleviated the inhibition induced by SMase. Thus, it is possible that ceramide, as derived from the hydrolysis of dietary SM, may regulate the process of chylomicron assembly in the distal jejunum and ileum, where SMase is localized. Evidence from recent studies suggested that ceramide also may play an important role in cholesterol efflux involving ATP-binding cassette transporter A1 (ABCA1) (38). At present, however, it is debatable whether ABCA1 is involved in the apical efflux of cholesterol into the lumen (38–40), thereby decreasing cholesterol absorption.

Milk and eggs are among the rich dietary sources of SM (10,41). The per capita consumption of SM in the United States is estimated to be 300–400 mg/d; milk (dairy products) and eggs provide 220 mg/d of this (10). It remains to be determined whether this level of SM intake significantly affects the intestinal absorption of cholesterol and other lipids in humans. In the present experiment, the dose of SM (10 µmol/h) was set to approximate the estimated intake of SM per meal from a typical diet. This amount is equivalent to 0.025 mg/kJ based on the rat’s energy intake of 297 kJ/d from the estimated food intake of 20 g (AIN-93G diet). For a human consuming 10.45 MJ (2500 kcal)/d, it would be equivalent to a daily intake of 264 mg SM, which approximates the estimated daily intake of SM (10). In the present study, a significant decrease in cholesterol absorption was observed at 2 h and thereafter. The amount of cholesterol absorbed may also depend not only on the absolute amount of SM intake but also on the ratio of SM/cholesterol consumed. In this experiment, the molar ratio of SM/cholesterol was 3.9. Previously, a single intragastric dose of milk SM (6.5 µmol), with varying molar ratios of cholesterol (SM/cholesterol = 0.5–2.6), was shown to be effective in lowering cholesterol absorption in rats, as measured by a dual-isotope plasma ratio method (5). The lowest absorption of cholesterol was observed at the molar ratio of SM/cholesterol, 1:1. This ratio falls within the range of SM and cholesterol intakes in humans, if the daily intakes of SM and cholesterol are estimated at 400 mg (500–600 µmol/d) (10) and 300 mg (780 µmol/d), respectively. Thus, based on the current data and available information, it is probable that a moderate daily intake of SM, as derived from milk and eggs, may lower the absorption of cholesterol and other lipids in humans.

In summary, the results of this study show that under in vivo conditions, milk and egg SM lower the intestinal absorption of cholesterol and fat. In addition, the absorption of -tocopherol is also decreased by both milk and egg SM. Data show that milk SM is more effective in inhibiting the absorption of the lipids. The inhibitory effect of SM appears to be associated with its higher degree of saturation and the longer chain length of its fatty acyl group. Such physical characteristics of SM may hinder the hydrolysis of SM and other lipids and slow the transfer of micellar lipids to the enterocyte for their uptake and absorption. Further studies are warranted to determine whether the high concentrations of SM in milk and egg yolk reduce the intestinal absorption of cholesterol and other lipids in humans. It remains to be determined whether SM from other food sources such as soybeans similarly influences intestinal lipid absorption. Whether a chronic high intake of SM influences the energy balance and vitamin E status is yet to be studied.

	FOOTNOTES

 
¹ Presented in part at Experimental Biology 03, April 2003, San Diego, CA [Noh, S. K. & Koo, S. I. (2003) Milk sphingomyelin (SM) is a more potent inhibitor than egg SM of intestinal cholesterol absorption in rats. FASEB J. 17: A754 (abs.)].

² Supported by USDA National Research Initiative Competitive Grants Program (#96-35200-3207).

³ Present address: Department of Food and Nutrition, Changwon National University, Changwon, Kyongnam, 641-773, Korea.

⁵ Abbreviations used: ABCA1, ATP-binding cassette transporter A1; lysoPC, lysophosphatidylcholine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PLA₂, phospholipase A₂; PS, phosphatidylserine; SM, sphingomyelin; SMase, sphingomyelinase; TG, triacylglycerol.

Manuscript received 21 June 2004. Initial review completed 28 June 2004. Revision accepted 12 July 2004.

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