The results of the current study indicate that in humans, dietary SM supplementation of 1 g/day for a diet containing 240 mg cholesterol/day does not affect cholesterol absorption, synthesis or serum lipids except for an effect of raising serum HDL-cholesterol. Current study represents the first definitive study to measure the lipid profile, cholesterol absorption and synthesis in humans under well controlled background diets. Studies in humans, to date, on the effect of SM on cholesterol metabolism or absorption have been limited despite its potential effect demonstrated in rodents and cell culture. Although there has been a human trial with administration of SM enriched formulated drink, study had been conducted under free living conditions without robust control on background diet .
Effect of dietary sphingolipids on plasma lipids was examined by Ohlsson et al.  in a parallel design of 48 healthy adults who consumed a sphingolipid enriched milk drink containing 975 mg sphingolipid with 700 mg as SM daily for 4 weeks. Consumption of sphingolipids in this amount had no effect on plasma lipids. Previous studies on effect of sphingolipids on postprandial lipid concentrations showed no change in plasma lipid profile. Although our study was only 2 weeks in duration and was 1 g SM/day with a cross over design, we found similar results with the exception of increased HDL-cholesterol with SM dietary enrichment.
Findings from studies with various animal models [13–17] have shown reductions in plasma total and LDL cholesterol along with reduction in intestinal cholesterol absorption. SM supplemented diet (0.5%) for 45 days reduced the hepatic lipids and plasma non HDL cholesterol levels in Zucker fatty rats . Hyperlipidemic APOE*3 Leiden mice consuming a Western diet supplemented with 0.2 and 0.4% sphingolipids for 3 weeks had reduced plasma cholesterol and triglycerides, whereas, lower supplementations (0.1%) had no effect on plasma lipids. When higher levels were fed (1%), cholesterol absorption was reduced by 50% in this animal model leading to a 57 and 58% reduction in plasma total cholesterol and triglycerides respectively . In male Sprague Dawley rats, cholesterol absorption was 19.5% after SM treatment compared to 37.6% in control animals .
Rodent studies have also suggested that the ratio between SM and cholesterol may also be a significant determinant for the inhibitory effect of SM on cholesterol absorption. Cholesterol absorption was reduced by different range of molar ratios of SM and cholesterol between 0.5 and 2.6 in rats . Intestinal cholesterol absorption was 17, 9 and 25% when rats treated with SM and cholesterol at 2.6:1, 1:1 and 0.5:1.0 ratio respectively, compared to 68% when treated with cholesterol alone without SM . However, our study used SM to cholesterol molar ratio of 1.0:0.3 which is closer to one of the molar ratios used in the animal study  but we found no significant change in either cholesterol absorption or synthesis.
It is disappointing that the previous findings in animal and cell culture studies could not be replicated in humans because of the potential that SM might be used as a functional food. There may be several reasons for these species/experimental condition differences in our findings. SM is not rapidly hydrolysed in intestine in rodents. SMase, which is responsible for digesting SM, is present in very low quantities in pancreatic juice and bile from liver of rats . SMase is an alkaline enzyme that is active at pH 9 and hence is inactive in gastric and duodenal lumen. Hydrolysis of SM in the upper small intestinal lumen is very deliberate, ineffective and incomplete which might be responsible for the inhibitory effect of cholesterol absorption by SM in animal models . In contrast, in humans the hydrolysis of SM is faster and more efficient than rodents as SMase is secreted into the bile . Recently it has been demonstrated that humans can digest and absorb most of the SM consumed in normal diets . It has also been reported that enzymes responsible for digestion of SM including intestinal alkaline SMase and ceramidase are well expressed in humans [25, 26]. Unlike animals, SMase is expressed both in the intestine as well as in liver [21, 23, 27] in humans. However, the digestion of sphingolipid by these enzymes remains poorly defined. The effects of these enzymes may be affected not only by the presence of other lipids but also the ratio between bile salt and SM as well as bile salt and ceramide [28, 29].
Although it has been reported that SM could bind to luminal cholesterol and resist solubilization into bile salt micelles [11, 12], we found no changes in the concentrations of luminal bile acids and cholesterol in humans in the current study. These findings suggest that the inhibitory effects of SM on cholesterol absorption in rodents is likely different than the effects in humans. Although this is the first definitive study of the effect of SM on cholesterol metabolism and absorption in humans, there were some limitations of current study: 1. The presence of multiple enzymes in the human gut including SMase were not analysed which may explain why we did not see the effects of SM in humans found in rodents. 2. Our sample size was small but given the minimal changes observed a larger sample would likely have shown similar results. 3. There may have been confounding problems with solubilizing SM with olive oil as this may not truly represent how SM might be present in the matrix of a high SM diet; however, this was only practicable way to ensure added SM in the diet. 4. The dose of SM may not have been sufficient to observe changes in cholesterol absorption/metabolism given the proposed higher efficiency of SMase in humans vs. rodents.
The current study included only healthy subjects. More studies may be needed to determine whether SM affects cholesterol absorption and plasma lipids in hyperlipidemic subjects. The dose of SM supplementation used in the current study was 1 g/day and was well tolerated as observed in a previous human study in humans consuming 975 mg per day of sphingolipid or 4 weeks . To determine an inhibitory effect on cholesterol absorption and reduction in plasma lipids, the dose could be increased provided that the increased dose does not cause any deleterious effects. Unfortunately, the cost of such supplementation may make additional supplement prohibitive and identifying foods with high SM content might be a more practicable approach.
In conclusion, consumption of 1 g/day of SM added to the diet in humans does not affect cholesterol absorption, synthesis and blood lipid profile except an increase in HDL. Hence, further studies are needed to investigate the safety and efficacy of SM consumption with optimal higher doses.