Micellar distribution of cholesterol and phytosterols after duodenal plant stanol ester infusion

Markku Nissinen, Helena Gylling, Matti Vuoristo, and Tatu A. Miettinen

Department of Medicine, Division of Internal Medicine, University of Helsinki, Helsinki FI-00029 HUCH, Finland


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Properties of the intestinal digestion of the dietary phytosterols, cholesterol and cholestanol, and the mechanisms by which phytosterols inhibit the intestinal absorption of cholesterol in healthy human subjects are poorly known. We have studied the hydrolysis of dietary plant sterol and stanol esters and their subsequent micellar solubilization by determining their concentrations in micellar and oil phases of the jejunal contents. Two liquid formulas with low (formula 1) and high (formula 2) plant stanol concentrations were infused via a nasogastric tube to the descending duodenum of 8 healthy human subjects, and intestinal contents were sampled for gas-liquid chromatographic sterol analysis 60 cm more distally. During the duodenal transit, phytosterol esters were hydrolyzed. This was especially profound for sitostanol, as its esterified fraction per milligram of sitosterol decreased 80% (P < 0.001) in formula 1 and 61% (P < 0.001) in formula 2. Contrary to that, esterified fraction of cholesterol per milligram of sitosterol was increased fourfold (P < 0.001) in formula 1 and almost sixfold (P < 0.001) in formula 2, whereas that of cholestanol remained unchanged. Percentages of esterified sterols and stanols in total intestinal fluid samples were higher after the administration of formula 2 than of formula 1. Esterified cholesterol and stanols accumulated in the oil phase, and free stanols replaced cholesterol in the micellar phase. At high intestinal plant stanol concentrations, cholesterol looses its micellar solubility possibly by replacement of its free fraction in the micellar phase by hydrolyzed plant stanols, which leads to a decreased intestinal absorption of cholesterol.

intestinal absorption of cholesterol; phytosterol esters; micelle formation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MICELLE FORMATION OF DIETARY lipids and their digestion products in the presence of bile acids and phospholipids is crucial for the intestinal absorption of cholesterol from the gut lumen to the intestinal epithelial cells (30, 31). Absorption of both free and esterified cholesterol has been suggested (3, 17, 29, 34) to be protein-mediated. Scavenger receptor class B type I in the small-intestine brush-border membrane was suggested to facilitate the uptake of dietary free and esterified cholesterol, triglycerides, and phospholipids (12). The percentage of absorbed dietary free cholesterol is higher than that of the esterified form because of its better micellar solubility (3). Thus to improve the cholesterol absorption, dietary esterified cholesterol should be hydrolyzed by bile acid-activated pancreatic cholesterol hydrolase. Contrary to cholesterol, dietary plant sterols (phytosterols), including sitosterol, stigmasterol, and campesterol, occur in diet in free, esterified, and glycosidic forms and are only weakly absorbed from the gut. Compared with cholesterol, these sterols have different intraluminal solubilization, different uptake, or intracellular processing by the enterocytes (3, 13, 28). Absorption of cholestanol, campesterol, and sitosterol is directly related to cholesterol absorption efficiency (24, 35). That dietary plant sterols lead to a reduction in serum cholesterol was first shown by Pollak (27), and their inhibitory effect on cholesterol absorption was evidenced by Grundy et al. (6). The plant stanols sitostanol and campestanol are 5alpha -saturated derivatives of sitosterol and campesterol. These stanols appeared to be almost unabsorbable, and they diminished both intestinal absorption and the serum level of cholesterol more effectively than their unsaturated parent plant sterols both in experimental animal studies (16, 32, 33) and human studies (2, 14, 15). Poorly soluble plant stanols were converted to highly fat soluble by their esterification with rapeseed oil fatty acids (39). This resulted in development of plant stanol ester margarine for serum cholesterol lowering. Daily consumption of rapeseed oil margarine (spread) with and without plant stanol esters revealed that the plant stanols inhibited cholesterol (and plant sterol) absorption, cholesterol elimination as neutral sterols, and, despite a compensatory increase in cholesterol synthesis, lowered serum total and low density lipoprotein cholesterol (7, 10, 11, 21). Effective impairment of cholesterol and plant sterol absorption by plant stanol esters apparently provides that the esters are hydrolyzed by intestinal sterol ester hydrolase and that released free plant stanols interfere subsequently with micellar solubilization of cholesterol and plant sterols. In addition, excessive amounts of plant stanols may interfere also with hydrolysis of dietary cholesterol and plant sterol esters during food digestion. However, although plant stanols are believed to interfere with micellar solubilization of cholesterol and other sterols, no studies are available on intestinal distribution of sterols in oil and micellar phases during ingestion of normal amounts of plant sterols or especially after consumption of large doses of stanol ester margarine. Therefore, we carried out an intestinal plant stanol ester perfusion study to clarify the hydrolysis of cholesterol, cholestanol, plant sterol and stanol esters, and their distribution to the micellar and oil phases in the upper part of the small intestine in healthy subjects. The studies were performed with stanol esters, but sterol esters might give similar results.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects. Eight healthy medical students (4 females, 4 males) with normal liver and thyroid function and with no evidence of diabetes or gastrointestinal disease or hypolipidemic medication volunteered for the study. Their body mass indexes were within normal limits (19.3-23.5 kg/m2). The study protocol followed principles of the Helsinki Declaration. Subjects were informed of the nature and purpose of the investigations and the study, which was approved by the Ethics Committee of the Department of Medicine, Helsinki University Central Hospital.

Experimental design. A triple-lumen radiopaque tube (model AN 20; H. W. Andersen Products) was used for the infusion of the test formulas and for the collection of the samples from the proximal jejunum for the sterol analysis. The study subjects fasted overnight. The tube was placed under X-ray control such that the proximal (infusion) outlet was in the middle of the second part of duodenum, i.e., adjacent to the ampulla of Vater. The second and third outlets were located 10 and 60 cm below the first one. Two kinds of liquid formulas were used (Table 1). The liquid formula 1 was prepared by sonication of 10 g of rapeseed oil margarine, one egg yolk, 4.5 g of glycerol-L-mono-oleate (Fluka), and 200 ml of water in a final volume of 218 ml. The liquid formula 2 was prepared by sonication of 0.9 g of plant stanols (sitostanol/campestanol = 3.5) as their esters (Raisio Group, Raisio, Finland) in 10 g of the rapeseed oil margarine, one egg yolk, 4.5 g of glycerol-L-mono-oleate, and 200 ml of water in a final volume of 218 ml. These formulas were infused at a constant rate of 50 ml/h through the proximal outlet of the tube by using an infusion pump (model 871-102; B. Braun, Melsungen, Germany). The liquid formula 1 was administered first for a period of 1 h. After an equilibration period of 40 min, followed by infusion of formula 2, infusion was started via the same route at the same speed for an additional period of 2.5 h. During both infusions, samples were aspirated at intervals of 15 min via the third lumen. Jejunal samples (5-10 ml) were placed in a water bath at 80°C for 5 min to destroy the lipase and hydrolase activities and then were stored at -20°C until analyzed.

                              
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Table 1.   Absolute values of total sterols and their ratios to sitosterol in test meal formulas and proximal jejunum

Chemical analysis. Separation of the oil and micellar phases and the sediment of the formulas and total intestinal fluid were obtained by ultracentrifugation of samples at a speed of 15,000 rpm for 1 h at 37°C. Samples from both the total intestinal fluid and, after ultracentrifugation, from the oil droplet on the surface of the ultracentrifuged sample and from the micellar phase were collected. After removal of the oil phase and most of the micellar phase, sediment in a measured amount of micellar phase was also sampled. The samples and those of the two formulas were extracted with chloroform-methanol, evaporated, and subjected to thin-layer chromatography in a diethyl ether/heptane (50:50) solution to separate free and esterified sterols, which were then eluated. Ester phases were saponified with 2 M KOH in 90% ethanol and nonsaponifiable lipids were extracted from the alkaline alcoholic-water medium with hexane. After the addition of the internal standard 5alpha -cholestane, the sterol fractions were silylated, and the sterols were quantified by gas-liquid chromatography on a 50-m long SE-30 capillary column (Ultra 2 column; Hewlett-Packard) with an automated electronic integrator (Sigma 10; Hewlett-Packard, Palo Alto, CA.) for measurement of peak areas (18-21).

Calculations. The esterification percentages of cholesterol, cholestanol, plant sterols (sitosterol and campesterol), stanols (sitostanol and campestanol), and their free, esterified, and total concentrations were calculated in formulas, total intestinal fluids, and in micellar, oil, and sediment fractions of aspirated samples from the proximal jejunum (third outlet). Because sitosterol was the largest plant sterol fraction in formula 1 and was probably negligibly absorbed during passage of the short upper intestinal segment, it was used as the internal marker to correct dilutions. Use of sitosterol as an unabsorbable marker in intestinal perfusion studies has been thoroughly examined and validated (4, 7, 8, 23). Thus the absolute values of sterols and their esterified forms were calculated as ratios to sitosterol to evaluate effects of the different intestinal plant stanol contents and the duodenal transit (Tables 1 and 2). The free sterols in micellar fractions of the upper jejunal samples were calculated as %portion of the total amount of the corresponding sterol (Fig. 1). The sterol composition of the intestinal micellar and oil phases was also calculated on a molar percent (M%) basis. Because the sterol concentrations in the sediment fractions were inconsistently different from those in the respective micellar phase, the difference between the total and micellar phases was considered to represent the oil phase. The term phytosterol in this text will mean saturated (5alpha ) or unsaturated (Delta 5) sterols or their mixture. Statistical analyses were carried out using Student's unpaired (Tables 1-3) and paired t-test (Tables 1-4 and Fig. 1). Logarithmic transformations were used with skewed distributions. The differences between the means were considered statistically significant if the P value was < 0.05. Mean ± SE values are given in the text, Tables 1-4, and Fig. 1.

                              
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Table 2.   Esterified sterols per sitosterol in test meal formulas and proximal jejunum



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Fig. 1.   Columns show percentage amounts of micellar and oil-free and esterified sterols in proximal jejunal aspirates of test meal formulas (F) 1 and 2. Concentrations (mg/dl) of sterols in total intestinal fluid samples are on the tops of the columns (mean ± SE). Free micellar sterols, percentage of total intestinal sterols, are marked within the columns (mean ± SE). Asterisks on the right hand side of the columns indicate statistical significances of esterification percentages of micellar and oil phase sterols between formulas 1 and 2, and those below the columns indicate total esters, percentage of total intestinal sterols. *P < 0.05, **P < 0.01, ***P < 0.001 from the respective value of formula 1 by paired t-test.


                              
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Table 3.   Sterol esters and esterification in test meal formulas and proximal jejunum


                              
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Table 4.   Concentration of jejunal free and esterified sterols in oil and micellar phases after infusion of formulas 1 and 2 test meals


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Test meal formulas. The liquid formulas 1 and 2 contained similar contents of cholesterol, whereas those of other sterols, especially that of campestanol and sitostanol, were higher in formula 2 than in formula 1 (Table 1). Contrary to the high esterification percentages of the phytosterols (64-92%) those of cholesterol (5.3%) and cholestanol (10.6%) were lower in formula 1 and the esterification percentage of stanols was higher than that of sterols in both formulas (Table 3). Owing to transesterification of the stanol preparation, esterification percentage of cholestanol (55.5%) and phytosterols (76-96%) were higher in formula 2 than in formula 1. In formula 1, absolute concentrations of cholesterol, campesterol, and sitosterol esters were similar (4-5 mg/dl), but the sum of phytosterol esters (10.4 mg/dl) was more than twice the content of cholesterol esters (Table 3). Ester contents of plant sterols, especially of campestanol and particularly of sitostanol, exceeded that of cholesterol in the formula 2 (Table 3). Cholesterol (90-94%), cholestanol (90-98%) and plant sterols and stanols (over 99%) were in the oil phase in both formulas 1 and 2, respectively.

Sterol levels in proximal jejunal contents. Comparing phytosterol contents in the intestinal fluid with those in formula 1 showed that the concentrations were diluted three to five times the highest dilutions (fivefold) being found for the plant stanols (for sitosterol 2.92 ± 0.66) (Table 1). Use of sitosterol as an unabsorbable marker showed that cholesterol per milligram of sitosterol was increased from 10.4 to 22.5 mg and that of cholestanol was about threefold in the proximal jejunum compared with those in formula 1, probably due to biliary secretion (Table 1). The respective phytosterol values tended to decrease, although the use of campesterol as the unabsorbable marker showed an opposite trend. After formula 2, the dilution of the phytosterols was two to three times, again the highest (threefold) for plant stanols (for sitosterol 2.18 ± 0.27; P = 0.06 vs. formula 1). Despite the slightly higher dilution after formula 1 infusion, jejunal cholesterol and cholestanol concentrations were similar in the two studies, whereas phytosterol levels were higher after formula 2 than formula 1 as in the infusates. Cholesterol standardized by infused sitosterol was increased, cholestanol and campesterol were unchanged, and plant stanols tended to decrease compared with the ratios in formula 2 (Table 1).

Sterol esters and their hydrolysis during duodenal transit. Comparison of the infused sterols with those in the intestinal fluids of the proximal jejunum (Table 3) revealed that jejunal levels of cholesterol and cholestanol esters were similar to the infused ones, whereas those of phytosterols were markedly decreased and the percentages of esterified cholesterol and cholestanol tended to increase and those of other esterified sterols were markedly decreased after infusion of formula 1. Thus 80% of campesterol and 60% of sitosterol esters had been hydrolyzed, respective values being 54% for campestanol and 58% for sitostanol. After formula 2, the concentration of jejunal esterified cholesterol was almost threefold higher than that of the infusate, those of the other sterols being markedly diluted. All the jejunal sterol ester concentrations were clearly higher after formula 2 than formula 1. The %ester of cholesterol was increased from 6.6% in the infusate to 26.4% in the intestinal contents, whereas those of other sterols were markedly decreased, indicating that 39-44% of the sterol esters, including the two stanol esters, had been hydrolyzed during the passage of the infusate through the first 60 cm of the upper small intestine. Owing to markedly high campestanol and sitostanol ester contents of the infusate, their mean absolute hydrolysis was highest from among the noncholesterol sterols.

The absolute amount of esterified cholesterol in relation to total sitosterol was increased in the proximal jejunum by a factor of almost four (P < 0.001) of that in formula 1 and by fivefold (P < 0.001) after formula 2, the actual increase being probably over fivefold higher by a similar sitosterol intake of formulas 1 and 2 (Table 2). For the four phytosterols, the esterification values were reduced by 58-82% after the formula 1 and less so by 39-61% after formula 2 (Table 2).

Sterols in oil and micellar phases of intestinal contents. Relative distributions of free and esterified phytosterols are shown for the oil and micellar phases in Fig. 1. Total concentration of each sterol in the intestinal fluid is shown on the top of each column. Respective concentrations of the free and esterified sterols in the oil and micellar phases are shown in Table 4.

Respective concentrations of intestinal cholesterol and cholestanol (about 1% cholesterol) were similar after infusion of the two formulas (Fig. 1). The relative distributions of their free and esterified forms were similar in the respective oil and micellar phases after infusion of formula 1, over two-thirds of sterols being found as free sterols in the micellar phase. Respective distributions of the two sterols were also similar after formula 2, but proportions of esterified sterols had markedly increased in the oil phase, and the respective amounts of free, but not esterified, micellar sterols had decreased. Micellar free cholesterol concentration was decreased by 16.2 ± 5.8 mg/dl (P = 0.06) (Table 4). However, micellar-free cholesterol comprised 82.7 ± 3.6% of total intestinal cholesterol in the formula 1 study and 56.2 ± 6.4% (P < 0.001) in the sitostanol ester-enriched formula 2 study. The respective values for cholestanol were 69.7 ± 7.0 vs. 45.5 ± 3.7% (P < 0.05) (Fig. 1). Distribution of the two plant stanols differed from that of the respective sterols mainly by surprisingly large micellar ester fractions after formula 1.

Infusion of formula 2 increased intestinal plant stanol concentrations >200 times and those of plant sterols 2-3 times compared with those after infusion of formula 1 (Tables 1 and 4, Fig. 1). As in the cases of cholesterol and cholestanol, the infusion of plant stanol ester-enriched formula markedly increased the oil phase fractions due to enlargement of all esterified phytosterols, especially of stanols, whereas the respective proportions of micellar fractions were decreased most consistently for free sterols but also for esters of phytosterols, except campesterol (Fig. 1). Despite the relative reduction of micellar sterols, the concentrations of micellar-free and esterified phytosterols were increased compared with the respective values after infusion of formula 1 (Table 4). The micellar concentration of esterified campesterol increased almost three times, the oil phase free campesterol increased four times, and the esterified one increased 16 times. The plant stanol levels increased up to hundreds of times in both phases by formula 2 vs. formula 1.

M% of intestinal sterols calculated for the sterols of Table 4 showed that M% of cholesterol was decreased in both the oil (from 87 to 24) and micellar phases (from 92 to 39) by formula 2. The respective values increased markedly for campestanol, both in the oil (from 0.1 to 18) and micellar (from 0.2 to 12) phases and especially for sitostanol, also in both the oil (0.9 to 52) and micellar (0.5 to 43) phases.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The major difference between the two test meal formulas was the 200 times higher concentrations of esterified plant stanols in formula 2 than formula 1. We found that the esterification percentage of cholesterol only tended to increase after infusion of formula 1, but it was significantly increased after the formula 2 infusion during the transit of the infusate from the proximal duodenum to the proximal jejunum (Table 3). Effective absorption of unesterified cholesterol in the upper small intestine could have increased the esterified percentage of cholesterol. However, calculation of absolute amounts of sterol esters in milligram per milligram of total sitosterol (plant stanols could have been better unabsorbable markers, but their baseline contents were low in formula 1) in the formulas and proximal jejunal contents showed, as in the earlier study (23) that the amount of esterified cholesterol was four times increased in the jejunal contents compared with that in infused formula. In the previous study (23), in contrast to the present one, infused cholesterol was mainly esterified (98%) and sitosterol mainly unesterified (95%); the respective increase of jejunal esterified cholesterol was only ~35%, whereas that of sitosterol was fourfold. Secretion of bile into the intestinal contents increased unesterified cholesterol fraction, because biliary cholesterol comprises ~97% of gallbladder bile sterols of, which virtually all are unesterified (20). Contrary to cholesterol, esters of the phytosterols of both formulas were significantly hydrolyzed in the upper intestine, whereas that of cholestanol tended to increase after infusion of formula 1. Absolute amount of sitosterol esters was reduced by ~60%, and those of other phytosterols were reduced by up to 80%, indicating that the plant sterol esters of our normal food are rapidly hydrolyzed after food intake already in the upper part of the small intestine. Analogous to cholesterol, cholestanol exhibited percentage of low ester in formula 1 but its relationship to sitosterol was increased fourfold in the intestinal contents. The method of analysis used here allows assessment of the relative contributions of different sterols to the total sterol composition, but it does not allow assessment of the absolute amounts of different sterols and stanols in the intestinal contents.

Infusion of stanol esters increased the absolute amount of cholesterol esters significantly by a factor of five, in contrast to fourfold after formula 1, but the increased sitosterol infusion also indicates a higher absolute increase of cholesterol esters. Infusion of formula 2 reduced esters of cholestanol and phytosterols somewhat less than after formula 1. Thus even in the presence of excessively large amounts of intestinal stanol esters, hydrolysis of minor sterol esters is effective, and stanol ester hydrolysis occurs rapidly already in the short, upper intestinal segment (Fig. 1, Table 3). The significant increase of cholesterol esters in relationship to sitosterol in the small intestine by the addition of a large stanol ester amount is a surprising finding suggesting an inverted reaction in ester hydrolysis. However, hydrolysis may also occur in the lower part at the small intestine, because esterified percentage of cholesterol is low in excreta of colectomized patients (25).

Esterified amounts of sterols in the oil fractions were markedly increased in the proximal jejunal contents after infusion of the plant stanol esters with concomitant marked absolute increase of free plant stanols and decrease of cholesterol in the micellar fractions. A decrease of the cholesterol content of the intestinal micelles after formula 2 infusion was also evident as calculated on an M% basis. Incorporation of free stanols into the duodenal and jejunal micelles may thus prevent entry of free cholesterol into micelles, resulting also in accumulation of unesterified cholesterol and other sterols in the oil phase. The increase of free sterols and fatty acids may retard hydrolysis of all sterol esters and could even contribute to increased formation of cholesterol esters, possibly by competition with cholesterol as a substrate of the pancreatic cholesterol esterase. Accordingly, sitostanol may enhance hydrolysis of various plant sterols over that of cholesterol, resulting in accumulation of esterified cholesterol. Replacement of cholesterol in the micellar phase by stanols is crucial in preventing cholesterol absorption because that occurs from the intestinal micellar phase (5). Solubilization of stanols, particularly in lecithin micelles, reduced cholesterol absorption by 37% in humans (26). Although Heinemann et al. (13) have shown that esterified cholesterol also is taken up by the enterocyte, the proportion of cholesterol absorbed in esterified form may be much less compared with that of free cholesterol because the esterified form has a poor solubility in the bile acid micelle (13, 15). In general, esterified cholesterol has been believed to be hydrolyzed before absorption (36, 38). Our findings on the increasing esterification percentage of cholesterol during the duodenal transit already at low, but especially at high, plant stanol concentrations suggest that the esters are only weakly solubilized into the micellar phase and that they accumulate in the oil phase and thus are less effectively absorbed into the enterocytes. Furthermore, the length of the side chain and hydrogenization of the unsaturated phytosterol Delta 5-double bond in the stanol molecule led to increased hydrophobicity and solubility into the micelle (1, 13) and, hence, enable the replacement of cholesterol from the micelle.

Cholestanol and plant stanols have a lower intestinal absorbability than cholesterol due to the saturated Delta 5-double bond of 5alpha -stanols (1, 37). Our results showed a higher esterification percentage for cholestanol in formula 2 than in formula 1, whereas that of cholesterol was the same in the two formulas. However, in the proximal jejunum, the relative distributions in oil and micellar-free and esterified fractions were similar to cholesterol. Additionally, the micellar-free sterol proportion of both cholesterol and cholestanol was lower with formula 2 than with formula 1.

In conclusion, our results suggest that the sitostanol ester is effectively hydrolyzed and entered into the bile acid micelles already during the duodenal transit. At a high sitostanol ester concentration, it is owing to its rapid hydrolysis that plant stanols replace cholesterol in the micelle and increase accumulation of cholesterol and its esters in the oil phase. These events may be essential for the reduction of cholesterol absorption caused by sitostanol ester.


    ACKNOWLEDGEMENTS

The authors thank Leena Kaipiainen, Orvokki Ahlroos, Pia Hoffström, Anne Honkonen, and Ritva Nissilä for expert technical assistance.


    FOOTNOTES

The study was supported with grants from the Helsinki University Central Hospital.

Address for reprint requests and other correspondence: T. A. Miettinen, Dept. of Medicine, University of Helsinki, PO Box 340, Helsinki FI-00029 HUCH, Finland.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpgi.00446.2001

Received 16 October 2001; accepted in final form 23 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 282(6):G1009-G1015
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