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© 2006 American Society for Nutrition J. Nutr. 136:2135-2140, August 2006

Biochemical, Molecular, and Genetic Mechanisms

Reduction of Cholesterol Absorption by Dietary Plant Sterols and Stanols in Mice Is Independent of the Abcg5/8 Transporter1,2

Torsten Plösch3,4,*, Janine K. Kruit3,4, Vincent W. Bloks4, Nicolette C. A. Huijkman4, Rick Havinga4, Guus S. M. J. E. Duchateau5, Yuguang Lin5 and Folkert Kuipers4

4 Center for Liver, Digestive and Metabolic Diseases, Laboratory of Pediatrics, University Medical Center Groningen, Groningen, The Netherlands and 5 Unilever Food and Health Research Institute, Vlaardingen, The Netherlands

* To whom correspondence should be addressed. E-mail: t.plosch{at}med.umcg.nl.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Dietary supplementation with plant sterols, stanols, and their esters reduces intestinal cholesterol absorption, thus lowering plasma LDL cholesterol concentration in humans. It was suggested that these beneficial effects are attributable in part to induction of genes involved in intestinal cholesterol transport, e.g., Abcg5 and Abcg8, via the liver X receptor (LXR), but direct proof is lacking. Male C57BL/6J mice were fed a purified diet (control), diets containing cholesterol (0.12 g/100 g) only, or in combination with either plant sterols or stanols (0.5 g/100 g) for 4 wk. Plant sterols and stanols dramatically increased neutral fecal sterol excretion (2.2 and 1.4-fold, respectively, compared with cholesterol-fed mice; P < 0.05). Cholesterol and cholesterol ester concentrations were higher in livers of mice fed cholesterol compared with controls (+135% and +925%; P < 0.05). Plant sterols and stanols completely prevented cholesterol accumulation as well as induction of LXR target genes in liver. Feeding plant sterols and stanols did not alter intestinal expression of Abcg5, Abcg8, or other LXR target genes nor of Npc1l1. Fractional cholesterol absorption in Abcg5–/– mice was reduced to the same extent by dietary plant sterols (49%) as in wild-type littermates (44%). Plant sterol and stanol-induced reduction of cholesterol absorption in mice is not associated with upregulation of intestinal LXR target genes nor is it influenced by Abcg5-deficiency. Our data indicate that dietary plant sterols and stanols inhibit cholesterol absorption within the intestinal lumen independently of LXR.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Dietary plant sterols and plant stanols have been recognized as efficient modulators of plasma LDL cholesterol concentrations in humans for decades (1,2). In contrast to cholesterol, these compounds are only poorly absorbed from the intestine. Based on their structural similarity to cholesterol, it was postulated that plant sterols and plant stanols physically interfere with intestinal cholesterol absorption, e.g., by interference with cholesterol micellization in the intestinal lumen (3–5).

Recently, proteins involved in cholesterol absorption as well as in intestinal sterol excretion were identified. Niemann-Pick C1-Like protein 1 (NPC1L1)6 is considered the target of the cholesterol absorption inhibitor ezetimibe and was shown to be responsible for the majority of cholesterol uptake into enterocytes (6,7). Recent findings indicate that plant sterols are also taken up by enterocytes via NPC1L1 (7). In the enterocyte, cholesterol is readily esterified by the action of acyl-CoA:cholesterol acyltransferase 2 (ACAT2) and released into lymph in association with chylomicrons. In contrast, plant sterols and stanols are effectively excreted back to the intestinal lumen by heterodimers of the ATP-binding cassette transporters, ABCG5 and ABCG8, present at the apical membrane of the enterocyte (8,9). ABCA1 is also highly expressed in the intestine, mainly at the basolateral domain of the cell membrane, but its role in sterol absorption is unclear (10–12). Expression of ABCA1, ABCG5, and ABCG8, but not of NPC1L1, is under control of a nuclear receptor, the liver-X-receptor (LXR), activated by oxysterols (13,14). Activation of LXR by synthetic ligands in mice was shown to increase fecal neutral sterol loss and to reduce fractional cholesterol absorption, as a consequence of increased Abcg5/Abcg8 expression, leading to enhanced cholesterol excretion back into the intestinal lumen (14,15).

The discovery that cholesterol absorption can be actively regulated at the level of the enterocyte opens the possibility that plant sterols and plant stanols, in addition to their postulated physicochemical effects (3–5), also influence cholesterol absorption by regulating expression of transport proteins. Specifically, plant sterols and stanols may act as LXR agonists either directly or after their conversion into oxyphytosterols or oxyphytostanols, respectively. Plant sterols and stanols were shown to act as LXR activators in in vitro experiments (16,17) and for very specific sterols, also in vivo in mice (18). Cell lines frequently lack ABCG5/ABCG8 expression and therefore may accumulate higher plant sterol and stanol levels than enterocytes in vivo.

To test whether plant sterols and stanols present in functional food items are able to induce LXR-activated gene expression in vivo, we fed C57BL/6 mice a purified diet virtually free of cholesterol and the same diet enriched with cholesterol (0.12 g/100 g), cholesterol and plant sterol fatty acid esters (0.12 and 0.83 g/100g), or cholesterol and plant stanol fatty acid esters (0.12 and 0.83 g/100 g) for 4 wk. The plant sterol and stanol fatty acid ester doses were equivalent to 0.5 g/100 g sterol or stanol, respectively. A purified diet with the established LXR agonist T0901317 (0.015 g/100 g) was used as a positive control for gene expression studies (19). Furthermore, we measured intestinal gene expression profiles and fractional cholesterol absorption in Abcg5 knock-out mice and their wild-type littermates fed both the cholesterol diet and the cholesterol diet enriched with plant sterols. Enterocytes in Abcg5 knockout mice are not protected against plant sterol accumulation; as a consequence, they should be more susceptible to LXR-mediated activation of gene expression by plant sterols.

The aim of this study was to determine whether the well-characterized inhibitory action of plant sterols and plant stanols on intestinal cholesterol absorption is dependent on the activation of LXR in different mouse models.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Animal experiments. Male, 3-mo-old C57BL/6J mice were purchased from Harlan. Mice were housed in temperature-controlled rooms (21°C) with 12-h light cycling and consumed a purified diet and water ad libitum. The composition of the purified diet (prepared by Unilever) is given in Table 1. All mice were fed the purified control diet for 2 wk (run-in period); then, they were assigned to 1 of 5 treatment groups (n = 6) based on their body weights and were fed the specific diets (control, cholesterol diet, plant sterol diet, plant stanol diet, T0901317 diet) for 4 wk. T0901317 was purchased from Cayman Chemicals.


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  		TABLE 1 Composition of purified diets1–3

 
At the end of the experiment, feces were collected for 24 h. Mice were anaesthetized by i.p. injection with Hypnorm (fentanyl/fluanisone, 1 mL/kg) and diazepam (10 mg/kg). Bile and tissues were collected as described (20). All experimental procedures were approved by the local Ethical Committee for Animal Experiments of the University of Groningen.

    Analytical procedures. Biliary bile salt concentrations were measured enzymatically (21). Phospholipids and cholesterol in bile were determined as described by Böttcher et al. (22) and Gamble et al. (23), respectively, after extraction according to Bligh and Dyer (24). The same extraction method was applied for hepatic lipids, after which commercially available kits were used for the determination of unesterified cholesterol (Free cholesterol C, 279–47106, Wako), and for total cholesterol and triglycerides (Cholesterol CHOD-PAP, 11489232–216, and Tri/GB, 12146029–216, respectively; Roche). Plasma lipids were measured using the same kits. Cholesterol ester concentrations were calculated from total and unesterified cholesterol concentrations.

Feces were weighed and homogenized to a powder. Aliquots of fecal powder were used for analysis of total neutral sterol and bile salt content according to Arca et al. (25) and Setchell et al. (26), respectively.

    Fractional cholesterol absorption in Abcg5–/– mice. Fractional cholesterol absorption in Abcg5–/– mice and wild-type littermates was measured using the plasma dual-isotope ratio method as previously described (27). Male Abcg5–/– mice and littermate controls were housed as described above. All mice were fed the purified diet for 2 wk (run-in period). Then, mice were fed the purified diet enriched in cholesterol or in cholesterol and plant sterols (Table 1) for 1 wk. Subsequently, mice were administered an i.v. injection of 2.4 µCi of 3H-cholesterol (NEN Life Science) dissolved in Intralipid (20%; Fresenius Kabi) and an oral dose of 1.2 µCi of 14C-cholesterol (Amersham Bioscience) dissolved in medium-chain triglyceride oil. 14C and 3H activity was measured by liquid scintillation counting. Blood samples obtained by retroorbital puncture 48 h after administration were used for the calculation of cholesterol absorption.

    RNA isolation and PCR procedures. Total RNA was extracted from frozen tissues with TriReagent (Sigma) and quantified photometrically. cDNA synthesis was performed using recombinant M-MLV reverse transcriptase (10 U/µL), the appropriate buffer, dNTPs (500 µmol/L), random nonamers (1 µmol/L), RNAse inhibitor (2 U/µL; all from Sigma) and total RNA (50 ng/µL). The reaction mix was incubated for 10 min at 25°C for primer annealing, 60 min at 37°C for synthesis, and 5 min at 94°C to denature the RT enzyme. Real-time quantitative PCR was performed as previously described (20). Primers (Invitrogen) and fluorogenic probes (Eurogentec) used in these studies were described elsewhere (Srebp1a, Srebp1c, Srebp2, Srb1, Acat2, Hmgcr, Cyp7a, Abca1, Abcg5, Abcg8, 18S rRNA (20); Ldlr (28); Npc1l1 (29). All data were subsequently normalized to 18S rRNA, which was analyzed in separate runs.

    Statistics. Statistical analyses were performed using SPSS 10.1 for Windows. Differences among the dietary groups (control, cholesterol, plant sterol, plant stanols) were evaluated using Kruskal-Wallis analysis followed by the Mann-Whitney-U-test. The positive control (T0901317 diet) was not included in the statistical analysis. Data presented are means ± SD. Differences with P < 0.05 were considered significant.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Fecal neutral sterol excretion. Growth rates of male C57BL/6J mice fed the purified diet with or without cholesterol, cholesterol and plant sterols, or cholesterol and plant stanols for 4 wk did not differ (data not shown), indicative of similar food intake in all groups. As anticipated, fecal neutral sterol excretion in mice fed the cholesterol diet was increased by 56% and that of bile salts by 131%; the latter is indicative of increased bile salt synthesis. The addition of plant sterols and stanols induced a massive further increase in fecal neutral sterol output compared with mice receiving the cholesterol diet only (2.2- and 1.4-fold for plant sterols and plant stanols, respectively; Fig. 1). Plant sterols reduced bile salt excretion by 31%. For comparison, treatment of mice fed the control diet with the LXR agonist T0901317 stimulated fecal neutral sterol and bile salt loss 3.6- and 1.7-fold, respectively, compared with mice fed the control diet only (data not shown).


Figure 1
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  		Figure 1  Fecal sterol output of C57BL/6 mice fed the control diet alone or supplemented with cholesterol with or without plant sterols or stanols for 4 wk. Neutral sterols do not include plant sterols and stanols. Values are means ± SD, n = 6. aDifferent from control; bdifferent from cholesterol, P < 0.05.

 
    Plasma and hepatic lipid composition. Plasma cholesterol, triglyceride, and phospholipid concentrations did not differ among the 4 groups (Supplemental Table 1). Nevertheless, mice fed the cholesterol diet had significantly greater concentrations of both unesterified cholesterol (+135%) and cholesterol ester (+925%) in their livers than those fed the control diet (Fig. 2). Together, this resulted in a hepatic cholesterol concentration that was 4-fold that of controls. Concurrently, the hepatic triglyceride concentration was 1.8-times greater in cholesterol-fed mice than in mice fed the control diet. In mice fed plant sterols or plant stanols, cholesterol and triglyceride concentrations were significantly lower than in cholesterol-fed mice and did not differ from those in mice fed the control diet. T0901317-treated mice had elevated hepatic triglyceride (5.2-fold) and total cholesterol concentrations (0.6-fold) compared with mice fed the control diet.


Figure 2
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  		Figure 2  Hepatic triglyceride (A) and cholesterol (B) concentrations in C57BL/6 mice fed the control diet alone or supplemented with cholesterol with or without plant sterols or stanols for 4 wk. Values are means ± SD, n = 6. aDifferent from control; bdifferent from cholesterol, P < 0.05.

 
Biliary cholesterol excretion is a major contributor to hepatic cholesterol turnover and an important determinant of fractional cholesterol absorption. Bile flow (Supplemental Table 2) and biliary bile salt output (not shown) did not differ among the groups. Hepatobiliary sterol output rates did not differ significantly between groups fed cholesterol with or without plant sterols and stanols because of large variations between individual mice.

    Hepatic and intestinal gene expression. To identify potential differences in LXR-mediated effects exerted by cholesterol and/or plant sterol and stanol feeding, gene expression patterns in liver tissue were determined (Table 2). Mice fed the control diet supplied with the synthetic LXR agonist T0901317 were used as a positive control. Feeding the cholesterol diet significantly upregulated the LXR target genes Abcg5 and Abcg8 (+194% and +143%, respectively) encoding the major regulator of hepatobiliary cholesterol excretion. In mice fed plant sterols or stanols, this upregulation was significantly less pronounced (+87% for Abcg5 and +94% for Abcg8 with sterols and +63% and +61% for Abcg5 and Abcg8, with stanols, respectively). Expression of Abcg5 and Abcg8 in the mice treated with T0901317 was increased by >300% compared with mice fed the control diet. A similar induction by cholesterol feeding, in parallel with reduction by plant sterols or stanols, occurred for Cyp7a1, the gene encoding cholesterol-7{alpha}-hydroxylase. Expression of Cyp7a1 is considered representative for bile salt synthesis. Concomitantly, the expression of the LXR target gene Srebp1c was increased by cholesterol feeding alone (+87%) and in combination with plant sterols and stanols (+89% and +87%, respectively). All other genes analyzed did not differ among groups.


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  		TABLE 2 Hepatic mRNA expression levels in male C57BL/6 mice fed a purified diet supplemented with cholesterol, plant sterols, or plant stanols for 4 wk12

 
Gene expression was measured in 3 segments along the lengths of the small intestine. The groups did not differ in the expression of Abcg5, Abcg8, Npc1l1 (Fig. 3), Srb1, Acat2, or Hmgcr (data not shown), indicating that treatment did not influence intracellular cholesterol homeostasis at the level of gene expressions. However, large interindividual differences were noticed. The expression of Abca1 was significantly greater than in controls in the medial section of the small intestine of mice fed the cholesterol or plant sterol diet (Fig. 3). Feeding the T0901317 diet induced 2- to 9-fold increases in the expression of Abcg5, Abcg8, and Abca1 along the whole axis of the small intestine compared with the control diet. T0901317 did not affect the expression of Npc1l1, Srb1, Acat2, or Hmgcr.


Figure 3
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  		Figure 3  Intestinal gene expression in C57BL/6 mice fed the control diet alone or supplemented with cholesterol with or without plant sterols or stanols for 4 wk. Expression was measured along 3 points of the small intestine (labeled proximal, mid, and distal) and corrected for that of 18S rRNA. T091317 (T09) was used as a positive control for LXR activation (not included in the statistical analysis). Values are means ± SD, n = 6. aDifferent from control, P < 0.05.

 
    Fractional cholesterol absorption in Abcg5–/– and wild-type mice. The Abcg5/Abcg8 heterodimer prevents accumulation of plant sterols in enterocytes and is thought to contribute to regulation of cholesterol absorption. Mice lacking Abcg5 (Abcg5–/–) should therefore, in principle, be prone to LXR activation upon consumption of a plant sterol diet. We measured intestinal gene expression levels and fractional cholesterol absorption in wild-type and Abcg5–/– mice fed cholesterol only or cholesterol and plant sterol esters (Fig. 4). Unexpectedly, plant sterols did not differentially affect fractional cholesterol absorption in wild-type and Abcg5–/– mice: plant sterol supplementation led to comparable significant reductions in fractional cholesterol absorption of 44% in wild-type mice and 49% in Abcg5–/– mice. Expression of typical LXR target genes did not differ between wild-type and Abcg5–/– mice (data not shown).


Figure 4
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  		Figure 4  Fractional cholesterol absorption in Abcg5–/– and wild-type mice fed a diet supplemented with cholesterol or cholesterol and plant sterols for 1 wk. Values are means ± SD, n = 5–6. aDifferent from cholesterol alone, P < 0.05.

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
Since the early 1950s, plant sterols and stanols were known to interfere with intestinal cholesterol absorption and to lower plasma cholesterol concentrations in humans (1,2). Several concepts were proposed to explain this effect (30). First, plant sterols and stanols may replace cholesterol from micelles in the intestinal lumen and therefore may lower the amount of cholesterol present in an absorbable form (5). Second, plant sterols and stanols may block uptake of cholesterol into the enterocyte or interfere with intracellular routing. This route may include the recently discovered ezetimibe-sensitive pathway involving NPC1L1 (6) and possibly aminopeptidase N (CD13) (31). Third, it was suggested that interference with the esterification machinery inside the enterocyte and inhibition of chylomicron formation might be responsible for the observed plasma cholesterol-lowering effects of plant sterols, as proposed almost 50 y ago (32).

Very recently, it was proposed that induction of intestinal genes involved in the control of cholesterol absorption may be involved (16–18, 30). During the last few years, it was shown that enterocytes actively excrete sterols back to the intestinal lumen by the action of the Abcg5/Abcg8 heterodimer. This process is regulated at the level of gene expression by the nuclear transcription factor LXR upon binding of oxysterols. Because plant sterols are able to activate LXR either directly or after oxidation to oxyphytosterols in in vitro studies (16–18), it is possible that the lowering of cholesterol absorption by plant sterols and stanols may also be due to LXR activation and subsequent increased expression of Abcg5/Abcg8 in enterocytes. Stigmasterol, a plant sterol present in many commercially available plant sterol formulations, was shown to activate LXR in the adrenal gland of mice lacking both Abcg5 and Abcg8 (33).

To test the hypothesis that the decrease in cholesterol absorption by plant sterols and stanols is related to induction of Abcg5/g8 expression mediated by LXR, we fed C57BL/6 mice a diet virtually free of sterols, enriched with cholesterol, or enriched with cholesterol and either plant sterols or stanols for 4 wk. Fecal neutral sterol excretion increased with the addition of plant sterols or stanols to the cholesterol diet, indicating the expected inhibitory effect on intestinal cholesterol absorption potentially enforced by an enhanced hepatobiliary clearance of cholesterol. The latter possibility, however, was excluded because hepatobiliary lipid excretion rates in all groups of mice were not affected by plant sterol or stanol feeding.

In contrast to the situation described in humans, mice did not have lower plasma cholesterol concentrations due to plant sterol and stanol supplementation but did have the expected reduction in cholesteryl ester concentration in the liver. Hepatic cholesteryl ester concentrations were shown to reflect intestinal cholesterol uptake in mice (34). We therefore conclude that supplementation of the cholesterol-containing diet with either plant sterols or stanols indeed reduces intestinal cholesterol absorption in mice.

To determine whether this reduction was associated with changes in the expression of LXR target genes in the intestine or the liver, gene expression profiles of those tissues were analyzed. Along the axis of the small intestine, no significant changes in gene expression were observed, with the exception of Abca1, which was upregulated by cholesterol feeding in the medial section of the small intestine. Plant sterol and stanol supplementation did not show any additional effect. As anticipated, treatment of mice with the synthetic LXR agonist T0901317 induced the expression of the LXR targets Abcg5, Abcg8, and Abca1 (20). Npc1l1, involved in intestinal cholesterol absorption, was not influenced by plant sterol and stanol supplementation nor by T0901317 treatment. Our observations are in line with the very recent findings of Calpe-Berdiel et al. (35), which were published while this work was in progress. These authors fed different mouse strains a Western-type diet with and without phytosterols and did not find differences in the intestinal expression of LXR target genes. We conclude that the observed effects of plant sterols and stanols on cholesterol absorption are not caused by LXR-mediated induction of intestinal gene expression in mice but must be the result of upstream events.

This conclusion is further supported by gene expression profiles in liver: feeding cholesterol alone did induce the expression of Abcg5 and Abcg8. Based on the assumption that LXR activation by plant sterol- and stanol-derived metabolites occurs, we expected an additional increase in Abcg5/g8 expression as a result of plant sterol or stanol supplementation. However, the opposite occurred: the addition of plant sterols or stanols partially prevented the effects usually associated with cholesterol feeding, i.e., it lowered the hepatic expression of Abcg5/g8 compared with mice fed cholesterol only.

Our data contrast with those of recently published in vitro studies demonstrating LXR activation by plant sterols in cell lines (16–18). Cell lines frequently differ from their source tissues in gene expression profiles. The commonly used CACO-2, HepG2 or HEK293 cell lines, for example, express only limited levels of Abcg5 and Abcg8, which makes them prone to the accumulation of plant sterols in vitro. This limits the conclusions that can be drawn from experiments in which cells were loaded with plant sterols and stanols (36). We speculated that in vivo, enterocytes do not react to plant sterols and stanols via LXR because they are largely protected from their accumulation by the action of Abcg5/g8. We therefore measured fractional cholesterol absorption in mice lacking Abcg5 which, as a consequence, hyperabsorb plant sterols and accumulate them in plasma and various tissues, including the intestinal mucosa (15). Moreover, mice lacking both Abcg5 and Abcg8 were shown to be prone to LXR activation by certain sterols, namely, stigmasterol, in selected tissues (33).

When Abcg5–/– mice and their wild-type littermates where fed cholesterol-rich diets, they did not differ in fractional cholesterol absorption, in line with previous studies (15). Moreover, the addition of plant sterols to the cholesterol diet reduced fractional cholesterol absorption to the same extent in Abcg5–/– mice and their wild-type littermates. This clearly demonstrates that the decline in cholesterol absorbed from the small intestine upon plant sterol supplementation is independent of the action of the Abcg5/Abcg8 heterodimer. Moreover, we did not detect differences in intestinal gene expression responses to plant sterols between Abcg5–/– and wild-type mice, providing additional evidence that the cholesterol-lowering effects of plant sterols and stanols are independent of LXR.

Based on our findings, we speculate that a combination of dietary plant sterol and stanol supplementation with ezetimibe would have cumulative inhibitory effects on intestinal cholesterol absorption in humans because plant sterols and stanols would putatively lower the relative amount of cholesterol available for absorption, whereas inhibition of NPC1L1 by ezetimibe would decrease total sterol uptake. Very recent data (37) demonstrate that a combination of dietary plant sterols and ezetimibe has no additional effect on plasma LDL cholesterol concentrations in human hypercholesterolemic patients compared with the monotherapies. However, plasma lathosterol concentrations were significantly higher upon treatment with plant sterols combined with ezetimibe compared with single treatments, which may indicate a greater reduction in the absorption of cholesterol, which is compensated for by a more pronounced increase in (hepatic) cholesterol synthesis.

Data presented in this study demonstrate that induction of Abcg5/Abcg8 transporter activity by LXR activation does not play a role in plant sterol- and stanol-induced reduction of intestinal cholesterol absorption in mice.


   ACKNOWLEDGMENTS
 
Elke Trautwein is kindly acknowledged for the useful discussions on the design and manuscript of the study. Wim Kloots was very instrumental in preparing the experimental diets. We are grateful to Renze Boverhof for fecal sterol analysis and to Paula Jansen and Dieter Lütjohann for plant sterol analyses and helpful discussions.


   FOOTNOTES
 
1 Supported in part by Unilever Food and Health Research Institute, Vlaardingen, The Netherlands and the Dutch Heart Foundation, grant 2004T048 to T.P. and 2001B043 to J.K.K. Back

2 Supplemental Tables 1 and 2 are available with the online posting of this paper at jn.nutrition.org. Back

3 These authors contributed equally. Back

6 Abbreviations used: Abca1, Abcg5, Abcg8: ATP-binding cassette transporter a1, g5, g8; Acat2: acyl-CoA:cholesterol acyltransferase 2; LXR: liver-X-receptor; Npc1l1: Niemann-Pick C1-Like 1 protein. Back

Manuscript received 27 February 2006. Initial review completed 24 April 2006. Revision accepted 11 May 2006.


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 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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