Effects of plant sterols and stanols on intestinal cholesterol metabolism: suggested mechanisms from past to present

Plant sterols and stanols are natural food ingredients found in plants. It was already shown in 1950 that they lower serum low-density lipoprotein cholesterol (LDL-C) concentrations. Meta-analysis has reported that a daily intake of 2.5 g plant sterols/stanols reduced serum LDL-C concentrations up to 10%. Despite many studies, the underlying mechanism remains to be elucidated. Therefore, the proposed mechanisms that have been presented over the past decades will be described and discussed in the context of the current knowledge. In the early days, it was suggested that plant sterols/stanols compete with intestinal cholesterol for incorporation into mixed micelles as well as into chylomicrons. Next, the focus shifted toward cellular processes. In particular, a role for sterol transporters localized in the membranes of enterocytes was suggested. All these processes ultimately lowered intestinal cholesterol absorption. More recently, the existence of a direct secretion of cholesterol from the circulation into the intestinal lumen was described. First results in animal studies suggested that plant sterols/stanols activate this pathway, which also explains the increased fecal neutral sterol content and as such could explain the cholesterol-lowering activity of plant sterols/stanols.     

Inhibition of cholesterol absorption by the combination of dietary plant sterols and ezetimibe: effects on plasma lipid levels

Consumption of plant sterols and treatment with ezetimibe both reduce cholesterol absorption in the intestine. However, the mechanism of action differs between the two treatments, and the consequences of combination treatment are unknown. Therefore, we performed a double-blind, placebo-controlled, crossover study for the plant sterol component with open-label ezetimibe treatment. Forty mildly hypercholesterolemic subjects were randomized to the following treatments for 4 weeks each: 10 mg/day ezetimibe combined with 25 g/day control spread; 10 mg/day ezetimibe combined with 25 g/day spread containing 2.0 g of plant sterols; 25 g/day spread containing 2.0 g of plant sterols; and placebo treatment consisting of 25 g/day control spread. Combination treatment of plant sterols and ezetimibe reduced low density lipoprotein cholesterol (LDL-C) by 1.06 mmol/l (25.2%; P < 0.001) compared with 0.23 mmol/l (4.7%; P = 0.006) with plant sterols and 0.94 mmol/l (22.2%; P < 0.001) with ezetimibe monotherapy. LDL-C reduction conferred by the combination treatment did not differ significantly from ezetimibe monotherapy (-0.12 mmol/l or -3.5%; P = 0.13). Additionally, the plasma lathosterol-to-cholesterol ratio increased with all treatments. Sitosterol and campesterol ratios increased after plant sterol treatment and decreased upon ezetimibe and combination therapy. Our results indicate that the combination of plant sterols and ezetimibe has no therapeutic benefit over ezetimibe monotherapy in subjects with mild hypercholesterolemia.     

Regulation of cholesterol metabolism by dietary plant sterols

Renewal has occurred in the use of plant sterols for the treatment of hypercholesterolemias. A novel development was to convert plant sterols to corresponding stanols and esterify them to fat soluble form. In contrast to the crystalline plant sterols or stanols, plant stanol esters can be easily consumed during normal food intake in soluble form in different fat-containing food constituents when they have a potent cholesterol-lowering effect, shown in normo- and hypercholesterolemic men and women without or with coronary heart disease, children and diabetes. Cholesterol lowering is approximately 10% for total and 15% for LDL cholesterol, with the respective values for stanol ester margarine (2-3 g/day stanols) being 15% and 20%. Stanol esters reduce cholesterol absorption efficiency by up to 65%, increase cholesterol elimination in feces as cholesterol itself, usually not as bile acids, and stimulate cholesterol synthesis. Serum beta-carotene level is lowered, but no fat malabsorption or lowering of serum fat soluble vitamins have been observed. In contrast to plant sterols, stanols and their esters are minimally absorbed and they reduce serum plant sterol concentrations, also preventing statin-induced increase of plant sterols. Stanol ester margarine has been included in dietary treatment of hypercholesterolemia followed by the addition of drug treatment in resistant cases.     

Sterols regulate development and gene expression in Arabidopsis

Sterols are important not only for structural components of eukaryotic cell membranes but also for biosynthetic precursors of steroid hormones. In plants, the diverse functions of sterol-derived brassinosteroids (BRs) in growth and development have been investigated rigorously, yet little is known about the regulatory roles of other phytosterols. Recent analysis of Arabidopsis fackel (fk) mutants and cloning of the FK gene that encodes a sterol C-14 reductase have indicated that sterols play a crucial role in plant cell division, embryogenesis, and development. Nevertheless, the molecular mechanism underlying the regulatory role of sterols in plant development has not been revealed. In this report, we demonstrate that both sterols and BR are active regulators of plant development and gene expression. Similar to BR, both typical (sitosterol and stigmasterol) and atypical (8, 14-diene sterols accumulated in fk mutants) sterols affect the expression of genes involved in cell expansion and cell division. The regulatory function of sterols in plant development is further supported by a phenocopy of the fk mutant using a sterol C-14 reductase inhibitor, fenpropimorph. Although fenpropimorph impairs cell expansion and affects gene expression in a dose-dependent manner, neither effect can be corrected by applying exogenous BR. These results provide strong evidence that sterols are essential for normal plant growth and development and that there is likely a BR-independent sterol response pathway in plants. On the basis of the expression of endogenous FK and a reporter gene FK::beta-glucuronidase, we have found that FK is up-regulated by several growth-promoting hormones including brassinolide and auxin, implicating a possible hormone crosstalk between sterol and other hormone-signaling pathways.     

Dietary intake of plant sterols stably increases plant sterol levels in the murine brain

Plant sterols such as sitosterol and campesterol are frequently administered as cholesterol-lowering supplements in food. Recently, it has been shown in mice that, in contrast to the structurally related cholesterol, circulating plant sterols can enter the brain. We questioned whether the accumulation of plant sterols in murine brain is reversible. After being fed a plant sterol ester-enriched diet for 6 weeks, C57BL/6NCrl mice displayed significantly increased concentrations of plant sterols in serum, liver, and brain by 2- to 3-fold. Blocking intestinal sterol uptake for the next 6 months while feeding the mice with a plant stanol ester-enriched diet resulted in strongly decreased plant sterol levels in serum and liver, without affecting brain plant sterol levels. Relative to plasma concentrations, brain levels of campesterol were higher than sitosterol, suggesting that campesterol traverses the blood-brain barrier more efficiently. In vitro experiments with brain endothelial cell cultures showed that campesterol crossed the blood-brain barrier more efficiently than sitosterol. We conclude that, over a 6-month period, plant sterol accumulation in murine brain is virtually irreversible.     

Common sequence variations in ABCG8 are related to plant sterol metabolism in healthy volunteers

Polymorphisms in the ATP binding cassette (ABC) transporters ABCG5 and ABCG8 are related to plasma plant sterol concentrations. It is not known whether these polymorphisms are also associated with variations in serum plant sterol concentrations during interventions affecting plant sterol metabolism. We therefore decided to study changes in serum plant sterol concentrations with ABCG5/G8 polymorphisms after consumption of plant stanol esters, which decrease plasma plant sterol concentrations. Cholesterol-standardized serum campesterol and sitosterol concentrations were significantly associated with the ABCG8 T400K genotype, as were changes in serum plant sterol concentrations after consumption of plant stanols. The reduction of -57.1 +/- 38.3 10(2) x micromol/mmol cholesterol for sitosterol in TT subjects was significantly greater compared with the -36.0 +/- 18.7 reduction in subjects with the TK genotype (P = 0.021) and the -16.9 +/- 13.0 reduction in subjects with the KK genotype (P = 0.047). Changes in serum campesterol concentrations showed a comparable association. No association with serum LDL cholesterol was found. Genetic variation in ABCG8 not only explains cross-sectional differences in serum plant sterol concentrations but also determines a subject's responsiveness to changes in serum plant sterols during interventions known to affect plant sterol metabolism.     

Plant sterols: Diversity,biosynthesis, and physiological functions

Sterols, which are isoprenoid derivatives, are structural components of biological membranes. Special attention is now being given not only to their structure and function, but also to their regulatory roles in plants. Plant sterols have diverse composition; they exist as free sterols, sterol esters with higher fatty acids, sterol glycosides, and acylsterol glycosides, which are absent in animal cells. This diversity of types of phytosterols determines a wide spectrum of functions they play in plant life. Sterols are precursors of a group of plant hormones, the brassinosteroids, which regulate plant growth and development. Furthermore, sterols participate in transmembrane signal transduction by forming lipid microdomains. The predominant sterols in plants are β-sitosterol, campesterol, and stigmasterol. These sterols differ in the presence of a methyl or an ethyl group in the side chain at the 24th carbon atom and are named methylsterols or ethylsterols, respectively. The balance between 24-methylsterols and 24-ethylsterols is specific for individual plant species. The present review focuses on the key stages of plant sterol biosynthesis that determine the ratios between the different types of sterols, and the crosstalk between the sterol and sphingolipid pathways. The main enzymes involved in plant sterol biosynthesis are 3-hydroxy-3methylglutaryl-CoA reductase, C24-sterol methyltransferase, and C22-sterol desaturase. These enzymes are responsible for maintaining the optimal balance between sterols. Regulation of the ratios between the different types of sterols and sterols/sphingolipids can be of crucial importance in the responses of plants to stresses.     

Plant sterol or stanol esters retard lesion formation in LDL receptor-deficient mice independent of changes in serum plant sterols

Statins do not always decrease coronary heart disease mortality, which was speculated based on increased serum plant sterols observed during statin treatment. To evaluate plant sterol atherogenicity, we fed low density lipoprotein-receptor deficient (LDLr(+/-)) mice for 35 weeks with Western diets (control) alone or enriched with atorvastatin or atorvastatin plus plant sterols or stanols. Atorvastatin decreased serum cholesterol by 22% and lesion area by 57%. Adding plant sterols or stanols to atorvastatin decreased serum cholesterol by 39% and 41%. Cholesterol-standardized serum plant sterol concentrations increased by 4- to 11-fold during sterol plus atorvastatin treatment versus stanol plus atorvastatin treatment. However, lesion size decreased similarly in the sterol plus atorvastatin (-99% vs. control) and the stanol plus atorvastatin (-98%) groups, with comparable serum cholesterol levels, suggesting that increased plant sterol concentrations are not atherogenic. Our second study confirms this conclusion. Compared with lesions after a 33 week atherogenic period, lesion size further increased in controls (+97%) during 12 more weeks on the diet, whereas 12 weeks with the addition of plant sterols or stanols decreased lesion size (66% and 64%). These findings indicate that in LDLr(+/-) mice 1) increased cholesterol-standardized serum plant sterol concentrations are not atherogenic, 2) adding plant sterols/stanols to atorvastatin further inhibits lesion formation, and 3) plant sterols/stanols inhibit the progression or even induce the regression of existing lesions.     

Serum sterols during stanol ester feeding in a mildly hypercholesterolemic population

We investigated the changes of cholesterol and non-cholesterol sterol metabolism during plant stanol ester margarine feeding in 153 hypercholesterolemic subjects. Rapeseed oil (canola oil) margarine without (n = 51) and with (n = 102) stanol (2 or 3 g/day) ester was used for 1 year. Serum sterols were analyzed with gas-liquid chromatography. The latter showed a small increase in sitostanol peak during stanol ester margarine eating. Cholestanol, campesterol, and sitosterol proportions to cholesterol were significantly reduced by 5-39% (P < 0.05 or less for all) by stanol esters; the higher their baseline proportions the higher were their reductions. The precursor sterol proportions were significantly increased by 10- 46%, and their high baseline levels predicted low reduction of serum cholesterol. The decrease of the scheduled stanol dose from 3 to 2 g/day after 6-month feeding increased serum cholesterol by 5% (P < 0. 001) and serum plant sterol proportions by 8-13% (P < 0.001), but had no consistent effect on precursor sterols. In twelve subjects, the 12-month level of LDL cholesterol exceeded that of baseline; the non-cholesterol sterol proportions suggested that stimulated synthesis with relatively weak absorption inhibition contributed to the non-responsiveness of these subjects. In conclusion, plant stanol ester feeding lowers serum cholesterol in about 88% of subjects, decreases the non-cholesterol sterols that reflect cholesterol absorption, increases the sterols that reflect cholesterol synthesis, but also slightly increases serum plant stanols. Low synthesis and high absorption efficiency of cholesterol results in the greatest benefit from stanol ester consumption.     

Dietary plant sterols accumulate in the brain

Dietary plant sterols and cholesterol have a comparable chemical structure. It is generally assumed that cholesterol and plant sterols do not cross the blood-brain barrier, but quantitative data are lacking. Here, we report that mice deficient for ATP-binding cassette transporter G5 (Abcg5) or Abcg8, with strongly elevated serum plant sterol levels, display dramatically increased (7- to 16-fold) plant sterol levels in the brain. Apolipoprotein E (ApoE)-deficient mice also displayed elevated serum plant sterol levels, which was however not associated with significant changes in brain plant sterol levels. Abcg5- and Abcg8-deficient mice were found to carry circulating plant sterols predominantly in high-density lipoprotein (HDL)-particles, whereas ApoE-deficient mice accommodated most of their serum plant sterols in very low-density lipoprotein (VLDL)-particles. This suggests an important role for HDL and/or ApoE in the transfer of plant sterols into the brain. Moreover, sitosterol upregulated apoE mRNA and protein levels in astrocytoma, but not in neuroblastoma cells, to a higher extend than cholesterol. In conclusion, dietary plant sterols pass the blood-brain barrier and accumulate in the brain, where they may exert brain cell type-specific effects.     

Increased incorporation of dietary plant sterols and cholesterol correlates with decreased expression of hepatic and intestinal Abcg5 and Abcg8 in diabetic BB rats

The aim of this study was to determine the impact of dietary plant sterols and stanols on sterol incorporation and sterol-regulatory gene expression in insulin-treated diabetic rats and nondiabetic control rats. Diabetic BioBreeding (BB) and control BB rats were fed a control diet or a diet supplemented with plant sterols or plant stanols (5 g/kg diet) for 4 weeks. Expression of sterol-regulatory genes in the liver and intestine was assessed by real-time quantitative polymerase chain reaction. Diabetic rats demonstrated increased tissue accumulation of cholesterol and plant sterols and stanols compared to control rats. This increase in cholesterol and plant sterols and stanols was associated with a marked decrease in hepatic and intestinal Abcg5 (ATP-binding cassette transporter G5) and Abcg8 (ATP-binding cassette transporter G8) expressions in diabetic rats, as well as decreased mRNA levels of several other genes involved in sterol regulation. Plant sterol or plant stanol supplementation induced the accumulation of plant sterols and stanols in tissues in both rat strains, but induced a greater accumulation of plant sterols and stanols in diabetic rats than in control rats. Surprisingly, only dietary plant sterols decreased cholesterol levels in diabetic rats, whereas dietary plant stanols caused an increase in cholesterol levels in both diabetic and control rats. Therefore, lower expression levels of Abcg5/Abcg8 in diabetic rats may account for the increased accumulation of plant sterols and cholesterol in these rats.     

Sitosterolemia.

Sitosterolemia is a rare inherited lipid storage disease characterized chemically by the accumulation of plant sterols and 5 alpha-saturated stanols in plasma and tissues. Very low cholesterol synthesis due to a deficiency of HMG-CoA reductase associated with increased intestinal plant sterol absorption and slow hepatic sterol removal are major biochemical features. Because cholesterol synthesis cannot up-regulate, bile acid malabsorption mobilizes body sterols for bile acid synthesis and dramatically lowers plasma and monocyte sterol concentrations and may halt the progression of the atherosclerotic process.     

The replacement of cholesterol by phytosterols and the increase of total sterol content in model erythrocyte membranes

The activity of phytosterols on human organism includes the ability of these compounds to incorporate into membranes. In the consequence the plant sterols are able to increase total sterol concentration in membrane or/and to replace cholesterol molecules. The aim of this work was to compare the influence of both these effects on the properties of model erythrocyte membranes. Moreover, the interactions between the plant sterols (β-sitosterol and stigmasterol) and saturated–monounsaturated phosphatidylcholine were investigated and the condensing and ordering potency of these phytocompounds on membrane phospholipids were thoroughly analyzed. It was found that the addition of the plant sterols into model membrane modifies the condensation, ordering and interactions in the system. Moreover, the replacement of mammalian sterol by phytosterol more strongly influences the model system than even a 10% increase of total sterol concentration induced by the incorporation of the plant sterol, at constant content of cholesterol. The investigated plant sterols at their lower concentration in the mixed system are of similar effect on its properties. At higher content stigmasterol was found to modify the properties of model membrane more strongly than β-sitosterol.     

Increased plasma post-heparin diamine oxidase activity and plant sterol levels in streptozotocin diabetic rat.

Plasma post-heparin diamine oxidase (DAO) activity and plasma levels of plant sterols were examined in streptozotocin diabetic rats fed with chow containing plant sterols, to investigate the enzyme activity in relation to the morphological changes of small intestine as well as sterol absorption in the diabetic rats. Diabetic rats showed increased small intestinal mass and surface area compared with control rats. Plasma post-heparin DAO activity and plant sterol level were also increased more than 2.5-fold in the diabetic rats. Insulin treatment improved these abnormalities. Plasma DAO activity correlated to both the small intestinal hyperplastic change and plasma plant sterol levels. These results indicate that plasma post-heparin DAO activity may be used as a marker of intestinal hypertrophy as well as ability to absorb dietary sterols.     

Genetic basis of sitosterolemia

The molecular mechanisms regulating the amount of dietary cholesterol retained by the body, as well as the body's ability to exclude other dietary sterols selectively, are poorly understood. An average Western diet will contain approximately 250-500 mg of dietary cholesterol and approximately 200-400 mg of non-cholesterol sterols, of which plant sterols are the major constituents. Approximately 50-60% of dietary cholesterol is absorbed and retained by the normal human body, but less than 1% of the non-cholesterol sterols are retained. There thus exists a subtle mechanism that allows the body to distinguish between cholesterol and non-cholesterol sterols. In sitosterolemia, a rare autosomal recessive disorder, affected individuals hyperabsorb and retain not only cholesterol but also all other sterols, including plant and shellfish sterols from the intestine. Consequently, patients with this disease have very high levels of plant sterols in the plasma, and develop tendon and tuberous xanthomas, accelerated atherosclerosis, and premature coronary artery disease. The STSL locus has been mapped to human chromosome 2p21. Mutations in two tandem ABC genes, ABCG5 and ABCG8, encoding sterolin-1 and -2, respectively, are now known to be mutant in sitosterolemia. The identification of these genes should now lead to a better understanding of the molecular mechanism(s) governing the highly selective absorption and retention of cholesterol by the body. Indeed, it is the very existence of this disease that has given credence to the hypothesis that there is a molecular pathway that regulates dietary cholesterol absorption and sterol excretion by the body.     

A two-carbon switch to sterol-induced autophagic death

Although both cholesterol and plant sterols are abundant in our diets, our intestinal epithelial cells selectively and efficiently rid the body of plant sterols. However, a rare mutation in plant sterol excretion in humans results in the accumulation of plant sterols, particularly sitosterol, in the plasma and tissues. Sitosterol differs from cholesterol only in an extra ethyl group on the sterol side chain. Significantly, sitosterolemia is associated with accelerated atherothrombotic vascular disease, notably myocardial infarction. An important process that promotes atherothrombosis is advanced lesional macrophage death, leading to plaque necrosis. One of the causes of atherosclerotic macrophage death is sterol-induced cytotoxicity. We therefore compared the effects of excess intracellular sitosterol vs. cholesterol on macrophage death. Whereas excess cholesterol kills macrophages by caspase-dependent apoptosis, sitosterol kills macrophages by a caspase-independent pathway involving necroptosis and autophagy. The finding that an ethyl group on the sterol side chain fundamentally alters the way cells respond to excess sterols adds new insight into the mechanisms of sterol-induced cell death and may provide at least one explanation for the excess atherosclerotic heart disease in patients with sitosterolemia.     

Response of obligate heterozygotes for phytosterolemia to a low-fat diet and to a plant sterol ester dietary challenge

Twelve obligate heterozygotes from two kindreds were ascertained through phytosterolemic probands homozygous for molecular defects in the ATP binding cassette (ABC) half transporter, ABCG8. The response of these heterozygotes to a Step 1 diet low in fat, saturated fat, and cholesterol, and to 2.2 g daily of plant sterols (as esters) was determined in Protocol I (16 weeks) and Protocol II (28 weeks) during three consecutive feeding periods: Step 1/placebo spread; Step 1/plant sterol spread; and Step 1/placebo spread (washout). At baseline, half the heterozygotes had moderate dyslipidemia and one-third had mildly elevated campesterol and sitosterol levels. On the Step 1/placebo spread, mean LDL cholesterol decreased significantly, 11.2% in Protocol I (n = 12), and 16.0% in Protocol II (n = 7). Substitution with plant sterol spread produced a significant treatment effect on LDL levels in Protocols I and II. Conversely, the mean levels of campesterol and sitosterol increased 119% and 54%, respectively, during the use of plant sterol spread for 6 weeks in Protocol I, an effect mirrored for 12 weeks in Protocol II. During the placebo spread washouts, LDL levels increased, while those of plant sterols decreased to baseline levels in both protocols. In conclusion, phytosterolemic heterozygotes respond well to a Step 1 diet, and their response to a plant sterol ester challenge appears similar to that observed in normals.     

Effect of a liver X receptor agonist on deposition and lymphatic absorption of plant sterols in stroke-prone spontaneously hypertensive rats having a mutation in ATP-binding cassette transporter G5

The effects were compared of T0901317, a liver X receptor agonist, on deposition in the liver and serum and lymphatic absorption of plant sterols in stroke-prone spontaneously hypertensive rats (SHRSPs) having a missense mutation in Abcg5, which codes for ATP-binding cassette transporter (ABC) G5, with those in Wistar rats. Both strains were pair-fed for 7 d with a 0.5% plant sterol diet with or without 5 mg/kg of body weight of T0901317. The deposition of plant sterols in the liver and serum was higher in SHRSPs than in Wistar rats. A significant reduction of plant sterol deposition was observed in Wistar rats, but not in SHRSPs when T0901317 was given. Both strains were then fed for 7 d with a control diet with or without T0901317. The lymphatic absorption of plant sterols was reduced to almost half the normal level by the T0901317 treatment. However, no difference in absorption was apparent between SHRSPs and Wistar rats regardless of the T0901317 treatment. These results suggest that the plant sterol deposition in SHRSPs was not necessarily caused by the increased absorption of plant sterols.     

Sitosterol-containing lipoproteins trigger free sterol-induced caspase-independent death in ACAT-competent macrophages

Sitosterolemia is a disease characterized by very high levels of sitosterol and other plant sterols and premature atherothrombotic vascular disease. One theory holds that plant sterols can directly promote atherosclerosis, but the mechanism is not known. Unesterified, or "free," cholesterol (FC) is a potent inducer of macrophage death, which causes plaque necrosis, a precursor to atherothrombosis. FC-induced macrophage death, however, requires dysfunction of the sterol esterifying enzyme acyl-coenzyme A-cholesterol acyltransferase (ACAT), which likely occurs slowly during lesion progression. In contrast, plant sterols are relatively poorly esterified by ACAT, and so they may cause macrophage death and plaque necrosis in an accelerated manner. In support of this hypothesis, we show here that macrophages incubated with sitosterol-containing lipoproteins accumulate free sterols and undergo death in the absence of an ACAT inhibitor. As with FC loading, sitosterol-induced macrophage death requires sterol trafficking to the endoplasmic reticulum, and sitosterol-enriched endoplasmic reticulum membranes show evidence of membrane protein dysfunction. However, whereas FC induces caspase-dependent apoptosis through activation of the unfolded protein response and JNK, sitosterol-induced death is caspase-independent and involves neither the unfolded protein response nor JNK. Rather, cell death shows signs of necroptosis and autophagy and is suppressed by inhibitors of both processes. These data establish two new concepts. First, a relatively subtle change in sterol structure fundamentally alters the type of death program triggered in macrophages. Understanding the basis of this alteration should provide new insights into the molecular basis of death pathway signaling. Second, sitosterol-induced macrophage death does not require ACAT dysfunction and so may occur in an accelerated fashion. Pending future in vivo studies, this concept may provide at least one mechanism for accelerated plaque necrosis and atherothrombotic disease in patients with sitosterolemia.     

Effects of diet and metamorphosis upon the sterol composition of the butterfly Morpho peleides

Whole body sterol metabolism in insects has seldom been studied. We were able to design an appropriate study at a butterfly farm in Belize. We collected six larvas of butterfly (Morpho peleides), their food (leaves of Pterocarpus bayessii), and their excretions. In addition, six adult butterflies were collected. The sterols of the diet, the larva, and adult butterfly were analyzed by gas-liquid chromatography. The structures of these sterols were identified by digitonin precipitation, GC-MS, and NMR. Four sterols (cholesterol, campesterol, stigmasterol, and sitosterol) and a sterol mixture were found in the food, the body, and the excreta of the larva. The tissue sterol content of the larva was 326 microg. They consumed 276 microg of sterols per day. Their excretion was 185 microg per day as sterols. The total tissue sterol contents of the larva and butterfly were similar, but they had different sterol compositions, which indicated interconversion of sterols during development. There was a progressive increase in the cholesterol content from larva to butterfly and a decrease in the content of sitosterol and other plant sterols, which were likely converted to cholesterol. Our data indicated an active sterol metabolism in butterfly larva. Diet played an important role in determining its sterol composition. During metamorphosis, there was an interconversion of sterols. This is the first paper documenting the fecal sterol excretion in insects as related to dietary intakes.