Sterol utilization in honey bees fed a synthetic diet: Analysis of prepupal sterols

The sterols of prepupal honey bees, Apis mellifera L., from brood reared by workers fed chemically-defined synthetic diets containing cholesterol, campesterol, sitosterol, stigmasterol, 24-methylenecholesterol, or no sterol over a 12-week period were isolated, identified, and quantified. The major sterol present in each prepupal sample was 24-methylenecholesterol, but significant levels of sitosterol and isofucosterol were also present in every case, as was a very small percentage of desmosterol (usually < 1%). This is the first report of isofucosterol being identified in the sterols of the honey bee. A considerably larger percentage of each dietary sterol was found in prepupae reared by workers fed that particular sterol in the diet. This was most dramatic in the case of the cholesterol diet in which case cholesterol content increased to as much as 17.2% of the prepupal sterols, whereas cholesterol had not exceeded 2.2% in samples from other diet regimens. However, stigmasterol comprised no more than 6.3% of the total sterols in any sample from prepupae fed the stigmasterol diet. The preponderance of 24-methylenecholesterol in all prepupae, regardless of the dietary sterol provided to the workers, as well as the lesser quantities of sitosterol and isofucosterol present in all samples, suggest a unique system of utilization and metabolism of these dietary sterols by the worker bees. Apparently they make available to the brood varying amounts of unchanged dietary sterol plus considerable and fairly constant portions of 24-methylenecholesterol, sitosterol, and isofucosterol drawn from their own sterol pools.     

Changes in the levels and composition of sterols in different tissues of Lolium temulentum plants during floral development

Free, esterified and glycosylated sterols were analysed separately from the shoot apices, leaves, leaf sheaths and stems of Lolium temulentum L. (strain Ceres) plants during floral development. Short-day grown plants (50 days old) were induced to flower by exposure to a single long day. The four major sterols found by GC-MS analysis were sitosterol, cholesterol, campesterol and stigmasterol. The sterol levels in the shoot apex were much higher than those in the leaf, leaf sheath and stem. A much greater proportion of cholesterol was found in the shoot apex than in other tissues and this may reflect a specific association of cholesterol with meristematic and/or reproductive tissues.
During the inductive treatment, the sterol levels decreased in all four tissues. The major effect during early differentiation was the occurrence of transient increases in the free and esterified sterol levels in the leaf and the stem tissues. The steryl ester content peaked 24 h before the appearance of double ridges, followed by a peak in free sterol content at the double ridge stage. Similar changes could not be detected in the shoot apices. This is the first report of the sterol composition of developing shoot apices, and the results emphasize the dynamic nature of sterol metabolism during reproductive growth of L. temulentum.     

Die quantitative verteilung der sterine und sterinderivate in organen von Solanum dulcamara

The following sterols were found in the roots, stems, leaves, unripe and ripe fruits of Solanum dulcamara: cholesterol, sitosterol, stigmasterol, campesterol, brassicasterol, isofucosterol and 24-methylenecholesterol. The most abundant components are cholesterol, sitosterol and stigmasterol (77–84%). In all parts of the plant the sterols are present in the free form and as esters, glycosides and acylated glycosides. The total sterol content and the content of combined forms were determined photometrically. In the leaves 58% of the sterols were found in the form of glycoside (26%), acylated glycoside (29%) and ester (2%). In the roots only 25% of the sterol were found in combined form. In the other organs the ratio of free and combined sterols was intermediate. In all cases, the ester fraction was the least.     

Comparison of sterols of pollens,honeybee workers,and prepupae from field sites

Sterols from pollen collected by foraging honeybees, Apis mellifera L, at seven field sites were compared with the sterols of foraging adults and/or prepupae collected from colonies at each site. Invariably, the composition of prepupal sterols was comparable to that found in previous cage studies using chemically defined diets containing various dietary sterols: 24-methyl-enecholesterol was the major sterol; sitosterol and isofucosterol were present in lesser, but significant amounts; and a trace amount of cholesterol was identified in each sample. This occurred even though some of the pollen sterols contained little 24-methylenecholesterol, sitosterol, or isofucosterol and a preponderance of certain other sterols, such as δ⁷-stigmasten-3β-ol and δ^7,24(28)-campestadien-3β-ol in goldenrod and corn pollens, respectively. Thus the selective transfer and utilization of sterols in honeybees that have been demonstrated in cage studies with artificial diets were also shown to occur under field conditions.     

STEROL PROFILES IN 18 MACROALGAE OF THE PORTUGUESE COAST1

The sterol profiles of dominant macroalgae occurring in the western Portuguese coast were evaluated. An analytical procedure, involving alkaline hydrolysis and extraction followed by separation by reversed‐phase HPLC–diode array detection (HPLC–DAD), was optimized for the study of their sterols composition. The validated methodology is short in analysis time (as the compounds are determined in <20 min), sensitive, reproducible, and accurate. It was then successfully applied to the determination of campesterol, cholesterol, desmosterol, ergosterol, fucosterol, stigmasterol, and β‐sitosterol in 18 species (three Chlorophyta, five Rhodophyta, and 10 Phaeophyta). The profiles obtained for the several macroalgal species were considerably different. C29 sterols were predominant in Phaeophyta and Chlorophyta (71%–95% of total sterol content), while in Rhodophyta cholesterol content is significantly higher (34%–87%). Among the studied species, Asparagopsis armata Harv. contained the lowest sterol amount (555 mg · kg^?1 dry weight), and Cystoseira tamariscifolia (Huds.) Papenf. the highest one (6,502 mg · kg^?1 dry weight). Data obtained may be helpful in identifying suitable marine sources of sterols, with potential applications in the food and pharmaceutical industries.     

4-Desmethylsterol composition of citrus rootstocks of different salt exclusion capacity

Fibrous roots from seedlings of three citrus rootstocks (Rungpur lime, Kharna khatta and Etrog citron) grown hydroponically for 6 weeks in the presence or absence of 50 mM NaCl were analysed for their content of free, esterified, and glycosidic sterols. Leaf chloride analyses indicated that Rangpur lime was a good Cl- excluder and the other two rootstocks were Cl- accumulators.
On a dry weight basis and in the absence of NaCI only the free 4-desmethyIsterol levels showed significant rootstock differences. Kharna khatta had the highest, and Rangpur lime the lowest, free stcrol levels. Sitostcrol was the major component of all sterol fractions of Rangpur lime, the esterified fraction of the other rootstocks, and the glycosidic sterol fraction of Kharna khatta. The ratio of sitosterol to stigmasterol was highest in Rangpur lime and lowest in Etrog citron in all cases and the ratio of apos;more-planar apos; to apos;less-planar apos; free sterols was highest in Etrog citron and lowest in Rangpur lime.
Treatment with 50 mM NaCI resulted in an increase in free sterol levels in Rangpur lime and a decrease in Kharna khatta. Steryl ester levels were unaffected in Rangpur lime but were significantly reduced in the other rootstocks. In all three sterol fractions the sitosterol/stigmasterol ratio was decreased. A decrease in the ratio of apos;more-planarapos; to apos;less-planarapos; free sterols was observed only in Kharna khatta and, more notably, Etrog citron. Salt-induced changes in the apos;more-planar apos; to apos;less-planar apos; free sterol ratio correlated well with salt exclusion capacity.     

Sterols of bryophytes

Sterol compositions of six species of bryophytes were studied. The major sterols identified were campesterol, 22-dihydrobrassicasterol, sitosterol, clionasterol, stigmasterol, cholesterol, 24-methyl-5,22-cholestadienol and 24-methyl-5,7,22-cholestatrienol. The quantitative determinations of the α- and β-epimers of 24-methyl and 24-ethylcholesterols were made based on 220 MHz ¹H NMR spectroscopy and capillary gas chromatography. Sterol compositions of bryophytes from other studies are reviewed, and possible sterol biosynthetic pathways in bryophytes are discussed.     

Effects of the growth retardant tetcyclacis on the sterol composition of oat (Avena sativa)

The norbornenodiazetine plant growth regulator tetcyclacis, when applied to roots of Avena sativa, caused a substantial increase in the cholesterol content of the shoots. Amounts of the C-24 alkylated sterols campesterol, stigmasterol and sitosterol all declined. A similar alteration in the sterol profile was observed for a plasma membrane preparation from the shoots. Changes in the sterol composition of root tissue were much less pronounced.     

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.     

Sterols of organs involved in brood food production and of royal jelly in honey bees

The sterols of the organs (hypopharyngeal and mandibular glands and honey stomachs) involved in worker and queen honey bee brood food production, royal jelly and intact nurse bees were analyzed to obtain information on the selective transfer of specific sterols from one generation to the next. No appreciable increase in the percentage of 24-methylenecholesterol, relative to the total sterols isolated from intact honey bee (Apis mellifera L.), prepupae or adults, was found in the hypopharyngeal or mandibular glands or the honey stomachs from nurse bees reared in colonies fed a chemically-defined diet supplemented with 24-methylenecholesterol. The sterols of these organs contained higher levels of cholesterol than did the sterols of whole body extracts. The other major sterols, sitosterol and isofucosterol, occurred at relative concentrations comparable to whole body extracts. Also, there were higher levels of cholesterol in the sterols from glandular tissues of nurse bees maintained on pollen and sucrose solution than in sterols isolated from intact insects. In a separate study, royal jelly collected over a 6-day period had much higher relative percentages of 24-methylenecholesterol and lower levels of sitosterol and isofucosterol than did the pollen fed to these colonies. The sterols of nurse bees in the latter study had an intermediate concentration of 24-methylenecholesterol. The significance of these findings relative to the unique selective transfer of specific sterols from the diet or from endogenous sterol pools of the nurse bees from generation to generation in the honey bee is discussed.     

Ezetimibe normalizes metabolic defects in mice lacking ABCG5 and ABCG8

The ATP binding cassette transporters ABCG5 (G5) and ABCG8 (G8) limit the accumulation of neutral sterols by restricting sterol uptake from the intestine and promoting sterol excretion into bile. Humans and mice lacking G5 and G8 (G5G8-/-) accumulate plant sterols in the blood and tissues. However, despite impaired biliary cholesterol secretion, plasma and liver cholesterol levels are lower in G5G8-/- mice than in wild-type littermates. To determine whether the observed changes in hepatic sterol metabolism were a direct result of decreased biliary sterol secretion or a metabolic consequence of the accumulation of dietary noncholesterol sterols, we treated G5G8-/- mice with ezetimibe, a drug that reduces the absorption of both plant- and animal-derived sterols. Ezetimibe feeding for 1 month sharply decreased sterol absorption and plasma levels of sitosterol and campesterol but increased cholesterol in both the plasma (from 60.4 to 75.2 mg/dl) and the liver (from 1.1 to 1.87 mg/g) of the ezetimibe-treated G5G8-/- mice. Paradoxically, the increase in hepatic cholesterol was associated with an increase in mRNA levels of HMG-CoA reductase and synthase. Together, these results indicate that pharmacological blockade of sterol absorption can ameliorate the deleterious metabolic effects of plant sterols even in the absence of G5 and G8.     

Increased sitosterol absorption, decreased removal, and expanded body pools compensate for reduced cholesterol synthesis in sitosterolemia with xanthomatosis

We measured the turnover and absorption of sitosterol and cholesterol, along with plasma sterol and lipoprotein concentrations, in one control and two subjects with sitosterolemia with xanthomatosis. All individuals consumed the same diet which contained approximately 500 mg/day of cholesterol and 250 mg/day of sitosterol. Sterol absorption was measured by the plasma dual-isotope ratio method and turnover by plasma isotope-kinetic analysis. In two sitosterolemic subjects, 28% and 63% of the sitosterol and 69% and 49% of the cholesterol were absorbed, respectively, compared to 4% of the sitosterol and 44% of the cholesterol in the control. As expected, plasma sitosterol specific activities decayed much more rapidly than cholesterol in the control subject. In contrast, plasma sitosterol and cholesterol specific activity-time curves were similar and decayed more slowly in the sitosterolemic subjects. In the control subject, the total sitotterol pool was 290 mg and was linearly related to low absorption (18 mg/day); whereas the total sitosterol pool was 17 times (4800 mg) and 13 times (3500 mg) larger, respectively, in the sitosterolemic subjects and was expanded out of proportion to increased absorption because of decreased removal. Daily cholesterol turnover and synthesis were markedly reduced in the sitosterolemic subjects. In four sitosterolemic subjects, plasma concentrations of total sterols, low density lipoproteins, and apolipoprotein B were increased, while those of high density lipoproteins and apolipoprotein A-I were low to normal. The low density lipoproteins were very similar to those of normal control subjects in density distribution, peak flotation rate, sterol-to-protein (apolipoprotein B) ratio, particle size, and morphology. These results demonstrate in patients with sitosterolemia with xanthomatosis that: 1) the absorption of sitosterol and cholesterol is enhanced; 2) tissue recognition between cholesterol and sitosterol is lost; 3) total exchangeable sitosterol pools are expanded out of proportion to absorption because of decreased excretion; 4) plasma sterol and lipoprotein concentrations favor tissue deposition; and 5) cholesterol synthesis is diminished. We postulate that the changes in sitosterol metabolism (increased absorption, loss of tissue sterol structural recognition, expanded pools, and hepatic retention) are a response to reduced cholesterol synthesis in these subject.     

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.     

The selective uptake of cholesterol by the rat tapeworm Hymenolepis diminuta (Cestoda)

1. The sterols of Hymenolepis diminuta are almost exclusively cholesterol or similar C-27 sterols; the free sterols of its environment (the lumen of the rat intestine) are cholesterol and various phytosterols. 2. During incubation of tapeworms with mixed micelles of taurocholate, glyceryl monooleate, and equimolar [3H]cholesterol and [14C]beta-sitosterol, the uptake of cholesterol is 40 times more rapid than the uptake of sitosterol. 3. Following uptake, the desorption of labeled sitosterol is six times more rapid than that of cholesterol. 4. We did not detect the esterification of absorbed sterols or the conversion of absorbed sitosterol of cholesterol. 5. The highly selective uptake of cholesterol and the moderately selective desorption of phytosterols can account for the selective accumulation of C-27 sterol by the tapeworm.     

The neutral sterols of Megalotomus quinquespinosus (say) (hemiptera: Alydidae) and identification of makisterone a as the major free ecdysteroid

Last-stage nymphs of the broad-headed bug, Megalotomus quinquespinosus contain the C₂₈ ecdysteroid makisterone A as their major ecdysteroid. No ecdysone or 20-hydroxyecdysone was detected in whole body extracts analyzed by high performance liquid chromatography and radioimmune assay. Analyses of the neutral sterols of this phytophagous hemipteran revealed that the sterol composition of the nymphs was highly reflective of their dietary sterols. The most abundant nymphal sterols were sitosterol (46.6%), Δ⁷-stigmastenol (13.8%) and spinasterol (13.4%). Cholesterol accounted for only 0.2% of the total sterols and indicates that this species is incapable of converting 24-alkyl sterols to cholesterol.     

Sterol composition of chitosomes from yeast cells of Mucor rouxii: Comparison with whole cells

Abstract The free sterol composition of chitosomes and whole yeast cells of Mucor rouxii was determined by gas-liquid chromatography (GLC). The results obtained showed that, in contrast to whole cells, chitosomes are particularly rich in ergosterol: 87% vs. 29% of total free sterols. Three other sterols were present in varying amounts in both chitosomes and whole cells. These were identified by GLC as cholesterol, stigmasterol and sitosterol. These results indicate that the yeast cells preferentially accumulate ergosterol in chitosomes providing these microvesicles with a unique composition that may be important for the structure of the organelle.     

The fate of radiolabelled C28 and C29 phytosterols in the honey bee

The fate of radiolabelled campesterol, sitosterol and 24-methylenecholesterol fed in chemically-defined diets to honey bee (Apis mellifera L.) workers was determined. At various intervals, sterols of prepupae, newly emerged adults and queens were analyzed qualitatively, quantitatively and radiochemically and it was determined that there was not sufficient radioactivity associated with cholesterol and/or desmosterol in any of the samples to verify that any of the three C₂₈ and C₂₉ sterols was dealkylated and converted to cholesterol. Similarly, there was no evidence for the conversion of campesterol or sitosterol to 24-methylenecholesterol. It was concluded that the major portion of the sterols incorporated into the tissues of the brood larvae originated from the worker bees used to establish the colony. There is good evidence supporting the premise that the workers can make available sterols from their endogenous pools to the nutrient in the hive and that they can replenish these sterols with those from the artificial diet. The queen is also able to replenish sterols utilized in egg production from those obtained by the workers from the artificial diet, and at the end of nine weeks queens contained more than four times as much sterol, on a ‘μg sterol per g fresh weight’ basis, than was found in fertile queens at the beginning of the test period.     

Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS

We have developed a highly sensitive and specific method for the analysis of serum sterol profiles. Sterols in 1 mul of dried serum were derivatized into picolinyl esters (3beta-picolinate) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the electrospray ionization (ESI) mode. In addition to cholesterol, 19 cholesterol precursors, cholestanol, campesterol, sitosterol, and sitostanol were identified simultaneously. Quantitative analyses for the picolinyl esters of 11 available sterols were performed, and detection limits were found to be less than 1 pg on-column. Reproducibilities and recoveries of 8 noncholesterol sterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.6% to 8.2% and 2.5% to 16.5%, respectively. The recovery experiments were performed using 1 mul aliquots of normal human serum spiked with 1 ng to 6 ng of sterols, and recoveries of the sterols ranged from 88.1% to 102.5% with a mean recovery of 98.1%. The present method provides reliable and reproducible results for the identification and quantification of neutral sterols, especially in small volumes of blood samples, which is useful for serological diagnosis of inherited disorders in cholesterol metabolism and for noninvasive evaluation of cholesterol biosynthesis and absorption in humans.     

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.

Addendum to:

Sitosterol-Containing Lipoproteins Trigger Free Sterol-Induced Caspase-Independent Death in ACAT-Competent Macrophages: Implications for Sterol Structure-Dependent Mechanisms of Cell Death and for Atherosclerotic Vascular Disease in Sitosterolemia

L. Bao, Y. Li, S.X. Deng, D. Landry and I. Tabas

J Biol Chem 2006; In press     

Sterols in hardening winter wheat

The main sterols in winter wheat crowns and roots were sitosterol and campesterol, with significant amounts of stigmasterol and traces of cholesterol. The main groups of sterol-containing lipids were free sterols, steryl glucosides, steryl esters and esterified steryl glucosides. Sterol analysis within each group showed little difference between them. Steryl esters were relatively rich in cholesterol and poor in stigmasterol. Free sterols were rich in stigmasterol. Low temperature caused an increase in sterol content but had little effect on sterol composition and sterol to lipid P ratio. There was some increase in steryl esters and some decrease in free sterols. Cholesterol and stigmasterol decreased in the steryl ester and free sterol fractions, respectively. There was little evidence for involvement of sterols in winter wheat frost hardening.