A preliminary investigation into the unsaponifiable fraction of donkey milk: Sterols of animal origin, phytosterols, and tocopherols.

We investigated the main sterols, phytosterols, and the α- and γ-tocopherol content in donkey milk during the first 2 mo of lactation. Cholesterol was the main sterol in milk (mean ± standard deviation = 0.97 ± 0.443 g/100 g of fat). Lanosterol was the main minor sterol of animal origin, followed by desmosterol (0.003 ± 0.001 and 0.001 ± 0.001 g/100 g of fat, respectively). Of the phytosterols, ß-sitosterol was the main sterol of vegetal origin in donkey milk (0.005 ± 0.002 g/100 g of fat), but lower levels of campesterol, brassicasterol, and stigmasterol were also recorded. Mean levels of α- and γ-tocopherol were 0.01 ± 0.007 and 0.003 ± 0.001 g/100 g of fat, respectively. We observed no significant changes in sterol or tocopherol content during the first 2 mo of lactation. The presence of lanosterol in donkey milk is of particular interest, because lanosterol is a potential drug and has important physiological effects. The presence of phytosterols, which are considered nutraceutical molecules, enhances the nutritional quality of donkey milk fat for consumers.

Simplified LC-MS Method for Analysis of Sterols in Biological Samples.

We developed a simple and robust liquid chromatographic/mass spectrometric method (LC-MS) for the quantitative analysis of 10 sterols from the late part of cholesterol synthesis (zymosterol, dehydrolathosterol, 7-dehydrodesmosterol, desmosterol, zymostenol, lathosterol, FFMAS, TMAS, lanosterol, and dihydrolanosterol) from cultured human hepatocytes in a single chromatographic run using a pentafluorophenyl (PFP) stationary phase. The method also avails on a minimized sample preparation procedure in order to obtain a relatively high sample throughput. The method was validated on 10 sterol standards that were detected in a single chromatographic LC-MS run without derivatization. Our developed method can be used in research or clinical applications for disease-related detection of accumulated cholesterol intermediates. Disorders in the late part of cholesterol synthesis lead to severe malformation in human patients. The developed method enables a simple, sensitive, and fast quantification of sterols, without the need of extended knowledge of the LC-MS technique, and represents a new analytical tool in the rising field of cholesterolomics.

Sterols in infant formulas: validation of a gas chromatographic method.

Sterols are components present in the fat fraction of infant formulas (IFs). Their characterization is therefore of interest, though there are no official reference methods for their analysis in these matrices. AIM: To validate a gas chromatographic method with flame ionization detection for the determination of animal (cholesterol and desmosterol) and plant sterols (brassicasterol, campesterol, stigmasterol, ß-sitosterol and sitostanol) found in IFs. All correlation coefficients obtained for the calibration curves of sterols studied were >0.99. Limits of detection (<1 µg/100 mL) and quantification (<4 µg/100 mL) are suitable for sterols determination in IFs. The within-assay precision ranged from 1.6% to 8.8%, while the between-assay precision was <10% for most of sterols. Accuracy was satisfactory and was calculated by recovery assays (ranging 93-108%). The analytical parameters obtained showed the suitability of the proposed method for the determination of sterols in IFs.

Analysis of bioactive oxysterols in newborn mouse brain by LC/MS.

Unesterified cholesterol is a major component of plasma membranes. In the brain of the adult, it is mostly found in myelin sheaths, where it plays a major architectural role. In the newborn mouse, little myelination of neurons has occurred, and much of this sterol comprises a metabolically active pool. In the current study, we have accessed this metabolically active pool and, using LC/MS, have identified cholesterol precursors and metabolites. Although desmosterol and 24S-hydroxycholesterol represent the major precursor and metabolite, respectively, other steroids, including the oxysterols 22-oxocholesterol, 22R-hydroxycholesterol, 20R,22R-dihydroxycholesterol, and the C(21)-neurosteroid progesterone, were identified. 24S,25-epoxycholesterol formed in parallel to cholesterol was also found to be a major sterol in newborn brain. Like 24S- and 22R-hydroxycholesterols, and also desmosterol, 24S,25-epoxycholesterol is a ligand to the liver X receptors, which are expressed in brain. The desmosterol metabolites (24Z),26-, (24E),26-, and 7α-hydroxydesmosterol were identified in brain for the first time.

Cholesterol and desmosterol in two sperm populations separated on Sil-Select gradient.

Sperm lipids are important for sperm viability, maturity and function. This study aimed to identify cholesterol and desmosterol composition of human spermatozoa of two sperm populations separated on Sil-Select gradient. Forty-eight males were divided into four groups namely healthy men (n = 13), asthenozoospermia (n = 11), asthenoteratozoospermia (n = 10) and oligoasthenoteratozoospermia (n = 14). Sperm cholesterol and desmosterol were estimated in two human sperm population separated by centrifugation in a discontinuous Sil-Select gradient. The results showed that cholesterol and desmosterol were the major sterols in human spermatozoa. Spermatozoa recovered from upper/lower layer interface (fraction I) had low fertilization potential, while those from the base (fraction II) had high fertilization potential. Median values of cholesterol and desmosterol in fraction I were 2.55 micromol and 0.77 micromol/10(9) spermatozoa and in fraction II were 1.16 micromol and 0.27 micromol/10(9) spermatozoa. Cholesterol/desmosterol ratio was significantly higher in fraction II than I (4.8 vs. 3.2, p < 0.01). Cholesterol, desmosterol, total phospholipids and sterols/phospholipids were negatively correlated with sperm concentrations, sperm motility, linear velocity, normal sperm morphology and acrosome reaction percentage whereas cholesterol/desmosterol ratio was positively correlated with these parameters. It is concluded that the difference in sterol composition of sperm subpopulations separated on Sil-Select gradient suggests that composition of sterols is related to sperm functions.

Plant stanol ester spreads as components of a balanced diet for pregnant and breast-feeding women: evaluation of clinical safety.

Clinical safety of consuming plant stanol ester spreads during pregnancy and lactation, the impact on maternal and infant serum and breast-milk cholesterol and the ratios (micromol/mmol of cholesterol) of synthesis and absorption markers were evaluated. Pregnant women (n 21) were randomised to control and dietary intervention groups, the intervention including advice to follow a balanced diet and to consume spreads enriched with plant stanol esters. Participants were followed during and after pregnancy and their infants up to 1 year of age. A mean 1.1 (sd 0.4) g consumption of plant stanols during pregnancy and 1.4 (sd 0.9) g 1 month post-partum increased sitostanol and the markers for cholesterol synthesis, lathosterol, lathosterol/campesterol and lathosterol/sitosterol, and reduced a marker for cholesterol absorption, campesterol, in maternal serum. In breast milk, desmosterol was lower in the intervention group, while no differences were detected between the groups in infants' serum. Plant stanol ester spread consumption had no impact on the length of gestation, infants' growth or serum beta-carotene concentration at 1 and 6 months of age, but the cholesterol-adjusted serum beta-carotene concentration was lowered at 1 month in the intervention group. Plant stanol ester spread consumption appeared safe in the clinical setting, except for potential lowering of infants' serum beta-carotene concentration, and was reflected in the markers of cholesterol synthesis and absorption in mothers' serum, encouraging further studies in larger settings.

Increased biosynthesis and accumulation of cholesterol in maternal plasma, but not amniotic fluid in pre-eclampsia.

Preeclampsia is one of the most serious complications during pregnancy, defined as development of hypertension during late pregnancy affecting other organ systems (proteinuria, thrombocytopenia, renal insufficiency, liver involvement, cerebral symptoms or pulmonary edema). Preeclampsia is known to be associated with significant dyslipidemia, but the cause or mechanism of this metabolic aberration is not clear. Quantitative analysis of cholesterol precursors and metabolites can reveal metabolic signatures of cholesterol, and provide insight into cholesterol biosynthetic and degradation pathways. We undertook this study to compare the metabolic signatures of cholesterol in serum and amniotic fluid collected from women who delivered in the late preterm period. Matching serum and amniotic fluid samples were collected from women who delivered in the late preterm period (34-0/7-36-6/7 weeks), had undergone amniocentesis within 3 days of delivery, had no evidence of rupture of membranes or intra-amniotic infection/inflammation, and who had not received antenatal corticosteroid prior to amniocentesis. Patients were classified into 3 groups according to the etiology of their preterm birth: Group 1, preeclampsia; Group 2, spontaneous preterm labor; Group 3, other maternal medical indications for iatrogenic preterm birth. Quantitative metabolite profiling of cholesterols was performed using gas chromatography-mass spectrometry. A total of 39 women were included in the analysis (n = 14 in Group 1, n = 16 in Group 2, n = 9 in Group 3). In maternal blood, patients in Group 1 had significantly higher ratios of cholesterol/desmosterol and cholesterol/7-dehydrocholesterol (which represent 24- and 7-reductase enzyme activity, respectively) than those in Group 3 (p < 0.05 for each), which suggests increased cholesterol biosynthesis. In contrast, patients in Group 1 had significantly decreased ratios of individual cholesterol esters/cholesterol and total cholesterol esters/cholesterol than those in Groups 3 (p < 0.01 for each), suggesting increased reverse cholesterol transport. No differences in cholesterol ratios were found in amniotic fluid among the 3 groups. In conclusion, the metabolic signatures of cholesterol suggest increased cholesterol biosynthesis and accumulation in the maternal blood (but not amniotic fluid) of women with preeclampsia.

HPLC method for quantification and characterization of cholesterol and its oxidation products in eggs.

A new method was developed for the simultaneous determination of cholesterol and its oxidation products in eggs, using HPLC with UV and refractive index (RI) detectors, and HPLC interfaced with atmospheric pressure chemical ionization coupled to MS (HPLC-APCI-MS). The best conditions for direct saponification of the sample and extraction of the non-saponifiable material were defined using complete factorial designs with central points. The method showed accuracy and precision with a detection limit between 0.002 and 0.079 microg/g. The oxides cholest-5-ene-3beta,20alpha-diol and cholest-5-ene-3beta,25-diol identified by HPLC-UV-RI were not confirmed by HPLC-APCI-MS.

Desmosterolosis presenting with multiple congenital anomalies and profound developmental delay.

Desmosterol (cholesta-5,24-dien-3beta-ol) is a minor sterol that forms as an intermediate in the cholesterol biosynthetic pathway when the 24-unsaturated sterol bond is reduced as the last step rather than earlier in the conversion of lanosterol to cholesterol. In 1998, FitzPatrick et al. reported a premature infant who died shortly after birth and had marked tissue elevations of desmosterol and a strikingly abnormal phenotype. We describe here the first living patient with desmosterolosis and show biochemical evidence in plasma and cultured lymphoblasts for an autosomal recessive deficiency of 24-dehydrocholesterol reductase (DHCR24). The infant has severe microcephaly, agenesis of the corpus callosum, downslanting palpebral fissures, micrognathia, submucous cleft palate, clubfoot, and a persistent patent ductus arteriosus. Plasma sterol quantification in the patient at age 2 years demonstrated a normal cholesterol level, but a 100-fold increased level of desmosterol (60 mcg/ml; nl 0.5 +/- 0.3 mcg/ml (SD)) suggesting deficient activity of 24-dehydrocholesterol (desmosterol) reductase (DHCR24). Both parents had mildly increased levels of desmosterol in plasma (mother: 1.4 mcg/ml; father: 1.8 mcg/ml), consistent with heterozygosity for DHCR24 deficiency. Analysis of sterol metabolism in cultured transformed lymphoblasts showed a 100-fold increased level of desmosterol and a moderately decreased level of cholesterol in the patient's cells and a 10-fold elevation of desmosterol in the mother's cells. At the age of 3.5 years, the patient stands but does not walk, uses a 5-word vocabulary, and lacks any major medical problems. This unique patient broadens the spectrum of inborn errors of cholesterol biosynthesis and suggests additional candidate clinical phenotypes associated with abnormal cholesterol metabolism.

Quantitative analysis of desmosterol, cholesterol and cholesterol sulfate in semen by high-performance liquid chromatography.

A simple, rapid and accurate method to separate and quantify cholesterol, desmosterol and cholesterol sulfate in human spermatozoa and seminal plasma (SP) is described. This high-performance liquid chromatographic procedure is based on reversed-phase chromatography on a Inertsil ODS2 5 microm silica column with a binary gradient of mixtures of chloroform-methanol and chloroform-methanol-water as the mobile phase at a flow-rate of 0.25 ml/min. Sterols are separated with good resolution and high reproducibility. The eluted sterols are quantified using a light-scattering (mass) detector. As little as 64, 64 and 68 pmol of cholesterol, desmosterol and cholesterol sulfate, respectively, can be quantified under these conditions. Cholesterol is the predominant sterol both in spermatozoa (107+/-7 nmol/10(8) spermatozoa) and SP (0.83+/-0.10 micromol/ml) whereas the concentrations of desmosterol were 38+/-6 nmol/10(8) in spermatozoa and 0.18+/-0.02 micromol/ml in SP. Cholesterol sulfate represents about 6% of total cholesterol in the spermatozoa and SP. In conclusion, this method offers interesting perspectives for the quantitative analysis of these sterols not only in semen, but also in other biological samples.

Intermediates of stigmasterol metabolism in Spodoptera littoralis.

Stigmasterol-24,28-epoxide, 22E-stigmasta-5,22,24(28E)-trien-3 beta-ol, and 22E-cholesta-5,22,24-trien-3 beta-ol were identified as normal metabolites of [3H]stigmasterol in Spodoptera littoralis larvae. Relative concentrations of all three of these metabolites increased when a diazasterol inhibitor was fed in combination with stigmasterol in the artificial diet. Identification of these sterols as intermediates in the conversion of stigmasterol to cholesterol in this insect indicates that intermediates analogous to fucosterol and fucosterol-24,28-epoxide in the conversion of sitosterol to cholesterol are produced in the metabolism of stigmasterol. This is the first published identification of stigmasterol-24,28-epoxide and 22E-stigmasta-5,22,24(28E)-trien-3 beta-ol as intermediates in this pathway in an insect.

Analysis of cholesterol and desmosterol in cultured cells without organic solvent extraction.

Cultured cell sterols such as cholesterol and desmosterol are usually extracted into organic solvents before they are quantified with cholesterol esterase and oxidase. A method to quantify these cultured cell sterols using cholesterol enzymes without prior organic solvent extraction is described. In this method, a suspension or monolayer of cultured L-M, U-937, or PC-12 cells is digested with 0.1% sodium dodecyl sulfate (SDS), and the digest treated with microbial cholesterol enzymes. The quantity of oxidized sterols produced by the reaction can be measured easily with high-pressure liquid chromatography, when a mixture of sterols is present, or by the production of hydrogen peroxide when only one sterol is present. This method is easier and safer to use than solvent extraction and can greatly expedite the quantitation of cultured cell sterols. Preliminary data show that other lipids such as choline phospholipids, triglycerides, and fatty acids can also be directly quantified in SDS cell digest by using specific enzymes to transform these lipids into hydrogen peroxides.

HPLC analysis of desmosterol, 7-dehydrocholesterol, and cholesterol.

A simple and sensitive method to analyze mixtures of desmosterol, 7-dehydrocholesterol and cholesterol is described. The method involves the oxidative conversion of the sterols with cholesterol oxidase, followed by high performance liquid chromatographic (HPLC) analysis. A C18 reversed phase column (3 microns, 75 X 4.6 mm) and a mixture of methanol and acetonitrile (1:1, v/v) at a rate of 1 ml/min are used to separate the sterols. The eluted sterols are quantified by measuring UV absorption at 240 nm. As little as 10 pmoles of sterol can be quantified under these conditions.

Identification of cholesta-7,24-dien-3 beta-ol and desmosterol in hamster cauda epididymal spermatozoa.

The sterol composition of hamster cauda epididymal spermatozoa was remarkably different from that of several other mammalian spermatozoa. Desmosterol and cholesta-7,24-dien-3 beta-ol account for as much as 90% of the total sterols. Cholesterol and desmosterol are the major components of mouse cauda epididymal spermatozoa, and rabbit, boar and bull ejaculated spermatozoa. Cholesta-7,24-dien-3 beta-ol was not detected. Furthermore, cholesterol was the main sterol in hamster caput epididymal spermatozoa, while only a trace amount of desmosterol was detected and cholesta-7,24-dien-3 beta-ol was hardly detected at all. The sterol content of cauda and caput epididymal spermatozoa was 0.17 +/- 0.05 mumol/10(8) spermatozoa. During maturation, the desmosterol and cholesta-7,24-dien-3 beta-ol levels increase and the cholesterol level decreases. Cholesta-7,24-dien-3 beta-ol appears as a sterol in mature spermatozoa and seems to be a characteristic sterol of hamster cauda epididymal spermatozoa.

Sterols of the phylum zygomycota: phylogenetic implications.

The sterol composition of 42 fungal species representing six of the eight orders of the Zygomycota was determined using gas-liquid chromatography-mass spectrometry to assess whether the distribution of major sterols in this phylum has taxonomic or phylogenetic relevance. Ergosterol, 22-dihydroergosterol, 24-methyl cholesterol, cholesterol, and desmosterol were detected as the major sterols among the species studied. Ergosterol was the major sterol of the Dimargaritales, Zoopagales, and 13 of the 14 Mucorales families included in this study. Desmosterol appeared to be the characteristic sterol of the Mortierellaceae (Mucorales), 24-Methyl cholesterol was the major sterol of the Entomophthorales genera Entomophthora, Conidiobolus and Basidiobolus, but cholesterol was the sole sterol detected in Delacroixia coronatus. The Kickxellales species analyzed in this study were characterized by 22-dihydroergosterol as the major sterol. These results suggest that certain orders of the Zygomycota may be distinguished on the basis of major sterol. Also, if sterol structure has phylogenetic implications, then orders might be arranged in the order Kickxellales (C28 delta 5,7)-->Dimargaritales, Zoopagales and Mucorales (C28 delta 5,7,22) on the basis of evolution of the predominant and presumably most competent sterol, ergosterol. Although the Entomophthorales would be expected to be more primitive than the above orders based on the predominance of C28 delta 5, it is not apparent from these data that members of the Zygomycota with ergosterol or its precursors as major sterols evolved from this taxon or the Chytridiomycota.

[Separate measurement of fluoroanalogues of desmosterol, cholesterol, 7-dehydrocholesterol by thin-layer and gas-liquid chromatography]. / Razdl'noe opredelenie ftoranalogov desmosterina, kholesterina, 7-degidrokholesterina pri pomoshchi tonkosloinoi i gazozhidkostnoi khromatigrafuu

An attempt was made to develop a new method for separate measurement of sterols of similar chemical structure--desmosterol, cholesterol, 7-dehydrocholesterol--by means of thin-layer and gas-liquid chromatography. Fluoroanalogues of the sterols were well separated in the adsorbent thin layer. This facilitated their further identification, quantitation and accumulation. Similar results were obtained with the aid of gas liquid chromatography.

[Sterol composition of Palaemon adspersus]. / O sterinovom sostave krevetok Palaemon adspersus.

The sterol composition of Palaemon adspersus is studied. It is established that the nonsaponifiable fraction of Palaemon adspersus contains cholesterol, 7-dehydrocholesterol, desmosterol, substance "240" and inconsiderable amounts of other still nonidentified sterols.

[Sterols of several Black Sea crustaceans]. / Issledovanie sterinov u nekotorykh rakoobraznykh Chernogo moria.

Sterol content has been investigated in lower (Artemia salina) and higher crustaceans--the isopods Idotea baltica and S phaeroma pulchellum, decapod Palaemon adspersus and several amphipods. In lower crustaceans, as well as in isopods and amphipods, no 7-dehydrocholesterol was found. This fact may be associated with peculiarities of feeding of these animals. It confirms the absence of sterol synthesis in some of the crustaceans reported in the literature. Cholesterol, demosterol and 7-dehydrocholesterol were found in shrimps.