Comparison of organic and deep eutectic solvents based dispersive liquid-liquid microextraction for the analysis of phytosterols in cow milk combined with high-performance liquid chromatography-ultraviolet detector.

In the present work, a dispersive liquid-liquid microextraction approach has been developed for extraction of four phytosterols (stigmasterol, ß-sitosterol, campesterol, and brassicasterol) from cow milk samples using organic and deep eutectic solvents and the results were critically compared. The extracted analytes were determined using high performance liquid chromatography. In the developed method, carbon tetrachloride and choline chloride:p-chlorophenol deep eutectic solvent were selected to use as the best extraction solvent. Effective parameters and validation data were studied for both methods, independently. Under optimum conditions, limits of detection and quantification were within the ranges of 0.3-0.9 and 1.0-3.0 ng/mL for organic solvent based dispersive liquid-liquid microextraction and 0.09-0.32 and 0.3-1.0 ng/mL for deep eutectic solvent based dispersive liquid-liquid microextraction, respectively. Good coefficient of determinations and relative standard deviations obtained for the methods were ≥0.994 and ≤7.6%, respectively. The introduced method was performed on different milk samples for the determination of target analytes using both solvents and the results were analyzed statistically by the t-test.

Amendment to Quality Parameters for Rice Bran Oil in the Codex Standard for Named Vegetable Oils.

The objective of this study is to review the compliance of fatty acid compositions of Thai and India rice bran oil and level of desmethylsterols of Thai crude rice bran oil with the Codex Standard for Named Vegetable Oil (Codex Stan 210-1999). Fatty acid compositions of 90 samples of Thai and India refined rice bran oil were analyzed by capillary gas liquid chromatography. The results indicated that the contents of the C14:0, C18:2, C22:0 and C24:0 possible fall outside the range of Codex Stan 210-1999. In addition, sterol profile content of 40 samples of crude rice bran oil from Thai refinery plants were studied. The test results of major compositions of desmethylsterols are in good agreement with CODEX STAN 210-1999 except for Brassicasterol and other desmethylsterols. Accordingly, these data were proposed to corporate into the codex standard. Consequently, Codex agreed to amend the fatty acid composition of C14:0, C18:2, C22:0 and C24:0 from ND to 1.0, 21 to 42, ND to 1.0 and ND to 0.9 % and broaden level of Brassicasterol and other desmethylsterols to "ND-0.3" and to "7.5-12.8" accordingly.

Comparative study of fatty acid and sterol profiles for the investigation of potential milk fat adulteration.

Milk fat adulteration is a common issue in Central Asia. To assess the current situation in the commercial milk market, 17 milk samples were checked for fatty acid (FA) and sterol profiles to detect potential adulteration using multivariate analysis. Analysis of FA and sterols was performed using gas chromatography with flame ionization detection and gas chromatography with mass-spectrometric detection, respectively. Cluster analysis of FA profiles revealed 3 types of milk samples: (1) samples containing a higher proportion of short-chain FA, (2) samples containing a higher proportion of long-chain FA, and (3) samples with significant amounts of C18 FA. Analysis of sterols showed that samples included (1) milk fat containing 100% cholesterol, sometimes with traces of phytosterols, (2) milk fat with high proportions of ß-sitosterol and campesterol, and (3) milk fat containing high proportions of brassicasterol. We found significant relationships between FA profiles and sterol profiles. The profiles were compared with vegetable oil patterns reported in the literature. More than 50% of the samples appeared to be counterfeited. We conclude that identification of adulteration in milk can be based solely on determination of sterol patterns.

Sewage contamination of Amazon streams crossing Manaus (Brazil) by sterol biomarkers.

Sewage pollution is a principal factor of decreasing water quality, although it has not been considered a real impact in Amazonia that is still considered a pristine environment around the world. Thus, this study aimed to assess the levels of sewage contamination in sediments from three streams crossing Manaus - a Brazilian city of 2,403,796 inhabitants in the heart of the Amazon rain forest. Cholesterol, cholestanol, brassicasterol, ergosterol, stigmasterol, ß-sitosterol, campesterol, stigmastanol, coprostanol, and epicoprostanol levels were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). The fecal indicator, coprostanol, was found in high concentrations (509-12 830 ng g-1) and high relative proportions (21-54%) in all samples collected in the Mindu stream that crosses many heavily populated districts of the city, and in the Quarenta stream that crosses the Industrial District of Manaus. The sediments of the Tarumã-Açu stream also presented coprostanol; however, concentrations (

Metal ions accelerated phytosterol thermal degradation on Ring A & Ring B of steroid nucleus in oils.

This study aimed to investigate the effect of metal ions on the degradation of phytosterols in oils. The oil was heated at 180°C for 1h with/without addition of Fe3+, Fe2+, Cu2+, Mn2+, Zn2+, Na+, Al3+ and Mg2+. Variations of ß-sitosterol, stigmasterol, campesterol, brassicasterol and their degradation products were confirmed by the GC-MS analysis. In general, the increase of the metal ion concentration resulted in more phytosterol degradation, and the ability of metal ions following decreasing order: Fe3+>Fe2+>Mn2+≥Cu2+≥Zn2+>Na+≥Mg2+>Al3+. Metal ions significantly induced phytosterol autoxidation on C5, C6 and C7 on Ring B of steroid nucleus at even a low concentration, and induced dehydration on the C3 hydroxyl to form dienes and trienes at high concentration. The metal ions in oils are accounted for increasing phytosterol degradation, which decreases food nutritional quality and gives rise to the formation of undesirable compounds.

Prenatal diagnosis of holoprosencephaly associated with Smith-Lemli-Opitz syndrome (SLOS) in a 46,XX fetus.

OBJECTIVE: To show the importance of measuring cholesterol precursor levels in amniotic fluid in all pregnancies with ultrasound features (such as holoprosencephaly) suggestive of Smith-Lemli-Opitz syndrome (SLOS), after exclusion of chromosomal anomalies. CASE REPORT: A 28-year-old woman, gravida 1 para 0, performed chorionic villus sampling for fetal karyotyping at 13 weeks of gestation due to positive combined first trimester screening in a fetus with increased nuchal translucency and suspected holoprosencephaly. The result was normal - 46,XX. The diagnosis of alobar holoprosencephaly was confirmed at 15 weeks of gestation, and cardiac and limb defects were also identified. Thus, a syndromic cause was considered, specifically a chromosomal microdeletion syndrome or a monogenic entity such as SLOS. The latter was confirmed by measuring 7-dehydrocholesterol (7DHC) and 8-dehydrocholesterol (8DHC) in amniotic fluid. Molecular analysis of DHCR7 gene identified a homozygous mutation in intron 8, c.964-1G>C, providing molecular confirmation for this diagnosis. CONCLUSION: The differential diagnosis of holoprosencephaly is broad. Identification of the cause of holoprosencephaly aids in establishing the prognosis and is essential to ascertain the mode of inheritance for adequate genetic counseling.

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.

Study of variation of tocochromanol and phytosterol contents in black and yellow seeds of Brassica napus L. doubled haploid populations.

BACKGROUND: In the study, an analysis of tocopherols, plastochomanol-8 and phytosterols was conducted using DH lines obtained from F1 hybrids of reciprocal crosses between yellow- and black-seeded lines. METHODS: The biological material for the study consisted of two DH populations of winter oilseed rape obtained from F1 hybrids of reciprocal crosses between two DH lines: yellow- and black-seeded. Seed color was determined using a ColorFlex spectrophotometer. Fat content was determined via pulsed NMR. The levels of tocopherols, and plastochromanol-8 are analyzed using HPLC. Phytosterol contents and composition were determined by the GC method. RESULTS: The fat content of the black-seeded parental line was 49% and this was higher than that of the yellow-seeded parental line (44%). The fat content of DH line populations ranged from 44 to 51%. Total tocopherol content ranged from 460 to 602 mg/kg and the α-T/γ-T ratio was from 0.66 to 1.09. In parental lines H2-26 and Z-114 the total tocopherol content was 534 and 525 mg/kg, but the α-T/γ-T ratios were 0.81 and 1.21, respectively. The yellow-seeded parental line (Z-114) was characterized by a higher PC-8 content (81 mg/kg) than the H2-26 black-seeded parental line (58 mg/kg). The largest part of the total phytosterol content in seeds of both populations was ß-sitosterol from 976 to 2148 mg/kg, followed by campasterol, from 636 to 1364 mg/kg, and brassicasterol from 375 to 678 mg/kg. The total tocopherol content ranged from 462 to 595 mg/kg (population HxZ) and from 460 to 602 mg/kg (population ZxH). Significantly positive correlations were observed between the seed color with α-T (r = 0.38, p < 0.01), γ-T (r = -0,34, p < 0.01) and PC-8 content (r = 0.29, p < 0.01). Correlations between the seed color with total tocopherol and total phytosterol content were not noted. CONCLUSIONS: Considering the range of genetic variation among doubled haploids of two populations, selected DH lines may be good parents for further breeding programs focused on increasing the amount and improving the quality of oilseed rapeseed oil. However, further studies will also be made to determine the influence of the environment on bioactive compounds in rapeseed oil. Cross direction of parental DH lines: yellow- and black-seeded has some influence, albeit not statistically significant, on the diversity of doubled haploid in their populations with regard to average fat, tocochromanol and phytosterol content.

Trends in prenatal diagnosis of non-specific multiple malformations disorders with reference to the own experience and research study on Smith-Lemli-Opitz syndrome.

AIM OF THE STUDY: Biochemical diagnosis of fetuses with multiple malformations--an attempt to determine the frequency of prenatal Smith-Lemli-Opitz syndrome. Discussion on trends in prenatal diagnosis of non-specific multiple malformations disorders. MATERIAL AND METHODS: A total of 117 fetal samples were obtained. They were analyzed with gas chromatography/mass spectrometry (GC/MS) method to assess the concentration of 7-dehydrocholesterol (7DHC) and 8-dehydrocholesterol (8DHC) in amniotic fluid samples and (or) to establish 7-dehydroestriol/estriol and 8-dehydropregnanetriol/pregnanetrio ratios in maternal urine. RESULTS: In 4 cases Smith-Lemll-Opitz syndrome was confirmed. CONCLUSIONS: Biochemical GC/MS sterol analyses of amniotic fluid or maternal urinary metabolites toward Smith- Lemli-Opitz syndrome, as cheap tests, should be performed in all pregnancies with suggestive ultrasound features (holoprosencephaly and(or) atrioventricular canal and(or) genital anomalies), especially when nuchal translucency is increased >3 mm, and after exclusion of chromosomal aberration in routine karyotyping or even arrayCGH.

Characterization of oilseed lipids from "DHA-producing Camelina sativa": a new transformed land plant containing long-chain omega-3 oils.

New and sustainable sources of long-chain (LC, ≥C20) omega-3 oils containing DHA (docosahexaenoic acid, 22:6ω3) are required to meet increasing demands. The lipid content of the oilseed of a novel transgenic, DHA-producing land plant, Camelina sativa, containing microalgal genes able to produce LC omega-3 oils, contained 36% lipid by weight with triacylglycerols (TAG) as the major lipid class in hexane extracts (96% of total lipid). Subsequent chloroform-methanol (CM) extraction recovered further lipid (~50% polar lipid, comprising glycolipids and phospholipids) and residual TAG. The main phospholipid species were phosphatidyl choline and phosphatidyl ethanolamine. The % DHA was: 6.8% (of total fatty acids) in the TAG-rich hexane extract and 4.2% in the polar lipid-rich CM extract. The relative level of ALA (α-linolenic acid, 18:3ω3) in DHA-camelina seed was higher than the control. Major sterols in both DHA- and control camelina seeds were: sitosterol, campesterol, cholesterol, brassicasterol and isofucosterol. C16-C22 fatty alcohols, including iso-branched and odd-chain alcohols were present, including high levels of iso-17:0, 17:0 and 19:0. Other alcohols present were: 16:0, iso-18:0, 18:0 and 18:1 and the proportions varied between the hexane and CM extracts. These iso-branched odd-chain fatty alcohols, to our knowledge, have not been previously reported. These components may be derived from wax esters, or free fatty alcohols.

Quantitative analysis of phytosterols in edible oils using APCI liquid chromatography-tandem mass spectrometry.

Previous methods for the quantitative analysis of phytosterols have usually used GC-MS and require elaborate sample preparation including chemical derivatization. Other common methods such as HPLC with absorbance detection do not provide information regarding the identity of the analytes. To address the need for an assay that utilizes mass selectivity while avoiding derivatization, a quantitative method based on LC-tandem mass spectrometry (LC-MS-MS) was developed and validated for the measurement of six abundant dietary phytosterols and structurally related triterpene alcohols including brassicasterol, campesterol, cycloartenol, ß-sitosterol, stigmasterol, and lupeol in edible oils. Samples were saponified, extracted with hexane and then analyzed using reversed phase HPLC with positive ion atmospheric pressure chemical ionization tandem mass spectrometry and selected reaction monitoring. The utility of the LC-MS-MS method was demonstrated by analyzing 14 edible oils. All six compounds were present in at least some of the edible oils. The most abundant phytosterol in all samples was ß-sitosterol, which was highest in corn oil at 4.35 ± 0.03 mg/g, followed by campesterol in canola oil at 1.84 ± 0.01 mg/g. The new LC-MS-MS method for the quantitative analysis of phytosterols provides a combination of speed, selectivity and sensitivity that exceed those of previous assays.

An ultra performance liquid chromatographic method for determining phytosterol uptake by Caco-2 cells.

A simple method for the determination of cellular uptake of phytosterols by Caco-2 cells has been developed by ultra performance liquid chromatography with ultraviolet detection (UPLC-UV). UPLC-UV was established using an ODS column, acetonitrile/H(2)O (9:1, v/v) as a mobile phase, and a detection wavelength at 210 nm. As analytes, ß-sitosterol, campesterol, stigmasterol, and brassicasterol were selected based on the abundance in foods and the similarity of their structures. A linear relation was observed between the peak area and the amount of sterol injected from 50 to 2000 pmol (r>0.999) with a relative standard deviation (RSD) of less than 2.5% (n=6). This method was applied to the determination of cellular uptake of phytosterols by Caco-2 cells. Recovery tests showed that phytosterols were extracted from the cell lysates by chloroform and determined by UPLC-UV with a recovery rate of more than 80.2% and an RSD of less than 11.3% (n=3). When Caco-2 cells were incubated with phytosterols at 37°C, their uptake was increased with time in a concentration-dependent manner. This method will be useful for the simultaneous determination of cellular phytosterols in an in vitro intestine model.

Phytosterols accumulation in the seeds of Linum usitatissimum L.

A comparative study was performed to determine the free sterols content and composition during the development of three varieties of linseed (H52, O116 and P129). Seed samples were collected at regular intervals from 7 to 60 days after flowering (DAF). Ten compounds were identified: cholesterol, campesterol, brassicasterol, stigmasterol, beta-sitosterol, Delta5-avenasterol, cycloartenol; 24-methylene cycloartanol, obtusifoliol, citrostadienol. The maximum level of 4-desmethylsterols (1,515 mg/100g oil) was reached at 7 DAF in P129 variety. H52 had the highest level of 4-4 dimethylsterols (355 mg/100g oil) at 28 DAF. The greatest amount of 4-monomethylsterols (35 mg/100g oil) was detected in H52 at 14 DAF. During linseed development, beta sitosterol (830 mg/100g oil) was the major 4-desmethylsterols, followed by campesterol (564 mg/100g oil) and stigmasterol (265 mg/100g oil). Some of these compounds followed nearly the same accumulation pattern during linseed maturation.

Precholesterol sterols accumulate in lipid rafts of patients with Smith-Lemli-Opitz syndrome and X-linked dominant chondrodysplasia punctata.

Systemic fetal dysmorphogenesis in disorders of postsqualene cholesterol biosynthesis is thought to be caused by disruption of Hedgehog signaling. Because precholesterol sterols such as 7-dehydrocholesterol and lathosterol can replace cholesterol in the activation of Hedgehog proteins, it is currently believed that cholesterol deficiency-related Hedgehog signaling block occurs further downstream, probably at the level of Smoothened. Experimentally, such a block in Hedgehog signaling occurs at sterol levels of <40 mug/mg protein. Recently, we studied autopsy material from 2 infants with fatal cholesterol biosynthetic disorders (Smith-Lemli-Opitz syndrome and X-linked dominant chondrodysplasia punctata) in which the hepatic cholesterol levels were far greater. In this study, we demonstrate abnormal accumulation of sterol precursors of cholesterol in membrane lipid rafts (detergent resistance membranes) prepared from liver tissues of these 2 infants: 8-dehydrocholesterol and 7-dehydrocholesterol in lipid rafts of the infant with Smith-Lemli-Opitz syndrome and cholest-8(9)-ene-3beta-ol in lipid rafts of the infant with X-linked dominant chondrodysplasia punctata. We suggest that such alterations in the lipid raft sterol environment may affect the biology of cells and the development of fetuses with cholesterol biosynthetic disorders.

Sterol profiling of amniotic fluid: a routine method for the detection of distal cholesterol synthesis deficit.

OBJECTIVES: Smith Lemli Opitz syndrome (SLOS) caused by a deficit of 3beta-hydroxysterol-Delta7 reductase was the first sterol deficit described with multiple malformations. The lack of specificity of many morphological abnormalities detected by ultrasound and their frequency have justified routine screening of amniotic fluid (AF) for sterols by GC-MS. The examination contributes to an improved knowledge of the sterol status in the fluid. METHODS: A series of sterol profiles is collated here. Accumulation of 7- and 8-dehydrocholesterol are diagnostic for SLOS. However, a number of other sterols have also been detected by GC-MS in control AF and their presence may be confusing. RESULTS AND CONCLUSIONS: In addition to cholesterol, the level of which varies as function of the gestational age, lathosterol is present together with trace amounts of 7- and 8-dehydrocholesterol and other precursors such as desmosterol, lanosterol, and dimethylsterol. Phytosterols are also present in 70% of AF samples that have been tested. Besides SLOS, GC-MS examination of amniotic fluid can detect various sterol deficits associated with malformations (lathosterolosis, desmosterolosis, X-linked chondrodysplasia, and particular Antley-Bixler syndrome). Practical conclusions support GC-MS as a routine method to investigate skeletal and central nervous system malformations.

Sterol composition of gonad, muscle and digestive gland of Pecten maximus from Málaga (South Spain).

Sterol composition and content and their seasonal variations over 18 months were investigated in adductor muscle, digestive gland and gonads of Pecten maximus. Sterols were isolated by Silicagel 60 thin layer chromatography and identified by gas chromatography/mass spectrometry. Eleven sterols were identified, with cholesterol, brassicasterol, 24-methylenecholesterol and 22-trans-dehydrocholesterol being the principal components. The same sterols were found in all three tissues independent of season. The relative amounts of each sterol present in each tissue differed. Total sterol levels in gonad and muscle were higher than in digestive gland. Statistically significant differences (P<0.05) were found between the concentrations of each of the sterols isolated from the gonad or muscle and digestive gland. The seasonal variations in the sterol content of the gonad seem be related to the reproductive cycle, while the sterol content of the digestive gland appears to be linked to diet, mainly diatoms or dinoflagellates. The muscle sterol content showed minor changes throughout the year.

Neutral sterols of rat epididymis. High concentrations of dehydrocholesterols in rat caput epididymidis.

Phospholipids and sterols are known to have multiple functions in reproductive tissue of mammals. High concentrations of the cholesterol precursor desmosterol have been described in testis, epididymis, and spermatozoa of various species. These findings and the recent discovery of some cholesterol precursors as meiosis-activating sterols suggest important functions of cholesterol precursors in fertility. Many sterol intermediates appear from the 19-step conversion of lanosterol, the first sterol synthesized in the cascade of cholesterol synthesis, to cholesterol. The biochemical basis of the genetically inherited Smith-Lemli-Opitz syndrome has been described as a defective conversion of 7-dehydrocholesterol to cholesterol. Since this discovery, interest has focused on this special cholesterol precursor. Here, we report high concentrations of 7- and 8-dehydrocholesterol in caput epididymidis and spermatozoa derived from caput epididymidis of Sprague-Dawley and Wistar rats, which comprised up to 30% of total sterols. In contrast to caput epididymidis, 7- and 8-dehydrocholesterol were barely detected in cauda epididymidis or testis. Desmosterol increased several times from caput to cauda epididymidis. This is the first report of the natural appearance of high concentrations of dehydrocholesterols in mammalian tissue, and it underlines the putative importance of cholesterol precursors in reproductive tissue.

Testicular concentration of meiosis-activating sterol is associated with normal testicular descent.

In the cryptorchid stallion, spermatogenesis is arrested at various levels before the completion of meiosis. In men, infantile cryptorchidism is also often associated with oligo- and azoospermia during adulthood. An impairment of spermatogenesis might be reflected in the level of locally produced factors. Formerly, a meiosis-activating sterol (T-MAS) has been isolated in murine and bovine testes. This sterol possesses the potential to trigger resumption of meiosis in cultured mouse oocytes, indicating that it might play an important role in the regulation of the meiotic process in the female gamete. The function of T-MAS in the testis is still unclear, but T-MAS may be associated with spermatogenesis. The objectives of this study were 1) to demonstrate the presence of T-MAS in equine testes, 2) to compare the contents of T-MAS in testicular tissue of stallions with complete and incomplete testicular descent and 3) to compare testicular T-MAS concentration before and after puberty Testes were collected from 16 normal and cryptorchid stallions submitted for castration and stored at -80 degrees C until the content of T-MAS was measured quantitatively with an HPLC-assay. In stallions > or = 2 years of age, the content of T-MAS was higher (P < 0.001) in normal testes (19.3+/-1.1 microg T-MAS/g, n=7) than in inguinally (4.1+/-2.4 microg T-MAS/g, n=4) or abdominally located testes (1.6+/-0.2 microg T-MAS/g, n=2). The contents of T-MAS in normal testes from stallions < 2 years of age (2.8+/-1.5 microg T-MAS/g, n=4) was lower than in normal testes from stallions > or =2 years of age (P < 0.001) From the present study it can be concluded that T-MAS is present in equine testicular tissue. Furthermore, the present study demonstrates that the production of T-MAS in testicular tissue is, concurrently with spermatogenesis, associated with normal testicular descent and is temporarily related to the onset of puberty.

Accurate detection of Smith-Lemli-Opitz syndrome carriers by measurement of the rate of reduction of the ergosterol C-7 double bond in cultured skin fibroblasts.

The activity of ergosterol delta 7-reductase (3 beta-hydroxysteroid delta 7-reductase) was measured in cultured skin fibroblasts from 7 controls, 10 Smith-Lemli-Opitz syndrome (SLOS) patients, and 10 parents (obligate carriers). The fibroblasts were exposed to delipidated medium supplemented with lovastatin for 24 h and the enzyme activity was determined by incubating cell-free homogenate with ergosterol (ergosta-5,7,22-trien-3 beta-ol) and measuring the mass of brassicasterol (ergosta-5,22-dien-3 beta-ol) formed by gas chromatography-mass spectrometry with selected-ion monitoring. In carriers, the activity was significantly lower than in controls (22 +/- 2 vs 65 +/- 10 pmol/min per mg protein, p < 0.0005), and no overlap was observed. The mean activity in carriers' fibroblasts was more than 100 times higher than in patients' cells (0.2 pmol/min per mg protein). The use of ergosterol avoids the many problems caused by the instability and lack of availability of radiolabelled 7-dehydrocholesterol. The present method makes it possible to discriminate SLOS carriers from both controls and patients using a commercially available substrate and common analytical equipment.