Compounds with estrogenic potencies and their adverse effects in surface waters have received much attention. Both anthropogenic and natural compounds contribute to overall estrogenic activity in freshwaters. Recently, estrogenic potencies were also found to be associated with cyanobacteria and their blooms in surface waters. The present study developed and compared the solid phase extraction and LC-MS/MS analytical approaches for determination of phytoestrogens (8 flavonoids - biochanin A, coumestrol, daidzein, equol, formononetin, genistein, naringenin, apigenin - and 5 sterols - ergosterol, β-sitosterol, stigmasterol, campesterol, brassicasterol) and cholesterol in water. The method was used for analyses of samples collected in stagnant water bodies dominated by different cyanobacterial species. Concentrations of individual flavonoids ranged from below the limit of detection to 3.58 ng/L. Sterols were present in higher amounts up to 2.25 μg/L. Biological potencies of these phytoestrogens in vitro were characterized using the hERα-HeLa-9903 cell line. The relative estrogenic potencies (compared to model estrogen - 17β-estradiol) of flavonoids ranged from 2.25E-05 to 1.26E-03 with coumestrol being the most potent. None of the sterols elicited estrogenic response in the used bioassay. Estrogenic activity was detected in collected field water samples (maximum effect corresponding to 2.07 ng/L of 17β-estradiol equivalents, transcriptional assay). At maximum phytoestrogens accounted for only 1.56 pg/L of 17β-estradiol equivalents, contributing maximally 8.5% of the total estrogenicity of the water samples. Other compounds therefore, most likely of anthropogenic origin such as steroid estrogens, are probably the major drivers of total estrogenic effects in these surface waters.
- MeSH
- chemické látky znečišťující vodu analýza MeSH
- cholestadienoly MeSH
- cholesterol analogy a deriváty MeSH
- estradiol analýza MeSH
- estrogeny analýza MeSH
- estron analýza MeSH
- fytoestrogeny analýza MeSH
- fytosteroly MeSH
- genistein analýza MeSH
- HeLa buňky MeSH
- isoflavony analýza MeSH
- lidé MeSH
- receptory pro estrogeny metabolismus MeSH
- sinice účinky léků metabolismus MeSH
- sitosteroly analýza MeSH
- sladká voda MeSH
- steroly analýza MeSH
- tandemová hmotnostní spektrometrie MeSH
- voda MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Naturally occurring acylated β-sitosteryl glucosides have been investigated for their novel properties. The synthetic protocol based on the literature data was improved and optimized. The main improvement consists in employing systems of ionic liquids combined with organic solvents in lipase-mediated esterification of (3β)-stigmast-5-en-3-yl β-d-glucopyranoside to get (3β)-stigmast-5-en-3-yl 6-O-acyl-β-d-glucopyranosides. Maximum yields of these products were achieved with Candida antarctica lipase B immobilized on Immobead 150, recombinant from yeast, in absolute THF and in the presence of either ionic liquid [1-butyl-3-methyl imidazolium tetrafluoroborate ([BMIM]BF4) or 1-butyl-3-methyl imidazolium hexafluorophosphate ([BMIM]PF6)] employed. Pharmacological activity of (3β)-stigmast-5-en-3-yl 6-O-acyl-β-d-glucopyranosides was studied in tests on MCF7 tumor cell lines; the compounds displayed moderate activity which was higher than the activity of β-sitosterol. Supramolecular characteristics were discovered at (3β)-stigmast-5-en-3-yl 6-O-dodecanoyl-β-d-glucopyranoside that formed supramolecular polymer through multiple H-bonds in a methanol/water system (60/40). Its formation was confirmed by the independent UV-vis measurements during certain time period, by variable temperature DOSY-NMR measurement in deuteriochloroform, and visualized by transmission electron microscopy (TEM) and atomic force microscopy (AFM) showing chiral helical structures and complex superassembly systems based on fibrous supramolecular polymer. In contrary, no such properties have been observed for the other two (3β)-stigmast-5-en-3-yl 6-O-acyl-β-d-glucopyranosides under the given experimental conditions.
- MeSH
- iontové kapaliny chemie MeSH
- lidé MeSH
- lipasa metabolismus MeSH
- magnetická rezonanční spektroskopie MeSH
- MFC-7 buňky MeSH
- mikroskopie atomárních sil MeSH
- sitosteroly chemie metabolismus farmakologie MeSH
- transmisní elektronová mikroskopie MeSH
- viabilita buněk účinky léků MeSH
- vodíková vazba MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The presented work describes the development and validation of a rapid UHPLC-UV/CAD method using a core-shell particle column for the separation and quantitative analysis of seven plant sterols and stanols. The phytosterols (ergosterol, brassicasterol, campesterol, fucosterol, stigmasterol, and β-sitosterol) and the phytostanol stigmastanol were separated and analyzed in 8.5min. The sample pre-treatment procedure was optimized to be less time-consuming than any other published method, especially due to no need of derivatization, evaporation and even reconstitution step. The chromatographic separation was performed on the Kinetex 1.7μ Phenyl-hexyl column (100×2.1mm) with a mobile phase acetonitrile/water according to the gradient program at a flow rate of 0.9mLmin-1and a temperature of 60°C. A tandem connection of PDA and CAD (Corona Charged Aerosol Detector) was used and both detection techniques were compared. The method was validated using saponification as a first step in sample pre-treatment and an universal CAD as the detector. Recoveries for all analyzed compounds were between 95.4% and 103.4% and relative standard deviation ranged from 1.0% to 5.8% for within-day and from 1.4% to 6.7% for between-day repeatability. The limits of detection were in the range of 0.4-0.6μgmL-1for standard solutions and 0.3-1.2μgmL-1for phytosterols in real samples. Although several gradient programs and different stationary phases were tested, two compounds, campesterol and campestanol, were not separated. Their peak was quantified as a sum of both analytes.
Úvod: Sitosterolémie je vzácné, autozomálně recesivně dědičné onemocnění, způsobené abnormním transportem fytosterolů ve střevě a játrech při poruše transportního proteinu ABCG5/ ABCG8. Projevuje se významnou hyperfytosterolémií, u dětí většinou i hypercholesterolémií, xantomy, hemolytickou anémií, makrotrombocytopenií, abnormální krvácivostí, splenomegalií, předčasnou aterosklerózou, artritidou, neprospíváním, hepatopatií. Výjimečný nález normální cholesterolémie při hyperfytosterolémii komplikuje diagnostiku, stejně tak nález normální hladiny fytosterolů u kojených dětí. Pacienti se sitosterolémií dobře reagují na dietu s restrikcí cholesterolu a fytosterolů v kombinaci s léčbou ezetimibem. Předkládáme výsledky klinických, biochemických a molekulárně genetických analýz u našeho pacienta se sitosterolémií. Kazuistika: Tříletý chlapec je sledován od 7 měsíců věku pro hypercholesterolémii (16–25,6 mmol/l), zjištěnou náhodně. Rodinná anamnéza byla negativní. Od 1 roku věku došlo k propadu v hmotnostně-délkovém poměru ze 75. na 8,8. percentil. Ve věku 20 měsíců byly zjištěny významně zvýšené sérové hladiny sitosterolu (13x nad normu) a kampesterolu (4x nad normu). Molekulárně genetická analýza prokázala, že pacient je složeným heterozygotem pro dříve popsanou patogenní mutaci p.Arg446* a dosud nepublikovanou, pravděpodobně patogenní variantu p.Arg419Cys v genu ABCG5. Oba rodiče jsou zdraví přenašeči. Závěr: U 3letého chlapce byla potvrzena diagnóza sitosterolémie s mutacemi v genu ABCG5. Jde o první potvrzený případ sitosterolémie v ČR. Chlapec je v současnosti léčen nízkocholesterolovou a nízkofytosterolovou dietou a ezetimibem. Během 16 měsíců léčby se téměř normalizovala hladina celkového cholesterolu a LDL cholesterolu, významně poklesla koncentrace fytosterolů. Sitosterolémii je potřeba brát v úvahu v rámci diferenciální diagnostiky závažné hypercholesterolémie u dětí.
Background: Sitosterolemia is a rare, autosomal recessive inherited disorder of lipid metabolism, associated with an abnormal transport of plant sterols in a small intestine and a liver due to malfunction of a transport protein ABCG5/ABCG8. Disorder is associated with significant hyperphytosterolemia and also with hypercholesterolemia in most pediatric patients, tuberous and tendinous xanthomas, hemolytic anemia, macrothrombocytopenia, abnormal bleeding, splenomegaly, premature atherosclerosis, arthritis, failure to thrive and hepatopathy. A rare finding of normocholesterolemia and normal levels of phytosterols in breastfed infants might result in a delay in diagnosis. Patients with sitosterolemia respond well to a low cholesterol and a low plant sterol diet in combination with ezetimibe therapy. Here, we report clinical, biochemical and molecular genetic data of a boy with sitosterolemia. Case report: 3 years old boy has been monitored since 7 months of age for hypercholesterolemia (16–25.6 mmol/l), found accidentally. Family history was negative. Since one year of age a failure to thrive was documented, weight/lenght ratio dropped from 75th to 8.8th percentile. At the age of 20 months laboratory tests showed significantly increased levels of serum sitosterol (13 times above normal level) and campesterol (4 times above normal level). Molecular genetic analysis revealed compouned heterozygosity for a previously described pathogenic mutation p.Arg446* and another, up to now unpublished, probably pathogenic variant p.Arg419Cys in the ABCG5 gene. Both parents were confirmed as healthy mutation carriers. Conclusion: We presented the first Czech patient with sitosterolemia carrying mutations in ABCG5. Patient is on a low-cholesterol and a low-plant sterol diet and ezetimibe therapy. During 16 months of therapy, level of total cholesterol and also of LDL cholesterol almost normalized and serum concentration of plant sterols decreased significantly. Therefore, sitosterolemia should be considered in the differential diagnosis of severe hypercholesterolemia in children.
- Klíčová slova
- sitosterolémie, fytosterolémie,
- MeSH
- ABC transportér podrodiny G, člen 5 genetika MeSH
- anticholesteremika MeSH
- cholesterol krev MeSH
- ezetimib aplikace a dávkování terapeutické užití MeSH
- fytosteroly krev MeSH
- hypercholesterolemie diagnóza terapie MeSH
- lidé MeSH
- nemoci střev MeSH
- neprospívání MeSH
- předškolní dítě MeSH
- sitosteroly krev MeSH
- vrozené poruchy metabolismu tuků MeSH
- výsledek terapie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- Publikační typ
- kazuistiky MeSH
- práce podpořená grantem MeSH
- MeSH
- cholesterol krev MeSH
- desmosterol krev MeSH
- hypercholesterolemie * diagnóza MeSH
- lidé MeSH
- pilotní projekty MeSH
- retrospektivní studie MeSH
- sitosteroly krev MeSH
- steroly klasifikace metabolismus MeSH
- těhotenství * metabolismus MeSH
- Check Tag
- lidé MeSH
- těhotenství * metabolismus MeSH
- ženské pohlaví MeSH
- MeSH
- cholesterol izolace a purifikace metabolismus MeSH
- desmosterol izolace a purifikace metabolismus MeSH
- HDL-cholesterol izolace a purifikace metabolismus MeSH
- hypercholesterolemie * diagnóza etnologie MeSH
- lanosterol izolace a purifikace metabolismus MeSH
- LDL-cholesterol izolace a purifikace metabolismus MeSH
- lidé MeSH
- retrospektivní studie * MeSH
- sitosteroly izolace a purifikace metabolismus MeSH
- statistika jako téma MeSH
- steroly izolace a purifikace metabolismus MeSH
- těhotenství * fyziologie metabolismus MeSH
- triglyceridy izolace a purifikace metabolismus MeSH
- Check Tag
- lidé MeSH
- těhotenství * fyziologie metabolismus MeSH
- ženské pohlaví MeSH
β-Sitosterol and betulinic acid were used in designing their conjugates with selected polyamines bearing either an amide bond, or an ester and an amide bond simultaneously in the target molecule. The synthesized compounds were subjected to basic cytotoxic and antimicrobial tests. The synthetic protocol is described separately for each of the three series of the target amides, because each series of compounds required a different synthetic approach. The cytotoxicity was tested on cells derived from human T-lymphoblastic leukemia, breast adenocarcinoma and cervical cancer, and compared with the tests on normal human fibroblasts. Most of the target compounds (5a-5c, 11a-11c and 16a-16c) showed medium to high cytotoxicity (0.7-7.8 μM), however, in some cases the compounds showed high cytotoxicity even toward normal human fibroblasts (11a-11c). Two compounds of this series (11c and 16c) also displayed antimicrobial activity with high and selective microbe specificity. The compound 11c was potent against Escherichia coli (minimal inhibition concentration (MIC) 6.25 μg mL(-1), i.e. 9.75 nM mL(-1)) and Staphylococcus aureus (MIC 12.5 μg mL(-1), i.e. 19.5 nM mL(-1)), and showed medium activity against Pseudomonas aeruginosa. The compound 16c was highly active against Enterococcus faecalis and S. aureus (both, MIC 3.125 μg mL(-1), i.e. 4.22 nM mL(-1)), both Gram-positive bacteria, however showed only weak activity against E. coli and no activity against P. aeruginosa, both Gram-negative bacteria, which indicates possible microbe specificity of 16c. Comparing β-sitosterol-based series (5a-5c) and betulinic acid series (11a-11c and 16a-16c) of the target compounds, the latter one gave more promising structures. The compounds 11c and 16c showed effects which may be described as multifarious activity (pleiotropic effects).
- MeSH
- antibakteriální látky chemie farmakologie MeSH
- Escherichia coli účinky léků MeSH
- HeLa buňky MeSH
- lidé MeSH
- MFC-7 buňky MeSH
- mikrobiální testy citlivosti MeSH
- polyaminy chemie toxicita MeSH
- Pseudomonas aeruginosa účinky léků MeSH
- sitosteroly chemie farmakologie MeSH
- Staphylococcus aureus účinky léků MeSH
- triterpeny chemie farmakologie MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
A new method, reversed phase liquid chromatography with off-line surface-assisted laser desorption/ionization mass spectrometry (RPLC-SALDI MS) for the determination of brassicasterol (BR), cholesterol (CH), stigmasterol (ST), campesterol (CA) and β-sitosterol (SI) in oil samples has been developed. The sample preparation consisted of alkaline saponification followed by extraction of the unsaponificable fraction with diethyl ether. The recovery of the sterols ranged from 91 to 95% with RSD less than 4%. Separation of the five major sterols on a C18 column using methanol-water gradient was achieved in about 10min. An on-line UV detector was employed for the initial sterol detection prior to effluent deposition using a laboratory-built spotter with 1:73 splitter. Off-line SALDI MS was then applied for mass determination/identification and quantification of the separated sterols. Ionization of the nonpolar analytes was achieved by silver ion cationization with silver nanoparticles used as the SALDI matrix providing limits of detection 12, 6 and 11fmol for CH, ST and SI, respectively. Because of the incorporated splitter, the effective limits of detection of the RPLC-SALDI MS analysis were 4, 3 and 4pmol (or 0.08, 0.06 and 0.08μg/mL) for CH, ST and SI, respectively. For quantification, 6-ketocholestanol (KE) was used as the internal standard. The method has been applied for the identification and quantification of sterols in olive, linseed and sunflower oil samples. The described off-line coupling of RPLC to SALDI MS represents an alternative to GC-MS for analysis of nonpolar compounds.
- MeSH
- cholestadienoly chemie izolace a purifikace MeSH
- cholesterol analogy a deriváty chemie izolace a purifikace MeSH
- chromatografie s reverzní fází metody normy MeSH
- fytosteroly chemie izolace a purifikace MeSH
- ketocholesteroly chemie izolace a purifikace MeSH
- lněný olej analýza chemie MeSH
- oleje rostlin analýza chemie MeSH
- referenční standardy MeSH
- sitosteroly chemie izolace a purifikace MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody normy MeSH
- stigmasterol chemie izolace a purifikace MeSH
- stříbro chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Účel studie: Cílem této pilotní studie bylo popsat změny v lipidovém metabolizmu u pacientů s akutním krvácením do gastrointestinálního traktu (GIT) a posoudit možný vliv hemodiluce a změn v procesu získávání cholesterolu (syntéza a absorpce). Materiál a metody: U dvanácti pacientů s akutním krvácením do GIT byly stanoveny hodnoty lipidového metabolizmu (celkový, LDL a HDL cholesterol, triglyceridy) v den 0, 3 a 6. Ve stejných termínech byly stanoveny hladiny ukazatelů cholesterolové syntézy (skvalen, lathosterol) a absorpce (sitosterol, kampesterol). Výsledky byly hodnoceny srovnáním s kontrolním souborem a porovnáním nemocných s cirhózou a bez postižení jater. Současně byl hodnocen vztah mezi lipidy a hodnotami hemoglobinu, hematokritu a spotřebou transfuzí. Výsledky: Hladiny celkového cholesterolu byly sníženy významně (p < 0,001), HDL a LDL nevýznamně a triglyceridy byly v normě. Hladiny lathosterolu byly sníženy signifikantně (p < 0,05), skvalenu, kampesterolu a sitosterolu nesignifikantně. Porovnání pacientů s cirhózou a bez ní přineslo statisticky významný rozdíl v hladinách cholesterolu a lathosterolu (p < 0,05). Korelace nepřinesly statisticky významné nálezy. Závěr: U pacientů s akutním krvácením do horního GITu byly prokázány změny v metabolizmu lipidů včetně alterace procesu syntézy i absorpce. Hypocholesterolemii proto nelze vysvětlit pouze krevní ztrátou. Je nutné provést další podrobnější studie.
Purpose of the study: The aim of this pilot study was to describe changes in lipid metabolism in patients with acute gastrointestinal (GI) bleeding and to assess possible influence of hemodilution and changes in the cholesterol synthesis/absorption process. Material and methods: In 12 patients with gastrointestinal bleeding, the levels of lipid metabolism (total, LDL and HDL cholesterol and triglycerides) were determined on days 0, 3 and 6. On the same days, levels of the cholesterol synthesis indexes (lathosterol, squalene) and absorption (campesterol, sitosterol) were determined. The results were evaluated in comparison with the control set, and cirrhotic and non-cirrhotic patients were compared. At the same time, correlations of lipids and haemoglobin, hematocrit levels anduse of transfusions were evaluated. Results: The patients had significantly lower levels of total cholesterol (p < 0.001). Decrease of LDL and HDL cholesterol was not significant. Concentrations of triglycerides were normal. Decrease of lathosterol was significant (p < 0.05), but decrease in squalene, campesterol and sitosterol levels was insignificant. Comparing patients with cirrhosis and without cirrhosis showed statistically significant difference in levels of cholesterol and lathosterol (p < 0.05). Correlations did not show any statistically significant findings. Conclusion: In patients with acute GI bleeding, changes is lipid metabolism including alteration of the process of synthesis and absorption were proved. Therefore, hypocholesterolemia cannot be explained by blood loss only. However, more detailed studies need to be carried out. 30. 1. 2013
- MeSH
- akutní nemoc MeSH
- časové faktory MeSH
- cholesterol * analogy a deriváty krev metabolismus MeSH
- fytosteroly krev MeSH
- gastrointestinální krvácení * krev metabolismus MeSH
- HDL-cholesterol krev metabolismus MeSH
- hematokrit MeSH
- hemodiluce MeSH
- jaterní cirhóza komplikace MeSH
- kritický stav MeSH
- LDL-cholesterol krev metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- lipidy MeSH
- pilotní projekty MeSH
- sitosteroly krev MeSH
- skvalen krev MeSH
- statistika jako téma MeSH
- steroly MeSH
- studie případů a kontrol MeSH
- triglyceridy krev metabolismus MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
