The minimization is currently expanding in all fields of human life. The smaller size of analytical instruments and their higher sensitivity enables an analysis of small amounts of a biological material with a very low consumption of chemicals, what is economically and also environmentally benefi cial. A patient friendly and minimal invasive sample collection is therefore more than required. The dried blood spot (DBS) sampling standardly used in the newborn screening (NS) may be an option. The sample collection is simple, non-invasive, does not require trained medical personnel assistance and also storing and transportation of these samples is much easier in comparison with the whole blood samples. The DBS sampling method is used in the therapeutic drug monitoring or in the diagnosis of infectious diseases and its usage is still spreading. In some cases, it is still only a research object, but it shows a big potential for a future use. It could completely replace the whole venous blood collection in some cases, for example in the metabolic screening of diabetic patients or monitoring of treatment response, and it can overall simplify the sample collection and the transportation process (Tab. 1, Ref. 152).
Polycyklické aromatické uhlovodíky (PAU) představují skupinu významných kontaminantů pracovního i životního prostředí. Při expozici PAU v pracovním prostředí se vedle inhalační expoziční cesty může významně uplatňovat i expozice dermální. Stávající experimentální údaje o intenzitě a rychlosti penetrace látek do systémové cirkulace jsou zatím omezené. Předkládaný článek se zabývá metodickou a interpretační problematikou transepidermální absorpce PAU in vitro. Byla sledována intenzita (Flux) a rychlost (Lag time) penetrace naftalenu, fenanthrenu, pyrenu a benzo[a]pyrenu přes epidermální membránu z prasečího boltce. Experiment byl prováděn ve vertikálních statických difuzních komůrkách dle Franze (n = 32) a jako rozpouštědlo byl použit isopropyl-myristát. Parametr Flux (nmol/cm2/hod) dosáhl hodnot 95,7 ± 45,5 u naftalenu, 19,5 ± 8,7 u fenanthrenu, 4,38 ± 1,98 u pyrenu a 0,21 ± 0,08 u benzo[a]pyrenu. Parametr Lag time (hod) hodnot 0,26 ± 0,17 u naftalenu, 2,12 ± 0,41 u fenanthrenu, 3,25 ± 0,50 u pyrenu a 11,2 ± 4,08 u benzo[a]pyrenu. Hodnota parametru Flux klesala s molární hmotností PAU, zatímco hodnota parametru Lag time s molární hmotností PAU stoupala. Množství PAU, které za daný časový úsek penetrovalo přes epidermální membránu, se pohybovalo mezi 0,24 % (benzo[a]pyren) až 0,84 % (fenanthren) aplikované dávky. Penetrace PAU přes epidermální membránu vykazovala, v porovnání s experimenty na plné kůži, nižší stupeň variability dat. Z výsledků vyplývá, že použití epidermální membrány by mohlo zpřesňovat jak odhad vnitřní dávky PAU po dermální expozici, tak i odhad souvisejícího zdravotního rizika v rámci konzervativního expozičního scénáře. Experimenty s epidermální membránou jsou však časově i experimentálně náročné, bez možnosti objektivní kontroly integrity epidermální membrány, což může vést k finanční náročnosti testování, ztrátám vzorků a v neposlední řadě i k nárůstu rozdílů hodnot dat, získaných v různých laboratořích.
Polycyclic aromatic hydrocarbons (PAHs) are a group of significant contaminants in the occupational and living environment. In addition to the inhalation route of exposure in the occupational environment, there is also significant the dermal route of exposure. Existing experimental data on the intensity and rate of penetration of these substances into systemic circulation are still limited. The presented paper is focused on methodological and interpretative issues of trans-epidermal absorption of PAHs in vitro. In this study, we assessed the intensity (Flux) and rate (Lag time) of penetration of naphthalene, phenanthrene, pyrene and benzo[a]pyrene through the epidermal membrane derived from a pig ear. The experiment was performed using static vertical Franz diffusion cells (n = 32) and isopropyl myristate was used as a solvent. Flux (nmol/cm2/hour) reached 95.7 ± 45.5 in the case of naphthalene, 19.5 ± 8.7 of phenanthrene, 4.38 ± 1.98 of pyrene and 0.21 ± 0.08 of benzo[a]pyrene. Lag time (hour) was 0.26 ± 0.17 in the case of naphthalene, 2.12 ± 0.41 of phenanthrene, 3.25 ± 0.50 of pyrene and 11.2 ± 4.08 of benzo[a]pyrene. The Flux value decreased with the molecular weight of PAHs, while the Lag time increased with the molecular weight of PAHs. The amount of PAHs that penetrated through the epidermal membrane in a given time period ranged between 0.24% (benzo[a]pyrene) and 0.84% (phenanthrene) of the applied dose. The penetration of PAHs through the epidermal membrane showed a lower degree of data variability compared to full thickness skin experiments. The results suggest that the use of the epidermal membrane could define more accurately both the estimation of the internal dose of PAHs after dermal exposure and the estimation of the associated health risk within a conservative exposure scenario. However, experiments with using the epidermal membrane are time consuming and experimentally demanding, with no option of an objective control of the integrity of the epidermal membrane, which can lead to costly testing, loss of samples and, finally, an increase in the differences in data values obtained in different laboratories.
- MeSH
- epidermis anatomie a histologie patofyziologie MeSH
- interpretace statistických dat MeSH
- kožní absorpce * fyziologie MeSH
- modely u zvířat MeSH
- polycyklické aromatické uhlovodíky * škodlivé účinky MeSH
- prasata MeSH
- techniky in vitro * metody statistika a číselné údaje MeSH
- ušní boltec MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
A method for the determination of selected amino acids in culture medium using HPLC with fluorescence detection is described. Twenty hours after intra-cytoplasmic sperm injection, one randomly selected zygote was transferred to the culture medium. After incubation (72 h after fertilization), the culture medium in which the embryo was incubated and blank medium was immediately stored at -80°C. Filtered medium samples were derivatized with ortho-phthalaldehyde (naphthalene-2,3-dicarboxaldehyde), forming highly fluorescent amino acids derivatives. Reverse-phase columns (LichroCART, Purospher STAR RP18e or Ascentis Express C18 ) were used for the separation. The derivatives were analyzed by gradient elution with a mobile phase containing ethanol and sodium dihydrogen phosphate. The analytical performance of this method is satisfactory for all amino acids; the intra-assay coefficients of variation were <10% and quantitative recoveries were between 95.5 and 104.4%. Changes in the levels of selected amino acids before and after human embryo cultivation were observed. After embryo incubation, the levels of all amino acids in the medium were increased, apart from aspartate and asparagine. After the cultivation of some embryos, amino acids which were not part of the medium were detected. Low amino acids turnover was observed in some embryos.
- MeSH
- aminokyseliny analýza metabolismus MeSH
- embryo savčí chemie metabolismus MeSH
- fertilizace in vitro MeSH
- kultivace embrya * MeSH
- kultivační média chemie metabolismus MeSH
- lidé MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- zygota chemie metabolismus MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
A method is described for the determination of fatty acids in dried sweat spot and plasma samples using gas chromatography with flame ionization detection. Plasma and dried sweat spot samples were obtained from a group of blood donors. The sweat was collected from each volunteer during exercise. Sweat was spotted onto collection paper containing butylated hydroxytoluene. Fatty acids were derivatized with acetyl chloride in methanol to form methyl esters of fatty acids. The fatty acids in dried sweat spot samples treated with butylated hydroxytoluene and stored at -20°C were stable for 3 months. Our results indicate that sweat contains, among fatty acids with short chain, also fatty acids with long chain and unsaturated fatty acids. Linear relationships between percentage content of selected fatty acids in dried sweat spot and plasma were observed.
- MeSH
- chromatografie plynová * MeSH
- estery analýza MeSH
- lidé MeSH
- mastné kyseliny analýza MeSH
- plamínková ionizace * MeSH
- pot chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The aim of this study was to find some relationship between amino acid metabolism and the embryo morphokinetic parameters studied via time-lapse analysis. Study included 48 human embryo samples and their culture media. Two groups of embryos were identified: embryos reached the 8-cell stage on day 3 (n=34) and embryos failed to develop at any point during the incubation (n=14). Amino acids levels were measured on day 3 of embryo development; using time-lapse analysis, the precise timing of embryo cleavage, synchrony of division, grade of fragmentation etc. were established. No statistically significant differences between dividing and arresting embryos were observed in terms of amino acids production/consumption and turnover. Amino acids which were part of the culture medium did not exhibit any statistically significant correlation with kinetic parameters with the exception of the grade of fragmentation on day 3; there were negative correlation with glutamate, and positive with glutamine, glycine and taurine. In some dividing and in some arresting embryos appeared new amino acids which strongly correlated with each other, with methionine, but not with any other amino acid that is a regular part of the culture medium.
- MeSH
- aminokyseliny metabolismus MeSH
- embryo savčí metabolismus MeSH
- embryonální vývoj * MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
I přes zvyšující se úspěšnost léčby neplodnosti metodami asistované reprodukce zůstává nadále problém vybrat nejkvalitnější embryo, které má v sobě potenciál dalšího vývoje a implantace. V současnosti je výběr embrya založen především na morfologických znacích. Tento přístup je subjektivní, proto je snahou nalézt jinou, objektivnější a robustnější metodu, vhodnou k výběru embrya. Metabolismus embrya představuje jednu z možností. Tato neinvazivní metoda umožňuje pozorovat změny v hladinách různých metabolitů v kultivačním médiu před inkubací a po inkubaci jednoho embrya. Nejčastěji diskutovanými látkami jsou sacharidy a aminokyseliny jako hlavní komponenty kultivačních médií. Sacharidy slouží převážně jako zdroj energie, zatímco aminokyseliny jsou využívány k syntéze proteinů a nukleotidů, působí jako antioxidanty, osmolyty a pufry. Bylo navrženo několik metod, které hodnotí metabolický profil embrya a je mnoho teorií, jak podle metabolismu embrya vybrat to nejvhodnější k transferu. Klíčová slova: aminokyseliny, metabolismus embrya, výběr embrya po IVF, sacharidy
Despite the increasing success of infertility treatment methods of assisted reproduction, it still remains a problem how to select the best embryo that has the potential for further development and implantation. At the present time, embryo selection is based especially on morphological criteria. This approach is subjective; therefore there is a trend to find another more objective and robust method for embryo selection. Embryo metabolism can be used as an indicator of viability. This non-invasive method allows observing changes in the levels of different metabolites in culture medium before and after incubation of the only one embryo. The most mentioned substances are carbohydrates and amino acids as important components of culture medium. Carbohydrates serve predominantly as energy sources, whereas amino acids are precursors of protein and nucleotides, antioxidants, osmolytes, pH regulators etc. Several methods have been proposed for evaluating of embryo metabolic profile of embryo. There are many hypotheses for embryo selection according its metabolic profile. Keywords: amino acids, embryo metabolism, embryo selection in IVF, carbohydrates
- Klíčová slova
- metabolismus embrya, výběr embrya,
- MeSH
- aminokyseliny metabolismus MeSH
- asistovaná reprodukce MeSH
- embryo savčí * metabolismus MeSH
- embryonální vývoj MeSH
- kultivace embrya MeSH
- kultivační média * chemie metabolismus MeSH
- lidé MeSH
- metabolismus lipidů MeSH
- metabolismus sacharidů MeSH
- nakládání s embryem * MeSH
- přenos embrya MeSH
- Check Tag
- lidé MeSH
