Spectrophotometric detection
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Prostate cancer is the most common malignant tumor in men, whose incidence significantly differs geograph-ically and increases with age. For rapid diagnostics, new tumor markers with higher prognostic relevance are still being sought. A very promising candidate molecule is the amino acid sarcosine. The aim of this work was to design a highly sensitive photometric detection system for the sarcosine determination. An original, yet unpublished, methodology for determining the amino acid sarcosine using Trinder's reaction has been proposed. Absorbance dependence on sarcosine concentration at 546 nm is linear over a total range of 0–1000 μM (r≥ 0.99) with limit of detection (LOD) of 6 μM and limit of quantification (LOQ) of 20 μM. The suggested procedure allows to ana-lyze the content of sarcosine in real urine specimens at micromolar concentrations.
A sensitive capillary electrophoretic method featuring spectrophotometric detection using a commercial Z-cell was devised for the assay of 8-hydroxy-2'-deoxyguanosine (8OHdG) in human urine. Solid-phase extraction (SPE) based on hydrophilic-lipophilic-balanced RP sorbent was utilized for urine sample pretreatment and analyte preconcentration. The separation was carried out in conventional fused-silica capillaries employing a Z-cell with hydrodynamic sample injection (at 50 mbar for 12 s). The BGE (pH* 9.2, adjusted with 1 M NaOH) contained 0.15 M boric acid and 10% v/v ACN. The detection wavelength was 282 nm. The calibration curve for 8OHdG (measured in spiked urine) was linear in the range 10-1000 ng/mL; R(2) = 0.9993. The LOD was 3 ng/mL (11 nmol/L) of 8OHdG. Determination of the 8OHdG urinary levels was possible even in healthy individuals.
Butyrylcholinesterase (BChE) is an enzyme presented in quite high level in blood plasma where it participates in detoxification reactions. Due to fact that the enzyme is constituted in livers, it is a marker of liver parenchyma function. It can be used for diagnosis of poisoning for e.g., nerve agents or carbofuran and intoxication by some drugs such as rivastigmine. The present experiment is devoted for the creation of new spectrophotometric tests for assay of BChE activity in biological samples. Standard Ellman's method was compared with use of 2,6-dichloroindophenol acetate and indoxylacetate as chromogenic substrates. Maximal velocities and Michaelis constants were calculated for the substrates. Considering calibration, 2,6-dichloroindophenol acetate provided the lowest limit of detection: 1.20 × 10(-9)kat and a long linear range. All methods were verified using pooled human plasma samples and tested for potential interferents. 2,6-dichloroindophenol acetate is recommended as suitable substrate for BChE assay in clinical diagnostics.
- MeSH
- 2,6-dichlorindofenol chemie MeSH
- acetáty MeSH
- biologické markery krev MeSH
- butyrylcholinesterasa krev MeSH
- indoly chemie MeSH
- játra enzymologie MeSH
- kinetika MeSH
- lidé MeSH
- limita detekce MeSH
- spektrofotometrie metody MeSH
- substrátová specifita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Procedures for the extraction-spectrophotometric determination of tris(2-chloroethyl)amine, an alkylating agent known as a drug as well as a chemical warfare agent (nitrogen mustard HN-3), with 7 acid-base indicators of a triphenylmethane lactone type, phthaleins, were developed. Representatives of phthaleins without an oxygen bridge (thymolphthalein, o-cresolphthalein, naphtholphthalein) and with an oxygen bridge (fluorescein, 2',7'-dichlorofluorescein, eosin B and eosin Y) were used. The methods were based on the formation of ion pair complexes. Chloroform was used as a non-polar solvent for an extraction. The conditions to determine were optimized for the optimal pH of the buffer and the concentration of a phthalein as a reagent. The dependence on the reaction time in a water phase and the stoichiometry of extraction products were studied. The detection limits and the limits of the determination of separate procedures and conditional extraction constants were determined. Comparison with the spectrophotometric method of the group determination of alkyl halides and acyl halides using alkaline ethanol-water solution of thymolphthalein, the so-called T-135 agent, was conducted. While studying the selectivity, the possible interference of bis(2-chloroethyl)sulphide and 3 nitrogen mustards in the proposed procedures were verified. Copyright © 2016 John Wiley & Sons, Ltd.
- MeSH
- alkylační látky analýza izolace a purifikace MeSH
- chemické bojové látky analýza izolace a purifikace MeSH
- fenolftaleiny chemie MeSH
- koncentrace vodíkových iontů MeSH
- limita detekce MeSH
- pufry MeSH
- sloučeniny dusíkatého yperitu analýza izolace a purifikace MeSH
- spektrofotometrie metody MeSH
- voda analýza MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
Cíl studie: Cílem metodického sdělení bylo zavedení a validace dostupné a praktické enzymatické metody stanovení Dlaktátu v krevní plazmě a moči. Zavedená metoda byla využita ke stanovení koncentrací D-laktátu v plazmě v souboru 117 pacientů v perioperační péči. Typ studie: Jedná se o metodické sdělení s uvedením výsledků prospektivní observační klinické studie. Název a sídlo pracoviště: Centrum pro výzkum a vývoj, Fakultní nemocnice Hradec Králové Materiál a metody: Krevní plazma je deproteinována ultrafiltrací, moč je analyzována přímo. Vlastním principem metody jsou dvě spřažené enzymové reakce katalyzované D-laktát dehydrogenázou při redukci nikotinamid-adenin-dinukleotidu a Dglutamát- pyruvát transaminázou. Měřena je konečná koncentrace redukovaného nikotinamid-adenin-dinukleotidu při 340 nm. Soubor pacientů studie byl tvořen 117 pacienty podstupujícími resekci tlustého střeva nebo konečníku pro operabilní karcinom kolorekta. Odběry byly prováděny předoperačně, dvě hodiny po operaci a první až čtvrtý pooperační den. Výsledky: V plazmě bylo dosaženo preciznosti CV 1,5 - 4,9 % podle koncentrace. Kvantifikační limit metody je na koncentraci 10 μmol/l. Detekční limit metody je na koncentraci 5 μmol/l. Metoda je lineární v rozsahu 10 μmol/l až 1 mmol/l. Bazální předoperační hodnoty 33,4 (26,4; 39,7) μmol/l stouply již za dvě hodiny po ukončení operace na koncentraci 90,2 (78; 101) μmol/l, která dosáhla vrcholu v první pooperační den a následně došlo v průběhu dalších tří dní k návratu k původním hodnotám. Závěr: Uvedená metoda může být vzhledem ke své dostupnosti, robustnosti i jednoduchosti snadno zavedena v biochemické laboratoři, kde se vyskytne požadavek na provádění tohoto vyšetření.
Objective: The aim of the study was to develop and validate an easily available and robust enzymatic method for determination of D-lactate in blood plasma and urine. The developed method was used for the evaluation of plasmatic D-lactate levels in patients in perioperative care. Design: The article describes the methodology and presents the results of a prospective, observational study. Settings: Center for Research and Development, University Hospital Hradec Králové Material and Methods: Blood plasma was deproteinated by ultrafiltration; urine may be analysed directly. The principle of the method is the coupled reaction of D-lactate dehydrogenase reducing nicotinamide adenine dinucleotide and D-glutamate pyruvate transaminase. The end-point concentration of nicotinamide adenine dinucleotide is determined as absorbance at 340 nm. The patient cohort consisted of 117 patients operated for colorectal cancer. The blood samples were collected preoperatively, 2 hours after operation, and daily from the first to the fourth postoperative day. Data are presented as median (interquartile range). Results: The precision achieved in plasma was 1.5 – 4.9 %. A quantification limit of 10 μmol/L and detection limit of 5 μmol/l were found. The method is linear from 10 μmol/L to 1 mmol/L. Basal preoperative values of 33.4 (26.4; 39.7) μmol/L increased within two hours postoperatively to 90.2 (78; 101) μmol/L, and D-lactate concentration peaked on the first postoperative day. The concentration returned to basal values on the fourth day. Conclusion: The described method may be used widely in clinical chemistry laboratories due to its availability, robustness and simplicity, wherever D-lactate testing is required.
- Klíčová slova
- D-laktát,
- MeSH
- acidóza * diagnóza MeSH
- analýza moči metody MeSH
- biochemická analýza krve metody MeSH
- kyselina mléčná * biosyntéza krev moč škodlivé účinky MeSH
- lidé MeSH
- perioperační péče MeSH
- reprodukovatelnost výsledků MeSH
- spektrofotometrie MeSH
- syndrom krátkého střeva patofyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- hodnotící studie MeSH
- klinická studie MeSH
- práce podpořená grantem MeSH
