Aging is a complex process of organism decline in physiological functions. There is no clear theory explaining this phenomenon, but the most accepted one is the oxidative stress theory of aging. Biomarkers of oxidative stress, substances, which are formed during oxidative damage of phospholipids, proteins, and nucleic acids, are present in body fluids of diseased people as well as the healthy ones (in a physiological concentration). 8-iso prostaglandin F2α is the most prominent biomarker of phospholipid oxidative damage, o-tyrosine, 3-chlorotyrosine, and 3-nitrotyrosine are biomarkers of protein oxidative damage, and 8-hydroxy-2(')-deoxyguanosine and 8-hydroxyguanosine are biomarkers of oxidative damage of nucleic acids. It is thought that the concentration of biomarkers increases as the age of people increases. However, the concentration of biomarkers in body fluids is very low and, therefore, it is necessary to use a sensitive analytical method. A combination of HPLC and MS was chosen to determine biomarker concentration in three groups of healthy people of a different age (twenty, forty, and sixty years) in order to find a difference among the groups.
- MeSH
- biologické markery metabolismus MeSH
- dinoprost analogy a deriváty metabolismus MeSH
- guanosin analogy a deriváty metabolismus MeSH
- lidé MeSH
- oxidační stres * MeSH
- poškození DNA MeSH
- proteiny chemie metabolismus MeSH
- stárnutí * MeSH
- tyrosin analogy a deriváty metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
A sensitive and precise method for simultaneous quantification of cysteinyl leukotrienes (=cys LTs) - leukotriene C4 (=LTC4), leukotriene D4 (=LTD4) and leukotriene E4 (=LTE4) - essential biomarkers of bronchial asthma present in exhaled breath condensate (=EBC) was developed. An immunomagnetic molecular probe was prepared by anchoring cysteinyl leukotrienes antibody on the surface of functionalized monodispersed magnetic particles and used to selectively isolate cys LTs from biological matrices - EBC, plasma and urine. Immobilization and the immunoaffinity capture procedures were optimized to maximize the amount of separated cys LTs, which were detected "off-beads" after acidic elution by UHPLC-ESI-MS/MS operated in a multiple reaction monitoring mode. The developed method was characterized with high precision ≤13.6% (intra-day precision determined as RSD) and ≤14.5% (inter-day precision determined as RSD), acceptable accuracy ≤18.5% (determined as RE), and high recovery of immunoseparation (≥93.1%) in aforementioned biological matrices. The applicability of the method was demonstrated on EBC, plasma and urine clinical samples of patients with various subtypes of bronchial asthma (occupational, steroid-resistant, moderate with and without corticosteroids therapy) and healthy subjects where reasonable differences in cys LTs concentration levels were found. Combining extremely selective immunomagnetic separation with highly sensitive and precise detection step, the developed method was used to aid diagnosis, predict the most effective therapy, and monitor the response to treatment. The detection of elevated inflammatory mediators (cys LTs) in EBC of subjects with relatively asymptomatic asthma and normal pulmonary function tests could offer a novel way for monitoring the lung inflammation and perhaps initiating treatment in an earlier stage.
- MeSH
- bronchiální astma diagnóza farmakoterapie patofyziologie MeSH
- dechové testy MeSH
- glukokortikoidy terapeutické užití MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- imunomagnetická separace metody MeSH
- leukotrien C4 analýza MeSH
- leukotrien D4 analýza MeSH
- leukotrien E4 analýza MeSH
- lidé středního věku MeSH
- lidé MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- studie případů a kontrol MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- klinické zkoušky kontrolované MeSH
- práce podpořená grantem MeSH
- validační studie MeSH
A rapid and precise method for the identification and quantification of cysteinyl leukotrienes (leukotriene C(4), leukotriene D(4) and leukotriene E(4)), essential markers of bronchial asthma, in exhaled breath condensate was developed. The protocol consists of immunoaffinity separation and a detection step, liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In particular, the selected reaction monitoring mode was used for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method was characterized with a high precision (≤ 7.7%, determined as RSD), an acceptable accuracy (90.4-93.7%, determined as recovery), a low limit of detection (≤ 2 pg/ml EBC) and a low limit of quantification (≤ 10 pg/ml EBC). It was compared to other simple, clinically appropriate combinations of pre-treatment methods (solid phase extraction and lyophilization) with LC/MS. Finally, the method (a combination of immunoaffinity separation with LC-MS) was successfully tested in a clinical study where a significant difference was found in the concentration levels of cysteinyl leukotrienes between patients with occupational bronchial asthma and healthy subjects.
- MeSH
- bronchiální astma diagnóza MeSH
- chromatografie kapalinová metody MeSH
- cystein analýza MeSH
- dechové testy metody MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- imunoanalýza metody MeSH
- leukotrieny analýza MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- vydechnutí MeSH
- Check Tag
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
A sensitive assay method was developed for a parallel, rapid and precise determination of dopamine and its metabolites, homovanillic acid, 3-methoxytyramine and 3,4-dihydroxyphenylacetic acid, from brain microdialysates. The method consisted of a pre-treatment step, freeze-drying (lyophilization), to concentrate dopamine and its metabolites from the microdialysates, and a detection step using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In particular, the reaction monitoring mode was selected for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method was characterized by the following parameters: the precision of the developed method was determined as ≥88.6% for dopamine, ≥89.9% for homovanillic acid, ≥86.1% for 3-methoxytyramine and ≥88.1% for 3,4-dihydroxyphenylacetic acid; the mean accuracy was determined as ≥88.2% for dopamine, ≥88.3% for homovanillic acid, ≥85.9% for 3-methoxytyramine and ≥88.6% for 3,4-dihydroxyphenylacetic acid. The developed method was compared to (1) other combinations of pre-treatment methods (solid phase extraction and nitrogen stripping) with LC-MS and (2) another detection method, liquid chromatography, with electrochemical detection. The novel developed method using combination of lyophilization with LC-ESI-MS/MS was tested on real samples obtained from the nucleus accumbens of rat pups after an acute methamphetamine administration. It was proven that the developed assay could be applied to both a simultaneous analysis of all four substrates (dopamine, homovanillic acid, 3-methoxytyramine and 3,4-dihydroxyphenylacetic acid) in microdialysis samples acquired from the rat brain and the monitoring of their slight concentration changes on a picogram level over time following methamphetamine stimulus.
- MeSH
- chromatografie kapalinová metody MeSH
- dopamin analogy a deriváty analýza metabolismus MeSH
- krysa rodu rattus MeSH
- kyselina 3,4-dihydroxyfenyloctová analýza metabolismus MeSH
- kyselina homovanilová analýza metabolismus MeSH
- lineární modely MeSH
- lyofilizace MeSH
- methamfetamin aplikace a dávkování MeSH
- mikrodialýza MeSH
- nucleus accumbens chemie metabolismus MeSH
- potkani Wistar MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- stabilita léku MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A method was developed for rapid and accurate determination of cysteinylleukotrienes (LTC4, LTD4, LTE4), essential biomarkers of bronchial asthma, in exhaled breath condensate. The method consists of an immunoseparation procedure combined with selective and sensitive detection by LC-ESI-MS in the multiple reaction monitoring mode (MRM) and using a stable-isotopedilution assay. The precision of the method was around 94 %. The mean accuracy was higher than 90 % for the content of cysteinylleukotrienes in the concentrate up to 250 pg ml-1. The method was tested on patients with occupational bronchial asthma and compared with healthy subjects. The differences between the two groups were significant.
Sensitive assay method was developed for a parallel, rapid and precise determination of the most prominent oxidative stress biomarkers: 8-iso-prostaglandin F(2alpha), malondialdehyde and 4-hydroxynonenal. The method consisted of a pre-treatment part a solid-phase extraction, for rapid and effective isolation of biomarkers from body fluids (exhaled breath condensate, plasma and urine) and the detection method LC-ESI-MS/MS, where the selected reaction monitoring mode was used for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method was characterized by the following parameters: the imprecision was below 14.3%, the mean inaccuracy was determined to be lower than 13.1%. The method was tested on samples obtained from patients diagnosed with asbestosis, pleural hyalinosis or silicosis, i.e. occupational lung diseases caused by fibrogenic dusts, inducing oxidative stress in the respiratory system, and then compared to samples from healthy subjects. The difference in concentration levels of biomarkers between the two groups was perceptible in all the body fluids (the difference observed in an exhaled breath condensate was statistically most significant).
- MeSH
- aldehydy analýza krev moč MeSH
- azbest toxicita MeSH
- biologické markery analýza krev moč MeSH
- dinoprost analogy a deriváty analýza krev moč MeSH
- extrakce na pevné fázi metody MeSH
- lidé středního věku MeSH
- lidé MeSH
- malondialdehyd analýza krev moč MeSH
- oxid křemičitý toxicita MeSH
- oxidační stres MeSH
- plicní nemoci etiologie krev metabolismus moč MeSH
- senioři MeSH
- studie případů a kontrol MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- tělesné tekutiny chemie metabolismus MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- senioři MeSH
- Publikační typ
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Rapid and precise method for the determination of 8-iso-prostaglandin F(2alpha), an essential marker of the oxidative stress, in exhaled breath condensate (EBC) was developed. The protocol consisted of stable isotope dilution, immunoseparation combined with selective and sensitive LC-ESI-MS/MS operated in multiple reaction monitoring (MRM) mode. The imprecision of the developed method was below 8.8%, the parameter of mean inaccuracy was determined as <9.6% (0-250pg of 8-iso-prostaglandin F(2alpha)/ml EBC). The limit of detection (LOD) was 1 pg/ml EBC and limit of quantification (LOQ) 5 pg/ml EBC. A significant difference in 8-iso-prostaglandin F(2alpha) content between the group of asbestosis patients and healthy volunteers was found.
- MeSH
- dechové testy MeSH
- dinoprost analogy a deriváty analýza MeSH
- financování organizované MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- kalibrace MeSH
- lidé MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- validační studie MeSH
