Sample acidification
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OBJECTIVES: Stability of concentrations of urinary stone-related metabolites was analyzed from samples of recurrent urinary stone formers to assess necessity and effectiveness of urine acidification during collection and storage. METHODS: First-morning urine was collected from 20 adult calcium-stone forming patients at Tomas Bata Hospital in the Czech Republic. Urine samples were analyzed for calcium, magnesium, inorganic phosphate, uric acid, sodium, potassium, chloride, citrate, oxalate, and urine particles. The single-voided specimens were collected without acidification, after which they were divided into three groups for storage: samples without acidification ("NON"), acidification before storage ("PRE"), or acidification after storage ("POST"). The analyses were conducted on the day of arrival (day 0, "baseline"), or after storage for 2 or 7 days at room temperature. The maximum permissible difference (MPD) was defined as ±20 % from the baseline. RESULTS: The urine concentrations of all stone-related metabolites remained within the 20 % MPD limits in NON and POST samples after 2 days, except for calcium in NON sample of one patient, and oxalate of three patients and citrate of one patient in POST samples. In PRE samples, stability failed in urine samples for oxalate of three patients, and for uric acid of four patients after 2 days. Failures in stability often correlated with high baseline concentrations of those metabolites in urine. CONCLUSIONS: Detailed procedures are needed to collect urine specimens for analysis of urinary stone-related metabolites, considering both patient safety and stability of those metabolites. We recommend specific preservation steps.
- MeSH
- analýza moči metody MeSH
- dospělí MeSH
- koncentrace vodíkových iontů MeSH
- kyselina močová moč MeSH
- lidé středního věku MeSH
- lidé MeSH
- močové kameny * moč MeSH
- odběr biologického vzorku metody MeSH
- recidiva * MeSH
- sbírání vzorku moči metody MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
In patients with testicular germ cell tumours (TGCT), sperm cryopreservation prior to anti-cancer treatment represents the main fertility preservation approach. However, it is associated with a low sperm recovery rate after thawing. Since sperm is a high-energy demanding cell, which is supplied by glycolysis and oxidative phosphorylation (OXPHOS), mitochondrial dysfunctionality can directly result in sperm anomalies. In this study, we investigated the bioenergetic pattern of cryopreserved sperm of TGCT patients in comparison with normozoospermic samples using two state-of-the-art methods: the Extracellular Flux Analyzer (XF Analyzer) and two-photon fluorescence lifetime imaging microscopy (2P-FLIM), in order to assess the contributions of OXPHOS and glycolysis to energy provision. A novel protocol for the combined measurement of OXPHOS (oxygen consumption rate: OCR) and glycolysis (extracellular acidification rate: ECAR) using the XF Analyzer was developed together with a unique customized AI-based approach for semiautomated processing of 2P-FLIM images. Our study delivers optimized low-HEPES modified human tubal fluid media (mHTF) for sperm handling during pre-analytical and analytical phases, to maintain sperm physiological parameters and optimal OCR, equivalent to OXPHOS. The negative effect of cryopreservation was signified by the deterioration of both bioenergetic pathways represented by modified OCR and ECAR curves and the derived parameters. This was true for normozoospermic as well as samples from TGCT patients, which showed even stronger damage within the respiratory chain compared to the level of glycolytic activity impairment. The impact of cryopreservation and pathology are supported by 2P-FLIM analysis, showing a significant decrease in bound NADH in contrast to unbound NAD(P)H, which reflects decreased metabolic activity in samples from TGCT patients. Our study provides novel insights into the impact of TGCT on sperm bioenergetics and delivers a verified protocol to be used for the assessment of human sperm metabolic activity, which can be a valuable tool for further research and clinical andrology.
- MeSH
- dospělí MeSH
- energetický metabolismus * MeSH
- germinální a embryonální nádory * metabolismus patologie MeSH
- glykolýza * MeSH
- kryoprezervace * metody MeSH
- lidé MeSH
- mitochondrie metabolismus MeSH
- oxidativní fosforylace * MeSH
- spermie * metabolismus MeSH
- spotřeba kyslíku fyziologie MeSH
- testikulární nádory * metabolismus patologie MeSH
- uchování spermatu metody MeSH
- zachování plodnosti metody MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Saxitoxins (STXs) are potent neurotoxins produced by marine dinoflagellates or freshwater cyanobacteria known to cause acute and eventually fatal human intoxications, which are classified as paralytic shellfish poisonings (PSPs). Rapid analysis of STXs in blood plasma can be used for a timely diagnosis and confirmation of PSPs. We developed a fast and simple method of STX extraction based on plasma sample acidification and precipitation by acetonitrile, followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Our approach provides the results ≤30 min, with a limit of detection of 2.8 ng/mL and a lower limit of quantification of 5.0 ng/mL. Within-run and between-run precision experiments showed good reproducibility with ≤15% values. Standard curves for calibration were linear with correlation coefficients ≥0.98 across the assay calibration range (5-200 ng/mL). In an interlaboratory analytical exercise, the method was found to be 100% accurate in determining the presence or absence of STX in human plasma specimens, with recovery values of 86-99%. This simple method for STX determination in animal or human plasma can quickly and reliably diagnose STX exposures and confirm suspected PSP cases to facilitate patient treatment or expedite necessary public health or security actions.
Citrate buffers are commonly utilized in the field of biomolecule stabilization. We investigate their applicability in the frozen state within a range of initial pHs (2.5 to 8.0) and concentrations (0.02 to 0.60 M). Citrate buffer solutions subjected to various cooling and heating temperatures are examined in terms of the freezing-induced acidity changes, revealing that citrate buffers acidify upon cooling. The acidity is assessed with sulfonephthalein molecular probes frozen in the samples. Optical cryomicroscopy combined with differential scanning calorimetry was employed to investigate the causes of the observed acidity changes. The buffers partly crystallize and partly vitrify in the ice matrix; these processes influence the resulting pH and allow designing the optimal storage temperatures in the frozen state. The freezing-induced acidification apparently depends on the buffer concentration; at each pH, we suggest pertinent concentration, at which freezing causes minimal acidification.
BACKGROUND: Recommendations on the optimal preservation of 24 h urine for the metabolic work-up in urolithiasis patients are very heterogeneous. In case two such tests with different storage condition recommendations are being analysed, multiple collections would be needed, challenging especially elderly and very young patients. We therefore aimed to evaluate the stability of urine constituents under different storage conditions. MATERIAL AND METHODS: We collected urine samples from ten healthy volunteers and prepared aliquots to be stored either at room temperature or 4 °C. Some aliquots were preserved using hydrochloric acid prior to storage, some thereafter, some using the BD Urine preservation tube and some were not preserved at all. Storage duration was 0, 24, 48 or 72 h. In all samples calcium, magnesium, phosphorus, creatinine, oxalate, citrate and uric acid were measured and compared to the according reference sample. RESULTS: We could not find any significant deviation for any of the analytes and preanalytical treatment conditions compared to the associated reference sample. CONCLUSION: Preservation of 24 h urine for the metabolic evaluation in stone formers might not be necessary for sample storage up to 72 h.
- MeSH
- hořčík MeSH
- koncentrace vodíkových iontů MeSH
- kyselina citronová MeSH
- lidé MeSH
- rizikové faktory MeSH
- senioři MeSH
- urolitiáza * diagnóza moč MeSH
- vápník MeSH
- Check Tag
- lidé MeSH
- senioři MeSH
- Publikační typ
- časopisecké články MeSH
Introduction: Kidney stone formers can have higher oxalate and phosphate salt amounts in their urine than healthy people and we hypothesized that its acidification may be useful. The study aims to compare results of urine concentrations of calcium, magnesium, and inorganic phosphorus in the midstream portion of first voided morning urine samples without (FMU) and with post-collection acidification (FMUa) in kidney stone patients. Materials and methods: This is a prospective single center study. A total of 138 kidney stone patients with spot urine samples were included in the study. Urine concentrations of calcium, magnesium and inorganic phosphorus were measured with and without post-collection acidification. Acidification was performed by adding 5 μL of 6 mol/L HCl to 1 mL of urine. Results: The median age (range) of all participants was 56 (18-87) years. The median paired differences between FMU and FMUa concentrations of calcium, magnesium, and inorganic phosphorus were: - 0.040 mmol/L, 0.035 mmol/L, and 0.060 mmol/L, respectively. They were statistically different: P < 0.001, P < 0.001, P = 0.004, respectively. These differences are not clinically significant because biological variations of these markers are much higher. Conclusions: No clinically significant differences in urinary calcium, magnesium, and inorganic phosphorus concentrations between FMU and FMUa in patients with kidney stones were found.
- MeSH
- elektrolyty MeSH
- fosfor MeSH
- hořčík * moč MeSH
- koncentrace vodíkových iontů MeSH
- ledvinové kameny * MeSH
- lidé středního věku MeSH
- lidé MeSH
- prospektivní studie MeSH
- vápník MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
A tutorial and spreadsheet for the validation and bottom-up uncertainty evaluation of quantifications performed by instrumental methods of analysis based on linear weighted calibrations is presented. The developed tool automatically assesses if calibrator values uncertainty is negligible given instrumental signal precision, assesses signal homoscedasticity by the Levene's test, guides the selection of weighting factors and evaluates the fitness of the regression model to define the calibration curve. The spreadsheet allows the use of the linear weighted regression model without the need for collecting many replicate signals of calibrators and sample by taking previously developed detailed models of signal precision variation in the calibration interval after adjustments to the daily precision conditions. This tool was successfully applied to the determination of the mass concentration of Cd, Pb, As, Hg, Co, V and Ni in a nasal spray by ICP-MS after samples dilution and acidification. The developed uncertainty models were checked through the analysis of nasal sprays after spiking with known analyte concentration levels. The metrological compatibility between estimated and reference analyte levels for 95% or 99% confidence level supports uncertainty model adequacy. The spiked samples were quantified from many replicate signals but uncertainty evaluation from duplicate calibrator and sample signals was assessed by randomly selecting calibrators and sample signals and by numerically defining a minimum acceptable success rate of the compatibility tests. The developed model was proven adequate to quantify the uncertainty of the studied measurements.
- MeSH
- kalibrace MeSH
- lineární modely MeSH
- nejistota MeSH
- nosní spreje * MeSH
- spektrální analýza MeSH
- Publikační typ
- časopisecké články MeSH
Stabilized cementitious aggregates AG were produced from wood ashes containing ∼10,000 mg kg-1 As, Cr and Cu, then amended to two agricultural pasture soils. Metal(loid) leaching (column tests), mobility (pore water extracts) and uptake to ryegrass was determined, comparing raw ashes with aggregates. Risk modeling was applied to selected data to inform wider discussion of the experimental results. Under rapid leaching (7 h) AG 2 (pre-strengthened with CO2) outperformed AG 1 in suppressing soluble metal(loid) removal. During prolonged leaching (12d) both aggregates were susceptible to mild dissolution/release of metal(loid)s upon acidification. Pore water sampled from the pot test indicated that Cr was generally most mobile, As least so, reduced furthest by AG 2. Risk modelling, based on pot experimental data, demonstrated soil specific accumulation of As in beef muscle and milk, being furthest reduced (compared to the raw ash addition) by AG 2 in soil A, but increased in soil B by the same treatment. The results of this study indicate that a reduction in soluble As, Cr and Cu can be achieved through cementitious aggregation of wood ashes, though the extent is metal(loid) specific when amended to soils. Pre-testing under local soil conditions before field application would be required to ensure that metal(loid) mobility remained suppressed.
- Publikační typ
- časopisecké články MeSH
Background: Monitoring the acidity of cheese is an important control mechanism in various stages of manufacture, including aging. Acid development in cheesemaking is essential to cheese flavor, texture, and safety. Objective: The aim of the work was to develop and validate calibration models by using NIR spectroscopy, which allows for the monitoring of changes in cheese acidity (pH and titration acidity) during cheese ripening. Methods: Cheeses were analyzed by an FT-NIR spectrometer. Each of the samples was analyzed three times, and for calibration, an average spectrum was used. A partial least-squares regression was used to develop calibration models. The constructed calibration models were validating by full cross-validation. Results: Calibration models were created with a high correlation coefficient for the following cheese pH levels: blue cheese (0.966), Olomouc curd read smear cheese (0.984), and fresh goat cheese (0.980). Results of the calibration of titratable acidity are functional for fresh goat cheese (0.953) and mozzarella (0.999). Conclusions: The results of these new calibration methods showed the possibility of NIR technology for the fast determination of pH and titratable acidity. Highlights: Detection of cheese acidity using FT-NIR spectrometry enables rapid evaluation of the process of lactic acidification in particular cheese technological operations, including the maturing of cheeses.
Sobuzoxane (MST-16) is an approved anticancer agent, a pro-drug of bisdioxopiperazine analog ICRF-154. Due to the structural similarity of ICRF-154 to dexrazoxane (ICRF-187), MST-16 deserves attention as a cardioprotective drug. This study presents for the first time UHPLC-MS/MS assay of MST-16, ICRF-154 and its metabolite (EDTA-diamide) in cell culture medium, buffer, plasma and cardiac cells and provides data on MST-16 bioactivation under conditions relevant to investigation of cardioprotection of this drug. The analysis of these compounds that differ considerably in their lipophilicity was achieved on the Zorbax SB-Aq column using a mixture of aqueous ammonium formate and methanol as a mobile phase. The biological samples were either diluted or precipitated with methanol, which was followed by acidification for the assay of MST-16. The method was validated for determination of all compounds in the biological materials. The application of the method for analysis of samples from in vitro experiments provided important findings, namely, that (1) MST-16 is quickly decomposed in biological environments, (2) the cardiac cells actively metabolize MST-16, and (3) MST-16 readily penetrates into the cardiac cells and is converted into ICRF-154 and EDTA-diamide. These data are useful for the in-depth examination of the cardioprotective potential of this drug.
- MeSH
- EDTA chemie MeSH
- kardiomyocyty cytologie metabolismus MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- piperaziny analýza MeSH
- potkani Wistar MeSH
- protinádorové látky analýza metabolismus MeSH
- razoxan analogy a deriváty chemie metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
