BACKGROUND: Exposure to benzophenone-1 (BP-1) and benzophenone-3 (BP-3), widely used as UV filters in personal care products, has been associated with adverse health effects. However, epidemiological evidence is limited and inconclusive, particularly in vulnerable populations such as teenagers. OBJECTIVE: To examine the relation between BP-1 and BP-3 concentrations and obesity, cardiometabolic biomarkers, and asthma/allergy outcomes in European teenagers, including possible sex-specific associations. METHODS: A multi-country cross-sectional study was conducted using pooled data from six aligned studies from the Human Biomonitoring for Europe Initiative (HBM4EU). Sociodemographic data, cardiometabolic biomarkers, and asthma/allergy outcomes were collected through questionnaires. Anthropometric data and BMI z-scores were calculated (n = 1339). Plasma/serum cardiometabolic biomarkers and asthma/allergy outcomes were available for a subsample (n = 173-594). Urinary BP-1 and BP-3 concentrations were adjusted for creatinine dilution using the traditional standardization (trad.) and the covariate-adjusted creatinine standardization (CAS) method. Generalized additive models, linear, logistic, and multinomial mixed models were applied, and sex-interaction terms were tested. RESULTS: Each natural log-unit increase in urinary BP-3 (CAS) concentrations was associated with higher odds of obesity in the whole population (OR: 1.20; 95%CI: 1.04-1.38). Sex-specific associations were also found with BP-1 (CAS) and BP-3 (CAS) concentrations, which were associated with higher odds of obesity in male teenagers (OR: 1.25; 95% CI: 1.01-1.55; OR: 1.34; 95%CI: 1.09-1.65, respectively). Linear mixed models showed consistent findings toward higher BMI z-scores. A negative association was found between BP-1 (CAS) concentration and serum adiponectin levels in females (% change per loge-unit increase: -3.73, 95%CI: -7.32, -0.10). BP-3 (CAS) concentrations were also associated with higher odds of non-food allergies in males (OR: 1.27; 95%CI: 1.00-1.63). Traditional creatinine adjustment showed similar or slightly attenuated estimates compared to the CAS method. CONCLUSIONS: BP-1 and BP-3 exposure was cross-sectionally associated with higher odds of obesity in European male teenagers, highlighting the need to update regulations and keep exposure levels as low as practically achievable. Longitudinal studies are needed to confirm these findings.
- MeSH
- Hypersensitivity * epidemiology MeSH
- Benzophenones * toxicity urine adverse effects MeSH
- Biomarkers blood MeSH
- Biological Monitoring MeSH
- Asthma * epidemiology chemically induced MeSH
- Humans MeSH
- Adolescent MeSH
- Obesity * epidemiology chemically induced MeSH
- Sunscreening Agents * adverse effects MeSH
- Cross-Sectional Studies MeSH
- Environmental Exposure * MeSH
- Check Tag
- Humans MeSH
- Adolescent MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Europe MeSH
Applications like drug development need simple and streamlined methods to process samples from 96-well cell culture plates for gene expression measurements. Unfortunately, current options are expensive for such processing. Therefore, our aim was to develop a method that would allow streamlined analysis of mRNA from 96-well cell culture plates while being relatively cheap and simple. We developed a method based on the qPCR 'Cells-to-cDNA' approach and validated it against commercially available kits using the same approach or spin columns-based RNA purification. For this purpose, we conducted a series of comparisons of gene expression from peripheral blood mononuclear cells, SK-HEP-1 and U-87 cell cultures in 96-well plates. Our final method involved lysing cells with 25-100 μl solution of 0.5% SDS, 10 mM DTT, 1 mg ml-1 proteinase K dissolved in water, 1 h incubation at 50°C, followed by proteinase K inactivation at 90°C for 5 min and lysate neutralization with 1 : 1 dilution by 20% Tween 20 solution. Reverse transcription and qPCR were carried out using standard methods. This method showed a mean reduction of Ct ± s.d. value by 2.4 ± 1.3 compared with the 'Cells-to-cDNA' kit and by 1.4 ± 0.5 compared with the RNA purification kit with lower variability.
- MeSH
- Cost-Benefit Analysis MeSH
- Cell Culture Techniques methods economics MeSH
- DNA, Complementary * genetics MeSH
- Real-Time Polymerase Chain Reaction methods MeSH
- Leukocytes, Mononuclear cytology metabolism MeSH
- Humans MeSH
- RNA, Messenger genetics metabolism MeSH
- Cell Line, Tumor MeSH
- Gene Expression Profiling methods economics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.
- MeSH
- Genetic Variation MeSH
- Genotype MeSH
- Humans MeSH
- Multiplex Polymerase Chain Reaction MeSH
- Polymorphism, Restriction Fragment Length MeSH
- DNA, Protozoan genetics MeSH
- Pregnancy MeSH
- Toxoplasma * genetics MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Check Tag
- Humans MeSH
- Pregnancy MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
OBJECTIVES: Both dimethylarginines are widely bound to chronic kidney disease (CKD). This study was focused to validate published LC-MS/MS method and compared the measured data with an immunoassay. DESIGN AND METHODS: The analysis was performed on a Dionex UltiMate 3000 UHPLC-Standard (Thermo Fisher Scientific, Waltham, Massachusetts, USA) with an amaZon SL ion trap (Bruker, Billerica, Massachusetts, USA). Comparison was evaluated by using Passing Bablok regression and Bland Altman plot. Healthy volunteers (n = 40) were used for validation and as control group to patients group (n = 40) with different stages of CKD. RESULTS: The results in healthy controls determined by the LC-MS/MS (ELISA) method were 0.52 ± 0.0892 with 95 % CI: 0.49-0.55 (0.61 ± 0.1213 with 95 % CI: 0.57-0.64) μmol/L for AD MA and 0.56 ± 0.0810 with 95 % CI: 0.53-0.58 (0.62 ± 0.0752 with 95 % CI: 0.57-0.65) μmol/L for SDMA. In the same way, the patient group values determined by the LC-MS/MS (ELISA) method were 0.82 ± 0.1604 with 95 % CI: 0.75-0.88 (1.06 ± 0.3002 with 95 % CI: 0.94-1.19) μmol/L and 2.14 ± 0.8778 with 95 % CI: 1.47-2.58 (1.65 ± 0.5160 with 95 % CI: 1.40-1.98) μmol/L for ADMA and SDMA, respectively. The correlation between the methods, expressed as the Spearman correlation coefficient (R), was 0.858 (0.8059) for ADMA (p < 0.0001) and 0.895 (0.9607) for SDMA (p < 0.0001). CONCLUSIONS: ADMA levels determined by the immunoassay were almost 30 % overestimated, in contrast to SDMA levels, which were 3 % underestimated. According to our findings, a better correlation could be obtained by simple sample dilution.
- Publication type
- Journal Article MeSH
OBJECTIVES: The analysis of organic acids in urine is an important part of the diagnosis of inherited metabolic disorders (IMDs), for which gas chromatography coupled with mass spectrometry is still predominantly used. METHODS: Ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for urinary organic acids, acylcarnitines and acylglycines was developed and validated. Sample preparation consists only of dilution and the addition of internal standards. Raw data processing is quick and easy using selective scheduled multiple reaction monitoring mode. A robust standardised value calculation as a data transformation together with advanced automatic visualisation tools are applied for easy evaluation of complex data. RESULTS: The developed method covers 146 biomarkers consisting of organic acids (n=99), acylglycines (n=15) and acylcarnitines (n=32) including all clinically important isomeric compounds present. Linearity with r2>0.98 for 118 analytes, inter-day accuracy between 80 and 120 % and imprecision under 15 % for 120 analytes were achieved. Over 2 years, more than 800 urine samples from children tested for IMDs were analysed. The workflow was evaluated on 93 patient samples and ERNDIM External Quality Assurance samples involving a total of 34 different IMDs. CONCLUSIONS: The established LC-MS/MS workflow offers a comprehensive analysis of a wide range of organic acids, acylcarnitines and acylglycines in urine to perform effective, rapid and sensitive semi-automated diagnosis of more than 80 IMDs.
- MeSH
- Chromatography, Liquid methods MeSH
- Child MeSH
- Humans MeSH
- Metabolic Diseases * MeSH
- Organic Chemicals MeSH
- Workflow MeSH
- Tandem Mass Spectrometry * methods MeSH
- Check Tag
- Child MeSH
- Humans MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: Color stability is a crucial performance parameter for dental restorations, and limited research exists on how surface preparation methods affect it. The purpose of this study was to test the color stability of three resins intended for 3D printing, which can be used to make dentures or crowns in A2 and A3 colors. MATERIALS AND METHODS: Samples were prepared in the form of incisors; the first group was not subjected to any treatment after curing and washing with alcohol, the second was covered with light-curing varnish, and the third was polished in a standard way. Then, the samples were placed in solutions of coffee, red wine, and distilled water and stored in the laboratory. After 14, 30, and 60 days, color changes were measured (presented as Delta E) compared to material stored in the dark. RESULTS: The greatest changes were observed for samples that were not polished, then were placed in red wine dilutions (ΔE = 18.19 ± 0.16). Regarding the samples covered with varnish, during storage, some parts detached, and the dyes penetrated inside. CONCLUSIONS: 3D-printed material should be polished as thoroughly as possible to limit the adhesion of dyes from food to their surface. Applying varnish may be a temporary solution.
- Publication type
- Journal Article MeSH
OBJECTIVES: To develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify 41 different purine and pyrimidine (PuPy) metabolites in human urine to allow detection of most known disorders in this metabolic pathway and to determine reference intervals. METHODS: Urine samples were diluted with an aqueous buffer to minimize ion suppression. For detection and quantification, liquid chromatography was combined with electrospray ionization, tandem mass spectrometry and multiple reaction monitoring. Transitions and instrument settings were established to quantify 41 analytes and nine stable-isotope-labeled internal standards (IS). RESULTS: The established method is precise (intra-day CV: 1.4-6.3%; inter-day CV: 1.3-15.2%), accurate (95.2% external quality control results within ±2 SD and 99.0% within ±3 SD; analyte recoveries: 61-121%), sensitive and has a broad dynamic range to quantify normal and pathological metabolite concentrations within one run. All analytes except aminoimidazole ribonucleoside (AIr) are stable before, during and after sample preparation. Moreover, analytes are not affected by five cycles of freeze-thawing (variation: -5.6 to 7.4%), are stable in thymol (variation: -8.4 to 12.9%) and the lithogenic metabolites also in HCl conserved urine. Age-dependent reference intervals from 3,368 urine samples were determined and used to diagnose 11 new patients within 7 years (total performed tests: 4,206). CONCLUSIONS: The presented method and reference intervals enable the quantification of 41 metabolites and the potential diagnosis of up to 25 disorders of PuPy metabolism.
A sample preparation method involving tandem implementation of protein precipitation and salting-out homogenous liquid-liquid extraction was developed for the determination of beta-blockers in serum. The entire procedure was automated using a computer-controlled syringe pump following the Lab-In-Syringe approach. It is based on the denaturation of serum proteins with acetonitrile followed by salt-induced phase separation upon which the proteins accumulate as a compact layer at the interphase of the solutions. The extract is then separated and diluted in-syringe before being submitted to online coupled UHPLC-MS/MS. A 1 mL glass syringe containing a small stir bar for solution mixing at up to 3000 rpm, was used to deal with sample volumes as small as 100 μL. A sample throughput of 7 h-1 was achieved by performing the chromatographic run and sample preparation procedure in parallel. Linear working ranges were obtained for all analytes between 5 and 100 ng mL-1, with LOD values ranging from 0.4 to 1.5 ng mL-1. Accuracy values in the range of 88.2-106% and high precision of <11% RSD suggest applicability for routine analysis that can be further improved using deuterated standards.
So far there is no internationally accepted, standardized method for MIC determination of natural substances such as essential oils (EOs). The aim of this study was to elucidate how much the MIC values obtained from various studies using different culture media are comparable. The median MICs for cinnamon essential oil (EO) obtained by broth dilution were 517, 465 and 517 μg/mL for Mueller-Hinton Broth (MHB), Tryptone Soya Broth (TSB) and Brain Heart Infusion (BHI), respectively. The MIC values for oregano EO were significantly (p < 0.001) lower in MHB than in highly nutritious media; the median MICs were 616 μg/mL for MHB and 474 μg/mL for TSB and BHI. This statistically significant difference was noted for all the pathogens studied (Salmonella Enteritidis, Escherichia coli O157, Listeria monocytogenes, Staphylococcus aureus). In the presence of oregano EO lag phase was also much less prolonged in MHB (by 6-17%) than in the other media (by 92-189%). Some components of EOs may bind to starch in MHB; since the phenomenon seems to be selective and EO dependent, the use of MHB for comparison of antimicrobial properties of various EOs thus cannot be recommended.
- Publication type
- Journal Article MeSH
At present, therapeutic drug monitoring is the standard in pharmacotherapy using medications with a narrow therapeutic index or showing serious adverse effects, such as in the case of ibrutinib. A technique commonly used for this purpose is liquid chromatography-tandem mass spectrometry combined with isotope dilution in sample processing. Although this method provides a high degree of reliability, its use can be complicated with some specific factors and does not guarantee trouble-free analysis. This paper is focused on investigating issues related to the differential adsorption of ibrutinib and its D4, D5 and 13C6 isotopically labeled analogues combined with instrument-specific carry-over. The results of the research point out the significantly different adsorption behavior of ibrutinib in fluidics of LC-MS compared with that of its D4, D5 and 13C6 stable isotope labeled analogues, showing preferential adsorption of non-labeled compound. The investigation also pointed to a strong affinity of ibrutinib to polymeric surfaces under specific conditions, which has to be taken into consideration during sample preparation and analysis. Our work opens a new field for the discussion of scarcely reported problem related to the use of stable isotope labeled internal standards in LC-MS/MS analysis.
