The aim of this study was to develop and validate methods for the determination of vitamins B2, B9, E and A in serum using liquid chromatography with mass spectrometry (MS) detection. Vitamin analysis was performed using an ultra performance liquid chromatography combined with tandem MS. The compounds were separated on a BEH C18 RP column (2.1 × 100 mm, 1.7 μm) using a gradient elution with an analysis time of 10 min. Sample preparation included protein precipitation with ethanol. The concentration range in human serum was as follows: riboflavin 5-1000 nmol/L, folic acid 2.5-250 nmol/L, α-tocopherol 0.5-100 μmol/L and all-trans-retinol 25-2500 nmol/L. Accuracy and precision were validated according to Food and Drug Administration guidelines, with coefficients of variation ranging from 3.1-11.7% and recoveries from 94.4-107.5%. Routine monitoring of the complex range of vitamins in bariatric medicine is still not common. This is despite the fact that patients are at risk for glitch deficits, especially of a neurological nature. An analytical method that allows for the complex measurement of both water-soluble and fat-soluble vitamins is important and necessary for the clinical monitoring of bariatric patients. The method we have described could benefit both clinical practice and nutritional research.
- MeSH
- alfa-tokoferol * krev MeSH
- bariatrická chirurgie MeSH
- chromatografie kapalinová metody MeSH
- kyselina listová * krev MeSH
- lidé MeSH
- limita detekce MeSH
- lineární modely MeSH
- reprodukovatelnost výsledků MeSH
- riboflavin * krev MeSH
- tandemová hmotnostní spektrometrie * metody MeSH
- vitamin A * krev MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Pathogen reduction technology (PRT) is increasingly used in the preparation of platelets for therapeutic transfusion. As the Czech Republic considers PRT, we asked what effects PRT may have on the recovery and function of platelets after cryopreservation (CP), which we use in both military and civilian blood settings. STUDY DESIGN AND METHODS: 16 Group O apheresis platelets units were treated with PRT (Mirasol, Terumo BCT, USA) before freezing; 15 similarly collected units were frozen without PRT as controls. All units were processed with 5-6% DMSO, frozen at - 80 °C, stored > 14 days, and reconstituted in thawed AB plasma. After reconstitution, all units were assessed for: platelet count, mean platelet volume (MPV), platelet recovery, thromboelastography, thrombin generation time, endogenous thrombin potential (ETP), glucose, lactate, pH, pO2, pCO2, HCO3, CD41, CD42b, CD62, Annexin V, CCL5, CD62P, and aggregates > 2 mm and selected units for Kunicki score. RESULTS: PRT treated platelet units had lower platelet number (247 vs 278 ×109/U), reduced thromboelastographic MA (38 vs 62 mm) and demonstrated aggregates compared to untreated platelets. Plasma coagulation functions were largely unchanged. CONCLUSIONS: Samples from PRT units showed reduced platelet number, reduced function greater than the reduced number would cause, and aggregates. While the platelet numbers are sufficient to meet the European standard, marked platelets activation with weak clot strength suggest reduced effectiveness.
- MeSH
- konzervace krve MeSH
- kryoprezervace MeSH
- kyselina mléčná MeSH
- lidé MeSH
- riboflavin farmakologie MeSH
- separace krevních složek * MeSH
- thrombin MeSH
- trombocyty fyziologie MeSH
- ultrafialové záření * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
OBJECTIVES: Age-related macular degeneration (AMD) is the leading cause of irreversible blindness among older adults in developed countries. Although many risk factors are known, the pathogenesis of AMD is still unclear. However, oxidative stress probably plays a vital role in the process of AMD. The increasing prevalence of AMD, risk of vision loss, limited treatment of dry form, expensive treatment of wet form, and decreased quality of life are factors that lead to considering modifiable risk factors of AMD, such as nutrition. This is the first study describing the relationship between dietary habits, dietary nutrient intake and AMD in the Czech Republic. METHODS: In this research, a total of 93 cases with AMD and 58 controls without AMD and cataracts participated. All participants were ophthalmologically examined at the Clinic of Eye Treatments at the University Hospital Brno. Data were collected using a pre-tested self-report questionnaire in a face-to-face interview. Food consumption frequency was assessed by an 18-item semiquantitative food-frequency questionnaire (FFQ). Dietary nutrient intakes were calculated from a 24-hour recall. RESULTS: Patients with AMD compared with controls had significantly higher consumption of legumes and lower consumption of meat products, salt and salty products. In men, we found statistically significant differences in alcohol consumption. The case group consumed alcoholic beverages more frequently (median: 2 times a week) than the control group (median: 1-3 times a month). No differences in alcohol consumption were found in women. In comparison to the case group, the control group had a significantly higher dietary intake of energy (5,783.8 vs. 4,849.3 kJ/day; p = 0.002), proteins (65.3 vs. 52.3 g/day; p = 0.002), fats (57.6 vs. 49.4 g/day; p = 0.046), saturated fatty acids (21.7 vs. 18.9 g/day; p = 0.026), carbohydrates (150.4 vs. 127.1 g/day; p = 0.017), dietary fibre (13.2 vs. 11.3 g/day; p = 0.044), vitamin B2 (1.0 vs. 0.9 mg/day; p = 0.029), vitamin B3 (13.9 vs. 10.0 mg/day; p = 0.011), pantothenic acid (3.5 vs. 2.8 mg/day; p = 0.001), vitamin B6 (1.3 vs. 1.0 mg/day; p = 0.001), potassium (1,656.5 vs. 1,418.0 mg/day; p = 0.022), phosphorus (845.4 vs. 718.7 mg/day; p = 0.020), magnesium (176.5 vs. 143.0 mg/day; p = 0.012), copper (1.0 vs. 0.8 mg/day; p = 0.011), and zinc (7.1 vs. 6.1 mg/day; p = 0.012) counted from a 24-hour recall. CONCLUSIONS: According to FFQ, dietary habits in the patients with AMD and controls were similar. In men from the case group, we found statistically significant higher alcohol consumption. According to a 24-hour recall, the controls achieved recommended dietary intakes rather than cases. In comparison to the case group, the control group had a significantly higher dietary intake of energy, proteins, fats, saturated fatty acids, carbohydrates, dietary fibre, vitamin B2, vitamin B3, pantothenic acid, vitamin B6, potassium, phosphorus, magnesium, copper, and zinc.
- MeSH
- dieta MeSH
- dietní tuky MeSH
- draslík MeSH
- energetický příjem MeSH
- fosfor MeSH
- hořčík * MeSH
- kvalita života MeSH
- kyselina pantothenová MeSH
- lidé MeSH
- makulární degenerace * epidemiologie chemicky indukované MeSH
- mastné kyseliny MeSH
- měď MeSH
- niacinamid MeSH
- potravní vláknina MeSH
- přijímání potravy MeSH
- riboflavin MeSH
- senioři MeSH
- stravovací zvyklosti MeSH
- studie případů a kontrol MeSH
- vitamin B6 MeSH
- zinek MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- dítě MeSH
- keratokonus * diagnóza terapie MeSH
- lidé MeSH
- pachymetrie rohovky metody MeSH
- riboflavin farmakologie terapeutické užití MeSH
- rohovková topografie metody MeSH
- transplantace rohovky * metody MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
BACKGROUND: Pathogen reduction technology (PRT) may improve the safety of RBCs for transfusion. As the Czech Republic considers PRT, we asked what effects riboflavin and UV light PRT pre-freezing has on the post-thaw recovery and properties of cryopreserved RBCs (CRBCs) after deglycerolization and liquid storage. STUDY DESIGN AND METHODS: 24 Group O whole blood (WB) units were leukoreduced and then treated with riboflavin and UV light PRT (Mirasol, Terumo BCT, USA) before cryopreservation (T-CRBC); 20 similarly-collected units were untreated controls (C-CRBC). Units were processed to RBCs and then cryopreserved with 40% glycerol (wt/vol), frozen at -80°C, stored >118 days, reconstituted as deglycerolized RBC units in AS-3, and stored at 4 ± 2°C for 21 days. One treated unit sustained massive hemolysis during the post-thaw wash process and was removed from data analysis. The remaining units were assessed pre-PRT, post-PRT, and post-thaw-wash on days 0, 7, 14, and 21 for hematocrit, volume, hemoglobin per transfusion unit, pH, % hemolysis, hemoglobin in the supernatant, potassium, phosphorus, NH3 , osmolality, ATP, and 2,3-diphosphoglycerate. RESULTS: PRT with leukoreduction caused a 5% loss of RBC followed by a 24% freeze-thaw-wash related loss for a total 28% loss but treated units contained an average of 45 g of hemoglobin, meeting European Union guidelines for CRBC. T-CRBCs displayed higher post-wash hemolysis, potassium, and ammonia concentrations, and lower ATP at the end of storage. CONCLUSIONS: Cryopreserved RBCs from Riboflavin and UV light-treated WB meet the criteria for clinical use for 7 days after thawing and provide additional protection against infectious threats.
- MeSH
- adenosintrifosfát MeSH
- draslík analýza MeSH
- erytrocyty MeSH
- hemoglobiny analýza MeSH
- hemolýza * MeSH
- konzervace krve MeSH
- kryoprezervace MeSH
- lidé MeSH
- riboflavin farmakologie MeSH
- ultrafialové záření * MeSH
- zmrazování MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Protein phosphorylation is a common phenomenon in human flavoproteins although the functional consequences of this site-specific modification are largely unknown. Here, we evaluated the effects of site-specific phosphorylation (using phosphomimetic mutations at sites S40, S82 and T128) on multiple functional aspects as well as in the structural stability of the antioxidant and disease-associated human flavoprotein NQO1 using biophysical and biochemical methods. In vitro biophysical studies revealed effects of phosphorylation at different sites such as decreased binding affinity for FAD and structural stability of its binding site (S82), conformational stability (S40 and S82) and reduced catalytic efficiency and functional cooperativity (T128). Local stability measurements by H/D exchange in different ligation states provided structural insight into these effects. Transfection of eukaryotic cells showed that phosphorylation at sites S40 and S82 may reduce steady-levels of NQO1 protein by enhanced proteasome-induced degradation. We show that site-specific phosphorylation of human NQO1 may cause pleiotropic and counterintuitive effects on this multifunctional protein with potential implications for its relationships with human disease. Our approach allows to establish relationships between site-specific phosphorylation, functional and structural stability effects in vitro and inside cells paving the way for more detailed analyses of phosphorylation at the flavoproteome scale.
- MeSH
- antioxidancia metabolismus MeSH
- flavinadenindinukleotid chemie MeSH
- flavoproteiny metabolismus MeSH
- fosforylace MeSH
- lidé MeSH
- mutace MeSH
- NAD(P)H dehydrogenasa (chinon) * metabolismus MeSH
- nádory * genetika MeSH
- proteasomový endopeptidasový komplex metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Sperm metabolism is fundamental to sperm motility and male fertility. Its measurement is still in its infancy, and recommendations do not exist as to whether or how to standardize laboratory procedures. Here, using the sperm of an insect, the common bedbug, Cimex lectularius, we demonstrate that standardization of sperm metabolism is required with respect to the artificial sperm storage medium and a natural medium, the seminal fluid. We used fluorescence lifetime imaging microscopy (FLIM) in combination with time-correlated single-photon counting (TCSPC) to quantify sperm metabolism based on the fluorescent properties of autofluorescent coenzymes, NAD(P)H and flavin adenine dinucleotide. Autofluorescence lifetimes (decay times) differ for the free and protein-bound state of the co-enzymes, and their relative contributions to the lifetime signal serve to characterize the metabolic state of cells. We found that artificial storage medium and seminal fluid separately, and additively, affected sperm metabolism. In a medium containing sugars and amino acids (Grace's Insect medium), sperm showed increased glycolysis compared with a commonly used storage medium, phosphate-buffered saline (PBS). Adding seminal fluid to the sperm additionally increased oxidative phosphorylation, likely reflecting increased energy production of sperm during activation. Our study provides a protocol to measure sperm metabolism independently from motility, stresses that protocol standardizations for sperm measurements should be implemented and, for the first time, demonstrates that seminal fluid alters sperm metabolism. Equivalent protocol standardizations should be imposed on metabolic investigations of human sperm samples.
- MeSH
- flavinadenindinukleotid * MeSH
- mikroskopie fluorescenční multifotonová MeSH
- motilita spermií MeSH
- NADP * MeSH
- spermie účinky léků MeSH
- štěnice MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Obligate symbiotic bacteria associated with the insects feeding exclusively on vertebrate blood are supposed to complement B vitamins presumably lacking in their diet. Recent genomic analyses revealed considerable differences in biosynthetic capacities across different symbionts, suggesting that levels of B vitamins may vary across different vertebrate hosts. However, a rigorous determination of B vitamins content in blood of various vertebrates has not yet been approached. A reliable analytical method focused on B vitamin complex in blood can provide valuable informative background and understanding of general principles of insect symbiosis. In this work, a chromatographic separation of eight B vitamins (thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, biotin, folic acid, and cyanocobalamine), four B vitamin derivatives (niacinamide, pyridoxal-5-phosphate, 4-pyridoxic acid, and tetrahydrofolic acid), and 3 stable isotope labelled internal standards was developed. Detection was carried out using dual-pressure linear ion trap mass spectrometer in FullScan MS/MS and SIM mode. Except for vitamin B9 (tetrahydrofolic acid), the instrument quantitation limits of all analytes were ranging from 0.42 to 5.0 μg/L, correlation coefficients from 0.9997 to 1.0000, and QC coefficients from 0.53 to 3.2%. Optimization of whole blood sample preparation step was focused especially on evaluation of two types of protein-precipitation agents: trichloroacetic acid and zinc sulphate in methanol. The best results were obtained for zinc sulphate in methanol, but only nine analytes were successfully validated. Accuracy of the procedure using this protein-precipitating agent was ranging from 89 to 120%, precision from 0.5 to 13%, and process efficiency from 65 to 108%. The content of B vitamins in whole blood samples from human and various vertebrates is presented as an application example of this newly developed method.
- MeSH
- chromatografie kapalinová metody MeSH
- kyselina listová analýza MeSH
- lidé MeSH
- methanol MeSH
- riboflavin analýza MeSH
- síran zinečnatý MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- thiamin analýza MeSH
- vitamin B komplex * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ciele: Vyhodnotenie viditeľnosti a hĺbky demarkačnej línie v stróme rohovky u očí s keratokónusom 1 mesiac a 3 mesiace po akcelerovanom rohovkovom cross-linkingu (ACXL) s abráziou s použitím prednosegmentovej optickej koherentnej tomografie (AS OCT). Materiál a metódy: Práca analyzuje skupinu 34 očí s keratokónusom 1 mesiac a 3 mesiace po ACXL (9mW/cm2počas 10min). Skupinu bola klasifikovaná na základe ABCD klinickej klasifikácie keratokónusu podľa Belina a Duncana. AS OCT (Zeiss Cirrus 500, Anterior Segment Premier module) bola použitá na posúdenie viditeľnosti a presnej hĺbky demarkačnej línie v stróme rohovky. Výsledky: Demarkačná línia bola 1 mesiac po ACXL viditeľná u 76,5 % očí s priemernou hĺbkou 238.13 ±20.36 μm a 3 mesiace po ACXL u 100 % očí s priemernou hĺbkou 263,43 ±12,59 μm. Štatistická analýza skupiny nepreukázala signifikantný vzťah medzi štádiom ochorenia a viditeľnosťou demarkačnej línie, avšak v súbore bola tendencia k vyššiemu veku (> 30 rokov) u tých očí, kde bola demarkačná línia viditeľná v porovnaní s očami, kde bola demarkačná línia len čiastočne viditeľná 3 mesiace po ACXL. Nezistili sme rozdiel v priemernej, ani v maximálnej hĺbke línie pri porovnaní 1 mesiac a 3 mesiace po zákroku. V súbore nebol zaznamenaný žiadny prípad progresie ochorenia 3 mesiace po ACXL. Záver: Naša štúdia naznačuje, že hodnotenie demarkačnej línie v stróme rohovky je spoľahlivejšie 3 mesiace po ACXL v porovnaní s hodnotením 1 mesiac po zákroku. Zároveň sme zaznamenali tendenciu k vyššiemu veku pacientov v prípade očí, kde bola demarkačná línia zreteľne viditeľná 3 mesiace po ACXL. Nepotvrdili sme vzťah medzi štádiom keratokónusu a hĺbkou línie, ani rozdiel v jej priemernej a maximálnej hĺbke 1 mesiac a 3 mesiace po zákroku.
Objectives: Evaluation of the visibility and depth of the demarcation line in the corneal stroma in eyes with keratoconus 1 month and 3 months after epi-off accelerated corneal cross-linking (ACXL) using anterior segment optical coherence tomography (AS OCT). Material and Methods: This study analyses a group of 34 eyes with keratoconus 1 month and 3 months after ACXL (9 mW/cm2 for 10 min). The group was classified based on the ABCD clinical classification of keratoconus according to Belin and Duncan. AS OCT (ZeissCirrus 500, Anterior Segment Premier module) was used to assess the visibility and exact depth of the demarcation line in the corneal stroma. Results: The demarcation line was visible 1 month after ACXL in 76.5% of eyes with a mean depth of 238.13 ±20.36 μm and 3 months after ACXL in 100% of eyes with a mean depth of 263.43 ±12.59 μm. Statistical analysis of the group did not show a significant relationship between the disease stage and the demarcation line visibility; however, there was a trend towards higher age (>30 years) in the group in those eyes where the demarcation line was visible vs. partially visible 3 months after ACXL. We found no difference in the mean and maximum line depth when comparing 1 month and 3 months after the procedure. There were no cases of disease progression 3 months after ACXL in the group. Conclusion: Our study suggests that the assessment of the demarcation line in the corneal stroma is more reliable 3 months compared to 1 month after ACXL. We also observed a trend towards higher patient age in eyes where the demarcation line was clearly visible 3 months after ACXL. We did not confirm a relationship between the stage of keratoconus and the depth of the line, nor a difference in its mean and maximum depth 1 month and 3 months after the procedure.
