Global DNA methylation in rats´ liver is not affected by hypercholesterolemic diet.

Increased plasma cholesterol levels are listed between the major atherosclerosis risk factors. The final plasma cholesterol levels result from the interplay between the genetic and environmental (diet, physical activity) factors. Little is known, how dietary factor influence epigenetics. We have analyzed, if an over-generation feeding of rat with cholesterol influences total liver-DNA methylation, and if total liver-DNA methylation differ between the different rat strains (Prague hereditary hypercholesterolemic rats, Prague hereditary hypertriglyceridemic rats and Wistar Kyoto rats). The animals were feed with high fat (additional 5 % over normal capacity) high cholesterol (2 %) diet for 14 days. DNA methylation in the liver tissue in different generations was analyzed using the liquid chromatography coupled with tandem mass spectrometry. We have not observed any significant changes in total liver-DNA methylation over the 9 generations of animals feed by fat/cholesterol enriched diet. Additionally, there were no differences in DNA methylation between different rat strains. In animal model, the dietary changes (hypercholesterolemic diet) not significantly influence the total DNA methylation status within the liver.

Evaluation of the DOAC-Stop Procedure by LC-MS/MS Assays for Determining the Residual Activity of Dabigatran, Rivaroxaban, and Apixaban.

The effect of direct oral anticoagulants (DOACs) on laboratory tests dependent on the production of their targets, factor IIa and factor Xa (FXa), is a well-known problem and can cause both false positive and negative results. Therefore, the correct interpretation of tests performed in patients receiving DOACs is necessary to avoid misclassification and subsequent clinical consequences. However, even with significant experience, there are situations where it is not possible to assess the influence of some methods. Particularly important is the situation in the diagnosis of lupus anticoagulants using the dilute Russell viper venom timetest, which is based on direct FXa activation. A very promising solution to this situation is offered by the DOAC laboratory balancing procedure DOAC-Stop. For evaluating the effectiveness of this procedure, 60 (20 apixaban, 20 dabigatran, and 20 rivaroxaban) patients treated with DOACs were enrolled. All patient samples were analyzed for the presence of individual DOAC types and subsequently subjected to the DOAC-Stop procedure.We evaluated its effectiveness by our own high-performance liquid chromatography-coupled tandem mass spectrometrymethod, which simultaneously sets all high-sensitivity DOACs. Unlike coagulation tests based on the determination of the residual effects of DOACs on target enzymes, which is complicated by extensive interindividual variation, this methodology is highly specific and sensitive.The DOAC-Stop procedure eliminated dabigatran from 99.5%, rivaroxaban from 97.9%, and apixaban from 97.1% of participants in our group. Residual amounts did not exceed 2.7 ng/mL for dabigatran, 10.9 ng/mL for rivaroxaban, or 13.03 ng/mL for apixaban, which are safe values that do not affect either screening or special coagulation tests.

Equine atypical myopathy: A metabolic study.

Atypical myopathy (AM) is a potentially fatal disease of grazing horses. It is reportedly caused by the ingestion of sycamore seeds containing toxic hypoglycin A. In order to study metabolic changes, serum and urine samples from nine horses with atypical myopathy and 12 control samples from clinically healthy horses were collected and then analysed using a high-performance liquid chromatography coupled with tandem mass spectrometry; serum metabolic profiles as the disease progressed were also studied. Metabolic data were evaluated using unsupervised and supervised multivariate analyses. Significant differences were demonstrated in the concentrations of various glycine conjugates and acylcarnitines (C2-C26). Moreover, the concentrations of purine and pyrimidine metabolites, vitamins and their degradation products (riboflavin, trigonelline, pyridoxate, pantothenate), and selected organic and amino acids (aspartate, leucine, 2-oxoglutarate, etc.) were altered in horses with AM. These results represent a global view of altered metabolism in horses with atypical myopathy.

Isotope dilution direct injection mass spectrometry method for determination of four tyrosine kinase inhibitors in human plasma.

BACKGROUND: Therapeutic drug monitoring is recommended for the optimal management of patients with several malignant diseases. The aim of this study was to develop and validate an isotope dilution direct injection mass spectrometry method for the high throughput determination of tyrosine kinase inhibitors in plasma from leukemic and cancer patients. METHODS: The plasma for analysis was deproteinated by methanol and the centrifuged supernatant was directly injected to mass spectrometer without separation step. Multiple reaction monitoring modes on a hybrid triple quadrupole - linear ion trap mass spectrometer (5500 QTRAP) were used for the detection and quantification of imatinib, nilotinib, lapatinib, and dasatinib. RESULTS: We developed a fast method with analysis time of 55 s and 19s in multiple injection setting. The method was successfully validated and applied to the patient plasma samples. In order to overcome insufficient sensitivity of dasatinib, multiple reaction monitoring cube mode in linear ion trap (MRM(3)) was successfully applied. The limits of quantification were in the range 1.0-5.5 ng/ml. Imprecisions were lower than 6.9% and the accuracy of the quality control samples ranged between 99.0 and 107.9%. CONCLUSIONS: Isotope dilution direct injection mass spectrometry method allows high-throughput therapeutic drug monitoring of tyrosine kinase inhibitors in plasma. The method offers low-cost analyses as a result of its speed and the exclusion of separation step and can be advantageously used in routine clinical practice. The method can be applied on various drugs and biochemical markers with the use of triple quadrupole instruments.

Oligodendroglia from ADSL-deficient patient produce SAICAribotide and SAMP.

Succinylpurines accumulate in the body fluids of patients with adenylosuccinate lyase (ADSL) deficiency but their source in the cerebrospinal fluid remains obscure. Study based on the incorporation of 13C-stable isotope-labeled glycine into cultured oligodendroglia from ADSL-deficient patient and the measurement of labeled products by LC/MS/MS showed total intracellular concentrations of succinylpurines from 45 to 99µmol/l and so these results suggest that these cells can be the source of the compounds in vivo.

ITPase activity in dry blood spots is comparable with that in fresh erythrocytes.

Inosine triphosphate pyrophosphohydrolase (ITPase) catalyzing the pyrophosphohydrolysis of inosine triphosphate, deoxyinosine triphosphate and xanthosine triphosphate is involved in the metabolism and tolerance of thiopurine drugs. ITPase activity plays an important role in the prediction of toxicity to thiopurine therapy. Activities in dry blood spots were compared with fresh erythrocytes. Samples were incubated with inosine triphosphate, then inosine monophosphate was determined by a capillary electrophoresis method. Calculated enzyme activities obtained from dry blood spots were in good accordance with activity in fresh erythrocytes.

Analysis of intracellular nucleotides by capillary electrophoresis-mass spectrometry.

A pilot study using capillary electrophoresis with mass spectrometry for the analysis of nucleotides in human erythrocytes is presented. Erythrocytes were incubated with 5-amino-4-imidazolecarboxamide riboside in order to mimic situation in defect of purine metabolism--AICA-ribosiduria. Characteristic AICA-ribotides together with normal nucleotides were separated by capillary electrophoresis in acetate buffer (20 mmol/L, pH 4.4) and identified on line by mass spectrometry.

Synthesis and mass spectrometric fragmentation characteristics of imidazole ribosides-analogs of intermediates of purine de novo synthetic pathway.

Two inherited deficiencies have been described in purine de novo synthesis pathway. Both the defects are diagnosed by detecting ribosides--dephosphorylated substrates of the enzymes--in patient's urine. We describe here a synthesis and mass spectrometric fragmentation of ribosides potentially of diagnostic importance for defects in the second part of the pathway. All the species, except 5-amino-4-imidazolesuccinocarboxamideriboside can be synthesized from the commercially available 5-amino-4-imidazolecarboxamideriboside by chemical methods. Fragmentation spectra of the compounds were obtained by the ion trap mass spectrometry. During fragmentation an opening of the imidazole ring was not observed for any of the compounds but loss of its substituents in the form of small molecules (NH3, CO2, CO) is the major route of fragmentation. The ribose moiety cleaves off molecule(s) of water, undergoes a cross-ring cleavage or breaks away as a whole.

Fast analysis of antibacterial isothiazolones by capillary electrophoresis.

Some technical aspects influencing the total time of CE analysis are discussed. A high throughput electrophoretic system based on micellar electrokinetic chromatography (MEKC) is demonstrated as an example. A short capillary, strong electric field, alkaline buffer (pH 9.5) generating strong electroosmotic flow, and parallel hydrodynamic pressure allow the separation of two uncharged isothiazolone derivatives within 45 s.

Capillary electrophoresis for detection of inherited disorders of purine and pyrimidine metabolism.

BACKGROUND: Measurement of purine and pyrimidine metabolites presents complex problems for separations currently performed by HPLC and thin-layer chromatography in clinical practice. We developed a novel capillary electrophoresis method for this purpose. METHODS: Separations were performed in 60 mmol/L borate-2-amino-2-methyl-1-propanol-80 mmol/L sodium dodecyl sulfate (pH 9.6) at 35 degrees C. RESULTS: The conditions reported allowed separation of all diagnostic metabolites from major urinary constituents in an analysis time of 3 min and with a separation efficiency of 220 000 theoretical plates/m. The clinically important metabolites were detectable at concentrations of 0.85-4.28 micromol/L. The method was linear over the range 5-500 micromol/L (r >0.99). The within-run and intra- and interday imprecision (CV) was <5%. Characteristic abnormalities were detected in the electropherograms of urine samples from patients with purine and pyrimidine enzyme deficiencies. We provide the electrophoretic and spectral characteristics of many intermediates in purine and pyrimidine metabolism and describe common artifacts from medication and ultraviolet-absorbing compounds. CONCLUSION: Capillary electrophoresis is a valuable screening tool in the detection of inborn errors of purine and pyrimidine metabolism.