Determination of dabigatran, rivaroxaban and apixaban by ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) and coagulation assays for therapy monitoring of novel direct oral anticoagulants.

BACKGROUND: Three novel direct oral anticoagulants (DOACs) have recently been registered by the Food and Drug Administration and European Medicines Agency Commission: dabigatran, rivaroxaban, and apixaban. To quantify DOACs in plasma, various dedicated coagulation assays have been developed. OBJECTIVE: To develop and validate a reference ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) method and to evaluate the analytical performance of several coagulation assays for quantification of dabigatran, rivaroxaban, and apixaban. METHODS: The developed UPLC-MS/MS method was validated by determination of precision, accuracy, specificity, matrix effects, lower limits of detection, carry-over, recovery, stability, and robustness. The following coagulation assays were evaluated for accuracy and precision: laboratory-developed (LD) diluted thrombin time (dTT), Hemoclot dTT, Pefakit PiCT, ECA, Liquid anti-Xa, Biophen Heparin (LRT), and Biophen DiXal anti-Xa. Agreement between the various coagulation assays and UPLC-MS/MS was determined with random samples from patients using dabigatran or rivaroxaban. RESULTS: The UPLC-MS/MS method was shown to be accurate, precise, sensitive, stable, and robust. The dabigatran coagulation assay showing the best precision, accuracy and agreement with the UPLC-MS/MS method was the LD dTT test. For rivaroxaban, the anti-factor Xa assays were superior to the PiCT-Xa assay with regard to precision, accuracy, and agreement with the reference method. For apixaban, the Liquid anti-Xa assay was superior to the PiCT-Xa assay. CONCLUSIONS: Statistically significant differences were observed between the various coagulation assays as compared with the UPLC-MS/MS reference method. It is currently unknown whether these differences are clinically relevant. When DOACs are quantified with coagulation assays, comparison with a reference method as part of proficiency testing is therefore pivotal.

Rapidly evolving liver decompensation with some remarkable features 14 years after biliopancreatic derivation: a case report and literature review.

Because of the rising incidence of obesity the use of bariatric surgery is also increasing. For the obese it is the only treatment with a proven long-term benefit on weight, comorbidities including non alcoholic steatohepatitis, and long-term mortality. There are, however, several reports on hepatic complications after bariatric surgery leading to malabsorption. The risk of liver decompensation or cirrhosis is one of the reasons jejunoileal bypass has been abandoned. Hepatic complications following Roux-en-Y gastric bypass and biliopancreatic derivation (BPD) are also reported but never beyond 2 years of follow-up. There is only one confirmed case of development of cirrhosis following BPD which presented 10 months after surgery. We present a case of a 39-year-old patient who developed rapidly evolving, and ultimately fatal, liver decompensation in previously unknown cirrhosis, 14 years after BPD. This is the first report of a severe hepatic complication such a long time after a BPD. Existing literature on hepatic complications after bariatric surgery is discussed as are 2 coincidental findings of pronounced ductular reaction on histology and autoimmune haemolytic anaemia.

Autocrine production of prostaglandin F2alpha enhances phenotypic transformation of normal rat kidney fibroblasts.

We have used normal rat kidney (NRK) fibroblasts as an in vitro model system to study cell transformation. These cells obtain a transformed phenotype upon stimulation with growth-modulating factors such as retinoic acid (RA) or transforming growth factor-beta (TGF-beta). Patch-clamp experiments showed that transformation is paralleled by a profound membrane depolarization from around -70 to -20 mV. This depolarization is caused by a compound in the medium conditioned by transformed NRK cells, which enhances intracellular Ca2+ levels and thereby activates Ca2+-dependent Cl- channels. This compound was identified as prostaglandin F2alpha (PGF2alpha) using electrospray ionization mass spectrometry. The active concentration in the medium conditioned by transformed NRK cells as determined using an enzyme immunoassay was 19.7 +/- 2.5 nM (n = 6), compared with 1.5 +/- 0.1 nM (n = 3) conditioned by nontransformed NRK cells. Externally added PGF2alpha was able to trigger NRK cells that had grown to density arrest to restart their proliferation. This proliferation was inhibited when the FP receptor (i.e., natural receptor for PGF2alpha) was blocked by AL-8810. RA-induced phenotypic transformation of NRK cells was partially (approximately 25%) suppressed by AL-8810. Our results demonstrate that PGF2alpha acts as an autocrine enhancer and paracrine inducer of cell transformation and suggest that it may play a crucial role in carcinogenesis in general.

Antioxidant supplementation and exercise-induced oxidative stress in the 60-year-old as measured by antipyrine hydroxylates.

The effects of 12 weeks of antioxidant supplementation on exercise-induced oxidative stress were investigated in older adults (60 (SE 1) years; BMI 26 (SE 1) kg/m(2)). Subjects were randomly divided in two groups: supplementation (n 11) with 100 mg dl-alpha-tocopheryl acetate, 200 mg ascorbic acid, and 2 mg beta-carotene, and placebo (n 9). Before and after the 12 week supplementation period, subjects cycled for 45 min at submaximal intensity (50 % maximal workload capacity). Antipyrine was used as marker for oxidative stress. Antipyrine reacts quickly with hydroxyl radicals to form para- and ortho-hydroxyantipyrine. The latter metabolite is not formed in man through the mono-oxygenase pathway of cytochrome P450. Daily supplementation significantly increased plasma concentrations of alpha-tocopherol and beta-carotene in the supplemented group (Delta 14.4 (SE 3.2) and 0.4 (se 0.1) micromol/l; P<0.001 and P<0.01). No significant differences, within and between groups, were observed in the exercise-induced increase in the ratios para- and ortho-hydroxyantipyrine to antipyrine. In addition, supplementation did not affect the exercise-induced increase in thiobarbituric acid reactive substances in plasma. In conclusion, in 60-year-old subjects antioxidant supplementation had no effect on the exercise-induced increase in oxidative stress as measured by free radical products of antipyrine.

Non-aqueous capillary electrophoresis using non-dissociating solvents. Application to the separation of highly hydrophobic oligomers.

The application of non-aqueous capillary electrophoresis for the separation of very hydrophobic oligomers has been studied. N-Phenylaniline oligomers having degrees of polymerisation (n) of 2, 4, 6, and 8 were taken as model compounds. Capillary electrophoresis could be performed using a mixture of non-aqueous solvents with a high percentage of solvents with a low dielectric constant. These solvents, such as tetrahydrofuran (THF), chloroform or dichloromethane, are needed to solubilise the hydrophobic solutes in the electrolyte. The composition of the solvent mixture and the nature of the acid added to the electrolyte, which is needed to obtain electrophoretic motion of the N-phenylaniline oligomers, are discussed in detail. Next, other parameters such as ionic strength, injection time, electric field, and temperature were investigated too and their influence on the separation is discussed as well. The existence of a reversed (anodic) electroosmotic flow in a fused-silica capillary containing a THF-methanol mixture under acidic conditions is reported.

Separation and characterization of oligomers by reversed-phase high-performance liquid chromatography: a study on well-defined oligothiphenes.

Reversed-phase high-performance liquid chromatography (RP-HPLC) was used for the separation of 3-hexylthiophene oligomers in the range of 3 to 30 monomeric units, while systematically varying stationary and mobile phases. A set of different columns was chosen, covering a broad range of silica types, pore sizes and bonding chemistry. Mobile phases of tetrahydrofuran (THF) combined with water, acetonitrile (ACN) or methanol (MeOH) were used. Although differences between columns were small, a higher selectivity correlated with a lower hydrophobicity parameter from the Galushko column test. The model of Jandera, based on the linear solvent strength model of Snyder, was used to describe the retention of the oligomers in gradient mode. This gave information about selectivities on different stationary phases similar to the hydrophobicity parameter. Contrary to the stationary phase, the mobile phase had a major influence on the selectivity. The THF-water combination gave much higher selectivities compared to THF combined with MeOH or ACN. Using the aqueous mobile phase even enabled separation of different isomers. Determination of thermodynamic parameters for the model compounds showed that retention of the different isomers was mainly determined by the orientation of the side chains at both ends of the chain. An additional repeating unit in the middle of the polymer backbone gave a similar contribution to retention, irrespective of the orientation of its side chain. Three model isomers were separated by preparative RP-HPLC and identified by proton nuclear magnetic resonance spectroscopy. The combination of subsequent preparative size-exclusion chromatography, RP-HPLC and matrix-assisted laser desorption ionization time-of-flight mass spectrometry enabled the identification of the two major oligomeric series in the sample as the regioregular product with one bromine end group and, in smaller amounts, a regioirregular product with two bromine end groups. reserved.

Off-line size-exclusion chromatographic fractionation-matrix-assisted laser desorption ionization time-of-flight mass spectrometry for polymer characterization. Theoretical and experimental study.

The parameters affecting the fractionation performance in size-exclusion chromatography (SEC) of broad polymer samples were investigated. Some equations were derived which enable the prediction of polydispersity (PD) in an SEC fraction. Good agreements were obtained between the calculated data and the experimental values. Based on these equations, SEC fractionation conditions were optimized. In the off-line SEC-matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS), two different modes can be employed, i.e., using MALDI-MS to provide an absolute calibration curve for SEC, or using SEC as a sample preparation step for MALDI-MS measurements. It was demonstrated that it is more reliable to use the latter combination, because most problems inherent in SEC can be circumvented. Some guidelines for the optimization of off-line SEC fractionation-MALDI-TOF-MS were given. It was found that under optimized conditions normally only a few SEC fractions are already sufficient to separate a highly polydisperse sample into portions of low PD that can accurately be measured by MALDI-TOF-MS.

Epidermal growth factor receptor activation in androgen-independent but not androgen-stimulated growth of human prostatic carcinoma cells.

These studies were undertaken to assess the relative expression and autocrine activation of the epidermal growth factor receptor (EGFR) in normal and transformed prostatic epithelial cells and to determine whether EGFR activation plays a functional role in androgen-stimulated growth of prostate cancer cells in vitro. EGFR expression was determined by Western blot analysis and ELISA immunoassays. Immunoprecipitation of radiophosphorylated EGFR and evaluation of tyrosine phosphorylation was used to assess EGFR activation. The human androgen-independent prostate cancer cell lines PC3 and DU145 exhibited higher levels of EGFR expression and autocrine phosphorylation than normal human prostatic epithelial cells or the human androgen-responsive prostate cancer cell line LNCaP. PC3 and DU145 cells also showed higher levels of autonomous growth under serum-free defined conditions. Normal prostatic epithelial cells expressed EGFR but did not exhibit detectable levels of EGFR phosphorylation when cultured in the absence of exogenous EGF. Addition of EGF stimulated EGFR phosphorylation and induced proliferation of normal cells. LNCaP cells exhibited autocrine phosphorylation of EGFR but did not undergo significant proliferation when cultured in the absence of exogenous growth factors. A biphasic growth curve was observed when LNCaP cells were cultured with dihydrotestosterone (DHT). Maximum proliferation occurred at 1 nM DHT with regression of the growth response at DHT concentrations greater than 1 nM. However, neither EGFR expression nor phosphorylation was altered in LNCaP cells after androgen stimulation. In addition, DHT-stimulated growth of LNCaP cells was not inhibited by anti-EGFR. These studies show that autocrine activation of EGFR is a common feature of prostatic carcinoma cells in contrast to normal epithelial cells. However, EGFR activation does not appear to play a functional role in androgen-stimulated growth of LNCaP cells in vitro.

A conserved family of cellular genes related to the baculovirus iap gene and encoding apoptosis inhibitors.

The baculovirus inhibitor of apoptosis gene, iap, can impede cell death in insect cells. Here we show that iap can also prevent cell death in mammalian cells. The ability of iap to regulate programmed cell death in widely divergent species raised the possibility that cellular homologs of iap might exist. Consistent with this hypothesis, we have isolated Drosophila and human genes which encode IAP-like proteins (dILP and hILP). Like IAP, both dILP and hILP contain amino-terminal baculovirus IAP repeats (BIRs) and carboxy-terminal RING finger domains. Human ilp encodes a widely expressed cytoplasmic protein that can suppress apoptosis in transfected cells. An analysis of the expressed sequence tag database suggests that hilp is one of several human genes related to iap. Together these data suggest that iap and related cellular genes play an evolutionarily conserved role in the regulation of apoptosis.

CD30 contains two binding sites with different specificities for members of the tumor necrosis factor receptor-associated factor family of signal transducing proteins.

CD30 is a member of the tumor necrosis factor (TNF) receptor family of proteins. CD30 can regulate proliferation of lymphocytes and may also play an important role in human immunodeficiency virus replication. However, little is known about CD30 signal transduction. We performed a yeast two-hybrid library screen with the cytoplasmic domain of CD30 and isolated multiple independent cDNAs encoding human tumor necrosis factor receptor-associated factor (TRAF) 1, TRAF2, and CRAF1 (TRAF3). The ability of TRAF1, TRAF2, and CRAF1 to associate with CD30 was confirmed using an in vitro coprecipitation assay, further demonstrating that the interaction was specific and direct. The TRAF-binding domain of CD30 was mapped to the COOH-terminal 36 amino acid residues, which contained two independent binding sites. CRAF1 bound only a single site, which contained the sequence PEQET, whereas TRAF1 and TRAF2 were capable of binding to either the PEQET site or an additional downstream domain. These data indicate that the TRAF protein binding pattern of CD30 differs from other TNF receptor family members and suggest that signaling specificity through TNF receptor family proteins may be achieved through differences in their abilities to bind TRAF proteins.