Profiling primaquine metabolites in primary human hepatocytes using UHPLC‐QTOF‐MS with 13C stable isotope labeling

Therapeutic efficiency and hemolytic toxicity of primaquine (PQ), the only drug available for radical cure of relapsing vivax malaria are believed to be mediated by its metabolites. However, identification of these metabolites has remained a major challenge apparently due to low quantities and their reactive nature. Drug candidates labeled with stable isotopes afford convenient tools for tracking drug‐derived metabolites in complex matrices by liquid chromatography‐tandem mass spectrometry (LC‐MS‐MS) and filtering for masses with twin peaks attributable to the label. This study was undertaken to identify metabolites of PQ from an in vitro incubation of a 1:1 w/w mixture of ¹³C₆‐PQ/PQ with primary human hepatocytes. Acquity ultra‐performance LC (UHPLC) was integrated with QTOF‐MS to combine the efficiency of separation with high sensitivity, selectivity of detection and accurate mass determination. UHPLC retention time, twin mass peaks with difference of 6 (originating from ¹³C₆‐PQ/PQ), and MS‐MS fragmentation pattern were used for phenotyping. Besides carboxy‐PQ (cPQ), formed by oxidative deamination of PQ to an aldehyde and subsequent oxidation, several other metabolites were identified: including PQ alcohol, predictably generated by oxidative deamination of PQ to an aldehyde and subsequent reduction, its acetate and the alcohol's glucuronide conjugate. Trace amounts of quinone‐imine metabolites of PQ and cPQ were also detected which may be generated by hydroxylation of the PQ/cPQ quinoline ring at the 5‐position and subsequent oxidation. These findings shed additional light on the human hepatic metabolism of PQ, and the method can be applied for identification of reactive PQ metabolites generated in vivo in preclinical and clinical studies. Copyright © 2013 John Wiley & Sons, Ltd.     

Lévy driven moving averages and semimartingales

The aim of the present paper is to study the semimartingale property of continuous time moving averages driven by Lévy processes. We provide necessary and sufficient conditions on the kernel for the moving average to be a semimartingale in the natural filtration of the Lévy process, and when this is the case we also provide a useful representation. Assuming that the driving Lévy process is of unbounded variation, we show that the moving average is a semimartingale if and only if the kernel is absolutely continuous with a density satisfying an integrability condition.     

Spectral Representation of Gaussian Semimartingales

The aim of the present paper is to characterize the spectral representation of Gaussian semimartingales. That is, we provide necessary and sufficient conditions on the kernel K for X _t =∫ K _t (s) dN _s to be a semimartingale. Here, N denotes an independently scattered Gaussian random measure on a general space S. We study the semimartingale property of X in three different filtrations. First, the ℱ ^X -semimartingale property is considered, and afterwards the ℱ ^X,∞-semimartingale property is treated in the case where X is a moving average process and ℱ _t ^X,∞=σ(X _s :s∈(−∞,t]). Finally, we study a generalization of Gaussian Volterra processes. In particular, we provide necessary and sufficient conditions on K for the Gaussian Volterra process ∫ _−∞ ^t K _t (s) dW _s to be an ℱ ^W,∞-semimartingale (W denotes a Wiener process). Hereby we generalize a result of Knight (Foundations of the Prediction Process, 1992) to the nonstationary case.     

Revisiting the Perfluorinated Trityl Cation

Although ultimately not isolable for X-ray structural characterization, the free perfluorinated trityl cation was shown to be observable in neat triflic acid, which represents milder conditions than previous reports of this cation in “magic acid” or oleum. A triflate-bound species could be generated in organic solvents using stoichiometric amounts of triflic acid and was shown to be synthetically viable for hydride abstraction from Et₃SiH. It was demonstrated that the para-position on the -C₆F₅ rings is the primary point of attack for decomposition of the cation.     

A two-dimensional gel protein database of noncultured total normal human epidermal keratinocytes: identification of proteins strongly up-regulated in psoriatic epidermis

A two-dimensional (2-D) gel database of proteins from noncultured total normal human epidermal keratinocytes has been established. A total of 1449 [35S]methionine labelled proteins (1112 isoelectric focusing, 337 nonequilibrium pH gradient electrophoresis) were resolved and recorded using computer assisted (PDQ-SCAN and PDQUEST software) 2-D gel electrophoresis. By matching the protein patterns of total keratinocytes and transformed human amnion cells (master database; Celis et al., Leukemia 1988, 2, 561-602) as well as by 2-D immunoblotting and microsequencing of keratinocyte proteins, it was possible to identify 72 polypeptides in the keratinocyte database. The database also includes data on polypeptides that are synthesized at a higher level by keratinocytes enriched in basal cells, and on six secreted proteins which are produced, albeit at a reduced rate, by normal keratinocytes and that are strongly up-regulated in psoriatic epidermis (Celis et al., FEBS Letters, in press).     

Reference points for comparisons of two-dimensional maps of proteins from different human cell types defined in a pH scale where isoelectric points correlate with polypeptide compositions

A highly reproducible, commercial and nonlinear, wide-range immobilized pH gradient (IPG) was used to generate two-dimensional (2-D) gel maps of [³⁵S]methionine-labeled proteins from noncultured, unfractionated normal human epidermal keratinocytes. Forty one proteins, common to most human cell types and recorded in the human keratinocyte 2-D gel protein database were identified in the 2-D gel maps and their isoelectric points (pI) were determined using narrow-range IPGs. The latter established a pH scale that allowed comparisons between 2-D gel maps generated either with other IPGs in the first dimension or with different human protein samples. Of the 41 proteins identified, a subset of 18 was defined as suitable to evaluate the correlation between calculated and experimental pI values for polypeptides with known composition. The variance calculated for the discrepancies between calculated and experimental pI values for these proteins was 0.001 pH units. Comparison of the values by the t-test for dependent samples (paired test) gave a p-level of 0.49, indicating that there is no significant difference between the calculated and experimental pI values. The precision of the calculated values depended on the buffer capacity of the proteins, and on average, it improved with increased buffer capacity. As shown here, the widely available information on protein sequences cannot, a priori, be assumed to be sufficient for calculating pI values because post-translational modifications, in particular N-terminal blockage, pose a major problem. Of the 36 proteins analyzed in this study, 18–20 were found to be N-terminally blocked and of these only 6 were indicated as such in databases. The probability of N-terminal blockage depended on the nature of the N-terminal group. Twenty six of the preteins had either M, S or A as N-terminal amino acids and of these 17–19 were blocked. Only 1 in 10 proteins containing other N-terminal groups were blocked.     

Short-term culturing of low-grade superficial bladder transitional cell carcinomas leads to changes in the expression levels of several proteins involved in key cellular activities

Fresh, superficial transitional cell carcinomas (TCCs) of low-grade atypia (3 grade I, Ta; 6 grade II, Ta), as well as primary cultures derived from them were labeled with [35S]methionine for 16 h, between 2 and 6 days after inoculation. Whole protein extracts were subjected to IEF (isoelectric focusing) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by autoradiography. Proteins were identified by a combination of proteomic technologies that included microsequencing, mass spectrometry, 2-D PAGE immunoblotting and comparison with the bladder TCC protein database available on the internet (http://biobase.dk/cgi-bin/celis). Comparison of the IEF 2-D gel protein profiles of fresh tumors and their primary cultures showed that the overall expression profiles were strikingly similar, although differing significantly in the levels of several proteins whose rate of synthesis was differentially regulated in at least 85% of the tumor/culture pairs as a result of the short-term culturing. Most of the proteins affected by culturing were upregulated and among them we identified components of the cytoskeleton (keratin 18, gelsolin and tropomyosin 3), a molecular chaperone (hsp 28), aldose reductase, GST pi, metastasin, synuclein, the calreticulin precursor and three polypeptides of unknown identity. Only four major proteins were downregulated, and these included two fatty acid-binding proteins (FABP:FABP5 and A-FABP) which are thought to play a role in growth control, the differentiation-associated keratin 20, and the calcium-binding protein annexin V. Proteins that were differentially regulated in only some of the cultured tumors included alpha-enolase, triosphosphate isomerase, members of the 14-3-3 family, hnRNPs F and H, PGDH, hsp (heat-shock protein) 60, BIP, the interleukin-1 receptor antagonist, the nucleolar protein B23, as well as several proteins of yet unknown identity. The suitability of in vitro bladder tumor culture models to study complex biological phenomena such as malignancy and invasion is discussed.     

Density fluctuations on mm and Mpc scales

We will in this Letter report on suggestive similarities between density fluctuation power versus wavenumber on small (mm) and large (Mpc) scales. The small scale measurements were made in fusion plasmas and compared to predictions from classical fluid turbulence theory. The data is consistent with the dissipative range of 2D turbulence. Alternatively, the results can be fitted to a functional form that cannot be explained by turbulence theory. The large scale measurements were part of the Sloan Digital Sky Survey galaxy redshift examination. We found that the equations describing fusion plasmas also hold for the galaxy data. The comparable dependency of density fluctuation power on wavenumber in fusion plasmas and galaxies might indicate a common origin of these fluctuations.     

MRI detection of tumor in mouse lung using partial liquid ventilation with a perfluorocarbon-in-water emulsion

Transverse relaxation time (T(2*))-weighted (1)H-MRI of mouse lungs has been performed using partial liquid ventilation (PLV) with a perfluorocarbon (PFC)-in-water emulsion as a contrast modality for lung MRI. Significant sensitivity enhancement in MRI of mouse lungs has been demonstrated with the protocol. The results show that the T(2*) value in lung is approximately proportional to the infusion dose up to a dose of 5 ml/kg body weight (BW) (4.5 g PFC/kg BW) and becomes essentially constant beyond this dosage. T(2*) maps of lungs have been calculated and T(2*) in lungs is in the range of 10-35 ms with this technique, which is an order of magnitude greater than the T(2*) value of mouse lungs without using a PFC-in-water emulsion. T(2*)-weighted (1)H-MR images of mouse lungs have been obtained with good quality under our experimental conditions. We have applied this technique to detect tumors in mouse lungs. Our technique can detect small lung tumors of B16 melanoma, about 1 mm in diameter, in mice. With its significant MR sensitivity enhancement and technical simplicity, T(2*)-weighted (1)H-MRI using PLV with PFC-in-water emulsion offers a promising approach to investigate lung cancers using rodent models.     

A comprehensive protein resource for the study of bladder cancer: http://biobase.dk/cgi-bin/celis

In our laboratories we are exploring the possibility of using proteome expression profiles of fresh bladder tumors (transitional cell carcinomas, TCCs; squamous cell carcinomas, SCCs) and random biopsies as fingerprints to subclassify histopathological types and as a starting point to search for protein markers that may form the basis for diagnosis, prognosis, and treatment. Ultimately, the goal of these studies is to identify signaling pathways and components that are affected at various stages of bladder cancer progression and that may provide novel leads in drug discovery. Here we present our ongoing efforts to establish comprehensive two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) databases of TCCs and SCCs which are being constructed based on the proteomic and immunohistochemical analysis of hundreds of fresh tumors, random biopsies and cystectomies received shortly after operation (http://biobase.dk/cgi-bin/celis).