Biomedical subjects

A C Laan

Publications and source records attributed to A C Laan.

7 recordsLinked to original sources

PubMed2006

The determination of gemcitabine and 2'-deoxycytidine in human plasma and tissue by APCI tandem mass spectrometry.

A fast, sensitive and accurate method for the determination of gemcitabine (difluorodeoxycytidine; dFdC) and deoxycytidine (CdR) in human plasma/tissue was developed using LC-MS/MS techniques. Effectiveness of the method is illustrated with the analysis of plasma from a phase I trial of dFdC administered as a 24h infusion. The method was developed using (15)N(3) CdR as an internal standard across the concentration range of 1-500ng/ml, using a cold alcohol-protein precipitation followed by desorption with freeze drying. Sample clean-up for LC-MS/MS analysis was performed by an innovative liquid/liquid back extraction with ethyl acetate and water. Chromatography was performed using a Chrompak-spherisorb-phenyl-column (3.1mmx200mm, 5microm) with a 50mM formic acid: acetonitrile (9:1) mobile phase eluted at 1ml/min. Extracted samples were observed to be stable for a minimum of 48h after extraction when kept at 4 degrees C. Detection was performed using an atmospheric pressure chemical ionization (APCI) source and mass spectrometric positive multi-reaction-monitoring-mode (+MRM) for dFdC (264 m/z; 112 m/z), CdR (228 m/z; 112 m/z), and (15)N(3) CdR (231 m/z; 115 m/z) at an ion voltage of +3500V. The accuracy, precision and limit-of-quantitation (LOQ) were as follows: dFdC: 99.8%, +/-7.9%, 19nM; CdR: 100.0%, +/-5.3%, 22nM, linear range LOQ to 2microM. During 24h infusion dFdC levels were detected with no interference from either CdR or difluorodeoxyuridine (dFdU). CdR co-eluted with dFdC but selectivity demonstrated no "crosstalk" between the compounds. In conclusion the analytical assay was very sensitive, reliable and robust for the determination of plasma and tissue concentrations of dFdC and CdR.

Chromatography, High Pressure Liquid↗

PubMed2006

The role of thymidine phosphorylase and uridine phosphorylase in (fluoro)pyrimidine metabolism in peripheral blood mononuclear cells.

Thymidine phosphorylase (TP) and uridine phosphorylase (UP) catalyze the (in)activation of several fluoropyrimidines, depending on their catalytic activity and substrate specificity. Blood cells are the first compartment exposed to most anticancer agents. The role of white blood cells in causing toxic side effects and catalyzing drug metabolism is generally underestimated. Therefore we determined the contribution of the white blood cell compartment to drug metabolism, and we investigated the activity and substrate specificity of TP and UP for the (fluoro)pyrimidines thymidine (dThd), uridine (Urd), 5'-deoxy-5-fluorouridine (5' dFUrd) and 5-fluorouracil (5FU) in peripheral blood mononuclear cells (PBMC) and undifferentiated monocytes and differentiated monocytes: macrophages and dendritic cells. PBMC had an IC50 of 742 microM exposed to 5'dFUrd, increasing to > 2000 microM when both TP and UP activities were inhibited. Total phosphorolytic activity was higher with dThd than with Urd, 5'dFUrd or 5FU. Using a specific TP inhibitor (TPI) and UP inhibitor (BAU) we concluded that dThd and Urd were preferentially converted by TP and UP, respectively, while 5'dFUrd and 5FU were mainly converted by TP (about 80%) into 5FU and FUrd, respectively. 5FU was effectively incorporated into RNA. dThd conversion into thymine was highest in dendritic cells (52.6 nmol thymine/h/10(6) cells), followed by macrophages (two-fold) and undifferentiated monocytes (eight-fold). TPI prevented dThd conversion almost completely. In conclusion, PBMC were relatively insensitive to 5'dFUrd, and the natural substrates dThd and Urd were preferentially converted by TP and UP, respectively. TP and UP were both responsible for converting 5'dFUrd/5FU into 5FU/FUrd, respectively.

Cells, Cultured↗

PubMed2006

Analysis of deoxycytidine accumulation in gemcitabine treated patients.

Deoxycytidine (CdR) analogs are increasingly popular as chemotherapeutic agents and their effectiveness can be linked to the direct competition with active forms of endogenous CdR. A tandem mass spectrometric assay was developed to determine the plasma concentrations of CdR. Plasma extracts were prepared by protein precipitation and an ethyl acetate/water back extraction, and then separated chromatographically. Detection parameters were optimized for multi-reaction monitoring (MRM) tandem mass spectrometry and assay efficiency was improved using 15N3 CdR as an isotopic internal standard. Preliminary results from a gemcitabine trial are shown which indicate that CdR concentrations increase systemically during infusion, from about 5 nM to 78 nM after hepatic artery infusion and to 102 nM after systemic infusion for 24 hours. The developed assay demonstrated good sensitivity and selectivity for CdR.

Antimetabolites, Antineoplastic↗

PubMed2003

Rapid disappearance of deoxyribose-1-phosphate in platelet derived endothelial cell growth factor/thymidine phosphorylase overexpressing cells.

Platelet derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) catalyzes the phosphorolysis of thymidine (TdR) to thymine and deoxyribose-1-phosphate (dR-1-P) and has a pro-angiogenic effect for which dR-1-P may be responsible. Using a purine nucleoside phosphorylase based assay it was found that TdR incubation did not increase dR-1-P accumulation in colon cancer cell line Colo320 and its PD-ECGF/TP transfected variant Colo320TP1. The assay was linear up to 25,000pmol dR-1-P with complete recovery of dR-1-P from cellular extracts. There was a huge discrepancy between thymine production and the measured dR-1-P level, 0.05% of the expected value for dR-1-P was found, indicating that there was a rapid disappearance of dR-1-P. However, in cellular extracts, TdR incubation increased dR-1-P, measurable by trapping, which was inhibited by a thymidine phosphorylase inhibitor. dR-1-P directly added to cellular extracts disappeared within 5-10min. In conclusion, large amounts of dR-1-P are produced by Colo320TP1 cells, which rapidly disappear thus not resulting in a net accumulation of dR-1-P in these cells.

Cell Line↗

PubMed1995

Binocular correspondence and visual direction.

Two classic theories of direction vision, one by Hering, the other by Wells, are expressed in mathematical form and compared. The Hering disparity field differs considerably from the Wells disparity field, but if both are scaled for the change of acuity with eccentricity their differences are much more subtle. This explains why it is hard to determine which theory predicts direction perception best, although the tests favour Hering's theory. It is proved that Wells's construction (his rule 3) follows directly from his first two rules and Aguillonius's assumption that the horopter in the fixation plane is a frontoparallel line. Wells's theory is clearly outdated and does not mesh well with modern three-dimensional geometry of binocular vision, which Hering's theory does. Moreover, Wells inextricably mixes distance and direction vision right from the start, whereas Hering properly treats the two-dimensional manifold of directions and the depth-gauging principles separately. The use of terms such as 'Wells-Hering' rules should be discouraged and both Wells and Hering should be remembered separately for their clearly distinct and independent contributions. The work of Hering is still relevant to modern theory and praxis of binocular vision. The extension of Hering's approach to vertical disparities is treated for stimuli in frontoparallel planes. It is shown that acuity-scaled vertical-disparity information sampled at a single glance is below resolution beyond about arm's length. It can only be used if eye movements are allowed. Throughout, the simplest derivations of the geometrical relations that it was possible to find are given, so that the review of binocular geometry might also be of some didactical use. Finally it is indicated in which direction it might be necessary to modernise the concept of binocular correspondence.

History, 18th Century↗

PubMed1989

Deformation-corrected computer-aided three-dimensional reconstruction of immunohistochemically stained organs: application to the rat heart during early organogenesis.

The application of a computer-assisted, three-dimensional reconstruction procedure for serial sections to embryonic rat hearts during the period of cardiac looping and compartmentalization is described. The procedure relies on immunohistochemical staining for the introduction of selective contrast and on episcopic and diascopic images of each of the sections for alignment and correction of compression due to sectioning. Episcopic (reference) images are taken from the embedding block just before the cutting of a slice and are still aligned and undeformed. Diascopic images are taken from the sections after immunohistochemical processing and, hence, contain selective contrast but are deformed and no longer aligned. The three-dimensional images are visualized as shaded voxel models. This approach allowed the unequivocal delineation of the developing myocardium and the inspection of its changing architecture both from the outside and from within. Furthermore, it allowed a quantification of myocardial volume. Because standardized and hence comparable views of three different stages were generated, changes in the shape of the cardiac loop, the atria, and the ventricles as well as changes in the position of the atrioventricular canal and interventricular foramen could be accurately described. Characteristic changes in the position of both the right ventricle and the atrioventricular canal that are essential for the formation of a correctly functioning four-chambered heart could be observed. These changes in shape occur while the myocardial size increases dramatically.

Animals↗

PubMed1986

Bivariate linear models in neurobiology: problems of concept and methodology.

Bivariate linear models, used to describe morphological and functional characteristics between two sets of observations, are examined both in concept and in application. This paper focuses on the underlying assumptions and statistics of the method most frequently used: ordinary linear regression, principal axis and standard major axis. It is shown how the choice of method should depend on: the purpose of the analysis and the a priori assumptions regarding the residual variance. It appears that none of the methods has a universal application. Differences among the models discussed are illustrated by a bivariate morphometric analysis of cerebrocortical regions in primates.

Animals↗