Molecular imaging of small molecule drugs in animal tissues using laser desorption postionization mass spectrometry.

Localization and quantification of the target drug in tissues is a key indicator of efficacy in drug discovery. In contrast to established methods that require matrices and complex sample pretreatment steps, matrix-free and low cost in situ analysis of small molecule drugs by mass spectrometry (MS) remains challenging. Here, we present a novel approach, laser desorption postionization (LDPI), which is coupled to a linear time-of-flight (TOF) MS and used to image the distribution of acriflavine (ACF) directly from a histological section of mouse kidney without any matrix or sample pretreatment. The identification of the mass peaks assigned to ACF was further confirmed by DESI-MS/MS. Moreover, the matrix effect from the tissue section was explored, showing minimal desorption and ionization suppression in the LDPI-MS process. LDPI-MS imaging (LDPI-MSI) was performed on 30 µm kidney sections from mice 15 min postdose that were dosed with 30 mg kg-1 of ACF by monitoring the fragment ion at m/z 209. The LDPI-MS image revealed a global view of the distribution of ACF in the kidney compartments (pelvis, medulla, and cortex). Estimated concentrations of ACF residue in mouse kidney were obtained by LDPI-MSI and LC-MS/MS and a 12.1% difference in measured tissue concentration was found. These results suggest that the use of LDPI-MS in small molecule drug localization and quantification directly from biological tissue at the same time is favorable.

Analysis of epithelial cell shedding and gaps in the intestinal epithelium.

The intestinal barrier is formed by a monolayer of columnar epithelial cells. This barrier is effectively maintained despite the high turnover of epithelial cells in the gut. Defects in the mechanism by which barrier function is maintained are believed to play a central role in the pathogenesis of inflammatory bowel disease (IBD). Proinflammatory cytokines such as TNF-α and IFN-γ are often elevated in inflamed tissue of patients with IBD. In fact, anti-TNF-α therapy is routinely administered to patients with Crohn's disease. We have previously demonstrated that intestinal epithelial cells are shed from the intestine leaving a 'gap' in the epithelium that is able to maintain barrier function. The rate of cell shedding and barrier permeability is substantially increased by the administration of TNF-α. Loss of barrier function at the site of a gap may provide a site of entry for disease-causing bacteria.

Influencia de diferentes derivados lácteos sobre el crecimiento de Listeria monocytogenes en medios selectivos / Influence of different dairy products on the growth kinetic of Listeria monocytogenes

Se estudió la influencia del añadido de crema de leche y leche parcialmente descremada sobre la cinética de crecimiento de Listeria monocytogenes en caldos de enriquecimiento para listerias, conteniendo diferentes concentraciones de acriflavina (15 y 7,5 mg/l). El crecimiento de Listeria monocytogenes en los caldos de enriquecimiento sufrió un retardo atribuible, al menos parcialmente, a la presencia de acriflavina. El añadido de crema de leche o leche parcialmente descremada al caldo de enriquecimiento que contiene 7,5 mg/l de acriflavina produjo un alargamiento de la fase de adaptación, pero las cosechas máximas alcanzadas a las 48 h no mostraron diferencias significativas. En presencia de 15 mg/l de acriflavina, se observó una pérdida inicial de la viabilidad de los cultivos, que fue potenciada por el agregado de crema de leche o leche parcialmente descremada al caldo de enriquecimiento. Además, la leche descremada produjo una disminución de la velocidad máxima de crecimiento que impidió alcanzar la cosecha máxima dentro de las 48 h. Los resultados obtenidos sugieren la necesidad de validar la metodología de recuperación de L. monocytogenes para cada producto, pues la eficiencia de recuperación podría ser afectada por la composición del mismo, sobre todo cuando la carga microbiana es baja (AU)

Influencia de diferentes derivados lácteos sobre el crecimiento de Listeria monocytogenes en medios selectivos / Influence of different dairy products on the growth kinetic of Listeria monocytogenes

Se estudió la influencia del añadido de crema de leche y leche parcialmente descremada sobre la cinética de crecimiento de Listeria monocytogenes en caldos de enriquecimiento para listerias, conteniendo diferentes concentraciones de acriflavina (15 y 7,5 mg/l). El crecimiento de Listeria monocytogenes en los caldos de enriquecimiento sufrió un retardo atribuible, al menos parcialmente, a la presencia de acriflavina. El añadido de crema de leche o leche parcialmente descremada al caldo de enriquecimiento que contiene 7,5 mg/l de acriflavina produjo un alargamiento de la fase de adaptación, pero las cosechas máximas alcanzadas a las 48 h no mostraron diferencias significativas. En presencia de 15 mg/l de acriflavina, se observó una pérdida inicial de la viabilidad de los cultivos, que fue potenciada por el agregado de crema de leche o leche parcialmente descremada al caldo de enriquecimiento. Además, la leche descremada produjo una disminución de la velocidad máxima de crecimiento que impidió alcanzar la cosecha máxima dentro de las 48 h. Los resultados obtenidos sugieren la necesidad de validar la metodología de recuperación de L. monocytogenes para cada producto, pues la eficiencia de recuperación podría ser afectada por la composición del mismo, sobre todo cuando la carga microbiana es baja

Liquid chromatographic determination of acriflavine and proflavine residues in channel catfish muscle.

A liquid chromatographic (LC) method was developed for determination of acriflavine (ACR) and proflavine (PRO) residues in channel catfish muscle. Residues were extracted with acidified methanol solution, and extracts were cleaned up with C18 solid-phase extraction columns. Residue concentrations were determined on an LC cyano column, with spectrophotometric detection at 454 nm. Catfish muscle was individually fortified with ACR (purified from commercial product) and PRO at concentrations of 5, 10, 20, 40, and 80 ppb (5 replicates per level). Mean recoveries from fortified muscle at each level ranged from 86 to 95%, with relative standard deviations (RSDs) of 2.5 to 5.7%. The method was applied to incurred residues of ACR and PRO in muscle after waterborne exposure of channel catfish to commercial acriflavine (10 ppm total dye for 4 h). RSDs for incurred residues of ACR and PRO were in the same range as those for fortified muscle. Low residue concentrations (< 1% of exposure water concentration) suggested poor absorption of ACR and PRO in catfish.

The effect of dietary factors on intradentinal dye penetration in the rat.

Fluorescent dye injected systemically into rats penetrated the dentinal tubules of molar teeth in a dynamic fashion. The presence of dye was established using histological and fluorescence microscopy techniques. The rate of intradentinal dye penetration was dependent on dietary factors: it was high in rats chronically fed Purina rat chow and low in rats fed a cariogenic, high-sucrose diet. In addition, parotidectomized rats showed low levels of intradentinal dye penetration, even though they were maintained on Purina chow. One and 2 ml of plasma from Purina-fed rats were effective in stimulating the dye penetration in intact and parotidectomized rats, whereas 2 and 4 ml of plasma from rats fed a high-sucrose diet were ineffective when infused in either intact or parotidectomized animals. The results suggest that rats fed Purina chow have a significantly higher titre of a circulating, dye penetration stimulating factor than animals fed a high sucrose diet. This circulating factor could be the equivalent of the parotid hormone isolated from porcine tissue. It is suggested that dietary factors may affect secretion of a parotid hormone and thereby regulate the rate of dentinal fluid movement. There is therefore the prospect of a functional relationship between diet, the regulation of dentinal fluid flow by an endocrine system and dental health.

Reversed-phase chromatography of novel biologically effective aminoacridine-N-glycosides by OPLC.

The reversed-phase chromatographic behavior of novel biologically active aminoacridine-N-glycosides is studied. The chromatographic experiments are performed with overpressurized layer chromatography. Weak ion pairs are formed with methanesulfonic acid, but only at low concentrations of the ion-pairing reagent. The retention seems to involve a reversed-phase mechanism. The base compounds only slightly modify the retention, while the number and polarity of the substituents have larger effects. The pH dependence of the retention is very typical for the aminoacridine-N-glycosides, and it plays an important role in the separation. The monoglycosides are completely separated from the corresponding base compounds, as are the diglycosides from the monoglycosides, on RP-2, RP-8, and RP-18 layers with eluents containing 30 to 60% acetonitrile and at least 0.005 M ammonium carbonate at pH 4 to 6.

A moving spot high resolution scanning microspectrometer using focused laser illumination: illustrations with acriflavin stained sephadex beads and human fibroblast nuclei.

We describe a high resolution moving spot scanning microspectrometer, capable of absorption or fluorescence detection, using focused laser illumination which is moved over the sample by rotating the laser beam direction prior to focusing. This rotation is achieved by reflecting the beam from mirrors mounted on bending mode piezoelectric transducers which, when bent by an applied voltage, cause the mirrors to rotate. The images of optically thin samples are analyzed by considering the convolution of the focused spot intensity distribution with the absorbance of a uniformly stained spherical particle. This analysis is verified experimentally with data from acriflavin stained Sephadex beads. Data from acriflavin-Feulgen stained human fibroblasts indicate that the efficiency of this type of nuclear staining is about 2 to 3 dye molecules incorporated per 100 nucleotide pairs. Quantitative data on fading of acriflavin fluorescence in stained fibroblasts indicate that fading is negligible in the time required to record the microscope images.