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Fine Mapping the Spatial Distribution and Concentration of Unlabeled Drugs within Tissue Micro-Compartments Using Imaging Mass Spectrometry

Readouts that define the physiological distributions of drugs in tissues are an unmet challenge and at best imprecise, but are needed in order to understand both the pharmacokinetic and pharmacodynamic properties associated with efficacy. Here we demonstrate that it is feasible to follow the in vivo transport of unlabeled drugs within specific organ and tissue compartments on a platform that applies MALDI imaging mass spectrometry to tissue sections characterized with high definition histology. We have tracked and quantified the distribution of an inhaled reference compound, tiotropium, within the lungs of dosed rats, using systematic point by point MS and MS/MS sampling at 200 µm intervals. By comparing drug ion distribution patterns in adjacent tissue sections, we observed that within 15 min following exposure, tiotropium parent MS ions (mass-to-charge; m/z 392.1) and fragmented daughter MS/MS ions (m/z 170.1 and 152.1) were dispersed in a concentration gradient (80 fmol-5 pmol) away from the central airways into the lung parenchyma and pleura. These drug levels agreed well with amounts detected in lung compartments by chemical extraction. Moreover, the simultaneous global definition of molecular ion signatures localized within 2-D tissue space provides accurate assignment of ion identities within histological landmarks, providing context to dynamic biological processes occurring at sites of drug presence. Our results highlight an important emerging technology allowing specific high resolution identification of unlabeled drugs at sites of in vivo uptake and retention.     

Paleoceanographic interpretations of coccoliths and oxygen-isotopes from the sediment surface of the southwest Indian Ocean

The distribution of forty-four coccolithophore species in one hundred deep-sea core-tops from the southwest Indian Ocean is described. Three coccolith assemblages have been recognised (Maputo, Agulhas Current and deep water) by the relative abundances of four ecologically significant coccolithophore species (Gephyrocapsa oceanica, Emiliania huxleyi, Calcidiscus leptoporus and Umbilicosphaera sibogae). Their biogeographical distribution appears to be related to water temperature, nutrient concentration and dissolution.The degree of preservation of coccoliths and foraminifera indicates that the carbonate lysocline lies somewhere between 3500 and 4000 m, resulting in the concentration of dissolution-resistant microfossils below this depth.Stable oxygen isotope ratios of the planktonic foraminiferal species Globigerinoides sacculifer range between −1.5 to −1.0‰ PDB (equal to 22.8–25.1°C) and occur in a narrow band on the sea floor beneath the “A” route of the Agulhas Current.These values are about 0.5 per mil PDB lighter than samples analyzed on either side of this band and can be explained by the Agulhas Current's elevated temperature at the ocean surface of 2–3°C. Thus an oxygen isotope imprint of the Agulhas Current exists beneath it on the sea floor.The Agulhas Current is probably the major factor influencing sedimentation, sediment-distribution patterns and geological features in the study area. At present it is voluminous and fast flowing, possibly eroding sediments up to 2500 m below the surface.The oxygen-isotope ratios and nannoplankton counts obtained in this study indicate, however, that the majority of samples are most probably recent or at least not older than 85,000 years. This implies that sediments are accumulating on the ocean floor and that the Agulhas Current does not have a pronounced erosional influence, at least in areas from which cores were retrieved for this study.     

Accurate MS-based Rab10 Phosphorylation Stoichiometry Determination as Readout for LRRK2 Activity in Parkinson's Disease

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Highlights

• •MS-based clinical assay that accurately determines phospho Rab10 occupancy.
• •Stable isotope labeled phosphopeptide injected as a standard with endogenous tryptic phospho Rab peptide for accurate ratio determination.
• •Determination of pRab levels in neutrophils of Parkinson disease patients.
• •Relevance of pRab levels as marker of PD.

     

Large Alterations in Ganglioside and Neutral Glycosphingolipid Patterns in Brains from Cases with Infantile Neuronal Ceroid Lipofuscinosis/Polyunsaturated Fatty Acid Lipidosis

Lipid composition was studied on cerebral tissue from nine children who had died of a progressive encephalopathy called the infantile form of neuronal ceroid lipofuscinosis (INCL) or polyunsaturated fatty acid lipidosis (PFAL). In the terminal stage of the disease, the concentrations of all lipid classes were found to be significantly reduced in the cerebral and cerebellar cortex and white matter. The concentration of gangliosides of the cerebral cortex was 15% and that of cerebrosides (galactosylceramide) in white matter 0.2-5% of the normal values for the children's ages. The reduction of gangliosides mainly affected those of the gangliotetraose series, particularly GD1a. The fatty acids of the linolenic acid series were strongly reduced in ethanolamine and serine phosphoglycerides. A very large increase up to 100-fold of oligoglycosphingolipids of the globo series and two fucose-containing lipids of the neolacto series was found in the forebrain of the three advanced cases examined. The brain tissue also contained very high concentrations of mono-, di-, and trisialogangliosides of the lacto and neolacto series, gangliosides with type 1 chain dominating. The structures of the gangliosides were tentatively identified by gas chromatography-mass spectrometry and monoclonal antibodies with carefully determined epitope specificity. The gangliosides and neutral glycosphingolipids had very similar fatty acid composition, consisting of about 40% stearic acid and 40% C24-acids.     

Altitudinal changes in the incidence of crassulacean acid metabolism in vascular epiphytes and related life forms in Papua New Guinea

Summary The occurrence of Crassulacean acid metabolism (CAM), as judged from ¹³C values, was investigated in epiphytes and some related plant species at a series of sites covering the approximate altitudinal range of epiphytes in Papua New Guinea. Comprehensive collections were made at each site and the occurrence of water storage tissue and blade thickness was also determined. Some 26% of epiphytic orchids from a lowland rainforest (2–300 m.a.s.l) showed ¹³C values typical of obligate CAM and possessed leaves thicker than 1 mm. A second group of orchids, mostly with succulent leaves, possessed intermediate ¹³C values between -23 and -26% and accounted for 25% of the total species number. Some species of this group may exhibit weak CAM or be facultative CAM plants. The remainder of the lowland rainforest species appeared to be C₃ plants with ¹³C values between -28 and -35%. and generally possessed thin leaves. Obligate CAM species of orchids from a lower montane rainforest (1175 m.a.s.l) comprised 26% of the species total and mostly possessed thick leaves. The remainder of the species were generally thin-leaved with ¹³C values between -26 and -35%. largely indicative of C₃ photosynthesis. Orchids with intermediate ¹³C values were not found in the lower montane rainforest. Obligate CAM appeared to be lacking in highland epiphytes from an upper montane rainforest and subalpine rainforest (2600–3600 m.a.s.l). However the fern, Microsorium cromwellii had a ¹³C value of -21.28%. suggesting some measure of CAM activity. Other highland ferns and orchids showed more negative °¹³C values, up to-33%., typical of C₃ photosynthesis. The highland epiphytic orchids possessed a greater mean leaf thickness than their lowland C₃ counterparts due to the frequent occurrence of water storage tissue located on the adaxial side of the leaf. It is suggested that low daytime temperatures in the highland microhabitats is a major factor in explaining the absence of CAM. The increased frequency of water storage tissue in highland epiphytes may be an adaptation to periodic water stress events in the dry season and/or an adaptation to increased levels of UV light in the tropicalpine environment.     

The relationship between turgor pressure and titratable acidity in mesophyll cells of intact leaves of a Crassulacean-acid-metabolism plant,Kalanchoe daigremontiana Hamet et Perr

Day/night changes in turgor pressure (P) and titratable acidity content were investigated in the (Crassulacean-acid-metabolism (CAM) plant Kalanchoe daigremontiana. Measurements of P were made on individual mesophyll cells of intact attached leaves using the pressure-probe technique. Under conditions of high relative humidity, when transpiration rates were minimal, changes in P correlated well with changes in the level of titratable acidity. During the standard 12 h light/12 h dark cycle, maximum turgor pressure (0.15 MPa) occurred at the end of the dark period when the level of titratable acidity was highest (about 300 eq H⁺·g^-1 fresh weight). A close relationship between P and titratable acidity was also seen in leaves exposed to perturbations of the standard light/dark cycle. (The dark period was either prolonged, or else only CO₂-free air was supplied in this period). In plants deprived of irrigation for five weeks, diurnal changes in titratable acidity of the leaves were reduced (H=160 eq H⁺·g^-1 fresh weight) and P increased from essentially zero at the end of the light period to 0.02 MPa at the end of the dark period. Following more severe water stress (experiments were made on leaves which had been detached for five weeks), P was zero throughout day and night, yet small diurnal changes in titratable acidity were still measured. These findings are discussed in relation to a hypothesis by Lüttge et al. 1975 (Plant Physiol. 56,613-616) for the role of P in the regulation of acidification/de-acidification cycles of plants exhibiting CAM.Abbreviations CAM crassulacean acid metabolism - FW fresh weight - P turgor pressure