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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.     

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.     

Influence of vasoactive intestinal polypeptide (VIP) on splanchnic and central hemodynamics in healthy subjects

The influence of VIP, a potent vasodilator, on central hemodynamics, splanchnic blood flow and glucose metabolism was studied in six healthy subjects. Teflon catheters were inserted into an artery, a femoral vein and a right-sided hepatic vein. A Swan-Ganz catheter was introduced percutaneously and its tip placed in the pulmonary artery. Determinations of cardiac output, systemic, pulmonary arterial and hepatic venous pressures as well as splanchnic blood flow were made in the basal state and at the end of two consecutive 45 min periods of VIP infusion at 5 and 10 ng/kg/min, respectively. Arterial blood samples for analysis of glucose, FFA, insulin and glucagon were drawn at timed intervals. VIP infusion at 5 ng/kg/min resulted in an increase in cardiac output (55%) and heart rate (25%) as well as a reduction in mean systemic arterial pressure (15%) and vascular resistance (45%). With the higher rate of VIP infusion heart rate tended to rise further while cardiac output and arterial pressure remained unchanged. At 15 min after the end of VIP infusion the above variables had returned to basal levels. Splanchnic blood flow and free hepatic venous pressure did not change significantly. Arterial concentrations of glucose, FFA, insulin and glucagon increased during VIP infusion. At 15 min after the end of infusion the glucose levels were still significantly higher than basal (20%). Net splanchnic glucose output did not change in response to VIP infusion. It is concluded that VIP exerts a potent vasodilatory effect resulting in augmented cardiac output and lowered systemic blood pressure and vascular resistance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assay and properties of 25-hydroxyvitamin D3 23-hydroxylase. Evidence that 23,25-dihydroxyvitamin D3 is a major metabolite in 1,25-dihydroxyvitamin D3-treated or fasted guinea pigs.

Incubation of 25-hydroxyvitamin D3 with kidney cortex mitochondria from 1,25-dihydroxyvitamin D3-treated guinea pigs resulted in the formation of 23,25-dihydroxyvitamin D3 as the major product. The identity of the product was verified by g.c.-m.s. and quantification was performed by h.p.l.c. The rates of the reaction were in the range 1.0-1.8 pmol/min per mg of mitochondrial protein (at 37 degrees C), which were 5-10 times the rates of formation of 24,25-dihydroxyvitamin D3. In mitochondrial preparations from untreated guinea pigs, the rate of 23-hydroxylation was below detection limit (0.02 pmol/min per mg of mitochondrial protein). Fasting the animals for 24 h induced the 23-hydroxylase almost as efficiently as treatment with 1,25-dihydroxyvitamin D3, with a concomitant depression of the 1 alpha-hydroxylase. The 23-hydroxylase reaction required oxidizable substrate, was decreased by low O2 partial pressures and inhibited by CO or the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. It was stimulated by the respiratory-chain inhibitors rotenone, antimycin A and KCN. These results indicate that the guinea-pig renal mitochondrial 23-hydroxylase is a cytochrome P-450 and that the reducing equivalents are primarily supplied by NADPH via the energy-dependent transhydrogenase.     

Neonatal screening for congenital adrenal hyperplasia using 17-hydroxyprogesterone assay in filter paper blood spots

Screening of infants for congenital adrenal hyperplasia (CAH) using filter paper blood samples collected on the 5th day of life was performed with a radioimmunoassay for 17-hydroxyprogesterone without extraction with organic solvents. A total of 153,000 newborns were screened and 12 cases of CAH were detected (1:12,800). With recall levels related to gestational age, the recall rate could be lowered to 0.05%.     

Nitrogen in shoot litter,root litter and root exudates from nitrogen-fixingAlnus incana

Summary A pot experiment withAlnus incana (L.) Moench growing in sand was set up to compare the amounts of nitrogen released from plants shoot litter with that released below ground as root litter and/or root exudation. No nitrogen fixation by free-living microorganisms was found in the sand and the increased nitrogen content of the plant + soil system was therefore due to nitrogen fixation byFrankia in the alder root-nodules. Most of the nitrogen released from the plants was in the nitrogen-rich leaf and other shoot litter. Only small amounts of nitrogen were found in the drainage water from the pots and were recorded as increased nitrogen content of the sand.     

Exceptionally high copy numbers of a staphylococcal plasmid in Bacillus subtilis revealed by a sandwich hybridization technique

Abstract The copy number of a pUB110 derivative, pKTH10, containing the α-amylase gene from Bacillus amyloliquefaciens , was determined, using an assay based on a sandwich hybridization technique. In this method, a known gene on the plasmid is hybridized between two non-overlapping fragments of that same gene, cloned into separate vectors. One fragment is used as a radiolabelled probe and the other bound to a filter, forming a three-component, 'sandwich' hybrid when the relevant gene is present in the sample. Since the hybridization can only take place in the presence of the relevant gene, the amount of radioactivity binding to the filters will be proportional to the concentration of this gene in the sample. We utilized the α-amylase gene on the plasmid to form the sandwich hybrid. The copy number was of a totally different magnitude from what has previously been reported, and ranged from 2500 copies/viable cell in early logrithimic growth phase to about 500 in late stationary phase.