Lipid Coverage in Nanospray Desorption Electrospray Ionization Mass Spectrometry Imaging of Mouse Lung Tissues.

Lipids are a naturally occurring group of molecules that not only contribute to the structural integrity of the lung preventing alveolar collapse but also play important roles in the anti-inflammatory responses and antiviral protection. Alteration in the type and spatial localization of lipids in the lung plays a crucial role in various diseases, such as respiratory distress syndrome (RDS) in preterm infants and oxidative stress-influenced diseases, such as pneumonia, emphysema, and lung cancer following exposure to environmental stressors. The ability to accurately measure spatial distributions of lipids and metabolites in lung tissues provides important molecular insights related to lung function, development, and disease states. Nanospray desorption electrospray ionization (nano-DESI) and other ambient ionization mass spectrometry techniques enable label-free imaging of complex samples in their native state with minimal to absolutely no sample preparation. However, lipid coverage obtained in nano-DESI mass spectrometry imaging (MSI) experiments has not been previously characterized. In this work, the depth of lipid coverage in nano-DESI MSI of mouse lung tissues was compared to liquid chromatography tandem mass spectrometry (LC-MS/MS) lipidomics analysis of tissue extracts prepared using two different procedures: standard Folch extraction method of the whole lung samples and extraction into a 90% methanol/10% water mixture used in nano-DESI MSI experiments. A combination of positive and negative ionization mode nano-DESI MSI identified 265 unique lipids across 20 lipids subclasses and 19 metabolites (284 in total) in mouse lung tissues. Except for triacylglycerols (TG) species, nano-DESI MSI provided comparable coverage to LC-MS/MS experiments performed using methanol/water tissue extracts and up to 50% coverage in comparison with the Folch extraction-based whole lung lipidomics analysis. These results demonstrate the utility of nano-DESI MSI for comprehensive spatially resolved analysis of lipids in tissue sections. A combination of nano-DESI MSI and LC-MS/MS lipidomics is particularly useful for exploring changes in lipid distributions during lung development, as well as resulting from disease or exposure to environmental toxicants.

Plasma Protein Turnover Rates in Rats Using Stable Isotope Labeling, Global Proteomics, and Activity-Based Protein Profiling.

Protein turnover is important for general health on cellular and organism scales providing a strategy to replace old, damaged, or dysfunctional proteins. Protein turnover also informs of biomarker kinetics, as a better understanding of synthesis and degradation of proteins increases the clinical utility of biomarkers. Here, turnover rates of plasma proteins in rats were measured in vivo using a pulse-chase stable isotope labeling experiment. During the pulse, rats (n = 5) were fed 13C6-labeled lysine ("heavy") feed for 23 days to label proteins. During the chase, feed was changed to an unlabeled equivalent feed ("light"), and blood was repeatedly sampled from rats over 10 time points for 28 days. Plasma samples were digested with trypsin and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant was used to identify peptides and proteins and quantify heavy/light lysine ratios. A system of ordinary differential equations was used to calculate protein turnover rates. Using this approach, 273 proteins were identified, and turnover rates were quantified for 157 plasma proteins with half-lives ranging 0.3-103 days. For the ∼70 most abundant proteins, variability in turnover rates among rats was low (median coefficient of variation: 0.09). Activity-based protein profiling was applied to pooled plasma samples to enrich serine hydrolases using a fluorophosphonate (FP2) activity-based probe. This enrichment resulted in turnover rates for an additional 17 proteins. This study is the first to measure global plasma protein turnover rates in rats in vivo, measure variability of protein turnover rates in any animal model, and utilize activity-based protein profiling for enhancing turnover measurements of targeted, low-abundant proteins, such as those commonly used as biomarkers. Measured protein turnover rates will be important for understanding of the role of protein turnover in cellular and organism health as well as increasing the utility of protein biomarkers through better understanding of processes governing biomarker kinetics.

Evaluation of non-invasive biomonitoring of 2,4-Dichlorophenoxyacetic acid (2,4-D) in saliva.

The objective of this study was to evaluate the potential for non-invasive biomonitoring of 2,4-Dichlorophenoxyacetic acid (2,4-D) in saliva. Using an in vitro rat salivary gland epithelial cell (SGEC) system, a collection of experiments investigating chemical protein binding, temporal and directional transport, as well as competitive transport with para-aminohippuric acid (PAH), a substrate for renal organic anion transporters, was conducted to identify cellular transport parameters required to computationally model salivary transport of 2,4-D. Additionally, a physiological protein gradient was implemented to mimic physiologically relevant concentrations of protein in rat plasma and saliva, and under these conditions the transfer of 2,4-D was markedly slower, driven by increased protein binding (i.e. reduced free 2,4-D species available to cross salivary barrier). The rate of transfer was directly proportional to the amount of unbound 2,4-D and demonstrated no indication of active transport. An in vivo assessment of 2,4-D exposure in rats revealed non-linear protein binding in plasma, indicating saturated protein binding and increased levels of unbound 2,4-D species at higher doses. A strong correlation between 2,4-D concentrations in saliva and unbound 2,4-D in plasma was observed (Pearson correlation coefficient = 0.95). Saliva:plasma 2,4-D ratios measured in vivo (0.0079) were consistent within the linear protein binding range and expected 2,4-D levels from occupational exposures but were significantly different than ratios measured in vitro (physiological conditions) (0.034), possibly due to 2,4-D concentrations in saliva not being at equilibrium with 2,4-D concentrations in blood, as well as physiological features absent in in vitro settings (e.g. blood flow). We demonstrated that 2,4-D is consistently transported into saliva using both in vitro and in vivo models, making 2,4-D a potential candidate for human non-invasive salivary biomonitoring. Further work is needed to understand whether current sensor limits of detection are sufficient to measure occupationally relevant exposures.

Lipidomics reveals dramatic lipid compositional changes in the maturing postnatal lung.

Lung immaturity is a major cause of morbidity and mortality in premature infants. Understanding the molecular mechanisms driving normal lung development could provide insights on how to ameliorate disrupted development. While transcriptomic and proteomic analyses of normal lung development have been previously reported, characterization of changes in the lipidome is lacking. Lipids play significant roles in the lung, such as dipalmitoylphosphatidylcholine in pulmonary surfactant; however, many of the roles of specific lipid species in normal lung development, as well as in disease states, are not well defined. In this study, we used liquid chromatography-mass spectrometry (LC-MS/MS) to investigate the murine lipidome during normal postnatal lung development. Lipidomics analysis of lungs from post-natal day 7, day 14 and 6-8 week mice (adult) identified 924 unique lipids across 21 lipid subclasses, with dramatic alterations in the lipidome across developmental stages. Our data confirmed previously recognized aspects of post-natal lung development and revealed several insights, including in sphingolipid-mediated apoptosis, inflammation and energy storage/usage. Complementary proteomics, metabolomics and chemical imaging corroborated these observations. This multi-omic view provides a unique resource and deeper insight into normal pulmonary development.

Evaluation of Cuprimine® and Syprine® for decorporation of radioisotopes of cesium, cobalt, iridium and strontium.

Cuprimine® and Syprine® are therapeutics approved by the USFDA to treat copper overload in Wilson Disease (a genetic defect in copper transport) by chelation and accelerated excretion of internally-deposited copper. These oral therapeutics are based on the respective active ingredients D-penicillamine (DPA) and N,N'-bis (2-aminoethyl) -1,2-ethanediamine dihydrochloride (Trien). Cuprimine is considered the primary treatment, although physicians are increasingly turning to Syprine as a first-line therapy. Both drugs exhibit oral systemic activity and low toxicity; their biological effects and safety are established. Previous in vivo studies using a rodent animal model established the decorporation potential of Cuprimine and Syprine for (60)Co and (210)Po. Currently these studies are being expanded to evaluate the in vivo decorporation efficacy of these drugs for several additional radionuclides. In this report, results of this investigation are discussed using the radionuclides (137)Cs, (60)Co, (192)Ir and (85)Sr. Short-term 48-h pilot studies were undertaken to evaluate DPA and Trien for their in vivo decorporation potential using male Wistar-Han rats. In these studies, a radionuclide solution was administered to the animals by intravenous (IV) injection, followed by a single IV dose of either DPA or Trien. Control animals received the radionuclide alone. Results show effective decorporation of (60)Co by DPA within the time frame evaluated. DPA and Trien were also modestly effective in decorporation of (137)Cs and (85)Sr, respectively. The study did not find DPA or Trien effective for decorporation of (192)Ir. Based on these encouraging findings, further studies to evaluate the dose-response profiles and timing of the chelator administration post exposure to radionuclides are warranted.

Biomaterials for the decorporation of (85)Sr in the rat.

Although four stable isotopes of strontium occur naturally, Sr is produced by nuclear fission and is present in surface soil around the world as a result of fallout from atmospheric nuclear weapons tests. It can easily transfer to humans in the event of a nuclear/radiological emergency or through the plant-animal-human food chain causing long-term exposures. Strontium is chemically and biologically similar to calcium, and is incorporated primarily into bone following internal deposition. Alginic acid (alginate) obtained from seaweed (kelp) extract selectively binds ingested strontium in the gastrointestinal tract blocking its systemic uptake and reducing distribution to bone in rats, while other natural polysaccharides including chitosan and hyaluronic acid had little in vivo affinity for strontium. Alginate exhibits the unique ability to discriminate between strontium and calcium and has been previously shown to reduce intestinal absorption and skeletal retention of strontium without changing calcium metabolism. In our studies, the effect of commercially available alginate on intestinal absorption of strontium was examined. One problem associated with alginate treatment is its limited solubility and gel formation in water. The aqueous solubility of sodium alginate was improved in a sodium chloride/sodium bicarbonate electrolyte solution containing low molecular weight polyethylene glycol (PEG). Furthermore, oral administration of the combined alginate/electrolyte/PEG solution accelerated removal of internal strontium in rats when compared to treatment with individual sodium alginate/electrolyte or electrolyte/PEG solutions. Importantly, both alginate and PEG are nontoxic, readily available materials that can be easily administered orally in case of a national emergency when potentially large numbers of the population may require medical treatment for internal depositions. Our results suggest further studies to optimize in vivo decorporation performance of engineered alginate material via modification of its chemical and physicochemical properties are warranted.

Aminothiol receptors for decorporation of intravenously administered (60)Co in the rat.

This report provides a comparison of the oral decorporation efficacy of L-glutathione (GSH), L-cysteine (Cys), and a liposomal GSH formulation (ReadiSorb) toward systemic (60)Co to that observed following intravenous administration of GSH and Cys in F344 rats. Aminoacid L-histidine (His) containing no thiol functionality was tested intravenously to compare in vivo efficacy of the aminothiol (GSH, Cys) chelators with that of the aminoimidazole (His) chelator. In these studies, (60)Co was administered to animals by intravenous injection, followed by intravenous or oral gavage doses of a chelator repeated at 24-h intervals for a total of 5 doses. The results suggest that GSH and Cys are potent decorporation agents for (60)Co in the rat model, although the efficacy of treatment depends largely on the systemic availability of the chelator. The intravenous route of administration of GSH or Cys was most effective in reducing tissue (60)Co levels and in increasing excretion of radioactivity compared to control animals. Liposomal encapsulation was found to markedly enhance the oral bioavailability of GSH compared to non-formulated GSH. The oral administration of liposomal GSH reduced (60)Co levels in nearly all tissues by 12-43% compared to that observed for non-formulated GSH. Efficacy of oral Cys was only slightly reduced in comparison with intravenous Cys. Further studies to optimize the dosing regimen in order to maximize decorporation efficiency are warranted.

Evaluation of Cuprimine and Syprine for decorporation of (60)Co and (210)Po.

The acknowledged risk of deliberate release of radionuclides into local environments by terrorist activities has prompted a drive to improve novel materials and methods for removing internally deposited radionuclides. These decorporation treatments will also benefit workers in the nuclear industry, should an exposure occur. Cuprimine and Syprine are oral therapeutics based on the active ingredients D-penicillamine and N,N'-bis-(2-aminoethyl)-1,2-ethanediamine dihydrochloride, respectively. These therapeutic drugs have been used for several decades to treat Wilson's disease, a genetic defect leading to copper overload, by chelation and accelerated excretion of internally deposited copper. Studies were undertaken to evaluate these FDA-approved drugs for the in vivo decorporation of radioactive cobalt (Co) and polonium (Po) using male Wistar-Han rats. In these studies, Co or Po was administered to animals by IV injection, followed by oral gavage doses of either Cuprimine or Syprine. Control animals received the radionuclide alone. For Co studies, animals received a single dose of Cuprimine or Syprine, while for Po studies animals were repeatedly dosed at 24-h intervals for a total of 5 doses. Results show that Syprine significantly increased urinary elimination and skeletal concentrations of Co compared to controls. While Cuprimine had little effect on total excretion of Co, the skeletal, kidney, liver, muscle, and stomach tissues had significantly lower radioactivity compared to control animals. The low overall excretion of Po made it difficult to reliably measure urinary or fecal radioactivity and draw a definitive conclusion on the effect of Cuprimine or Syprine treatment on excretion. However, Cuprimine treatment was effective at reducing spleen levels of Po compared to controls. Similarly, Syprine treatment produced statistically significant reductions of Po in the spleen and skeletal tissues compared to control animals. Based on these promising findings, further studies to evaluate the dose-response pharmacokinetic profiles for decorporation are warranted.

Investigation of chitosan for decorporation of 60Co in the rat.

With the increased threat of terrorist release of radioactive materials, there is a need for non-toxic decorporation agents to treat internal contamination with radionuclides. In this study, low molecular weight chitosan was evaluated for decorporation of radioactive cobalt (60Co). The affinity of chitosan for Co(II) was tested in vitro using spectrophotometric and potentiometric titration techniques. For in vivo studies, the effect of chitosan on ingested 60Co was evaluated using F344 rats administered a single dose followed by oral chitosan. Chitosan was also evaluated for systemic decorporation of 60Co following intravenous injection with repeated chitosan administration over 5 d. Control animals received 60Co without chelation treatment. Excreta and tissues were collected for analysis using gamma-counting techniques. Results from in vitro experiments confirmed the binding of Co(II) to chitosan, with the postulated formation of a mixed cobalt-chitosan-hydroxide complex species; a stability constant was calculated for this complex. For in vivo studies, oral administration of chitosan significantly reduced systemic absorption of orally administered 60Co as evidenced by an increase in fecal elimination and decrease in urinary elimination. However, oral administration of chitosan lactate slightly decreased fecal excretion of 60Co. Further, oral administration of chitosan significantly reduced 60Co levels in kidney, liver, and skeleton compared to control animals receiving 60Co alone. By the i.v. route, chitosan slightly reduced levels of 60Co in tissues compared to controls, although statistically significant reductions were only observed for blood and kidney. Overall, this commercially available chitosan oligosaccharide exhibited promising potential; further studies are warranted to evaluate the optimal dosing regimen and chemical modifications to increase effectiveness.