Determination of Caffeine as a Marker for Septic Tank Contamination of Wilgreen Lake

Wilgreen Lake in Richmond, Kentucky, has been listed by both the state and the Environmental Protection Agency as an “impaired” lake due to excess nutrients, which may be in part contributed by domestic septic systems. Caffeine can be used as an anthropogenic marker to estimate the contribution of septic tank effluent to the lake. We have modified existing analytical methods to produce a viable method for the determination of caffeine in environmental water samples and applied the method to water samples collected from Wilgreen Lake. The modified method allows determination of caffeine in a concentration range of 75 to 10,000 ng/L in the water samples. Waters Oasis® HLB solid phase extraction cartridges are used to clean up and concentrate the water samples, which are then analyzed by liquid chromatography-tandem mass spectrometry. A Waters XTerra MS C18 column (3.5 µm film thickness, 2.1 x 100 mm column dimensions) is utilized in the separation. Carbon-13 labeled caffeine is added to all samples prior to extraction and serves as an internal standard. The parameters of the optimized method and results of the application of this method to water samples collected from Wilgreen Lake will be presented

Nano-Sampling and Reporter Tools to Study Metabolic Regulation in Zebrafish

In the past years, evidence has emerged that hallmarks of human metabolic disorders can be recapitulated in zebrafish using genetic, pharmacological or dietary interventions. An advantage of modeling metabolic diseases in zebrafish compared to other “lower organisms” is the presence of a vertebrate body plan providing the possibility to study the tissue-intrinsic processes preceding the loss of metabolic homeostasis. While the small size of zebrafish is advantageous in many aspects, it also has shortcomings such as the difficulty to obtain sufficient amounts for biochemical analyses in response to metabolic challenges. A workshop at the European Zebrafish Principal Investigator meeting in Trento, Italy, was dedicated to discuss the advantages and disadvantages of zebrafish to study metabolic disorders. This perspective article by the participants highlights strategies to achieve improved tissue-resolution for read-outs using “nano-sampling” approaches for metabolomics as well as live imaging of zebrafish expressing fluorescent reporter tools that inform on cellular or subcellular metabolic processes. We provide several examples, including the use of reporter tools to study the heterogeneity of pancreatic beta-cells within their tissue environment. While limitations exist, we believe that with the advent of new technologies and more labs developing methods that can be applied to minimal amounts of tissue or single cells, zebrafish will further increase their utility to study energy metabolism

Reversed-Phase Liquid Chromatography Tandem Mass Spectrometry And Elisa Determination Of Caffeine As A Marker For Sewage Contamination Of Wilgreen Lake

The primary objective of this study was to determine the concentration of caffeine in Wilgreen Lake and its tributaries in order to establish the contribution of domestic wastewater effluents to the lake\u27s impairment. This was achieved by collecting samples from different locations and depth profiles from the lake and its tributaries, and analyzing them using liquid chromatography-tandem mass spectrometry coupled to an Electrospray Ionization (ESI) Interface. An isotope dilution method using 13C3-labeled caffeine as the internal standard was adapted from known methods and employed to determine and correct the recovery of native caffeine in water samples. The presence in the lake of seven other human pharmaceutical compounds (carbamazepine, sulfamethoxazole, trimethoprim, cotinine, paraxanthine, acetaminophen and fluoxetine) belonging to various therapeutic categories in the lake was also investigated using the LC-MS/MS method of analysis. Since caffeine exists in surface waters at low concentrations, solid phase extraction (SPE) was used to pre-concentrate the samples to achieve concentrations detectable by the instrumentation. SPE also served as a sample clean-up step to minimize matrix effects. A multi-point standard calibration curve was generated using peak area ratios of the most abundant product ions from standards containing caffeine in the range of 6.25 - 2500 ng/ml and a stable 13C labeled isotope of caffeine as the internal standard. 13C3-atrazine was used as an injection standard. Using the developed method, caffeine was detected in the range of 66 - 417 ng/L in water samples collected from various sites in Wilgreen Lake. The recoveries of 13C-labeled caffeine were from 40 to 126%, while caffeine\u27s recovery ranged from 105-116%. A secondary objective was to analyze caffeine using a recently manufactured caffeine Enzyme-Linked Immunosorbent Assay (ELISA) kit, and compare its effectiveness in the determination of caffeine in surface water samples to LC-MS/MS. Results from ELISA showed high concentrations of caffeine in the lake water samples ranging from 537 - 1080 ng/L but its performance was inferior to LC-MS/MS analysis; the assay had a lower reliability, sensitivity and precision in the analyses. The ELISA method has the advantages of quick assay requiring only a small amount of sample, relatively low expense, and simple technique. LC-MS/MS offers advantages in terms of simultaneous determinations of several analytes present in water samples, high sensitivity, and high quantitative reliability

Single-Cell Metabolomics of the Frog (Xenopus laevis ) Embryo by Capillary Electrophoresis Mass Spectrometry

Metabolic characterization of single embryonic cells offers a direct snapshot of molecular events, raising the potential to understand biochemical processes that underlie healthy or pathological status during embryogenesis. To enable single-cell metabolic characterization for cell and developmental biology, several technological developments are needed. In this dissertation, we lay down the foundation for highly selective and sensitive analytical advances based on mass spectrometry (MS), that are compatible with the limited material afforded by single cells, and hence enable unbiased quantitation of diverse types of small biomolecules in the cells. The work presented herein shows the outcome of my efforts to develop strategies and methodologies based on capillary electrophoresis-MS to investigate chemical heterogeneity between single embryonic cells during early stages of embryonic development. Additionally, the work features my contributions to the fundamentals of cell biology, and how metabolite activity is involved during early embryogenesis. Chapter 1 discusses the basic biological and technological motivations for this dissertation. Chapter 2 introduces current mass spectrometry-based techniques, including chemical separation by capillary electrophoresis, for the metabolic characterization of single cells. Chapter 3 presents our development of single-cell capillary electrophoresis mass spectrometry for embryos. This chapter also discusses our discovery of metabolite-induced cell fate changes in the embryo of the vertebrate frog Xenopus laevis. Chapter 4 details a multi-solvent extraction strategy to identify metabolites deeper into the single-cell metabolome. Additionally, we utilize this approach to uncover previously unknown metabolic cell heterogeneity between cells that occupy the left vs. right sides of the early developing X. laevis embryo. Chapter 5 is dedicated to the development of a microprobe strategy to measure cell metabolism in situ in the live embryo. We demonstrate, for the first time, single-cell metabolomics in both the spatial and temporal dimension of embryonic development. Chapter 6 injects the concept of spatially resolved metabolomics of multiple cells in the same freely developing embryo, focusing on the left-right developmental axis. Chapter 7 reflects on the current state of metabolic single-cell mass spectrometry, spanning from technical challenges to new investigative potential in basic and translational research

Altering metabolite distribution at Xenopus cleavage stages affects left–right gene expression asymmetries

The left–right (L–R) axis of most bilateral animals is established during gastrulation when a transient ciliated structure creates a directional flow of signaling molecules that establish asymmetric gene expression in the lateral plate mesoderm. However, in some animals, an earlier differential distribution of molecules and cell division patterns initiate or at least influence L–R patterning. Using single-cell high-resolution mass spectrometry, we previously reported a limited number of small molecule (metabolite) concentration differences between left and right dorsal-animal blastomeres of the eight-cell Xenopus embryo. Herein, we examined whether altering the distribution of some of these molecules influenced early events in L–R patterning. Using lineage tracing, we found that injecting right-enriched metabolites into the left cell caused its descendant cells to disperse in patterns that varied from those in control gastrulae; this did not occur when left-enriched metabolites were injected into the right cell. At later stages, injecting left-enriched metabolites into the right cell perturbed the expression of genes known to: (a) be required for the formation of the gastrocoel roof plate (foxj1); (b) lead to the asymmetric expression of Nodal (dand5/coco); or (c) result from asymmetrical nodal expression (pitx2). Despite these perturbations in gene expression, we did not observe heterotaxy in heart or gut looping at tadpole stages. These studies indicate that altering metabolite distribution at cleavage stages at the concentrations tested in this study impacts the earliest steps of L–R gene expression that then can be compensated for during organogenesis

Single-cell mass spectrometry reveals small molecules that affect cell fates in the 16-cell embryo.

Spatial and temporal changes in molecular expression are essential to embryonic development, and their characterization is critical to understand mechanisms by which cells acquire different phenotypes. Although technological advances have made it possible to quantify expression of large molecules during embryogenesis, little information is available on metabolites, the ultimate indicator of physiological activity of the cell. Here, we demonstrate that single-cell capillary electrophoresis-electrospray ionization mass spectrometry is able to test whether differential expression of the genome translates to the domain of metabolites between single embryonic cells. Dissection of three different cell types with distinct tissue fates from 16-cell embryos of the South African clawed frog (Xenopus laevis) and microextraction of their metabolomes enabled the identification of 40 metabolites that anchored interconnected central metabolic networks. Relative quantitation revealed that several metabolites were differentially active between the cell types in the wild-type, unperturbed embryos. Altering postfertilization cytoplasmic movements that perturb dorsal development confirmed that these three cells have characteristic small-molecular activity already at cleavage stages as a result of cell type and not differences in pigmentation, yolk content, cell size, or position in the embryo. Changing the metabolite concentration caused changes in cell movements at gastrulation that also altered the tissue fates of these cells, demonstrating that the metabolome affects cell phenotypes in the embryo