Search

Elevated levels of endogenous cardiotonic steroids (CTS), such as telocinobufagin (TCB) and marinobufagin (MBG), are observed in patients with chronic kidney disease (CKD). The characteristic six-membered δ-lactone ring structure of CTS is critical for binding to the Na+ /K+-ATPase and subsequent signaling. Paraoxonases (PONs) belong to a family of hydrolytic enzymes that can regulate levels of δ-lactone ring structures, like those found in CTS, rendering them inactive. Clinical studies have reported decreased lactonase activity of PONs in CKD patients and its association with cardiovascular morbidity and mortality. PON’s activity is diminished in CKD, yet it is unclear whether it is mechanistically linked to increased CTS levels. This research aimed to enhance the knowledge regarding CTS by exploring their regulation in the body and association with PON enzymes, whose exact physiological substrate(s) is/are still unknown. To bridge these knowledge gaps, this research describes novel methods for extraction, detection, and quantification of CTS using highly specific, sensitive, and accurate analytical techniques, such as ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS) as well as HPLC-photodiode array (PDA).In the first project, a protocol was developed for the extraction, detection and quantification of TCB and MBG. Solid-phase extraction (SPE) with hydrophilic-lipophilic balanced (HLB) cartridges was used for the extraction of CTS from an aqueous solution. The methods were optimized to extract and quantify TCB and MBG from the urine of rats with different genotypes; wild type (WT) and PON3 knockout (KO) subjected to different diets (normal chow and high salt) for 8 weeks. The high salt-treated rats serve as a model for studying CKD, while the wild type rats on normal chow act as controls. The results showed that the urine of SS-PON3 KO rats contained higher CTS levels than urine from SS-WT rats for both control and high-salt samples, suggesting its role in CTS regulation.The second project aimed to investigate the role of the enzymes PON1 and PON3 in the degradation of the TCB. TCB was administered at a dose of 100 µg/kg/day subcutaneously (s.q.) for 4 weeks to male salt-sensitive rats with normal PON1 and PON3 levels (WT), as well as to rats lacking either PON1 or PON3. After 4 weeks, the levels of TCB in urine were measured using gamabufutalin (GBT) as an internal standard and utilizing SPE and UHPLC-MS. The results showed that rats lacking PON1 or PON3 had higher TCB levels in their urine compared to the WT rats. These findings suggest that PON1 and PON3 may play a role in the degradation of TCB in vivo. Additionally, a potential metabolite of TCB, called 3-epitelocinobufagin, was putatively identified based on various data, including its retention time, charge, exact mass, and fragmentation data, which matched information in the literature.In the third project, experiments were conducted to investigate TCB hydrolysis and determine the degradation products in vitro and in vivo. In the in vitro experiments, TCB was incubated individually with recombinant PON1 and PON3 enzymes in a buffer. The conversion of TCB from the lactone form to the hydroxy acid form was monitored. Sample purification using SPE was carried out, and elution fractions from the TCB-PONs mixture and control samples were analyzed using HPLC-PDA and UHPLC-MS. Furthermore, experiments were conducted using rat urine samples to determine the degradation products of TCB in vivo. These urine samples were extracted and analyzed using LC-MS/MS. The potential degradation products were analyzed and compared with those obtained from the in vitro studies and alkaline hydrolysis products. Additionally, some preliminary experiments using UPLC interfaced with ion mobility spectrometry MS (UPLC-IMS-MS) were performed to monitor the TCB and MBG and their degradation products.The research investigated whether TCB is a potential endogenous substrate for PON1 and PON3 using LC-PDA and LC-MS. Understanding this physiological correlation could have implications for future clinical investigations and potential treatments for CKD.

Healthcare-associated infections (HCAIs) affect 1.7 million hospitalized patients each year, resulting in over 98,000 deaths and anywhere from $28.4 to $45 billion in treatment costs. Furthermore, it has been established that more than 80% of these infections are caused by biofilms While HCAIs can be bacterial, viral, fungal, or parasitic, there are between 12 and 17 species cause 80 to 87% of all HCAIs. Among the most commonly isolated microorganisms are Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus). Thus, biofilms are of importance not only due to their prevalence in HCAIs but also due to the increased antibiotic resistance that can be observed in comparison to their planktonic counterparts. Due to the importance of understanding biofilm interaction not only between bacterial species, but also interactions within the host, this research was focused on biofilm interactions using an in vitro co-culture with macrophage-like cells. Due to their prevalence in HCAIs, biofilms of S. aureus, S. epidermidis, and P. aeruginosa were explored. To combat antibiotic resistant microorganisms, research into alternative therapeutic treatments is of importance. One such alternative is cationic antimicrobial peptides. Currently, hCAP-18/LL-37 is the only known human cathelicidin with a broad spectrum anti-microbial response. Its antimicrobial and immunomodulatory properties make it of particular interest in antibiotic resistance research. This dissertation focuses on the development of a method to rapidly detect and quantify LL-37 in an in vitro co-culture setting.

Due to the increasing need to accurately estimate drug use in a community, it may be important to analyze wastewater. When the wastewater samples being tested are not able to be analyzed immediately after collection, understanding the storage effects on a drug panel’s stability in wastewater will lead to a better estimation of drug use. Here, a stability study included storing samples at 22ºC, 4ºC, and -20ºC for 0, 24, and 72 hours. Time and temperature stability effects were investigated on a drug panel consisting of 6-monoacetylmorphine (6- MAM), alprazolam, amphetamine, benzoylecgonine, buprenorphine, caffeine, clonazepam, cocaine, codeine, fentanyl, hydrocodone, 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine, morphine, norfentanyl, norhydrocodone, noroxycodone, oxycodone, and tetrahydrocannabinol (THC) in wastewater during storage. Chemical substances were extracted from the wastewater using solid-phase extraction followed by analysis using Liquid Chromatography with tandem Mass spectrometry (LC-MS/MS). Only eight of these drugs were detected from the fresh wastewater samples. The findings after storage indicated that only three of the eight drugs were stable after 24 hours, and only two were stable after 72 hours. In terms of temperature, the most significant decreases in mean concentrations were observed at 22ºC. Both 4ºC and -20ºC demonstrated similar stability, though not for every drug.

Microcystins (MCs) are secondary metabolites generated by cyanobacteria, which can be present in drinking water sources during harmful algal blooms (HABs). MCs are potent liver toxins that inhibit the function of protein phosphatases 1 and 2A (PP1 and PP2A) by binding to the enzymes’ active sites. Large MC doses lead to acute liver failure, but prolonged exposure to low levels of MCs may be more prevalent and pernicious. The effects of such exposure in humans are not well understood and are generally extrapolated from animal models. Further complicating their study, over 275 MC congeners have been discovered, though many remain unidentified. This dissertation advances MC research by describing novel extraction and data analysis methods coupled with liquid chromatography-mass spectrometry (LC-MS) for quantitative and qualitative analyses of MCs. A method was developed to extract and quantify MCs from mouse liver with limits of quantification (LOQs) lower than previously reported. MCs were extracted from 40-mg liver samples using 85:15 (v:v) CH3CN:H2O containing 200 mM ZnSO4 and 1% formic acid. Solid-phase extraction with a C18 cartridge was used for sample cleanup. MCs were detected and quantified using LC-orbitrap-MS with simultaneous MS/MS detection of the 135.08 m/z fragment from the conserved Adda amino acid for structural confirmation. The method was used to extract six MCs (MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, and MC-LW) and the MC-LR cysteine adduct (MC-LR-Cys), which can be created from MC-LR in vivo by the glutathione detoxification pathway, from spiked liver tissue. Matrix-matched internal standard calibration curves were constructed for each MC (R2 ≥ 0.993), with LOQs between 0.25 ng per g of liver tissue (ng/g) and 0.75 ng/g for MC-LR, MC-RR, MC-YR, MC-LA, and MC-LR-Cys, and 2.5 ng/g for MC-LF and MC-LW. The protocol was applied to extract and quantify MC-LR and MC-LR-Cys from the liver of mice that had been gavaged with 50 μg or 100 μg of MC-LR per kg bodyweight and were euthanized 2 h, 4 h, or 48 h after final gavage. C57Bl/6J (wild type, control) and Leprdb/J (sample) mice were used as a model to study non-alcoholic fatty liver disease (NAFLD). The Leprdb/J mice were relatively inefficient in metabolizing MC-LR into MC-LR-Cys, which is an important defense mechanism against MC-LR exposure. Trends were also observed as a function of MC-LR gavage amount and time between final MC-LR gavage and euthanasia/organ harvest. In the second project, putative identification of novel MCs was achieved in water originating from HAB of 2020 using ultra-high-performance (UHP) LC-high resolution (HR) MS and a new bottom-up sequencing strategy. Maumee River water samples were collected during a HAB and analyzed with simultaneous HRMS and MS/MS. Unidentified ions with characteristic MC fragments (135 and 213 m/z) were recognized as possible novel MC congeners. An innovative workflow, involving separation and detection of MC ions and their fragments by ultrahigh pressure (UPLC)-orbitrap-MS, MS/MS and MS/MS/MS, was developed for putative identification of those ions. Two codes were written using the Python programming language. The first predicts molecular composition from exact mass measurements by generating potential structures using 59 amino acids that have been found in known MCs. The second assigns fragment ions after multistage MS (MS/MS and MSn) for structural confirmation. Peptide fragmentation is well-characterized, but existing software for rapid fragment ion identification is not suitable for MCs due to their cyclic structure and unique amino acids. The developed workflow was used for putative identification of two unknown congeners found in Lake Erie: MC-HarR and MC-E(OMe)R. The method was also used to putatively identify eight MCs for which standards are not available, and was able to distinguish between isomeric MCs such as [Asp3] MC-YR and [Dha7] MC-YR.

Dissolved organic carbon (DOC) is the largest exchangeable pool of organic carbon in the ocean, and is similar in size to atmospheric carbon. DOC is formed during surface ocean primary production by marine phytoplankton and can survive on average >6000 14C years in the deep ocean. This residence time greatly exceeds that of dissolved inorganic carbon (DIC), and indicates that a majority of DOC is refractory, or unreactive. Even though previous estimates of refractory DOC (RDOC) abundance is ~95% of total DOC, the mechanisms that facilitate its formation, cycling, and sinks are still poorly understood. A better understanding of these mechanisms is important because the ocean’s role in the global carbon cycle, and global physical climate, may link closely to the strength of the biological pump and DOC storage. This dissertation research characterizes the stable and radiocarbon isotopic compositions (13C and ∆14C) of solid-phase extracted DOC (SPE-DOC), which is representative of RDOC. First, the mass and isotopic composition of extraneous carbon (Cex) in the SPE method is analyzed. This study identifies that one SPE resin commonly used to extract DOC from seawater (Bond Elut PPL) elutes SPE-DOC with isotopic heterogeneity. Incomplete elution may exclude terrestrial-like organic matter that elutes later. An updated protocol including an extended elution is developed to ensure no fractionation of SPE-DOC occurs. This updated protocol is used to characterize SPE-DOC 13C and ∆14C values at the highest latitudinal resolution to date, using samples from three GO-SHIP Repeat Hydrography Cruises spanning the central and eastern Pacific Ocean and a portion of the western Indian Ocean. SPE-DOC 13C and ∆14C values are significantly lower than those for total DOC. Low 13C values relative to total DOC indicate preferential respiration of large biomolecules with high 13C values. An additional product of this heterotrophic respiration is carboxy-rich alicyclic matter, which is known to be extremely recalcitrant (C-rich and low in oxygen and hydrogen). These low SPE-DOC 13C and ∆14C values point to the microbial carbon pump (MCP) as a major driver of the marine carbon cycle through the removal of biomolecules with high 13C, and addition of refractory products that resist degradation. This increases DOC residence time and drives low ∆14C values. The large meridional span in this ∆14C dataset also allows for the first regional estimates of RDOC abundance. These estimates match the simple yet powerful “two-pool” model of DOC cycling proposed in 1987, in which the deep ocean is a well-mixed pool of old RDOC, and the surface is a mixture of RDOC from depth and labile DOC from recent photosynthesis. Although the importance of the MCP, and the two-pool model are not new ideas, sampling has been spatially and temporally sparse. This dissertation research provides a high-resolution isotopic perspective from under-sampled regions in the open ocean that supports these concepts.

This research study successfully completed three objectives: 1) validate liquid-liquid, supported-liquid, and solid-phase extractions for the quantitation of three barbiturates (amobarbital, butalbital, and phenobarbital) in blood and urine using liquid chromatography-tandem mass spectrometry; 2) to compare the efficiency and effectiveness among methods in accomplishing extraction of barbiturates under the laboratory setting at Boston University School of Medicine; and 3) to report all the analytical data to RTI International for interlaboratory comparison. For the validation study, a six-point linear calibration model (20-2000 ng/mL) with inversely weighted concentration (1/x) was reproducible in all three sample preparation methods for both blood and urine with r2 greater than or equal to 0.994. Bias and precision evaluated from three controls throughout the range of the curve were within ±20% and ±20%CV, respectively. Neither carryover nor interference was observed. Detection limits were evaluated down to 5 ng/mL depending on the extraction procedure. Samples were able to be diluted up to 50 times prior to instrumental analysis. Samples were stable on autosampler at room temperature up to 72 hours after their initial analysis. Recovery of barbiturates from blood and urine all ranged from 45% to 86%. The effect of ionization suppression or enhancement was found to have minimal impact on the validation. For choosing the most suitable method quantifying barbiturates, efficiency and effectiveness were studied. Efficiency evaluates the time and ease of sample preparation required to prepare a sample for analysis. Supported-liquid extraction was found to be the most efficient method for extracting barbiturates as it required the least amount of time to perform and could be easily automated with minimal training. Effectiveness is an assessment of one’s ability to selectively recover target analyte at a reasonably low concentration. By considering a method’s recovery, extract cleanliness, detection limits, and reproducibility, liquid-liquid extraction was the best at quantifying barbiturates in blood and supported-liquid extraction was the most suitable method for extracting barbiturates from urine. For interlaboratory comparison, all the data collected has been reported to RTI International. These findings can be used for examining the overall reliability and reproducibility of the validated methods. Results obtained can also be used to explore the possibility for streamlining sample preparation in the forensic laboratory, and hence reducing the case backlog.

An in-depth analysis of orange wines was conducted to assess factors affecting the phenolic composition. Orange wines were produced from Pinot Gris juice and table grape skins from September 2017 to March 2018. Factors included in this experiment were different ratios of skin contact to juice, additions of sulfur dioxide and aging. Basic wine data was analysed along with methylcellulose precipitable tannin, fractionation by solid-phase extraction and mean degree of polymerisation via acid catalysis in the presence of excess phloroglucinol. Increasing rates of skin addition resulted in a higher tannin composition in wine, along with an increase in pH and a decrease in TA and alcohol percentage. The use of SO2 limited tannin development, mean degree of polymerisation and affected the proportions observed in fractions two and three, but protected total phenolics from oxidation over time, especially those present in fraction one. The use of SO2 also resulted in orange wines with a lesser degree of browning. Orange wine phenolics develop in a similar way to red wine phenolics and are affected by similar winemaking conditions, however further study is required to confirm results.

Organic micropollutants (MPs) occur ubiquitously in the aquatic environment at trace concentrations, which presents great challenges for environmental monitoring. Solid-phase extraction (SPE) is a widely-used sample preparation procedure that concentrates MPs in environmentally derived water samples. SPE relies on columns packed with an adsorbent material that captures MPs from water samples. Loaded SPE adsorbents can then be eluted with organic solvents to release the captured MPs. The conventional adsorbent used for SPE is a hydrophilic-lipophilic balanced material available from a variety of commercial vendors, often referred to as HLB. These HLB adsorbents feature stability at extreme pHs and high retention of polar and apolar organic MPs. The primary disadvantage of HLB adsorbent materials is price, which often makes sample preparation the most expensive step in an environmental monitoring workflow. In this research, novel porous β-cyclodextrin polymers (P-CDP) were evaluated as an alternative SPE adsorbent. Cyclodextrins are sustainably produced macrocycles of glucose, featuring a hydrophobic interior cavity that forms host-guest complexes with thousands of organic molecules. P-CDP is a polymer of cyclodextrin and is synthesized in one-step, making it relatively inexpensive to produce. The objective of this research was to develop an SPE procedure using P-CDP and compare its performance to a conventional SPE procedure using HLB. A set of 189 MPs were included in the study and the P-CDP method was optimized to maximize efficiency in MP capture and MP release. The optimized P-CDP method exhibits moderate absolute recovery (≥60%) for 135 (75%) MPs and high absolute recovery (≥80%) for 121 (67%) MPs. The conventional HLB method exhibits moderate absolute recovery for 130 (72%) MPs and high absolute recovery for 109 (61%) MPs. The results of this research demonstrate that P-CDP could be used as a cost-effective SPE adsorbent.

Gangliosides are membrane lipids with a complex sugar head containing sialic acid (polar) and a ceramide moiety made up of a sphingosine and a fatty acid (non-polar). Typically, gangliosides are part of the ganglion, a group of nerve cell bodies where they act as neuroprotective agents, support, and maintenance of the mature neuronal cells. Glaucoma is an age-related neurodegenerative disorder of the eye that leads to blindness. Retinal ganglion cells (RGCs) in retinal tissue of the eye, degeneration is seen followed by the optic nerve head damage. From immunohistochemistry studies with cholera toxin-B, gangliosides which are part of the RGCs were lost during the RGCs degeneration. Non-neuronal cells, astrocytes, which do not express gangliosides in normal retinal tissue surprisingly express/uptake gangliosides in correlation to the RGCs degeneration. Astrocyte expression/uptake of gangliosides can play protective, adverse or combined effects at different times. An identification and quantitation of individual gangliosides that are expressed/uptake provide an insight into understanding the pathophysiology of glaucoma.A specific, sensitive and quantitative liquid-chromatography-mass spectrometry (LC-MS/MS) analytical techniques were developed to identify heterogeneous molecular species of gangliosides. Of all of them, new LC-MS/MS method using a phenyl-hexyl reverse-phase chromatography was successful in resolving eight major gangliosides and ten minor gangliosides. An optimized ganglioside isolation technique was developed which is a combination of liquid-liquid extraction for lipid phase separation followed by a solid-phase extraction for desalting and removal of debris. This new LC-MS/MS method was validated for linearity, repeatability, matrix effect and recovery studies. Age studies (young vs old mice) were performed for the first time to correlate the changes of gangliosides in retinal tissue level in comparison to the superior colliculus of the mid-brain using the new LC-MS/MS method in normal control mice (C57BL/6J) versus glaucoma model mice (DBA/2J). LC-MS/MS results show an increase of major gangliosides in the glaucoma retinal tissue from old to young age mice groups whereas little change in the superior colliculus. Nevertheless, these gangliosides changes were statistically insignificant as p-value is > 0.05. The optic nerve studies using electron microscopy was undertaken to establish the glaucoma disease progression. Electron microscopy study results show no change in the optic nerve count which represents no glaucoma in old age animal samples. Thus, a non-correlation was seen with the ganglioside changes compared to optic nerve counts. A greater animal sample with much older age provides a better understanding of gangliosides role in glaucoma.

The first two projects describe new applications of flow injection capillary electrophoresis (CE). The first study focuses on the determination of lactate or oxalate using injected lactate oxidase (LO) and peroxidase with UV detection. Two reactions, catalyzed by (LO) and peroxidase, are initiated by a single injection of the enzymes and the substrate 2,2’-azino-bis(3-ethylene-thiazoline-6-sulfonic acid) (ABTS) into the capillary previously filled with the sample (lactate or lactate-oxalate mixture) and the running buffer containing NADH. The oxidized ABTS product upon reaction with NADH is converted to NAD+, which is detected at 266 nm with a sample throughput of 7 min including wash steps. Linearity for lactate is established from 0.0025 – 1mM and serum samples are analyzed with an average recovery of 101%. This method can also be applied to the determination of oxalate as an inhibitor of LO. The second topic describes the determination of the concentration of cardiolipin using CE with on-line N-nonylacridine orange (NAO) dye interaction and spectrophotometric detection at 497 nm. Other phospholipids do not interfere and a detection limit of 0.05 ìM was found. In a blind study, actual mitochondrial cell membrane samples in the µL range, taken from cells before or after UV light exposure, are analyzed using the CE method. The last two topics describe preconcentration of pharmaceuticals and phospholipids with nano-electrospray ionization mass spectrometry (nano-ESI MS). An analytical method is developed for the quantitative determination of dicyclomine in serum with cyclopentolate as the internal standard by off-line nano-ESI MS with reversed phase mini-solid phase extraction (mini-SPE). Different length (0.5cm, 1cm) reversed phase (C4 and C18) mini-SPE cartridges are prepared by packing gel-loader pipette tips for optimization of the preconcentration effect. A factor of 100 is possible using the 1 cm C18 or C4 cartridge. Spiked serum samples are quantitatively determined using the C18 cartridge. Preconcentration of phospholipids is first studied for nano-ESI MS by weak anion exchange SPE. With aminopropyl SPE, detection limits are lowered 2-2000 times for different phospholipids as compared to no SPE. With aminopropyl mini-SPE and mixed mode C18/aminopropyl (1:1) mini-SPE, the detection limits for eight different phospholipids are lowered another factor of 5 better (about 0.05 to 5 pmol/ìL) than those for weak anion exchange SPE. Linear calibration curves are able to be established.