Pharmacokinetic modeling of cortisol binding to dietary fiber in the gastrointestinal tract

Abstract only availableCortisol is a glucocorticoid hormone produced in the adrenal cortex during stressful situations. The purpose of this project was to determine whether cortisol binds to dietary fiber and to design a pharmacokinetic model to predict whether or not fiber has a significant binding capacity in the human gastrointestinal tract. Studies have shown that estrogen binds to dietary fiber. Coumesterol, a cholesterol derived steroid structurally similar to estrogen, is also thought to bind to dietary fiber. The fluorescence of coumestrol bound to oat, wheat and psyllium fiber was analyzed in order to determine the binding capacities of each. This indicates that steroids have different binding capacities important in the pharmacokinetic model. This model would provide useful information capable of predicting physiological changes due to changes in dietary habits as well as medicines such as antibiotics that may alter steroid secretion. If steroids do have a recycling route and dietary fiber has a significant binding capacity in the human body then an increase in dietary fiber may result in a decrease in cortisol.NSF-REU Program in Biosystems Modeling and Analysi

Extracellular Protons Regulate the Extracellular Cation Selectivity of the Sodium Pump

The effects of 0.3–10 nM extracellular protons (pH 9.5–8.0) on ouabain-sensitive rubidium influx were determined in 4,4′-diisocyanostilbene-2, 2′-disulfonate (DIDS)-treated human and rat erythrocytes. This treatment clamps the intracellular H. We found that rubidium binds much better to the protonated pump than the unprotonated pump; 13-fold better in rat and 34-fold better in human erythrocytes. This clearly shows that protons are not competing with rubidium in this proton concentration range. Bretylium and tetrapropylammonium also bind much better to the protonated pump than the unprotonated pump in human erythrocytes and in this sense they are potassium-like ions. In contrast, guanidinium and sodium bind about equally well to protonated and unprotonated pump in human red cells. In rat red cells, protons actually make sodium bind less well (about sevenfold). Thus, protons have substantially different effects on the binding of rubidium and sodium. The effect of protons on ouabain binding in rat red cells was intermediate between the effects of protons on rubidium binding and on sodium binding. Remarkably, all four cationic inhibitors (bretylium, guanidinium, sodium, and tetrapropylammonium) had similar apparent inhibitory constants for the unprotonated pump (∼5–10 mM). The Kd for proton binding to the human pump, with the empty transport site facing extracellularly is 13 nM, whereas the extracellular transport site loaded with sodium is 9.5 nM, and with rubidium is 0.38 nM. In rat red cells there is also a substantial difference in the Kd for proton binding to the sodium-loaded pump (14.5 nM) and the rubidium-loaded pump (0.158 nM). These data suggest that important rearrangements occur at the extracellular pump surface as the pump moves between conformations in which the outward facing transport site has sodium bound, is empty, or has rubidium bound and that guanidinium is sodium-like and bretylium and tetrapropylammonium are rubidium-like

Encapsulation of FITC to monitor extracellular pH: a step towards the development of red blood cells as circulating blood analyte biosensors

A need exists for a long-term, minimally-invasive system to monitor blood analytes. For certain analytes, such as glucose in the case of diabetics, a continuous system would help reduce complications. Current methods suffer significant drawbacks, such as low patient compliance for the finger stick test or short lifetime (i.e., 3–7 days) and required calibrations for continuous glucose monitors. Red blood cells (RBCs) are potential biocompatible carriers of sensing assays for long-term monitoring. We demonstrate that RBCs can be loaded with an analyte-sensitive fluorescent dye. In the current study, FITC, a pH-sensitive fluorescent dye, is encapsulated within resealed red cell ghosts. Intracellular FITC reports on extracellular pH: fluorescence intensity increases as extracellular pH increases because the RBC rapidly equilibrates to the pH of the external environment through the chloride-bicarbonate exchanger. The resealed ghost sensors exhibit an excellent ability to reversibly track pH over the physiological pH range with a resolution down to 0.014 pH unit. Dye loading efficiency varies from 30% to 80%. Although complete loading is ideal, it is not necessary, as the fluorescence signal is an integration of all resealed ghosts within the excitation volume. The resealed ghosts could serve as a long-term (>1 to 2 months), continuous, circulating biosensor for the management of diseases, such as diabetes

Development of an assay to measure cortisol using a standard glucose meter

Abstract only availableWhen an animal is stressed many changes occur, such as altered behavior and reduced resistance to disease, and these effect population performance (Millspaugh 2004). Cortisol is a stress hormone produced by the adrenal glands to regulate cardiovascular function, and energy utilization. Clinicians and wildlife biologists monitor cortisol levels in animals to determine their stress levels . Current tests must be done in a research lab; I want to develop a test that can be used at home or in the field. Four methods were compared: a glucose meter, FOX (ferric-xylenol orange complex) assay, o-dianisidine assay, and a lanthanide-based luminescent sensing probe, all of which detect H2O2, which is produced by glucose oxidase. The glucose oxidase will be used as a label for cortisol in an immunoassay. I assessed the sensitivity of the four methods. O-dianisidine assay detected 2.5 µM H2O2, lanthanide detected 5 µM H2O2, FOX detected 25µM H2O2, the glucose meter detected 500µM H2O2. I have found that the OneTouch Sure Step® glucose meter detects H2O2 in the absence of glucose. The optimal method for field/home testing has to have great sensitivity and also be easy to quantitate. Even though the meter had the lowest sensitivity, it is the most convenient and inepensive approach for quantitation. We are using modeling and other approaches to develop the best method(s) for cortisol measurement that will not only aid conservation biology, but it can also be used to monitor patient hormonal levels for treatment of endocrine disorders including Cushing's syndrome.NSF-REU Program in Biosystems Modeling and Analysi

Modeling sodium pump degradation and removal: Sorting out your dirty laundry [abstract]

Faculty Mentor: Dr. Mark Milanick, Medical Pharmacology and PhysiologyAbstract only availableWe live in a society where time management and energy efficiency are important productivity skills. A prime example of this is sorting through ones clothes: is it more time and energy-effective to search through a pile of clothes to pick out the dirty articles than to just put the whole pile in the wash? One may apply this same question, on a much smaller scale, to the human cell: what's the best strategy for removing damaged sodium pumps? Is it more time and energy-effective to search through the Na pumps to find the damaged ones than to just to remove all the pumps periodically? Na pumps are synthesized in the Endoplasmic Reticulum (ER), moved to the Golgi, then to the Plasma Membrane (PM), then to the early and late endosomal compartments, and finally degraded in the lysosome. Four cases of Na pump trafficking were studied: the control case, where each step is random and reversible, except for translation to the golgi and from the late endosome to the lysosome; the first case, which was an extension of the control with one irreversible step from the early endosome to the late endosome; the second case, which was an extension of the control with one irreversible step from the plasma membrane to the early endosome; and a final case, which was a combination of the control case and the first two cases. These cases were modeled using Microsoft Excel and Visual Basic Editor. Seven numerical values (0-6) signifying various stages of protein trafficking were randomly assigned to each protein at each time interval. In order to evaluate all four cases, five trials were completed. During each trial, one hundred proteins were studied for each of the four cases, and a bar graph was generated each time to show the distribution of times for the proteins to reach degradation (the final stage, 0). After having found that identifying damaged Na pumps significantly decreased the amount of time on average that it takes for a pump to reach degradation, we are developing models to additionally study the energy cost-affectivity of this process. So it is more time-effective to sort through your clothes for the dirty laundry, but does it ultimately cost more energy

The reactive nitrogen species peroxynitrite is a potent inhibitor of renal Na-K-ATPase activity

Peroxynitrite is a reactive nitrogen species produced when nitric oxide and superoxide react. In vivo studies suggest that reactive oxygen species and, perhaps, peroxynitrite can influence Na-K-ATPase function. However, the direct effects of peroxynitrite on Na-K-ATPase function remain unknown. We show that a single bolus addition of peroxynitrite inhibited purified renal Na-K-ATPase activity, with IC50 of 107 ± 9 μM. To mimic cellular/physiological production of peroxynitrite, a syringe pump was used to slowly release (∼0.85 μM/s) peroxynitrite. The inhibition of Na-K-ATPase activity induced by this treatment was similar to that induced by a single bolus addition of equal cumulative concentration. Peroxynitrite produced 3-nitrotyrosine residues on the α, β, and FXYD subunits of the Na pump. Interestingly, the flavonoid epicatechin, which prevented tyrosine nitration, was unable to blunt peroxynitrite-induced ATPase inhibition, suggesting that tyrosine nitration is not required for inhibition. Peroxynitrite led to a decrease in iodoacetamidofluorescein labeling, implying that cysteine modifications were induced. Glutathione was unable to reverse ATPase inhibition. The presence of Na+ and low MgATP during peroxynitrite treatment increased the IC50 to 145 ± 10 μM, while the presence of K+ and low MgATP increased the IC50 to 255 ± 13 μM. This result suggests that the EPNa conformation of the pump is slightly more sensitive to peroxynitrite than the E(K) conformation. Taken together, these results show that peroxynitrite is a potent inhibitor of Na-K-ATPase activity and that peroxynitrite can induce amino acid modifications to the pump