Thomas J. van ‘t Erve

An assay for the rate of removal of extracellular hydrogen peroxide by cells

Cells have a wide range of capacities to remove extracellular hydrogen peroxide. At higher concentrations of extracellular H2O2 (micromolar) the rate of removal can be approximated by a rate equation that is first-order in the concentration of H2O2 and cell density. Here we present a method to determine the observed rate constant for the removal of extracellular H2O2 on a per cell basis. In the cells examined, when exposed to 20 μM H2O2, these rate constants (kcell) range from 0.46×10−12 s−1 cell−1 L for Mia-PaCa-2 cells (human pancreatic carcinoma) to 10.4×10−12 s−1 cell−1 L for U937 cells (human histiocytic lymphoma). For the relatively small red blood cell kcell=2.9×10−12 s−1 cell−1 L. These rate constants, kcell, can be used to compare the capacity of cells to remove higher levels of extracellular H2O2, as often presented in cell culture experiments. They also provide a means to estimate the rate of removal of extracellular H2O2, rate=−kcell [H2O2] (cells L−1), and the half-life of a bolus of H2O2. This information is essential to optimize experimental design and interpret data from experiments that expose cells to extracellular H2O2.

The heritability of metabolite concentrations in stored human red blood cells.

The degeneration of red blood cells (RBCs) during storage is a major issue in transfusion medicine. Family studies in the 1960s established the heritability of the RBC storage lesion based on poststorage adenosine triphosphate (ATP) concentrations. However, this critical discovery has not been further explored. In a classic twin study we confirmed the heritability of poststorage ATP concentrations and established the heritability of many other RBC metabolites.

The concentration of glutathione in human erythrocytes is a heritable trait

Glutathione (GSH) is a ubiquitous, redox-active, small molecule that is critical to cellular and organism health. In red blood cells (RBCs), the influence of the environment (e.g., diet and lifestyle) on GSH levels has been demonstrated in numerous studies. However, it remains unknown if levels of GSH are determined principally by environmental factors or if there is a genetic component, i.e., heritability. To investigate this we conducted a twin study. Twin studies are performed by comparing the similarity in phenotypes between mono- and dizygotic twin pairs. We determined the heritability of GSH, as well as its oxidation product glutathione disulfide (GSSG), the sum of GSH equivalents (tGSH), and the status of the GSSG/2GSH couple (marker of oxidation status, Ehc) in RBCs. In our study population we found that the estimated heritability for the intracellular concentration of GSH in RBCs was 57 %; for GSSG it was 51 %, tGSH 63 %, and Ehc 70 %. We conclude that a major portion of the phenotype of these traits is controlled genetically. We anticipate that these heritabilities will also be reflected in other cell types. The discovery that genetics plays a major role in the innate levels of redox-active species in RBCs is paradigm shifting and opens new avenues of research in the field of redox biology. Inherited RBC antioxidant levels may be important disease modifiers. By identifying the relative contributions of genes and the environment to antioxidant variation between individuals, new therapeutic strategies can be developed. Understanding the genetic determinants of these inherited traits may allow personalized approaches to relevant therapies.

The heritability of hemolysis in stored human red blood cells

The transfusion of red blood cells (RBCs) with maximum therapeutic efficacy is a major goal in transfusion medicine. One of the criteria used in determining stored RBC quality is end‐of‐storage hemolysis. Between donors, a wide range of hemolysis is observed under identical storage conditions. Here, a potential mechanism for this wide range is investigated. We hypothesize that the magnitude of hemolysis is a heritable trait. Also, we investigated correlations between hemolysis and RBC metabolites; this will establish pathways influencing hemolysis as future targets for genetic analysis.

Moles of a Substance per Cell Is a Highly Informative Dosing Metric in Cell Culture

Background The biological consequences upon exposure of cells in culture to a dose of xenobiotic are not only dependent on biological variables, but also the physical aspects of experiments e.g. cell number and media volume. Dependence on physical aspects is often overlooked due to the unrecognized ambiguity in the dominant metric used to express exposure, i.e. initial concentration of xenobiotic delivered to the culture medium over the cells. We hypothesize that for many xenobiotics, specifying dose as moles per cell will reduce this ambiguity. Dose as moles per cell can also provide additional information not easily obtainable with traditional dosing metrics. Methods Here, 1,4-benzoquinone and oligomycin A are used as model compounds to investigate moles per cell as an informative dosing metric. Mechanistic insight into reactions with intracellular molecules, differences between sequential and bolus addition of xenobiotic and the influence of cell volume and protein content on toxicity are also investigated. Results When the dose of 1,4-benzoquinone or oligomycin A was specified as moles per cell, toxicity was independent of the physical conditions used (number of cells, volume of medium). When using moles per cell as a dose-metric, direct quantitative comparisons can be made between biochemical or biological endpoints and the dose of xenobiotic applied. For example, the toxicity of 1,4-benzoquinone correlated inversely with intracellular volume for all five cell lines exposed (C6, MDA-MB231, A549, MIA PaCa-2, and HepG2). Conclusions Moles per cell is a useful and informative dosing metric in cell culture. This dosing metric is a scalable parameter that: can reduce ambiguity between experiments having different physical conditions; provides additional mechanistic information; allows direct comparison between different cells; affords a more uniform platform for experimental design; addresses the important issue of repeatability of experimental results, and could increase the translatability of information gained from in vitro experiments.