Direct spectrophotometric measurement of supra-physiological levels of ascorbate in plasma

Witmer, Jordan R; Wetherell, Bailey Jordan; Wagner, Brett A.; Du, Juan; Cullen, Joseph J.; Buettner, Garry R.

doi:10.1016/j.redox.2016.02.004

Cited by 20 publications

(16 citation statements)

References 28 publications

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“…Total ascorbate in NSCLC subject’s plasma was quantified as previously described (Witmer et al, 2016). Briefly, plasma samples were measured spectrophotometrically (265 nm; ε = 13,000 M −1 cm −1 ) using an Implen Nanophotometer P-330 with a 250× dilution lid, path length = 4.00 × 10 −3 cm.…”

Section: Methodsmentioning

confidence: 99%

O2⋅− and H2O2-Mediated Disruption of Fe Metabolism Causes the Differential Susceptibility of NSCLC and GBM Cancer Cells to Pharmacological Ascorbate

Schoenfeld¹,

Sibenaller²,

Mapuskar³

et al. 2017

Cancer Cell

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123

158

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SUMMARY Pharmacological ascorbate has been proposed as a potential anti-cancer agent when combined with radiation and chemotherapy. The anti-cancer effects of ascorbate are hypothesized to involve the autoxidation of ascorbate leading to increased steady-state levels of H2O2; however, the mechanism(s) for cancer cell-selective toxicity remain unknown. The current study shows that alterations in cancer cell mitochondrial oxidative metabolism resulting in increased levels of O2.− and H2O2 are capable of disrupting intracellular iron metabolism, thereby selectively sensitizing non-small-cell lung cancer (NSCLC) and glioblastoma (GBM) cells to ascorbate through pro-oxidant chemistry involving redox-active labile iron and H2O2. In addition, preclinical studies and clinical trials demonstrate the feasibility, selective toxicity, tolerability, and potential efficacy of pharmacological ascorbate in GBM and NSCLC therapy.

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Section: Methodsmentioning

confidence: 99%

O2⋅− and H2O2-Mediated Disruption of Fe Metabolism Causes the Differential Susceptibility of NSCLC and GBM Cancer Cells to Pharmacological Ascorbate

Schoenfeld¹,

Sibenaller²,

Mapuskar³

et al. 2017

Cancer Cell

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123

158

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“…To date, the Implen Nanophotometer has not been used to quantify plasma ascorbate in canines (49). A recent study confirmed the ability to use UV spectroscopy to rapidly quantify high concentrations (up to 35 mM) of human plasma ascorbate, with minimal processing (27). Using this same assay on our canine plasma samples detected ascorbate concentrations that corresponded with those found in human and murine studies (7, 17, 21).…”

Section: Discussionmentioning

confidence: 84%

“…Plasma ascorbate quantification was conducted at The University of Iowa using two different assay systems as previously described (27, 28). Briefly, a plate reader (SpectraMax® M3, Molecular Devices, Sunnyvale, CA) was used to measure baseline and physiological levels of ascorbate present in the plasma samples (28).…”

Section: Methodsmentioning

confidence: 99%

“…To quantify the high pharmacological levels of ascorbate a second assay using an Implen Nanophotometer (Implen GmbH, Munich, Germany) microvolume UV-Vis spectrometer was used. With little required sample processing, this assay, developed at The University of Iowa (27), allows rapid assessment of ascorbate concentrations in plasma ranging from 2.9 to 35 mM.…”

Section: Methodsmentioning

confidence: 99%

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In vitro Cytotoxicity and Pharmacokinetic Evaluation of Pharmacological Ascorbate in Dogs

et al. 2019

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Background: High-dose, pharmacological ascorbate (P-AscH−) is preferentially cytotoxic to human cancer cells in vitro. Investigations on the efficacy of P-AscH− as an adjuvant treatment for multiple human cancers are on-going, but has yet to be formally investigated in dogs. The primary objectives of this study were to determine the pharmacokinetic (PK) profile of P-AscH− in healthy Beagle dogs and the effects of P-AscH− on canine osteosarcoma cells in vitro.Methods: Eight purpose-bred, healthy, spayed female Beagle dogs, between 20 and 21 months old, and 8–10 kg were administered two doses of P-AscH− (550 or 2,200 mg/kg) via intravenous infusion over 6 h, on separate days. Plasma ascorbate concentrations were measured at 12 time points during and after infusion for PK analysis using nonlinear mixed-effects (NLME) and non-compartmental analysis (NCA). Clonogenic assays were performed on 2 canine osteosarcoma cell lines (D-17 and OSCA-8) and canine primary dermal fibroblasts after exposure to high concentrations of ascorbate (75 pmoles/cell).Results: Plasma ascorbate levels in the dogs peaked at approximately 10 mM following 2,200 mg/kg and returned to baseline 6–8 h after dosing. Minor adverse effects were seen in two dogs. Ascorbate (75 pmoles/cell) significantly decreased survival in both the osteosarcoma cell lines (D-17 63% SD 0.010, P = 0.005; OSCA-8 50% SD 0.086, P = 0.026), compared to normal fibroblasts (27% SD 0.056).Conclusions: Pharmacological ascorbate is preferentially cytotoxic to canine-derived cancer cells. High levels of ascorbate can be safely administered to dogs. Further studies are needed to determine the effects of P-AscH− on canine patients.

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“…Supraphysiological levels of ascorbate were quantified by direct ultraviolet (UV) spectroscopy using an Implen P330 UV/Visible NanoPhotometert®(Westlake Village, CA) with a CCD array detector using a 0.04 mm cap (dilution factor = 250) as described by Witmer et al (19). All samples were measured in triplicate.…”

Section: [Asch − ] Measurementsmentioning

confidence: 99%

Assessment of the Stability of Supraphysiological Ascorbate in Human Blood: Appropriate Handling of Samples from Clinical Trials for Measurements of Pharmacological Ascorbate

Petronek¹,

Wagner²,

Hollenbeck³

et al. 2019

Radiation Research

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Based on encouraging results from several early-phase clinical trials, there is renewed interest in the use of pharmacological ascorbate (i.e., intravenous administration resulting in .> ≈ 10 mM plasma ascorbate concentrations) in combination with standard-of-care cancer treatments including radiation and/or chemotherapy. Under normal, healthy physiological conditions, humans maintain plasma ascorbate concentrations in the range of 40-80 μM.However, in vivo antitumor activity requires supraphysiological plasma concentrations on the order of ≈20 mM. The stability of ascorbate in whole blood has been well studied. The goal of this work was to determine the appropriate handling methods of blood samples, after treatment with pharmacological ascorbate, which allow for the optimal measurement of ascorbate in plasma for dosing verification. Our findings indicate that ascorbate concentrations (mM) are relatively stable in whole blood collected in sodium heparin tubes and stored on ice (or at 4°C) for up to 24 h. After 24 h, ascorbate levels in plasma are relatively stable at 4°C for up to 72 h. At −20°C, plasma concentrations are relatively stable for 2-3 weeks, while at −80°C, ascorbate concentrations in plasma are stable for at least one month. In contrast, patient samples showed better stability when stored as whole blood compared to plasma at 4°C but increasing hemolysis over time may significantly skew ascorbate measurements. Additionally, patient samples can be reliably stored as plasma at −20°C for up to three weeks in either a frost-containing or frost-free environment. This information can guide the collection, processing and storage of clinical samples after pharmacological ascorbate infusions amenable to multi-center clinical trials.

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Direct spectrophotometric measurement of supra-physiological levels of ascorbate in plasma

Cited by 20 publications

References 28 publications

O2⋅− and H2O2-Mediated Disruption of Fe Metabolism Causes the Differential Susceptibility of NSCLC and GBM Cancer Cells to Pharmacological Ascorbate

O2⋅− and H2O2-Mediated Disruption of Fe Metabolism Causes the Differential Susceptibility of NSCLC and GBM Cancer Cells to Pharmacological Ascorbate

In vitro Cytotoxicity and Pharmacokinetic Evaluation of Pharmacological Ascorbate in Dogs

Assessment of the Stability of Supraphysiological Ascorbate in Human Blood: Appropriate Handling of Samples from Clinical Trials for Measurements of Pharmacological Ascorbate

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