Peroxiredoxin 2 oxidation reveals hydrogen peroxide generation within erythrocytes during high-dose vitamin C administration.

Intravenous infusion of high dose (>10 g) vitamin C (IVC) is a common alternative cancer therapy. IVC results in millimolar levels of circulating ascorbate, which is proposed to generate cytotoxic quantities of H2O2 in the presence of transition metal ions. In this study we report on the in vitro and in vivo effects of millimolar ascorbate on erythrocytes. Addition of ascorbate to whole blood increased erythrocyte intracellular ascorbate approximately 35-fold. Within 10 min of ascorbate addition, we detected increased oxidation of erythrocyte peroxiredoxin 2 (Prx2), a major thiol antioxidant protein and a sensitive marker of H2O2 production. Up to 50% of Prx2 was present in the oxidised form after 60 min. The presence of extracellular catalase, removal of plasma or the addition of a metal chelator did not prevent ascorbate-induced Prx2 oxidation, suggesting that the H2O2 responsible for Prx2 oxidation was generated within the erythrocyte. Ascorbate is known to increase the rate of haemoglobin autoxidation and H2O2 production. Through spectral monitoring of oxidised haemoglobin we estimated a generation rate of 15 µM H2O2/min inside erythrocytes. We also investigated changes in erythrocyte ascorbate concentration and Prx2 oxidation following IVC infusion in a cohort of patients with cancer. Plasma ascorbate levels ranged from 7.8 to 35 mM immediately post infusion, while erythrocyte ascorbate levels reached 1.5-3.4 mM 4 h after beginning infusion. Transient oxidation of erythrocyte Prx2 was observed. We conclude that erythrocytes accumulate ascorbate during IVC infusion, providing a significant reservoir of ascorbate, and this ascorbate increases H2O2 generation within the cells. The consequence of increased erythrocyte Prx2 oxidation warrants further investigation.

Initial Evidence of Variation by Ethnicity in the Relationship between Vitamin C Status and Mental States in Young Adults.

Higher fruit and vegetable intake has been associated with improved mood, greater vitality, and lower stress. Although the nutrients driving these benefits are not specifically identified, one potentially important micronutrient is vitamin C, an important co-factor for the production of peptide hormones, carnitine and neurotransmitters that are involved in regulation of physical energy and mood. The aim of our study was to investigate the cross-sectional relationship between blood plasma vitamin C status and mood, vitality and perceived stress. A sample of 419 university students (aged 18 to 35; 67.8% female) of various ethnicities (49.2% European, 16.2% East Asian, 8.1% Southeast/Other Asian, 9.1% Maori/Pasifika, 11.5% Other) provided a fasting blood sample to determine vitamin C status and completed psychological measures consisting of the Profile of Mood States Short Form (POMS-SF), the vitality subscale of the Rand 36-Item Short Form (SF-36), and the Perceived Stress Scale (PSS). Participants were screened for prescription medication, smoking history, vitamin C supplementation, fruit/juice and vegetable consumption, kiwifruit allergies, excessive alcohol consumption and serious health issues, and provided age, gender, ethnicity, and socioeconomic status information, which served as covariates. There were no significant associations between vitamin C status and the psychological measures for the sample overall. However, associations varied by ethnicity. Among Maori/Pasifika participants, higher vitamin C was associated with greater vitality and lower stress, whereas among Southeast Asian participants, higher vitamin C was associated with greater confusion on the POMS-SF subscale. These novel findings demonstrate potential ethnicity-linked differences in the relationship between vitamin C and mental states. Further research is required to determine whether genetic variation or cultural factors are driving these ethnicity differences.

Low Vitamin C Status in Patients with Cancer Is Associated with Patient and Tumor Characteristics.

Vitamin C (ascorbate) acts as an antioxidant and enzyme cofactor, and plays a vital role in human health. Vitamin C status can be affected by illness, with low levels being associated with disease due to accelerated turnover. However, robust data on the ascorbate status of patients with cancer are sparse. This study aimed to accurately measure ascorbate concentrations in plasma from patients with cancer, and determine associations with patient or tumor characteristics. We recruited 150 fasting patients with cancer (of 199 total recruited) from two cohorts, either prior to cancer surgery or during cancer chemo- or immunotherapy. A significant number of patients with cancer had inadequate plasma ascorbate concentrations. Low plasma status was more prevalent in patients undergoing cancer therapy. Ascorbate status was higher in women than in men, and exercising patients had higher levels than sedentary patients. Our study may prompt increased vigilance of ascorbate status in cancer patients.

Vitamin C Administration by Intravenous Infusion Increases Tumor Ascorbate Content in Patients With Colon Cancer: A Clinical Intervention Study.

The use of high dose ascorbate infusions in cancer patients is widespread, but without evidence of efficacy. Several mechanisms whereby ascorbate could affect tumor progression have been proposed, including: (i) the localized generation of cytotoxic quantities of H2O2; (ii) ascorbate-dependent activation of the 2-oxoglutarate-dependent dioxygenases that control the hypoxia-inducible factors (HIFs) and that are responsible for the demethylation of DNA and histones; (iii) increased oxidative stress induced by dehydroascorbic acid. We hypothesize that the dysfunctional vasculature of solid tumors results in compromised delivery of ascorbate to poorly perfused regions of the tumor and that this ascorbate deficit acts as an additional driver of the hypoxic response via upregulation of HIFs. Using a randomized "therapeutic window of opportunity" clinical study design we aimed to determine whether ascorbate infusions affected tumor ascorbate content and tumor biology. Patients with colon cancer were randomized to receive infusions of up to 1 g/kg ascorbate for 4 days before surgical resection (n = 9) or to not receive infusions (n = 6). Ascorbate was measured in plasma, erythrocytes, tumor and histologically normal mucosa at diagnostic colonoscopy and at surgery. Protein markers of tumor hypoxia or DNA damage were monitored in resected tissue. Plasma ascorbate reached millimolar levels following infusion and returned to micromolar levels over 24 h. Pre-infusion plasma ascorbate increased from 38 ± 10 µM to 241 ± 33 µM (p < 0.0001) over 4 days and erythrocyte ascorbate from 18 ± 20 µM to 2509 ± 1016 µM (p < 0.005). Tumor ascorbate increased from 15 ± 6 to 28 ± 6 mg/100 g tissue (p < 0.0001) and normal tissue from 14 ± 6 to 21 ± 4 mg/100 g (p < 0.001). A gradient of lower ascorbate was evident towards the tumor centre in both control and infusion samples. Lower expression of hypoxia-associated proteins was seen in post-infusion tumors compared with controls. There were no significant adverse events and quality of life was unaffected by ascorbate infusion. This is the first clinical study to demonstrate that tumor ascorbate levels increase following infusion, even in regions of poor diffusion, and that this could modify tumor biology. CLINICAL TRIAL REGISTRATION: ANZCTR Trial ID ACTRN12615001277538 (https://www.anzctr.org.au/).

Vitamin C and immune cell function in inflammation and cancer.

Vitamin C (ascorbate) is maintained at high levels in most immune cells and can affect many aspects of the immune response. Intracellular levels generally respond to variations in plasma ascorbate availability, and a combination of inadequate intake and increased turnover during severe stress can result in low plasma ascorbate status. Intracellular ascorbate supports essential functions and, in particular, acts as an enzyme cofactor for Fe- or Cu-containing oxygenases. Newly discovered enzymes in this family regulate cell metabolism and epigenetics, and dysregulation of their activity can affect cell phenotype, growth and survival pathways, and stem cell phenotype. This brief overview details some of the recent advances in our understanding of how ascorbate availability can affect the hydroxylases controlling the hypoxic response and the DNA and histone demethylases. These processes play important roles in the regulation of the immune system, altering cell survival pathways, metabolism and functions.

Appropriate Handling, Processing and Analysis of Blood Samples Is Essential to Avoid Oxidation of Vitamin C to Dehydroascorbic Acid.

Vitamin C (ascorbate) is the major water-soluble antioxidant in plasma and its oxidation to dehydroascorbic acid (DHA) has been proposed as a marker of oxidative stress in vivo. However, controversy exists in the literature around the amount of DHA detected in blood samples collected from various patient cohorts. In this study, we report on DHA concentrations in a selection of different clinical cohorts (diabetes, pneumonia, cancer, and critically ill). All clinical samples were collected into EDTA anticoagulant tubes and processed at 4 °C prior to storage at -80 °C for subsequent analysis by HPLC with electrochemical detection. We also investigated the effects of different handling and processing conditions on short-term and long-term ascorbate and DHA stability in vitro and in whole blood and plasma samples. These conditions included metal chelation, anticoagulants (EDTA and heparin), and processing temperatures (ice, 4 °C and room temperature). Analysis of our clinical cohorts indicated very low to negligible DHA concentrations. Samples exhibiting haemolysis contained significantly higher concentrations of DHA. Metal chelation inhibited oxidation of vitamin C in vitro, confirming the involvement of contaminating metal ions. Although EDTA is an effective metal chelator, complexes with transition metal ions are still redox active, thus its use as an anticoagulant can facilitate metal ion-dependent oxidation of vitamin C in whole blood and plasma. Handling and processing blood samples on ice (or at 4 °C) delayed oxidation of vitamin C by a number of hours. A review of the literature regarding DHA concentrations in clinical cohorts highlighted the fact that studies using colourimetric or fluorometric assays reported significantly higher concentrations of DHA compared to those using HPLC with electrochemical detection. In conclusion, careful handling and processing of samples, combined with appropriate analysis, is crucial for accurate determination of ascorbate and DHA in clinical samples.

A randomized steady-state bioavailability study of synthetic versus natural (kiwifruit-derived) vitamin C.

Whether vitamin C from wholefoods has equivalent bioavailability to a purified supplement remains unclear. We have previously showed that kiwifruit provided significantly higher serum and tissue ascorbate levels than synthetic vitamin C in a genetically vitamin C-deficient mouse model, suggesting a synergistic activity of the whole fruit. To determine if these results are translatable to humans, we carried out a randomized human study comparing the bioavailability of vitamin C from kiwifruit with that of a vitamin C tablet of equivalent dosage. Thirty-six young non-smoking adult males were randomized to receive either half a gold kiwifruit (Actinidia Chinensis var. Hort 16A) per day or a comparable vitamin C dose (50 mg) in a chewable tablet for six weeks. Ascorbate was monitored weekly in fasting venous blood and in urine, semen, leukocytes, and skeletal muscle (vastus lateralis) pre- and post-intervention. Dietary intake of vitamin C was monitored using seven day food and beverage records. Participant ascorbate levels increased in plasma (P < 0.001), urine (P < 0.05), mononuclear cells (P < 0.01), neutrophils (P < 0.01) and muscle tissue (P < 0.001) post intervention. There were no significant differences in vitamin C bioavailability between the two intervention groups in any of the fluid, cell or tissue samples tested. Overall, our study showed comparable bioavailability of synthetic and kiwifruit-derived vitamin C.

The bioavailability of vitamin C from kiwifruit.

Vitamin C is an essential component of the diet for humans, and an adequate intake is important not only for the prevention of scurvy but also to limit the risk of developing chronic diseases such as heart disease and cancer. To achieve a regular and adequate intake, daily consumption of fresh fruit and vegetables is recommended. The vitamin C content of food varies widely, however, and plasma levels generally reflect the amount consumed, regardless of its origin. Kiwifruit are one of the premier dietary sources of vitamin C, with all commercially important varieties having high content, and with one serving delivering the bulk of the recommended dietary intake. Recent studies have shown that the addition of kiwifruit to a marginal vitamin C diet markedly improves plasma vitamin C levels and can increase them to both healthy and optimal levels.

Bioavailability of vitamin C from kiwifruit in non-smoking males: determination of 'healthy' and 'optimal' intakes.

Vitamin C is an essential nutrient in humans and must be obtained through the diet. The aim of this study was to determine vitamin C uptake in healthy volunteers after consuming kiwifruit (Actinidia chinensis var. Hort. 16A), and to determine the amount of fruit required to raise plasma vitamin C to 'healthy' (i.e. >50 µmol/l) and 'optimal' or saturating levels (i.e. >70 µmol/l). Leucocyte and urinary vitamin C levels were also determined. A total of fifteen male university students with below average levels of plasma vitamin C were selected for the study. Weekly fasting blood samples were obtained for a 4-week lead-in period and following supplementation with, sequentially, half, one, two and three Gold kiwifruit per d for 4-6 weeks each, followed by a final 4-week washout period. The results showed that addition of as little as half a kiwifruit per d resulted in a significant increase in plasma vitamin C. However, one kiwifruit per d was required to reach what is considered healthy levels. Increasing the dose of kiwifruit to two per d resulted in further increases in plasma vitamin C levels as well as increased urinary output of the vitamin, indicating that plasma levels were saturating at this dosage. Dividing the participants into high and low vitamin C groups based on their baseline plasma and leucocyte vitamin C levels demonstrated that it is critical to obtain a study population with low initial levels of the vitamin in order to ascertain a consistent effect of supplementation.

Mitochondrial peroxiredoxin 3 is more resilient to hyperoxidation than cytoplasmic peroxiredoxins.

The Prxs (peroxiredoxins) are a family of cysteine-dependent peroxidases that decompose hydrogen peroxide. Prxs become hyperoxidized when a sulfenic acid formed during the catalytic cycle reacts with hydrogen peroxide. In the present study, Western blot methodology was developed to quantify hyperoxidation of individual 2-Cys Prxs in cells. It revealed that Prx 1 and 2 were hyperoxidized at lower doses of hydrogen peroxide than would be predicted from in vitro data, suggesting intracellular factors that promote hyperoxidation. In contrast, mitochondrial Prx 3 was considerably more resistant to hyperoxidation. The concentration of Prx 3 was estimated at 125 microM in the mitochondrial matrix of Jurkat T-lymphoma cells. Although the local cellular environment could influence susceptibility, purified Prx 3 was also more resistant to hyperoxidation, suggesting that despite having C-terminal motifs similar to sensitive eukaryote Prxs, other structural features must contribute to the innate resilience of Prx 3 to hyperoxidation.

Inhibition of receptor-mediated apoptosis upon Bcl-2 overexpression is not associated with increased antioxidant status.

Bcl-2 is reported to augment the antioxidant capacity of cells and this is hypothesized to contribute to the anti-apoptotic activity of this oncoprotein. We generated a number of stable Jurkat cell lines expressing varying levels of Bcl-2, and showed a strong correlation between Bcl-2 levels and resistance to Fas-mediated apoptosis. While individual differences could be detected, there was no overall correlation between Bcl-2 and the expression and activity of superoxide dismutases, catalase, glutathione peroxidases, thioredoxin, thioredoxin reductases, and peroxiredoxins. Cells transfected with Bcl-2 averaged 70% more glutathione than parental cells, but there was no correlation between glutathione and resistance to apoptosis. This challenges the hypothesis that the anti-apoptotic properties of Bcl-2 are linked to a global increase in antioxidant status.

Oxidation of mitochondrial peroxiredoxin 3 during the initiation of receptor-mediated apoptosis.

It is hypothesized that activation of death receptors disrupts the redox homeostasis of cells and that this contributes to the induction of apoptosis. The redox status of the peroxiredoxins, which are extremely sensitive to increases in H2O2 and disruption of the thioredoxin system, were monitored in Jurkat T lymphoma cells undergoing Fas-mediated apoptosis. The only detectable change during the early stages of apoptosis was oxidation of mitochondrial peroxiredoxin 3. Increased H2O2 triggers peroxiredoxin overoxidation to a sulphinic acid; however during apoptosis peroxiredoxin 3 was captured as a disulfide, suggesting impairment of the thioredoxin system responsible for maintaining peroxiredoxin 3 in its reduced form. Peroxiredoxin 3 oxidation was an early event, occurring within the same timeframe as increased mitochondrial oxidant production, caspase activation and cytochrome c release. It preceded other major apoptotic events including mitochondrial permeability transition and phosphatidylserine exposure, and glutathione depletion, global thiol protein oxidation and protein carbonylation. Peroxiredoxin 3 oxidation was also observed in U937 cells stimulated with TNF-alpha. We hypothesize that the selective oxidation of peroxiredoxin 3 leads to an increase in mitochondrial H2O2 and that this may influence the progression of apoptosis.

Phenethyl isothiocyanate triggers apoptosis in Jurkat cells made resistant by the overexpression of Bcl-2.

Isothiocyanates are a class of naturally occurring chemopreventive agents known to be effective at triggering apoptosis. In this study, we show that whereas overexpression of the oncoprotein Bcl-2 renders Jurkat T-lymphoma cells resistant to a range of cytotoxic agents, phenethyl isothiocyanate is able to overcome the inhibitory action of Bcl-2 and trigger apoptosis. A 50-fold increase in Bcl-2 expression shifted the dose-response curve, with an increase in the phenethyl isothiocyanate LD(50) from 7 to 15 micromol/L, but there was still a complete loss in cell viability at doses in excess of 20 micromol/L. At these concentrations, cytotoxicity was strongly associated with caspase activation, phosphatidylserine exposure, and morphologic changes characteristic of apoptosis. Cytotoxicity was inhibited by treatment of the cells with a broad-spectrum caspase inhibitor. A structure-activity analysis showed that the phenethyl and benzyl isothiocyanates were most effective at triggering apoptosis in cells overexpressing Bcl-2 whereas phenyl isothiocyanate and benzyl thiocyanate had no proapoptotic activity. Allyl isothiocyanate also had limited efficacy despite its ability to trigger apoptosis in the parental Jurkat cell line. From this information, we propose that isothiocyanates modify a key cysteine residue in an apoptosis regulatory protein and that the aromatic side chain facilitates access to the target site. An in-depth investigation of the cellular targets of the aromatic isothiocyanates is warranted.

The chemopreventive agent phenethyl isothiocyanate sensitizes cells to Fas-mediated apoptosis.

The chemopreventive properties of the isothiocyanates have been attributed to their ability to inhibit phase I enzymes that activate procarcinogens, induce phase II protective enzymes and trigger apoptosis in transformed cells. In this study we provide evidence for a new mechanism of chemoprevention, wherein sublethal doses of phenethyl isothiocyanate (PEITC) sensitize cells to Fas-mediated apoptosis. The phenomenon was observed in the Fas-resistant T24 bladder carcinoma cell line and in Jurkat T cells overexpressing the anti-apoptotic protein Bcl-2. Caspase-3-like activity was increased up to 20-fold of that observed with either PEITC or anti-Fas antibody alone. While PEITC activated ERK, JNK and p38, inhibitors of these MAP kinases did not block apoptosis. PEITC transiently depleted cellular glutathione, providing a putative mechanism for sensitizing the cells to apoptosis. However, lowering glutathione with buthionine sulfoximine did not mimic the effect of PEITC. Instead, we propose that PEITC promotes apoptosis by directly modifying intracellular thiol proteins. The ability of PEITC to sensitize cells to receptor-mediated apoptosis provides an additional mechanism to explain its chemopreventive properties.

Diphenyleneiodonium triggers the efflux of glutathione from cultured cells.

Diphenyleneiodonium (DPI) is a broad-spectrum flavoprotein inhibitor commonly used to inhibit oxidant production by the NADPH oxidase of phagocytic and nonphagocytic cells. A previous study has shown that DPI can sensitize T24 bladder carcinoma cells to Fas-mediated apoptosis. We observed DPI to deplete intracellular reduced glutathione (GSH) in T24 cells and a range of other primary and transformed cell types. The effect was immediate, with 50% loss of intracellular GSH within 2 h of treatment with DPI. The glutathione was quantitatively recovered in the extracellular medium, indicating that efflux was occurring. The loss of GSH was blocked with bromosulfophthalein, an inhibitor of the canalicular GSH transporters. We conclude that DPI induces a dramatic efflux of cellular GSH from T24 cells via a specific transport channel. This provides a potential mechanism for its proapoptotic effect, and it also has important implications for the regulation of glutathione homeostasis in cells.