Search

The most common and aggressive brain cancer, glioblastoma, is characterized by hypoxia and poor survival. The pro-tumour transcription factor, hypoxia-inducible factor (HIF), is regulated via HIF-hydroxylases that require ascorbate as cofactor. Decreased HIF-hydroxylase activity triggers the hypoxic pathway driving cancer progression. Tissue ascorbate accumulates via the sodium-dependent vitamin C transporter-2 (SVCT2). We hypothesize that glioblastoma cells rely on SVCT2 for ascorbate accumulation, and that knockout of this transporter would disrupt the regulation of the hypoxic pathway by ascorbate. Ascorbate uptake was measured in glioblastoma cell lines (U87MG, U251MG, T98G) by high-performance liquid chromatography. CRISPR/Cas9 was used to knockout SVCT2. Cells were treated with cobalt chloride, desferrioxamine or 5% oxygen, with/without ascorbate, and key hypoxic pathway proteins were measured using Western blot analysis. Ascorbate uptake was cell line dependent, ranging from 1.7 to 11.0 nmol/10 6 cells. SVCT2-knockout cells accumulated 90%-95% less intracellular ascorbate than parental cells. The hypoxic pathway was induced by all three stimuli, and ascorbate reduced this induction. In the SVCT2-knockout cells, ascorbate had limited effect on the hypoxic pathway. This study verifies that intracellular ascorbate is required to suppress the hypoxic pathway. As patient survival is related to an activated hypoxic pathway, increasing intra-tumoral ascorbate may be of clinical interest., (© 2024 The Author(s). Journal of Cellular Biochemistry published by Wiley Periodicals LLC.)

The treatment of metastatic melanoma has been revolutionised by immunotherapy, yet a significant number of patients do not respond, and many experience autoimmune adverse events. Associations have been reported between patient outcome and monocyte subsets, whereas vitamin C (ascorbate) has been shown to mediate changes in cancer-stimulated monocytes in vitro. We therefore investigated the relationship of ascorbate with monocyte subsets and epigenetic modifications in patients with metastatic melanoma receiving immunotherapy. Patients receiving immunotherapy were compared to other cancer cohorts and age-matched healthy controls. Ascorbate levels in plasma and peripheral blood-derived mononuclear cells (PBMCs), monocyte subtype and epigenetic markers were measured, and adverse events, tumour response and survival were recorded. A quarter of the immunotherapy cohort had hypovitaminosis C, with plasma and PBMC ascorbate levels significantly lower than those from other cancer patients or healthy controls. PBMCs from the immunotherapy cohort contained similar frequencies of non-classical and classical monocytes. DNA methylation markers and intracellular ascorbate concentration were correlated with monocyte subset frequency in healthy controls, but correlation was lost in immunotherapy patients. No associations between ascorbate status and immune-related adverse events or tumour response or overall survival were apparent.

Vitamin C-rich foods can improve mood; however, the timecourse of these benefits is unknown. This study utilised intensive longitudinal smartphone surveys from a three-armed placebo-controlled trial to determine mood-related changes following supplementation with vitamin C (250 mg tablet/d), kiwifruit (2 SunGold™ kiwifruit/d) or a placebo (1 tablet/d). Secondary data were analysed from the KiwiC for Vitality trial (Trial ID: ACTRN12617001031358). Adults ( n 155, 63 % female, aged 18-35 years) with low plasma vitamin C (<40 μmol/l) completed a 14-d lead-in, 28-d intervention and 14-d washout. Participants self-reported vitality (SF-36), mood (POMS total mood disturbance), flourishing (flourishing scale), sleep quality, sleep quantity and physical activity every second day using smartphone surveys. Plasma vitamin C, measured fortnightly, reached saturation after 2 weeks of vitamin C or kiwifruit supplementation. Kiwifruit supplementation improved vitality and mood within 4 days, peaking around 14-16 days, and improved flourishing from day 14. Vitamin C marginally improved mood until day 12. Incremental AUC analyses revealed significant overall effects of kiwifruit consumption on vitality and mood compared with placebo, which were stronger than effects for vitamin C tablets, but attenuated when adjusting for covariates. Sensitivity analyses of participants with low baseline vitamin C status revealed improved mood (vitamin C and kiwifruit) and flourishing (kiwifruit only). This is the first study to use intensive smartphone surveys to model the day-to-day timecourse of mood-related states following vitamin C intervention and highlights the value of using smartphone surveys to reveal the temporal changes in mood-related outcomes following nutrient supplementation.

Our previous work in guinea pigs revealed that low vitamin C intake during preconception and pregnancy adversely affects fertility, pregnancy outcomes, and foetal and neonatal growth in a sex-dependent manner. To investigate the long-term impact on offspring, we monitored their growth from birth to adolescence (four months), recorded organ weights at childhood equivalence (28 days) and adolescence, and assessed physiological parameters like oral glucose tolerance and basal cortisol concentrations. We also investigated the effects of the timing of maternal vitamin C restriction (early vs. late gestation) on pregnancy outcomes and the health consequences for offspring. Dunkin Hartley guinea pigs were fed an optimal (900 mg/kg feed) or low (100 mg/kg feed) vitamin C diet ad libitum during preconception. Pregnant dams were then randomised into four feeding regimens: consistently optimal, consistently low, low during early pregnancy, or low during late pregnancy. We found that low maternal vitamin C intake during early pregnancy accelerated foetal and neonatal growth in female offspring and altered glucose homeostasis in the offspring of both sexes at an age equivalent to early childhood. Conversely, low maternal vitamin C intake during late pregnancy resulted in foetal growth restriction and reduced weight gain in male offspring throughout their lifespan. We conclude that altered vitamin C during development has long-lasting, sex-specific consequences for offspring and that the timing of vitamin C depletion is also critical, with low levels during early development being associated with the development of a metabolic syndrome-related phenotype, while later deprivation appears to be linked to a growth-faltering phenotype.

We recently developed a novel keratin-derived protein (KDP) rich in cysteine, glycine, and arginine, with the potential to alter tissue redox status and insulin sensitivity. The KDP was tested in 35 human adults with type-2 diabetes mellitus (T2DM) in a 14-wk randomised controlled pilot trial comprising three 2×20 g supplemental protein/day arms: KDP-whey (KDPWHE), whey (WHEY), non-protein isocaloric control (CON), with standardised exercise. Outcomes were measured morning fasted and following insulin-stimulation (80 mU/m 2 /min hyperinsulinaemic-isoglycaemic clamp). With KDPWHE supplementation there was good and very-good evidence for moderate-sized increases in insulin-stimulated glucose clearance rate (GCR; 26%; 90% confidence limits, CL 2%, 49%) and skeletal-muscle microvascular blood flow (46%; 16%, 83%), respectively, and good evidence for increased insulin-stimulated sarcoplasmic GLUT4 translocation (18%; 0%, 39%) vs CON. In contrast, WHEY did not effect GCR (-2%; -25%, 21%) and attenuated HbA1c lowering (14%; 5%, 24%) vs CON. KDPWHE effects on basal glutathione in erythrocytes and skeletal muscle were unclear, but in muscle there was very-good evidence for large increases in oxidised peroxiredoxin isoform 2 (oxiPRX2) (19%; 2.2%, 35%) and good evidence for lower GPx1 concentrations (-40%; -4.3%, -63%) vs CON; insulin stimulation, however, attenuated the basal oxiPRX2 response (4%; -16%, 24%), and increased GPx1 (39%; -5%, 101%) and SOD1 (26%; -3%, 60%) protein expression. Effects of KDPWHE on oxiPRX3 and NRF2 content, phosphorylation of capillary eNOS and insulin-signalling proteins upstream of GLUT4 translocation Akt Ser437 and AS160 Thr642 were inconclusive, but there was good evidence for increased IRS Ser312 (41%; 3%, 95%), insulin-stimulated NFκB-DNA binding (46%; 3.4%, 105%), and basal PAK-1 Thr423 /2 Thr402 phosphorylation (143%; 66%, 257%) vs WHEY. Our findings provide good evidence to suggest that dietary supplementation with a novel edible keratin protein in humans with T2DM may increase glucose clearance and modify skeletal-muscle tissue redox and insulin sensitivity within systems involving peroxiredoxins, antioxidant expression, and glucose uptake., Competing Interests: Declaration of competing interest We were unable to get the Elsevier online DOI working, so this document covers declarations., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Ascorbate plays a vital role as a co-factor for a superfamily of enzymes, the 2-oxoglutarate dependent dioxygenases (2-OGDDs), which govern numerous pathways in cancer progression, including the hypoxic response and the epigenetic regulation of gene transcription. Ascorbate uptake into most cells is through active transport by the sodium-dependent vitamin C transporter 2 (SVCT2). The aims of this study were to determine the kinetics of ascorbate uptake and retention by breast cancer cell lines under various oxygen conditions, and to investigate the role of SVCT2 in mediating ascorbate uptake and intracellular trafficking. Human MDA-MB231 cells accumulated up to 5.1 nmol ascorbate/10 6 cells, human MCF7 cells 4.5 nmol/10 6 cells, and murine EO771 cells 26.7 nmol/10 6 cells. Intracellular ascorbate concentrations decreased rapidly after reaching maximum levels unless further ascorbate was supplied to the medium, and there was no difference in the rate of ascorbate loss under normoxia or hypoxia. SVCT2 was localised mainly to subcellular compartments, with the nucleus apparently containing the most SVCT2 protein, followed by the mitochondria. Much less SVCT2 staining was observed on the plasma membrane. Our data showed that careful management of the doses and incubation times with ascorbate in vitro allows for an approximation of in vivo conditions. The localisation of SVCT2 suggests that the distribution of ascorbate to intracellular compartments is closely aligned to the known function of ascorbate in supporting 2-OGDD enzymatic functions in the organelles and with supporting antioxidant protection in the mitochondria.

Identifying how specific nutrients can impact fertility, pregnancy, and neonatal outcomes will yield important insights into the biological mechanisms linking diet and reproductive health. Our study investigates how dietary vitamin C intake affects various fertility parameters and pregnancy and neonatal outcomes in the guinea pig, a natural model of vitamin C dependency. Dunkin Hartley guinea pigs were fed an optimal (900 mg/kg feed) or low (100 mg/kg feed) vitamin C diet ad libitum for at least three weeks prior to mating and throughout pregnancy. We found that animals receiving the low vitamin C diet had an increased number of unsuccessful matings, a higher incidence of foetal reabsorption, and, among pregnancies resulting in delivery at term, produced fewer offspring. Neonates from mothers on the low vitamin C diet had significantly decreased plasma vitamin C concentrations at birth and exhibited mild growth impairments in a sex-dependent manner. We conclude that a diet low of vitamin C induces a state of subfertility, reduces overall fecundity, and adversely impacts both pregnancy outcomes and growth in the offspring. Our study provides an essential foundation for future investigations to determine whether these findings translate to humans. If so, they could have important clinical implications for assisted reproductive technologies and nutritional recommendations for couples trying to conceive, pregnant women, and breastfeeding mothers.

Necroptosis is a form of regulated cell death triggered by various host and pathogen-derived molecules during infection and inflammation. The essential step leading to necroptosis is phosphorylation of the mixed lineage kinase domain-like protein by receptor-interacting protein kinase 3. Caspase-8 cleaves receptor-interacting protein kinases to block necroptosis, so synthetic caspase inhibitors are required to study this process in experimental models. However, it is unclear how caspase-8 activity is regulated in a physiological setting. The active site cysteine of caspases is sensitive to oxidative inactivation, so we hypothesized that oxidants generated at sites of inflammation can inhibit caspase-8 and promote necroptosis. Here, we discovered that hypothiocyanous acid (HOSCN), an oxidant generated in vivo by heme peroxidases including myeloperoxidase and lactoperoxidase, is a potent caspase-8 inhibitor. We found HOSCN was able to promote necroptosis in mouse fibroblasts treated with tumor necrosis factor. We also demonstrate purified caspase-8 was inactivated by low concentrations of HOSCN, with the predominant product being a disulfide-linked dimer between Cys360 and Cys409 of the large and small catalytic subunits. We show oxidation still occurred in the presence of reducing agents, and reduction of the dimer was slow, consistent with HOSCN being a powerful physiological caspase inhibitor. While the initial oxidation product is a dimer, further modification also occurred in cells treated with HOSCN, leading to higher molecular weight caspase-8 species. Taken together, these findings indicate major disruption of caspase-8 function and suggest a novel mechanism for the promotion of necroptosis at sites of inflammation., Competing Interests: Conflict of interest J. M. M. contributes to the development of necroptosis inhibitors in collaboration with Anaxis Pharma Pty Ltd. G.S.S. consults for Genentech Inc. All other authors declare they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

We have read the Comment article by Vorland et al. [...].

Gliomas are incurable brain cancers with poor prognosis, with epigenetic dysregulation being a distinctive feature. 5-hydroxymethylcytosine (5-hmC), an intermediate generated in the demethylation of 5-methylcytosine, is present at reduced levels in glioma tissue compared with normal brain, and that higher levels of 5-hmC are associated with improved patient survival. DNA demethylation is enzymatically driven by the ten-eleven translocation (TET) dioxygenases that require ascorbate as an essential cofactor. There is limited data on ascorbate in gliomas and the relationship between ascorbate and 5-hmC in gliomas has never been reported. Clinical glioma samples (11 low-grade, 26 high-grade) were analysed for ascorbate, global DNA methylation and hydroxymethylation, and methylation status of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter. Low-grade gliomas contained significantly higher levels of ascorbate than high-grade gliomas (p = 0.026). Levels of 5-hmC were significantly higher in low-grade than high-grade glioma (p = 0.0013). There was a strong association between higher ascorbate and higher 5-hmC (p = 0.004). Gliomas with unmethylated and methylated MGMT promoters had similar ascorbate levels (p = 0.96). One mechanism by which epigenetic modifications could occur is through ascorbate-mediated optimisation of TET activity in gliomas. These findings open the door to clinical intervention trials in patients with glioma to provide both mechanistic information and potential avenues for adjuvant ascorbate therapy., (© 2022. The Author(s).)

Glioblastoma multiforme is a challenging disease with limited treatment options and poor survival. Glioblastoma tumours are characterised by hypoxia that activates the hypoxia inducible factor (HIF) pathway and controls a myriad of genes that drive cancer progression. HIF transcription factors are regulated at the post-translation level via HIF-hydroxylases. These hydroxylases require oxygen and 2-oxoglutarate as substrates, and ferrous iron and ascorbate as cofactors. In this retrospective observational study, we aimed to determine whether ascorbate played a role in the hypoxic response of glioblastoma, and whether this affected patient outcome. We measured the ascorbate content and members of the HIF-pathway of clinical glioblastoma samples, and assessed their association with clinicopathological features and patient survival. In 37 samples (37 patients), median ascorbate content was 7.6 μg ascorbate/100 mg tissue, range 0.8 - 20.4 μg ascorbate/100 mg tissue. In tumours with above median ascorbate content, HIF-pathway activity as a whole was significantly suppressed (p = 0.005), and several members of the pathway showed decreased expression (carbonic anhydrase-9 and glucose transporter-1, both p < 0.01). Patients with either lower tumour HIF-pathway activity or higher tumour ascorbate content survived significantly longer than patients with higher HIF-pathway or lower ascorbate levels (p = 0.011, p = 0.043, respectively). Median survival for the low HIF-pathway score group was 362 days compared to 203 days for the high HIF-pathway score group, and median survival for the above median ascorbate group was 390 days, compared to the below median ascorbate group with 219 days. The apparent survival advantage associated with higher tumour ascorbate was more prominent for the first 8 months following surgery. These associations are promising, suggesting an important role for ascorbate-regulated HIF-pathway activity in glioblastoma that may impact on patient survival., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Burgess, Crake, Phillips, Morrin, Royds, Slatter, Wiggins, Vissers, Robinson and Dachs.)

The maternal diet during pregnancy is a key determinant of offspring health. Early studies have linked poor maternal nutrition during gestation with a propensity for the development of chronic conditions in offspring. These conditions include cardiovascular disease, type 2 diabetes and even compromised mental health. While multiple factors may contribute to these outcomes, disturbed epigenetic programming during early development is one potential biological mechanism. The epigenome is programmed primarily in utero, and during this time, the developing fetus is highly susceptible to environmental factors such as nutritional insults. During neurodevelopment, epigenetic programming coordinates the formation of primitive central nervous system structures, neurogenesis, and neuroplasticity. Dysregulated epigenetic programming has been implicated in the aetiology of several neurodevelopmental disorders such as Tatton-Brown-Rahman syndrome. Accordingly, there is great interest in determining how maternal nutrient availability in pregnancy might affect the epigenetic status of offspring, and how such influences may present phenotypically. In recent years, a number of epigenetic enzymes that are active during embryonic development have been found to require vitamin C as a cofactor. These enzymes include the ten-eleven translocation methylcytosine dioxygenases (TETs) and the Jumonji C domain-containing histone lysine demethylases that catalyse the oxidative removal of methyl groups on cytosines and histone lysine residues, respectively. These enzymes are integral to epigenetic regulation and have fundamental roles in cellular differentiation, the maintenance of pluripotency and development. The dependence of these enzymes on vitamin C for optimal catalytic activity illustrates a potentially critical contribution of the nutrient during mammalian development. These insights also highlight a potential risk associated with vitamin C insufficiency during pregnancy. The link between vitamin C insufficiency and development is particularly apparent in the context of neurodevelopment and high vitamin C concentrations in the brain are indicative of important functional requirements in this organ. Accordingly, this review considers the evidence for the potential impact of maternal vitamin C status on neurodevelopmental epigenetics.

Background/aims: Maintenance of whole-body ascorbate levels and distribution is mediated via sodium-dependent vitamin C transporters (SVCTs). The kidney is one of a few organs that express both SVCT1 and SVCT2. Recent evidence suggests that accumulation of ascorbate may be different in tumour compared to normal tissue, but data on SVCT levels in tumours is sparse., Methods: The role of the two SVCT isoforms in ascorbate uptake in renal cell carcinoma (RCC) was investigated in vitro and in clinical samples. In three human RCC cell lines, we investigated SVCT protein levels and cellular location in response to ascorbate supplementation and withdrawal. In clinical RCC samples (n=114), SVCT patterns of staining and protein levels were analysed and compared to ascorbate levels., Results: In cell culture, transporter levels and cellular location were not modified by ascorbate availability at any time up to 8h, although basal SVCT2 levels governed maximal ascorbate accumulation. In clinical samples, SVCT1 protein levels in papillary RCC (pRCC) were similar to matched normal renal cortex, but were increased in clear-cell RCC (ccRCC). Native SVCT2 (72 kDa) was significantly decreased in both pRCC and ccRCC tissues compared to cortex (p<0.01), whereas a modified form of SVCT2 (100 kDa) was significantly increased (p<0.001). There was no association between the transporters (SVCT1, native or modified SVCT2) and ascorbate concentrations in either normal or tumour tissues. SVCT1 and SVCT2 displayed diffuse cytoplasmic staining in both pRCC and ccRCC tumour cells, with cortex showing distinct membrane staining for SVCT1., Conclusion: We observed a re-distribution of ascorbate transporters in tumour tissue compared to normal cortex and a shift from native to modified SVCT2 in cell culture and clinical samples. Data presented here show that SVCT protein levels do not appear to predict intracellular ascorbate accumulation in RCC., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Loss-of-function mutations in the DNA demethylase TET2 are associated with the dysregulation of hematopoietic stem cell differentiation and arise in approximately 10% of de novo acute myeloid leukemia (AML). TET2 mutations coexist with other mutations in AML, including TP53 mutations, which can indicate a particularly poor prognosis. Ascorbate can function as an epigenetic therapeutic in pathological contexts involving heterozygous TET2 mutations by restoring TET2 activity. How this response is affected when myeloid leukemia cells harbor mutations in both TET2 and TP53 is unknown. Therefore, we examined the effects of ascorbate on the SKM-1 AML cell line that has mutated TET2 and TP53 . Sustained treatment with ascorbate inhibited proliferation and promoted the differentiation of these cells. Furthermore, ascorbate treatment significantly increased 5-hydroxymethylcytosine, suggesting increased TET activity as the likely mechanism. We also investigated whether ascorbate affected the cytotoxicity of Prima-1 Met , a drug that reactivates some p53 mutants and is currently in clinical trials for AML. We found that the addition of ascorbate had a minimal effect on Prima-1 Met -induced cytotoxicity, with small increases or decreases in cytotoxicity being observed depending on the timing of treatment. Collectively, these data suggest that ascorbate could exert a beneficial anti-proliferative effect on AML cells harboring both TET2 and TP53 mutations whilst not interfering with targeted cytotoxic therapies such as Prima-1 Met ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Smith-Díaz, Magon, McKenzie, Hampton, Vissers and Das.)

Gliomas are a heterogeneous group of cancers that predominantly arise from glial cells in the brain, but may also arise from neural stem cells, encompassing low-grade glioma and high-grade glioblastoma. Whereas better diagnosis and new treatments have improved patient survival for many cancers, glioblastomas remain challenging with a highly unfavorable prognosis. This review discusses a super-family of enzymes, the 2-oxoglutarate dependent dioxygenase enzymes (2-OGDD) that control numerous processes including epigenetic modifications and oxygen sensing, and considers their many roles in the pathology of gliomas. We specifically describe in more detail the DNA and histone demethylases, and the hypoxia-inducible factor hydroxylases in the context of glioma, and discuss the substrate and cofactor requirements of the 2-OGDD enzymes. Better understanding of how these enzymes contribute to gliomas could lead to the development of new treatment strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Crake, Burgess, Royds, Phillips, Vissers and Dachs.)

Tumour-associated macrophages (TAMs) are ubiquitously present in tumours and commonly associated with poor prognosis. In immune cells, ascorbate affects epigenetic regulation, differentiation and phenotype via its co-factor activity for the 2-oxoglutarate dependent dioxygenase enzymes. Here, we determined the effect of ascorbate on TAM development in response to tumour microenvironmental cues. Naïve murine bone marrow monocytes were cultured with Lewis Lung Carcinoma conditioned media (LLCM) or macrophage colony-stimulating factor (MCSF) to encourage the development into tumour-associated macrophages. Cells were stimulated with hypoxia (1% O 2 ), with or without ascorbate (500 µM) supplementation. Cells and media were harvested for gene, cell surface marker and protein analyses. LLCM supported bone marrow monocyte growth with >90% of cells staining CD11b + F4/80 + , indicative of monocytes/macrophages. LLCM-grown cells showed increased expression of M2-like and TAM genes compared to MCSF-grown cells, which further increased with hypoxia. In LLCM-grown cells, ascorbate supplementation was associated with increased F4/80 cell surface expression, and altered gene expression and protein secretion. Our study shows that ascorbate modifies monocyte phenotype when grown under tumour microenvironmental conditions, but this was not clearly associated with either a pro- or anti-tumour phenotype, and reflects a complex and nuanced response of macrophages to ascorbate. Overall, ascorbate supplementation clearly has molecular consequences for TAMs, but functional and clinical consequences remain unknown.

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% Māori/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 Māori/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.

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 H 2 O 2 ; (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/)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dachs, Gandhi, Wohlrab, Carr, Morrin, Pullar, Bayer, Eglinton, Robinson and Vissers.)

Consumption of vitamin C-rich fruits and vegetables has been associated with greater feelings of vitality. However, these associations have rarely been tested in experimental trials. The aim of the current study was to test the effects of eating a vitamin C-rich food (kiwifruit) on subjective vitality and whether effects are driven by vitamin C. Young adults (n = 167, 61.1% female, aged 18–35 years) with plasma vitamin C < 40 µmol/L were allocated to three intervention conditions: kiwifruit (2 SunGold™ kiwifruit/day), vitamin C (250 mg tablet/day), placebo (1 tablet/day). The trial consisted of a two-week lead-in, four-week intervention, and two-week washout. Plasma vitamin C and vitality questionnaires (total mood disturbance, fatigue, and well-being) were measured fortnightly. Self-reported sleep quality and physical activity were measured every second day through smartphone surveys. Nutritional confounds were assessed using a three-day food diary during each study phase. Plasma vitamin C reached saturation levels within two weeks for the kiwifruit and vitamin C groups. Participants consuming kiwifruit showed a trend of improvement in mood disturbance, significantly decreased fatigue, and significantly improved well-being after two weeks of the intervention. Improvements in well-being remained elevated through washout. Consumption of vitamin C tablets alone was associated with improved well-being after two weeks, and additionally improved mood and fatigue for participants with consistently low vitamin C levels during lead-in. Diet records showed that participants consuming kiwifruit reduced their fat intake during the intervention period. Intervention effects remained significant when adjusting for condition allocation groupings, age, and ethnicity, and were not explained by sleep quality, physical activity, BMI, or other dietary patterns, including fat intake. There were no changes in plasma vitamin C status or vitality in the placebo group. Whole-food consumption of kiwifruit was associated with improved subjective vitality in adults with low vitamin C status. Similar, but not identical changes were found for vitamin C tablets, suggesting that additional properties of kiwifruit may contribute to improved vitality.

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.

Plasma vitamin C concentrations fluctuate in response to recent dietary intake; therefore levels are typically determined in the fasting state. Erythrocyte ascorbate concentrations have been shown to be similar to plasma levels, but little is known about the kinetics of ascorbate accumulation in these cells. In this study, we investigated ascorbate uptake into erythrocytes after dietary supplementation with vitamin C and compared it to changes in plasma ascorbate concentrations. Seven individuals with baseline fasting plasma vitamin C concentrations ≥ 50 µmol/L were depleted of vitamin C-containing foods and drinks for one week, and then supplemented with 250 mg vitamin C/day in addition to resuming their normal diet. Fasting or steady-state plasma ascorbate concentrations declined to almost half of their baseline concentration over the week of vitamin C depletion, and then returned to saturation within two days of beginning supplementation. Erythrocyte ascorbate concentrations exhibited a very similar profile to plasma levels, with values ~76% of plasma, and a strong linear correlation (r = 0.89, p < 0.0001). Using a pharmacokinetic study design in six individuals with baseline fasting plasma vitamin C concentrations ≥50 µmol/L, we also showed that, unlike plasma, which peaked between 2 and 4 h following ingestion of 200 mg of vitamin C, erythrocyte ascorbate concentrations did not change in the six hours after supplementation. The data from these two intervention studies indicate that erythrocyte ascorbate concentration provides a stable measure of steady-state plasma ascorbate status and could be used to monitor ascorbate status in healthy non-fasting individuals.

Neutrophils contribute to low oxygen availability at inflammatory sites through the generation of reactive oxidants. They are also functionally affected by hypoxia, which delays neutrophil apoptosis. However, the eventual fate of neutrophils in hypoxic conditions is unknown and this is important for their effective clearance and the resolution of inflammation. We have monitored the survival and function of normal human neutrophils exposed to hypoxia over a 48 h period. Apoptosis was delayed, and the cells remained intact even at 48 h. However, hypoxia promoted significant changes in neutrophil morphology with the appearance of many new cytoplasmic vesicles, often containing cell material, within 5 hours of exposure to low O 2 . This coincided with an increase in LC3B-II expression, indicative of autophagosome formation and an autophagy-like process. In hypoxic conditions, neutrophils preferentially lost myeloperoxidase, a marker of azurophil granules. Short-term (2 h) hypoxic exposure resulted in sustained potential to generate superoxide when O 2 was restored, but the capacity for oxidant production was lost with longer periods of hypoxia. Phagocytic ability was unchanged by hypoxia, and bacterial killing by neutrophils in both normoxic and hypoxic conditions was substantially diminished after 24 hours. However, pre-exposure to hypoxia resulted in an enhanced ability to kill bacteria by oxidant-independent mechanisms. Our data provide the first evidence for hypoxia as a driver of neutrophil autophagy that can influence the function and ultimate fate of these cells, including their eventual clearance and the resolution of inflammation., (©2019 Society for Leukocyte Biology.)

Scope: Blueberry polyphenols are thought to confer cardiovascular health benefits, but have limited bioavailability. They undergo extensive metabolism and their phenolic acid metabolites are likely to be the mediators of bioactivity. The effect of blueberry-derived phenolic acids on one aspect of inflammation, monocyte adhesion to vascular endothelial cells, is investigated., Methods and Results: The major blueberry-derived phenolic acids in human plasma are identified and quantified. Three test mixtures representing compounds present at 0-4 h (Early), 4-24 h (Late), or 0-24 h (Whole) are used to investigate the effect on adhesion of monocytes to tumor necrosis factor alpha (TNFα)-activated endothelial cells. The Late mixture reduces monocyte adhesion, but there is no effect of the Early or Whole mixtures. Exclusion of syringic acid from each mixture results in inhibition of monocyte adhesion. Exposure to the phenolic acid mixtures has no effect on the endothelial surface expression of adhesion molecules intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or E-selectin, suggesting that other molecular mechanisms are responsible for the observed effect., Conclusion: This study shows that physiological concentrations of blueberry polyphenol metabolites can help maintain cardiovascular health by regulating monocyte adhesion to the vascular endothelium., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The transcription factor hypoxia inducible factor (HIF) -1 drives tumor growth and metastasis and is associated with poor prognosis in breast cancer. Ascorbate can moderate HIF-1 activity in vitro and is associated with HIF pathway activation in a number of cancer types, but whether tissue ascorbate levels influence the HIF pathway in breast cancer is unknown. In this study we investigated the association between tumor ascorbate levels and HIF-1 activation and patient survival in human breast cancer., Methods: In a retrospective analysis of human breast cancer tissue, we analysed primary tumor and adjacent uninvolved tissue from 52 women with invasive ductal carcinoma. We measured HIF-1α, HIF-1 gene targets CAIX, BNIP-3 and VEGF, and ascorbate content. Patient clinical outcomes were evaluated against these parameters., Results: HIF-1 pathway proteins were upregulated in tumor tissue and increased HIF-1 activation was associated with higher tumor grade and stage, with increased vascular invasion and necrosis, and with decreased disease-free and disease-specific survival. Grade 1 tumors had higher ascorbate levels than did grade 2 or 3 tumors. Higher ascorbate levels were associated with less tumor necrosis, with lower HIF-1 pathway activity and with increased disease-free and disease-specific survival., Conclusions: Our findings indicate that there is a direct correlation between intracellular ascorbate levels, activation of the HIF-1 pathway and patient survival in breast cancer. This is consistent with the known capacity of ascorbate to stimulate the activity of the regulatory HIF hydroxylases and suggests that optimisation of tumor ascorbate could have clinical benefit via modulation of the hypoxic response.

Hypoxia-inducible transcription factors (HIFs) drive angiogenesis and cancer cell growth, contributing to an aggressive tumor phenotype. HIF-α protein levels and activity are controlled at the post-translational level by HIF hydroxylases. Hydroxylated HIF-α is recognized by the von Hippel Lindau (VHL) tumor suppressor and targeted for degradation. The HIF hydroxylases are members of the iron and 2-oxoglutarate-dependent dioxygenases, which require ascorbate as cofactor for activity. Clear cell renal cell carcinomas (ccRCC) harbor mutations in the VHL gene, whereas papillary RCC (pRCC) have a functional VHL. These natural occurring VHL variants in RCC enable the testing, in clinical samples, of the hypothesis that ascorbate modulates HIF-α levels through its role as a cofactor for the HIF hydroxylases. We measured ascorbate, HIF-1α, and HIF-2α protein and HIF downstream targets BNIP3, CA9, cyclin D1, GLUT1, and VEGF (combined to generate the HIF pathway score) in VHL-defective ccRCC ( n = 73) and VHL-proficient pRCC human tumor tissue ( n = 41). HIF and ascorbate levels were increased in ccRCC and pRCC tumors compared to matched renal cortex. HIF-1 and total HIF pathway activation scores were decreased with higher ascorbate in pRCC tumors (Spearman r = -0.38, p < 0.05 and r = -0.35, p < 0.05). This was not evident for ccRCC tumors. In mechanistic studies in vitro , ascorbate influenced HIF-1 activity in VHL-proficient, but not VHL-defective ccRCC cells. Our results indicate that ccRCC, which lacks a functional VHL, does not respond to ascorbate-mediated modulation of the HIF response. This contrasts with the demonstrated association between ascorbate content and the HIF pathway observed in pRCC and other tumors with a functional VHL. The results support a role for ascorbate as a modulator of HIF activity and tumor aggression in cancer types with a functional hypoxic response.

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., (© 2018 The Author(s).)

Micronutrient status is thought to impact on psychological mood due to the role of nutrients in brain structure and function. The aim of the current study was to investigate the association of vitamin C status with mood state in a sample of male tertiary students. We measured fasting plasma vitamin C levels as an indicator of vitamin C status, and subjective mood was determined using the Profile of Mood States (POMS) questionnaire. One hundred and thirty-nine male students aged 18 to 35 years were recruited from local tertiary institutes in Christchurch, New Zealand. The average plasma vitamin C concentration was 58.2 ± 18.6 (SD) µmol/L and the average total mood disturbance score was 25.5 ± 26.6 (possible score -32 to 200 measuring low to high mood disturbance, respectively). Plasma vitamin C concentration was inversely correlated with total mood disturbance as assessed by POMS (r = -0.181, p < 0.05). Examination of the individual POMS subscales also showed inverse associations of vitamin C status with depression, confusion, and anger. These findings suggest that high vitamin C status may be associated with improved overall mood in young adult males.

Whether vitamin C (ascorbate) has a role to play as an anti-cancer agent has been debated for decades. Ascorbate has been used by cancer patients in an unregulated environment, either as a dietary supplement or in pharmacological doses administered by infusion, with numerous reports of clinical benefit, but in the absence of rigorous clinical trial data. The design of appropriate clinical trials has been hindered by a lack of understanding of the mechanism(s) of action that would inform the choice of effective dose, timing of administration and likely responsive cancer models. More recently, expanded understanding of the biological activities of ascorbate has led to a number of plausible hypotheses for mechanisms of anti-cancer activity. Prominent among these are the generation of significant quantities of hydrogen peroxide by the autoxidation of supra-physiological concentrations of ascorbate and stimulation of the 2-oxoglutarate-dependent dioxygenase family of enzymes (2-OGDDs) that have a cofactor requirement for ascorbate. Hydrogen peroxide generation is postulated to generate oxidative stress that preferentially targets cancer cells. The 2-OGDDs include the hydroxylases that regulate the hypoxic response, a major driver of tumor survival, angiogenesis, stem cell phenotype and metastasis, and the epigenetic histone and DNA demethylases. The latter are of particular interest, with recent studies suggesting a promising role for ascorbate in the regulation of the ten-eleven translocase (TET) DNA demethylases in hematological cancers. Support for these proposed mechanisms has come from many in vitro studies, and xenograft animal models have consistently shown an anti-cancer effect of ascorbate administration. However, decisive evidence for any particular mechanism(s) of action is not yet available from an in vivo setting. With a number of early phase clinical trials currently underway, evidence for potential mechanism(s) of action is required to inform the most appropriate study design and choice of cancer model. Hopefully such information will result in sound clinical data that will avert adding any further controversy to this already contentious debate.

Scope: Blueberry consumption is believed to confer a cardiovascular health advantage, but the active compounds and effects require characterization. This study aims to identify the polyphenol metabolites in plasma after blueberry juice intake and determine their bioactivity on endothelial cells., Methods and Results: Three healthy individuals are recruited to obtain profiles of bioavailable plasma polyphenol metabolites following intake of blueberry juice. Of 33 phenolic compounds screened, 12 aglycone phenolic acids are detected and their maximum plasma concentrations and circulation time determined. Using this information, the effect of three physiologically relevant mixtures of blueberry-derived phenolic acids is investigated for their ability to induce nuclear factor erythroid 2-related factor 2 (Nrf2)-nuclear translocation and downstream gene expression in human endothelial cells. Pretreatment with the phenolic acids for 18 h results in a significant upregulation of the Nrf2-regulated antioxidant response proteins heme oxygenase 1 (HO-1) and glutamate-cysteine ligase modifier subunit (GCLM), following 6 h exposure to 2.5 μm H 2 O 2 ., Conclusion: Physiologically relevant concentrations of blueberry-derived aglycone phenolic acids can induce Nrf2-regulated antioxidant response proteins in vascular endothelial cells in response to low μm concentrations of H 2 O 2 . Our results represent an advance over previous studies that have used single compounds or high concentrations in cell-based investigations., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The inflammatory and metabolic derangements of obesity in pregnant women generate an adverse intrauterine environment, increase pregnancy complications and adverse fetal outcomes and program the fetus for obesity and metabolic syndrome in later life. We hypothesized that epigenetic modifications in placenta including altered DNA methylation/hydroxymethylation may mediate these effects. Term placental villous tissue was collected following cesarean section from lean (prepregnancy BMI<25) or obese (BMI>30) women. Genomic DNA was isolated, methylated and hydroxymethylated DNA immunoprecipitated and hybridized to the NimbleGen 2.1M human DNA methylation array. Intermediate metabolites in placental tissues were measured by HPLC-ESI-MS, ascorbate levels by reverse phase HPLC and gene expression by RT-PCR. Differentially methylated and hydroxymethylated regions occurred across the genome, with a 21% increase in methylated but a 31% decrease in hydroxymethylated regions in obese vs lean groups. Whereas increased methylation and decreased methylation was evident around transcription start sites of multiple genes in the GH/CSH and PSG gene clusters on chromosomes 17 and 19 in other areas there was no relationship. Increased methylation was associated with decreased expression only for some genes in these clusters. Biological pathway analysis revealed the 262 genes which showed reciprocal differential methylation/ hydroxymethylation were enriched for pregnancy, immune response and cell adhesion-linked processes. We found a negative relationship for maternal BMI but a positive relationship for ascorbate with α-ketoglutarate a metabolite that regulates ten eleven translocase (TET) which mediates DNA methylation. We provide evidence for the obese maternal metabolic milieu being linked to an altered DNA methylome that may affect placental gene expression in relation to adverse outcomes.

The primary function of the skin is to act as a barrier against insults from the environment, and its unique structure reflects this. The skin is composed of two layers: the epidermal outer layer is highly cellular and provides the barrier function, and the inner dermal layer ensures strength and elasticity and gives nutritional support to the epidermis. Normal skin contains high concentrations of vitamin C, which supports important and well-known functions, stimulating collagen synthesis and assisting in antioxidant protection against UV-induced photodamage. This knowledge is often used as a rationale for the addition of vitamin C to topical applications, but the efficacy of such treatment, as opposed to optimising dietary vitamin C intake, is poorly understood. This review discusses the potential roles for vitamin C in skin health and summarises the in vitro and in vivo research to date. We compare the efficacy of nutritional intake of vitamin C versus topical application, identify the areas where lack of evidence limits our understanding of the potential benefits of vitamin C on skin health, and suggest which skin properties are most likely to benefit from improved nutritional vitamin C intake., Competing Interests: The authors declare no conflict of interest. Zespri International, a partial funder, had no influence on the selection of material to cover, nor on the focus and interpretation of the studies reviewed.

A cohort of 50-year-olds from Canterbury, New Zealand ( N = 404), representative of midlife adults, undertook comprehensive health and dietary assessments. Fasting plasma vitamin C concentrations ( N = 369) and dietary vitamin C intake ( N = 250) were determined. The mean plasma vitamin C concentration was 44.2 µmol/L (95% CI 42.4, 46.0); 62% of the cohort had inadequate plasma vitamin C concentrations (i.e., <50 µmol/L), 13% of the cohort had hypovitaminosis C (i.e., <23 µmol/L), and 2.4% had plasma vitamin C concentrations indicating deficiency (i.e., <11 µmol/L). Men had a lower mean plasma vitamin C concentration than women, and a higher percentage of vitamin C inadequacy and deficiency. A higher prevalence of hypovitaminosis C and deficiency was observed in those of lower socio-economic status and in current smokers. Adults with higher vitamin C levels exhibited lower weight, BMI and waist circumference, and better measures of metabolic health, including HbA1c, insulin and triglycerides, all risk factors for type 2 diabetes. Lower levels of mild cognitive impairment were observed in those with the highest plasma vitamin C concentrations. Plasma vitamin C showed a stronger correlation with markers of metabolic health and cognitive impairment than dietary vitamin C.

Objectives: To develop and test the effectiveness of a mobile-phone based ecological momentary intervention (EMI) to increase fruit and vegetable (FV) consumption in low-consuming young adults., Methods: A two-week randomised controlled trial of low-FV consuming young adults ages 18-25 years (n = 171) compared three conditions: ecological momentary intervention (EMI), fruit and vegetable intervention (FVI), and a diet-as-usual control (ANZCTRN12615000183583). Participants in the EMI condition were sent two targeted text messages a day for 13 days and were asked to increase daily FV consumption to at least five servings. These messages were designed, using the Health Action Process Approach, to address salient beliefs identified as effective in a preliminary focus group investigation. Participants in the FVI condition were provided two servings of FV a day (carrots, kiwifruit or oranges, and apples) to eat in addition to their current diet. Control participants ate their normal diet. Participants reported their daily servings of FV each evening during the study using a smartphone-delivered survey. Blood samples testing plasma vitamin C and total carotenoids were taken pre- and post-intervention as an objective biomarker of FV intake., Results: Participants in the EMI and FVI conditions reported higher daily servings of FV - approximately +1 serving per day more compared to control (EMI = 3.7 servings/day; FVI = 3.7 servings/day; Control = 2.8 servings/day) and approximately +1.2 servings compared to baseline. Increases in objective biomarkers for the experimental conditions supported the validity of self-reported FV consumption., Conclusions: Our results provide initial proof of concept that EMI strategies (with minor financial assistance) are as effective as giving FV in increasing FV consumption in educated, low-consuming young adults., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Despite recent evidence for an anti-tumour role for high-dose ascorbate, potential mechanisms of action are still unclear. At mM concentrations that are achieved with high-dose intravenous administration, autoxidation of ascorbate can generate cytotoxic levels of H 2 O 2 . Ascorbate is also a required co-factor for the hydroxylases that suppress the transcription factor hypoxia-inducible factor (HIF-1). HIF-1 supports an aggressive tumour phenotype and is associated with poor prognosis, and previous studies have shown that optimizing intracellular ascorbate levels down-regulates HIF-1 activation. In this study we have simultaneously measured ascorbate concentrations and the HIF-1 pathway activity in tumour tissue following high dose ascorbate administration, and have studied tumour growth and physiology. Gulo -/- mice, a model of the human ascorbate dependency condition, were implanted with syngeneic Lewis lung tumours, 1g/kg ascorbate was administered into the peritoneum, and ascorbate concentrations were monitored in plasma, liver and tumours. Ascorbate levels peaked within 30min, and although plasma and liver ascorbate returned to baseline within 16h, tumour levels remained elevated for 48h, possibly reflecting increased stability in the hypoxic tumour environment. The expression of HIF-1 and its target proteins was down-regulated with tumour ascorbate uptake. Elevated tumour ascorbate levels could be maintained with daily administration, and HIF-1 and vascular endothelial growth factor protein levels were reduced in these conditions. Increased tumour ascorbate was associated with slowed tumour growth, reduced tumour microvessel density and decreased hypoxia. Alternate day administration of ascorbate resulted in lower tumour levels and did not consistently decrease HIF-1 pathway activity. Levels of sodium-dependent vitamin C transporters 1 and 2 were not clearly associated with ascorbate accumulation by murine tumour cells in vitro or in vivo. Our results support the suppression of the hypoxic response by ascorbate as a plausible mechanism of action of its anti-tumour activity, and this may be useful in a clinical setting., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Humans are unable to synthesise ascorbate (Vitamin C) due to the lack of a functional gulonolactone oxidase (Gulo), the enzyme that catalyses the final step in the biosynthesis pathway. Ascorbate is a vital micronutrient required for many biological functions, including as a cofactor for metalloenzymes that regulate the transcription factor hypoxia-inducible factor-1 (HIF-1), which governs cell survival under hypoxia. In most animals, ascorbate is made in liver cells. This study aimed to restore ascorbate synthesis to human hepatocellular carcinoma HepG2 cells and determine the effect of internally produced ascorbate on HIF-1 activation. HepG2 cells were gene-modified with a plasmid encoding the mouse Gulo cDNA, tested for genomic incorporation by PCR and ascorbate synthesis by high performance liquid chromatography. Levels of HIF-1 protein were measured using Western blotting. Gulo-modified HepG2 cells showed increased adherence compared to control HepG2 cells. A PCR-positive clone synthesised ascorbate when the Gulo substrate, l-gulono-1,4-lactone, was supplied. Intracellular ascorbate concentrations reached 5% of saturation levels (6 nmol/10⁶ cells). Addition of ascorbate or gulonolactone reduced HIF-1 accumulation in the Gulo clone, but also in parental HepG2 cells. Our data confirm the requirement for a number of factors in addition to Gulo in the ascorbate biosynthesis pathway in human cells.

Some cells, including neutrophils, accumulate high intracellular ascorbate concentrations, which suggests that they have an important function in these cells. In this study we have used L-gulono-γ-lactone oxidase (Gulo)-/- mice, which are unable to synthesize ascorbate, to generate ascorbate-deficient neutrophils and have used these to investigate the effect of ascorbate on neutrophil function. Peritoneal neutrophils from ascorbate-deficient animals had normal morphology and respiratory burst activity but failed to undergo spontaneous apoptosis, determined by morphology and the surface expression of phosphatidylserine. Initially, there was increased cell survival, but death eventually occurred by necrosis within 48 h. Neutrophils persisted in thioglycollate-induced inflammation in Gulo-/Ȓ mice with the later appearance of necrotic cells, suggesting that apoptosis was also affected in vivo. Also, ascorbate-deficient neutrophils were not recognized by macrophages in an in vitro assay for phagocytosis, providing further evidence for defective apoptosis and clearance. Neutrophils from Gulo-/- mice had elevated levels of hypoxia-inducible factor (HIF)-1α, a transcription factor regulated by Fe 2 + -dependent hydroxylases which require ascorbate for optimal activity. HIF-1α has been shown previously to inhibit neutrophil apoptosis under hypoxic conditions. Our results suggest that in ascorbate deficiency, up-regulation of HIF-1α blocks neutrophil apoptosis under normoxic conditions and that this represents a novel and important function for vitamin C in inflammatory cells., (© 2007 Society for Leukocyte Biology.)