STUDY OF ROLE OF CIRCULATING ANTIOXIDANTS IN DEVELOPMENT OF SENILE CATARACT

Introduction: Senile cataract is the commonest worldwide cause of treatable blindness, most often due to excess reactive oxygen species [ROS]. Anti-oxidant vitamins namely beta-carotene, ascorbate and tocopherol and enzymes like superoxide dismutase (SOD), constitute rst line defenses against ROS assault, while malondialdehyde (MDA) levels indicate the total burden of lipid peroxidation in-vivo. Objectives: We aimed to compare the levels of above ve analytes in senile cataract patients in contrast to apparently healthy controls and also among smoking and non-smoking sub groups of both cases and controls. Methods: A hospital-based case-control study, was conducted with 102 cases of senile cataract and 102 control subjects, following strict inclusion and exclusion criteria. Recruited individuals were sub-categorized into smokers and non-smokers. After overnight fasting (12 hours), 10 ml blood was drawn aseptically. Serum and plasma were separated and used for biochemical estimations of all ve analytes, following established protocols. Levels were compared between cases and controls as well as between the smoking and non-smoking sub-sections of both groups. Results: Signicantly lower levels of plasma ascorbate and serum tocopherol were seen in cases as compared to controls (P=0.0078 and P<0.0001 respectively). Signicantly lower levels of serum beta carotene (P<0.0001), tocopherol (P<0.0001), plasma ascorbate (P<0.0001), and SOD (P<0.0001). Signicantly higher level of serum MDA (P= 0.0494) was seen in the smokers, as compared to non-smokers Conclusions: Lowered serum tocopherol and plasma ascorbate were signicant factors leading to senile cataract. Furthermore, smoking was found crucial in loss of anti-oxidant defenses and subsequent development of cataract.

Neutrophils Isolated from Septic Patients Exhibit Elevated Uptake of Vitamin C and Normal Intracellular Concentrations despite a Low Vitamin C Milieu

Vitamin C (ascorbate) plays an important role in neutrophil function and is accumulated by the cells either directly via vitamin C transporters (SVCT) or indirectly following oxidation to dehydroascorbic acid. Septic patients are known to have significantly depleted plasma ascorbate status, but little is known about the ascorbate content of their circulating cells. Therefore, we assessed the ascorbate concentrations of plasma, leukocytes and erythrocytes from septic patients and compared these to healthy controls. Non-fasting blood samples were collected from healthy volunteers (n = 20) and critically ill patients with sepsis (n = 18). The ascorbate content of the plasma and isolated neutrophils and erythrocytes was measured using HPLC and plasma myeloperoxidase concentrations were determined using ELISA. Ex vivo uptake of ascorbate and dehydroascorbic acid by neutrophils from septic patients was also assessed. Neutrophils isolated from septic patients had comparable intracellular ascorbate content to healthy volunteers (0.33 vs. 0.35 nmol/106 cells, p > 0.05), despite significantly lower plasma concentrations than the healthy controls (14 vs. 88 µmol/L, p < 0.001). In contrast, erythrocytes from septic patients had significantly lower intracellular ascorbate content than healthy controls (30 vs. 69 µmol/L, p = 0.002), although this was 2.2-fold higher than the matched plasma concentrations in the patients (p = 0.008). Higher concentrations of myeloperoxidase, a source of reactive oxygen species, were observed in the septic patients relative to healthy controls (194 vs. 14 mg/mL, p < 0.0001). In contrast to neutrophils from healthy volunteers, the neutrophils from septic patients demonstrated elevated uptake of extracellular ascorbate. Overall, neutrophils from septic patients exhibited comparable intracellular ascorbate content to those from healthy controls, despite the patients presenting with hypovitaminosis C. The mechanisms involved are currently uncertain, but could include increased generation of dehydroascorbic acid in septic patients, enhanced basal activation of their neutrophils or upregulation of their vitamin C transporters.

Reduced Plasma Ascorbate and Increased Proportion of Dehydroascorbic Acid Levels in Patients Undergoing Hemodialysis

Ascorbate functions as an electron donor and scavenges free radicals. Dehydroascorbic acid (DHA), the oxidized form of ascorbate, is generated as a result of these reactions. While low plasma ascorbate levels have been reported in hemodialysis patients worldwide, no studies have measured DHA because it is not generalized. In this study, we aimed to clarify whether plasma ascorbate levels are low in dialysis patients and whether plasma ascorbate levels fluctuate before and after dialysis. Moreover, we applied our previously established method to measure the plasma ascorbate and DHA levels in chronic kidney disease (CKD) stage G3–G5 non-hemodialysis-dependent patients, and pre- and post-dialysis plasma ascorbate and DHA levels in CKD stage G5D hemodialysis patients. The sample size was calculated using G-power software. The pre-dialysis plasma total ascorbate levels, including DHA, were significantly (56%) lower in hemodialysis patients than in non-hemodialysis-dependent CKD patients. After dialysis, there was a 40% reduction in the plasma total ascorbate levels. Hemodialysis increased the post-dialysis plasma proportions of DHA from 37% to 55%. The study results demonstrated lower plasma total ascorbate levels in hemodialysis patients compared with in non-hemodialysis-dependent CKD patients; these low levels in hemodialysis patients were further reduced by hemodialysis and increased DHA proportion.

Low Plasma Ascorbate Levels in Type 2 Diabetic Patients With Adequate Dietary Vitamin C

Background Dietary intake of antioxidative vitamin C plays a protective role in the prevention of oxidative damage in diabetics, demanding increased requirement of vitamin C. Hyperglycemia results in impaired uptake of vitamin C in the cell. The present study was conducted to compare the plasma ascorbate levels in type 2 diabetic patients and controls consuming adequate dietary vitamin C. Methodology Fifty consented type 2 diabetes mellitus (T2DM) patients who were on treatment with oral hypoglycemic drugs and consuming adequate vitamin C in diet were taken in the study and 50 healthy controls equitably matched for age, gender between 40 and 70 years, and dietary intake of vitamin C were compared. Dietary intake of vitamin C was estimated by a food frequency questionnaire. Subjects consuming more than 35 mg/d of vitamin C were included in the study. Fasting blood sugar was estimated by glucose oxidase and peroxidase method and estimation of ascorbic acid was done by using 2, 4 dinitro phenyl hydrazine method. Result The mean ± standard deviation levels of plasma ascorbate levels in diabetic subjects were 0.22 ± 0.12 mg/dL, which were significantly lower as compared with controls with plasma ascorbate level of 0.47 ± 0.15 mg/dL. In diabetic subjects, insignificant positive correlation was observed between these parameters with r-value 0.168 and p-value 0.245. Conclusion This study concludes that even with the recommended dietary intake of vitamin C low plasma ascorbate levels were found among T2DM patients, which necessitates increased demand and dietary advice to diabetic patients on consuming foods rich in vitamin C more than the recommended daily allowance.

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 &lt; 0.0001) over 4 days and erythrocyte ascorbate from 18 ± 20 µM to 2509 ± 1016 µM (p &lt; 0.005). Tumor ascorbate increased from 15 ± 6 to 28 ± 6 mg/100 g tissue (p &lt; 0.0001) and normal tissue from 14 ± 6 to 21 ± 4 mg/100 g (p &lt; 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 RegistrationANZCTR Trial ID ACTRN12615001277538 (https://www.anzctr.org.au/).

191 Effects of injectable vitamin C at weaning and prior to transit on growth performance and immune function of beef steers

Ninety-one early-weaned (65 ± 11 d) Angus steers (92 ± 4 kg) were blocked by age to a 2 × 2 factorial examining effects of injectable vitamin C (VC) at weaning and/or prior to transport to the feedlot on antibody titers and growth performance. Injections (20 mL/steer) of VC (250 mg sodium ascorbate/mL) or saline (SAL) were given at time of weaning on d 0 (WEAN) and/or prior to a 6 hr trucking event to a feedlot on d 49 (TRANS). Steers were given booster vaccinations on d 0. Steers were weighed on d 0, 1, 14, 48, 49, 64, 106, and 107. Blood was collected (12 steers/treatment) on d 0, 1, 2, 14, 49 (pre- and post-transit), 50, and 51. Data were analyzed via Proc-Mixed of SAS (experimental unit = steer; n = 22–23/treatment) with fixed effects of block, WEAN, TRANS, and WEAN × TRANS. Plasma ascorbate concentrations for weaning (d 0, 1, and 2) and transit (d 49-pre-trucking, 49-post-trucking, 50, and 51) were analyzed as repeated measures (repeated effect = day). Plasma ascorbate concentrations were greater on d 1 and 2 for steers that received VC at weaning (VC = 19.6, SAL = 8.8 ± 1.26 µM; WEAN × day P &lt; 0.01). Similarly, ascorbate concentrations were greater on d 49 post-trucking, 50, and 51 for steers that received VC pre-transit (TRANS × day P = 0.01). Treatments did not affect bodyweight or average daily gain throughout the trial (P &gt; 0.32). There were no effects of treatment on serum Bovine Viral Diarrhea Virus type 2 antibody titers on d 14 or 51 (P &gt; 0.33). An injection of VC administered to early weaned beef steers at weaning or pre-transit increases plasma ascorbate concentrations but does not improve growth performance or antibody response to vaccination booster.

392 Vitamin C and transit stress in beef cattle

Transportation of cattle by road is unavoidable in beef production due to segmentation of the industry. Psychological and physical stress associated with transit can negatively impact cattle health and performance upon arrival at their destination. Thus, investigation of resiliency or recovery-based nutritional strategies to mitigate transit stress are warranted. Because oxidative stress is likely linked to transportation stress, one such strategy is antioxidants such as vitamin C (VC). Seventy-two Angus-cross steers (356 ± 18 kg) were used to determine the effects of a pre- versus post-transit VC injection on 56 d feedlot performance. Steers were randomly assigned to intramuscular injection treatments (24 steers/treatment): saline pre- and post-transit (CON), VC (Vet One; 250 mg sodium ascorbate/mL; 5 g sodium ascorbate/steer) pre-transit and saline post-transit (PRE), or saline pre-transit and VC post-transit (POST). Steers were transported for ~18 h (1,675 km) prior to sorting into pens equipped with GrowSafe bunks (6 steers/pen). Blood was collected from 12 steers/treatment on d 0, 1, 2, and 7 for analysis of plasma ascorbate concentrations. Plasma ascorbate concentrations were decreased by ~10% immediately post-transit for CON and POST-steers but increased for PRE-steers; regardless of treatment, concentrations were similar to pre-transit values by d 7 (treatment × day; P &lt; 0.01). Steers that received VC at either timepoint (pre- or post-transit) exhibited greater dry matter intake from d 31–57 and d 1–57 compared to CON-steers (P ≤ 0.02). However, PRE-steers exhibited the greatest average daily gain from d 7–31 and d 1–57 (P ≤ 0.05), resulting in PRE-steers being heaviest on d 30/31 (P = 0.03) and tending to be heaviest on d 56/57 (P = 0.07). Based on these data, timing of nutritional intervention is vital for optimal effectiveness. Furthermore, nutritional strategies that improve antioxidant status prior to long-distance transit events may positively influence post-transit performance.

Vitamin C administration by intravenous infusion increases tumour ascorbate content in patients with colon cancer: a clinical intervention study

Background: The use of high dose ascorbate infusions in cancer patients is widespread, but without evidence of efficacy. Several mechanisms whereby ascorbate could affect tumour progression have been proposed, including (i) the localised 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 and (iii) increased oxidative stress induced by dehydroascorbic acid. We hypothesise that the dysfunctional vasculature of solid tumours results in compromised delivery of ascorbate to poorly perfused regions of the tumour and that this ascorbate deficit acts as an additional driver of the hypoxic response via upregulation of HIFs. Using a randomised ‘therapeutic window of opportunity’ clinical study we aimed to determine whether ascorbate infusions affected tumour ascorbate content and tumour biology.Methods: Patients with colon cancer were randomised to receive infusions of up to 1 g/kg ascorbate for four days before surgical resection (n=9) or to not receive infusions (n=6). Ascorbate was measured in plasma, erythrocytes, tumour and histologically normal mucosa at diagnostic colonoscopy and at surgery. Protein markers of tumour hypoxia or DNA damage were monitored in resected tissue.Results: Plasma ascorbate reached millimolar levels following infusion and returned to micromolar levels over 24 h. Pre-infusion plasma ascorbate increased from 38 ± 10 mM to 241 ± 33 mM (p<0.0001) over four days and erythrocyte ascorbate from 14 ± 15 mM to 2004 ± 814 mM (p<0.005). Tumour ascorbate increased from 15 ± 6 to 28 ± 6 mg/100g tissue (p<0.0001) and normal tissue from 14 ± 6 to 21 ± 4 mg/100g (p<0.001). A gradient of lower ascorbate was evident towards the tumour centre in both control and infusion samples. Lower expression of hypoxia-associated proteins was seen in post-infusion tumours compared with controls. There were no significant adverse events and quality of life was unaffected by ascorbate infusion.Conclusions: This is the first clinical study to demonstrate that tumour ascorbate levels increase following infusion, even in regions of poor diffusion, and that this could modify tumour biology. Trial registration: ANZCTR Trail ID ACTRN12615001277538

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.

Evaluating extremely low plasma ascorbate levels and reduction of plasma ascorbate levels by dialysis in Japanese hemodialysis patients

Background: Low plasma ascorbate levels in hemodialysis patients have been reported worldwide; hence, many end-stage kidney disease patients are forced to restrict their diets, especially potassium-rich fruits and vegetables, to prevent hyperkalemia. In this study, we aimed to clarify whether plasma ascorbate levels are low in Japanese dialysis patients and whether plasma ascorbate levels fluctuate before and after dialysis. In addition, we aimed to clarify whether there are clinical test items that have a causal relationship with plasma ascorbate levels.Methods: Plasma ascorbate levels in 27 chronic kidney disease (CKD) stage G3–G5 patients (mean age 84 years) and pre- and post-dialysis plasma ascorbate levels in 19 CKD stage G5D hemodialysis patients (mean age 79 years) were determined using high-performance liquid chromatography and electrochemical detection.Results: Pre-dialysis plasma ascorbate levels in hemodialysis patients (12.0 ± 1.4 µM) were significantly lower (by 56%) than those in CKD stage G3–G5 patients (27.1 ± 2.7 µM). After dialysis, there was a 40% reduction in plasma ascorbate levels. Moreover, pre-dialysis ascorbate levels correlated significantly with plasma potassium levels.Conclusions: The study results indicate that Japanese hemodialysis patients have lower plasma ascorbate levels than CKD stage G3–G5 patients and that these low plasma ascorbate levels in hemodialysis patients were further reduced by hemodialysis. To avoid the development of scurvy in hemodialysis patients, it is necessary to take sufficient ascorbate from supplements or medicines.