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Effects of intravenous administration of ascorbic acid (vitamin C) on oxidative status in healthy adult horses

Corresponding Author

Sandra D. Taylor

[email protected]

orcid.org/0000-0002-8807-5554

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana, USA

Correspondence

Sandra D. Taylor, Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, 625 Harrison Street, West Lafayette, IN 47907, USA.

Email: [email protected]

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Kelsey A. Hart,

Kelsey A. Hart

orcid.org/0000-0003-4331-6470

Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

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Sarah Vaughn,

Sarah Vaughn

orcid.org/0000-0002-8347-7247

Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

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Shyla C. Giancola,

Shyla C. Giancola

Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

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Priscila B. S. Serpa,

Priscila B. S. Serpa

orcid.org/0000-0001-6073-2172

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana, USA

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Andrea P. Santos,

Andrea P. Santos

orcid.org/0000-0002-1037-970X

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana, USA

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Sandra D. Taylor,

Corresponding Author

Sandra D. Taylor

[email protected]

orcid.org/0000-0002-8807-5554

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana, USA

Correspondence

Sandra D. Taylor, Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, 625 Harrison Street, West Lafayette, IN 47907, USA.

Email: [email protected]

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Kelsey A. Hart,

Kelsey A. Hart

orcid.org/0000-0003-4331-6470

Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

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Sarah Vaughn,

Sarah Vaughn

orcid.org/0000-0002-8347-7247

Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

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Shyla C. Giancola,

Shyla C. Giancola

Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

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Priscila B. S. Serpa,

Priscila B. S. Serpa

orcid.org/0000-0001-6073-2172

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana, USA

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Andrea P. Santos,

Andrea P. Santos

orcid.org/0000-0002-1037-970X

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana, USA

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First published: 10 November 2023

https://doi.org/10.1111/jvim.16934

Citations: 3

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Abstract

Background

Ascorbic acid (AA) is an antioxidant that might be beneficial for adjunctive treatment of sepsis in horses. The optimal dose and effects on oxidative status are unknown.

Hypothesis

Ascorbic acid administration will increase plasma AA concentrations and decrease determinants of reactive oxygen metabolites (dROM), basal and stimulant-induced intraerythrocytic reactive oxygen species (ROS) concentrations, and stimulant-induced neutrophil ROS production, and increase plasma antioxidant capacity (PAC) in a dose-dependent manner.

Animals

Eight healthy horses.

Methods

Randomized placebo-controlled crossover study. Each horse received 4 single-dose IV treatments including AA at 25, 50, and 100 mg/kg and saline (placebo) with each treatment separated by ≥1 week. Blood was collected at baseline, 2 and 6 hours for assessment of plasma dROM and PAC via photometer, intraerythrocytic ROS by flow cytometry, and stimulant-induced neutrophil ROS by a fluorometric assay. Plasma AA concentrations were measured by high-performance liquid chromatography/electrochemical detection.

Results

Ascorbic acid at 100 mg/kg resulted in decreased dROM 2 hours after treatment (P = .03, 95% CI 5.51-121.2, point estimate 63.3). There was no effect of AA on basal or stimulant-induced intraerythrocytic ROS (P = .88, 95% CI −0.156 to 0.081, point estimate −0.037; P = .93, 95% CI −0.123 to 0.112, point estimate −0.006, respectively), basal or stimulant-induced neutrophil ROS (P ≥ .12, 95% CI −644.9 to 56.2, point estimate −294.4), or PAC (P ≥ .64, 95% CI −1567 to 463.4, point estimate −552.0) at any dose or timepoint. Plasma AA concentrations increased in a dose-dependent manner.

Conclusions and Clinical Importance

High-dose administration of AA might provide antioxidant benefits in horses.

Abbreviations

AA: ascorbic acid
dROM: determinants of reactive oxygen metabolites
ECD: electrochemical detection
FRAS-5: free radical analytical system-5
HPLC: high-performance liquid chromatography
MFI: median fluorescence intensity
PAC: plasma antioxidant capacity
ROS: reactive oxygen species
SVCT: sodium-dependent vitamin C transporter

1 INTRODUCTION

Oxidative stress is integral to the pathogenesis of sepsis, with overproduction of reactive oxygen species (ROS) leading to endothelial disruption, decreased vascular tone, and increased vascular permeability.^{1, 2} Progressive oxidative damage can result in organ failure and death.^{3, 4} Antioxidant mechanisms that mitigate oxidative damage include enzymatic defenses such as glutathione peroxidase, superoxide dismutase and catalase, and non-enzymatic defenses, including ascorbic acid (AA; vitamin C), α-tocopherol (vitamin E), and reduced glutathione.^{5, 6} Oxidative stress develops when these endogenous defense mechanisms are overwhelmed.

Ascorbic acid offers benefits for adjunctive antioxidant treatment of sepsis. Antioxidant effects of AA are mediated through scavenging of ROS and activation of other scavengers.^7-9 Ascorbic acid inhibits enzymes that promote ROS production, protecting endothelial integrity and mitigating hypotension and shock.¹⁰ In addition, AA is a co-factor for enzymes that synthesize vasopressin and catecholamines, critical players in maintaining tissue perfusion during sepsis.^11-13 Ascorbic acid is also directly anti-inflammatory and bacteriostatic, improving neutrophil chemotaxis, oxidative killing of bacteria, and T lymphocyte function in mice exposed to lipopolysaccharide (LPS; endotoxin).^{14, 15} Plasma AA concentrations are lower in septic people.^16-20 In animal models of sepsis, intravenous (IV) administration of AA supports vasomotor responses and organ function, and decreases vascular permeability.^21-23 In septic human patients, pro-inflammatory biomarkers are lower in those who received AA, as was the need for vasopressor support.^{24, 25} However, the impact of AA on survival in humans with sepsis is variable. A systematic review and meta-analysis of randomized controlled trials concluded that adjunctive AA therapy did not improve outcome in septic patients, although doses administered in those trials were low to moderate.²⁶ In contrast, so-called “high-dose” AA was associated with improved outcome in similar trials.^{27, 28}

Despite advances in therapeutic approaches, sepsis remains a common cause of morbidity and death in neonatal and adult horses.^29-32 There is minimal data on the use of exogenous AA in adult horses and foals, but the oral bioavailability of AA is poor while IV administration results in expected increases in plasma AA concentrations.^{33, 34} In an experimental trial where 32 healthy horses received a sublethal dose of LPS, endogenous plasma AA concentrations decreased 1 hour after LPS infusion in 83% of horses, suggesting that AA depletion might contribute to the deleterious effects of endotoxemia.³⁵ Furthermore, AA supplementation was associated with attenuated neutropenia after LPS infusion, suggesting that exogenous AA might support innate immune responses during sepsis.³⁵

The effects of AA on equine oxidant responses are poorly characterized, and ideal dosing protocols are unknown. Therefore, the objective of this study was to determine the dose-dependent effects of AA on plasma derivatives of reactive oxygen metabolites (dROM), plasma antioxidant capacity (PAC), intraerythrocytic and stimulant-induced neutrophil ROS production after single-dose administration to healthy adult horses. We hypothesized that AA administration would decrease dROM, intraerythrocytic and neutrophil ROS production, and increase PAC in a dose-dependent fashion. Further objectives were to assess adverse effects after AA administration at increasing doses, and measure plasma AA concentration after exogenous administration.

2 METHODS

2.1 Animals and study design

A randomized placebo-controlled crossover study was performed in 8 healthy adult horses obtained from a university teaching herd. Horses were determined to be healthy based on known medical history over the past year or longer and normal physical examination findings immediately before and during the course of the sampling period. Five mares, 2 geldings, and 1 stallion (median age 14.9 years; range 13-24 years) were included in the study. Three breeds were represented, including 5 Quarter horses, 2 Thoroughbreds, and 1 Paint horse. The median weight of the horses was 527 kg (range 483-598 kg); horses were weighed before the start of the study to ensure accurate dosing. Each horse received 1 of 4 AA doses, and assigned in random order using an online randomization program (https://www.randomlists.com/team-generator). During each of the 4 dosing phases, horses were individually housed in box stalls for 12-18 hours before sampling and for the entire 6-hour sampling period. All horses had free access to fresh water and grass hay. Based on pharmacokinetic data of AA in horses,³³ each dosing phase was separated by a 1-week washout period, during which time the horses were housed on pasture. This study was approved by the university's Institutional Animal Care and Use Committee (protocol # A2019-01-019).

2.2 Sample collection and drug administration

On the morning of each dosing phase, a baseline physical examination was performed and approximately 35 mL of whole blood was collected by jugular venipuncture (baseline, T0h). An intravenous catheter was aseptically placed in the contralateral jugular vein for drug administration. Following the collection of baseline blood samples (T0h), the respective AA dose or saline was administered through the IV catheter over approximately 10 minutes.³⁵ The 4 dosing phases included 25 mg/kg AA (Ascor, McGruff Pharmaceuticals, Santa Ana, California, USA), 50 mg/kg AA, and 100 mg/kg AA, each diluted in 500 mL 0.9% sodium chloride, and 500 mL 0.9% sodium chloride (placebo).^35-37 The lowest dose (25 mg/kg) was chosen based on an experimental trial in 32 horses that reported modest anti-endotoxic benefits of AA, with the higher doses expected to increase the likelihood of conferring antioxidant effects in healthy horses.³⁵ Whole blood was collected again at T2h and T6h based on suppression of induced ROS in equine neutrophils after AA exposure in an in vitro study (Hart KA. Unpublished observation, 2019), and the elimination half-life of AA.³³ At each sampling time point (T0h, T2h, and T6h), whole blood was aliquoted into heparinized blood tubes (for plasma oxidative marker assays and measurement of plasma AA concentrations), EDTA-coated blood tubes (for intraerythrocytic ROS assays), and ACD-coated blood tubes (for neutrophil assays). Heparinized samples were protected from light, centrifuged and plasma was collected and frozen in amber tubes at −80°C within 1 hour of collection to minimize the impacts of handling artifact on oxidative markers.

2.3 Plasma oxidative markers

A photometer (FRAS-5, Innovatics Laboratories, Inc., Philadelphia, Pennsylvania, USA) was used to measure oxidative stress in 2 ways following validation for use in horses. Plasma concentrations of dROM were measured to reflect ROS concentrations and PAC was measured to reflect antioxidant potential as described previously in horses.^38-40 Validation of the photometer for use in horses was performed by spiking aliquots of the same equine blood sample with various concentrations of hydrogen peroxide (H₂O₂; 0.001%, 0.002%, 0.005%, and 0.01%) to act as known concentrations of reactive oxygen metabolites.⁴¹ The samples demonstrated a dose-dependent increase in dROM values. To validate the PAC assay, aliquots of the same plasma sample were spiked with AA, a known antioxidant, at concentrations of 1.56, 3.125, 6.25, 12.5, and 25 mM. Samples demonstrated a dose-dependent response until appearing to reach saturation at 12.5 mM. A range of normal baseline values in healthy Quarter Horses was also determined by assessing dROM in 45 healthy horses (3-20 years, mean 12.5 ± 5.0; 13 mares, 32 geldings). Values for dROM and PAC were expressed as arbitrary Carratelli units (U Carr) and Cornelli units (U Cor), respectively, because of the chemical heterogeneity of free radicals generated during ROS breakdown (1 U Carr = 0.08 mg/dL of H₂O₂; 1 U Cor = 1.4 μmol/L of AA).⁴² In this group of horses, the mean dROM value was 103 ± 20.7 U Carr and the mean PAC value was 2881 ± 313.9 U Cor.

2.4 Intraerythrocytic ROS assays

Flow cytometry was used to detect intraerythrocytic ROS using a BD Accuri C6 Plus flow cytometer (Becton, Dickinson and Co., Franklin Lakes, New Jersey, USA). This assay utilizes 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA) that readily crosses cell membranes and is cleaved by intracellular esterases to form 2′-7′-dichlorodihydrofluorescein (H₂DCF). Subsequent oxidation of H₂DCF by intracellular ROS produces a highly fluorescent product, which is detected by flow cytometry. This assay has been recently validated in horses⁴³ and has confirmed the presence of increased intraerythrocytic ROS in whole blood from horses administered sublethal IV LPS compared to baseline.⁴⁰ Samples were immediately refrigerated after collection and were shipped cold overnight on ice to the laboratory. Upon arrival, samples were centrifuged at 3000 × g for 5 minutes at 4°C. The erythrocyte pellet was collected and diluted in phosphate saline buffer (PBS), pH 7.4; 137 mM NaCl (EMD Chemicals Inc, Savannah, Georgia, USA), 10 mM Na₂HPO₄ (Sigma-Aldrich, St Louis, Missouri, USA), 2.7 mM KCl (Mallinckrodt Specialty Chemicals Co., Saint Louis, Missouri, USA), and 1.8 mM KH₂PO₄ (Mallinckrodt Specialty Chemicals Co.) supplemented with 1% w/v albumin (PBSA, Fisher Scientific, Pittsburg, Pennsylvania, USA). Samples were incubated with 50 μM DCFH-DA (Sigma-Aldrich, St Louis, Missouri, USA) or DMSO (vehicle control, American Bioanalytical, Natick, Massachusetts, USA) for 20 minutes at 37°C. Cells were either left unstimulated to represent the basal oxidative status of erythrocytes (ie, intracellular ROS concentration) or were stimulated with 1.82 mM H₂O₂ (Sigma-Aldrich, St Louis, Missouri, USA) for 20 minutes at room temperature to induce generation of ROS within erythrocytes; this latter method was used to test the intraerythrocytic antioxidant capacity. Samples were then quenched with 300 μL 1% PBSA and immediately analyzed by flow cytometry. ROS-dependent fluorescence was detected by green fluorescence with an excitation wavelength of 488 nm with gating around erythrocytes only. All samples were run in triplicate, and the mean of the median fluorescence intensity (MFI) was recorded.

2.5 Stimulant-induced neutrophil ROS assay

ACD-anticoagulated blood samples from baseline (T0h), T2h, and T6h were stored at 20-22°C for 18-24 hours after collection, for assessment of neutrophil ROS production the day following collection to ensure consistent timing with the above intraerythrocytic ROS assay. Peripheral blood neutrophils were isolated with density-gradient centrifugation over Ficoll-Paque (1.077 g/mL, GE Healthcare, Uppsala, Sweden) as described.⁴⁴ Briefly, neutrophil viability and concentration were assessed with 0.04% trypan blue solution, and cell viability was greater than 95% for all animals. Neutrophils were then suspended in complete media (RPMI 1640 without phenol red [Mediatech, Manasses, Virginia, USA] + 10% heat-inactivated low endotoxin fetal bovine serum [Hyclone, Logan, Utah, USA] + 1% L-glutamine + 0.1% gentamicin) to a concentration of 3 × 10⁶ cells/mL, and plated in 96-well flat bottom sterile plates at 100 μL cell suspension/well.

Neutrophil ROS response to Staphylococcus aureus whole cell antigen (SAA; prepared in the UGA College of Veterinary Medicine Applied Immunology Laboratory as described previously^{45, 46}) at a 1:500 dilution in media, or to phorbol myristate acetate (PMA, 10⁻⁷ M; Molecular Probes, Eugene, Oregon, USA) was measured using a previously described fluorometric assay.⁴⁷ Cells were exposed to media alone for assessment of basal ROS production, and to SAA or PMA for 2 hours. All samples were run in quadruplicate and averaged. Stimulant-induced ROS production was normalized against basal ROS production by subtracting basal ROS from stimulant-induced ROS measurements. Preliminary studies were performed with neutrophils obtained from 2 additional horses to determine the impact of this overnight storage protocol on induced ROS production. In a preliminary study, no difference in induced ROS production was found in neutrophils stimulated immediately after collection or after short-term storage as detailed above (data not shown).

2.6 Plasma AA concentrations

Plasma AA concentration was measured in heparinized plasma collected at T0h, T2h, and T6h via high-performance liquid chromatography and electrochemical detection following a previously described protocol⁴⁸ to which specific validated modifications were applied.⁴⁹

2.7 Statistical analysis

For analysis of dROM, PAC, neutrophil ROS production, and plasma AA concentrations, normality of the data was assessed via Shapiro-Wilk's test. These data met criteria for the assumption for normality, so parametric analyses were used. Baseline concentrations of dROM, PAC, and baseline neutrophil ROS production at T0h (before drug administration) were subtracted from the responses in these variables at T2h and T6h, and these response-baseline values were used for analysis. One-way repeated measures ANOVA were used to compare dROM and PAC concentrations among saline and AA doses at each timepoint after dosing. Two-way repeated measures ANOVA was utilized to evaluate for effects of AA dose and time before and after dosing on neutrophil ROS production and plasma AA concentrations. All multiple pair-wise comparisons were performed using the method of Šidák. For intraerythrocytic ROS analysis, raw data were exported to Flow Jo V10.5.3 software (Flow Jo, Ashland, Oregon, USA). Data were tested for normality with a D'Agostino & Pearson test. Two-way repeated measures ANOVA and multiple comparison tests were performed as described above. Alpha was set at 0.05. All statistical analyses were conducted using GraphPad Prism 8.0.2 and 10.0.0 (GraphPad, San Diego, California, USA).

3 RESULTS

3.1 Tolerability of AA administration in horses

AA administration was well tolerated during and after infusion by all horses at all doses, and no adverse effects were noted in any animal during AA administration or throughout the 4-week study period.

3.2 Effects of AA administration on plasma oxidative markers (dROM and PAC)

At T2h, plasma dROM significantly decreased from T0h in response to AA administration at a dose of 100 mg/kg (P = .03, 95% confidence interval [CI] 5.51 to 121.2 [point estimate 63.3] U Carr; Figure 1). There was no effect of AA administration at 25 or 50 mg/kg on dROM at T2h or T6h (P ≥ .14; 95% CI −8.73 to 106.9 [point estimate 49.1] and −56.1 to 59.6 [point estimate 1.8] U Carr, respectively). Plasma antioxidant capacity did not differ significantly at T2h or T6h at any AA dose (P ≥ .64, 95% CI −1567-463.4 [point estimate −552.0] and −999.9 to 1031 [point estimate 15.5] U Cor, respectively; Figure 2).

Details are in the caption following the image — **FIGURE 1**
Open in figure viewer PowerPoint

Mean ± 95% confidence interval plasma concentration of derivatives of reactive oxygen metabolites (dROM) in Carratelli units (U Carr) in 8 horses at 2 and 6 hours after receiving 25, 50 or 100 mg/kg of ascorbic acid or an equivalent volume of saline intravenously once. Data shown are dROM responses at 2 and 6 hours minus the baseline dROM concentration immediately before ascorbic acid/saline administration. Each circle represents data from an individual horse. “*” Denotes a significant (P < .05) difference between doses at a specific time-point.

3.3 Effects of AA administration on basal and stimulant-induced intraerythrocytic ROS concentration

There was no significant difference among the AA doses (25, 50, and 100 mg/kg AA; mean MFI 2.402, 2.404, and 2.420, respectively) on quantity of basal (unstimulated) intraerythrocytic ROS concentration in comparison to saline (mean MFI 2.365) as measured by flow cytometry (P = .88; 95% CI −0.156 to 0.081 [point estimate −0.037], −0.158 to 0.079 [point estimate −0.039], and −0.173 to 0.064 [point estimate −0.054], respectively; Figure 3). The quantity of intraerythrocytic ROS after stimulation with H₂O₂ as an indirect measurement of antioxidant capacity was also not significantly different at any AA dose (P = .93, CI −0.123 to 0.112 [point estimate −0.006], −0.146 to 0.089 [point estimate −0.028], and −0.127 to 0.108 [point estimate −0.009], respectively). The mean MFI of stimulated cells for saline, 25, 50, and 100 mg/kg AA were 3.778, 3.784, 3.807, and 3.788, respectively (Figure 3).

3.4 Effects of AA administration on stimulant-induced neutrophil ROS production

When compared to saline treatment, AA administration at any dose (25, 50, and 100 mg/kg) did not significantly impact stimulant-induced neutrophil ROS production after exposure to SAA (P > .12, 95% CI −644.9 to 56.2 [point estimate −294.4] to −346.7 to 354.4 [point estimate 3.9] arbitrary fluorescence units) or PMA (P ≥ .67, 95% CI −1383 to 575.5 [point estimate −403.5] to −965.0 to 993.4 [point estimate −14.3] arbitrary fluorescence units, respectively) at any time point (Figure 4).

3.5 Effects of AA administration on plasma AA concentrations

Plasma AA concentrations before and after saline and AA administration are shown in Figure 5. At T2h, plasma AA concentrations increased significantly from T0h concentrations in a dose-dependent fashion from before administration concentrations for 25, 50, and 100 mg/kg AA doses (P < .001 for all comparisons, 95% CI −132.0 to −65.5 [point estimate −98.8], −276.5 to −210.0 [point estimate −243.3], and −641.8 to −575.4 [point estimate −608.6] μmol/L, respectively). Plasma AA concentrations at T6h remained significantly higher than before administration concentrations for all 3 AA doses (P < .02, 95% CI −70.7 to −4.28 [point estimate −37.5], −91.3 to −24.9 [point estimate −58.1], and −138.5 to −72.0 [point estimate −105.3] μmol/L, respectively). A significant effect of AA dose was only present for the 100 mg/kg dose at this time point, as compared to the 25 mg/kg dose (P ≤ .001, 95% CI −106.4 to −27.3 [point estimate −66.9] μmol/L) and the 50 mg/kg dose (P = .01, 95% CI −87.3 to −8.2 [point estimate −47.8] μmol/L).

4 DISCUSSION

Intravenous administration of AA at 100 mg/kg IV resulted in decreased dROM at 2 hours after administration, with dROM returning to baseline levels by 6 hours. This suggests that endogenous, circulating ROS were decreased by AA but only at the highest dose tested. In contrast, intraerythrocytic concentrations and stimulant-induced neutrophil ROS production were not affected by time or AA dose. This might have been because of insensitivity of these tests to detect oxidative stress in healthy horses. It is possible that in sick horses with higher circulating markers of oxidative stress, lower doses of AA (25 and 50 mg/kg) might reduce dROM levels as effectively as the high dose (100 mg/kg) did in this group of healthy animals, and that intraerythrocytic ROS concentrations and stimulant-induced neutrophil ROS production might also be impacted by AA administration. In human studies, high-dose AA (>50 mg/kg) administered IV attenuates inflammation and reduce mortality in septic patients.^{27, 37, 50} In preliminary studies, 1 of the authors observed attenuation of stimulant-induced ROS produced by isolated equine neutrophils after ex vivo exposure to the same stimulants examined here in the presence of increasing concentrations of AA (Hart KA. Unpublished observation, 2018). In a separate study investigating the effects of in vivo AA alone or in combination with hydrocortisone on clinical signs and inflammatory markers after experimental endotoxemia in horses, the relatively low dose of AA administered (25 mg/kg) was likely insufficient to confer clinical protection.³⁵ Recent clinical trials in human sepsis have found that low to moderate (<50 mg/kg) doses of AA administered IV did not improve outcome,^51-54 despite earlier preliminary studies that suggested otherwise.³⁶ Taken together, this suggests that further investigation of high-dose AA administration in septic patients is warranted.

The transport of AA across erythrocyte membranes is mediated by the sodium-dependent transmembrane protein SVCT in some species.⁵⁵ However, human erythrocytes lack this transporter, resulting in intraerythrocytic concentrations of AA that are similar to plasma and many-fold lower than in other tissues.⁵⁶ Although this information is not available for horses, it is possible that the unchanged concentrations of intraerythrocytic ROS over time, despite exogenous AA administration, was because of a lack of AA transport into erythrocytes. Since high-dose (100 mg/kg) AA resulted in decreased dROM 2 hours after administration and erythrocytes play a key role in balancing the redox state of the blood, the use of AA in horses with sepsis might help scavenge excess extracellular ROS because of erythrocytic free radical buffering capacity.

Administration of AA did not decrease stimulant-induced neutrophil ROS production, the significance of which is unknown. It is possible that in the current study, peak AA concentrations (ie, within 5 minutes of IV AA administration), might have dampened neutrophil ROS responses, but this was not measured until 2 hours after administration. Additionally, we assessed stimulant-induced ROS production in isolated neutrophils that were suspended in media, after removal of the horses' endogenous plasma containing increased AA concentrations. Furthermore, administration of IV AA 1 hour after IV LPS infusion in horses protected against LPS-induced neutrophil depletion compared to controls that did not receive AA.³⁵ Although stimulant-induced neutrophil ROS production was not measured in that study, it is possible that less oxidative burst and subsequent destruction of neutrophils was conferred by AA administration.

Plasma antioxidant capacity was not affected by AA administration at any dose or timepoint. It is important to note that these variables were not measured at the expected peak plasma concentration of AA immediately after IV administration.³³ It is also possible that administration of AA might have affected plasma concentrations or activity of other endogenous antioxidants that contribute to the overall antioxidant capacity assessed here.^{57, 58}

The AA administered IV is labeled for IM use but was given IV in the current study. This formulation was chosen based on the absence of adverse effects when administered IV to horses in a previous study,³⁵ and the cost effectiveness of the formulation compared to AA labeled for IV use. No adverse effects of IV AA administration were observed throughout the study period, even at the highest dose. A limitation of the study was that CBCs and biochemistry panels were not performed at baseline or after AA administration. However, in the previous study, IV AA administration did not negatively affect any clinicopathological variables,³⁵ and the horses used in this study have remained healthy for >2 years after data collection. Safety of high-dose AA administered IV to humans has been reported in several early phase clinical trials.^{24, 59-61} However, since data collection for this study, acute kidney injury was reported in human patients treated with high-dose AA.⁶² Renal function should therefore be critically assessed in similar forthcoming studies in horses.

Plasma concentrations of AA increased in a dose-dependent manner, as expected. Although a pharmacokinetic study was not done, plasma concentrations remained elevated from baseline concentrations 6 hours after administration of all 3 doses. Baseline plasma AA concentrations were similar to those reported in adult horses in a separate study.³⁵ Repeated dosing at q 6-8 hours intervals or constant rate infusion is more likely to mimic a clinical situation as described in people,^{36, 51} and might affect plasma drug concentrations at steady state, but further study is needed to determine an ideal dosing interval.

Limitations of this study include the small sample size, the lack of clinicopathological data before and after AA administration (as stated above), and single dose administration. In addition, the current study was performed on healthy adult horses that were expected to have a relatively low level of plasma oxidative stress compared to sick horses.

In sum, IV administration of high-dose (100 mg/kg) AA resulted in decreased oxidative stress in healthy horses, and might warrant evaluation in septic horses and foals.

ACKNOWLEDGMENT

This study was supported by the USDA National Institute of Food and Agriculture (Hatch project # 1016369). The authors thank Natalie Norton, Loralei Branch, and Meredith Colby for their assistance with animal care and sample processing for this study.

CONFLICT OF INTEREST DECLARATION

Authors declare no conflict of interest.

OFF-LABEL ANTIMICROBIAL DECLARATION

Authors declare no off-label use of antimicrobials.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION

Approved by the University of Georgia's IACUC.

HUMAN ETHICS APPROVAL DECLARATION

Authors declare that human ethics approval was not necessary for this study.

REFERENCES

1Parihar A, Parihar MS, Milner S, Bhat S. Oxidative stress and anti-oxidative mobilization in burn injury. Burns. 2008; 34: 6-17.
10.1016/j.burns.2007.04.009
PubMed Web of Science® Google Scholar
2Goode HF, Cowley HC, Walker BE, Howdle PD, Webster NR. Decreased antioxidant status and increased lipid peroxidation in patients with septic shock and secondary organ dysfunction. Crit Care Med. 1995; 23: 646-651.
10.1097/00003246-199504000-00011
CAS PubMed Web of Science® Google Scholar
3Motoyama T, Okamoto K, Kukita I, Hamaguchi M, Kinoshita Y, Ogawa H. Possible role of increased oxidant stress in multiple organ failure after systemic inflammatory response syndrome. Crit Care Med. 2003; 31: 1048-1052.
10.1097/01.CCM.0000055371.27268.36
CAS PubMed Web of Science® Google Scholar
4Ware LB, Fessel JP, May AK, Roberts LJ. Plasma biomarkers of oxidant stress and development of organ failure in severe sepsis. Shock. 2011; 36: 12-17.
10.1097/SHK.0b013e318217025a
CAS PubMed Web of Science® Google Scholar
5Galley HF, Howdle PD, Walker BE, Webster NR. The effects of intravenous antioxidants in patients with septic shock. Free Radic Biol Med. 1997; 23: 768-774.
10.1016/S0891-5849(97)00059-2
CAS PubMed Web of Science® Google Scholar
6Kunimoto F, Morita T, Ogawa R, Fujita T. Inhibition of lipid peroxidation improves survival rate of endotoxemic rats. Circ Shock. 1987; 21: 15-22.
CAS PubMed Web of Science® Google Scholar
7Koekkoek WA, van Zanten AR. Antioxidant vitamins and trace elements in critical illness. Nutr Clin Pract. 2016; 31: 457-474.
10.1177/0884533616653832
CAS PubMed Web of Science® Google Scholar
8van den Berg JJ, Kuypers FA, Roelofsen B, et al. The cooperative action of vitamins E and C in the protection against peroxidation of parinaric acid in human erythrocyte membranes. Chem Phys Lipids. 1990; 53: 309-320.
10.1016/0009-3084(90)90028-P
PubMed Web of Science® Google Scholar
9Berger MM, Oudemans-van Straaten HM. Vitamin C supplementation in the critically ill patient. Curr Opin Clin Nutr Metab Care. 2015; 18: 193-201.
10.1097/MCO.0000000000000148
CAS PubMed Web of Science® Google Scholar
10McKinnon RL, Lidington D, Tyml K. Ascorbate inhibits reduced arteriolar conducted vasoconstriction in septic mouse cremaster muscle. Microcirculation. 2007; 14: 697-707.
10.1080/10739680701410389
CAS PubMed Web of Science® Google Scholar
11Treschan TA, Peters J. The vasopressin system: physiology and clinical strategies. Anesthesiology. 2006; 105: 599-612. quiz 639–540.
10.1097/00000542-200609000-00026
CAS PubMed Web of Science® Google Scholar
12Carr AC, Shaw GM, Fowler AA, Natarajan R. Ascorbate-dependent vasopressor synthesis: a rationale for vitamin C administration in severe sepsis and septic shock? Crit Care. 2015; 19: 418.
10.1186/s13054-015-1131-2
PubMed Web of Science® Google Scholar
13Dillon PF, Root-Bernstein RS, Lieder CM. Antioxidant-independent ascorbate enhancement of catecholamine-induced contractions of vascular smooth muscle. Am J Physiol Heart Circ Physiol. 2004; 286: H2353-H2360.
10.1152/ajpheart.00968.2003
CAS PubMed Web of Science® Google Scholar
14De la Fuente M, Victor VM. Ascorbic acid and N-acetylcysteine improve in vitro the function of lymphocytes from mice with endotoxin-induced oxidative stress. Free Radic Res. 2001; 35: 73-84.
10.1080/10715760100300611
CAS PubMed Web of Science® Google Scholar
15Wilson JX. Evaluation of vitamin C for adjuvant sepsis therapy. Antioxid Redox Signal. 2013; 19: 2129-2140.
10.1089/ars.2013.5401
CAS PubMed Web of Science® Google Scholar
16Prauchner CA. Oxidative stress in sepsis: pathophysiological implications justifying antioxidant co-therapy. Burns. 2017; 43: 471-485.
10.1016/j.burns.2016.09.023
PubMed Web of Science® Google Scholar
17Borrelli E, Roux-Lombard P, Grau GE, et al. Plasma concentrations of cytokines, their soluble receptors, and antioxidant vitamins can predict the development of multiple organ failure in patients at risk. Crit Care Med. 1996; 24: 392-397.
10.1097/00003246-199603000-00006
CAS PubMed Web of Science® Google Scholar
18Schorah CJ, Downing C, Piripitsi A, et al. Total vitamin C, ascorbic acid, and dehydroascorbic acid concentrations in plasma of critically ill patients. Am J Clin Nutr. 1996; 63: 760-765.
10.1093/ajcn/63.5.760
CAS PubMed Web of Science® Google Scholar
19Nathens AB, Neff MJ, Jurkovich GJ, et al. Randomized, prospective trial of antioxidant supplementation in critically ill surgical patients. Ann Surg. 2002; 236: 814-822.
10.1097/00000658-200212000-00014
PubMed Web of Science® Google Scholar
20Marcus SL, Dutcher JP, Paietta E, et al. Severe hypovitaminosis C occurring as the result of adoptive immunotherapy with high-dose interleukin 2 and lymphokine-activated killer cells. Cancer Res. 1987; 47: 4208-4212.
CAS PubMed Web of Science® Google Scholar
21Armour J, Tyml K, Lidington D, Wilson JX. Ascorbate prevents microvascular dysfunction in the skeletal muscle of the septic rat. J Appl Physiol. 1985; 2001(90): 795-803.
Google Scholar
22Wu F, Wilson JX, Tyml K. Ascorbate protects against impaired arteriolar constriction in sepsis by inhibiting inducible nitric oxide synthase expression. Free Radic Biol Med. 2004; 37: 1282-1289.
10.1016/j.freeradbiomed.2004.06.025
CAS PubMed Web of Science® Google Scholar
23Fisher BJ, Kraskauskas D, Martin EJ, et al. Attenuation of sepsis-induced organ injury in mice by vitamin C. JPEN J Parenter Enteral Nutr. 2014; 38: 825-839.
10.1177/0148607113497760
CAS PubMed Web of Science® Google Scholar
24Fowler AA 3rd, Syed AA, Knowlson S, et al. Phase I safety trial of intravenous ascorbic acid in patients with severe sepsis. J Transl Med. 2014; 12: 32.
10.1186/1479-5876-12-32
CAS PubMed Web of Science® Google Scholar
25Zabet MH, Mohammadi M, Ramezani M, Khalili H. Effect of high-dose ascorbic acid on vasopressor's requirement in septic shock. J Res Pharm Pract. 2016; 5: 94-100.
10.4103/2279-042X.179569
CAS PubMed Web of Science® Google Scholar
26Chen CY, Chiu CT, Lee HS, Lai CC. The impact of vitamin C-containing treatment on the mortality of patients with sepsis: a systematic review and meta-analysis of randomized controlled trials. J Infect Public Health. 2022; 15: 1514-1520.
10.1016/j.jiph.2022.11.015
PubMed Web of Science® Google Scholar
27Patel JJ, Ortiz-Reyes A, Dhaliwal R, et al. IV vitamin C in critically ill patients: a systematic review and meta-analysis. Crit Care Med. 2022; 50: e304-e312.
10.1097/CCM.0000000000005320
CAS PubMed Web of Science® Google Scholar
28Lv SJ, Zhang GH, Xia JM, Yu H, Zhao F. Early use of high-dose vitamin C is beneficial in treatment of sepsis. Ir J Med Sci. 2021; 190: 1183-1188.
10.1007/s11845-020-02394-1
CAS PubMed Web of Science® Google Scholar
29Cohen ND. Causes of and farm management factors associated with disease and death in foals. J Am Vet Med Assoc. 1994; 204: 1644-1651.
10.2460/javma.1994.204.10.1644
CAS PubMed Web of Science® Google Scholar
30Peek SF, Semrad S, McGuirk SM, et al. Prognostic value of clinicopathologic variables obtained at admission and effect of antiendotoxin plasma on survival in septic and critically ill foals. J Vet Intern Med. 2006; 20: 569-574.
10.1111/j.1939-1676.2006.tb02898.x
PubMed Web of Science® Google Scholar
31Arroyo MG, Slovis NM, Moore GE, Taylor SD. Factors associated with survival in 97 horses with septic pleuropneumonia. J Vet Intern Med. 2017; 31: 894-900.
10.1111/jvim.14679
CAS PubMed Web of Science® Google Scholar
32Bertin FR, Reising A, Slovis NM, Constable PD, Taylor SD. Clinical and clinicopathological factors associated with survival in 44 horses with equine neorickettsiosis (Potomac horse Fever). J Vet Intern Med. 2013; 27: 1528-1534.
10.1111/jvim.12209
CAS PubMed Web of Science® Google Scholar
33Loscher W, Jaeschke G, Keller H. Pharmacokinetics of ascorbic acid in horses. Equine Vet J. 1984; 16: 59-65.
10.1111/j.2042-3306.1984.tb01855.x
CAS PubMed Web of Science® Google Scholar
34Snow DH, Frigg M. Bioavailability of ascorbic acid in horses. J Vet Pharmacol Ther. 1990; 13: 393-403.
10.1111/j.1365-2885.1990.tb00794.x
CAS PubMed Web of Science® Google Scholar
35Anderson MJ, Ibrahim AS, Cooper BR, Woolcock AD, Moore GE, Taylor SD. Effects of administration of ascorbic acid and low-dose hydrocortisone after infusion of sublethal doses of lipopolysaccharide to horses. J Vet Intern Med. 2020; 34: 2710-2718.
10.1111/jvim.15896
PubMed Web of Science® Google Scholar
36Marik PE, Khangoora V, Rivera R, Hooper MH, Catravas J. Hydrocortisone, vitamin C, and thiamine for the treatment of severe sepsis and septic shock: a retrospective before-after study. Chest. 2017; 151: 1229-1238.
10.1016/j.chest.2016.11.036
PubMed Web of Science® Google Scholar
37Feng F, Yang H, Yang W, Li M, Chang X, Chen Y. Effect of vitamin C in critically ill patients with sepsis and septic shock: a meta-analysis. Sci Prog. 2021; 104:36850421998175.
10.1177/0036850421998175
CAS PubMed Web of Science® Google Scholar
38Shono S, Gin A, Minowa F, Okubo K, Mochizuki M. The oxidative stress markers of horses-the comparison with other animals and the influence of exercise and disease. Animals (Basel). 2020; 10: 10.
Web of Science® Google Scholar
39Sgorbini M, Bonelli F, Marmorini P, et al. Maternal and neonatal evaluation of derivated reactive oxygen metabolites (d-ROMs) and biological antioxidant potential in the horse. Theriogenology. 2015; 83: 48-51.
10.1016/j.theriogenology.2014.07.041
CAS PubMed Web of Science® Google Scholar
40Taylor SD, Serpa PBS, Santos AP, et al. Effects of intravenous administration of peripheral blood-derived mesenchymal stromal cells after infusion of lipopolysaccharide in horses. J Vet Intern Med. 2022; 36: 1491-1501.
10.1111/jvim.16447
PubMed Web of Science® Google Scholar
41Vaughn SA, Lemons MB, Norton NA, Hart KA. Associations between systemic oxidative stress and endocrine parameters in horses and ponies. Global Equine Endocrine Symposium 2020.
Google Scholar
42Celi P, Sullivan M, Evans D. The stability of the reactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP) tests on stored horse blood. Vet J. 2010; 183: 217-218.
10.1016/j.tvjl.2008.09.018
CAS PubMed Web of Science® Google Scholar
43Serpa PBS, Woolcock A, Taylor SD, Pires dos Santos A. Validation of a flow cytometric assay to detect intraerythrocytic reactive oxygen species in horses. Vet Clin Pathol. 2021; 50: 20-27.
10.1111/vcp.12976
PubMed Web of Science® Google Scholar
44Hart KA, Barton MH, Ferguson DC, et al. Serum free cortisol fraction in healthy and septic neonatal foals. J Vet Intern Med. 2011; 25: 345-355.
10.1111/j.1939-1676.2010.0667.x
CAS PubMed Web of Science® Google Scholar
45Ryman V, Nickerson S, Hurley D, et al. Influence of horn flies (Haemotobia irritans) on teat skin condition, intramammary infection, and serum anti-S. aureus antibody titres in holstein heifers. Res Vet Sci. 2013; 95: 343-346.
10.1016/j.rvsc.2013.04.017
CAS PubMed Web of Science® Google Scholar
46Nace E, Nickerson S, Kautz F, et al. Modualtion of innate immune function and phenotype in bred dairy heifers during the periparturient period induced by feeding an immunostimulant for 60 days prior to delivery. Vet Immunol Immunopathol. 2014; 161: 240-250.
10.1016/j.vetimm.2014.08.013
CAS PubMed Web of Science® Google Scholar
47Donovan DC, Jackson CA, Colahan PT, et al. Assessment of exercise-induced alterations in neutrophil function in horses. Am J Vet Res. 2007; 68: 1198-1204.
10.2460/ajvr.68.11.1198
CAS PubMed Web of Science® Google Scholar
48Li X, Franke AA. Fast HPLC-ECD analysis of ascorbic acid, dehydroascorbic acid and uric acid. J Chromatogr B Analyt Technol Biomed Life Sci. 2009; 877: 853-856.
10.1016/j.jchromb.2009.02.008
CAS PubMed Web of Science® Google Scholar
49Clark ZD, Frank EL. Development and implementation of an HPLC-ECD method for analysis of vitamin C in plasma using single column and automatic alternating dual column regeneration. Pract Lab Med. 2016; 6: 25-37.
10.1016/j.plabm.2016.09.001
PubMed Google Scholar
50Xia G, Fan D, He Y, Zhu Y, Zheng Q. High-dose intravenous vitamin C attenuates hyperinflammation in severe coronavirus disease 2019. Nutrition. 2021; 91-92:111405.
10.1016/j.nut.2021.111405
CAS PubMed Web of Science® Google Scholar
51Lamontagne F, Masse MH, Menard J, et al. Intravenous vitamin C in adults with sepsis in the intensive care unit. N Engl J Med. 2022; 386: 2387-2398.
10.1056/NEJMoa2200644
CAS PubMed Web of Science® Google Scholar
52Chang P, Liao Y, Guan J, et al. Combined treatment with hydrocortisone, vitamin C, and thiamine for sepsis and septic shock: a randomized controlled trial. Chest. 2020; 158: 174-182.
10.1016/j.chest.2020.02.065
CAS PubMed Web of Science® Google Scholar
53Hussein AA, Sabry NA, Abdalla MS, Farid SF. A prospective, randomised clinical study comparing triple therapy regimen to hydrocortisone monotherapy in reducing mortality in septic shock patients. Int J Clin Pract. 2021; 75:e14376.
10.1111/ijcp.14376
CAS PubMed Web of Science® Google Scholar
54Hwang SY, Ryoo SM, Park JE, et al. Combination therapy of vitamin C and thiamine for septic shock: a multi-centre, double-blinded randomized, controlled study. Intensive Care Med. 2020; 46: 2015-2025.
10.1007/s00134-020-06191-3
CAS PubMed Web of Science® Google Scholar
55Rivas CI, Zuniga FA, Salas-Burgos A, et al. Vitamin C transporters. J Physiol Biochem. 2008; 64: 357-375.
10.1007/BF03174092
CAS PubMed Web of Science® Google Scholar
56May JM, Qu ZC, Qiao H, Koury MJ. Maturational loss of the vitamin C transporter in erythrocytes. Biochem Biophys Res Commun. 2007; 360: 295-298.
10.1016/j.bbrc.2007.06.072
CAS PubMed Web of Science® Google Scholar
57Traber MG, Stevens JF. Vitamins C and E: beneficial effects from a mechanistic perspective. Free Radic Biol Med. 2011; 51: 1000-1013.
10.1016/j.freeradbiomed.2011.05.017
CAS PubMed Web of Science® Google Scholar
58Koracevic D, Koracevic G, Djordjevic V, Andrejevic S, Cosic V. Method for the measurement of antioxidant activity in human fluids. J Clin Pathol. 2001; 54: 356-361.
10.1136/jcp.54.5.356
CAS PubMed Web of Science® Google Scholar
59Ou J, Zhu X, Lu Y, et al. The safety and pharmacokinetics of high dose intravenous ascorbic acid synergy with modulated electrohyperthermia in Chinese patients with stage III-IV non-small cell lung cancer. Eur J Pharm Sci. 2017; 109: 412-418.
10.1016/j.ejps.2017.08.011
CAS PubMed Web of Science® Google Scholar
60Polireddy K, Dong R, Reed G, et al. High dose parenteral ascorbate inhibited pancreatic cancer growth and metastasis: mechanisms and a phase I/IIa study. Sci Rep. 2017; 7: 17188.
10.1038/s41598-017-17568-8
PubMed Web of Science® Google Scholar
61Trankle CR, Puckett L, Swift-Scanlan T, et al. Vitamin C intravenous treatment in the setting of atrial fibrillation ablation: results from the randomized, double-blinded, placebo-controlled CITRIS-AF pilot study. J Am Heart Assoc. 2020; 9:e014213.
10.1161/JAHA.119.014213
PubMed Web of Science® Google Scholar
62McCune TR, Toepp AJ, Sheehan BE, et al. High dose intravenous vitamin C treatment in Sepsis: associations with acute kidney injury and mortality. BMC Nephrol. 2021; 22: 387.
10.1186/s12882-021-02599-1
CAS PubMed Web of Science® Google Scholar

Citing Literature

Volume38, Issue1

January/February 2024

Pages 460-468

Effects of intravenous administration of ascorbic acid (vitamin C) on oxidative status in healthy adult horses