Effectiveness of IV Vitamin C in Reducing Oxidative Stress Associated With Free Flap Surgery

The Efficacy of Perioperative Parenteral Ascorbic Acid in Reducing Oxidative Stress Among Patients Undergoing Free Flap Reconstructive Surgery: A Prospective Multicenter Randomized Controlled Pilot Trial

Ischemia and reperfusion injury during free flap reconstructive surgery creates a state of increased oxidative stress that can adversely affect the flap outcomes. Ascorbic acid (AA) had been proven to have beneficial effect on end-organ protection and flap survival from ischemia-reperfusion injury via its antioxidant properties. The investigators hypothesise that perioperative parenteral ascorbic acid treatment may reduce oxidative stress among participants undergoing free flap reconstructive surgery along with reduction in inflammatory markers, improved rate of flap viability and wound healing at both donor and recipient sites.

Temporary cessation of blood supply to the flap tissues from clamping of donor vascular pedicle (ischemia) followed by restoration of flap tissue perfusion (reperfusion) after micro-anastomosis are inevitable in any free flap surgery. In combination, the ischemia and reperfusion process during free flap tissue transfers induce a state of increased oxidative stress, which may lead to complications such as flap failure and non-healing wounds at either donor or recipient sites. The negative impacts of which include additional wounds from flap loss, higher costs and increased duration of hospital stay. Previous studies had demonstrated the beneficial effects of ascorbic acid in end-organ protection against ischemia and reperfusion injury. In addition, parenteral ascorbic acid has been shown to be remarkably safe even at high dose in both clinical and nonclinical models. Nonetheless, the data on efficacy of ascorbic acid in free flap survival in human is very limited. The aims of this prospective, multicentre, double-blind, randomized, placebo-controlled pilot study are to measure the extent of oxidative stress in participants undergoing free flap reconstructive surgery before and after administration of parenteral ascorbic acid; and to evaluate its efficacy on modulation of inflammation, flap viability and wound healing. Eligible participants will be randomized to receive 1 gram of parenteral ascorbic acid and 0.9% normal saline (as placebo) 8 hourly for 7 days (from pre-operative day 2 until post-operative day 5). Blood sampling will be performed on day 0 (pre-operative), day 3 (post-operative day 1) and day 5 (post-operative day 3) of intravenous ascorbic acid or placebo infusion for measurement of i) oxidative stress biomarkers, including isoprostane level, gene expression of glutamate-cystein ligase (GCL) and total glutathione level) ii) inflammatory markers, including leucocytes count and gene expression of TNF-α and IL-1. Post-operative outcomes of free flap surgery, up to post-operative 14 days, including flap viability, wound healing at both donor and recipient sites and duration of ICU and hospital stay will be evaluated. The investigators estimate that a total sample of 28 participants (14 on each arm) will be necessary for 80% power to detect a 33% oxidative stress reduction with medium effect size (0.5) at 5% level of significance (α) between treatment (intravenous ascorbic acid) and placebo group (0.9% normal saline). A total of 34 participants are required to account for 20% of dropouts. Primary analysis of this study utilizes an intention-to-treat approach and includes all randomized participants undergoing elective free flap reconstructive surgery. The mean difference between the baseline (pre-operative) and post-operative oxidative stress and inflammatory levels will be analyzed and compared between the intravenous ascorbic acid and placebo group using analysis of variance (ANOVA) for all normally distributed dataset whilst the non-parametric Kruskal-Wallis test is used, if otherwise. The effect size of such difference will be determined and compared. Subsequently, correlation between reduction of oxidative stress and post-operative flap outcomes in the intravenous ascorbic acid group will be evaluated. The secondary outcomes such as flap viability (percentage of flap necrosis), wound healing at both recipient and donor sites (percentage of wound dehiscence and percentage of skin graft failure to take/loss), duration of hospital and ICU stay and wound infection rate will be presented as mean with standard deviation (SD) or median with interquartile range (IQR) based on their normality distribution and are compared with Student's t-test or Mann-Whitney U test.

Intravenous ascorbic acid 1 gram 8 hourly (3 grams per day) over 15 minutes for 7 days since pre-operative day 1 until post-operative day 5.

Intravenous 0.9% normal saline 8 hourly bolus infusion over 15 minutes for 7 days since pre-operative day 1 until post-operative day 5.

Plasma isoprostane level will be analyzed using a competitive ELISA assay and expressed in unit of pg/ml.

The expression of glutamate cysteine ligase (GCL) will be quantitated using real time quantitative polymerase chain reaction (qRT-CR). The differences in gene expression, expressed as fold-change, will be calculated using the 2 -DΔCt algorithm where GAPDH will be used as the housekeeping gene.

Total glutathione level (GSSG + GSH) will be determined using a Glutathione Assay Kit, which will be expressed in unit of µM.

Leucocytes count will be measured through flow cytometry White blood cells Differential Fluorescence (WDF) scattergram using Sysmex XN-10 (Sysmex Corporation TM , Kobe, Japan) and expressed in unit of X10^9/L.

The expression of TNF-α will be quantitated using real time quantitative polymerase chain reaction (qRT-CR). The differences in gene expression, expressed as fold-change, will be calculated using the 2 -DΔCt algorithm where GAPDH will be used as the housekeeping gene.

The expression of IL-1 will be quantitated using real time quantitative polymerase chain reaction (qRT-CR). The differences in gene expression, expressed as fold-change, will be calculated using the 2 -DΔCt algorithm where GAPDH will be used as the housekeeping gene.

Flap viability is the survival of flap without tissue loss. For flap viability assessment, total flap loss is defined as complete necrosis (death) of flap (100% flap loss), whilst partial flap loss refers to incomplete necrosis of flap. By using simple tracing technique, the percentage of flap necrosis is calculated as: [Necrotic flap surface area (cm^2)/Total flap surface area (cm^2)] × 100%

Surgical wound dehiscence is defined as separation of the margins of a closed surgical incision that has been made in skin, with or without exposure or protrusion of underlying tissue, organs or implants. Wound dehiscence is assessed upon suture removal. It is categorized as either partial (1-99%) or complete (100%), depending on the depth/thickness of skin layers involvement. Partial wound dehiscence is defined as separation of wound edge up to level of epidermis and dermis layer of sutured wounds, whereas complete wound dehiscence refers to complete separation of wound edge involving the full thickness of skin. The percentage of wound dehiscence in relative to the total wound size is calculated.

Skin graft recipient area is inspected at post-operative day 5 for assessment of graft take. Graft loss is categorized as partial (1-99%) or complete (100%). The percentage of skin graft failure to take/loss is calculated as: [Graft failure to take or loss surface area (cm^2)/Total skin graft area surface area (cm^2)] X 100%

Wound infection is defined by presence of clinical sign and symptoms of infection associated with wound, including: i) Local signs: Erythema - localized or spreading (cellulitis); Pus/purulent or haemopurulent exudate; Abscess; Swelling/induration; Local warmth; Malodour; Crepitus; Dehiscence; Unexpected pain or tenderness. ii) Systemic signs: Malaise; Loss of appetite; Pyrexia or hypothermia; Tachycardia; Tachypnoea; Elevated C-reactive protein (CRP); Elevated or suppressed white blood cell count; Sepsis or Septic Shock.

Inclusion Criteria: - Adults (age 18 years and older, male or female) who are planned for elective free flap reconstructive surgery. Exclusion Criteria: Hypersensitivity to vitamin C Oliguria (urine output <400mL/day) or anuria (urine output <100mL/day) Renal failure (serum creatinine level ≥175.0 %mol/L) Hemodialysis Renal calculi Thalassemia Glucose-6-phosphate dehydrogenase (G6PD) deficiency Unfit for surgery Pregnancy or lactating Hemochromatosis Hyperoxaluria

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