This phase I trial focused on the safety of administering intravenous ascorbic acid to patients with severe sepsis. The intravenous route of administration was chosen in this trial in order to achieve high ascorbic acid plasma levels. Padayatty and colleagues showed that high-level ascorbic acid plasma concentrations could only be achieved by intravenous administration [15]. Prior human studies employing pharmacologic ascorbic acid dosing report no adverse events. Nathens et al. administered 1 gram of ascorbic acid every 8 hours for 28 days to surgically critically ill patients with no ill effects [16]. Tanaka et al. administered 66 mg/kg/hour for 24 hours to patients with 50% surface area burns with no adverse events [17]. Hoffer et al. intravenously administered up to 90 grams of ascorbic acid 3 times weekly to patients with advanced malignancy with no adverse events [30]. The dosing protocols we chose for this trial arose out of our preclinical work.

No patient in the low or high dose ascorbic acid treatment arms of this study suffered any identifiable adverse event. As noted above, the one instance in which ascorbic acid infusion was halted for a cardiac rhythm disturbance was determined to be artifact by the Division of Cardiology. Thus, a pharmacologic ascorbic acid treatment strategy in critically ill patients with severe sepsis appears to be safe.

Prior studies show that patients with severe sepsis exhibit significantly reduced plasma ascorbic acid levels upon admission to intensive care [31]. The mean initial plasma ascorbic acid level for all septic patients in this study was 17.9 ± 2.4 μM compared to normal human plasma levels of 50 – 70 μM (Figure 1). Prior studies [13, 14, 26], and the current study show that subnormal plasma ascorbic acid levels are a predictable feature in patients with severe sepsis. Importantly, Placebo patients exhibited no change in plasma ascorbic acid levels throughout the 4-day study period despite receiving full ICU standard of care practice for severe sepsis (Figure 1). Ascorbic acid depletion in sepsis results from ascorbic acid consumption by the reduction of plasma free iron, ascorbic acid consumption by the scavenging of aqueous free radicals (peroxyl radicals), and by the destruction of the oxidized form of ascorbic acid dehydroascorbic acid[14]. Sepsis further inhibits intracellular reduction of dehydroascorbic acid, producing acute intracellular ascorbic acid depletion. Sepsis-induced ascorbic acid destruction permits uncontrolled oxidant activity which amplifies tissue injury [14, 32, 33]. Ascorbic acid treated patients in this study exhibited rapid and sustained increases in plasma ascorbic acid levels using an intermittent every six hours administration protocol (Figure 1).

SOFA scores are robust indicators of mortality during critical illness. SOFA score increases during the first 48 hours of ICU care predict a mortality rate of at least 50% [26]. In this study, the extent of organ failure accompanying patients with severe sepsis was high with an average SOFA score for all patients equal to 11.4 ± 3 confirming that multiple organ failure was present at enrollment. Given that the mean plasma ascorbic acid levels on admission were subnormal (17.9 ± 2.4 μM), a mean initial SOFA score of 11.4 ± 3 in patients with severe sepsis was not surprising. This study is in agreement with other studies which show that plasma ascorbic acid levels in severe sepsis correlate inversely with the incidence of multiple organ failure [13]. We showed that the addition of ascorbic acid to standard of care practice (i.e., fluid resuscitation, antibiotics, vasopressor medication) for patients with severe sepsis significantly reduced organ injury. Ascorbic acid treated patients exhibited prompt and sustained reductions in SOFA scores during the 4-day treatment regimen unlike placebo controls where SOFA scores slowly increased over time. SOFA score reduction was most remarkable in patients receiving the high dose ascorbic acid infusion (Figure 2).

C-reactive protein (CRP) [19] and procalcitonin (PCT) [20] levels are known to correlate with the overall extent of infection and higher levels of both have both been linked to higher incidences of organ injury and death in the critically ill. CRP in circulation has a short half-life of approximately 19 hours. Thus, the kinetics of CRP make it a useful monitor for tracking the inflammatory response produced by infection, and the response to antibiotic treatment. Lobo et al. reported that patients with CRP levels greater than 10 mg/dL at ICU admission exhibited significantly higher rates of multiple organ failure as well as higher mortality rates [34]. A decrease in CRP levels in Lobo’s study after 48 hours was associated with a mortality rate of only 15.4%, while a persistently high CRP level was associated with a mortality rate of 60.9%. Both low and high dose ascorbic acid infusion in this trial promptly reduced serum CRP levels in septic patients (Figure 3A). Thus, the findings in this study support the findings of Lobo et al. with descending CRP levels being associated with lower mortality rate and reduced levels or organ failure. Jensen and colleagues found that high maximal procalcitonin levels were an early independent predictor of all-cause mortality in a 90-day follow-up period after intensive care unit admission [20]. Karlsson and colleagues [21] showed that mortality in patients with severe sepsis was lower in those patients in whom procalcitonin concentrations fell by more than 50% at 72 hours with respect to initial values. Infusion of ascorbic acid into patients with severe sepsis in this study reduced serum procalcitonin levels by greater than 50% (Figure 3B). Thrombomodulin is an endothelial cell bound molecule that captures thrombin holding it adjacent to protein C bound to its receptor (endothelial protein C receptor). Elevated soluble TM in the circulation indicates endothelial cell injury [35]. Lin et al. reported that increased TM levels correlated with the extent of organ failure and mortality in patients with sepsis [36]. In the current study, thrombomodulin levels in patients randomized to placebo began to rise at approximately 36 hours into the study period, indicating sepsis-induced endothelial injury (Figure 4). Patients randomized to receive either dose of ascorbic acid exhibited no subsequent rise in plasma thrombomodulin. Though our patient numbers were small, these early results suggest that intravenous ascorbic acid acts to attenuate the proinflammatory state of sepsis and perhaps attenuates the development of endothelial injury.

On the basis of this study and our prior preclinical studies, we speculate as to the pleiotropic mechanisms by which ascorbic acid would be beneficial in sepsis. Ascorbic acid is rapidly taken up by endothelial cells in millimolar quantities where it scavenges reactive oxygen species and increases endothelial nitric oxide synthase-derived nitric oxide by restoring tetrahydrobiopterin content, thus, increasing bioavailable nitric oxide. As we and others have shown in basic investigations [10, 11, 37], by inhibiting NFκB activation, ascorbic acid could potentially attenuate the “cytokine storm” that arises due to NFκB driven genes known to be activated in sepsis. Septic ascorbic acid-deficient neutrophils fail to undergo normal apoptosis. Rather, they undergo necrosis thereby releasing hydrolytic enzymes in tissue beds, thus contributing to organ injury. We speculate that intravenous ascorbic acid acts to restore neutrophil ascorbic acid levels. Repletion of ascorbic acid in this way allows for normal apoptosis, thus, preventing the release of organ damaging hydrolytic enzymes. A multitude of biological mechanisms are active in patients with sepsis and they promote multiple organ injury and death.

Tens of thousands of lives are lost across the world annually due to severe sepsis [1, 38–40]. Multiple treatment trials have failed to measurably improve outcomes. The majority of trials have singly eliminated certain proinflammatory mediators which research has suggested promotes tissue damage. The single mediator approach has largely been unsuccessful. The results from this small phase I safety trial suggest that administering ascorbic acid in pharmacological dosages to critically ill patients with sepsis is safe and that it may provide adjunctive therapy in the treatment of severe sepsis. A larger phase II proof-of-concept trial is needed.