Endogenous Metabolite

Cytotoxic reactive oxygen species have a role in mediating neuronal damage during ischemic stroke. The oxidized version of the antioxidants, dehydroascorbic acid (DHA), penetrates the brain by facilitated transport even if ascorbic acid (AA) and vitamin C cannot cross the blood-brain barrier (BBB) [1].

Neuronal injury in ischemic stroke is partly mediated by cytotoxic reactive oxygen species. Although the antioxidant ascorbic acid (AA) or vitamin C does not penetrate the blood–brain barrier (BBB), its oxidized form, dehydroascorbic acid (DHA), enters the brain by means of facilitative transport. We hypothesized that i.v. DHA would improve outcome after stroke because of its ability to cross the BBB and augment brain antioxidant levels. Reversible or permanent focal cerebral ischemia was created by intraluminal middle cerebral artery occlusion in mice treated with vehicle, AA, or DHA (40, 250, or 500 mg/kg), either before or after ischemia. Given before ischemia, DHA caused dose-dependent increases in postreperfusion cerebral blood flow, with reductions in neurological deficit and mortality. In reperfused cerebral ischemia, mean infarct volume was reduced from 53% and 59% in vehicle- and AA-treated animals, respectively, to 15% in 250 mg/kg DHA-treated animals (P < 0.05). Similar significant reductions occurred in nonreperfused cerebral ischemia. Delayed postischemic DHA administration after 15 min or 3 h also mediated improved outcomes. DHA (250 mg/kg or 500 mg/kg) administered at 3 h postischemia reduced infarct volume by 6- to 9-fold, to only 5% with the highest DHA dose (P < 0.05). In contrast, AA had no effect on infarct volumes, mortality, or neurological deficits. No differences in the incidence of intracerebral hemorrhage occurred. Unlike exogenous AA, DHA confers in vivo, dose-dependent neuroprotection in reperfused and nonreperfused cerebral ischemia at clinically relevant times. As a naturally occurring interconvertible form of AA with BBB permeability, DHA represents a promising pharmacological therapy for stroke based on its effects in this model of cerebral ischemia [1].

Recent development in electronics has enabled the use of non-thermal plasma (NTP) to strictly direct oxidative stress in a defined location at near-physiological temperature. In preclinical studies or human clinical trials, NTP promotes blood coagulation, wound healing with disinfection, and selective killing of cancer cells. Although these biological effects of NTP have been widely explored, the stoichiometric quantitation of free radicals in liquid phase has not been performed in the presence of biocompatible reducing agents, which may modify the final biological effects of NTP. Here we quantitated hydroxyl radicals, a major reactive oxygen species generated after NTP exposure, by electron paramagnetic resonance (EPR) spectroscopy using two distinct spin-trapping probes, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO), in the presence of thiols or antioxidants. l-Ascorbic acid (AsA) at 25-50 μM concentrations (physiological concentration in the serum) significantly scavenged these hydroxyl radicals, whereas dithiothreitol (DTT), reduced glutathione (GSH), and N-acetyl-cysteine (NAC) as thiols were required in millimolar concentrations to perform scavenging activities. l-Dehydroascorbic acid (DHA), an oxidized form of AsA, necessitated the presence of 25-50 μM DTT or sub-millimolar concentrations of GSH and NAC for the scavenging of hydroxyl radicals and failed to scavenge hydroxyl radicals by itself. These results suggest that the redox cycling of AsA/DHA via thiols and cellular AsA metabolism are important processes to be considered while applying NTP to cells and tissues. Further studies are warranted to elucidate the interaction between other reactive species generated by NTP and biomolecules to promote biological and medical applications of NTP [2].

Murine Model of Cerebral Ischemia.[1]
For experiments examining the effect of focal cerebral ischemia on the ability of DHA to cross the BBB and protect cerebral tissue, we used an intraluminal murine model of transient (45 min) or permanent (24 h) right middle cerebral artery occlusion (MCAO). All studies were performed in accordance with an institutionally approved protocol and guidelines provided by the American Academy of Accreditation of Laboratory Animal Care. Normothermic, anesthetized C57BL/6J mice (aged 6–8 weeks and weighing 20–25 g) underwent perioperative, bilateral transcranial measurements of cortical cerebral blood flow by using a straight 0.7-mm laser Doppler probe (model PF 303) at previously described landmarks.[1]
In Vivo BBB Transport Studies. [1]
Nine mice were subjected to 2 h of focal ischemia or a sham operation in which the arteriotomy was performed but no occluding suture was placed, and they were immediately killed to assess transport across the BBB of ascorbate (250 mg/kg, n = 3), DHA (n = 3), and sucrose (n = 3), as measured by radiation scintillation counting using dorsal penile vein injections of 5 μCi of 14C-AA (l-[1-14C]-AA, specific activity, 6.6 mCi/mmol, DuPont/NEN), 14C-DHA generated by incubating 14C-AA with ascorbate oxidase (derived from Cucurbita species, Sigma), 1 unit/1.0 mmol l-ascorbate, or 3H-sucrose ([fructose-1-3H]sucrose, specific activity 20.0 Ci/mmol, DuPont/NEN), as described. The brains were harvested, homogenized in 70% methanol, and prepared for scintillation spectrometry or HPLC. HPLC was performed on the methanol fraction with 1 mmol/liter EDTA added. HPLC samples were separated on a Whatman strong anion exchange Partisil 10 SAX (4.6 × 25 cm) column. A Whatman-type WCS solvent-conditioning column was used, and the eluents were monitored with a Beckman System Gold liquid chromatograph with a diode array detector and radioisotope detector arranged in series. AA was monitored by UV absorbance at 265 nm and by radioactivity. DHA exhibits no absorbance at 265 nm and was monitored by radioactivity only.

[1]. Dehydroascorbic acid, a blood-brain barrier transportable form of vitamin C, mediates potentcerebroprotection in experimental stroke. Proc Natl Acad Sci U S A. 2001 Sep 25;98(20):11720-4.

L-Dehydroascorbic acid is a dehydroascorbic acid with an L-configuration. It is a coenzyme and a mouse metabolite. It is both vitamin C and a dehydroascorbic acid. Functionally, it is related to L-ascorbic acid. It is the conjugate acid of L-dehydroascorbic acid. Dehydroascorbic acid is produced by the oxidation of ascorbic acid. This reaction is reversible, but dehydroascorbic acid can also undergo irreversible hydrolysis to produce 2,3-diketogulonic acid. Both dehydroascorbic acid and ascorbic acid are known as vitamin C, but the latter is the predominant form in humans. In vivo, both dehydroascorbic acid and ascorbic acid have similar antiviral activities, but dehydroascorbic acid also has neuroprotective effects. Currently, dehydroascorbic acid is an experimental drug with no known approved indications.
Dehydroascorbic acid is a metabolite of Escherichia coli (K12 strain, MG1655 strain).
Dehydroascorbic acid has also been reported in chili peppers, canine roses, and other organisms with relevant data.
It is a reversible oxidized form of ascorbic acid, a lactone of 2,3-diketogulonic acid, and exhibits antiscurvy activity in humans after oral administration.
Pharmacological Indications

Currently, there are no approved indications for dehydroascorbic acid, but it has potential therapeutic uses in certain viral infections and ischemic stroke patients.
Mechanism of Action

Although dehydroascorbic acid and ascorbic acid have similar effects, their mechanisms of action appear to differ. The exact mechanism of action is still under investigation, but some mechanisms have been elucidated. Regarding the antiviral effect of dehydroascorbic acid against herpes simplex virus type 1, studies have shown that dehydroascorbic acid acts after viral DNA replication, preventing the assembly of progeny viral particles.
Pharmacodynamics

Dehydroascorbic acid has similar biological activities to ascorbic acid. Both compounds have been shown to have antiviral activity against herpes simplex virus type 1, influenza A virus, and poliovirus type 1, with dehydroascorbic acid showing a stronger effect. Furthermore, unlike ascorbic acid, dehydroascorbic acid can cross the blood-brain barrier and be converted to ascorbic acid in the brain, thus remaining there. This is significant because one study found that after ischemic stroke, dehydroascorbic acid exerts neuroprotective effects by reducing infarct volume, neurological deficits, and mortality.

C6H6O6

174.1

174.016

C, 41.39; H, 3.47; O, 55.13

490-83-5

440667

Light yellow to yellow solid

1.7±0.1 g/cm3

389.3±38.0 °C at 760 mmHg

228 °C (dec.)(lit.)

170.0±20.3 °C

0.0±2.0 mmHg at 25°C

1.570

-2.32

2

6

2

12

244

2

O1C(C(C([C@@]1([H])[C@]([H])(C([H])([H])O[H])O[H])=O)=O)=O

SBJKKFFYIZUCET-JLAZNSOCSA-N

InChI=1S/C6H6O6/c7-1-2(8)5-3(9)4(10)6(11)12-5/h2,5,7-8H,1H2/t2-,5+/m0/s1

L-threo-2,3-Hexodiulosonic acid gamma-lactone

L Dehydroascorbic acid; L-Dehydroascorbic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~287.17 mM)
H2O : ~5 mg/mL (~28.72 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (14.36 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (14.36 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (14.36 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 12.5 mg/mL (71.79 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)