rexresearch.com


Douglas KITT

DeHydroAscorbic Acid









https://www.youtube.com/watch?v=YHKBhz7OCB4
   
Learn how to make do-it-yourself DHAA (dehydroascorbic acid)

The discoverer claims it surpasses absorption limits of other forms of vitamin C and sites a number of studies so indicating.

There are different types of ports on the cells in the body, and one of the ports is for vitamin C.

The form of vitamin C disclosed in the video and on the web site is absorbed through the glucose ports on the cells, and there are 4-5 times more glucose ports than ascorbic acid/vitamin C ports in the cells, so much higher absorption rates are achieved with this form (it also takes energy to absorb vitamin C through the vitamin C ports, but it doesn't take energy to absorb through the glucose ports). (Absorption into blood stream from digestive tract and from blood stream into cells in the body).  The cells can then convert this form of vitamin C back to the common form.





https://www.recverin.com

About Us

Launched in 2010, ReCverin LLC was founded to make available the finest Vitamin C products for skin care and dietary supplementation. Based on our experience with stable Vitamin C solutions, and our patented discovery of stable solutions of dehydroascorbic acid, we proudly offer ReCverin 50/50™ and ReCverin C™.

ReCverin LLC
944 E. 3300 S.
Salt Lake City, UT 84106
(801) 556-6424

ReCverin C™ contains 10% L-ascorbic acid as a stabilized solution in pure, vegetable glycerin. It provides the moisturizing, collagen-stimulating and antioxidant power of L-ascorbic acid in an ultra-pure, fragrance-free, moisturizing base with no harsh chemical preservatives. For firmer, smoother, more radiant-looking skin, apply daily before any other skincare product to the face, neck, hands, arms, and other exposed skin.

Stratum corneum (the dead outer layer of skin) poses a significant barrier to absorption of the common form of Vitamin C called L-ascorbic acid. Therefore L-ascorbic acid serums are most effective for persons who also practice some type of exfoliation (which refers to physical or chemical techniques for removing stratum corneum). Nevertheless, daily topical use of a high-strength serum like ReCverin C™ has been shown to increase the Vitamin C concentration in skin to higher levels than can be achieved by oral ingestion of Vitamin C, even in persons who don't exfoliate. The pH of an L-ascorbic acid serum has been shown to be a critical factor; absorption is much improved in serums with pH values of 3.0 or lower. ReCverin C™ is formulated at pH 2.7 for best absorption.

ReCverin C™ compares to many competing high-strength Vitamin C serums, with these important differences:

ReCverin C™ is extremely stable. It will easily retain greater than 95% of its stated L-ascorbic acid concentration for a year when stored at typical room temperatures, and it will remain crystal clear and colorless. With ReCverin C™, you are free to ignore any concerns about stability and/or yellowing of your Vitamin C skin serum!

ReCverin C™ does not contain emulsifiers, detergents, preservatives, colorants or fragrances. Formulated in pure vegetable glycerin, it does not require any unnatural chemical agents that can contribute to skin sensitivity or other problems. Glycerin is the natural humectant found in sebum, the fluid that skin normally secretes onto its own surface. This water-soluble, skin-identical moisturizer has been used for centuries for its cosmetic effects in smoothing and moisturizing the skin, and also for its soothing, beneficial effects on rough, dry and irritated skin.

ReCverin C™ is economical. The two-ounce bottle contains 2 or 4 times more serum than a bottle of most competing products.

Please be aware that some people experience tingling or irritation after applying high-strength L-ascorbic acid solutions. For most people, if they experience them at all, these effects are mild, temporary, and completely tolerable. But those with very sensitive skin can have difficulty using high-strength L-ascorbic acid.

We highly recommend our premiere product ReCverin 50/50™ for those with sensitive skin, for those who don't regularly exfoliate, and, in fact, for anyone who is seeking maximum absorption of Vitamin C.

ReCverin C™ Use and Storage Guidelines

Apply daily to the face, neck, arms, hands and other exposed skin. Avoid direct contact with eyes.

We suggest using 3-5 drops for the face, and a similar application rate for other, similar-sized skin areas.

Apply to clean skin before any other products, and massage in thoroughly.

ReCverin C™ can be used directly as supplied, or it may be mixed with water immediately before applying. We suggest applying to moist skin or mixing with a little water by placing a few drops of water and product in the palm and rubbing the hands together. Water combines with the humectant glycerin base to create a deep-penetrating moisturizer that absorbs quickly and gives a skin texture that many people prefer. Vary the proportions to the consistency you desire.

ReCverin C™ blends nicely with most lotions. You can mix a few drops with another lotion immediately before applying, or you can apply another lotion immediately after applying our serum.

Please keep in mind that ReCverin C™ is carefully formulated to stabilize Vitamin C. One of the secrets to its stability is that it contains no water. We recommend that you do not premix with water or lotions for later use.

Store at room temperature or below. For best results, use within 1 year.

A Special Note for Do-It-Yourselfers

Making do-it-yourself skin care products is a popular hobby. We respect and admire those who blend their own in that quest for the perfect, individualized composition of ingredients! Both ReCverin C™ and ReCverin 50/50™ can be utilized as a starting base or component of the water-soluble fraction of your own formulas.

A particularly popular composition among DIYers contains Vitamin C, Vitamin E, and Ferulic Acid, commonly known as a "CEF Serum," and there are many different and varied recipes. To demonstrate how our products can be used by DIY folks, we have prepared a video that shows how an excellent and easy CEF Serum can be prepared featuring ReCverin C™. This recipe focuses on maintaining a water-free composition to preserve the remarkable stability of the Vitamin C component as provided in ReCverin C™.


https://www.youtube.com/watch?v=cwwdk9scG64&feature=youtu.be

DIY CEF Vitamin C Skin Serum for Beginners

See how to make an economical, effective and elegant skin serum with vitamin C, vitamin E, and ferulic acid.
Ingredients:
1 bottle ReCverin C TM or ReCverin 50/50 TM from www.ReCverin.com
1/8 tsp ferulic acid
18 drops vitamin E oil
18 drops polysorbate 80
(from a supplier of ingredients for DIY skin care like Lotioncrafter or Skin Essentials Actives)



https://www.researchgate.net/publication/225274699_Topical_Dehydroascorbic_Acid_Oxidized_Vitamin_C_Permeates_Stratum_Corneum_More_Rapidly_Than_Ascorbic_Acid?ev=prf_pub_p2

Topical Dehydroascorbic Acid (Oxidized Vitamin C) Permeates Stratum Corneum More Rapidly Than Ascorbic Acid

Abstract

Topical application of vitamin C has an established history of use in skincare. A large body of literature from clinical and laboratory studies supports a scientific basis for its use in improving both the appearance and health of the skin. Ascorbic acid (AA) is the naturally-occurring chemical form of vitamin C that is most familiar, and it is commonly used in topical products. But AA has limited permeation through the stratum corneum, and this has led to the use of very high concentrations that are associated with side effects such as tingling, irritation and redness in some people. Dehydroascorbic acid (DHAA) is the other naturally-occurring form of vitamin C, and has chemical properties that suggest its skin permeation rate would be higher than AA. In this study, the rates of AA and DHAA permeation were compared by a clinically relevant, in vivo method on human subjects. Specifically, a solution containing equal parts of AA and DHAA was applied in amounts and for time periods likely to be achieved in common use of a topical product by consumers. The amount absorbed was determined by subtracting the amount recoverable in skin washings. The results show that DHAA permeates stratum corneum at a rate up to 12 times faster than AA. This supports the concept that lower concentrations of DHAA in topical preparations can enhance skin vitamin C levels with less potential for side effects…

DHAA is a more lipophilic compound than AA, and it is not ionized in aqueous solution [11]. Both of these properties suggest that DHAA would permeate the stratum corneum more easily [12]. The aim of the present study was to compare the rate of AA versus DHAA absorption in order to assess whether or not DHAA actually does permeate more easily. A solution containing both AA and DHAA in approximately equal concentrations was used to compare their absorption rates in vivo, using a simple, non-invasive technique on human subjects. To assess permeation into the stratum corneum, the solution was applied to delineated skin surfaces and allowed 0, 2 or 4 hours for absorption. Each skin surface was then washed with deionized water, and the skin washings were collected and measured for AA and DHAA content. The amount absorbed at 2 and 4 hours was determined by subtraction from the amount present in the 0 hour washings. The aim of the study was achieved, since measurable differences in AA versus DHAA absorption were observed, and the DHAA absorption rate was found to be significantly greater than that of AA…





http://www.sciencedirect.com/science/article/pii/S2213231715300045
doi:10.1016/j.redox.2015.11.003
Redox Biology, Volume 7, April 2016, Pages 8–13

l-dehydroascorbic acid can substitute l-ascorbic acid as dietary vitamin C source in guinea pigs

Henriette Frikke-Schmidt, Pernille Tveden-Nyborg, Jens Lykkesfeldt,

Highlights

Dehydroascorbic acid is an effective vitamin C source in guinea pigs.
Like in humans, efficient recycling of vitamin C has evolved in guinea pigs.
The guinea pig is a better model of human vitamin C homeostasis than rat and mouse.

Abstract

Vitamin C deficiency globally affects several hundred million people and has been associated with increased morbidity and mortality in numerous studies. In this study, bioavailability of the oxidized form of vitamin C (l-dehydroascorbic acid or DHA)—commonly found in vitamin C containing food products prone to oxidation—was studied. Our aim was to compare tissue accumulation of vitamin C in guinea pigs receiving different oral doses of either ascorbate or DHA. In all tissues tested (plasma, liver, spleen, lung, adrenal glands, kidney, muscle, heart, and brain), only sporadic differences in vitamin C accumulation from ascorbate or DHA were observed except for the lowest dose of DHA (0.25 mg/ml in the drinking water), where approximately half of the tissues had slightly yet significantly less vitamin C accumulation than from the ascorbate source. As these results contradicted data from rats, we continued to explore the ability to recycle DHA in blood, liver and intestine in guinea pigs, rats and mice. These investigations revealed that guinea pigs have similar recycling capacity in red blood cells as observed in humans, while rats and mice do not have near the same ability to reduce DHA in erythrocytes. In liver and intestinal homogenates, guinea pigs also showed a significantly higher ability to recycle DHA compared to rats and mice. These data demonstrate that DHA in guinea pigs—as in humans—is almost as effective as ascorbate as vitamin C source when it comes to taking up and storing vitamin C and further suggest that the guinea pig is superior to other rodents in modeling human vitamin C homeostasis.



Exp Neurobiol. 2015 Mar;24(1):41-54. English.
http://dx.doi.org/10.5607/en.2015.24.1.41

Dehydroascorbic Acid Attenuates Ischemic Brain Edema and Neurotoxicity in Cerebral Ischemia: An in vivo Study

Juhyun Song, Joohyun Park, Jae Hwan Kim, Ja Yong Choi, Jae Young Kim, Kyoung Min Lee, and Jong Eun Lee

Abstract

Ischemic stroke results in the diverse phathophysiologies including blood brain barrier (BBB) disruption, brain edema, neuronal cell death, and synaptic loss in brain. Vitamin C has known as the potent anti-oxidant having multiple functions in various organs, as well as in brain. Dehydroascorbic acid (DHA) as the oxidized form of ascorbic acid (AA) acts as a cellular protector against oxidative stress and easily enters into the brain compared to AA. To determine the role of DHA on edema formation, neuronal cell death, and synaptic dysfunction following cerebral ischemia, we investigated the infarct size of ischemic brain tissue and measured the expression of aquaporin 1 (AQP-1) as the water channel protein. We also examined the expression of claudin 5 for confirming the BBB breakdown, and the expression of bcl 2 associated X protein (Bax), caspase-3, inducible nitric oxide synthase (iNOS) for checking the effect of DHA on the neurotoxicity. Finally, we examined postsynaptic density protein-95 (PSD-95) expression to confirm the effect of DHA on synaptic dysfunction following ischemic stroke. Based on our findings, we propose that DHA might alleviate the pathogenesis of ischemic brain injury by attenuating edema, neuronal loss, and by improving synaptic connection.

INTRODUCTION

Ischemic stroke is the second leading cause of death worldwide accompanied by severe disability [1]. Cerebral ischemia and reperfusion injury leads to damage of brain tissues, inflammation as a result of the blood-brain barrier (BBB) disruption, oxidative damage [2], and apoptosis [3]. Brain tissue is highly vulnerable to oxidative damage because of its high use of oxygen [4] under cerebral ischemia. Cerebral ischemia leads to loss of tight junction proteins in brain endothelium, BBB disruption, and finally brain edema [5]. Brain edema leads to an imbalance in energy demand and influences on the postsynaptic effects of glutamate [6] and interruption of synaptic transmission in the penumbra after stroke [7, 8]. Overall, excitotoxicity, inflammation and oxidative stress caused by ischemic stroke plays a crucial role in the pathophysiology of ischemic stroke [9, 10]. To reduce the brain damage caused by cerebral ischemia, the solution for oxidative damage is the issue of the greatest importance. Vitamin C is the most important antioxidant for metabolic function of the brain [11, 12, 13] owing to its low redox potential which is capable of neutralizing diverse pro oxidants [14, 15, 16, 17]. Mainly, vitamin C could be found in its form such as ascorbic acid (AA) and dehydroascorbic acid (DHA) (AA's oxidized form) [18, 19]. According to earlier studies, lower levels of vitamin C are a risk factor of cerebral stroke [20, 21] and actually, decreased vitamin C levels has been demonstrated in patients with ischemic stroke [22]. Recent study demonstrated that the treatment of AA prevented the disruption of BBB and sustained the BBB integrity in the cortex [23]. Neuroprotection by DHA has been demonstrated in several recent studies in both in vitro and in vivo. In in vitro study, DHA has been reported that it inhibits mitochondrial damage and cell death against oxidative injury [24]. Specifically, DHA among vitamin C could crosses the BBB through glucose transporter 1 (GLUT1) [25] and prevents cell death against oxidative damage by increasing glutathione (GSH) levels through glucose transporters [26, 27]. In in vivo study, DHA have been reported to have protective effects as antioxidants in experimental neurological disease models such as stroke [19, 28, 29, 30]. DHA administration attenuates oxidative stress markers and inflammation in hyperglycemic stroke models [31]. However, the study on the role of DHA administration through intra-peritoneal route in ischemic stroke animal model focused on edema formation, neurotoxicity, and synaptic dysfunction has not yet been determined. In present study, we investigated DHA's beneficial effect after ischemic brain injury in in vivo study. Our results show that DHA is involved in the prevention of brain edema formation, neurotoxicity, and synaptic dysfunction following ischemia injury. Thus, we suggest that DHA might mitigate stroke-induced pathological alterations following cerebral ischemic stroke...

RESULTS

DHA reduced brain edema formation following cerebral ischemia

...The percentage of brain edema in the MCAO group was >12% whereas the percentage of brain edema after DHA treatment was <8% (Fig. 1C). Brain edema (%) was significantly reduced in the DHA group compared with the MCAO group. Our results indicate that the DHA treatment reduced brain edema formation after ischemic brain injury...

DHA reduced the expression of AQP-1 as the marker of vascular permeability following cerebral ischemia...
    
DHA attenuates the cell damage against neurotoxicity following cerebral ischemia…

DHA prevents the damage of post synaptic plasticity following cerebral ischemia…

DISCUSSION

Ischemic stroke causes the blockage of cerebral blood vessels in the regions of brain, which can lead to human disability and death [36]. Subsequently, the blockage of blood vessels following stroke leads to diverse pathophysiologies including brain edema, neuronal loss, and cognitive dysfunction [37, 38, 39, 40]. Cerebral cortex, hippocampus, and corpus striatum in the brain are the most vulnerable regions against oxidative stress and hypoxic injury induced by cerebral ischemia [41]. Many studies has reported that vitamin C among antioxidants is generally neuroprotective in response to brain ischemic injury [42, 43, 44, 45]. Oral administration of AA to animal had demonstrated that it suppresses neuronal damage under cerebral ischemia-reperfusion [46]. Dehydroascorbic acid as AA's oxidized form [15, 18, 19] has been reported that it has a neuroprotective role [47] and is easily transported to the brain by mediating glucose transporter 1 (GLUT1) located in the endothelial cells of the BBB [48]. However, DHA did not fully be investigated in ischemic stroke animal model in spite of its advantages. We anticipated that DHA as an anti-oxidant may considerably affect on cerebral ischemia animal because it can rapidly pass through the brain than AA [25]. In present study, we investigated the neuroprotective effects of brain by DHA i.p administration in cerebral ischemia rat. First, we obtained the consequence that DHA treatment inhibits the brain edema formation in MCAO rat brain. Edema defined as an abnormal increase in brain water content is frequently observed in cerebral ischemia and also has a critical influence on morbidity and mortality [49]. Several studies reported that cerebral ischemia contributes to damage the integrity and permeability of the BBB [50, 51]. Aquaporin (AQP) is the water channel protein that facilitates water transport through cell membranes [52, 53]. Specifically AQP-1 is permeable only to water and is considered to participate in brain water homeostasis [54]. In addition, AQP 1 has been reported that it is involved in edema formation and cell death in the hippocampus following brain injury [55]. Following our results, we suggest that DHA may reduce osmotic water permeability following cerebral ischemia by inhibiting the expression of AQP-1. All BBB components have been reported to the association with the regulation of the BBB permeability including tight junctions of endothelial cells, glia cells [56, 57, 58]. The BBB is composed of the brain endothelial cells interconnected with transmembrane tight junction proteins such as claudin-5 [59] and regulates paracellular permeability [60, 61]. In present study, our results indicated that claudin 5 as a tight junction protein in DHA treated MCAO rat brain was evidently preserved against ischemic injury. According to our results, DHA may protect the BBB integrity by preserving tight junction protein in response to ischemic injury. Cerebral ischemia induces the neurotoxic environment in brain and it could result in the severe neuronal cell damage, so we investigated the cell death marker such as Bax [62, 63], caspase-3[64, 65], and iNOS [66, 67] in order to examine the protective effect of DHA against the neurotoxicity following ischemic stroke. In present study, DHA treatment reduced the expression of Bax and caspase-3 which is the marker of the mitochondrial cell death and iNOS in ischemic injured brain. Nitric oxide (NO) that causes neuronal cell death and exacerbates glutamate toxicity after cerebral ischemia [68] is synthesized by NO synthase such as iNOS [69]. Several studies demonstrated that inhibition of iNOS in cerebral ischemia improves neurological deficits and reduces infarct volume [70, 71]. In consideration of Figure 1 result, our finding suggested that DHA attenuates the expression of iNOS and it may be linked to reduced infarct volume and improved cell death against hypoxic injury. Additionally, NO formed by iNOS has been reported the implicated in neurodegeneration [69]. Judging from our result regarding the reduced iNOS expression, we suggest the possibility of DHA regarding the improvement of cognitive function against ischemic stroke although we did not check the production of NO and behavior test considering that AA improves the cognitive decline in Alzheimer's disease [72]. As mentioned earlier, several studies demonstrated that DHA prevents cell death against ischemic injury [19, 28, 29, 30]. However, previous studies have not yet been determined the effect of DHA on recovery of neuronal function in ischemia animal model. Therefore, we tried to examine the effect of DHA on neuronal function by measuring indirectly synaptic dysfunction in present study. In order to observe the effect of DHA on ischemia-induced synaptic connection alteration, we investigated the expression of PSD-95 protein in ischemic brain tissue. PSD-95 protein as a postsynaptic marker [73, 74] is a member of the membrane-associated guanylate kinase family of synaptic molecules and is localized at excitatory synapses [75]. Postsynaptic densities (PSD) proteins are involved in regulation of synaptic function and in the transduction of synaptic signals to the postsynaptic cell [76, 77, 78]. Especially, PSD-95 has been implicated in the regulation of ion-channel function, synaptic activity, and intracellular signaling and finally cognitive impairment [79, 80, 81]. In addition, PSD-95 protein is implicated in promoting synapse stability and makes synaptic contacts more stable in neurons [75]. Recent studies suggested that the PSD-95 protein improves the neurophysiologic phenomenon after ischemic stroke involving MCAO [82, 83]. Moreover, some researchers demonstrated that the decrease of PSD-95 protein immunoreactivity in the ischemic brain leads to a deficit of postsynaptic plasticity in the brain [84]. Several studies suggest that PSD-95's reduction is associated with cognitive impairment [85, 86, 87, 88]. Based on our results, our findings indicate that DHA induced the increase of PSD-95 protein immunoreactivity in ischemic stroke brain and DHA may improve the ischemic-induced synaptic plasticity dysfunction. In addition, although we did not check the memory function related behavior test such as water maze, we carefully expect that DHA may improve the learning and memory dysfunction following cerebral ischemia by promoting the neuron's synapse stability. Taken together, our findings suggest three points that 1) DHA is involved in the inhibition of AQP-1 expression and the preservation of claudin 5, ultimately resulting in the reduction of edema formation induced by cerebral ischemia, 2) DHA is associated with the decrease of Bax, cleaved caspase-3 and iNOS expression, ultimately resulting in the protection of cell death against neurotoxicity following cerebral ischemia, 3) DHA is linked to the preservation of PSD-95 protein expression, ultimately resulting in the improvement of neuron's synaptic connection in cerebral ischemia. The present study has some limitations fully to prove the beneficial effect of DHA against ischemic injury. However, we suggest that this study is worthy in that it provide the need of the further study of DHA on ischemic stroke. Taken together, we propose that the DHA might be beneficial to alleviate clinical pathologies that occur after ischemic stroke.



http://www.ncbi.nlm.nih.gov/pubmed/24460956
J Neurochem. 2014 May;129(4):663-71.
doi: 10.1111/jnc.12663. Epub 2014 Feb 19.

The oxidized form of vitamin C, dehydroascorbic acid, regulates neuronal energy metabolism.

Cisternas P, Silva-Alvarez C, Martínez F, Fernandez E, Ferrada L, Oyarce K, Salazar K, Bolaños JP, Nualart F.

Abstract

Vitamin C is an essential factor for neuronal function and survival, existing in two redox states, ascorbic acid (AA), and its oxidized form, dehydroascorbic acid (DHA). Here, we show uptake of both AA and DHA by primary cultures of rat brain cortical neurons. Moreover, we show that most intracellular AA was rapidly oxidized to DHA. Intracellular DHA induced a rapid and dramatic decrease in reduced glutathione that was immediately followed by a spontaneous recovery. This transient decrease in glutathione oxidation was preceded by an increase in the rate of glucose oxidation through the pentose phosphate pathway (PPP), and a concomitant decrease in glucose oxidation through glycolysis. DHA stimulated the activity of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the PPP. Furthermore, we found that DHA stimulated the rate of lactate uptake by neurons in a time- and dose-dependent manner. Thus, DHA is a novel modulator of neuronal energy metabolism by facilitating the utilization of glucose through the PPP for antioxidant purposes.



http://www.jbc.org/content/288/13/9092.full

Intestinal Dehydroascorbic Acid (DHA) Transport Mediated by the Facilitative Sugar Transporters, GLUT2 and GLUT8*

Christopher P. Corpe, Peter Eck, Jin Wang, Hadi Al-Hasani and Mark Levine

Background: The molecular identity of the intestinal vitamin C transporters is incomplete.
Results: Facilitative sugar transporters, GLUT2 and GLUT8, transport dehydroascorbic acid, the oxidized form of vitamin C.
Conclusion: Intestinal vitamin C absorption can occur via facilitative sugar transporters.
Significance: Vitamin C bioavailability may be inhibited by dietary factors, such as glucose and phytochemicals.
 
Abstract

Intestinal vitamin C (Asc) absorption was believed to be mediated by the Na+-dependent ascorbic acid transporter SVCT1. However, Asc transport across the intestines of SVCT1 knock-out mice is normal indicating that alternative ascorbic acid transport mechanisms exist. To investigate these mechanisms, rodents were gavaged with Asc or its oxidized form dehydroascorbic acid (DHA), and plasma Asc concentrations were measured. Asc concentrations doubled following DHA but not Asc gavage. We hypothesized that the transporters responsible were facilitated glucose transporters (GLUTs). Using Xenopus oocyte expression, we investigated whether facilitative glucose transporters GLUT2 and GLUT5–12 transported DHA. Only GLUT2 and GLUT8, known to be expressed in intestines, transported DHA with apparent transport affinities (Km) of 2.33 and 3.23 mm and maximal transport rates (Vmax) of 25.9 and 10.1 pmol/min/oocyte, respectively. Maximal rates for DHA transport mediated by GLUT2 and GLUT8 in oocytes were lower than maximal rates for 2-deoxy-d-glucose (Vmax of 224 and 32 pmol/min/oocyte for GLUT2 and GLUT8, respectively) and fructose (Vmax of 406 and 116 pmol/min/oocyte for GLUT2 and GLUT8, respectively). These findings may be explained by differences in the exofacial binding of substrates, as shown by inhibition studies with ethylidine glucose. DHA transport activity in GLUT2- and GLUT8-expressing oocytes was inhibited by glucose, fructose, and by the flavonoids phloretin and quercetin. These studies indicate intestinal DHA transport may be mediated by the facilitative sugar transporters GLUT2 and GLUT8. Furthermore, dietary sugars and flavonoids in fruits and vegetables may modulate Asc bioavailability via inhibition of small intestinal GLUT2 and GLUT8.



http://www.ingentaconnect.com/content/sp/ijmm/2008/00000022/00000004/art00018
International Journal of Molecular Medicine, Volume 22, Number 4, 2008, pp. 541-545(5)
http://dx.doi.org/10.3892/ijmm_00000053

Antiviral effects of ascorbic and dehydroascorbic acids in vitro

Furuya, Ayami; Uozaki, Misao; Yamasaki, Hisashi; Arakawa, Tsutomu; Arita, Mikio; Koyama, A. Hajime

Abstract:

In the present study, ascorbic acid weakly inhibited the multiplication of viruses of three different families: herpes simplex virus type 1 (HSV-1), influenza virus type A and poliovirus type 1. Dehydroascorbic acid, an oxidized form of ascorbic acid and hence without reducing ability, showed much stronger antiviral activity than ascorbic acid, indicating that the antiviral activity of ascorbic acid is due to factors other than an antioxidant mechanism. Moreover, addition of 1 mM Fe3+, which oxidizes ascorbic acid to dehydroascorbic acid and also enhances the formation of hydroxyl radicals by ascorbic acid in the culture media, strongly enhanced the antiviral activity of ascorbic acid to a level significantly stronger than that of dehydroascorbic acid. Although both ascorbic acid and dehydroascorbic acid showed some cytotoxicity, the degree of cytotoxicity of the former was 10-fold higher than the latter, suggesting that the observed antiviral activity of ascorbic acid with and without ferric ion is, at least in part, a secondary result of the cytotoxic effect of the reagent, most likely due to the free radicals. However, the possibility that oxidation of ascorbic acid also contributed to the antiviral effects of ascorbic acid exists, in particular in the presence of ferric ion, since dehydroascorbic acid exhibited a very strong antiviral activity. Characterization of the mode of antiviral action of dehydroascorbic acid revealed that the addition of the reagent even at 11 h post infection almost completely inhibited the formation of progeny infectious virus in the infected cells, indicating that the reagent inhibits HSV-1 multiplication probably at the assembly process of progeny virus particles after the completion of viral DNA replication.



http://onlinelibrary.wiley.com/doi/10.1002/art.21254/full
Arthritis & Rheumatism, Volume 52, Issue 9, pages 2676–2685, September 2005
DOI: 10.1002/art.21254

Dehydroascorbate transport in human chondrocytes is regulated by hypoxia and is a physiologically relevant source of ascorbic acid in the joint

Amy L. McNulty, Thomas V. Stabler, Thomas P. Vail, Gary E. McDaniel and Virginia B. Kraus*

Abstract

Objective


To evaluate the dehydroascorbate (DHA) transport mechanisms in human chondrocytes.

Methods


The transport of L-14C-DHA in human chondrocytes was analyzed under various conditions, including the use of RNA interference (RNAi), to determine the role of glucose transporter 1 (GLUT-1) and GLUT-3 in L-14C-DHA transport and to evaluate the effects of physiologically relevant oxygen tensions on L-14C-DHA transport. In order to estimate the contributions of reduced ascorbic acid (AA) and DHA to intracellular ascorbic acid (Asc), the quantities of AA and DHA were measured in synovial fluid samples from osteoarthritis (OA) patients and compared with the reported levels in rheumatoid arthritis (RA) patients.
Results

DHA transport in human chondrocytes was glucose-sensitive, temperature-dependent, cytochalasin B–inhibitable, modestly stereoselective for L-DHA, and up-regulated by low oxygen tension. Based on the RNAi results, GLUT-1 mediated, at least in part, the uptake of DHA, whereas GLUT-3 had a minimal effect on DHA transport. DHA constituted a mean 8% of the total Asc in the synovial fluid of OA joints, in contrast to 80% of the reported total Asc in RA joints.
Conclusion

We provide the first evidence that chondrocytes transport DHA via the GLUTs and that this transport mechanism is modestly selective for L-DHA. In the setting of up-regulated DHA transport at low oxygen tensions, DHA would contribute 26% of the total intracellular Asc in OA chondrocytes and 94% of that in RA chondrocytes. These results demonstrate that DHA is a physiologically relevant source of Asc for chondrocytes, particularly in the setting of an inflammatory arthritis, such as RA.



US8324269
STABLE COMPOSITIONS OF DEHYDROASCORBIC ACID

    
Inventor: KITT DOUGLAS / KITT JAY P    

Stable liquid compositions containing the oxidized form of vitamin C known as dehydroascorbic acid are provided. The compositions comprise dehydroascorbic acid and a pharmacologically acceptable liquid organic polyol solvent for said dehydroascorbic acid, wherein said polyol solvent comprises about 50% or greater of the total weight of said composition. The compositions are useful as dietary supplements, skin-enhancers, concentrates, or research solutions.

BACKGROUND

[0001] 1. Field of Invention

[0002] This invention relates to compositions of matter used as sources of vitamin C in dietary supplementation, skin care products, therapy, research, and manufacturing. More specifically, the invention relates to stable liquid compositions containing the oxidized form of vitamin C known as dehydroascorbic acid.

[0003] 2. Prior Art

[0004] Ever since the elucidation of the chemical structure of vitamin C in the mid-1930's it has been known that vitamin C occurs naturally as two different compounds, namely, ascorbic acid (AA) and an oxidized form of AA called dehydroascorbic acid (DHAA). It also is known that AA and DHAA are unstable compounds. In aqueous solutions, some factors which affect the rate of their destruction include the pH of the solution, and exposure to various metal ions, heat, light and air. It also is known that DHAA is considerably less stable than AA when subjected to comparable conditions. ‘Deutsch J C. Dehydroascorbic acid. Review Journal of Chromatography A, 881 (2000) 299-307 ’ (Deutsch), incorporated here by reference, states en-equivocally “DHA is more reactive and unstable in solution than AA.” Therefore, as a supplement to the diet, or as an ingredient of a topically applied product such as a skin lotion, AA has been the preferred chemical form of vitamin C because of its greater stability. In fact, we do not know of any commercially available dietary or topically applied product wherein DHAA specifically has been utilized as a substantial source of vitamin C.

[0005] Also known is that solid AA is far more easily dissolved in water than is solid DHAA, as noted in ‘Pecherer B J. The Preparation of Dehydro-L-ascorbic Acid and its Methanol Complex. Am Chem Soc 73 (1951) 3827-3830’ (Pecherer) and ‘Koliou E K and Ioannou P V. Preparation of dehydro-L-ascorbic acid dimer by air oxidation of L-ascorbic acid in the presence of catalytic amounts of copper(II) acetate and pyridine. Carbohydrate Research 340 (2005) 315-318 ’ (Koliou) which are incorporated here by reference. To prepare aqueous solutions of DHAA from the solid form requires prolonged mixing at temperatures well above 37 degrees centigrade. Thus solutions of DHAA are much more difficult to manufacture than solutions of AA. Also, since the conditions to solubilize it efficiently do not exist in the gut of human or other animals, substantial doubt exists about whether the dry, solid form of DHAA can be absorbed when ingested. These are also reasons why DHAA has not been utilized as the source of vitamin C for dietary supplements or topical products.

[0006] Around the same time as the chemical structures of AA and DHAA were elucidated in the mid-1930's, the antiscorbutic properties (ability to prevent the disease called scurvy) of both compounds were recognized and generally accepted as being equal or nearly so. The oxidation of AA to DHAA was shown to be reversible both in vitro and in biological systems, so the equivalence of the two compounds could easily be attributed to simple interconversion within an organism. Although a few early investigators did note some peculiar differences in the biological utilization of these two compounds, at least as essential dietary ingredients for humans and certain other species, AA and DHAA were generally considered bioequivalent. The dietary supplement and skin care product industries developed their products using AA (and various more stable derivatives of AA) because of the stability and solubility issues with DHAA, and DHAA has essentially been ignored and forgotten in these industries.

[0007] Since the mid-1930's, the volume of research in vitamin C has been enormous, and it is possible that no single subject in the field of biology has been the focus of more research and more scientific journal articles than vitamin C. And since about the mid-1990's, many new discoveries about DHAA have been made. Among these discoveries, those of particular pertinence to the present invention include those which demonstrate that, although the two compounds are equivalent in their antiscorbutic properties, AA and DHAA are not “bioequivalent” in any broad definition of the word. Specifically it is known today that AA and DHAA are absorbed by different mechanisms in the gut; that they accumulate differently in the various tissues of an animal when ingested; that they are absorbed into living cells by completely different mechanisms utilizing different receptors on the cell surface; that the cells of certain important tissues of the human body (e.g., brain) have a very high concentration of vitamin C but completely lack cell surface receptors for AA; that DHAA is absorbed into cells by the same receptors as glucose, which are present on every cell in the human body; that in human skin cells, DHAA is absorbed up to 5 times faster and to levels 2 times higher than is AA; that DHAA is almost instantly converted into AA once it has been absorbed into a cell; that both AA and DHAA have antiviral effects in vitro against viruses that cause disease in humans such as HSV-1 (herpes simplex virus type 1 that causes oral herpes and can cause genital herpes), influenza virus, and poliovirus; and that DHAA has much stronger antiviral effects than does AA. Literature that supports these statements, and is incorporated here by reference, includes ‘Savini et al. Dehydroascorbic acid uptake in a human keratinocyte cell line (HaCaT) is glutathione-independent. Biochem J 345 (2000) 665-672 ’ (Savini) and ‘Furuya et al. Antiviral effects of ascorbic and dehydroascorbic acids in vitro. Int J Mol Med 22 (2008) 541-545’ (Furuya).

[0008] Thus it can be seen that a solution of DHAA for oral ingestion or topical application, while being a source of vitamin C much like numerous other available products that contain AA, also can provide specific benefits and uses unavailable in any other product on the market today. What is needed is a stable liquid solution of DHAA in an orally and topically acceptable medium.

[0009] U.S. Pat. No. 5,140,043 (Dan) discloses topical compositions of ascorbic acid (or a reducing analog of ascorbic acid) in a water-(glycol or polyol) carrier, wherein the ratio of water to glycol/polyol carrier is high (e.g., at least 1:1). These solutions of Darr do not contain DHAA, and Darr is silent as to the stability of the non-reducing compound DHAA in this carrier. We have found that DHAA is not stable in polyol solutions containing such high concentrations of water, which points out that no assumptions about the chemical and physical behavior of DHAA in polyol solutions should be drawn from the behavior of AA in those solutions. While AA and DHAA share certain biological functions, they are two different molecules in regard to their physical and chemical behavior, including stability.

[0010] U.S. Pat. No. 6,197,813 (Hegenauer) discloses stable vitamin C compositions of mineral ascorbates in liquid organic polyol solvents having pH values of about 5 to 7, but is silent as to the stability of the non-mineral DHAA in those solvents. These compositions of Hegenaur do not contain DHAA. In fact, these compositions do not even contain a naturally-occurring form of vitamin C, and therefore if these compositions were applied to the skin, vitamin C would not be expected to be absorbed by either the ascorbic acid receptors or the glucose receptors of skin cells.

[0011] US Patent Application 2009/0016974 A1 (Pruche et al) discloses DHAA-containing compositions formed “in situ” from ascorbic acid via chemical oxidation and/or via enzymatic oxidation, and a two-component agent thereof. These compositions attempt to overcome the instability of DHAA by preparing it fresh as needed, but they require handling and mixing steps of the two-component agent. The two components must be stored separately. Chemical oxidizers are harsh and can be dangerous, and enzymes are unstable, thus these compositions are problematic in regard to safety and reliability. Since the two components are intended to be combined by the end user, the temperature of the reaction and other conditions necessary for reliable oxidation processes are beyond the control of the manufacturer. Without some separate indicator, the final consumer cannot be assured that the solution prepared by the two-component system actually contains DHAA, because the oxidation of AA to DHAA is not visually or otherwise simply detected. These compositions do not contain DHAA pre-prepared in a stable solution, and Pruche et al is silent as to the stability of DHAA in the disclosed compositions.

OBJECTS AND ADVANTAGES

[0012] Several objects and advantages of the present invention are:

a. To provide compositions containing DHAA in a stable form.
b. To provide stable DHAA-containing compositions for topical application to the skin of a human or animal as a source of highly absorbable vitamin C.
c. To provide compositions for topical application that are pharmacologically acceptable and pleasant to use.
d. To provide compositions for topical application that can be applied alone, or mixed with water to provide greater humidifying effect, or mixed with another skin care product to enhance the vitamin C content of that product.
e. To provide compositions containing DHAA for topical application that can also solubilize other skin-enhancing substances that are insoluble in water, such as vitamin E.
f. To provide stable DHAA-containing compositions for dietary supplementation of a human or animal as a source of highly absorbable vitamin C.
g. To provide compositions for dietary supplementation that are pharmacologically acceptable and pleasant to use.
h. To provide compositions for dietary supplementation that can be taken orally alone, or mixed with water or some other liquid, or applied to solid food.
i. To provide stable DHAA-containing concentrates for manufacturing of other products.
j. To provide stable DHAA-containing compositions that can be conveniently used in research, for example in chemical studies, or in microbial culture or tissue culture.
k. To provide stable DHAA-containing compositions that do not require the addition of chemical stabilizers or preservatives.

DRAWING FIGURES

[0024] FIGS. 1 to 8 show the DHAA stability of the various compositions described in Example 1 as compared with DHAA prepared similarly in water.

[0025] FIG. 9 shows the DHAA stability of the composition described in Example 2 as compared with DHAA prepared in water.

[0026] FIGS. 10 to 15 show the DHAA stability of the compositions described in Example 3 as compared with DHAA prepared similarly in water.



DESCRIPTION

[0027] We have discovered that DHAA is stable in solutions of pure polyol solvents and in solutions wherein the polyol content is greater than about 50 percent. By “stable” is meant that DHAA in these solutions deteriorates very slowly over a sufficient period of time that it can be stored and sold as a dietary supplement or as a skin care product, or as a concentrate for preparing or manufacturing them, with a reasonable shelf life. The solutions are made by oxidizing ascorbic acid that is first dissolved in a pure polyol solvent, or in water, or in some mixture of these liquids. The polyol concentration may be adjusted to about 50% or greater prior to oxidizing the AA or afterwards.

[0028] The solutions can also be made by oxidizing AA that is dissolved in an alcohol (e.g., ethanol), and then combining the DHAA-containing alcohol with a polyol solvent. If it is desired that the final solution does not contain alcohol, the alcohol can be removed by evaporating the alcohol from the polyol solvent solution using heat or vacuum, or both.

[0029] The solutions can also be made by dissolving solid DHAA in a pure polyol solvent, or in water, or in some mixture of these liquids. The polyol concentration may be adjusted to about 50% or greater prior to dissolving the DHAA or afterwards.

[0030] The organic polyol solvents are chosen for pharmaceutical and dietary acceptability, their ability to solubilize the AA and DHAA component, water content, and effect on the stability of the DHAA component. At present we prefer to employ commercially available glycerol which generally contains 5% or less water. In general, we prefer to minimize the water content of the solvent(s), consistent with economic and functional considerations. Other polyols which can be employed include propylene glycol, hexylene glycol, butylene glycol and the almost infinite molecular weight range of polyethylene glycols, as well as so-called sugar alcohols, e.g., sorbitol and xylitol, and mixtures thereof with other polyols.

[0031] These solutions can be prepared entirely with one polyol solvent, e.g., glycerol, or mixtures of polyol solvents. The final choice of solvent will depend on economics and other relevant factors.

[0032] Methods we have successfully applied for oxidizing the ascorbic acid include the use of halogen or ozone or oxygen/activated charcoal or Fenton's Reagent or ascorbic acid oxidase enzyme. All of these methods are known in the art, as are other methods; the previously cited references Pecherer and Koliou show typical applications of various methods for example. The method by which the oxidation is accomplished is not the determinant factor of the long term stability of the DHAA in the solution, and other methods of oxidation are within the scope of the invention.

[0033] AA concentration in solution is commonly measured as the reducing activity of the solution using starch-iodine titration methods that are well-known in the art. AA is also measured by ultra-violet spectrophotometry using a wavelength at which AA absorbs strongly and DHAA does not, typically about 265 nm. This method is also well known in the art. DHAA in solution can be converted into AA by reducing agents such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP), and its concentration is commonly measured spectrophotometrically as the difference in absorbance of a solution subjected to reduction by DTT or TCEP versus a similar solution that is not subjected to a reducing agent. These methods are also well-known in the art, but see Deutsch for examples. In the description, claims, and the following examples, DHAA in the compositions of the invention is the vitamin C that can be measured by the difference in absorbance at 262 nm using TCEP reducing agent.

[0034] The following embodiments are exemplary of the invention:

Example 1

[0035] In a preferred embodiment of the invention, AA dissolved in glycerol and/or water is oxidized using ozone to produce DHAA solutions. Water-based solutions and glycerol-based solutions may be combined to yield stable DHAA compositions having the desired polyol concentration.

[0036] A 15% AA solution in water was prepared by adding 15 grams AA per 100 mL purified water with stirring. A 15% solution of AA in glycerol was prepared by adding 15 grams AA per 100 mL pure USP glycerol and stirring with heat. A corona-discharge type ozone generator with feed-gas of pure oxygen was used to supply an oxidizing gas containing about 5% ozone, and each of the 15% AA solutions was subjected to oxidizing conditions by bubbling the oxidizing gas through the solution using a glass diffuser. The progress of AA oxidation in each solution was monitored by the disappearance of reducing activity as measured by starch-iodine titration. Each solution was subjected to the oxidizing conditions until all (>99%) of the original reducing activity had disappeared. The solution made with pure glycerol was labeled “100% Glycerol,” and the solution made with purified water was labeled “100% Water.” Portions of these two solutions were combined to produce solutions of various glycerol concentrations by weight, specifically “99% Glycerol,” “98% Glycerol,” “97% Glycerol,” “96% Glycerol,” “95% Glycerol”, “90% Glycerol,” and “50% Glycerol.” For example, 99 parts by weight of “100% Glycerol” was combined with 1 part by weight “100% Water” to produce the “99% Glycerol” solution.

[0037] Aliquots of each of the solutions prepared above were placed in translucent, screw-capped polyethylene vials and were stored at room temperature. No attempt was made to further protect the vials from ambient indoor light, and each vial contained a headspace of normal air. Each vial was periodically opened to remove a sample for stability testing over the next 229 days. The concentration of DHAA in each sample was measured by spectrometry on each testing day. The initial DHAA concentration of each solution on Day 1 was recorded and assigned a value of 100%, and the concentration on each subsequent stability test day was calculated as the percent remaining of the initial concentration.

[0038] FIGS. 1 through 8 show the results of stability testing of the various glycerol-containing solutions; each graph also shows the result of the “100% Water” solution for comparison. It can be seen that DHAA decomposes rapidly in water. By the time the water solution was tested at 20 days, less than 10 percent of the initial amount of DHAA remained. By contrast, DHAA is preserved very well in solutions containing high concentrations of glycerol. In pure glycerol for example, greater than 80% of the initial DHAA concentration remains even after approximately 8 months of storage at room temperature. As the glycerol concentration is reduced, stability is reduced, until only minor improvement is gained at 50% glycerol concentration.

Example 2

[0039] In another embodiment, a stable DHAA composition is produced by oxidation of AA dissolved in glycerol using exposure to activated charcoal and oxygen as the oxidation method.

[0040] A solution of AA in pure USP glycerol was subjected to oxidizing conditions by suspending activated charcoal in the solution and then bubbling pure oxygen through the solution. Oxidation of AA during this process was monitored by starch-iodine titration. After the desired amount of AA had been oxidized, the activated charcoal was removed from the solution by centrifugation and filtration. This solution was labeled “100% Glycerol.” A portion of the solution was then placed in a translucent, screw-capped polyethylene vial and was stored at room temperature. No attempt was made to further protect the vial from ambient indoor light, and the vial contained a headspace of normal air. The vial was periodically opened to remove a sample for stability testing over the next 191 days. The concentration of DHAA in the sample was measured by spectrometry on each testing day. The initial DHAA concentration of the solution on Day 1 was recorded and assigned a value of 100%, and the concentration on each subsequent stability test day was calculated as the percent remaining of the initial concentration.

[0041] FIG. 9 shows the results of the stability testing of this solution, and for comparison also shows the stability of a DHAA solution prepared in purified water (labeled “100% Water”). It can be seen that DHAA in glycerol produced by an alternative oxidation method shows excellent long-term stability.

Example 3

[0042] In another embodiment, stable DHAA compositions are produced by oxidizing AA dissolved in water using Fenton's Reagent as the oxidizing method, and then combining the water solution with propylene glycol such that the final concentration of polyol is 50% or greater.

[0043] AA was dissolved in purified water to give a highly concentrated solution, and then sufficient 30% hydrogen peroxide was added to oxidize about half of the AA. Iron to catalyze the reaction was provided by addition of ferrous sulfate. Oxidation of the AA was monitored by spectrometry until the expected amount of AA had been oxidized. This solution was labeled “100% Water.” Portions of this solution were combined with portions of pure, USP grade propylene glycol to produce solutions of “97% Propylene Glycol,” “95% Propylene Glycol,” “90% Propylene Glycol,” “80% Propylene Glycol,” “70% Propylene Glycol,” and “50% Propylene Glycol.” For example, 3 parts by volume of the “100% Water” solution were combined with 97 parts by volume propylene glycol to yield the “97% Propylene Glycol” solution.

[0044] Aliquots of each of the solutions prepared above were placed in translucent, screw-capped polyethylene vials and were stored at room temperature. No attempt was made to further protect the vials from ambient indoor light, and each vial contained a headspace of normal air. Each vial was periodically opened to remove a sample for stability testing over the next 31 days. The concentration of DHAA in each sample was measured by spectrometry on each testing day. The initial DHAA concentration of each solution was recorded and assigned a value of 100% (Day 0), and the concentration on each subsequent stability test day was calculated as the percent remaining of the initial concentration.

[0045] FIGS. 10 through 15 show the results of stability testing of the various propylene glycol-containing solutions; each graph also shows the result of the “100% Water” solution for comparison. It can be seen that DHAA decomposes rapidly in water; after only 5 days, less than 20 percent of the initial amount of DHAA remains. By contrast, DHAA is preserved very well in solutions containing high concentrations of propylene glycol. In fact, the DHAA concentration in many of these solutions actually increased significantly over time, a remarkable and unexpected discovery. We believe this phenomenon can be explained this way: residual AA continues to oxidize while the DHAA is stabilized and therefore accumulates in the solution. The spectrophotometric measurements support this explanation, but we do not wish to be bound by this explanation.

[0046] Example 3 demonstrates that stable DHAA compositions may be prepared using a third alternative oxidation method as compared with the first two examples, and also demonstrates that an alternative polyol solvent can be used.

CONCLUSION, RAMIFICATIONS, AND SCOPE

[0047] While the above description contains many specificities, these should not be construed as limitations on the scope of the invention, but rather as exemplification of preferred embodiments. The compositions can be prepared using various methods and ingredients as mentioned, and their equivalents. Polyol solvents are known to be antimicrobial in high concentrations and therefore the compositions of the invention generally do not require preservatives. Polyol solvents are also capable of dissolving substances that are not soluble in water, so are capable of solubilizing not only AA and DHAA but additional dietary or skin-enhancing ingredients such as vitamin E. Many polyol solvents are excellent skin-enhancing substances in their own right, such as glycerol which is commonly utilized in skin care products as a humectant. Many polyol solvents are not only safe for ingestion, but in fact have a pleasant, sweet flavor. Thus the compositions have favorable properties that are synergistic with their use as dietary supplements, skin-enhancers, concentrates, or research solutions. Accordingly, the scope of the invention should be determined not by the embodiments illustrated, but by the appended claims and their equivalents.



US6221904
Method for increasing the concentration of ascorbic acid in brain tissues of a subject

Inventor(s):     AGUS DAVID / VERA JUAN / GOLDE DAVID
Applicant(s):     SLOAN KETTERING INST CANCER

This invention provides a method for increasing the ascorbic acid concentration in brain tissues of a subject which comprises administering to the subject an amount of dehydroascorbic acid effective to increase the concentration of ascorbic acid in brain tissues. This invention also provides that the dehydroascorbic acid enters the tissues through the facilitative glucose transporter.

Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end the specification, preceding the claims.

BACKGROUND OF THE INVENTION

Numerous connections have been made between the generation and presence of oxidative free radicals in brain tissue and neurological disorders. For example, 1) Jenner (26) links oxidative stress to Parkinson's, Alzheimer's and Huntington's diseases. 2) Recent clinical studies have demonstrated that alpha-tocopherol (vitamin E) and selegiline (deprenyl), pharmacologic agents that have antioxidant activity, can slow the progression of moderately severe Alzheimer's disease (27). 3) Antioxidants such as vitamin C and vitamin E may have an important role in the treatment of diseases whose pathogenesis involves free radical formation and impaired antioxidant defenses in the aging population. Oxidative damage has been hypothesized as central to the neurodegenerative processes such as Alzheimer's disease (28). According to the free radical hypothesis, Alzheimer disease is an acceleration of the normal aging process in affected brain regions which become progressively more damaged by free radicals generated from metabolism. In Alzheimer's disease, the cerebral cortex seems to have increased antioxidant requirements, increased sensitivity to free radicals, and levels of the free radical defense enzymes, such as superoxide dismutase, that are reduced by 25-35% in the frontal cortex and hippocampus. The loss of hippocampal cholinergic neurons is a key feature of Alzheimer's disease and these neurons seem particularly vulnerable to the deleterious effects of free radicals on the muscarinic cholinergic receptor (29). 4) Antioxidants have been tested as drugs for Parkinson's disease (30), and it was found that selegiline, which may act as an antioxidant since it inhibits oxidative deamination, delays the onset of the disability (31). 5) Peyser et al. concluded that antioxidant therapy may slow the rate of motor decline early in the course of Huntington's disease (35). 6) According to Challem (32) free radicals and oxidative stress may be factors involved with the pathogenesis of Mad Cow disease. 7) The oxidative modification of low-density lipoprotein (LDL), termed lipid perioxidation has been shown to be an initiating event in atherosclerosis. Probucol, an antioxidant, is effective in reducing the rate of restenosis after balloon coronary angioplasty (36). Oxidized LDL has several detrimental effects on cells including brain cells such as cytotoxicity and vascular dysfunction.

Therefore, increasing the concentration of free-radical scavengers or antioxidants in brain tissue may provide therapeutic benefits to subjects suffering from neurodegenerative diseases. Sano et al. conclude (27) that the use of the antioxidants, selegiline or vitamin E may delay clinically important functional deterioration in patients with Alzheimer's disease. Their results are particularly significant because vitamin E does not cross the blood-brain barrier in large amounts, and still it has a measurable effect.

The enhancement of the antioxidant potential is useful in treating of many diseases. For example, the increase of antioxidant potential achieved by this invention will be able to treat stroke and neurovascular diseases. It is known that ischemic stroke is the most common neurologic disorder causing death or disability among adults. Strokes of all types rank third as a cause of death, surpassed only by heart disease and cancer. Ischemic stroke events account for approximately 85% of all strokes. Because no medical or surgical treatment has yet been established as reversing the effects of acute ischemic stroke, early identification and treatment of persons at the time they present with stroke is compelling, if such a treatment is efficacious. Currently, there are no approved treatments for stroke. The damage from stroke is caused by occlusion of a vessel, thereby restricting the delivery of oxygen in the blood to an area of the brain. Much of the damage is caused by damage from oxygen free radicals in the area served by the occluded vessel after reperfusion of the affected area (37). Thus, increasing the antioxidant potential of the brain may have beneficial effect on stroke and other neurovascular diseases.

Therefore, increasing vitamin C concentrations in the brain by providing dehydroascorbic acid to the subject could enhance antioxidant potential in the central nervous system and may be therapeutic in stroke and neurovascular diseases as described.

Researchers have proposed that atherosclerosis, and its deadly effects of heart attack and stroke, develops in relationship to oxidation of low-density lipoproteins (LDL) carrying cholesterol in the blood. The theory states that free radicals generated by the body's own immune cells oxidize LDL which is taken up by cells of the vascular intima initiating the atherosclerosis lesion. Ultraviolet and gamma radiation, cigarette smoke and other environmental pollutants, also cause oxidative damage to cells and vital compounds. The damage leads to the development of several chronic diseases including cancer and coronary heart disease (CHD). It was further proposed that antioxidants such as vitamin E and C and the carotenoids could prevent damage and the ensuing diseases. Many epidemiologic and animal studies have offered evidence to support the theory (33, 34). Recent studies demonstrated that the antioxidant proburol is effective in reducing the rate of restenosis after balloon coronary angioplasty (36).

Evidence suggests that the neuropathology of Huntington's disease, a neuropsychiatric disorder, results from excessive activation of glutamate-gated ion channels, which kills neurons by oxidative stress. It was reported that antioxidant therapy may slow the rate of motor decline early in the course of Huntington's disease (35).

Vitamin C enters cells, in vitro, through the facilitative glucose transporter GLUT1 in the form of dehydroascorbic acid and is retained intracellularly as ascorbic acid (1). In order to test the hypothesis that GLUT1 transport of dehydroascorbic acid is a primary physiological mechanism for tissue acquisition of vitamin C, we investigated the transport of vitamin C across the blood-brain barrier (BBB) in rodents. GLUT1 is expressed at the BBB on endothelial cells and is responsible for glucose entry into the brain. Ascorbic acid, the predominant form of vitamin C in blood, was incapable of crossing the BBB while dehydroascorbic acid readily entered the brain and was retained in the form of ascorbic acid. The transport of dehydroascorbic acid into the brain was competitively inhibited by D-glucose, but not by L-glucose. These findings define the transport of dehydroascorbic acid by GLUT1 as the mechanism by which the brain acquires vitamin C, and point to the oxidation of vitamin C as the important regulatory step in the accumulation of the vitamin by the brain.

Dehydroascorbic acid, the oxidized form of vitamin C, was previously found to be transported through the facilitative glucose transporters. Expression of GLUT1, GLUT2, and GLUT4 in Xenopus oocytes conferred the ability to take up dehydroascorbic acid which was retained intracellularly after it was reduced to ascorbic acid (1). It was also established that facilitative glucose transporters are involved in the transport and accumulation of vitamin C by normal human neutrophils and the myeloid leukemia cell line, HL60 (1-3). In these cells dehydroascorbic acid is transported across the cell membrane and accumulated in the reduced form, ascorbic acid, which is not transportable through the bidirectional glucose transporter (1-3). Ascorbic acid may be transported through a Na@+ -ascorbate co-transporter that is reported to be present in small intestine, kidney and adrenomedullary chromaffin cells (4). The co-transporter has not been molecularly characterized and no Na@+ -dependent ascorbic acid uptake in white blood cells has been found (2, 3).

GLUT1 is expressed on endothelial cells at the BBB and is responsible for glucose transport into the brain (5, 6). In the 1880's, Ehrlich found that intravenously injected aniline dyes colored all of the organs of experimental rabbits except the brain and the spinal cord (7, 8). This observation led to the eventual discovery that the BBB is comprised of a wall of capillaries forming an endothelial barrier between the blood and the brain, functioning primarily to regulate the transport of nutrients and waste products (9, 10). Several nutrient transporters have been identified at the BBB including GLUT1, a monocarboxylic acid transporter, neutral amino acid transporter, amine transporter, basis amino acid transporter, nucleoside transporter, and purine base transporter (11). Here it is shown in rodents that vitamin C cross the BBB through GLUT1 only in the oxidized form, dehydroascorbic acid, and is retained in the brain in the reduced form, ascorbic acid.

The present invention allows for the controlled introduction of the antioxidant vitamin C into brain tissue, which should serve as an important therapeutic method to treat and prevent various disorders associated with free radicals and oxidative damage.

SUMMARY OF THE INVENTION

This invention provides a method for increasing the ascorbic acid concentration in brain tissues of a subject which comprises administering to the subject an amount of dehydroascorbic acid effective to increase the concentration of ascorbic acid in brain tissues. This invention also provides the above-described method wherein the dehydroascorbic acid enters the tissues through the facilitative glucose transporter.

This invention also provides a method for treating neurodegenerative disease of a subject comprising administering to the subject an amount of dehydroascorbic acid effective to increase the antioxidant potential of brain tissues.

This invention finally provides a method for preventing neurodegenerative disease of a subject comprising administering to the subject an amount of dehydroascorbic acid effective to increase the antioxidant potential of brain tissues.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Dehydroascorbic acid is transported across the BBB and accumulates in the brain as ascorbic acid. (A) Balb/c mice (age 6-8 weeks) and (B) Fischer F344 rats (70-80 gram body weight) were injected into the tail vein with 5 .mu.Ci(mouse) or 10 .mu.Ci(rat) @14 C-ascorbic acid (L-[1-C]-ascorbic acid, specific activity, 6.6 mCi/mmol, Dupont NEN), @14 C-dehydroascorbic acid or H-sucrose ([fructose-1-@3 H]-sucrose, specific activity 20.0 Ci/mmol, Dupont NEN). Each group consists of 12 animals and the values are expressed as mean .±.SEM. (C)HPLC analysis of the methanol soluble fraction of the brain and (H) serum of a mouse injected with 20 .mu.Ci @14 C-dehydroascorbic acid and sacrificed at 5 min (injected material, hashed line). (C) Accumulation of vitamin C in the brain is in the form of ascorbic acid (.about.90&; retention time.apprxeq.11.80 min, solid line). (H) Radioactivity present in serum is in the form of ascorbic acid (>98%; retention time.apprxeq.11.80 min, solid line). (D) The initial kinetics and (E) 2 hr kinetics of accumulation of radioactivity in the brain of mice injected intravenously with @14 C-ascorbic acid (.circle-solid.), @14 C-dehydroascorbic acid (.box-solid.) or@3 H-sucrose (.largecircle.). (F) The initial kinetics and (G) 2 hr kinetics of radioactivity in the serum of mice injected intravenously with @14 C-ascorbic acid(.circle-solid.),@14 C-dehydroascorbic acid (.box-solid.) or @3 H-sucrose(.largecircle.). Each data set in (D) through (G) represents 4 mice.±.SEM.

FIG. 2 Specificity of the transport of dehydroascorbic acid through GLUT1 at the Balb/c mouse BBB. (A) @14 C-Dehydroascorbic acid (.box-solid.) entered the brain and its accumulation was blocked by increasing amounts of D-deoxyglucose which is transported through GLUT1. Transport of @3 H-leucine (.largecircle.) or @14 C-ascorbic acid (.circle-solid.) across the BBB was not affected by D-deoxyglucose. (B) L-glucose, which is not transported through GLUT1, had no effect on the transport of @14 C-dehydroascorbic acid. Transport of @3 H-leucine (.largecircle.) or @14 C-ascorbic acid (.circle-solid.) across the BBB was not affected by L-glucose. All experiments were carried out over a 30-second time course. Each data set included 4 mice and the data were expressed as mean .±.SEM. A mouse has a baseline serum glucose concentration of approximately 12 mM, which calculates to 2.67 mg glucose in the entire mouse based on the average plasma volume of the mouse. The amount of exogenous glucose administered in this experiment was based on this number and subsequent multiples to a maximum tolerable level.

FIG. 3 Brain digital autoradiography of rat with @14 C-labelled ascorbic acid, dehydroascorbic acid, D-deoxyglucose and sucrose. (A) Digital autoradiography was performed on a Fisher F344 rat (8 wks of age) 3 min after intravenous injection with 40 .mu.Ci of @14 C-dehydroascorbic acid, (B) 40 .mu.Ci @14 C-ascorbic acid and (C) 40 @14 .mu.Ci C-sucrose ([glucose-@14 C(U)]-sucrose, specific activity, 310 mCi/mmol, Dupont NEN). The area of the brain is denoted with an * in the figure. The photo-stimulated luminescence (PSL)/mm@2 ratio of brain/background counts for the dehydroascorbic acid-injected rat was 8.6.±.0.3 (mean of 3 sections.±.SEM). The PSL/mm@2 ratio in the ascorbic acid-injected rat was 1.5.±.0.1 and 1.4.±.0.1 in the sucrose-injected rat.

 


DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method for increasing the ascorbic acid concentration in brain tissues of a subject which comprises administering to the subject an amount of dehydroascorbic acid effective to increase the concentration of ascorbic acid in brain tissues. This invention also provides the above-described method wherein the dehydroascorbic acid enters the tissues through the facilitative glucose transporter.

This invention also provides a method for increasing the ascorbic acid concentration in brain tissues of a subject which comprises administering to the subject an amount of dehydroascorbic acid effective to increase the antioxidant potential of the brain tissues.

In an embodiment of this invention, the subject is a human. In a separate embodiment, the human subject has a neurodegenerative disease. Such neurodegenerative disease includes but is not limited to Alzheimer's Disease, Parkinson's Disease or other forms of presenile dementia.

In another embodiment, the subject has neurovascular disease. This invention is useful for treating or preventing stroke or neurovascular diseases.

The subject may carry genetic diseases with central nervous system manifestations. In an embodiment, the genetic disease is the Huntington's disease.

For a separate embodiment, the subject has schizophrenia. In a still another embodiment, the human subject has a behavioral disorder. Such behavioral disorder includes, but is not limited to dysthymia, involution depression, aggressiveness via dominance, hyperactivity, deprivation syndrome, separation anxiety, intermittent anxiety, instrumental sociopathy, stereotypies, phobia or socialization disorders.

As it will be easily appreciated by persons of skills in the art, this invention is applicable to both human and animal diseases which could be treated by antioxidants. This invention is intended to be used in husbandry and veterinary medicine.

In this invention, the dehydroascorbic acid may be administered orally, intravenously, subcutaneously, intramuscularly or by other routes or circumstances of administration by which the dehydroascorbic acid will not be hydrolyzed. Dehydroascorbic acid hydrolyses easily in aqueous solution. It is the intention of this invention to administer the dehydroascorbic acid in a stabilized form. It is known that dehydroascorbic acid is stable under low pH conditions. Accordingly, dehydroascorbic acid may be stored in low pH and then administered directly to a large vein of a subject. Alternatively, dehydroascorbic acid may be stored in powdered form and hydrated before administering to a subject.

Moreover, dehydroascorbic acid may be encapsulated in liposomes at low pH. The encapsulated dehydroascorbic acid will then be administered to a subject. In a preferred embodiment, the encapsulated dehydroascorbic acid is administered orally.

U.S. Pat. No. 4,822,816 describes uses of aldono-lactones and salts of L-threonic, L-xylonic and L-lyxonic to stabilize the dehydroascorbic acid. The content of U.S. Pat. No. 4,822,816 is hereby incorporated into this application by reference. Accordingly, this method provides another means for stabilization of the dehydroascorbic acid.

Finally, appropriate amounts of ascorbic acid and ascorbate oxidase may be administered together to a subject to produce an amount of dehydroascorbic acid effective to increase the concentration of ascorbic acid in the brain tissues of the subject. Ascorbate oxidase catalyzes oxidation of L-ascorbic acid, and it is commercially available. U.S. Pat. No. 5,612,208 describes a new ascorbate oxidase and its gene, the content of which is hereby incorporated into this application by reference. Accordingly, ascorbate oxidase may be produced by the recombinant DNA technology.

Using this invention, the brain tissues of a subject may be loaded with the maximum amount of ascorbic acid.

Dehydroascorbic acids may exist in various salt forms. It is the intention of this invention to encompass these forms. The salts upon hydration will generate dehydroascorbic acid.

This invention provides a method for treating or preventing dementia of a subject comprising administering to the subject an amount of dehydroascorbic acid effective to increase the concentration of ascorbic acid in brain tissues.

This invention also provides a method for treating or preventing neurodegenerative disease of a subject comprising administering to the subject an amount of dehydroascorbic acid effective to increase the antioxidant potential of the brain tissues.

This invention also provides a combination therapy wherein an effective amount of dehydroascorbic acid is administered with therapeutic agents for the neurodegenerative disease. The administration may be performed concomitantly or at different time points. When treating the Alzheimer's disease, the therapeutic agents include, but are not limited to, Estrogen, Vitamin E (alpha-tocopherol), Tacrine (Tetrahydroacridinamine), Selegiline (Deprenyl), and Aracept (Donepezil). With respect to the Parkinson's disease, the therapeutic agents include, but are not limited to, the anticholinergic class of drugs, clozapine, levodopa with carbidopa or benserazide, Selegiline (Deprenyl), and dopamine agonist class of drugs.

This invention provides a method for treating or preventing stroke or neurovascular disease or other diseases which can be caused by lipid perioxidation of a subject comprising administering to the subject an amount of dehydroascorbic acid effective to increase the concentration of ascorbic acid in brain tissues.

This invention also provides a method for treating or preventing stroke or neurovascular disease or other diseases which can be caused by lipid perioxidation of a subject comprising administering to the subject an amount of dehydroascorbic acid effective to increase the antioxidant potential of the brain tissues.

These diseases include, but are not limited to stroke, atherosclerosis and neurodegenerative disorders.

Moreover, this invention provide a method for treating or preventing central nervous system manifestations of genetic diseases. The conditions of the disease will be improved by increasing the antioxidant potential of the brain. Prevention of such central nervous system manifestations of genetic disease may even be prevented if the antioxidant potential of the brain maintain to be a high level. This genetic disease includes, but not limited to, Huntington's disease.

This invention provides a method for preventing or treating behavioral disorders of a subject comprising administering to the subject an amount of dehydroascorbic acid effective to increase the concentration of ascorbic acid in brain tissues. This invention finally provides a method for preventing or treating behavioral disorders of a subject comprising administering to the subject an amount of dehydroascorbic acid effective to increase the antioxidant potential of the brain tissues. Such behavioral disorder includes, but is not limited to dysthymia, involution depression, aggressiveness via dominance, hyperactivity, deprivation syndrome, separation anxiety, intermittent anxiety, instrumental sociopathy, stereotypies, phobia or socialization disorders.

When treating or preventing the behavioral disorders, dehydroascorbic acid may be used in combination with other drugs. They may be administered concomitantly or at different time points.

In another embodiment, the behavioral disorder is schizophrenia.

This invention will be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.

Experimental Details

Experimental Methods

Blood-brain barrier transport studies. @14 C-dehydroascorbic acid was generated in all experiments by incubating the @14 C-ascorbic acid with ascorbate oxidase, 1 unit/1.0 mmol L-ascorbate (derived from Cucurbita species, Sigma). Dithiothreitol (0.1 mmol/liter) was added to the vitamin C preparations as a reducing agent. Animals were sacrificed at various time points after injection by cervical dislocation of CO2 inhalation. The brain was then dissected out and homogenized in 70% methanol. Samples were processed for scintillation spectrometry or HPLC as described (2, 3). HPLC was performed on the methanol fraction with 1 mmol/L EDTA added (2, 3). Samples were stored at -70 DEG C. until analysis. HPLC samples were separated on a Whatman strong anion exchange Partisil 10 SAX (4.6-.times.25-cm) column (Whatman, Hillsboro, Oreg.). A Whatman-type WCS solvent-conditioning column was used and the eluates monitored with a Beckman System Gold liquid chromatograph (Beckman Instruments, Irvine, Calif.) with a diode array detector and radioisotope detector arranged in series. Ascorbic acid was monitored by absorbance at 265 nm and by radioactivity. Dehydroascorbic acid shows no absorbance at 265 nm and was monitored by radioactivity.

Digital autoradiography. Animals were sacrificed, frozen in a dry ice/hexane mixture and then embedded in .about.5% carboxymethylcellulose (Sigma Aldrich). The animal blocks were allowed to equilibrate for .about.12 hours at -20 DEG C. and the animals were sectioned in coronal cuts with a slice thickness of .about.40-45 .mu.m in a cryo-microtome (PMV), and tape lifted for direct exposure onto digital plates (23). The exposure time was approximately 72 hours. All digital plates were scanned on a Fuji Bas 5000 digital autoradiographic system (Fuji, Inc.) At 25 .mu.m resolution.

Calculation of the BBB permeability-surface area product. The amount of compound which crosses the BBB is dependent on two parameters defined by the following equation: ##EQU1##

where PS is the BBB permeability-surface area product and AUC is the plasma area under the concentration time-activity curve at a given time (t) after injection. A variant of the single intravenous injection technique termed the external organ technique was used to quantify the BBB PS product in anesthetized animals. The plasma and brain radioactivity was measured as decays per min (DPM)/.mu.l of serum (after the ascorbic acid or sucrose was solubilized from the cells in the presence of 70% methanol) which was equivalent to the integral of the plasma radioactivity. The BBB PS product is calculated:
% injected dose/gm of brain tissue=PS.times.AUC

where the variables are defined, as follows:
t=time ##EQU2##

The rats were anesthetized with a mixture of ketamine 90 mg/kg and xylazine 10 mg/kg anesthesia during the procedure. The xylazine causes a hyperglycemia and hypoinsulinemia in the animals with the serum glucose measured at approximately 280 mg/dl 30 min after induction of anesthesia (24, 25). This is almost three-fold higher than baseline glucose concentrations in the rats and affects transport through GLUT1 and therefore the PS calculations. Radiolabeled test compound (@3 H-sucrose, @14 C-ascorbic acid, @14 C-dehydroascorbic acid) was injected into a cannulated femoral vein in groups of 3 rats. Sucrose was used as a V0 marker (plasma volume marker). For 30 seconds (t) after injection arterial blood was collected by gravity from a catheter cannulated in the abdominal aorta and then the animal was sacrificed and the brain harvested.

Results and Discussions

Mice and rats were injected into the tail vein with @14 C-ascorbic acid, @14 C-dehydroascorbic acid or @3 H-sucrose. Three min after intravenous injection the animals were sacrificed, the brains harvested and the methanol soluble fraction counted by liquid scintillation. Approximately 4% of the dehydroascorbic acid (expressed as percent of injected dose (ID) per gram of brain tissue) was found in the brain after 3 min (FIGS. 1A and 1B). Injected ascorbic acid and sucrose yielded only trace radioactivity in the brain homogenate at 3 min, indicating that ascorbic acid could not pass the BBB. Because sucrose is not metabolized or transported it is used as a marker of plasma volume (12). The small amount of radioactivity present in the brain of the sucrose and ascorbic acid-injected animals was consistent with the radioactivity being present within the brain blood vessels. High-performance liquid chromatography (HPLC) analysis of the methanol (70%) fraction of the brain homogenate showed that the form of the vitamin C accumulated in the brain of dehydroascorbic acid-injected animal was >85% ascorbic acid (FIG. 1C). This result indicated that dehydroascorbic acid was transported across the BBB and retained as ascorbic acid in the brain.

Brain radioactivity, after dehydroascorbic acid injection, reached a maximum of 4.3% of ID/gram brain tissue at 3 min, decreased to 3.3% at 25 min, and remained at that level for up to 2 hours after injection (FIGS. 1D, 1E). Injection of sucrose and ascorbic acid resulted in a maximum brain accumulation of 0.4% ID/gram brain tissue at 15 to 30 seconds after injection (FIG. 1D). Brain radioactivity in the sucrose-injected animals decreased to <0.1% after 15 min, concomitant with the fall in serum radioactivity in these mice (FIGS. 1E, 1G). In ascorbic acid-injected mice there was an increase in brain radioactivity to 1.1% ID/gram brain tissue 2 hours after injection, a time period during which there was a decreasing amount of radioactivity in the serum (FIGS. 1E, 1G). The serum radioactivity concentration at 15 seconds after dehydroascorbic acid injection was 8% ID/gram serum, whereas the corresponding figure in mice injected with ascorbic acid was 27%. Thus dehydroascorbic acid was cleared from the circulation substantially faster than ascorbic acid (FIG. 1F). At the 3-min time point the radioactivity in the serum of the ascorbic acid and dehydroascorbic acid-injected animals was equivalent (FIG. 1G). Radioactivity remaining in the serum of the dehydroascorbic acid-injected animals at 5 min was associated with ascorbic acid (FIG. 1H).

Injected @14 C-ascorbic acid showed no measurable transport into the brain over the first 30-min, but some radioactivity accumulated in the brain at longer time periods. There are at least three potential explanations for this result. The first is that the ascorbic acid was metabolized in the interval time period and the counts in the brain represented transported radiolabeled metabolic breakdown products of ascorbic acid. Such an explanation is unlikely as the HPLC results demonstrated that the majority of the radioactivity in the dehydroascorbic acid-injected brain was eluted in radioactive peaks consistent with intact ascorbic acid. A second possibility is the presence of a small number of Na@+ -ascorbate cotransporters at the BBB or choroid plexus, which is unlikely since the accumulation of ascorbic acid did not occur linearly with time, as it would in this case, but only occurred after 30 min (13). The interpretation is that oxidation of ascorbic acid in the microenvironment occurred in vivo leading to the production of dehydroascorbic acid which was then transported across the BBB and retained in the brain as ascorbic acid.

The serum concentration of injected dehydroascorbic acid reached only 20 to 25% of the serum concentration of ascorbic acid or sucrose during the initial several minutes after injection. Sucrose has no transport mechanism, therefore its clearance from the serum was slow. Part of the clearance mechanisms for ascorbic acid and dehydroascorbic acid are through transport, the GLUTs in the case of dehydroascorbic acid and potentially a Na@+ -ascorbate cotransporter in the case of ascorbic acid (4). The rapid clearance of dehydroascorbic acid from the serum likely reflected the large number of glucose transporters available for transport.

The glucose transporter GLUT1 selectively transports D-glucose but not L-glucose. In order to confirm that dehydroascorbic acid passed the BBB through GLUTs, inhibition experiments were conducted with D- and L-glucose. 2-Deoxy-D-glucose (D-deoxyglucose) and D-glucose (data not shown) inhibited uptake of dehydroascorbic acid in the brain in a dose-dependent fashion up to 70%, whereas L-glucose and leucine had no effect (FIG. 2A). The uptake of leucine, which is not transported by GLUTs, but crosses the BBB largely through L system transporters and to a minor extent by the ASC system transporter (14), was not affected by increasing concentrations of L-glucose of D-deoxyglucose (FIG. 2B) nor were the serum concentrations of ascorbic acid, dehydroascorbic acid and leucine affected by increasing concentrations of D-deoxyglucose or L-glucose (data not shown). These results established that D-deoxyglucose inhibits dehydroascorbic acid from entering the brain through the glucose transporters but does not affect certain other transport systems or alter general BBB permeability by osmotic effects.

The external organ approach, utilizing serum as the external organ, was used to calculate the BBB permeability-surface areas product (PS) in the Fischer F344 rat (15). The calculated PS of @14 C-dehydroascorbic acid was 136.±.12(SEM).mu.l/min/gm brain tissue, @14 C-ascorbic acid was -0.44.±.0.24 .mu.l/min/gm brain tissue, and @3 H-D-deoxyglucose was 44.±.3.2 .mu.l/min/gm brain tissue. The difference in the BBB permeability-surface area products (PS) between ascorbic acid and dehydroascorbic acid illustrated the marked differences in the BBB transport between the redox states of vitamin C. The calculated PS of ascorbic acid was approximately 0 .mu.l/min/gm brain tissue at 30 seconds, similar to sucrose, which indicates no transport across the BBB. The PS of dehydroascorbic acid was 3-fold greater than D-deoxyglucose which corresponds with the difference in the Km values between the two compounds. The apparent Km of D-deoxyglucose for transport was 2.5 mM in HL60 cells compared with an apparent Km of 0.85 mM for dehydroascorbic acid in HL60 cells (2, 3).

Digital autoradiography of the brain of a rat injected with @14 C-dehydroascorbic acid and a rat injected with @14 C-ascorbic acid was performed to confirm the anatomical distribution of the injected compounds (FIG. 3). Autoradiographic evidence of activity accumulation in the brain was seen only in animals injected with dehydroascorbic acid. @14 C-sucrose was used as a marker of intravascular volume.

The results of this study established that the transport of vitamin C into the brain is mediated by GLUTs at the BBB which transport dehydroascorbic acid. Ascorbic acid itself is not transportable across the BBB. The glucose transport in vivo therefore was found to function comparably to in vitro models in that only the oxidized form of vitamin C, dehydroascorbic acid, was transportable (1-3). Dehydroascorbic acid was reduced to ascorbic acid after passing the BBB and was retained in the brain as ascorbic acid. This trapping mechanism allows for the accumulation of higher concentrations of vitamin C in the brain than in the blood. Overall, the findings point to the oxidation of ascorbic acid as being the critical step in the regulation of the accumulation of vitamin C in the brain.

The current recommended daily allowance of vitamin C is 60 mg daily and yields a steady-state plasma concentration of approximately 24 .mu.M in human volunteers (16). Only ascorbic acid is detected in the serum, with dehydroascorbic acid at trace serum levels or not measurable (17). The vitamin C injected in this study was approximately 500 .mu.M, which is 5-fold greater than the physiologic serum concentration of vitamin C in rodents (18). In this study, at physiologic glucose concentrations, dehydroascorbic acid transport through GLUT1 did occur. The serum concentration of glucose in normal rodents is approximately 10 mM yet there is still dehydroascorbic acid transport to the brain indicating that both dehydroascorbic acid and glucose are substrates of the GLUTs under physiologic conditions. This result is consistent with in vitro data demonstrating that a deoxyglucose concentration greater than 50 mM is necessary to block the transport of dehydroascorbic acid through GLUT1 (2, 3).

James Lind detailed the clinical description of scurvy in A Treatise of the Scurvy in 1772. He concluded his report of the autopsy results of scorbutic patients' "ravaged bodies" as follows, "What was very surprising, the brains of those poor creatures were always sound and entire . . . " (19). There thus appeared to be a mechanism for the accumulation and storage of ascorbic acid in the brain such that the brain would be the last organ depleted of vitamin C. The normal human brain has a vitamin C concentration of approximately 1 mM, 10 times the normal serum concentration (20). The precise role of vitamin C in the brain is uncertain, but ascorbic acid may be a cofactor of dopamine .beta.-hydroxylase and is thus involved in the biosynthesis of catecholamines. Vitamin C can also inhibit the peroxidation of membrane phospholipids and act as a scavenger of free radicals in the brain (21, 22). The results of this study demonstrate the physiological importance of vitamin C transport through GLUT1 in the form of dehydroascorbic acid and define the mechanism by which the brain obtains and retains vitamin C.

Recent data show that large quantities of vitamin C can be loaded into the brain. An experiment was done in which the carotid artery of a subject rat was cannulated with a catheter and 24 mg of dehydroascorbic acid was injected into the artery. The injected dehydroascorbic acid was spiked with a tracer amount of radioactive (@14 C-labeled) dehydroascorbic acid. The dehydroascorbic acid was infused over forty minutes and the brain was harvested. The amount of radioactive vitamin C was quantitated in the brain and total amount of injected vitamin C that accumulated in the brain was thus extrapolated. The experiment demonstrated that 2.6 mg of vitamin C accumulated in the brain of the subject rat during the forty minute injection period, which was approximately 11% of the injected dose. This shows that it is possible to achieve pharmacologic concentrations of vitamin C in the brains of subject animals. It is of note that the total vitamin C concentration in the normal adult rat brain is approximately 150 .mu.g. A log-fold greater Vitamin C than baseline normal concentration of Vitamin C was thus achieved.

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