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VITAMIN C

Introduction

It has been previously theorized that free­radical reactions led to the first life on Earth, and their ability to randomly cause mutations may have subsequently led to the evolution of life. One of the most efficient free­radical quenchers is ascorbate, which most animals manufacture endogenously. It is generally believed that, approximately 25 million years ago, an ancestor of the Anthropoidea primate suborder, which includes Homo sapiens, lost the ability to produce its own ascorbate, and all descending species inherited this genetic defect. The first of three hypotheses presented here proposes that a genetic defect, caused by either free radicals or a virus, deleted the gene needed by Anthropoidea to manufacture endogenous ascorbate. The second hypothesis proposes that this evolutionary accident permitted large numbers of free radicals to remain metabolically unquenched. The third hypothesis proposes that the presence of these excessive free radicals increased the likelihood of free­radical­induced genetic mutations, and these mutations propelled the evolution of Anthropoidea, leading to Homo sapiens. i

Antioxidants and ageing

The ageing process in skin is intimately related to the acute and chronic control of free radical formation.

The First Line of Defence The body has many antioxidant defences, many of which you know. They are classified in many ways.

I prefer to separate them into two major groups

The protein & enzyme group: Protein ­ transferrin, ferritin and ceruloplasmin. These are proteins that bind metals in the blood stream. Enzymes ­ superoxide dismutase, catalase, glutathione peroxide, glutathione transferase, thio­specific peroxidases and others.

and the vitamin & metabolite group: Vitamin ­ ascorbic acid, tocopherol and retinol ­ Vit C, E and A respectively.

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Metabolites ­ bilirubin, uric acid, glutathione, nicotinamide, dinucleotide, phospate complex, magnesium, manganese, zinc and lipoic acid.

Recent findings indicate the first molecular line of defence against free­radical lipid peroxidation damage in the body involves water­ soluble vitamin L­ascorbate (i.e., in salt form at physiological pH values).

Even in the presence of high levels of Vitamin E, in vitro experiments indicate L­ascorbate is consumed first because it reduces αtocopheroxyl radical back to atocopherol.

This can also be accomplished to a lesser extent by cysteine and glutathione.

Antioxidant activity after sun exposure

Among the non­enzymic antioxidants two different dose­response patterns were seen. Ascorbate was rapidly depleted at doses between 0 and 5 J/cm2 but was less affected between 5 and 25 J/cm2. In contrast, glutathione, ubiquinol/one, and alpha­tocopherol levels remained approximately equal to control levels between 0 and 5 J/cm2, then decreased to varying degrees from 5 to 25 J/cm2; ubiquinol was almost completely depleted, whereas alpha­tocopherol dropped only 30%.

Catalase activity decreased dramatically at doses above 5 J (to 11.8% of initial activity in epidermis and 27.7% in dermis at 25 J). The dramatic loss of catalase is almost entirely accounted for by direct destruction by the simulated solar light, but superoxide dismutase was unaffected by direct exposure; hence its destruction must be due to indirect effects, either mediated by free radicals or other harmful species formed upon irradiation. At low doses of UV light many components of the cutaneous antioxidant system were damaged, whereas at high doses all components were damaged and some were almost completely destroyed. ii

Impairment of cutaneous catalase and glutathione reductase activity was observed. Superoxide dismutase and glutathione peroxidase were not significantly influenced. Inhibition of catalase may render skin more susceptible to the damaging effects of hydrogen peroxide and its reaction products such as the hydroxyl radical.

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This partial impairment of the cutaneous antioxidant defence system by near ultraviolet/visible light, showing that the most susceptible component in skin is catalase, suggests possible pharmacological interventions. iii

Ultraviolet radiation to murine skin causes a large degree of destruction of enzymic and non­enzymic antioxidants immediately after irradiation. The pattern of recovery was different for each enzyme and for epidermis and dermis. The activities of superoxide dismutase and catalase decreased remarkably and recovered slowly. Super oxide dismutase in the dermis recovered full activity by 120 h and in the epidermis by 12 h. Catalase activity in both epidermis and dermis had returned to only 50% of control activity at 120 h, although the epidermis showed a temporary increase (to 93%) at 24 h. Glutathione peroxidase and glutathione reductase were slightly decreased immediately after irradiation, recovered to 100% at 3 h and then increased to 200­250% in both the epidermis and the dermis at various times; values had returned to 100% in epidermis by 120 h but remained elevated in dermis. iv

Iron can act as a catalyst for free radical oxidative reactions; chronic exposure of skin to UV radiation causes increased iron deposition. Using our spin trapping system, we have shown that topical application of the iron­chelator, Desferal, to a section of skin reduces the UV light­ induced POBN adduct radical signal. v

Ultraviolet radiation damage to the skin is due, in part, to the generation of reactive oxygen species. Vitamin C (L­ascorbic acid) functions as a biological co­factor and antioxidant due to its reducing properties. Topical application of vitamin C has been shown to elevate significantly cutaneous levels of this vitamin in pigs, and this correlates with protection of the skin from UVB damage as measured by erythema and sunburn cell formation. This protection is biological and due to the reducing properties of the molecule. Further, we provide evidence that the vitamin C levels of the skin can be severely depleted after UV irradiation, which would lower this organs’ innate protective mechanism as well as leaving it at risk of impaired healing after photo induced damage. In addition, vitamin C protects porcine skin from UVA­mediated phototoxic reactions (PUVA) and therefore shows promise as a broad­spectrum photo­protectant. vi

From the well­characterized enzymatic reactions involving ascorbic acid, it seems that the major function of ascorbate is to protect tissues

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from harmful oxidative products and to keep certain enzymes in their required reduced forms. However, it remains unclear how the deficiency of ascorbate leads to the pathological symptoms found in scurvy. vii

If Vit C is not in the daily diet, then within about six weeks we develop the signs of deficiency of Vit C, beginning with bruising, slowness to heal, bleeding gums and in its most serious form the skin condition called scurvy.

Vit C is a water­soluble vitamin and we do not have large stores of Vit C in our body. We cannot manufacture Vit C and are entirely dependent on dietary sources. Vit C helps the absorption of iron. However, supplemental iron in baby food may carry a potential risk of increasing free radical damage. viii Vit C plays an essential part in the incorporation of the amino acid proline into collagen and is involved in the formation of elastin. Vit C is also necessary for the formation of cartilage, dentine in the teeth and bone, and it maintains the integrity of our capillaries, and prevents them from bruising.

Recommended Daily Dietary Allowances (RDA) FOR ADULTS IS 45 MG. (HIGHER DOSES RECOMMENDED DURING PREGNANCY AND LACTATION and persons on prescription medication for schizophrenia or mood disorders) Ascorbic acid is the acidic form of vitamin C, and even when encapsulated can cause gastric upset or diarrhoea for some people. This can often be alleviated by consuming it with meals.

Especially if you have a stomach ulcer, use of an antacid, buffering agent, or a buffered form of vitamin C is suggested. Unbuffered ascorbic acid in the mouth may be harmful to tooth enamel.

It is well known that UVR causes a great influx of free radicals, even the energy produced by the conversion of ADP in ATP creates free radicals. In fact just staying alive creates free radicals.

All vitamin C products should be stored away from heat, light, and moisture although this is most important for pure ascorbic acid and ascorbyl palmitate powders.

How much vitamin C? When the daily dose is increased from 200 to 2500 mg (from 1.1 to 14.2 mmol) the mean steady state plasma concentration increases only from approximately 12 to 15 mg/L (from 68.1 to 85.2 mumol/L).

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The analysis indicates that both saturable gastrointestinal absorption and non­linear renal clearance act additively to produce the ceiling effect in plasma concentrations. As a consequence of this ceiling effect, there is no pharmacokinetic justification for the use of mega doses of ascorbic acid. ix

However, the consensus is that adverse effects do not occur in healthy subjects ingesting large amounts of vitamin C. x

Oral supplementation of 500 mg of ascorbic acid daily for two months alone without any other antioxidant does not appear to have protective effect on either in vitro lipoprotein oxidation resistance or in vivo lipid peroxidation in smoking men, but might even promote the formation of MDA. (Malonaldehyde). xi

In general, at low ascorbate concentrations, ascorbate is prone to be a pro­oxidant, and at high concentrations, it will tend to be an antioxidant. Hence there is a crossover effect. We propose that the "position" of this crossover effect is a function of the catalytic metal concentration. xii

Vitamin C also protects Vit A against oxidation, improves the benefits of B12 and compensates for deficiencies of pantothenic acid, another B vitamin. As we age, we show poor absorption and retention of many nutrients like Vit C and other water­soluble vitamins. Topical delivery of Vit C is much more efficient than massive doses or oral supplements. Experiments have proven that Vit C once absorbed into the skin, cannot be rubbed, washed or perspired off for three days. Vit C is destroyed by ULTRA VIOLET RADIATION. It is denatured in the skin by exposure to UVR and as with Vitamin A will probably develop a chronic deficiency of Vit C in all the areas of skin that are exposed to sunlight. The Vit C concentration in sun­exposed skin has been found to considerably lower than that in unexposed skin. Experiments also show that the skin gets considerably less damage if significant amounts of Vit C are applied topically before sun exposure.

What destroys Vit C. Smoking: Smokers and older persons have a greater need for Vit C. (each cigarette destroys 25 mg) 2 cigarettes will destroy the daily RDA of 45 mg).

UVR: All forms of ultra violet radiation will destroy Vit C, but it is particularly susceptible to UVA (sunbeds)

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Stress: We use up more Vit C when under physical and emotional stress. Oxygen destroys ascorbic acid. It is metabolised to dehydroascrobic acid generally and that is about 1/6 th as effective as ascorbic acid.

Skin conditions Vit C is best for: All forms of Environmental Defence Collagen loss Thin skin density (fragile) Diffused redness Healing

Vitamin C as an antioxidant. Vitamin C (Ascorbic acid) is an extremely powerful antioxidant but also has significant metabolic functions that make it particularly important for the normal action of skin.

Our skin certainly needs lots of vitamin C to counteract the dangers of free radicals. Because it is water­soluble, vitamin C cannot be stored for a significant period in the cell, and should be replaced daily. Since the body consists mainly of water, vitamin C is an important antioxidant for the cytoplasm of the cell (that is the liquid/gel that forms the main filler of the cell and in which the various structures like the mitochondria, Golgi apparatus, nucleus etc are cushioned. The paradox is that vitamin C seems to have an extremely important role in the protection of lipid membranes and the prevention of peroxidation of lipids. We now understand that ascorbic acid works in tandem with vitamin E and besides scavenging common free radicals, actually can also scavenge the free radical form of vitamin E that occurs when vitamin E neutralises a lipid based free radical. This free radical form of vitamin E is inactive and vitamin C can convert it into the antioxidant form of vitamin E, while itself being changed into a weak free radical. This weak free radical can itself be re­activated by either glutathione, which is a common amino acid found in cells, or by dihydrolipoic acid (alpha lipoic acid). Co­enzyme Q10 is also used in this mutual reactivation between these five special antioxidants that are normally found in the skin.

Nevertheless, we need good doses of vitamin C to protect the skin constantly so as to minimise photo damage. Research has shown us that when we supply adequate doses of vitamin C topically, we not only increase the antioxidant protection of the skin and reduce the dangers of developing sun burn cell, but we also stimulate the fibroblast to make more collagen and elastin, and persuade the melanocyte to make less melanin. For us, as skin therapists, an

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important sideline effect is that the vitamin A in the skin is better protected and less liable to being destroyed by free radicals.

Vitamin C and a healthy stratum corneum: Vitamin C plays an important part in the formation of stratum corneum barrier lipids. With the extensive production of lamellar bodies, their complete extrusion at the stratum granulosum/stratum corneum interface, and the formation of multiple broad lipid lamellar structures in the intercorneocyte space. Ascorbic acid ensures xiii

Vitamin C and the production of collagen: Ascorbic acid is essential to incorporate proline into collagen. Without this, defective collagen would be made. Vitamin C also has effects on the DNA of fibroblasts to promote the formation of collagen and elastin and the GAGs (glycoseaminoglycans).

Vitamin C and melanin formation: Ascorbic acid is the most powerful natural controller of melanin formation. First of all, melanin formation has to occur in an oxidising atmosphere because the enzymes that produce melanin can only work in under oxidising conditions. Because ascorbic acid is such a powerful antioxidant in the mitochondria, it creates a reducing (anti­oxidising) atmosphere and makes it almost impossible to make any melanin.

In addition, ascorbic acid inhibits melanin formation by specifically inhibiting the transformation of the melanin pre­cursor, tyrosine, into melanin by blocking the action of the enzyme tyrosinase.

Signicative increase of melanin concentrations in skin were found after 4, 5, 6 and 8 weeks of dietary antioxidant intake in both groups (p < 0.05). These results are discussed with regard to the redox control theory of melanocytes, which regulates the tyrosinase activity. xiv

VC­PMG suppressed melanin formation by tyrosinase and melanoma cells. In situ experiments demonstrated that VC­PMG cream was absorbed into the epidermis and that 1.6% remained 48 hours after application. The lightening effect was significant in 19 of 34 patients with chloasma or senile freckles and in 3 of 25 patients with normal skin. CONCLUSION: VC­PMG is effective in reducing skin hyperpigmentation in some patients. xv

As you can see there is great value in applying vitamin C topically but here is the crunch: vitamin C is a water­soluble molecule and we have to get it through the lipid based horny layer of the epidermis in order

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to get its benefits. The horny layer is a virtually solid lipid membrane that acts as an extremely effective barrier. The story becomes interesting as we learn about how scientists have managed to get around this obstacle. There is a lot of hype about vitamin C and if you read some advertisements then you would think that certain companies only have the magic formula to get vitamin C into the skin cell itself. Lets examine the chemistry of vitamin C.

Ascorbic acid is inexpensive and easy to obtain but unfortunately, it is unstable and does not last long. On the other hand scientists have managed to stabilise vitamin C by combining it with certain other chemicals but stable versions of Vitamin C are expensive and are usually trashed by companies selling ascorbic acid products. They say that you have to use natural ascorbic acid and nothing else will work the same.

First of all let’s deal with the myths about natural ascorbic acid. Ascorbic acid is one of the most important vitamins in our lives. The exact chemical configuration of natural vitamin C is described as l­ ascorbic acid. That means that if polarized light is passed through ascorbic acid crystals, then the light is twisted to the left. When we synthesize vitamin C then d­ascorbic acid is created and as you have probably guessed, it is labelled "d­" because it twists light to the right. In fact synthetic ascorbic acid comprises both d­ and l­ forms and they can be separated ­ of course this adds some expense, but not much. It just so happens that virtually all "natural" ascorbic acid that is used in the manufacture of cosmetics is synthesized, but it has been "purified" and made natural by excluding the d­ascorbic acid. Although this ascorbic acid is synthesized, it is true "natural" vitamin C.

Ascorbic acid is commercially available as a dry powder (technically called dehydro­ascorbic acid), which is relatively stable and is white in colour. When ascorbic acid powder is exposed to light and air, it slowly decomposes to oxidized ascorbic acid, which is yellowish­brown in colour. When ascorbic acid crystals are mixed in water, the solution, as its name implies, has an acidic pH. Under natural conditions the pH can easily be 2 or lower, depending on the concentration. Obviously the greater the saturation, the lower the pH. And the lower the pH, the more stable the ascorbic acid solution. Of course, the lower the pH the more irritant the solution is to the skin. A solution of vitamin C is much less stable than dry powdered ascorbic acid and rapidly decomposes to its oxidized form. I have found that under the best laboratory conditions, ascorbic acid gels even with so­called

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"stabilizers", only last at it’s intended concentration for a maximum of three weeks. Therefore, a solution of ascorbic acid, even in a gel, has a limited shelf life and once the solution has been made up, it should be used within three weeks to get the optimum levels of vitamin C. It is easy to see when ascorbic acid gel has become oxidized: it becomes a yellowish brown in colour. When the solution starts to turn a pale yellow, then one already has less than the original concentration of vitamin C. Many manufacturers claim to have 10% ascorbic acid in their product and because ascorbic acid is so unstable they must "overage" the product and add about 25% more, which means that they have 12.5% ascorbic acid in the final product immediately after manufacture. A light yellow colour develops within a few days and by then the concentration may have dropped to 10%. By four weeks, it is likely that the concentration has dropped to about 5%, which is only half of the concentration that was intended.

By three months, you can forget about achieving any enhanced vitamin C effects. So in other words, check the date of expiry on your ascorbic acid products. Also check the list of ingredients. I cannot understand why any manufacturer of ascorbic acid products would want to include a yellow brown colourant or any other colour into their product. There is no aesthetic reason, neither is there a functional, physiological reason. I leave you to decide what the motives could be. Therefore, check with your supplier and find out if the ascorbic acid product they sell you has any colour in it. If it does, then ask why and make sure that you get a detailed answer.

You need the freshest product that you can get, and it has to be the freshest possible by the time your client uses it. Under ideal conditions, the client should use up the ascorbic acid product before three weeks have passed after its manufacture. That sounds impossible but some very clever and ethical companies have arranged that the ascorbic acid products are sold unmixed to their clients. Sometimes separate sachets preserve the ascorbic acid crystals from exposure to light and air. The crystals are kept stable by packing them with nitrogen instead of air. Other companies have made special bottles that separate the fluid from the ascorbic acid crystals. When the client presses a plunger, the powder falls into the solution and after shaking, the final solution is made which ensures the freshest possible vitamin C. However, it does not ensure that the concentration is as strong as it should be. You really need a concentration of ascorbic acid at a minimum of 10% or more, to get the real benefits of ascorbic acid. I don’t think that you have to worry about the pH because the manufacturer is bound to make sure that the pH will be

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about 2. At this pH of 2, it will ensure the best penetration of the ascorbic acid through the horny layer into the skin and unfortunately, it will also sting! The pH is naturally low because ascorbic acid acts like an AHA (alpha hydroxy acid) and softens the glue between the cells of the horny layer and increases the penetration of vitamin C into the deeper layers of the skin. That, however, does not ensure the best penetration into the cell. Vitamin C passes with difficulty from the intercellular fluid into the cell wall because it is a water­soluble molecule. When it is oxidized, it can be taken into the cell itself masquerading as glucose, and can be converted into active vitamin C by glutathione and alpha lipoic acid. That, of course, uses up essential components of the antioxidant brigade and can weaken the total antioxidant protection mechanism.

So, in summary, vitamin C as ascorbic acid has a difficult task to get through into the skin and if it does then it cannot easily get into the cell itself where it has to work!

Although vitamin C is critical to human physiology, it is not clear how it is taken up into cells. The kinetics of cell and tissue accumulation of ascorbic acid in vitro indicate that the process is mediated by specific transporters at the cell membrane. Some experimental observations have linked the transport of ascorbic acid with hexose transport systems in mammalian cells, although no clear information is available regarding the specific role(s) of these transporters, if any, in this process.

These observations indicate that mammalian facilitative hexose transporters are a physiologically significant pathway for the uptake and accumulation of vitamin C by cells, and suggest a mechanism for the accumulation of ascorbic acid against a concentration gradient xvi

The significance of the functional linkage between vitamin C and adrenal glucocorticoid, which has been confirmed both in both the humans and rats in our laboratory, was discussed in the light of the historical development of vitamino­endocrinology. xvii

Topical vitamin C Topical L­ascorbic acid, when used in an appropriate vehicle and when initiated at an appropriate postoperative period, may decrease the degree and duration of erythema after cutaneous CO2 laser resurfacing. It is presumed that the anti­inflammatory effect of vitamin C is responsible for the clinical changes observed in this study. xviii

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Absorption of ascorbic acid The data clearly show that ascorbic acid uptake is inhibited instantly by glucose in a concentration dependent fashion. The results support the contention that local ascorbic acid deficiency in tissues could be a natural consequence of hyperglycaemia of whatever cause. The rate of ascorbic acid uptake under various conditions suggests that additional supplements of ascorbic acid might be helpful to individuals in averting deleterious effects of hyperglycaemia on tissue ascorbic acid supply. xix

1. Ascorbic acid was absorbed across the mucosa of the human mouth.

2. Omission of sodium ions from the medium decreased the absorption of ascorbic acid.

3. The presence of D­glucose, or 3­O­methyl­D­glucose, increased the absorption of ascorbic acid but D­fructose had little effect and D­mannitol had no effect.

4. Calcium ions also increased ascorbic acid absorption probably by a secondary effect on "Na+ fluxes.

5. Buccal mucosa was also permeable to dehydroascorbic acid and D­isoascorbic acid. xx

Many cell types transport vitamin C solely in its oxidized form, dehydroascorbic acid, through facilitative glucose transporters. These cells accumulate large intracellular concentrations of vitamin C by reducing dehydroascorbic acid to ascorbate, a form that is trapped intracellularly. Certain specialized cells can transport vitamin C in its reduced form, ascorbate, through a sodium­dependent co­transporter. We found that normal human melanocytes and human malignant melanoma cells are able to transport vitamin C using both mechanisms. xxi

The conditions used for glycation reactions in vitro rapidly oxidized ASA, but not glucose. The UVA­dependent generation of oxygen free radicals also oxidized ASA at a 10(3) faster rate than glucose. Superoxide anion and singlet oxygen were identified as the principal oxidants of ASA in this process. ASA may be the primary glycating agent in aging normal lenses. xxii

The degree to which antioxidant loss occurs in human skin after UV irradiation is unknown, as is the cascade of events that might occur. Human skin equivalents, a tissue culture model, were irradiated using a full solar UV spectrum (UVA and UVB, 280­400 nm) (0 to 16.8 J/cm2, 0­12 minimal erythemal dose, (MED), then incubated from 1 to 24 h. Ubiquinol was the most UV­light sensitive antioxidant and was

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depleted by 2.1 J/cm2 (1.5 MED, p < .004); ubiquinone decreased with 4.2 J/cm2 (3 MED, p < .0007). A linear decrease in alpha­ tocopherol occurred­­approximately 1.7 pmol tocopherol/cm2 surfaces were destroyed per J/cm2 UV­light. Urate was depleted by irradiation with 8.4 J/cm2 (6 MED), while ascorbate was depleted by 16.8 J/cm2 (12 MED). In conclusion, human skin equivalents respond to suberythemal levels of UV­irradiation by increasing production of PGE2; higher levels of UV­irradiation (at least 1 MED) were needed to deplete cellular antioxidants and induce immediately detectable oxidative damage xxiii .

The rate of ascorbate oxidation in the presence of vitamin E homologues was enhanced by a photosensitizer (riboflavin) but was not influenced by reactive oxygen radical quenchers, superoxide dismutase or 5,5­dimethyl­1­pyrroline­N­oxide. These experimental results suggest that the UV irradiation­induced ascorbate oxidation in murine skin homogenates is caused by photoactivated reactions rather than reactive oxygen radical reactions. xxiv

50 days after supplementation alpha­Toc keratinocyte levels were increased in groups (1) and (3), Asc concentrations were elevated in groups (2) and (3), and the a/gamma­Toc ratio increased in groups (1) and (3). The dose response curve of UVR induced erythema showed a significant flattening and the MED increased from 103 +/­ 29 mJ/cm2 (before supplementation) to 183 +/­ 35 mJ/cm2 (after supplementation) in group (3), while there were no significant changes in groups (1) and (2) after vitamin supplementation.

CONCLUSION: Alpha­Toc and Asc act synergistically in suppression of the sunburn reaction. xxv

L­Ascorbic acid was able to down regulate IL­1alpha mRNA expression in both UVA­irradiated and non­irradiated cells; however, IL­6 mRNA expression remained unaffected. The secretion of these cytokines was reduced nearly to normal in the presence of L­ascorbic acid. These findings indicate a major cell­protective effect of L­ascorbic acid on UVA­induced lipid peroxidation and the secretion of pro­inflammatory cytokines by UVA­irradiated human keratinocytes xxvi .

The skin possesses an elaborate antioxidant defence system to deal with UV­induced oxidative stress. However, excessive exposure to UV can overwhelm the cutaneous antioxidant capacity, leading to oxidative damage and ultimately to skin cancer, immunosuppression and premature skin ageing. Therefore, an interesting strategy for

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photoprotection is the support of the endogenous antioxidant system. This can be accomplished by induction or transdermal delivery of the various antioxidant enzymes, such as glutathione peroxidase, catalase, or superoxide dismutase. Supplementation of non­enzymatic antioxidants such as glutathione, alpha­tocopherol, ascorbate and beta­carotene was also found to be very effective in photo protection. Although treatments with single components of the antioxidant system were successful against a wide variety of photo damage, the balance between the different antioxidants in the skin is very important. In some studies, it was found that too much of a single component could even have deleterious effects. The most promising results were obtained in studies combining several compounds, often resulting in synergism of the protective effects. xxvii

A critical step in the escape from the carcinogenic potential of UV radiation is mediated by the protein p53. P53 activates growth arrest, allowing for DNA repair, and apoptosis, which removes damaged cells.

Here I show that p53 in cultured human skin fibroblasts is elevated after treatment with hydrogen peroxide, an oxidant produced in cells during exposure to solar UV radiation. Simulated solar UV radiation increased p53, and agents that scavenge active oxygen species, N­acetyl cysteine, ascorbate and alpha­tocopherol, inhibited the increase. The generation of DNA single strand breaks has been proposed to be an important step in the pathway leading to the increase in p53 initiated by a variety of cytotoxic agents. xxviii

Pre­clinical studies amply illustrate the photo protective properties of supplemented antioxidants, particularly RRR­alpha­tocopherol, L­ ascorbate and beta­carotene. However, clinical evidence that these antioxidants prevent, retard or slow down solar skin damage is not yet convincing. xxix

Topical application of combinations of both vitamins, or of melatonin with vitamins, enhanced the photo protective response. Better protection was obtained by using the combination of melatonin with both vitamins. xxx

Combined vitamins C and E reduce the sunburn reaction, which might indicate a consequent reduced risk for later sequelae of UV­induced skin damage. The increase of sunburn reactivity in the placebo group could be related to "priming" by the previous UV exposure. xxxi

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Photo protective effect of AA­2G on cytotoxicity of UVB in SCC cells was dose dependent (0.125­1 mM) and more effective than that of ascorbic acid (AsA) at 1 mM. This protection was completely abolished in the presence of an alpha­glucosidase inhibitor, castanospermine, indicating that release of AsA from this derivative was essential for reduction of the actinic injury. AA­2G significantly suppressed cytotoxicities of hydrogen peroxide and superoxide anion produced by xanthine and xanthine oxidase. In addition, AA­2G reduced UVB­ promoted formation of lipid peroxide and accumulation of lipofuscin, which is known to be a complex of cellular proteins and metabolites of lipid peroxide. These data suggest that AA­2G prevents the acute inflammation induced by UVB irradiation partly through scavenging reactive oxygen species and potentiating the anti oxidative activity of alpha­tocopherol. xxxii

Melatonin (N­acetyl­5­methoxytryptamine), vitamin E (alpha­ tocopherol) and vitamin C (ascorbic acid) were topically administered alone or in combination following UVR exposure as single applications (immediately or 30 min after irradiation, respectively) or as multiple applications (three times: 30 min, 1 h and 2 h after irradiation). The erythemal reaction was evaluated visually and non­invasively with bioengineering methods (skin colour and skin blood flow). RESULTS: No significant protective effect of melatonin or the vitamins when applied alone or in combination were obtained when antioxidants were applied after UVR exposure. No improved photo­protective effect was obtained when multiple applications were done.

CONCLUSION: UVR­induced skin damage is a rapid event, and antioxidants possibly prevent such damage only when present in relevant concentration at the site of action beginning and during oxidative stress. xxxiii

The effects of reactive oxygen species (ROS) on elastin molecules (tropoelastin) were studied in vitro.

ROS generated by ultraviolet A and hematoporphyrin rapidly degraded tropoelastin within 5 min. Their degradative activity was inhibited by the addition of NaN3. Treatment of tropoelastin with copper sulfate/ascorbic acid resulted in degradation of tropoelastin producing fragments of molecular weight 45, 30 and 10 kDa within 30 min.

ROS generated by copper­ascorbate seems to be unique in that it cleaves relatively specific sites of the tropoelastin molecule. Thus ROS may play a degradative role in elastin metabolism, which may cause

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the elastolytic changes or the deposition of fragmented elastic fibers in photoaged skin or age­related elastolytic disorders. xxxiv

Ascorbate and collagen formation

Ascorbic acid specifically increases type I and type III procollagen messenger RNA levels in human fibroblasts. xxxv

L­ascorbic acid is an essential cofactor for lysyl hydroxylase and prolyl hydroxylase, enzymes essential for collagen biosynthesis. In addition, L­ascorbic acid preferentially stimulates collagen synthesis in a manner, which appears unrelated to the effect of L­ascorbic acid on hydroxylation reactions. This reaction is stereospecific and unrelated to intracellular degradation of collagen. The effect apparently occurs at a transcriptional or translational level, since L­ascorbic acid preferentially stimulates collagen­specific mRNA. In addition, it stimulates lysyl hydroxylase activity but inhibits prolyl hydroxylase activity in human skin fibroblasts in culture. xxxvi

L­Ascorbic acid stimulates procollagen synthesis in cultured human skin fibroblasts without appreciably altering noncollagen protein synthesis. The effect is unrelated to intracellular degradation of newly synthesized procollagen. Levels of mRNA for pro alpha 1(I), pro alpha 2(I), and pro alpha 1(III), measured by hybridization with the corresponding cDNA probes, are elevated in the presence of ascorbic acid, whereas the level of mRNA for fibronectin is unchanged. Levels of functional mRNA for procollagen, measured in a cell­free translation assay, are specifically increased in the presence of ascorbic acid. Thus, ascorbic acid appears to control the expression of three different procollagen genes, each of which is located on a separate chromosome. It is proposed that intracellularly accumulated procollagen in ascorbate deficiency may lead to a translational repression of procollagen synthesis. Ascorbic acid may relieve this block by promoting hydroxyproline formation and, consequently, secretion of procollagen from the cell. The increased level of procollagen mRNA under the influence of ascorbic acid may be secondary to increased synthesis of procollagen polypeptides; the control point may be gene transcription or mRNA degradation. xxxvii

Ascorbate is required for hydroxylation of proline residues in procollagen and hydroxyproline stabilizes the collagen triple helical structure. Proteoglycan synthesis, which does not require ascorbate, also is decreased and both effects are correlated with the extent of

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weight loss during scurvy. The inhibitor appears to consist of two IGF­ binding proteins induced during vitamin C deficiency and starving and may be responsible for in vivo inhibition of collagen and proteoglycans. xxxviii

Procollagen biosynthesis and matrix deposition were studied in long­ term human skin fibroblast cultures exposed to ascorbic acid. Ascorbic acid specifically stimulated types I and III collagen synthesis, reaching a maximum at day 2 and maintaining a specific high rate of production until day 10 of ascorbate exposure, after which collagen production declined. The increased level of collagen synthesis after different exposure times could also be achieved by only brief treatment (10 h) of parallel scorbutic (ascorbic­acid­deficient) cultures with ascorbic acid. This brief exposure did not result in increased collagen mRNA, thus demonstrating that the ascorbate­induced increase in collagen synthesis at all stages of ascorbic acid exposure was due to post­ transcriptional mechanisms, most likely a rapid increase in type 1 collagen mRNA translational efficiency. This mechanism, rather than the transcriptional activation, was the primary response and is adequate to explain the ascorbate­induced increase in collagen synthesis.

These data also demonstrate that the presence of a collagenous extracellular matrix was not involved in this collagen biosynthetic regulation. During long­term exposure (18 days) to ascorbic acid, a substantial cross­linked collagenous matrix formed, following an approximately sigmoidal time course. The most rapid matrix deposition occurred during the later days of exposure when the rate of collagen synthesis was decreasing, suggesting that the presence of a pre­ existing matrix is important for further collagen accumulation. Procollagen was also efficiently processed to collagen during this phase, demonstrating that efficient procollagen processing is an important regulatory event in collagen matrix deposition. xxxix

Ascorbate has been shown to stimulate collagen synthesis in cultured human dermal fibroblasts by increasing transcription of the collagen genes. In the present studies, ascorbate stimulates lipid peroxidation at concentrations similar to those necessary to affect collagen synthesis. Molecules, which inhibit lipid peroxidation, such as propyl gallate, cobalt chloride, and alpha­naphthol, also inhibit collagen synthesis, suggesting a correlation between the two phenomena. Retinoic acid and some synthetic retinoids have previously been shown to inhibit collagen synthesis in cultured human dermal fibroblasts. In our studies two different retinoids, at similar concentrations, inhibit

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both ascorbate­stimulated lipid peroxidation and collagen synthesis. Since high concentrations of retinoids were required, the ability of retinoids to inhibit the oxidant effect of ascorbate, and not their receptor­mediated activity, may be responsible for their effect on collagen synthesis. xl

Thus, ascorbic acid appears to control the expression of three different procollagen genes, each of which is located on a separate chromosome. It is proposed that intracellularly accumulated procollagen in ascorbate deficiency may lead to a translational repression of procollagen synthesis. Ascorbic acid may relieve this block by promoting hydroxyproline formation and, consequently, secretion of procollagen from the cell. The increased level of procollagen mRNA under the influence of ascorbic acid may be secondary to increased synthesis of procollagen polypeptides; the control point may be gene transcription or mRNA degradation. xli

These studies indicate that the ascorbate­induced increase in type I procollagen synthesis is due to increased levels of type I procollagen mRNA and is independent of the level of hydroxylation of the procollagen. xlii

Transforming growth factor­beta (TGF­beta) is a prototype of a family of polypeptides that regulates cellular growth and phenotypic differentiation. TGF­beta injection induces angiogenesis and fibrosis locally and stimulates the synthesis of extracellular matrix proteins, fibronectin, collagens, and proteoglycans in vitro in many cell types. Ascorbate is also known to induce collagen synthesis and to promote wound healing. We report that in cultured human skin fibroblasts, ascorbate and TGF­beta synergistically enhance the biosynthesis of type I and III collagens and their steady­state mRNAs. TGF­beta alone has no enhancing effect on type III collagen synthesis. The cooperation between ascorbate and TGF­beta may be of significance in wound healing and in disorders of fibrosis. xliii

Ascorbate contributes to several metabolic processes including efficient hydroxylation of hydroxyproline in elastin, collagen, and proteins with collagenous domains, yet hydroxyproline in elastin has no known function. Prolyl hydroxylation is essential for efficient collagen production; in contrast, ascorbate has been shown to decrease elastin accumulation in vitro and to alter morphology of elastic tissues in vivo. Ascorbate doses that maximally stimulated collagen production (10­ 200 microM) antagonized elastin biosynthesis in vascular smooth muscle cells and skin fibroblasts, depending on a combination of dose

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and exposure time. Diminished elastin production paralleled reduced elastin mRNA levels, while collagen I and III mRNAs levels increased. We compared the stability of mRNAs for elastin and collagen I with a constitutive gene after ascorbate supplementation or withdrawal. Ascorbate decreased elastin mRNA stability, while collagen I mRNA was stabilized to a much greater extent. Ascorbate withdrawal decreased collagen I mRNA stability markedly (4.9­fold), while elastin mRNA became more stable. Transcription of elastin was reduced 72% by ascorbate exposure. Differential effects of ascorbic acid on collagen I and elastin mRNA abundance result from the combined, marked stabilization of collagen mRNA, the lesser stability of elastin mRNA, and the significant repression of elastin gene transcription. xliv

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Ascorbic acid (vitamin C) is a cofactor required for the function of several hydroxylases and monooxygenases. Its absence is responsible for scurvy, a condition related in its initial phases to a defective synthesis of collagen by the reduced function of prolylhydroxylase and production of collagen polypeptides lacking hydroxyproline, therefore, they are unable to assemble into stable triple­helical collagen molecules. In fibroblast cultures, vitamin C also stimulates collagen production by increasing the steady­state level of mRNA of collagen types I and III through enhanced transcription and prolonged half­life of the transcripts. The mRNA of collagen type I and type III were increased to a similar extent by vitamin C and that of three post­ translational enzymes, the carboxy­ and amino­procollagen proteinases and lysyloxidase similarly increased. The mRNA of decorin was also stimulated, but elastin, and fibrillin 1 and 2 were not modified by the vitamin. The expression of matrix metalloproteinases 1, 2, and 9 was not significantly changed, but an increased level of tissue inhibitor of matrix metalloproteinase 1 mRNA was observed without modification of tissue inhibitor of matrix metalloproteinase 2 mRNA. The stimulating activity of topical vitamin C was most conspicuous in the women with the lowest dietary intake of the vitamin and unrelated to the level of actinic damage. The results indicate that the functional activity of the dermal cells is not maximal in postmenopausal women and can be increased. xlv

The study of nitroxide radical interactions with tissue antioxidants and oxidants is of growing interest. Skin is a target organ of the EPR methodology and is frequently exposed to oxidative stress. We investigated the piperidine­type nitroxide 2,2,5,5­tetramethyl­4­ piperidin­1­oxyl (TEMPO) because it is skin permeable and readily accepts electrons in biological systems. Quantitative considerations

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indicate that the major reduction site of TEMPO in skin and skin cells is the cytosol ascorbate/glutathione redox cycle. We suggest that analysis of TEMPO radical scavenging by the EPR technique is a convenient method for measuring skin ascorbate and thiol­dependent antioxidant activity in vitro and in vivo. xlvi

The benefit of AA and PA supplementation could be due to the variations of the trace elements, as they are correlated to mechanical properties of the scars. xlvii

To make healthy collagen, lsyl hydroxylase, a vitamin C­dependent enzyme, is essential to convert lysyl residues to hydroxylysine on procollagen peptides. xlviii

Ascorbate stimulated the deposition of glycosaminoglycans into the insoluble matrix of normal fibroblasts xlix

These results suggest that collage polypeptide synthesis, posttranslational hydroxylations, and activities of the two hydroxylases are independently regulated by ascorbate. l

A similar increase in prolyl hydroxylase activity occurred when cells were incubated with ascorbate. Lysyl hydroxylase activity remained unaltered under these conditions. li

Old scars break open in scorbutic patients because (1) the rate of collagen degradation is greater in an old scar than it is in normal skin, and (2) the rate of collagen synthesis is diminished throughout the body in ascorbate deficiency. lii

Our results suggest that there are differences in collagen synthesis between photoaged and aged cells, depending on culture conditions. Responsiveness to ascorbic acid, TGF­beta and IFN­gamma related to collagen synthesis in photoaged and aged fibroblasts in collagen gel appears to be the same as in newborn fibroblasts, even though basal levels of collagen synthesis are downregulated in a photoaging­ or aging­dependent manner. liii

L­ascorbate (vitamin C) stimulated growth and collagen synthesis, as well as synthesis of non­collagenous proteins, with no significant effect on hyaluronic acid synthesis. Co­presence of epidermal growth factor and ascorbate gave additive effects on growth and protein synthesis of the cells. These results suggest that the two growth­promoting factors, epidermal growth factor and L­ascorbate, modulate metabolism of

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extracellular matrix components as well as cell growth in a quite different manner in human skin fibroblasts. liv

For a fructose concentration of 25 mM, we observed that in the absence of glucose, intracellular total proteins increased 1.5­fold and prolidase specific activity, 1.8­fold. For ascorbate, a broad optimum concentration was found (range 0.01 ­ 0.50 mM). Addition to cultures of 0.1 mM ascorbate increased total proteins 1.4­fold, and doubled prolidase activity. This investigation was prompted by our previous results [J. Metab. Dis. 1983, 6, 27­31], confirmed here, and suggesting that increased prolidase activity at confluency was due to a rise in cell density. lv

Ascorbate and nitroxides

The most convincing evidence for the involvement of vitamin C in cancer prevention is the ability of ascorbic acid to prevent formation of nitrosamine and of other N­nitroso compounds. lvi

UV radiation (300 nm) increases the ascorbate free radical (Asc.­) electron paramagnetic resonance (EPR) signal in human facial skin biopsies (340%). Visible light (lambda > 400 nm; 0.23 mW/cm2 UVA) also increased the Asc.­ signal in human skin samples (45%) indicating that human skin is more susceptible to free radical formation and that a chromophore for visible light may be present. These results provide the first direct evidence for UV radiation­induced free radical formation at near physiological temperatures in human skin and suggest that iron chelators may be useful as photo protective agents. lvii

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Topical antioxidants

ß­Carotene, a precursor of vitamin A, and ­tocopherol (vitamin E) are the main lipid­soluble non­enzymatic antioxidants and are mainly confined to cell membranes and low­density lipoprotein. Among water­ soluble antioxidants, ascorbate is the most efficient antioxidant. Table 4 summarizes the most important characteristics of these systems.

Experimentally, the topical application of antioxidants has shown promising results in preventing damage and the photo induced inflammatory response (63­66). lviii lix lx lxi lxii

Magnesium ascorbyl phosphate

We found, in an in vitro experiment, that MAP was converted to AS as it crossed the epidermis, but that AS­Na did not pass through the epidermis. Furthermore, MAP was also converted to AS in serum. These results suggest that the protective effect of MAP on UVB­ induced cutaneous damage is due to conversion of MAP to AS. lxiii

L­Ascorbic acid 2­phosphate (Asc 2­P), a long­acting vitamin C derivative, stimulated transcription of genes for pro alpha 1(I) and pro alpha 2(I) collagen in normal human skin fibroblasts after 8 h of treatment in the absence or in the presence of cycloheximide, indicating Asc 2­P stimulates transcription of type I collagen genes in the absence of protein synthesis. The transcriptional rate in these cells reached the maximum value after 40 h of treatment, and at that time it was three to four times higher than that of the control cells cultured in the absence of Asc 2­P. Steady­state levels of mRNAs for pro alpha 1(I) and pro alpha 2(I) chains were also increased to be three to four times higher than the control levels by treatment of the cells with Asc 2­P for 72 h. lxiv

The interaction between vitamin C and E, and other antioxidants

Ascorbate regenerates vitamin E by a nonenzymic mechanism, whereas glutathione regenerates vitamin E enzymatically. These studies suggest that significant interaction occurs between water­ and lipid­soluble molecules at the membrane­cytosol interface and that vitamin C may function in vivo to repair the membrane­bound oxidized vitamin E. lxv

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Vit C produced a clear cytoprotective effect on aged cells over the entire range of doses applied. The protection provided by Vit E, was less pronounced. lxvi

The results showed a modest protective effect of the vitamins when applied alone and a dose­dependent photo protective effect of melatonin. Topical application of combinations of both vitamins, or of melatonin with vitamins, enhanced the photo protective response. Better protection was obtained by using the combination of melatonin with both vitamins. The role of reactive oxygen species and oxygen­ derived free radicals, as well as potential sun screening properties of the employed antioxidants, are discussed in view of possible mechanisms to explain this elevated photo protective effect. lxvii

The literature associates low vitamin C levels in the blood to an increased relative risk of gastrointestinal cancer, including cancer of the oesophagus, mouth, stomach, and pancreases (Gey, 1993). The reason for its effect on cancer in the gastrointestinal tract is thought to be due to the pH of the stomach and vitamin C's nitrate trapping property. Below pH 6, as in the stomach, vitamin C may have an increased trapping ability for nitrates that are produced in the stomach and oesophagus and thus an increased protective effect at those sites. Vitamin E has a very similar mechanism of prevention to that of vitamin C, but is different in that vitamin E is a lipid soluble vitamin and has a longer life in the body so that blood levels reflect more chronic consumption of vitamin E.

Vitamins E and C as well as many other antioxidants are very effective inhibitors of skin tumor promotion. lxviii

Ascorbic acid, while by itself not cytoprotective synergized with quercetin, lowered the quercetin EC50 and prolonged the window for cytoprotection. The related flavonoids rutin and dihydroquercetin also decreased BSO­induced injury to dermal fibroblasts, albeit less efficaciously so than quercetin. The cytoprotective effect of rutin, but not that of dihydroquercetin, was enhanced in the presence of ascorbic acid. Further, quercetin rescued sensory ganglion neurons from death provoked by GSH depletion. Direct oxidative injury to this last cell type has not been previously demonstrated. The results show that flavonoids are broadly protective for cutaneous tissue­type cell populations subjected to a chronic intracellular form of oxidative stress. Quercetin in particular, paired with ascorbic acid, may be of therapeutic benefit in protecting neurovasculature structures in skin from oxidative damage. lxix

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Vitamin C Fact File

There is an enormous amount of literature on vitamin C intake and health in animals, cell cultures, and humans. Beyond its function in collagen formation, ascorbic acid is known to increase absorption of inorganic iron, to have essential roles in the metabolism of folic acid and of some amino acids and hormones, and to act as an antioxidant. lxx

Because ascorbic acid is water­soluble and the cell membranes are lipid structures, we have to find ways of persuading the cells to take up increased doses of vitamin C through the lipid membranes. There is another way to get the ascorbic acid into the cell itself: combine it with another molecule that is selectively taken into the cell. An example of this form of vitamin C is magnesium (or sodium) ascorbyl phosphate, which is also water­soluble but is taken up into cells much more effectively because the cells select the minerals, which they need for their normal metabolism. Unfortunately this is an expensive ingredient to work with. Can this be one reason why some manufacturers of the less expensive form of ascorbic acid tend to trash the more effective ascorbyl phosphate salts? Inside the cells, the vitamin C compound is easily converted to ascorbic acid and phosphate, and magnesium (or sodium). The advantage of using these solutions are that they are also more stable than conventional ascorbic acid and can last up to 200 days before there is any appreciable loss of activity. Lower concentrations (compared to ascorbic acid) are required to get the same amount of ascorbic acid into the cell itself. I have used both ascorbic acid and these stabilized salts of vitamin C. They are both useful and, depending on dose and means of administration, have similar results, but have a different client profile. People with sensitive skins cannot use ascorbic acid and so I use magnesium ascorbyl phosphate products for them. People with pigmentation problems should avoid any product that peels the skin significantly. Ascorbic acid has an exfoliant property so I generally recommend that clients with melasma or other pigmentation problems should rather use the more neutral ascorbyl phosphate salt. Both ascorbic acid and its salts are suitable for iontophoresis and sonophoresis and generally they can both be used for salon treatments for pigmentation. Your client will tell you if the ascorbic acid stings too much and in that case use magnesium ascorbyl phosphate.

I am pleased to tell you that wonderful strides have been made in vitamin C variants for cosmetics. The latest, which I believe will set

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the standards for vitamin C treatments, is ascorbyl tetra­isopalmitate, which is fat soluble and extremely stable. That means that there are four molecules of palmitic acid attached to the ascorbic acid molecule. The palmitic acid molecule is about the same weight as the ascorbic acid molecule so you can see that vitamin C forms only about a quarter of this large molecule. However, with only a tiny amount of vitamin C, this fat soluble form passes easily through the horny layer and enters the cell wall with great ease and you can get up to ten times more active vitamin C into the cell itself. As a result, there is more effective control of melanin formation, greater collagen deposition and more efficient antioxidant protection.

My experience with this wonderful molecule makes me believe that we have entered a new era in vitamin C treatments for the skin. However, bear in mind that this vitamin C is fat­soluble so it cannot be used for iontophoresis or sonophoresis.

Ascorbyl­tetra­isopalmitate, combined with vitamin A, produces rapid smoothening, without any irritation, and significant lightening of the skin (provided it is adequately protected from light. Of course if it is also combined with a wide antioxidant brigade and effective UV­A protection, then pigmented marks seem to melt away. Very few companies have used this new ingredient. Don’t confuse this molecule for the less effective ascorbyl palmitate, or ascorbyl di­palmitate, which are less effective at getting through the skin and into the cell itself. These ingredients have been used for many years. The fact remains that not nearly as much vitamin C gets inside the cell as with the tetra­isopalmitate. However, don’t avoid ascorbyl palmitate. It’s good, but just not as good as the current star ascorbyl tetra­ isopalmitate.

In summary, we would like to show you briefly how I use the various forms of vitamin C:

Ascorbic acid – I use it as fresh as possible and discard anything that is older than a month after production. I use this on people with tough, rough and wrinkled skin. I avoid using this on people prone to acne because the exfoliation can aggravate the acne. I have also used this for home vitamin C peels with great success.

Magnesium (or sodium) ascorbyl phosphate–I prefer to use this as fresh as possible and discard anything that is older than six to seven months after production. I use this on normal, sensitive, or pigmented skin. Can be used on acne but may initially aggravate the acne.

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Ascorbyl palmitate (ascorbyl di­palmitate) –Good stability so you don’t have to specially check the production date. Can be used on delicate skins and acne. I have never seen good changes and I cannot make a really active rejuvenating compound with it.

Ascorbyl tetra­isopalmitate (VCIP) – excellent stability but for the best results the product should not be older than 18 months after production. Gives the best levels of vitamin C inside the cells and can be used on delicate skins at high doses. I use it in preference to any other forms for pigmentation, wrinkles and even acne.

Types of Vit C found in cosmetic formulations. It is important to remember that the various types of vitamin C have been chosen for their greater stability than l­ascorbic acid. Whatever type of vitamin C is used, it eventually gets converted into ascorbic acid and that is how it expresses its effects. So ultimately, the amount of ascorbic acid inside the cell is what determines the effectiveness.

1. L Ascorbic Acid § l­Ascorbic Acid: Natural vitamin C: A water­soluble antioxidant which interacts with Vit E and other fat­soluble antioxidants, which are in the lipid rich areas of the cell. (Cell membrane) Together they bind to free radicals before they have a chance to damage the tissue. However, the paradox is that ascorbic acid is water­soluble and has great difficulty penetrating through the epidermis and through cell walls.

§ The second difficulty is that vitamin C is very unstable and solutions rapidly turn brown and are inactive. If you do buy a serum that does not change colour at all over the months, it probably has very little Vit C in it in the first place.

§ Despite claims of superior stabilization, all Vitamin C will lose strength after six months, (even if kept cool and away from direct sunlight). Some companies incorporate a calcium ion, to protect the ascorbic acid and keep it more stable.

2. Ascorbyl Palmitate § This is l­ascorbic acid with an added palmitate group to make the vitamin C more soluble in the lipid phase and allow better penetration through the skin and cell walls. The palmitate section is selectively absorbed into the cell

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and then esterase enzymes cleave off the palmitate moiety and l­ascorbic acid is set free inside the cell.

3. Ascorbyl di­palmitate § Similar to ascorbyl palmitate but more soluble in the lipid phases, and gets into cell walls easier than ascorbyl palmitate. Once again esterase enzymes have to separate the palmitic acid from the l­ascorbic acid before it can start being effective.

4. Magnesium ascorbyl phosphate (MAP or VCPMg) § This is a water­soluble Vit C that is not acidic and is more easily absorbed into the skin cell than ascorbic acid. This type of Vit C is converted by the metabolism into ascorbic acid plus the other important components such as magnesium and phosphorus. (Very important micronutrients).

The magnesium salt of ascorbyl­2­phosphate was found to be equivalent to ascorbic acid in stimulating collagen synthesis in these assays, while the sodium salt required at least a tenfold greater concentration to produce the same effect as ascorbic acid. These data support the use of magnesium ascorbyl­2­phosphate in experiments where stability of ascorbic acid is a concern, e.g. in long­term cultures or in in­ vivo studies. lxxi

5. Sodium ascorbyl phosphate § This is even more stable in water than VCPMg but research shows that the vitamin C is not as easily released inside the cell as compared to VCPMg.

Supplementation of the medium with Asc 2­P also accelerated procollagen processing to collagen and deposition of collagen in the cell layer. Among the acidic glycosaminoglycans (GAG), another major component of extracellular matrix (ECM), deposition of sulfated forms was increased by the additive. lxxii

6. Ascorbyl tetra­isopalmitate § The most effective form of vitamin C today for topical application. As its name implies, it has four palmitic acid ions attached to the l­ascorbic acid molecule. That makes it lipophilic and so it easily penetrates through the stratum

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corneum and is rapidly absorbed into the cell where the palmitic acid is separated from the ascorbic acid.

Topical vitamin C has been used as a PHOTOPROTECTANT as an additive to sunscreens (Thomas, 1991). The amount of vitamin C in the preparation was not specified.

Cathcart reported that topical ascorbic acid or sodium ascorbate has been effective in treating HERPES SIMPLEX and KAPOSI'S SARCOMA in AIDS patients. lxxiii The vitamin C was applied as a paste made by using ascorbic acid powder or sodium ascorbate powder and water. It was also applied as a solution of 20% ascorbic acid or sodium ascorbate in water used as a soak for 15 to 30 minutes 4 times daily. lxxiv

REFERENCES:

Slaga TJ & Bracken WM: The effects of antioxidants on skin tumor initiation and aryl hydrocarbon hydroxylase. Cancer Res 1977; 37:1631­1635.

Attempted use of vitamin C through iontophoresis in the treatment of circumscribed hyperpigmentations of the skin. Preliminary communication] Original Title Próby zastosowania jontoforezy z witzminy C w leczeniu ograniczonych przebarwien skóry. (Doniesienie tymczasowe) Author Ciecierski L Source Przegl Dermatol, 54: 4, 1967 Jul­Aug, 473­4 Abstract Abstract not available online. Language of Publication Polish Unique Identifier 68010245

Minocycline, a member of the tetracycline family of antibiotics, is widely used in the treatment of acne. Its use has been associated with intrinsic staining of adult human teeth, bones, and soft tissues. It causes blackening of the thyroid glands in both animals and humans. It has been determined that the pigment is the product of an oxidation reaction. Laboratory studies have shown that the pigment formation can be induced by exposure to ultraviolet light in the presence of air, and that an antioxidant, such as ascorbic acid (vitamin C), can block its formation Microscopic examination revealed extensive deposits of black pigment throughout the follicles of the minocycline group, whereas the group receiving both minocycline and vitamin C showed no sign of

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pigmentation and were indistinguishable from controls. It is suggested that patients on long­term minocycline medication be monitored for thyroid function. lxxv

. Further, we observed that for AA­2G, even on the 8th day of the culture, the amount of AA in the fibroblasts was virtually unchanged from the beginning of the experiment, whereas, in the case of adding AA and AA­2P, virtually no AA was detectable in the culture medium on the fifth day. These findings suggest that AA­2G is decomposed to AA by alpha­glucosidase in the cells. This AA promotes collagen synthesis, which is prolonged through AA­2G's sustained decomposition. lxxvi

Robert L. Goldemberg Free radicals: Efforts to Reduce Skin Aging Process by fighting free radicals. “Drug & Cosmetic Industry" Nov 1993 v153 n5 p48(3)

Free radicals often activate chain reactions known as cascades, sometimes polymerizing into larger molecules, sometimes degrading longer ones into short pieces. The latter effect, especially when it involves free radicals containing oxygen (such as the hydroxyl radical) is the most damaging physiologically, leading to destruction of fatty membranes, cell collapse, or DNA mutation, which occasionally leads to cancer.

Absorbing in the UVB portion of the solar spectrum, Vitamin E is transformed in the process, itself forming a free radical (tocopheroxyl), which can regenerate back to tocopherol via reaction with the skin’s ascorbic acid content (water soluble Vitamin C). Packer’s hypothesis is that as UV dosage is gradually increased, these two antioxidant defenses of the skin are overwhelmed. The free radicals, which then form in the skin, cause various types of cell damage, including lipid peroxidation and oxidative modification of dermal protein and its DNA. Packer has succeeded in measuring the depletion of cutaneous lipid soluble antioxidants (such as Vitamin E) after UVAB skin irratiation; he also showed the protective effect of sunscreens in this respect.

Dr. Martin Rieger has discussed the chemistry of oxidation and peroxidation processes in various of his publications, pointing out that melanin is also a free radical scavenger, and therefore somewhat "photo­protective" in a sense which is unrelated to its light­scattering ability. Rieger has also noted that PABA may play a role in DNA­ dimer formation.

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Petrolatum, of which Dr. Albert Kligman recently observed, "Although not a sunscreen, we found it surprisingly effective in suppressing ultraviolet­induced tumors in hairless mice. Morover, its daily use enabled their skins to avoid most of the structural changes which accompany old age."

It may interest some of you to learn that ­ via a process known as photophoresis ­ the blood of skin cancer patients can be temporarily by­passed out of the body and irradiated with UV light before being returned. This process reduces symptoms of skin cancer called mycosis fungoides; it increases overall survival rates and does not produce the side effects commonly associated with chemotherapy. Once again here, we see the ambivalent role of UV radiation in human physiology.

At the University of Texas (Austin) a group led by Adam Heller is producing titanium dioxide coated glass "bubbles” 50­80 microns in diameter, which are then made water repellent so that they float on oceanic oil spills. These "bubbles” then bind to floating hydrocarbons and act as photocatalysts, destroying the oil via a sunlight­driven reaction with the seawater. A cascade of such reactions results in converting the oil to carbon dioxide and water. The effect is supposed to be quite spectacular, as the titanox­coated beadlets apparently "bleach” the oil, taking on their own weight in oil each hour, the reaction being most efficient in turbulent seas, just when other methods of oil spill control fail. It has been suggested that fly ash from burning' coal ­ produced in the USA at the rate of 58 million tons/year, requiring costly land fills ­ could be coated with titanjura dioxide this way, providing a cheaper source of these photocatalytic "bubbles".

The hyperglycaemia­activated aldose reductase was inhibited by alpha­lipoic (thioctic) acid, O­phenanthroline and aldose reductase inhibitors (ARIs) including Zeopolastat (ZPLS), Sorbinil (SBN) and AL­ 1576. This study also examined ARIs for the ability to chelate metal ions. We found that ARIs suppress copper­dependent ascorbate oxidation, lipid peroxidation and hydrogen peroxide production in erythrocytes. lxxvii

These results suggest that UVA might impair the function of LC by suppressing a co­stimulatory molecule, ICAM­1, expression via an oxidation pathway, and this suppression is mitigated by antioxidants. lxxviii

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Genistein, a tyrosine kinase inhibitor, suppressed the effect of UVB irradiation and hydrogen peroxide on cyclooxygenase­2 induction in HaCat keratinocytes. Kirsi Isoherranen, Kari Punnonen, Christer Jansen, Pekka Uotila ULTRAVIOLET IRRADIATION INDUCES CYCLOOXYGENASE­2 EXPRESSION IN HACAT KERATINOCYTES AND IN HUMAN SKIN. Department of Clinical Chemistry and Dermatology, University of Turku, Turku, Finland.

1 Challem JJ Did the loss of endogenous ascorbate propel the evolution of Anthropoidea and Homo sapiens? Email address: 74543.2122@compuserve.com Med Hypotheses, 1997 May, 48:5, 387­92 ii Shindo Y, Witt E, Han D, Packer L Dose­response effects of acute ultraviolet irradiation on antioxidants and molecular markers of oxidation in murine epidermis and dermis. J Invest Dermatol 1994 Apr;102(4):470­475 iii Fuchs J; Huflejt ME; Rothfuss LM; Wilson DS; Carcamo G; Packer L Acute effects of near ultraviolet and visible light on the cutaneous antioxidant defence system. Photochem Photobiol, 50: 6, 1989 Dec, 739­44 iv Shindo Y; Witt E; Han D; Tzeng B; Aziz T; Nguyen L; Packer L Recovery of antioxidants and reduction in lipid hydroperoxides in murine epidermis and dermis after acute ultraviolet radiation exposure. Photodermatol Photoimmunol Photomed, 10: 5, 1994 Oct, 183­91 v Jurkiewicz BA, Buettner GR Ultraviolet light­induced free radical formation in skin: an electron paramagnetic resonance study. Photochem Photobiol 1994 Jan;59(1):1­4 vi Darr D; Combs S; Dunston S; Manning T; Pinnell S Topical vitamin C protects porcine skin from ultraviolet radiation­induced damage. Br J Dermatol, 127: 3, 1992 Sep, 247­53 vii Padh H Cellular functions of ascorbic acid. Biochem Cell Biol, 1990 Oct, 68:10, 1166­73 viii Almaas R; Rootwelt T; Oyasaeter S; Saugstad OD Supplemental ascorbic acid causes hydroxyl radical formation in iron­fortified infant nutrients in vitro. Ascorbic acid enhances hydroxyl radical formation in iron­fortified infant cereals and infant formulas. Eur J Pediatr, 1997 Jun, 156:6, 488­92 ix Blanchard J; Tozer TN; Rowland M Pharmacokinetic perspectives on megadoses of ascorbic acid [see comments] Am J Clin Nutr, 1997 Nov, 66:5, 1165­71 x Diplock AT Safety of antioxidant vitamins and beta­carotene. Am J Clin Nutr, 62: 6 Suppl, 1995 Dec, 1510S­1516S xi Nyyssönen K; Poulsen HE; Hayn M; Agerbo P; Porkkala Sarataho E; Kaikkonen J; Salonen R; Salonen JT Effect of supplementation of smoking men with plain or slow release ascorbic acid on lipoprotein oxidation. Eur J Clin Nutr, 1997 Mar, 51:3, 154­63 xii Buettner GR; Jurkiewicz BA Catalytic metals, ascorbate and free radicals: combinations to avoid. Radiat Res, 145: 5, 1996 May, 532­41 xiii Ponec M; Weerheim A; Kempenaar J; Mulder A; Gooris GS; Bouwstra J; Mommaas AM The formation of competent barrier lipids in reconstructed human epidermis requires the presence of vitamin C. J Invest Dermatol, 1997 Sep, 109:3, 348­55 xiv Postaire E; Jungmann H; Bejot M; Heinrich U; Tronnier H Evidence for antioxidant nutrients­ induced pigmentation in skin: results of a clinical trial. Biochem Mol Biol Int, 1997 Aug, 42:5, 1023­33 xv Kameyama K; Sakai C; Kondoh S; Yonemoto K; Nishiyama S; Tagawa M; Murata T; Ohnuma T; Quigley J; Dorsky A; Bucks D; Blanock; K Inhibitory effect of magnesium L­ascorbyl­2­ phosphate (VC­PMG) on melanogenesis in vitro and in vivo. J Am Acad Dermatol, 34: 1, 1996 Jan, 29­33

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xvi Vera JC; Rivas CI; Fischbarg J; Golde DW Mammalian facilitative hexose transporters mediate the transport of dehydroascorbic acid. Nature 1993 Jul 1;364(6432):79­82 xvii Kodama M; Inoue F; Kodama T; Kodama M Intraperitoneal administration of ascorbic acid delays the turnover of 3H­labelled cortisol in the plasma of an ODS rat, but not in the Wistar rat. Evidence in support of the cardinal role of vitamin C in the progression of glucocorticoid synthesis. In Vivo, 1996 Jan, 10:1, 97­102 xviii Alster TS; West TB Effect of topical vitamin C on postoperative carbon dioxide laser resurfacing erythema. Dermatol Surg, 1998 Mar, 24:3, 331­4 xix Padh H; Subramoniam A; Aleo JJ Glucose inhibits cellular ascorbic acid uptake by fibroblasts in vitro. Cell Biol Int Rep, 9: 6, 1985 Jun, 531­8 xx Sadoogh­Abasian F, Evered DF Absorption of vitamin C from the human buccal cavity. Br J Nutr 1979 Jul;42(1):15­20 xxi Spielholz C; Golde DW; Houghton AN; Nualart F; Vera JC Increased facilitated transport of dehydroascorbic acid without changes in sodium­dependent ascorbate transport in human melanoma cells Cancer Res 1997 Jun 15;57(12):2529­37 (ISSN: 0008­5472) xxii Giangiacomo A; Olesen PR; Ortwerth BJ Ascorbic acid and glucose oxidation by ultraviolet A­ generated oxygen free radicals. Invest Ophthalmol Vis Sci 1996 Jul;37(8):1549­56 xxiii Podda M; Traber MG; Weber C; Yan LJ; Packer L. UV­irradiation depletes antioxidants and causes oxidative damage in a model of human skin. Free Radic Biol Med, 1998 Jan, 24:1, 55­65 xxiv Kitazawa M, Podda M, Thiele J, Traber MG, Iwasaki K, Sakamoto K, Packer L Interactions between vitamin E homologues and ascorbate free radicals in murine skin homogenates irradiated with ultraviolet light. Photochem Photobiol 1997 Feb;65(2):355­365 xxv Fuchs J, Kern H Modulation of UV­light­induced skin inflammation by D­alpha­tocopherol and L­ascorbic acid: a clinical study using solar simulated radiation. Free Radic Biol Med 1998 Dec;25(9):1006­12 xxvi Tebbe B; Wu S; Geilen CC; Eberle J; Kodelja V; Orfanos CE L­ascorbic acid inhibits UVA­ induced lipid peroxidation and secretion of IL­1alpha and IL­6 in cultured human keratinocytes in vitro. J Invest Dermatol 1997 Mar;108(3):302­6 xxvii Steenvoorden DP, van Henegouwen GM The use of endogenous antioxidants to improve photoprotection. J Photochem Photobiol B, 1997 Nov, 41:1­2, 1­10 xxviii Vile GF. Active oxygen species mediate the solar ultraviolet radiation­dependent increase in the tumour suppressor protein p53 in human skin fibroblasts. Photochem Photobiol 1997 Feb;65(2):355­365 xxix Fuchs J Potentials and limitations of the natural antioxidants RRR­alpha­tocopherol, L­ ascorbic acid and beta­carotene in cutaneous photoprotection. Free Radic Biol Med 1998 Nov 1;25(7):848­73 xxx Dreher F, Gabard B, Schwindt DA, Maibach HI Topical melatonin in combination with vitamins E and C protects skin from ultraviolet­induced erythema: a human study in vivo. Br J Dermatol 1998 Aug;139(2):332­9 xxxi Eberlein König B; Placzek M; Przybilla B Protective effect against sunburn of combined systemic ascorbic acid (vitamin C) and d­alpha­tocopherol (vitamin E). J Am Acad Dermatol, 1998 Jan, 38:1, 45­8 xxxii Miyai E; Yanagida M; Akiyama J; Yamamoto I Ascorbic acid 2­O­alpha­glucoside­induced redox modulation in human keratinocyte cell line, SCC: mechanisms of photoprotective effect against ultraviolet light B. Biol Pharm Bull, 1997 Jun, 20:6, 632­6 xxxiii Dreher F, Denig N, Gabard B, Schwindt DA, Maibach HI Effect of Topical Antioxidants on UV­ Induced Erythema Formation when administered after Exposure. Dermatology 1999;198(1):52­55 xxxiv Hayashi A, Ryu A, Suzuki T, Kawada A, Tajima S In vitro degradation of tropoelastin by reactive oxygen species. Arch Dermatol Res 1998 Sep;290(9):497­500 xxxv Geesin JC, Darr D, Kaufman R et al: Ascorbic acid specifically increases type I and type III procollagen messenger RNA levels in human fibroblasts. J Invest Dermatol 1988; 90:420­424. xxxvi Pinnell SR Regulation of collagen biosynthesis by ascorbic acid: a review. Yale J Biol Med, 58: 6, 1985 Nov­Dec, 553­9 xxxvii Pinnel SR; Murad S; Darr D Induction of collagen synthesis by ascorbic acid. A possible mechanism. Arch Dermatol, 123: 12, 1987 Dec, 1684­6

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xxxviii Peterkofsky B. Ascorbate requirement for hydroxylation and secretion of procollagen: relationship to inhibition of collagen synthesis in scurvy. Am J Clin Nutr, 1991 Dec, 54:6 Suppl, 1135S­1140S xxxix Chan D, Lamande SR, Cole WG, Bateman JF Regulation of procollagen synthesis and processing during ascorbate­induced extracellular matrix accumulation in vitro. Biochem J 1990 Jul 1;269(1):175­181 xl Geesin JC, Gordon JS, Berg RA Retinoids affect collagen synthesis through inhibition of ascorbate­induced lipid peroxidation in cultured human dermal fibroblasts. Arch Biochem Biophys 1990 May 1;278(2):350­355 xli Pinnel SR, Murad S, Darr D Induction of collagen synthesis by ascorbic acid. A possible mechanism. Arch Dermatol 1987 Dec;123(12):1684­1686 xlii Takehara K, Grotendorst GR, Trojanowska M, Leroy EC Ascorbate effects on type I procollagen synthesis by human adult skin fibroblasts: different migration positions of type I procollagen chains on SDS polyacrylamide gel after incubation with ascorbate. Coll Relat Res 1987 Feb;6(6):455­466 xliii Appling WD; O'Brien WR; Johnston DA; Duvic M. Synergistic enhancement of type I and III collagen production in cultured fibroblasts by transforming growth factor­beta and ascorbate. FEBS Lett, 1989 Jul 3, 250:2, 541­4 xliv Davidson JM, LuValle PA, Zoia O, Quaglino D Jr, Giro M. Ascorbate differentially regulates elastin and collagen biosynthesis in vascular smooth muscle cells and skin fibroblasts by pretranslational mechanisms. J Biol Chem 1997 Jan 3;272(1):345­352 davidsjm@ctrvax.vanderbilt.edu xlv Nusgens BV; Humbert P; Rougier A; Colige AC; Haftek M; Lambert CA; Richard A; Creidi P; Lapiere CM Topically applied vitamin C enhances the mRNA level of collagens I and III, their processing enzymes and tissue inhibitor of matrix metalloproteinase 1 in the human dermis. J Invest Dermatol 2001 Jun;116(6):853­9 (ISSN: 0022­202X) xlvi Fuchs J, Groth N, Herrling T, Zimmer G Electron paramagnetic resonance studies on nitroxide radical 2,2,5,5­tetramethyl­4­piperidin­1­oxyl (TEMPO) redox reactions in human skin. Free Radic Biol Med 1997;22(6):967­976 xlvii Vaxman F; Olender S; Lambert A; Nisand G; Aprahamian M; Bruch JF; Didier E; Volkmar P; Grenier JF Effect of pantothenic acid and ascorbic acid supplementation on human skin wound healing process. A double­blind, prospective and randomized trial. Eur Surg Res, 1995, 27:3, 158­66 xlviii Pasquali M; Still MJ; Vales T; Rosen RI; Evinger JD; Dembure PP; Longo N; Elsas LJ Hydroxylysine is essential for the formation of the covalent pyridinium cross­links pyridinoline (Pyr) and deoxypyridinoline (Dpyr), among mature collagen molecules. Proc Assoc Am Physicians, 1997 Jan, 109:1, 33­41 xlix Edward M Ascorbate induced changes in glycosaminoglycan synthesis and distribution of normal and SV40­transformed fibroblasts. J Cell Sci 1986 Sep;85:217­229 l Murad S, Grove D, Lindberg KA, Reynolds G, Sivarajah A, Pinnell SR Regulation of collagen synthesis by ascorbic acid. Proc Natl Acad Sci U S A 1981 May;78(5):2879­2882 li Freiberger H, Grove D, Sivarajah A, Pinnell SR Procollagen I synthesis in human skin fibroblasts: effect on culture conditions on biosynthesis. J Invest Dermatol 1980 Nov;75(5):425­ 430 lii Cohen IK, Keiser HR Disruption of healed scars in scurvy ­­ the result of a disequilibrium in collagen metabolism. Plast Reconstr Surg 1976 Feb;57(2):213­215 liii Chung JH; Youn SH; Kwon OS; Cho KH; Youn JI; Eun HC Regulations of collagen synthesis by ascorbic acid, transforming growth factor­beta and interferon­gamma in human dermal fibroblasts cultured in three­dimensional collagen gel are photo aging and aging­independent. J Dermatol Sci, 1997 Sep, 15:3, 188­200 liv Hata R; Sunada H; Arai K; Sato T; Ninomiya Y; Nagai Y; Senoo H Regulation of collagen metabolism and cell growth by epidermal growth factor and ascorbate in cultured human skin fibroblasts [published erratum appears in Eur J Biochem 1988 Aug 15;175(3):679] Eur J Biochem, 173: 2, 1988 Apr 15, 261­7

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lv Myara I; Wolfrom C; Charpentier C; Gautier M; Lemonnier A Relationship between cell density and prolidase activity in human skin fibroblasts: effects of ascorbate and fructose. Biochimie, 66: 6, 1984 Jun, 445­9 lvi Birt DF Update on the effects of vitamins A, C, and E and selenium on carcinogenesis. Proc Soc Exp Biol Med 1986 Dec;183(3):311­320 lvii Jurkiewicz BA, Buettner GR EPR detection of free radicals in UV­irradiated skin: mouse versus human. Photochem Photobiol 1996 Dec;64(6):918­922 lviii A.C. Tedesco, L. Martínez2 and S. González2 Photochemistry and photobiology of actinic erythema: defensive and reparative cutaneous mechanisms. Braz J Med Biol Res, May 1997, Volume 30(5) 561­575 lix Mizushima Y, Hoshi K, Yamagawa A & Takana K (1991). Topical application of superoxide dismutase cream. Drugs under Experimental and Clinical Research, 17: 127­131. lx Hamanaka H, Miyachi Y & Imamura S (1990). Photoprotective effect of topically applied SOD on sunburn reaction in comparison with sunscreen. Journal of Dermatology, 17: 595­598. lxi Darr D, Combs S, Dunston S, Manning T & Pinnell S (1992). Topical vitamin C protects swine skin from ultra­violet radiation­induced damage. British Journal of Dermatology, 127: 247­253. lxii González S, Joshi PC & Pathak MA (1994). Polypodium leucotomos extract as an antioxidant agent in the therapy of skin disorders. Journal of Investigative Dermatology, 102: 651s lxiii Kobayashi S, Takehana M, Itoh S, Ogata E Protective effect of magnesium­L­ascorbyl­2 phosphate against skin damage induced by UVB irradiation. Photochem Photobiol 1996 Jul;64(1):224­228 lxiv Kurata S; Senoo H; Hata R Transcriptional activation of type I collagen genes by ascorbic acid 2­phosphate in human skin fibroblasts and its failure in cells from a patient with alpha 2(I)­chain­ defective Ehlers­Danlos syndrome. Exp Cell Res, 1993 May, 206:1, 63­71 lxv Chan AC Partners in defense, vitamin E and vitamin C. Can J Physiol Pharmacol, 1993 Sep, 71:9, 725­31 lxvi Farriol M; Mourelle M; Schwartz S Effect of vitamin C and vitamin E analog on aged fibroblasts. Rev Esp Fisiol, 50: 4, 1994 Dec, 253­7

lxvii Dreher F, Gabard B, Schwindt DA, Maibach HI Topical melatonin in combination with vitamins E and C protects skin from ultraviolet­induced erythema: a human study in vivo. Br J Dermatol 1998 Aug;139(2):332­9 lxviii Slaga TJ Inhibition of skin tumor initiation, promotion, and progression by antioxidants and related compounds. Crit Rev Food Sci Nutr, 35: 1­2, 1995, 51­7 lxix Skaper SD; Fabris M; Ferrari V; Dalle Carbonare M; Leon A Quercetin protects cutaneous tissue­associated cell types including sensory neurons from oxidative stress induced by glutathione depletion: cooperative effects of ascorbic acid. Free Radic Biol Med, 1997, 22:4, 669­ 78 lxx Gershoff SN Vitamin C (ascorbic acid): new roles, new requirements? Nutr Rev 1993 Nov;51(11):313­26 lxxi Geesin JC; Gordon JS; Berg RA Regulation of collagen synthesis in human dermal fibroblasts by the sodium and magnesium salts of ascorbyl­2­phosphate. Skin Pharmacol, 6: 1, 1993, 65­71

lxxii Hata R; Senoo H L­ascorbic acid 2­phosphate stimulates collagen accumulation, cell proliferation, and formation of a three­dimensional tissuelike substance by skin fibroblasts. J Cell Physiol, 138: 1, 1989 Jan, 8­16 lxxiii 1. Cathcart RF: Vitamin C in the treatment of acquired immune deficiency syndrome (AIDS). Med Hypotheses 1984; 14:423­433. lxxiv Silvetti AN: An effective method of treating long­enduring wounds and ulcers by topical applications of solutions of nutrients. J Dermatol Surg Oncol 1981; 7:501­508. lxxv Bowles WH Protection against minocycline pigment formation by ascorbic acid (vitamin C). J Esthet Dent 1998;10(4):182­6 lxxvi Kumano Y; Sakamoto T; Egawa M; Tanaka M; Yamamoto I Enhancing effect of 2­O­alpha­D­ glucopyranosyl­L­ascorbic acid, a stable ascorbic acid derivative, on collagen synthesis. Biol Pharm Bull, 1998 Jul, 21:7, 662­6

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lxxvii Ou P; Nourooz Zadeh J; Tritschler HJ; Wolff S Activation of aldose reductase in rat lens and metal­ion chelation by aldose reductase inhibitors and lipoic acid. Free Radic Res, 1996 Oct, 25:4, 337­46 lxxviii Masato Hatao, Ichiro Iwai, Tomohiro Kuwahara, Eiichiro Yagi, Kenji Yamaguchi, Masako Naganuma and Yoshimaru Kumano UVA­INDUCED IMMUNE SUPPRESSION THROUGH AN OXIDATIVE PATHWAY. J invest Dermatol 1999 Jan; 112(1):19­24