Ascorbate is consumed stoichiometrically in the uncoupled reactions catalyzed by prolyl 4-hydroxylase and lysyl hydroxylase.
The hydroxylation of proline and lysine residues by the collagen hydroxylases is coupled with a stoichiometric decarboxylation of 2-oxoglutarate. Ascorbate is virtually a specific requirement for these enzymes, but previous studies have demonstrated that it is not consumed during most catalytic cycles. Prolyl 4-hydroxylase and lysyl hydroxylase are known also to catalyze an uncoupled decarboxylation of 2-oxoglutarate in the absence of the peptide substrate. It is shown here that, unlike the complete hydroxylation reaction, the uncoupled decarboxylation reaction involves stoichiometric ascorbate consumption. This stoichiometric ascorbate consumption was also seen when the rate of the uncoupled prolyl 4-hydroxylase reaction was enhanced by the addition of poly(L-proline). Since collagen hydroxylases may catalyze occasional uncoupled reaction cycles even in the presence of the peptide substrates, the main function of ascorbate in these reactions in vivo is suggested to be that of reactivating the enzymes after such uncoupled cycles.
J Biol Chem. 1984 May 10;259(9):5403-5
Use of topical ascorbic acid and its effects on photodamaged skin topography.
OBJECTIVE: To determine the efficacy of topical ascorbic acid application in treating mild to moderate photodamage of facial skin using an objective, computer-assisted image analysis of skin surface topography and subjective clinical, photographic, and patient self-appraisal questionnaires. DESIGN: A 3-month, randomized, double-blind, vehicle-controlled study. SETTING: Facial plastic surgery private practice. PATIENTS: Nineteen evaluable volunteer sample patients aged between 36 and 72 years with Fitzpatrick skin types I, II, and III who were in good physical and mental health with mild to moderately photodamaged facial skin were considered for analysis. INTERVENTION: Coded, unmarked medications were randomly assigned to the left and right sides of each subject’s face, one containing the active agent, topical ascorbic acid (Cellex-C high-potency serum; Cellex-C International, Toronto, Ontario), the other, the vehicle serum (Cellex-C International). Three drops (0.5 mL) of each formulation were applied daily to the randomly assigned hemifaces over the 3-month study period. Treatment assignments were not disclosed to subjects, clinicians, or personnel involved in analyzing skin replicas. MAIN OUTCOME MEASURES: Specific clinical parameters were evaluated and graded on a 0- to 9-point scale (0, none; 1-3, mild; 4-6, moderate; and 7-9, severe). Reference photographs were used to standardize grading criteria. Overall investigator scores were compared with baseline and graded as excellent (much improved), good (improved), fair (slightly improved), no change, or worse. Patient self-appraisal questionnaires rated the degree of improvement (much improved, improved, slightly improved, no change, or worse) and reported adverse effects (burning, stinging, redness, peeling, dryness, discoloration, itching, and rash). Standard photographs were taken at baseline, including anteroposterior and left and right oblique views to facilitate subsequent clinical evaluations, and at the end of therapy for comparison. Optical profilometry analysis was performed on the skin surface replicas of the lateral canthal (crow’s feet) region, comparing baseline to end-of-study specimens. Using this computer-based system, the resulting image was digitally analyzed, and numeric values were assigned to reflect surface features. The parameters obtained included Rz, Ra, and shadows. These values provided objective data that document pretreatment and posttreatment texture changes proportional to the degree of wrinkling, roughness, and other surface irregularities. RESULTS: Optical profilometry image analysis demonstrated a statistically significant 73.7% improvement in the Ra and shadows north-south facial axis values with active treatment greater than vehicle control, as well as a trend for improvement in the Rz north-south facial axis parameter, showing a 68.4% greater improvement of active treatment vs vehicle control. Clinical assessment demonstrated significant improvement with active treatment greater than control for fine wrinkling, tactile roughness, coarse rhytids, skin laxity/tone, sallowness/yellowing, and overall features. Patient questionnaire results demonstrated statistically significant improvement overall, active treatment 84.2% greater than control. Photographic assessment demonstrated significant improvement, active treatment 57.9% greater than control. CONCLUSIONS: A 3-month daily regimen of topical ascorbic acid provided objective and subjective improvement in photodamaged facial skin. Skin replica optical profilometry is an objective method for quantification of the skin surface texture changes.
Arch Otolaryngol Head Neck Surg. 1999 Oct;125(10):1091-8
Topical ascorbic acid on photoaged skin. Clinical, topographical and ultrastructural evaluation: double-blind study vs. placebo.
Vitamin C is known for its antioxidant potential and activity in the collagen biosynthetic pathway. Photoprotective properties of topically applied vitamin C have also been demonstrated, placing this molecule as a potential candidate for use in the prevention and treatment of skin ageing. A topically applied cream containing 5% vitamin C and its excipient were tested on healthy female volunteers presenting with photoaged skin on their low-neck and arms in view to evaluate efficacy and safety of such treatment. A double-blind, randomized trial was performed over a 6-month period, comparing the action of the vitamin C cream vs. excipient on photoaged skin. Clinical assessments included evaluation at the beginning and after 3 and 6 months of daily treatment. They were performed by the investigator and compared with the volunteer self assessment. Skin relief parameters were determined on silicone rubber replicas performed at the same time-points. Cutaneous biopsies were obtained at the end of the trial and investigated using immunohistochemistry and electron microscopy. Clinical examination by a dermatologist as well as self-assessment by the volunteers disclosed a significant improvement, in terms of the ‘global score’, on the vitamin C-treated side compared with the control. A highly significant increase in the density of skin microrelief and a decrease of the deep furrows were demonstrated. Ultrastructural evidence of the elastic tissue repair was also obtained and well corroborated the favorable results of the clinical and skin surface examinations. Topical application of 5% vitamin C cream was an effective and well-tolerated treatment. It led to a clinically apparent improvement of the photodamaged skin and induced modifications of skin relief and ultrastructure, suggesting a positive influence of topical vitamin C on parameters characteristic for sun-induced skin ageing.
Exp Dermatol. 2003 Jun;12(3):237-44
Double-blind, half-face study comparing topical vitamin C and vehicle for rejuvenation of photodamage.
BACKGROUND: Aging of the population, in particular the “baby boomers,” has resulted in increased interest in methods of reversal of photodamage. Non-invasive treatments are in high demand, and our knowledge of mechanisms of photodamage to skin, protection of the skin, and repair of photodamage are becoming more sophisticated and complex. OBJECTIVE: The objective of this study is to determine if the topical use of a vitamin C preparation can stimulate the skin to repair photodamage and result in clinically visible differences, as well as microscopically visible improvement. METHODS: Ten patients applied in a double-blind manner a newly formulated vitamin C complex having 10% ascorbic acid (water soluble) and 7% tetrahexyldecyl ascorbate (lipid soluble) in an anhydrous polysilicone gel base to one-half of the face and the inactive polysilicone gel base to the opposite side. Clincial evaluation of wrinkling, pigmentation, inflammation, and hydration was performed prior to the study and at weeks 4, 8, and 12. Two mm punch biopsies of the lateral cheeks were performed at 12 weeks in four patients and stained with hematoxylin and eosin, as well as in situ hybridization studies using an anti-sense probe for mRNA for type I collagen. A questionnaire was also completed by each patient. RESULTS: A statistically significant improvement of the vitamin C-treated side was seen in the decreased photoaging scores of the cheeks (P = 0.006) and the peri-oral area (P = 0.01). The peri-orbital area improved bilaterally, probably indicating improved hydration. The overall facial improvement of the vitamin C side was statistically significant (P = 0.01). Biopsies showed increased Grenz zone collagen, as well as increased staining for mRNA for type I collagen. No patients were found to have any evidence of inflammation. Hydration was improved bilaterally. Four patients felt that the vitamin C-treated side improved unilaterally. No patient felt the placebo side showed unilateral improvement. CONCLUSION: This formulation of vitamin C results in clinically visible and statistically significant improvement in wrinkling when used topically for 12 weeks. This clinical improvement correlates with biopsy evidence of new collagen formation.
Dermatol Surg. 2002 Mar;28(3):231-6
Topical vitamin C: a useful agent for treating photoaging and other dermatologic conditions.
BACKGROUND: Cosmeceuticals containing antioxidants are among the most popular antiaging remedies. Topically applied antioxidants exert their benefits by offering protection from damaging free radicals produced when skin is exposed to ultraviolet light or allowed to age naturally. Vitamin C is a naturally occurring potent water-soluble antioxidant. Accordingly, it has been incorporated into a variety of cosmeceuticals designed to protect and rejuvenate photoaged skin. OBJECTIVE: This article reviews the scientific data and clinical studies supporting the use of topically applied vitamin C for treating photoaged skin. Other innovative uses for vitamin C cosmeceuticals are also discussed. CONCLUSION: A significant body of scientific research supports the use of cosmeceuticals containing vitamin C. Cutaneous benefits include promoting collagen synthesis, photoprotection from ultraviolet A and B, lightening hyperpigmentation, and improvement of a variety of inflammatory dermatoses. Because of the diverse biologic effects of this compound, topical vitamin C has become a useful part of the dermatologist’s armamentarium.
Dermatol Surg. 2005 Jul;31(7 Pt 2):814-7; discussion 818
Aging- and photoaging-dependent changes of enzymic and nonenzymic antioxidants in the epidermis and dermis of human skin in vivo.
This is a comprehensive study of the changes in major antioxidant enzymes and antioxidant molecules during intrinsic aging and photoaging processes in the epidermis and dermis of human skin in vivo. We show that the activities of superoxide dismutase and glutathione peroxidase are not changed during these processes in human skin in vivo. Interestingly, the activity of catalase was significantly increased in the epidermis of photoaged (163%) and naturally aged (118%) skin (n = 9), but it was significantly lower in the dermis of photoaged (67% of the young skin level) and naturally aged (55%) skin compared with young (n = 7) skin. The activity of glutathione reductase was significantly higher (121%) in naturally aged epidermis. The concentration of alpha-tocopherol was significantly lower in the epidermis of photoaged (56% of young skin level) and aged (61%) skin, but this was not found to be the case in the dermis. Ascorbic acid levels were lower in both epidermis (69% and 61%) and dermis (63% and 70%) of photoaged and naturally aged skin, respectively. Gluta thione concentrations were also lower. Uric acid did not show any significant changes. Our results suggest that the components of the antioxidant defense system in human skin are probably regulated in a complex manner during the intrinsic aging and photoaging processes.
J Invest Dermatol. 2001 Nov;117(5):1212-7
Dermal connective tissue metabolism in photoageing.
The term photoageing describes the clinical and histological cutaneous changes that are the consequence of repeated chronic sun exposures and are qualitatively different from those observed in chronological ageing. The connective tissue of the skin is composed mainly of collagen, glycosaminoglycans and elastin and, thus, alterations of these components in photoageing are briefly reviewed in the present article. Collagen changes in photoageing are partly explained by cross-links as well as the unbalanced regulation of collagen production and breakdown. Some visible skin changes can be induced by the consequence of dermal glycosaminoglycans, because the total amount, as well as the composition of the main disaccharide units, is significantly altered in the exposed sites of both aged people and photoaged mice. As for the mechanism of solar elastosis, increased elastin mRNA levels resulting from transcriptional up-regulation of the gene have been reported. Taken together, all components of the dermal connective tissue are affected by chronic actinic damage; however, further in vitro investigation is required to unmask the exact events in photoageing. With regard to this, our novel three-dimensional culture system should be of great help because it mimics the in vivo condition by self producing the extracellular matrices.
Australas J Dermatol. 1998 Feb;39(1):19-23
Skin aging 2003: recent advances and current concepts.
In developed countries, interest in cutaneous aging is in large part the result of a progressive, dramatic rise over the past century in the absolute number and proportion of the population who are elderly. The psychosocial, as well as physiologic, effects of skin aging on older persons have created a demand for a better understanding of the aging process and particularly for effective interventions. Skin aging is a complex process determined by the genetic endowment of the individual and by environmental factors. The appearance of old skin and the clinical consequences of skin aging have been well-known for centuries, but it is only in the past 50 years that mechanisms and mediators have been pursued systematically. Still, within a relatively short time, there has been tremendous progress, a progress greatly enhanced by basic gerontologic research using immunologic, biochemical, and in particular, molecular biologic approaches.
Cutis. 2003 Sep;72(3 Suppl):5-10; discussion 10
Regulation of collagen synthesis in human dermal fibroblasts by the sodium and magnesium salts of ascorbyl-2-phosphate.
Ascorbic acid has been shown to stimulate collagen synthesis in dermal fibroblasts by increasing the rate of transcription of collagen genes. Experiments involving the use of ascorbic acid require daily supplementation due to the instability of the molecule in aqueous solutions. In order to provide a more stable alternative to ascorbic acid, two salts of ascorbyl-2-phosphate, having a greater chemical stability than ascorbic acid, were tested for their ability to stimulate collagen synthesis in monolayer fibroblast cultures. The concentration and time dependence of their activities were compared with ascorbic acid. The magnesium salt of ascorbyl-2-phosphate 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. Solutions of either ascorbic acid or the ascorbyl-2-phosphate analogs (at 10 mM) in phosphate-buffered saline (PBS) were relatively stable as shown by their decay rates and their ability to stimulate collagen synthesis even after nine days in solution prior to testing their effects on cultured cells. Ascorbic acid was unstable at neutral pH compared to solutions of either sodium or magnesium ascorbyl-2-phosphate. These data support the use of magnesium ascorbyl-2-phosphate in experiments where stability of ascorbic acid is a concern, e.g. in long-term cultures or in in vivo studies.
Skin Pharmacol. 1993;6(1):65-71
L-ascorbic acid 2-phosphate stimulates collagen accumulation, cell proliferation, and formation of a three-dimensional tissuelike substance by skin fibroblasts.
Proliferation of human skin fibroblasts was stimulated significantly by the presence of L-ascorbic acid 2-phosphate (Asc 2-P). The presence of Asc 2-P (0.1-1.0 mM) in the culture medium for 3 weeks enhanced the relative rate of collagen synthesis to total protein synthesis 2-fold as well as cell growth 4-fold. Coexistence of L-azetidine 2-carboxylic acid (AzC), an inhibitor of collagen synthesis, attenuated both effects of Asc 2-P in a dose-
dependent manner. Supplemen-tation of the medium with Asc 2-P 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. Electron microscopic observations showed multilayered, rough endoplasmic reticulum-rich cells surrounded by dense ECM. These results indicate that Asc 2-P is useful in culture systems as a long-acting vitamin C derivative and also that it promotes reorganization of a three-dimensional tissuelike substance from skin fibroblasts in culture by stimulating collagen accumulation in the fibroblasts.
J Cell Physiol. 1989 Jan;138(1):8-16
Epidermal growth factor inhibits transcription of type I collagen genes and production of type I collagen in cultured human skin fibroblasts in the presence and absence of L-ascorbic acid 2-phosphate, a long-acting vitamin C derivative.
Recombinant human epidermal growth factor (EGF, 2-10 ng/ml) stimulated growth and production of non-collagenous proteins, but inhibited production of collagen by 60% in cultured human skin fibroblasts. Type analysis of the collagen produced indicated that inhibition of the collagen production observed was mainly a reflection of a reduction in type I collagen. The accumulation of pro alpha 1(I) and pro alpha 2(I) mRNAs and the transcriptional activity of these genes were determined in human skin fibroblasts in order to investigate site(s) of regulation of type I collagen production by human EGF in the absence and presence of L-ascorbic acid 2-phosphate (Asc 2-P), a long-acting vitamin C derivative. Human EGF (10 ng/ml) used alone reduced the steady state levels of mRNAs for pro alpha 1(I) and pro alpha 2(I) chains and transcriptional activity of these genes in vitro by 45%. Asc 2-P (0.2 mM) alone, on the other hand, raised production of type I collagen and the steady state levels of mRNAs for pro alpha 1(I) and pro alpha 2(I) collagen chains as well as stimulated transcriptional activity of these genes. Human EGF attenuated these stimulative effects of Asc 2-P. These results indicate that human EGF regulates type I collagen synthesis at the transcriptional level in cultured fibroblasts in the presence and absence of Asc 2-P. The possibility that human EGF plays a role as a regulator of type I collagen genes in vivo was discussed.
J Biol Chem. 1991 May 25;266(15):9997-10003
Ascorbic acid preferentially enhances type I and III collagen gene transcription in human skin fibroblasts.
Ascorbic acid is a potent stimulator for type I and III collagen expression in human skin fibroblasts; stimulation of type I and III collagen synthesis and their mRNA levels by ascorbic acid has been reported previously. Nuclear run-on experiments demonstrated that ascorbic acid enhanced the transcription of type I and III collagen genes 4- and 3.4-fold respectively, whereas transcription of type IV collagen was slightly stimulated (1.7-fold). The results suggest that ascorbic acid preferentially enhanced type I and III collagen transcription.
J Dermatol Sci. 1996 Mar;11(3):250-3
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.
Ascorbic acid (vitamin C) is a cofactor required for the function of several hydroxylases and monooxygenases. It is not synthesized in humans and some other animal species and has to be provided by diet or pharmacologic means. 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 triple-helical collagen molecules. In fibroblast cultures, vitamin C also stimulates collagen production by increasing the steady-state level of mRNA of collagen types I and III through enhanced transcription and prolonged half-life of the transcripts. The aim of the experimental work has been to evaluate the effect on dermal cells of a preparation of vitamin C topically applied on one side vs placebo on the other side of the dorsal face of the upper forearm of postmenopausal women. Biopsies were collected on both sides and the level of mRNA measured by non competitive reverse transcription-polymerase chain reaction made quantitative by the simultaneous transcription and amplification of synthetic RNA used as internal standards. 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 amino-procollagen 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.
J Invest Dermatol. 2001 Jun;116(6):853-9
Topically applied vitamin C increases the density of dermal papillae in aged human skin.
BACKGROUND: The influence of ageing on the density of the functional entities of the papillae containing nutritive capillaries, here in terms as the papillary index, and the effect of topically applied vitamin C were investigated by
confocal laser scanning microscopy (CLSM) in vivo. METHODS: The age dependency of the papillary index was determined by CLSM on 3 different age groups. Additionally, we determined the effect of a topical cream containing 3% vitamin C against the vehicle alone using daily applications for four months on the volar forearm of 33 women. RESULTS: There were significant decreases in the papillary index showing a clear dependency on age. Topical vitamin C resulted in a significant increase of the density of dermal papillae from 4 weeks onward compared to its vehicle. Reproducibility was determined in repeated studies. CONCLUSIONS: Vitamin C has the potential to enhance the density of dermal papillae, perhaps through the mechanism of angiogenesis. Topical vitamin C may have therapeutical effects for partial corrections of the regressive structural changes associated with the aging process.
BMC Dermatol. 2004 Sep 29;4(1):13
Enzymic and non-enzymic antioxidants in epidermis and dermis of human skin.
We measured enzymic and non-enzymic antioxidants in human epidermis and dermis from six healthy volunteers undergoing surgical procedures. Epidermis was separated from dermis by curettage and antioxidants were measured by high-performance liquid chromatography (HPLC) or standard spectrophotometric methods. The concentration of every antioxidant (referenced to skin wet weight) was higher in the epidermis than in the dermis. Among the enzymic antioxidants, the activities of superoxide dismutase, glutathione peroxidase, and glutathione reductase were higher in the epidermis compared to the dermis by 126, 61, and 215%, respectively. Catalase activity in particular was much higher (720%) in the epidermis. Glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase, which provide reduced nicotinamide adenine dinucleotide phosphate (NADPH), also showed higher activity in the epidermis than the dermis by 111% and 313%, respectively. Among the lipophilic antioxidants, the concentration of alpha-tocopherol was higher in the epidermis than the dermis by 90%. The concentration of ubiquinol 10 was especially higher in the epidermis, by 900%. Among the hydrophilic antioxidants, concentrations of ascorbic acid and uric acid were also higher in the epidermis than in the dermis by 425 and 488%, respectively. Reduced glutathione and total glutathione were higher in the epidermis than in the dermis by 513 and 471%. Thus the antioxidant capacity of the human epidermis is far greater than that of dermis. As the epidermis composes the outermost 10% of the skin and acts as the initial barrier to oxidant assault, it is perhaps not surprising that it has higher levels of antioxidants.
J Invest Dermatol. 1994 Jan;102(1):122-4