Pharmaceutical Research, Vol. 19, No. 7, July 2002 (© 2002)
Trommer, H.1; Böttcher, R.2; Pöppl, A.2; Hoentsch, J.2; Wartewig, S.3; Neubert, R. H. H.1,4
1 Martin-Luther-University
Halle-Wittenberg, School of Pharmacy, Institute of Pharmaceutics and
Biopharmaceutics, Wolfgang-Langenbeck-Straße 4, D-06120 Halle (Saale),
Germany.
2 University of Leipzig, Department of
Physics and Earth Sciences, Institute of Experimental Physics II, Linnéstraße 5,
D-04103 Leipzig, Germany.
3 Institute of Applied
Dermatopharmacy, Wolfgang-Langenbeck-Straße 4, D-06120 Halle (Saale),
Germany.
4
Purpose. The effects of ascorbic acid
on Stratum corneum lipid models following ultraviolet irradiation were studied
adding iron ions as transition metal catalysts.
Methods. Lipid peroxidation was quantified by the thiobarbituric
acid assay. The qualitative changes were studied on a molecular level by mass
spectrometry. To elucidate the nature of free radical involvement we carried out
electron paramagnetic resonance studies. The influence of ascorbic acid on the
concentration of hydroxyl radicals was examined using the spin trapping
technique. Moreover, we checked the vitamin's ability to react with stable
radicals.
Results. Ascorbic acid was found to have
prooxidative effects in all lipid systems in a concentration dependent manner.
The degradation products of ascorbic acid after its prooxidative action were
detected. The concentration of the hydroxyl radicals in the Fenton assay was
decreased by ascorbic acid. The quantification assay of
Conclusions. Considering human skin and its constant exposure to
UV light and oxygen, an increased pool of iron ions in irradiated skin and the
depletion of co-antioxidants, the administration of ascorbic acid in cosmetical
formulations or in sunscreens could unfold adverse effects among the Stratum
corneum lipids.
KEY WORDS: ascorbic acid; thiobarbituric acid
assay; mass spectrometry; EPR; oxidative stress.
Introduction
Ascorbic acid is a hydrophilic
hexuronic acid lacton. Because humans have lost the ability to synthesize
ascorbic acid, it must be provided as an essential micronutrient from food.
The most well-known biochemical function of ascorbic acid is the
protection of the prolyl and lysyl hydroxylases. This seems to be essential for
the synthesis of stable collagen molecules. Furthermore, ascorbic acid has been
shown to be a neuromodulator, an antiviral substance and immunostimulant, and a
radical scavenger.
Most of the chemical and biochemical properties
of ascorbic acid are related to its oxoenediol structure and can be explained by
its participation in biochemical redox processes. Ascorbic acid is a diabasic
acid and in aqueous solution a strong reducing compound.
Despite the
high number of publications dealing with the vitamin, and the relatively simple
chemical structure of the molecule, other properties of the molecule are not yet
fully understood. Conflicting data has lead to controversy in the literature,
and at present even the daily human intake is still under debate. Further
sources of controversy are the interactions of vitamin C with reactive oxygen
species (ROS), as both antioxidant and prooxidant properties have been reported.
Recently, genotoxic effects were suggested to be a result of the ability of
ascorbic acid to decompose lipid hydroperoxides to DNA damaging secondary
products raising the question why add ascorbic acid to drug formulation for
topical application.
In the skin, ascorbic acid is part of the
antioxidative network of the Stratum corneum and therefore, along with
lipophilic antioxidants, proteins, and other biomolecules, a target to the
oxidative stressors of the environment. A decrease in the concentration of
ascorbic acid was measured in murine Stratum corneum following ozone exposure.
In this paper, therefore, the mechanism of the redox behavior of
ascorbic acid is studied using Stratum corneum lipid model systems to test its
actions on exposure to UV light. As the biggest organ of the human body, the
skin is constantly exposed to both ultraviolet radiation and oxygen. The
degradation of ascorbic acid caused by UV-light was studied at a molecular level
using electrospray ionization mass spectrometry (ESI-MS). The advantages of the
soft electrospray ionization (ESI) avoiding early fragmentation and the ion trap
possibilities of full scan mass spectrometry (MS), MA/MS, and multiple state MS
experiments allowed the identification of the ascorbic acid decomposition
products as well as the lipid peroxidation products.
Electron
paramagnetic resonance (EPR) spectroscopy is being used more frequently in
pharmaceutical research because of the unique information which can be obtained
by EPR spectrums of both in vivo and in vitro experiments. We
used the spin trapping method by