\subsection{Oxidation of Bip and H$_{2}$Bip upon solar exposure}

Aqueous solutions of Bip and H$_{2}$Bip were exposed to solar radiation at noon in clear sky days in La Plata city and then they were analyzed using our HPLC device (see Subsection \ref{HPLC}). For both compounds a significant decrease in their concentrations was observed within a few minutes of exposure, which indicates that solar radiation is able to cause the degradation of the pterin derivatives. By means of HPLC analysis, carried out using commercial standards and considering published chromatographic data \citep{Vignoni2009,Vignoni2010}, dimeric products, Bip, Fop and Cap were detected in irradiated solutions of H$_{2}$Bip and Fop and Cap were detected in irradiated solutions of Bip.

To further investigate the photochemical reactions, the concentration profiles of reactants and products were determined upon solar exposure of the corresponding solutions. Figure \ref{consuptionprofile}a clearly shows that Bip was converted into Fop and, subsequently, this compound underwent oxidation to Cap. The sum of the concentrations of Bip, Fop and Cap remained constant during the experiments, which indicates that Fop and Cap are the main photoproducts, with no other substances being formed in significant amounts. Figure \ref{consuptionprofile}b, in turn, shows that H$_{2}$Bip was converted into Bip and other photoproducts with the same retention times and spectral features as those reported for the dimers of H$_{2}$Bip \citep{Vignoni2010,Vignoni2012}. 

These results are in good agreement with the pathways depicted in Figure \ref{scheme}. Therefore in this Section we have presented evidence that indicates that solar exposure of Bip and H$_{2}$Bip solutions, under outdoor conditions, leads to the exactly the same photochemical transformations as those reported for photolyses carried out under laboratory conditions using quasi-monochromatic light sources \citep{Vignoni2009, Vignoni2010}. In addition, it is worth mentioning that, as expected taking into account the comparison of sources described in the previous section, the photochemical reactions took place very fast, i.e. the sun is able to photoinduce significant chemical changes of pterins in a few seconds. 

In diseased skin cells micromolar concentrations of pterins have been determined, which is much higher than concentrations found in healthy cells; e.g., in human keratinocytes and cell cultures from suction blister roofs the concentration of total biopterin was determined to be in the range 4–93$\mu$M (41–950 pmol/mg of protein), depending on the cell type \cite{Schallreuter2001}; concentrations higher than 35$\mu$M (360 pmol/mg of protein) were determined for Cap \cite{Rokos2002}. In addition, fluorescence studies have determined that the concentration of pterins is not homogeneous inside the cells. Therefore the local concentrations could be even much higher than those noted above. Our experiments were performed with solutions of pterins at concentrations of the same order of magnitude as those reported for in vivo determinations. Consequently, our study strongly suggests that photodegradation of pterins can take place in cells upon solar irradiation.

In tissues affected by vitiligo non-enzymatic oxidation of H$_{4}$Bip by O$_{2}$ or other oxidants leads to the formation of H$_{2}$Bip \cite{Nixon_1980}. It is accepted that Bip is formed in vivo from H$_{2}$Bip by a non-enzymatic oxidation. However, the specific chemical process that leads to this conversión in human skin under pathological conditions is still unknown. It had been proposed that the formation of Bip results from the oxidation of H$_{2}$Bip by H$_{2}$O$_{2}$ \cite{Moore_2002}, but it was later demonstrated that this reaction yields dihydroxanthopterin (H$_{2}$Xap) as a product \cite{Laura_D_ntola_2008}. The same main product was obtained for the “autooxidation” of H$_{2}$Bip (reaction with O$_{2}$ in an air-equilibrated solution) \cite{D_ntola_2008}. Therefore the results presented so far suggest that the photochemical oxidation of H$_{2}$Bip upon solar radiation can be the pathway responsible for the formation of Bip in the affected tissues. Likewise, Cap found in the skin \cite{Rokos2002} very likely is produced by the oxidation of H$_{2}$Bip by solar radiation. It is worth mentioning that artificial sources with a significant UV component might also contribute to the oxidation of pterins in vivo. Moreover, if photochemistry of pterins occurs, reactive oxygen species, such as H$_{2}$O$_{2}$ and $^{1}$O$_{2}$ should be formed and photosensitized reactions and oxidative stress could be exacerbated in vivo, which, in turn, means that photochemistry of pterins could contribute to inactivation of enzymes and unspecific damages in other biomacromolecules.
