Senile graying of human hair has been the subject of intense research since ancient times. Reactive oxygen species have been implicated in hair follicle melanocyte apoptosis and DNA damage. Here we show for the first time by FT-Raman spectroscopy in vivo that human gray/white scalp hair shafts accumulate hydrogen peroxide (H(2)O(2)) in millimolar concentrations. Moreover, we demonstrate almost absent catalase and methionine sulfoxide reductase A and B protein expression via immunofluorescence and Western blot in association with a functional loss of methionine sulfoxide (Met-S=O) repair in the entire gray hair follicle. Accordingly, Met-S=O formation of Met residues, including Met 374 in the active site of tyrosinase, the key enzyme in melanogenesis, limits enzyme functionality, as evidenced by FT-Raman spectroscopy, computer simulation, and enzyme kinetics, which leads to gradual loss of hair color. Notably, under in vitro conditions, Met oxidation can be prevented by L-methionine. In summary, our data feed the long-voiced, but insufficiently proven, concept of H(2)O(2)-induced oxidative damage in the entire human hair follicle, inclusive of the hair shaft, as a key element in senile hair graying, which does not exclusively affect follicle melanocytes. This new insight could open new strategies for intervention and reversal of the hair graying process.

SummaryPatients with vitiligo accumulate up to 10−3 mol/L concentrations of H2O2 in their epidermis, which in turn affects many metabolic pathways in this compartment, including the synthesis and recycling of the cofactor (6R)‐l‐erythro‐5,6,7,8‐tetrahydrobiopterin (6BH4). De novo synthesis of 6BH4 is dependent on the rate‐limiting enzyme GTP cyclohydrolase I (GTPCHI) together with its feedback regulatory protein (GFRP). This step is controlled by 6BH4 and the essential amino acid l‐phenylalanine. In the study presented here we wanted to investigate whether H2O2 affects the GTPCHI/GFRP cascade in these patients. Our results demonstrated concentration‐dependent regulation of rhGTPCHI where 100 μmol/L H2O2 was the optimum concentration for the activation of the enzyme and >300 μmol/L resulted in a decrease in activity. Oxidation of GFRP and GTPCHI does not affect feedback regulation via l‐phenylalanine and 6BH4. In vitiligo a constant upregulation of 6BH4de novo synthesis results from epidermal build up of l‐phenylalanine that is not controlled by H2O2. Taking the results together, 6BH4de novo synthesis is controlled by H2O2 in a concentration‐dependent manner, but H2O2‐mediated oxidation does not affect the functionality of the GTPCHI/GFRP complex.

Oxidation of methionine residues by reactive oxygen (ROS) in protein structures leads to the formation of methionine sulfoxide which can consequently lead to a plethora of impaired functionality. The generation of methionine sulfoxide yields ultimately a diastereomeric mixture of the S and R sulfoxides. So far two distinct enzyme families have been identified. MSRA reduces methionine S-sulfoxide, while MSRB reduces the R-diastereomer. It has been shown that these enzymes are involved in regulation of protein function and in elimination of ROS via reversible methionine formation besides protein repair. Importantly, both enzymes require coupling to the NADPH/thioredoxin reductase/thioredoxin electron donor system. In this report, we show for the first time the expression and function of both sulfoxide reductases together with thioredoxin reductase in the cytosol as well as in the nucleus of epidermal melanocytes which are especially sensitive to ROS. Since this cell resides in the basal layer of the epidermis and its numbers and functions are reduced upon ageing and for instance also in depigmentation processes, we believe that this discovery adds an intricate repair mechanism to melanocyte homeostasis and survival.

The human epidermis has the full machinery for autocrine l-phenylalanine turnover to l-tyrosine in keratinocytes and melanocytes. Phenylalanine hydroxylase (PAH) activities increase linearly with inherited skin colour (skin phototype I–VI, Fitzpatrick classification) yielding eightfold more activities in black skin compared to white skin. Moreover, UVB irradiation (1 MED) significantly increases epidermal PAH activities 24h after exposure. Importantly, l-phenylalanine uptake and turnover in the pigment forming melanocytes is vital for initiation of melanogenesis. In this context it was shown that the uptake of this amino acid is regulated by calcium. The depigmentation disorder vitiligo provides a unique model to follow impaired l-phenylalanine turnover in the skin as well as in serum because affected individuals hold an impaired epidermal 6BH4 de novo synthesis/recycling and regulation including low epidermal PAH activities. After overnight fasting and oral loading with l-phenylalanine (100mg/kg body weight), 29.6% of 970 patients tested (n=287/970) yielded serum phenylalanine/tyrosine ratios ⩾4 and 35.3% (n=342/970) had mild to moderate hyperphenylalaninaemia (HPA), while 9.3% (n=90/970) had both serum l-phenylalanine levels ⩾2.0mg/dl and phe/tyr ratios ⩾4.0. Isolated HPA was found in 26% (n=252/970), whereas 20.3% had only increased ratios (n=197/970). None of the patients had phenylketonuria and the family history for this metabolic disease was negative. The IQ followed normal Gaussian distribution. In vitro l-phenylalanine uptake/turnover studies on primary epidermal melanocytes originating from these patients demonstrated a significantly decreased calcium dependent l-phenylalanine uptake and turnover compared to healthy control cells. Based on our observation, we would like to propose that phenylalanine uptake/turnover is under tight control by calcium which in turn could offer an additional novel mechanism in the aetiology of HPA.

The human skin holds the full machinery for pro-opiomelanocortin processing. The alpha-melanocyte-stimulating hormone (alpha-MSH)/melanocortin-1-receptor cascade has been implicated as a major player via the cAMP signal in the control of melanogenesis. Only very recently the beta-endorphin/mu-opiate receptor signal has been added to the list of regulators of melanocyte dendricity and melanin formation. In this context it was reported that (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)) can act as an allosteric inhibitor of tyrosinase, the key enzyme in melanogenesis, and this inhibition is reversible by both alpha- and beta-MSH. It was also shown earlier that 7BH(4), the isomer of 6BH(4), is twice as active in this inhibition reaction. However, as yet it is not known whether 7BH(4) is indeed present in loco in the melanosome. We here provide evidence that this isomer is present in this organelle in a concentration range up to 50 x 10(-6) M. Determination of beta-MSH in melanosomal extracts yielded 10 pg/mg protein. Moreover, we demonstrate reactivation of the 7BH(4)/tyrosinase inhibitor complex by beta-MSH, whereas alpha-MSH failed to do so. Furthermore, we show intra-melanosomal l-dopa formation from dopachrome by 7BH(4) in a concentration range up to 134 x 10(-6) M. Based on these results, we propose a new receptor-independent mechanism in the control of tyrosinase/melanogenesis by beta-MSH and the pterin 7BH(4).

Recently two alternative mechanisms have been put forward for the inhibition of tyrosinase by 6R-l-erythro 5,6,7,8-tetrahydrobiopterin (6BH(4)). Initially allosteric uncompetitive inhibition was demonstrated due to 1:1 binding of 10(-6)M 6BH(4) to a specific domain 28 amino acids away from the Cu(A) active site of the enzyme. Alternatively it was then shown that 10(-3)M 6BH(4) inhibit the reaction by the reduction of the product dopaquinone back to l-dopa. In the study presented herein we have used two structural analogues of 6BH(4) (i.e., 6,7-(R,S)-dimethyl tetrahydrobiopterin and 6-(R,S)-tetrahydromonapterin) confirming classical uncompetitive inhibition due to specific binding of the pyrimidine ring of the pterin moiety to the regulatory domain on tyrosinase. Under these conditions there was no reduction of l-dopaquinone back to l-dopa by both cofactor analogues. Inhibition of tyrosinase by 6BH(4) occurs in the concentration range of 10(-6)M after preactivation with l-tyrosine and this mechanism uncouples the enzyme reaction producing H(2)O(2) from O(2). Moreover, a direct oxidation of 6BH(4) to 7,8-dihydrobiopterin by tyrosinase in the absence of the substrate l-tyrosine was demonstrated. The enzyme was activated by low concentrations of H(2)O(2) (<0.3 x 10(-3)M), but deactivated at concentrations in the range 0.5-5.0 x 10(-3)M. In summary, our results confirm a major role for 6BH(4) in the regulation of human pigmentation.