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.

Several lines of evidence support an autoimmune basis for alopecia areata (AA), a common putative autoimmune hair loss disorder. However, definitive support is lacking largely because the identity of hair follicle (HF) autoantigen(s) involved in its pathogenesis remains unknown. Here, we isolated AA-reactive HF-specific antigens from normal human scalp anagen HF extracts by immunoprecipitation using serum antibodies from 10 AA patients. Samples were analyzed by LC-MALDI-TOF/TOF mass spectrometry, which indicated strong reactivity to the hair growth phase-specific structural protein trichohyalin in all AA sera. Keratin 16 (K16) was also identified as another potential AA-relevant target HF antigen. Double immunofluorescence studies using AA (and control sera) together with a monoclonal antibody to trichohyalin revealed that AA sera contained immunoreactivity that colocalized with trichohyalin in the growth phase-specific inner root sheath of HF. Furthermore, a partial colocalization of AA serum reactivity with anti-K16 antibody was observed in the outer root sheath of the HF. In summary, this study supports the involvement of an immune response to anagen-specific HFs antigens in AA and specifically suggests that an immune response to trichohyalin and K16 may have a role in the pathogenesis of the enigmatic disorder.