A quantitative histochemical study of sulphydryl and disulphide content during normal epidermal keratinization

Abstract

A quantitative histochemical study was carried out on the distribution of protein thiol and disulphide groups in normal human plantar epidermal tissue. Histochemical demonstration of reactive groups was achieved by addition of N-(4-aminophenyl) maleimide, subsequent diazotization and final coupling with a Nitro Red or chromotropic acid label as first described by Sippel. The quantitative reliability of the method was tested by absorption cytophotometry, and evaluated on the basis of the internal consistency of the results reported. Our histological observations and histophotometric data support accepted views on epidermal keratinization. A limited, though reproducible, amount of disulphide bonds was observed near the basement membrane. The free thiol concentration in basal and prickle cells was low and almost constant, but was higher in the granular cells, where deposition of sulphur-containing proteins on cell membranes is initiated. In Malpighian layers, disulphide cross-links only occurred just beneath the transition zone in thickened cell membranes. The staining pattern of the inner stratum corneum resembled a mosaic and was characterized by a Sharp rise of the disulphide content, which exceeded the decrease in free thiol groups. The free thiol concentration decreased further throughout the cornified layers whilst the disulphide content remained fairly constant. Staining of thiol and disulphide groups together corresponded, within the limits of the standard error, to the sum of the thiol and disulphide concentrations when they were assayed separately in living ahd horny cells. These results confirm that living cells are the main site of free thiol groups, while horny cells are the most prominent of site of disulphide cross-links.

Protein Oxidation in Human Stratum Corneum: Susceptibility of Keratins to Oxidation In Vitro and Presence of a Keratin Oxidation Gradient In Vivo

“…Another SC protein, recognized by immunoblotting using a monoclonal antibody against keratins 1, 10 and 11, µ110-120 kDa, was shown to be susceptible to carbonyl formation upon the same oxidant challenges. In analogy to the high degree of saturation of fatty acids in the SC (Wertz and Downing, 1991), the amount of disulfide cross-links in human SC is known to be many-fold higher than in lower epidermal layers (Broekaert et al, 1982). Similarly, Figure 4.…”

“…By using sequential tape strippings, a steep gradient with lowest levels of carbonyl groups in keratins from lower layers and highest in the upper layers was found (Figs 3c and 4). Importantly, this protein oxidation gradient is inversely correlated with the gradients of the anti-oxidant vitamin E (Thiele et al, 1998a), and free thiols (Broekaert et al, 1982) in human SC. There is in vitro and in vivo evidence from other biologic systems that protein oxidation can be counteracted by anti-oxidants such as vitamin E (Ibrahim et al, 1997) and thiols (Yan et al, 1996).…”

The Antioxidant Network of the Stratum corneum

“…By using sequential tape strippings, a steep gradient with the lowest levels of carbonyl groups in keratins from lower layers and the highest in the upper layers was found. Importantly, this protein oxidation gradient is inversely correlated with the gradients of the antioxidant vitamin E [18] and free thiols [37] in human SC. There is in vitro and in vivo evidence from other biological systems that protein oxidation can be counteracted by antioxidants such as vitamin E and thiols.…”

Oxidative Targets in the Stratum corneum

“…By using sequential tape-strippings, a steep gradient with lowest levels of carbonyl groups in keratins from lower layers and highest in the upper layers was found. Importantly, this protein oxidation gradient is inversely correlated with the gradients of the antioxidant vitamin E [7], and free thiols [19] in human SC. The inverse correlation with SC antioxidant levels on the one hand and the positive correlation with the levels of oxygen and oxidizing xenobiotics within the SC on the other, may account for the protein oxidation gradient in SC keratins.…”