N-Alkanes in Cultured Normal Human Keratinocytes
Within the group of neutral lipids, n-alkanes are regularly found in scales of normal human stratum corneum. In scales from patients with congenital ichthyosiform erythroderma (CIE) n-alkanes are increased by a factor of 5–10. As these n-alkanes are ingredients of emollients and components of air pollution, it is still under discussion whether n-alkanes found in stratum corneum are of exogenous or of endogenous origin. To obtain more infor-mation on the metabolic behavior of these lipids we investigated their content in cultured normal human keratinocytes and in one experiment under the influence of an aqueous dermatophytic extract.
Primary cultures were established according to Rheinwald and Green on lethally irradiated 3T3 cells. For experiments second- or third-passage keratinocytes were cultured in the serum-free keratinocyte growth medium (KGM from Clonetics). Lipids were extracted according to Bligh and Dyer and separated and analyzed by a micromethod (TLC/FID) or by convention¬al TLC after incorporation of radioactive precursors.
N-alkanes were regularly found in cultured normal human keratinocytes in a range of 325 ±6 μg/μg DNA, which corresponds to approximately 10% of total lipids. To rule out contaminations, each component of the analytical procedure from the culture medium to the chromatography equipment was tested for n-alkanes. Small amounts of n-alkanes were detected only in filters used to separate the extracted cells from the solvent extract. Trials to incorporate 14C-acetate or 14C-linoleic acid into n-alkanes failed. Hence, under our conditions, using just confluent cultures, synthesis of n-alkanes was not demonstrated. A dermatophytic extract from a strain of Trichophyton mentagrophytes induced an increase of all neutral lipid classes when incubated together with cultured normal human keratinocytes.
KeywordsStratum Corneum Neutral Lipid Sterol Ester Confluent Culture Essential Fatty Acid Deficiency
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