Abstract
Retinoids present in cultured human epidermal keratinocytes maintained in medium containing 25 nM retinol were identified and quantitated utilizing reverse-phase gradient high performance liquid chromatography. Total cell retinoid mass and composition averaged 12.59 pmol/mg cell protein +/- 1.86, (mean +/- S.D., n = 5) between strains and was constant with culture age. Long chain fatty acid esters of retinol and 3,4-didehydroretinol comprised greater than 95% of total retinoid, each contributing 77 and 23% of total esters, respectively. In mature stratified cultures, less abundant retinoids included unesterified retinol, 1%, and 3,4-didehydroretinol, 0.5%, retinoic acid, 0.5%, and a retinoid not previously reported in mammalian cells, 3,4-didehydroretinoic acid, 0.3%. This absolute and relative retinoid composition was essentially constant with culture age except that 3,4-didehydroretinoic acid was not detected in 3-4-day-old cultures (16-32 cell colonies), suggesting that keratinocyte differentiation may be related to endogenous changes in production of "active" retinoids. Separate pools of retinyl and 3,4-didehydroretinyl esters served as predominant sources of substrate for production of retinoic and 3,4-didehydroretinoic acids, respectively, suggesting that their production may be regulated independently.
Highlights
S.D., n = 5) between strains and was constant with culture age
Relative retinoid composition was essentially con- Active retinoids are thought to bind to specific nuclear stant with culture age except that 3,4-didehydroreti- retinoic acid receptors which in turn interact with specific noic acid was not detected in 3-4-day-old cultures
As variation between strains for the retinoid acids, the relative keratinocytedifferentiationisknownto be influenced by composition of retinoid alcohols varied considerably between exogenous retinoids, it was of interest to quantitate the reti- strains, ranging between 0.2 and 1%of total cell retinoid for Keratinocyte Retinol Metabolism
Summary
Identification of Keratinocyte Retinoids-Human epidermal keratinocytes maintained insubmerged culture for 14-16 days grow to form multilayered epithelial sheets (2-3 cell layers). 1 a n d 2 from two different gradient mobile phase systems depict typical retinoid elution profiles of extracts from these stratified keratinocyte cultures (see Table I for key to chromatograms). The most abundant retinoids presenwtere esters of long chain fatty acids of retinol and 3,4-didehydroretinol (peaks 5-11 ). Panel A , chromatogram of anextract of keratinocytes didehydroretinyl esters was similar except that no stearate harvested from 20 175-cmZflasks. The identityof the acyl species forthese chromatogram of a extract of keratinocytes harvested from1175-cm esters tions was further validated by collecting the ester from this gradient, transmethylating, and peak fracseparating flask which had beenincubated for 24 h prior to harvest with medium cgtorhanemtasinwinasg.
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