| Summary: | 碩士 === 國立成功大學 === 臨床藥學研究所 === 98 === Hormones influence the development and function of human skin which also produces hormones and neurotransmitters. The local formation of sex steroids provides autonomous control to human skin according to local needs. In addition, in post-menopausal women, all sex steroids made in the skin are from adrenal steroid precursors. Human sebocytes and keratinocytes express steroidogenic enzymes and cofactors. Hormones exhibit a wide range of biological activities on the skin and regulate cutaneous wound healing.
Stratum corneum (SC) is the outmost and the terminal differention layer of the skin. The endogenous steroids content of the human SC has never been reported on the literature. The aim of the present study was to investigate the distribution of endogenous steroids in human SC with respect to gender, age, anatomical site, and depth into SC, using a non-invasive sampling technique and a sensitive analytic method for quantitation of endogenous steroids.
A liquid chromatograph-tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous identification and quantitation of three endogenous steroids (hydrocortisone, cortisone, and testosterone) in human SC. SC was removed by sequential tape stripping and the samples were incubated with reporter lysis buffer followed by extraction with dichloromethane. The extractants were evaporated to dryness, resuspended in 0.1 mL methanol, and then detected using LC/MS/MS in the multiple reaction monitoring mode (MRM). The chromatography was operated on a reversed phase C18 column using a linear gradient of water/acetonitrile (95/5, v/v) and acetonitrile/water (95/5, v/v) in 15min. The detector’s response was linear for hydrocortisone, cortisone, and testosterone concentrations ranging from 0.3 to 50 ng/mL. Extraction recovery ranged from 20% to 40%. Limits of detection and limits of quantification for three steroids were in the ranges of 0.1-0.8 ng/mL and 0.3-1.0 ng/mL, respectively.
32 healthy volunteers participated in the study. The result showed that testosterone was detected in only one volunteer. The frequency of hydrocortisone detection was higher in the SC of the forearm, while cortisone was detected more frequently in the SC of the back from Group 1 subjects (years 20-35). However, cortisone was not observed in the back from Group 2 subjects (years 50-65). Amounts of hydrocortisone and cortisone in SC of forehead ranged 0.13-0.37 ng/cm2 and 0.02-0.20 ng/cm2, respectively. Amounts of hydrocortisone and cortisone in SC of forearm ranged 0.02-0.96 ng/cm2 and 0.03-0.12 ng/cm2, respectively. Amounts of hydrocortisone and cortisone in SC of back ranged 0.06-0.49 ng/cm2 and 0.02-0.06 ng/cm2, respectively. The amounts of hydrocortisone and cortisone were not statistically different between genders, age groups and anatomical sites. Statistically higher amounts of cortisone were found in the surface layer of SC than deeper layers in Group 2 subjects. Whether the amounts of hydrocortisone and cortisone found in SC are related to their plasma levels remains to be clarified in future studies.
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