We define previously unrecognized in vivo pathways of vitamin D ₃ (D3) metabolism generating novel D3‐hydroxyderivatives different from 25‐hydroxyvitamin D ₃ [25(OH)D3] and 1,25(OH) ₂ D3. Their novel products include 20‐hydroxyvitamin D ₃ [20(OH)D3], 22(OH)D3, 20,23(OH) ₂ D3, 20,22(OH) ₂ D3, 1,20(OH) ₂ D3,1,20,23(OH) ₃ D3, and 17,20,23(OH) ₃ D3 and were produced by placenta, adrenal glands, and epidermal keratinocytes. We detected the predominant metabolite [20(OH)D3] in human serum with a relative concentration ~20 times lower than 25(OH)D3. Use of inhibitors and studies performed with isolated mitochondria and purified enzymes demonstrated involvement of the steroidogenic enzyme cytochrome P450scc (CYP11A1) as well as CYP27B1 (1α‐hydroxylase). In placenta and adrenal glands with high CYP11A1 expression, the predominant pathway was D3 → 20(OH)D3 → 20,23(OH) ₂ D3 → 17,20,23(OH) ₃ D3 with further 1α‐hydroxylation, and minor pathways were D3 → 25(OH)D3 → 1,25(OH) ₂ D3 and D3 → 22(OH)D3 → 20,22(OH) ₂ D3. In epidermal keratinocytes, we observed higher proportions of 22(OH)D3 and 20,22(OH) ₂ D3. We also detected endogenous production of 20(OH)D3, 22(OH) D3, 20,23(OH) ₂ D3, 20,22(OH) ₂ D3, and 17,20,23(OH) ₃ D3 by immortalized human keratinocytes. Thus, we provide in vivo evidence for novel pathways of D3 metabolism initiated by CYP11A1, with the product profile showing organ/cell type specificity and being modified by CYP27B1 activity. These findings define the pathway intermediates as natural products/endogenous bioregulators and break the current dogma that vitamin D is solely activated through the sequence D3 → 25(OH)D3 → 1,25(OH) ₂ D3.—Slominski, A. T., Km, T.‐K., Shehabi, H. Z., Semak, I., Tang, E. K. Y., Nguyen, M. N., Benson, H. A. E., Korik, E., Janjetovic, Z., Chen, J., Yates, C. R., Postlethwaite, A. , Li, W., Tuckey, R. C. In vivo evidence for a novel pathway of vitamin D ₃ metabolism initiated by P450scc and modified by CYP27B1. FASEB J. 26, 3901–3915 (2012). www.fasebj.org

We investigated the cutaneous expression of genes and enzymes responsible for the multistep conversion of tryptophan to serotonin and further to melatonin. Samples tested were human skin, normal and pathologic (basal cell carcinoma and melanoma), cultured normal epidermal and follicular melanocytes, melanoma cell lines, normal neonatal and adult epidermal and follicular keratinocytes, squamous cell carcinoma cells, and fibroblasts from dermis and follicular papilla. The majority of the samples showed simultaneous expression of the genes for tryptophan hydroxylase, arylalkylamine N‐acetyltransferase (AANAT), and hydroxyindole‐Omethyltransferase (HIOMT). The products of AANAT activity were identified by RP‐HPLC with fluorimetric detection in human skin and in cultured normal and malignant melanocytes and immortalized keratinocytes; HIOMT activity was detected in human skin, keratinocytes, and melanoma cells. N‐acetylserotonin (NAS) was detected by RP‐HPLC in human skin extracts. NAS identity was confirmed further by LC/MS in keratinocytes. In conclusion, we provide evidence that the human skin expresses intrinsic serotonin and melatonin biosynthetic pathways.

Following up on our previous findings that the skin possesses steroidogenic activity from progesterone, we now show widespread cutaneous expression of the full cytochrome P450 side‐chain cleavage (P450scc) system required for the intracellular catalytic production of pregnenolone, i.e. the genes and proteins for P450scc enzyme, adrenodoxin, adrenodoxin reductase and MLN64. Functionality of the system was confirmed in mitochondria from skin cells. Moreover, purified mammalian P450scc enzyme and, most importantly, mitochondria isolated from placenta and adrenals produced robust transformation of 7‐dehydrocholesterol (7‐DHC; precursor to cholesterol and vitamin D3) to 7‐dehydropregnenolone (7‐DHP). Product identity was confirmed by comparison with the chemically synthesized standard and chromatographic, MS and NMR analyses. Reaction kinetics for the conversion of 7‐DHC into 7‐DHP were similar to those for cholesterol conversion into pregnenolone. Thus, 7‐DHC can form 7‐DHP through P450scc side‐chain cleavage, which may serve as a substrate for further conversions into hydroxy derivatives through existing steroidogenic enzymes. In the skin, 5,7‐steroidal dienes (7‐DHP and its hydroxy derivatives), whether synthesized locally or delivered by the circulation, may undergo UVB‐induced intramolecular rearrangements to vitamin D3‐like derivatives. This novel pathway has the potential to generate a variety of molecules depending on local steroidogenic activity and access to UVB.