Identification of 7-dehydrocholesterol, vitamin D3, 25(OH)-vitamin D3 and 1,25(OH)2-vitamin D3 in Solanum glaucophyllum cultures grown in absence of light

Solanum glaucophyllum contains the calciotropic hormone 1,25-dihydroxy-vitamin D-3 (1,25(OK)(2)D-3). The metabolic pathway leading to the formation of 1,25(OH)(2)D-3 in the plant is largely unknown. Specifically, there is controversy about the participation of a photolytic reaction in the generation of vitamin D-3 and its metabolites. To investigate the requirement for light, S. glaucophyllum tissue (callus) and cell suspension cultures grown under strict conditions of darkness were extracted with chloroform/methanol (1:2, v/v) followed by purification of the lipidic fraction by Sephadex LH-20 and highperformance liquid chromatography. HPLC peaks with elution times similar to those of authentic samples of 7-dehydrocholesterol, vitamin D-3, 25(OH)D-3 and 1,25(OK)(2)D-3 were detected. The presence of 1,25(OH)(2)D-3 was also evidenced by [H-3]1,25(OH)(2)D-3 competitive binding analysis using the chick hormone intestinal receptor. Furthermore, 7-dehydrocholesterol, vitamin D-3, 25(OH)D-3 and 1,25(OH)(2)D-3 were unequivocally identified by mass spectrometry. Incubation of control samples of 7-dehydrocholesterol under the same conditions as S. glaucophyllum cultures did not result in vitamin D-3 formation, excluding the influence of light in these experiments. The results suggest that a synthetic route of vitamin D-3 compounds independent of light operates in Solanum glaucophyllum cultured in vitro. (C) 1998 Elsevier Science B.V. All rights reserved.