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Stable maintenance of 5 α ‐reductase activity in long‐term subcultures of fibroblasts derived from the foreskin
Author(s) -
Nakae Kazumi,
Tsuji Motomu,
Kuraoka Tetsuro,
Cho Sooilu,
Cunha Gerald R.,
Shima Hiroki
Publication year - 2002
Publication title -
international journal of urology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.172
H-Index - 67
eISSN - 1442-2042
pISSN - 0919-8172
DOI - 10.1046/j.1442-2042.2002.00471.x
Subject(s) - foreskin , subculture (biology) , reductase , medicine , microbiology and biotechnology , cell culture , andrology , enzyme , biology , biochemistry , genetics
Background: There is up to a 50‐fold variation in control subjects in current assays of 5α‐reductase activity which makes interpretation difficult. It was therefore attempted in this study to establish an assay method which produced stable 5α‐reductase activity in long‐term subcultured foreskin fibroblasts. Methods: Foreskin fibroblasts were obtained from three boys with phimosis (control subjects), three patients with Reifenstein syndrome and one patient with 5α‐reductase deficiency (due to mutation L113P in exon 2 of the SRD5A2 gene). To maximize the number of cells in the DNA synthesis phase, cells were subcultured consistently to approximately 70% confluency. Thawed cells, frozen after the third subculture, were incubated for 24 h with [1β,2β‐ 3 H] testosterone. 5α‐Reductase activity was expressed as the sum of formed [ 3 H] 5α‐reduced metabolites (separated by thin‐layer chromatography). Results: The full range of 5α‐reductase activity in controls and patients with Reifenstein syndrome was 3.44–15.59 pmol/h per mg protein: a 4.53‐fold variation. The activity in the patient with 5α‐ reductase deficiency was 0.52 pmol/h per mg protein. Conclusion: By the cell culture methods used in this study, which aimed to increase the number of cells in the DNA synthesis phase, foreskin fibroblasts maintained a considerably stable level of 5α‐reductase activity during long‐term subculture. Therefore, this assay method can be used for differential diagnosis of 5α‐reductase deficiency from other relevant entities.