Stable maintenance of 5alpha-reductase activity in long-term subcultures of fibroblasts derived from the foreskin.
BACKGROUND
There is up to a 50-fold variation in control subjects in current assays of 5alpha-reductase activity which makes interpretation difficult. It was therefore attempted in this study to establish an assay method which produced stable 5alpha-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 5alpha-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 [1beta,2beta-3H] testosterone. 5alpha-Reductase activity was expressed as the sum of formed [3H] 5alpha-reduced metabolites (separated by thin-layer chromatography).


RESULTS
The full range of 5alpha-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 5alpha- reductase deficiency was 0.52 pmol/h per mg protein.


CONCLUSIONS
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 5alpha-reductase activity during long-term subculture. Therefore, this assay method can be used for differential diagnosis of 5alpha-reductase deficiency from other relevant entities.
Nakae
Kazumi
K
Department of Urology, Hyogo College of Medicine, Nishinomiya, Japan.
Tsuji
Motomu
M
Kuraoka
Tetsuro
T
Cho
Sooilu
S
Cunha
Gerald R
GR
Shima
Hiroki
H
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000032:analysis / Q000172:deficiency* / Q000378:metabolism*; D013734:Androgen-Insensitivity Syndrome / Q000378:metabolism* / Q000473:pathology; D001681:Biological Assay / Q000379:methods*; D018929:Cell Culture Techniques / Q000379:methods*; D002648:Child; D002675:Child, Preschool; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007223:Infant; D008297:Male; D010413:Penis / Q000378:metabolism* / Q000473:pathology; D012867:Skin / Q000378:metabolism* / Q000473:pathology; D013997:Time Factors
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase