Transactivation assay for determination of glucocorticoid bioactivity in human serum.
J Clin Endocrinol Metab
; 87(8): 3740-4, 2002 Aug.
Article
em En
| MEDLINE
| ID: mdl-12161504
ABSTRACT
We have developed a mammalian cell (COS-1) bioassay, which measures glucocorticoid bioactivity (GBA) directly from a small amount of human serum. The assay is based on the expression of human glucocorticoid receptor (GR) together with a coactivator protein and reporter plasmid containing GR response elements upstream of the luciferase gene. Ten microliters of human serum, in duplicate, are added directly to the cell culture medium, and GBA is derived from reporter gene activity. The assay differentiates between biopotencies of synthetic steroids, and importantly, mifepristone (RU486) is able to block glucocorticoid-induced response. The assay is sensitive (<15.6 nM cortisol in fetal calf serum) and precise, with the within- and between-assay coefficients of variation less than 8% and 10%, respectively. We measured serum GBA (bioassay) and cortisol (RIA) levels in 34 asthmatic children (age range, 5.7-14.2 yr) at baseline and after treatment with either inhaled budesonide (800 microg/d, n = 14), fluticasone propionate (500 microg/d, n = 14), or cromones (control group, n = 6). Pretreatment serum GBA and cortisol levels correlated strongly (r = 0.90, P < 0.0001, n = 34). Two months of treatment with inhaled budesonide resulted in excess GBA in circulation, which was not attributable to endogenous cortisol (P < 0.001). In the fluticasone propionate group, the presence of serum excess GBA was at the borderline of statistical significance (P < 0.08) after 2 months of inhalation therapy, and no excess GBA was detected in the cromone group. In conclusion, our bioassay enables measurement of mammalian cell response to bioactive glucocorticoids in circulation and provides a novel means to investigate patients receiving drugs acting through the GR.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Asma
/
Bioensaio
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Receptores de Glucocorticoides
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Glucocorticoides
Limite:
Animals
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Child
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Child, preschool
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Female
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Humans
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Male
Idioma:
En
Revista:
J Clin Endocrinol Metab
Ano de publicação:
2002
Tipo de documento:
Article