A sensitive thyrotropin (TSH) bioassay based on iodide uptake in rat FRTL-5 thyroid cells: comparison with the adenosine 3',5'-monophosphate response to human serum TSH and enzymatically deglycosylated bovine and human TSH.
A sensitive in vitro assay based on the uptake of 125I by the FRTL-5 rat thyroid cell line has been applied to the measurement of TSH bioactivity from different sources. In this bioassay various human pituitary reference preparations showed similar potencies; the limit of detection and the half-maximal response were 1.60 +/- 0.1 (+/- SE) and 9.70 +/- 0.40 microU/0.5 ml, respectively; however, when compared to pituitary TSH from other species, human TSH was 29- and 10-fold less biopotent than bovine and rat TSH, respectively. The iodide uptake response to TSH was inhibited by the presence of human serum in a dose-dependent fashion, but pretreatment of serum with 10% polyethylene glycol restored TSH activity. The iodide uptake response was compared to the stimulation of cAMP production in the measurement of serum TSH bioactivity from human samples after immunoaffinity purification. In the cAMP production bioassay, immunoaffinity-purified serum TSH showed increased bioactivity in patients with primary hypothyroidism and TSH-secreting pituitary tumor compared to that in normal subjects, while in the iodide uptake bioassay minimal differences were detected among the different groups. To investigate further structure-function relationships of TSH in FRTL-5 cells we studied the effects of deglycosylated purified pituitary bovine and human TSH on both bioassays. Using two new enzymes, peptide-N-glycosidase and endo-beta-N-acetylglucosaminidase F, we removed one carbohydrate chain from TSH alpha and all three chains from TSH, respectively. In the iodide uptake bioassay both enzymes induced a 2-fold decrease in TSH biopotency, while in the cAMP production bioassay this decrease was only present with peptide-N-glycosidase-treated TSH. In summary, 1) the iodide uptake bioassay system in FRTL-5 cells represents a valid and sensitive method for the measurement of TSH bioactivity from different sources and can be applied to serum samples with elevated TSH concentrations by simple pretreatment with polyethylene glycol without immunoaffinity purification; 2) the enzymatic removal of one carbohydrate chain from both bovine and human TSH significantly decrease their biological activity, assessed as cAMP production and iodide uptake response in FRTL-5 cells, while the removal of three carbohydrate chains induces a significant decrease only in the iodide uptake bioassay: and 3) the disparate results for cAMP and iodide uptake in both human samples and deglycosylated pituitary TSH suggest that in addition to cAMP, other second messengers may play a role in TSH action.
Nissim
M
M
Molecular, Cellular, and Nutritional Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Lee
K O
KO
Petrick
P A
PA
Dahlberg
P A
PA
Weintraub
B D
BD
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D001790:Blood Physiological Phenomena; D002417:Cattle; D000242:Cyclic AMP / Q000096:biosynthesis*; D006801:Humans; D007454:Iodides / Q000378:metabolism; D051381:Rats; D012016:Reference Values; D013959:Thyroid Diseases / Q000097:blood; D013961:Thyroid Gland / Q000166:cytology / Q000378:metabolism; D013972:Thyrotropin / Q000031:analogs & derivatives* / Q000032:analysis* / Q000097:blood / Q000494:pharmacology
D007454:Iodides; C053694:deglycosylated thyrotropin; D013972:Thyrotropin; D000242:Cyclic AMP