Bioactivity of human growth hormone in serum: validation of an in vitro bioassay.
GH, in clinical practice, is determined by RIA, but RIA estimates may not accurately reflect serum GH bioactivity. The available measures of GH bioactivity lack either sensitivity, specificity, or a physiologically relevant end point. The objective of this research was to develop a physiologically relevant GH bioassay which would not only measure the bioactivity of purified GH preparations, but would also have sufficient sensitivity to measure GH bioactivity in human serum. The method consisted of incubating murine 3T3-F442A adipocytes in serum-free medium containing BSA, 14C-glucose, and increasing concentrations of GH or test materials for 24 h, followed by measurement of conversion of glucose to lipid. Interference by nonspecific serum factors was reduced by the addition of 10 micrograms/liter insulin, 25 nM dexamethasone, and 37 nM estradiol to the medium. In the presence of 10 micrograms/liter insulin, 50 micrograms/liter insulin-like growth factor-1 did not alter the ability of GH to suppress lipid accumulation. Epinephrine and glucagon could suppress lipid accumulation but only at concentrations greatly in excess of the physiological range in serum. Twenty two thousand dalton hGH produced dose-dependent suppression of lipid accumulation which was linear between 0.625 and 10 micrograms/liter (r = 0.926; P = 0.0001) with a half-maximal response of 3.0 +/- 0.2 micrograms/liter (n = six experiments). The intra- and interassay coefficients of variation were 7% and 19%, respectively. The assay was specific for GH since addition of human PRL produced suppression of lipid accumulation only at concentrations where contamination of the preparation by GH became a significant factor. ACTH also suppressed lipid accumulation but only at doses of 1000 micrograms/liter or greater. Human placental lactogen and hLH, hFSH, and hTSH did not cross-react with GH in this assay. Addition of human serum did not alter the slope of ED50 of the GH dose-response curve. Pools of serum from prepubertal and pubertal boys and girls, subjects treated with arginine or insulin, a diabetic girl, and a boy with gigantism who had a serum GH content of 80 micrograms/liter by RIA and 40 micrograms/liter by bioassay, produced dose response curves parallel to that of the GH standard curve. Serum from patients with hypopituitarism did not produce significant suppression of lipid accumulation in any assay. Recovery of 5 micrograms/liter GH added to human serum was 94%. Twenty thousand dalton GH also suppressed lipid accumulation in this assay, but was 2-fold less potent than 22,000 dalton GH.(ABSTRACT TRUNCATED AT 400 WORDS)
Foster
C M
CM
Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor 48109.
Borondy
M
M
Padmanabhan
V
V
Schwartz
J
J
Kletter
G B
GB
Hopwood
N J
NJ
Beitins
I Z
IZ
D000273:Adipose Tissue / Q000187:drug effects* / Q000378:metabolism; D000293:Adolescent; D000818:Animals; D001681:Biological Assay; D001769:Blood; D002460:Cell Line; D003907:Dexamethasone / Q000494:pharmacology; D003920:Diabetes Mellitus / Q000097:blood; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D005947:Glucose / Q000378:metabolism; D006130:Growth Disorders / Q000097:blood; D013006:Growth Hormone / Q000097:blood* / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D050356:Lipid Metabolism; D008297:Male; D051379:Mice; D011786:Quality Control; D011863:Radioimmunoassay; D014424:Turner Syndrome / Q000097:blood
D007328:Insulin; D004958:Estradiol; D003907:Dexamethasone; D013006:Growth Hormone; D005947:Glucose