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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 11 4274-4279
Copyright © 2000 by The Endocrine Society

Original Studies

A Novel Specific Bioassay for Serum Human Growth Hormone

Mayumi Ishikawa, Atsuko Nimura, Reiko Horikawa, Noriyuki Katsumata, Osamu Arisaka, Mitsufumi Wada, Masaru Honjo and Toshiaki Tanaka

Department of Endocrinology and Metabolism, National Children’s Medical Research Center, (M.I., A.N., R.H., N.K., T.T.) Setagaya-ku, Tokyo 154-8509; The 1st Department of Internal Medicine, Toho University of School Medicine (M.I.), Ota-Ku, Tokyo 143-8541; Department of Pediatrics, Dokyo University School of Medicine (O.Y.), Shimotuka-gun, Ibaragi 321-0267; and Life Science Laboratories Mitsui Chemicals, Inc., (M.W., M.H.), Mobara, Chiba 297-0017, Japan

Address correspondence and requests for reprints to: Mayumi Ishikawa, Department of Endocrinology and Metabolism, National Children’s Medical Research Center, 3-35-31, Taishido, Setagaya-ku, Tokyo 154-8509, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human GH receptor (hGHR) was recently expressed on a Ba/F3 cell line, which is a mouse pro-B cell lymphoma that has been induced to become a cloned cell line (Ba/F3-hGHR). Using a Ba/F3-hGHR cell line, we have established a bioassay for serum hGH.

hGH stimulated cell proliferation in a dose-dependent manner in concentrations ranging from 1 ng to 100 ng/mL. Cell proliferation was not influenced by other hormones or growth factors in the bioassay, with the exception of insulin-like growth factor I (IGF-I) and GH binding protein. Free IGF-I significantly stimulated the proliferation of Ba/F3-hGHR cells at concentrations over 25.85 ng/mL in this bioassay system, but serum IGF-I did not stimulate cell proliferation because the sensitivity of cell proliferation was insufficient for free IGF-I in serum. GH binding protein, however, did suppress cell proliferation at the highest concentration (100 ng/mL), but did not at the average concentration (20 ng/mL). Human serum stimulated cell proliferation, which was completely suppressed by anti-GH antibody. The GH bioactivity of serum samples from normal children and patients with non-GH deficient short stature correlated strongly with the serum hGH concentration determined by immunoradiometric assay (IRMA) (r = 0.967, r = 0.924, P < 0.0001, respectively). The ratio of bioactivity/IRMA was 1.01 ± 0.26 in sera from normal children and 1.18 ± 0.24 and 1.00 ± 0.29 at basal values and peak values in GH stimulation tests, respectively, in sera from patients with non-GH deficient short stature. The bioactivity/IRMA ratio for the serum GH bioactivity of a patient who had biologically inactive GH caused by an amino acid substitution was 0.333 ± 0.056 (mean ± SD).

In conclusion, we established a new sensitive bioassay for hGH that is specific for hGH somatogenic action and is useful for screening of patients with short stature caused by biologically inactive hGH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS CLINICALLY useful to measure the bioactivity of serum human GH (hGH) to determine whether short stature is due to biologically inactive hGH, as suggested by Kowarski et al. (1). Various GH bioassays, both in vivo and in vitro have been proposed. The two most common types of in vivo bioassays of GH are body weight gain in hypophysectomized rats (2, 3, 4) or in dwarf "little" mice (5) and the tibial plate assay on hypophysectomized rats (6). In both in vivo bioassays, however, the response is easily influenced by stress and metabolic state, and the sensitivity is insufficient for measuring serum GH bioactivities.

In vitro GH bioassays suitable for clinical use include radioreceptor assays (7, 8), receptor modulation assays (9), and cell proliferation bioassays using the Nb2 cell line (10).

Recently, expression of the hGH receptor (hGHR) in the mouse pro-B cell lymphoma cell line. Ba/F3 has led to a cloned cell line (Ba/F3-hGHR) the proliferative response of which is hGH dose dependent (11). Using this cell line, we established in this study a bioassay system for hGH and measured hGH bioactivity in sera from normal children and patients with non-GH-deficient short stature.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hormone preparations

hGH and human PRL (hPRL) were provided by the Polypeptide Hormone Laboratory (University of Manitoba, Canada). Bovine GH was purchased from Biogenesis Ltd. (England, UK), and human IGF-1 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Insulin (Novorin R 40 IU/mL) was purchased from Novo Nordisk (Gentafte, Denmark). FSH (Fertinorm P) and hCG (Prophacy) were provided by Serono Japan (Tokyo, Japan). Fibroblast growth factor was purchased from Collaborative Research (Waltham, MA), and epidermal growth factor was purchased from Toyobo Co., Ltd. (Osaka, Japan). TSH was purchased from Zymed Laboratories Inc. (California), and L-thyroxine sodium was purchased from Teikoku Zouki (Tokyo, Japan). Hydrocortisone (Solu-cortef) was provided by Sumitomo-Pharmacia-Upjohn (Stockholm, Sweden). GHBP was provided by Novo Nordisk. GRF (GRF Sumitomo) was purchased from Sumitomo Pharmaceuticals (Tokyo, Japan). Anti-GH antibody (clone 5801) was provided from Oy Medix Biochemica Ab (Kaunianinen, Finland).

Cell cultures

Ba/F3-hGHR (11), which was established by Mitsui Chemicals, Inc. (Tokyo, Japan), was maintained as suspension cultures in 75 tissue culture flask (Falcon) Life Technologies, Inc., (Grand Island, NY) supplemented with FCS (10%; JRH Biosciences, Australia), 2-mercaptoethanol (2-ME) (50 µM; Nacalai Tesque, Inc., Kyoto, Japan), penicillin (50 U/mL), streptomycin (50 µg/mL; Life Technologies, Inc.), and hGH (10 nM) in an atmosphere of 5% CO2, 95% air at 37 C.

Bioassay with Ba/F3-cell line

Approximately 4–6 h before the start of the bioassays, the cells were washed twice with assay medium (RPMI 1680, supplemented with 5% FCS, 50 µM 2-ME, and antibiotics, without hGH) and were transferred to the assay medium and incubated for 4–6 h to slow down the rate of cell replication. After incubation, the cells were collected by centrifugation (3 min at 1000 rpm) and resuspended in the assay medium at a concentration of 1 x 105 cells/mL. Two-hundred microliter aliquots were distributed in each well of 96-well microplate (Nalge Nunc International, Roskilde, Denmark). Standard hGH was diluted with 0.01 M PBS supplemented with 0.1% BSA, (Sigma Chemical Co., St. Louis, MO) at each concentration (0–1000 µg/L). Samples were incubated at 56 C for 40 min to inactivate the serum. To each well was added 25 µL of standard or sample. The cultures were incubated in a CO2 incubator (5% CO2 + 95% air) for 48 h at 37 C. At the end of the incubation, the colorimetric end point was determined by an eluted stain bioassay (ESTA) described by Marshall et al. (12) and Ealey et al. (13) with a slight modification. Briefly, 20 µL MTT solution (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) (5 mg/mL in 0.01 M PBS; Sigma) were added to each well and incubated at 37 C for 4 h in a CO2 incubator. During this time, activated cells reduced the yellow MTT salt to purple formazan. The plate was centrifuged at 800 rpm for 10 min, and the stain was eluted into dimethyl sulfoxide (Nacalai Tesque, Inc.), of which 100 µL were added to each well. Bioactive responses were determined with a kinetic microplate reader (Molecular Devices, Menlo Park, CA), reading optical densities at the test wavelength of 550 nm and a reference wavelength of 650 nm to correct for differential scattering. All samples were assayed in duplicate (coefficient of variation, <12%), and a control serum with known bioactivity was used at every assay for the quality control.

In the blocking study, 25 µL anti-hGH antibody (diluted x50 or x100 with 0.01 M PBS containing with 0.1% BSA) were added to wells of standard or sample.

The influence of GHBP was determined by diluting the GH standard with GHBP, in concentrations of 20 or 100 ng/mL in 0.01 M PBS containing 0.1% BSA.

The bioactivity of other hormones and growth factors—bGH (0.1–1000 µg/L), PRL (0.1–1000 µg/L), human IGF-I (0.1–1000ng/mL), human insulin (0.1 µ IU/mL to 1.0 mIU/mL), hydrocortisone (10-1000 ng/mL), (25-1000 IU/L), FSH (100-100,000 IU/L), TSH (1–100 mU/L), L-thyroxin Na (80.4–643.5 nmol/L), epidermal growth factor (10–1000 ng/mL), fibroblast growth factor (10–1000 ng/mL), and GRF (0.1–1000 ng/mL)—was assayed by adding 25 µl to each well.

Bioassay with Nb2 cell line

Approximately 48 h before the start of the bioassays, the cells were transferred to the pre-assay medium (Fisher’s Medium supplemented with 1% horse serum, 50 µM 2-ME, and antibiotics) to slow down the rate cell of replication. After incubation, cells were collected by centrifugation (10 min at 800 rpm) and resuspended in assay medium (Fisher’s Medium supplemented with 10% horse serum, 50 µM 2-ME, and antibiotics) at a concentration of 1 x 105 cells/mL. Two-hundred microliter aliquots were distributed in each well of a 96-well microplate (Nalge Nunc International). Standard hGH was diluted with 0.01 M PBS supplemented with 0.1% BSA (Sigma) at each concentration. To each well were added 25 µl of standard or samples. The cultures were incubated in a CO2 incubator (5% CO2 x 95% air) for 48 h at 37 C. After incubation, the colorimetric end point was determined to be the same as the bioassay with the Ba/F3-hGHR cell line.

Subjects

Serum samples were obtained from 23 normal children (14 boys and 9 girls) aged 0 yr, 10 months to 20 yr, 6 months (9 yr, 10 months ± 3 yr, 11 months, mean ± SD) and 10 non-GH deficient short children (6 boys and 4 girls) aged 4 yr, 10 months to 13 yr, 6 months (8 yr, 3 months ± 2 yr, 11 months). Basal and peak hGH samples of GH provocative tests in non-GH-deficient short children (arginine tolerance test, GRH stimulation test, clonidine tolerance test) were also used in non-GH-deficient short children.

Serum samples from a patient with severe short stature caused by bioinactive GH, reported previously by Takahashi et al. (14), were obtained after GRF stimulation (Table 1Go). The concentration of GH and IGF-I in serum sample from a patient from acromegaly were 24.9 µg/L and 440 ng/mL, respectively. Another serum sample was collected from a patient with precocious puberty on TSH provocative test (TRH). The concentration of PRL and GH were 47.8 µg/L and 6.9 µg/L, respectively.


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  		Table 1. Immunoactivity and bioactivity in the patient with bioinactive GH

 
All samples were collected after obtaining informed consent.

Measurements

Immunoactivity of hGH in serum was determined by IRMA (Daiichi Radio Isotope, Japan). GHBP was measured by ligand-mediated immunofunctional assay, described previously (15, 16). IGF-I was measured by Somatomedin C-RIA kit (Chiron, Yuka Medias Company Ltd.).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Growth stimulatory activity of hGH

hGH stimulated Ba/F3-hGHR cell proliferation in a dose-dependent manner between 1 µg/L and 100 µg/L (Fig. 1Go), which was used as the standard curve for the following GH bioassay.



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  		Figure 1. Stimulation of the proliferation of Ba/F3-hGHR by hGH and other hormones or growth factors. Points are the mean values of triplicate (except IGF-I) or duplicate (IGF-I) wells (SD ± 10% of the mean).

 
None of the other hormones and growth factors, except IGF-I and bGH, showed any mitogenic activity. IGF-I stimulated the cell proliferation in a dose-dependent manner (Fig. 1Go), and the cell proliferation by IGF-I was suppressed by anti-IGF-1 antibody. The bGH increased cell proliferation at a concentration of more than 1000 ng/mL. The highest GHBP concentration (100 ng/mL) slightly suppressed the cell proliferation by hGH, but the average GHBP concentration (20 ng/mL) in the serum did not (Fig. 2Go).



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  		Figure 2. Effect of GHBP on the proliferation of Ba/F3-hGHR cell cultures. Points are the mean values of duplicate wells (SD ± 10% of the mean).

 
The growth stimulatory activity of hGH was completely blocked by anti-hGH antibody (Fig. 3Go).



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  		Figure 3. Effect of anti-GH antibody on the growth stimulatory activity of hGH. Ba/F3-hGHR cell cultures were incubated for 48 h with hGH in the presence or absence of anti-GH antibody. (The dilutions of the antibody were x50 or x100. The final concentrations of antibody were x5000 or x1000.)

 
Bioassay of hGH in human serum

Human serum stimulated cell growth in the dilution range of 6.25–100% (the final concentration in the wells was 0.69–11%). The dose-dependent cell growth paralleled that produced by the standards (Fig. 4Go). The anti-hGH antibody also completely inhibited the stimulatory activity of serum samples.



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  		Figure 4. Effect of human serum on the proliferation of Ba/F3-hGHR cell cultures after 48-h incubation with and the anti-hGH antibody.

 
Figure 5Go shows the correlation between bioactivity and immunoactivity in serum samples from normal children. There was a strong positive correlation between them, and the ratio of bioactivity to immunoactivity was 1.01 ± 0.26 (mean ± SD) (Fig. 5Go).



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  		Figure 5. A, Correlation between hGH bioactivity and immunoactivity in normal children. B, Ratio of bioassay/IRMA. Mean value of bioassay to IRMA ratio was 1.013 ± 0.260 (mean ± SD).

 
The bioactivity of samples from non-GH-deficient short children also showed a close correlation with immunoactivity. The mean ratios of bioactivity/IRMA at basal values and peak values in GH stimulation tests were 1.18 ± 0.24 and 1.00 ± 0.29 (mean ± SD), respectively (Fig. 6Go). There was no significant difference in the mean ratios of bioactivity/IRMA between basal values and peak values. The bioactivity of samples measured by Ba/F3-hGHR showed a close correlation with the bioactivity by Nb2 assay (Fig. 7Go).



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  		Figure 6. A, Correlation between hGH bioactivity and immunoactivity in short children. {circ}, basal values; •, peak values. B, Ratio of bioassay/IRMA at basal and peak values. Mean value of bioassay to IRMA ratio was 1.181 ± 0.243 or 0.997 ± 0.293 (mean ± SD), respectively.

 


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  		Figure 7. Correlation between hGH bioactivity measured with the Ba/F3-hGHR cell line and with the Nb2 cell line.

 
The bioactivity of samples from a patient with severe short stature caused by bioinactive GH were lower than immunoactivity (0.33 ± 0.06, mean ± SD) (Table 1Go). The bioactivity of samples from acromegaly were almost equal to the immunoactivity and the ratio of bioactivity/IRMA was 1.04. The cell growth of diluted serum samples from acromegalic patient paralleled with the standards (Fig. 8Go). The ratio of bioactivity/IRMA of hyperprolactinemia with precocious puberty, which was under the TRH stimulation test, was 1.5.



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  		Figure 8. Effect of serum from acromegaly of the proliferation of Ba/F3-hGHR cell line.

 
The mean GHBP concentrations in the sera from normal children and non-GH-deficient short children were lower than 20 ng/mL [5.1 ± 3.8 ng/mL (2–17.4 ng/mL) and 4.9 ± 3.4 ng/mL (0.8–9.4 ng/mL), respectively] did not correlate with bioactivity. IGF-I concentrations in the serum from normal children and non-GH-deficient short children were 216.9 ± 144.7 ng/mL (76–430 ng/mL) and 120.4 ± 64.9 ng/mL (43–210 ng/mL), respectively, and did not correlate with bioactivity, either.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Among the hGH bioassays suitable for the measurement of serum GH bioactivity, radioreceptor assays and receptor modulation assays only reflect the binding of hGH to its receptor (7, 8, 9, 17), whereas the cell proliferation assay reflects not only receptor binding but also intracellular function activated by hGH. Although the Nb2 bioassay does not need a radioactive ligand, it becomes more convenient and useful after ESTA system modification (12, 13). However, bioactivity in the Nb2 bioassay is mediated through the PRL receptor (10, 18). The bioactivity of hGH is measurable by the Nb2 bioassay because hGH is the only animal GH that has lactogenic activity. The Nb2 bioassay, therefore, measures not somatotrophic but lactogenic activity of hGH, and the addition of anti-hPRL antibody to block hPRL lactogenic activity is necessary to measure the hGH bioactivity in the serum (10). Another method to measure hGH bioactivity has been reported: for example, suppressive activity of hGH on lipid accumulation has been measured using cell lines with hGHR (19). However, this method is not sensitive enough to establish a bioassay system (20) and does not detect somatotrophic activity (19). The Ba/F3-hGHR cell line is both suitable and sensitive enough to serve as an hGH bioassay.

The Ba/F3-hGHR cell line proliferates dose-dependently when hGH is added, and cell proliferation is blocked by anti-hGH antibody. Cell proliferation is stimulated by no other hormone or growth factor but free IGF-I and bGH. Although free IGF-I did stimulate cell growth, the bioactivity of the serum sample did not correlate with total IGF-I concentrations but only with hGH concentration. In addition, the cell growth by diluted samples from acromegaly paralleled with the standards. Those demonstrate that the free IGF-I concentration is insufficient to affect the assay system under both normal and abnormal conditions, because most IGF-I binds to the binding proteins in serum (21, 22, 23). The cell proliferation was suppressed by anti-IGF-I antibody.

The other factor influencing cell proliferation is GHBP, which also influences the Nb2 bioassay (24). Yet cell proliferation is not disturbed significantly when the GHBP concentration is less than 20 ng/mL. Therefore, to measure hGH bioactivity when the GHBP concentration in serum is higher than 20 ng/mL, GHBP should be added to the standard samples at the same concentration as in serum samples. Bovine GH increases cell proliferation at a concentration of over than 1000 ng/mL, but does not influence assay system because a very low concentration of bGH was used in assay medium 5% FCS and 0.1% BSA/PBS).

In the samples determined by IRMA in our study, the bioactivity of serum samples from normal children and from non-GH-deficient short children is observed to be very close to the concentration of hGH. In patients with Turner syndrome and with non-GH-deficient short stature children, serum GH bioactivity measured by the suppression of lipid accumulation with GH on 3T3-F442A embryonic murine fibroblasts is reported to be higher than the GH concentration determined by RIA both in basal and peak values of the GH stimulation test (20). In healthy adult volunteers, serum GH bioactivity using the Nb2 bioassay is accompanied by bigger rises in bioactivity than in immunoactivity at the peak value of GH stimulation tests (25). Contrastingly, the bioactivity/immunoactivity ratio is normal in peak GH samples by the provocative test in non-GH-deficient short stature and acromegaly (26, 27). Because the bioactivity of the Ba/F3-hGHR cell line correlates with the bioactivity of the Nb2 cell line in normal and non-GH-deficient short children, this bioassay system confirms the Nb2 cell line bioassay system in use since the 1980s.

Only two cases with severe growth retardation caused by bioinactive hGH confirmed by DNA analysis have been reported (14, 28). We measured bioactivity in the serum of one of these cases by the Ba/F3-hGHR cell line assay. The patient was a 3-year-old girl whose height was 79.4 cm (3.6 SD below the mean for her age and sex). A heterozygous single-based substitution (AG) in exon 4 of the GH-1 gene was found; the mutation is located in binding site 2 of the GH molecule to hGHR (14). The recombinant mutant GH is less potent than the wild-type GH in phosphorylation of tyrosine residues in hGHR (14). This demonstrates that the bioactivity measured by the Ba/F3-hGHR cell line assay reflects the somatogenic activity of mutant hGH.

In summary, we have established a novel hGH bioassay that is specific for hGH, one that will be useful for screening of patients with short stature caused by biologically inactive hGH.

Received December 7, 1999.

Revised July 27, 2000.

Accepted August 1, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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