The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 11 4274-4279
Copyright © 2000 by The Endocrine Society
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.
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Abstract
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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.
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Introduction
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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.
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Materials and Methods
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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 1
). 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.
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.).
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Results
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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. 1), 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).
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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. 1
), 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. 2).
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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).
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The growth stimulatory activity of hGH was completely blocked by
anti-hGH antibody (Fig. 3).
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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.)
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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. 4). 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.
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Figure 5 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. 5).
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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).
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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. 6).
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. 7).
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Figure 6. A, Correlation between hGH bioactivity and
immunoactivity in short children.  , 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.
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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 1). 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. 8). The ratio of bioactivity/IRMA
of hyperprolactinemia with precocious puberty, which was under the TRH
stimulation test, was 1.5.
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.
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Discussion
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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.
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Abstract
Introduction
