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Original Studies |
Department of Medicine 2, Tokyo Women’s Medical University (T.T.), Tokyo 162-8666; Department of Medicine 1, Shimane Medical School (Y.K.), Izumo 693-8501; First Department of Medicine, Tokyo 143-8541 University School of Medicine (Y.M.), Tokyo 143-8541; Department of Internal Medicine 3, Kobe University School of Medicine (K.C.), Kobe 650-0017; Department of Neurosurgery, Nippon Medical School (A.T.), Tokyo 113-8603; Toho Medical School (M.I.), Tokyo 143-8541; and Institute of Biological Science, Mitsui Pharmaceuticals, Inc. (Y.H.), Mobara 297-0017, Japan
Address all correspondence and requests for reprints to: Dr. Toshio Tsushima, Department of Medicine, Tokyo Women’s Medical University, Kawadacho 8–1, Shinjukuku, Tokyo 162-8666, Japan. E-mail: tsushimt{at}ss.iij4u.or.jp
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Introduction |
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Materials and Methods |
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Recombinant 20K hGH was prepared as previously described (12), and recombinant 22K hGH (Genotropin) was obtained from Sumitomo Pharmaceuticals, Inc. (Osaka, Japan). Human PRL (hPRL) was purchased from UCB Bioproducts S.A (Brussels, Belgium). Human placental lactogen (hPL) was a product of Chemicon (Temecula, CA). Recombinant hGH-(44–191) (17K hGH) was provided by Dr. U. J. Lewis (La Jolla, CA).
mAbs
Three mAbs were selected on the basis of affinity and
specificity. The mAbs D05 and D14 were prepared in our laboratory, and
the mAb A36020047P, a product of BiosPacific, Inc. (Emeryville, CA),
preferentially recognized 22K hGH (Kd = 280
pmol/L for 22K hGH). The mAb D05 was specific to 20K hGH
(Kd = 440 pmol/L for 20K hGH). A36020047P and mAb
D05 were coated onto the microtiter plate as capture antibodies in the
assays for 22K hGH and 20K hGH, respectively. The mAb D14 bound both
20K and 22K hGH with equal affinity (Kd = 460 and
570 pmol/L, respectively) and did not compete with either D05 or
A36020047P, indicating that this mAb binds a different epitope from
that recognized by the other two mAbs. The isotype of D05 and
A36020047P was IgG1 
, whereas that of D14 was IgG2a . Horseradish
peroxidase (POD)-conjugated mAb D14 (POD-D14) with maleimide was
prepared as described previously (11).
Other reagents
8-Anilino-1-naphthalenesulfonic acid ammonium salt and 3,3',5,5'-tetramethylbenzidine were purchased from Sigma Chemical Co. (St. Louis, MO). Heterophilic blocking reagent (HBR) was a product of Scantibodies Laboratory (Santee, CA).
20K hGH ELISA
To each well of a 96-well microtiter plate (ELISA-PLATTE, Greiner, Frickenhauser, Germany), 0.06 mL phosphate-buffered saline (PBS; 137 mmol/L NaCl, 8.1 mmol/L Na2HPO4, 1.5 mmol/L KH2PO4, and 2.7 mmol/L KCl, pH 7.4) containing mAb D05 (40 mg/L) was added, and the plates were incubated for 2 days at 4 C. The wells were washed with wash buffer (10 mmol/L Tris-HCl buffer containing 0.5 mL/L Tween-20, pH 8.0) and blocked overnight at 4 C with 0.25 mL blocking reagent containing a mixture of milk proteins (Block Ace, Dainippon Pharmaceutical Co., Osaka, Japan). The plates were washed again, and the remaining wash buffer was removed by tapping the plates on a paper towel. The assay was started by adding 0.1 mL assay buffer (PBS supplemented with 1 mol/L NaCl, 10 mg/L HBR, and 10 g/L BSA, pH 7.0), 0.025 mL GH standard solution in calibration buffer (100 mmol/L sodium phosphate buffer containing 20 g/L BSA, 2 mmol/L ethylenediamine tetraacetate, and 100 mmol/L NaCl, pH 7.0) or serum sample. HBR was added to avoid interference to the ELISA by antimouse IgG, which is rarely present in human serum. The plates were incubated for 2 h at room temperature with constant shaking. After extensive washing, 0.1 mL POD-D14 (0.5 mg/L) in conjugate buffer (100 mmol/L sodium phosphate buffer containing 5 g/L BSA, 150 mmol/L NaCl, 0.5 g/L casein, 10 mL normal rabbit serum, and 0.2 g/L 8-anilino-1-naphthalenesulfonic acid ammonium salt, pH 6.5) was added, and the plates were incubated for another 2 h at room temperature with shaking. The wells were washed again, and 0.1 mL substrate solution (100 mmol/L citrate buffer containing 65 mg/L 3,3',5,5'-tetramethylbenzidine and 4 mmol/L H2O2, pH 3.8) was added. The plates were incubated for 30 min in at room temperature, and the reaction was terminated by adding 0.1 mL 500 mmol/L sulfuric acid. The absorbance was determined with a microtiter plate reader at 450 nm against a reference of 620 nm.
22K hGH ELISA
Microtiter plates were coated with the mAb A36020047P (10 mg/L in PBS) as described for the mAb D05. Other procedures were the same as those described above, except that the concentration of POD-014 was 0.05 mg/L.
Other measurements
The serum concentration of insulin-like growth factor I (IGF-I) was determined with a commercial kit (Kailon Co., Tokyo, Japan). Free T4 and TSH were measured with a RIA kit (Daiichi RI, Tokyo, Japan) and an immunoradiometric assay kit (Ortho Clinical Diagnostics Co., Tokyo, Japan), respectively.
Subjects
Serum concentrations of 20K and 22K hGH were determined in 508 normal adults (282 women, aged 20–75 yr, and 226 men, aged 20–79 yr). The blood samples were obtained with informed consent at the time of annual health check after an overnight fast. Patients with active acromegaly (n = 33), surgically treated acromegalics (inactive acromegaly) whose basal GH levels decreased to lower than 2 ng/mL (n = 11), and subjects with GH deficiency (GHD; n = 30), untreated Graves’ disease (n = 9; serum free T4, 4.5 ± 1.8 ng/dL), primary hypothyroidism (n = 10; free T4, 0.5 ± 0.3 ng/dL; TSH, 94.2 ± 102 mU/L), anorexia nervosa (n = 8; body mass index, 12.5 ± 1.3 kg/m2; IGF-I, 63 ± 31 ng/mL), and idiopathic short stature (n = 10; height score, -2.6 ± 0.7 SD) were also studied for GH concentrations in serum obtained after an overnight fast. Idiopathic short stature was defined as GH-independent short stature below the mean height -2.0 SD. They had no endocrine, metabolic, or chromosomal abnormalities and responded normally to GH provocative tests (peak GH, >10 ng/mL).
GHD was diagnosed by the absence or low response of GH (peak GH, <5 ng/mL) on at least two provocative tests, i.e. the insulin tolerance test, the GHRH test, and the clonidine test, and by low concentrations of serum IGF-I. Sixteen of the 30 GHD patients had childhood onset of disease and had received GH therapy during childhood (duration, 6.8 ± 2.5 yr). Five of them had received cranial irradiation because of germinoma. The duration of GH deficiency in the 14 adult-onset GHD patients was estimated to be 19.7 ± 13.5 yr, and they had not received GH replacement therapy. Two of the 30 GHD patients had isolated GH deficiency due to peripartal hypoxia, and the remaining 28 (4 Sheehan syndrome, 6 craniopharyngioma, 4 germinoma, 9 postoperative pituitary tumor, and 5 idiopathic) had multiple pituitary hormone deficiencies in various combinations and were receiving adequate adrenal, thyroid, and/or gonadal hormone replacement therapy.
The insulin tolerance test and GHRH test were performed in a subset of patients with suspected GHD, and the TRH test was performed in eight patients with active acromegaly.
Statistical analysis
The results were expressed as the mean ± SD unless otherwise noted. As fasting serum levels of both 20K and 22K hGH were not normally distributed, the values were log transformed before statistical analysis. Although the actual values are given in the text and tables, statistical significance are those of log-transformed data. The difference between groups was evaluated by ANOVA using the StatView package (Abacus Concepts, Berkeley, CA). The hGH values below the detection limit (5 pg/mL for 20K hGH and 50 pg/mL for 22K hGH) were calculated as 2.5 pg/mL for 20K hGH and 25 pg/mL for 22K hGH, respectively. Correlation between variables was estimated by Spearman rank correlation test. P < 0.05 was considered significant.
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Results |
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Typical standard curves for 20K and 22K hGH ELISAs are shown in
Fig. 1
. The limits of detection, the
concentration of hGHs giving an optical density (OD) greater than 3
SD above the OD of the blank, were 5 pg/mL for 20K hGH and
50 pg/mL for 22K hGH, respectively. The limit of detection in the 22K
hGH ELISA could be reduced to 5 pg/mL or less by modifying the
concentrations of captured antibody and labeled antibody; however, we
selected the conditions described above because the higher sensitivity
narrowed the assay range. 22K hGH, hGH fragment 44–191, hPRL, and hPL
did not cross-react in the ELISA for 20K hGH at concentrations up to
1000 ng/mL (Fig. 2). Likewise,
cross-reaction of hGH fragment 44–191, 20K hGH, hPL, and hPRL was
negligible in the 22K hGH ELISA. Serial dilution of three human sera
(diluted with calibration buffer) gave linear results down to the limit
of detection in both 20K and 22K ELISAs (data not shown). The recovery
of added recombinant 20K hGH (50, 100, and 200 pg/mL) into three human
sera was 88.2–126% in the 20K hGH ELISA, and that of added
recombinant 22K hGH (0.5, 1, and 2 ng/mL) was 81.0–108% in the 22K
hGH ELISA. In the 20K hGH ELISA, the intraassay coefficients of
variation (CVs; n = 10) were 3.7%, 3.0%, and 2.5% for sera with
20K hGH values of 13.4, 237, and 766 pg/mL, respectively, and the
interassay CVs (n = 5) were 5.2%, 2.8%, and 4.9% for serum
containing 44.7, 99.8, and 282 pg/mL, respectively. In the 22K hGH
ELISA, the intraassay CVs were 2.2%, 3.2%, and 4.6% at 0.22, 4.44,
and 9.3 ng/mL, and the interassay CVs (n = 5) were 3.2%, 3.7%,
and 2.6% for serum containing 1.54, 3.12, and 7.41 ng/mL,
respectively. In both assays, no interference was seen with hemoglobin
(up to 4.8 g/dL), free bilirubin (up to 14 mg/L), conjugated bilirubin
(up to 69 mg/L), rheumatoid factor (up to 5400 IU/L), chyle (up to 1900
hormazine turbidity), ascorbic acid (up to 1 g/L), or heparin (up to
0.5 g/L).
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The serum levels of 20K hGH and 22K hGH and the ratio of 20K hGH
to 20K plus 22K hGH in normal subjects and patients with various
endocrine disorders are summarized in Table 1. The mean serum 20K hGH level was
94 ± 176 pg/mL in 508 normal subjects, with a significantly
higher level of 117 ± 178 pg/mL (mean ± SD,
n = 282) in women than in men (64 ± 170 pg/mL; n =
226). Both 20K and 22K hGH concentrations were less than the assay
limit in 50 of 226 men and in 19 of 282 women. There were no subjects
with undetectable levels of 20K or 22K hGH alone. Similarly, 22K hGH
concentrations were significantly higher in women. Figure 3 shows a positive correlation between
serum 20K and 22K hGH concentrations in normal subjects (r =
0.956; P < 0.0001), suggesting that both species of
hGH are secreted in a parallel fashion.
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). The majority of normal adults (76%
of females and 61% of males) showed a ratio of 5–10%, and 19.8% of
women and 30% of men had a value below 5%. A ratio equal to or
greater than 10% was seen in only 9% of females and 13% of males. No
daily variation in the ratio of 20K to 22K plus 22K GH was found in 5
normal subjects (data not shown). There was no correlation between the
ratio and age, sex, body weight, body height, or body mass index. The
ratio of 20K to 22K hGH ratio did not correlate with percent body fat
mass in normal subjects (40 men and 40 women; data not shown).
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We then determined whether acute stimulation of hGH secretion altered
the ratio of 20K hGH. The concentrations of 20K and 22K hGH were
measured in serum obtained before and 30 and 60 min after the
administration of insulin (0.1 U/kg BW) or GHRH (100 µg) in patients
with suspected hypopituitarism. The TRH test was also performed in
seven patients with active acromegaly (Table 2). There was no significant difference
in the ratio of 20K to 20K plus 22K hGH before and after these
provocative tests.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our novel assay for 20K hGH described here was sufficiently sensitive to detect the 20K hGH isoform at a concentration as low as 5 pg/mL. Furthermore, there was no cross-reactivity among 22K hGH, hGH fragment 44–191 (17K hGH), hPRL, and hPL in the assay. We measured serum concentrations of 20K hGH in a large number of normal subjects and in patients with endocrine disorders. The basal 20K hGH concentration in the circulation was higher in women than in men, most likely due to the higher levels of estradiol, as reported for 22K hGH (15). The results in various endocrine disorders were also similar to those for 22K hGH; the levels were high in acromegalics and patients with anorexia nervosa and low in patients with GHD.
A previous study by Baumann et al. (6) using PAGE showed that the proportions of 22K, 20K, and other isoforms in human serum obtained after L-DOPA stimulation were 85%, 7%, and 5–10%, respectively.
In a subsequent study (16), the proportions were 83%, 11%, and 6%, respectively, in serum obtained after GHRH stimulation. In addition, the ratio of 20K hGH was approximately 20% in serum at the time of a secretory episode (7). Thus, circulating monomeric hGHs largely consist of 22K and 20K hGH, although Sinha and Jacobsen have recently reported that a 17-kDa fragment of hGH [hGH-(44–191)] circulates in blood at levels 1–2 times higher than those of 22K hGH (17). The mean ratio of 20K hGH to total (20K plus 22K) in normal subjects in this study was approximately 6.3%, which was slightly lower than that obtained by SDS-PAGE. The lower value in this study may be due to the difference in the specificity of the antibody employed.
We showed here that the 20K/total ratio did not correlate with body height, body weight, or BMI, suggesting that the proportion of 20K hGH is not the determinant for these variables. Although an in vitro study (12) has shown that 20K hGH has a statistically higher lipolytic activity at low concentrations (1 and 10 ng/mL) than 22K, we could not find any correlation between the percentage of 20K hGH and the percent body fat mass. Further studies are required to determine whether 20K is involved in the regulation of lipid and carbohydrate metabolism.
It is interesting to note that the proportion of 20K hGH was significantly higher in patients with active acromegaly, which is similar to the results recently reported by Boguszewski et al. (18). They showed that the proportion of non-22K hGH in serum was significantly higher than that in normal subjects and decreased to the normal levels after successful removal of pituitary tumors. Furthermore, the values correlated to tumor size, mean 24-h hGH concentration, and serum PRL, suggesting that GH-producing pituitary adenoma secretes a higher proportion of non-22K hGH, including 20K hGH. We were not able to correlate the proportion of 20K hGH to tumor size or mean 24-h hGH levels in this retrospective study, but there was a positive correlation between the 20K proportion and serum IGF-I levels, suggesting that the evaluation of 20K hGH isoform, like that of non-22K hGH, is useful in the follow-up of acromegalic patients. It should be noted, however, that the high proportion of 20K hGH was not specific to acromegaly. Higher values, comparable to those in active acromegalics, were noted in patients with anorexia nervosa, which is characterized by marked body weight loss and elevation of circulating hGH levels with very low levels of serum IGF-I during the active stage. The reason for the different relative levels in this disorder is not clear at present. Secretion of 20K hGH from the pituitary may be higher than that of 22K hGH. Alternatively, the metabolism of the variant could be altered in anorexia nervosa. It has been shown that the MCR of 20K hGH is slower than that of 22K hGH (19). The high concentrations of 22K hGH may further inhibit degradation of 20K hGH by competing for the degradation sites.
Recently, it has been reported that the proportion of non-22K hGH is elevated in a subset of children with non-GH-deficient short stature (20). The authors showed that in children born small for date, the proportion of non-22K hGH isoforms was directly correlated with the mean 24-h GH concentration and inversely correlated with the height SD score. In the present study, however, there was no difference in the proportion of 20K hGH isoform between normal subjects and 10 subjects with idiopathic short stature. It is possible that other hGH isoforms distinct from 20K hGH may contribute to the higher proportion of non-22K hGH observed in their study.
We have examined the proportion of 20K variant secreted in response to several GH provocative tests. In all three provocative tests (insulin tolerance, GHRH, and TRH tests), serum 20K hGH levels increased in parallel to those of 22K hGH, and the proportion of the 20K variant was fairly constant in the individual patients. Thus, the release of the 20K variant was not stimulus specific. The results confirmed those reported by others using SDS-PAGE (6, 7, 16) or the 22K GH exclusion assay (8). Taken together, the data presented here suggest that regulation of 20K GH secretion from pituitary is similar, if not identical, to that of 22K hGH.
In conclusion, we have established a simple ELISA specific to 20K hGH. With this assay, we showed that the ratio of 20K hGH to 20K plus 22K hGH is higher in active acromegalic patients and subjects with anorexia nervosa. The ratio in patients with GHD, thyroid disorders, and idiopathic short stature was not different from that in normal subjects. The 20K hGH ratio was fairly constant during GH provocative tests, confirming previous findings using other methods. The assay we developed was simple and sensitive enough to measure the circulating levels of the 20K hGH isoform without extraction and will prove useful for understanding the physiological or pathophysiological role of the 20K hGH isoform.
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Acknowledgments |
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Footnotes |
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Received June 29, 1998.
Accepted October 6, 1998.
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), 17K hGH (
),
hPRL (
), and hPL (
) were tested in the 20K hGH (left)
and 22K hGH (right) ELISAs.
