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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 1 98-104
Copyright © 1999 by The Endocrine Society

Original Studies

Serum Levels of 20-Kilodalton Human Growth Hormone (GH) Are Parallel Those of 22-Kilodalton Human GH in Normal and Short Children

Mayumi Ishikawa, Susumu Yokoya, Katsuhiko Tachibana, Yukihiro Hasegawa, Toshiaki Yasuda, Etsurou Tokuhiro, Yoshihide Hashimoto and Toshiaki Tanaka

National Children’s Medical Research Center (M.Y., T.T.), Tokyo 154-8509; Toranomon Hospital (S.Y.), Tokyo 105-0001; Kanagawa Children’s Medical Center (K.T.), Yokohama 232-0066; Tokyo Metropolitan Kiyose Children’s Hospital (Y.H.), Kiyose 204-0024; Chiba University (T.Y.), Chiba 260-0856; Odawara City Hospital (E.T.), Odawara 250-0055; and Mitsui Pharmaceuticals, Inc. (Y.H.), Mobara 297-0017, Japan

Address all correspondence and requests for reprints to: Dr. 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
 Subjects and Methods
 Results
 Discussion
 References
 
Twenty-kilodalton human GH (20K), which is one of the human GH (hGH) variants, is thought to be produced by alternative premessenger ribonucleic acid splicing. However, its physiological role is still unclear due to the lack of a specific assay. We have measured serum 20K and 22-kDa hGH (22K) by specific ELISAs to investigate the physiological role of 20K in children.

The subjects were 162 normal children, aged 1 month to 20 yr; 12 patients with GH deficiency (GHD), aged 11 months to 13 yr; 57 children with non-GHD short stature, aged 2–17 yr; and 13 girls with Turner’s syndrome, aged 5 months to 15 yr. Samples were collected at random from normal children and were collected after hGH provocative tests and 3-h nocturnal sleep from GHD, non-GHD short stature, and Turner’s syndrome children. The mean basal serum concentrations of 22K and 20K were 2.4 ± 2.8 ng/mL and 152.3 ± 184.0 pg/mL in normal boys and 2.5 ± 3.1 ng/mL and 130.6 ± 171.5 pg/mL in normal girls, respectively. The percentages of 20K (%20K) were 5.8 ± 2.1% and 6.0 ± 3.2% in 83 normal boys and 79 normal girls, respectively. There was no significant difference in %20K either among ages or between the prepubertal stage and the pubertal stage in normal boys and girls. The mean %20K values in basal samples of provocative tests in 12 patients with GHD, non-GHD short stature, and Turner’s syndrome were 6.5 ± 2.4%, 6.5 ± 3.8%, and 5.9 ± 3.2%, respectively. There was no significant difference in %20K among normal children and these growth disorders, and there was no significant difference in %20K throughout the hGH provocative tests and 3-h nocturnal sleep in these growth disorders. There was also no significant correlation between the percentage of 20K and the height SD score or body mass index in either normal children or subjects with these growth disorders.

In conclusion, the %20K is constant, regardless of age, sex, puberty, height SD score, body mass index, and GH secretion status. The regulation of serum 20K levels remains to be established.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
HUMAN GH (hGH) has several monomeric size variants (1, 2), such as 22-kDa hGH (22K), which is the most abundant form of pituitary GH; 20-kDa hGH (20K); 17-kDa hGH; and 5-kDa hGH. 20K is a single chain and 176 amino acids; it is different from 22K in that residues 32–46 are deleted (3). The variant 20K has been described as the second most abundant form of pituitary GH and is thought to be produced by alternative premessenger ribonucleic acid (pre-mRNA) splicing (4, 5).

It has been reported that some actions of 20K are different from those of 22K. The diabetogenic action, insulin-like action, and antiinsulin-like action of 20K are weaker than those of 22K (6). The binding of hGH receptor (7, 8) or GH-binding protein (9) by 20K is less than that by 22K. Due to the lack of a specific antibody and of a specific assay system for 20K, it has been difficult to determine quantities of 20K directly. Therefore, the function of 20K in vivo has not been well understood, although it is suggested that non-22K isomers may be related to the growth mechanism (10).

In this study, we measured both serum 20K and 22K and determined the percentage of 20K (%20K) in hGH in normal children and patients with diseases causing growth disorders, such as GH deficiency (GHD), non-GH-deficient short stature (non-GHD), and Turner’s syndrome.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Sera were collected from 162 normal children (aged 1 month to 20 yr), 12 patients with GHD (aged 11 months to 13 yr), 57 children with non-GHD short stature (aged 2–17 yr), and 13 children with Turner’s syndrome (aged 5 months to 15 yr). All samples were collected after obtaining informed consent. The patients were diagnosed as having GHD when peak GH responses to at least two provocative tests [except GH-releasing hormone (GRH) stimulation] were less than 10 ng/mL. The clinical characteristics of subjects are shown in Table 1Go. The height SD score in normal children was within ±2 SD compared with the Japanese cross-sectional standards for height (11). As there were no data about pubertal stage, normal boys aged younger than 11 yr and normal girls aged younger than 10 yr were arbitrarily defined as prepubertal, whereas boys and girls equals to or older than these ages were defined as pubertal. All samples from normal children were collected at random.


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  		Table 1. Clinical characteristics of subjects

 
Levels of 20K and 22K were measured in sera from normal children and in sera during provocative tests [arginine tolerance test (ATT), GRH stimulation test (GRH), clonidine tolerance test (CTT), and insulin tolerance test (ITT)] and 3-h nocturnal sleep (samples were collected every 20 min) from the patients with growth disorders.

The %20K was calculated as a percentage of total 22K and 20K with the equation 20K/(22K + 20K) x 100. Statistical analysis was carried out with Student’s t test, Pearson’s correlation coefficient, and ANOVA. The values are shown as the mean ± SD.

hGH assay

Both serum 20K and 22K were measured by specific enzyme-linked immunosorbent assays (ELISAs), respectively. Experimental details of these procedures have been reported previously (12). In the 20K ELISA, 0.1 mL assay buffer (150 mmol/L phosphate-buffered saline containing 1% BSA, 1 mol/L NaCl, and 10 mg/L heterophilic blocking reagent; Scantibodies Laboratory, Santee, CA) and 0.025 mL of standards (Mitsui Pharmaceuticals, Inc., Tokyo, Japan) (13) or serum samples were added to monoclonal anti-20K antibody (anti-hGH-antibody D05, Mitsui Pharmaceuticals, Inc., Tokyo, Japan)-precoated microtiter plates and incubated for 2 h at room temperature. After through washing (0.01 mol/L Tris-HCl, pH 8.0, containing 0.05% Tween-20), 0.1 mL (0.5 mg/L) peroxidase-labeled anti-20K monoclonal antibody (POD-D14, Mitsui Pharmaceuticals, Inc.) was added and incubated for 2 h at room temperature. After a further washing step, 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, and the plates were incubated for 30 min at room temperature. The absorbance were read at 450 nm (reference wave length was 620 nm) after stopping the enzyme reaction with 0.1 mL H2SO4. The detection limit was 5 pg/mL, and the cross-reactions with 22K, hPRL, and human placental lactogen were less than 0.1%. Samples with 20K values above 1 ng/mL were diluted with the zero standard.

In 22K ELISA, microtiter plates were precoated with the monoclonal anti-hGH antibody (anti-hGH antibody A36020047P, BiosPacific, Inc., Emeryville, CA). Other procedures were the same as described above, except that the concentration of POD-D14 was 0.05 mg/L. The detection limit was 50 pg/mL, and the cross-reactions with 20K, hPRL, and human placental lactogen were less than 0.1%. Samples with 22K values greater than 10 ng/mL were diluted with the zero standard.\.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Normal children

The mean basal serum concentrations of 22K and 20K were 2.4 ± 2.8 ng/mL and 152.3 ± 184.0 pg/mL in normal boys (Fig. 1AGo) and 2.5 ± 3.1 ng/mL and 130.6 ± 171.5 pg/mL in normal girls (Fig. 2AGo), respectively. The percentage of 20K in hGH was 5.8 ± 2.1% in normal boys and 6.0 ± 3.2% in normal girls (Figs. 1BGo and 2BGo). There was no significant difference in %20K among ages and between prepubertal and pubertal ages in either normal boys or normal girls.



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  		Figure 1. Serum 20K and 22K concentrations (A) and the percentage of 20K in hGH (B) in samples collected at random in normal boys (mean ± SD). There was no significant difference among ages in either 20K or 22K. The %20K was almost 6% of hGH, and there was no significant difference among any ages or between prepuberty and puberty.

 


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  		Figure 2. Serum 20K and 22K concentrations (A) and %20K in hGH (B) of samples collected at random in normal girls (mean ± SD). There was no significant difference among ages in 20K or 22K. The %20K was almost 6% of hGH, and there was no significant difference among any ages or between prepuberty and puberty.

 
Figure 3Go shows the correlation between the 20K and height SD score or body mass index (BMI) in boys. In neither boys nor girls was there a significant correlation between 20K and height SD score or BMI (data not shown).



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  		Figure 3. The correlation between %20K and height SD score (A) or BMI (B) in normal boys. There was no correlation between %20K and height SD score or BMI. The same result was recognized in normal girls and patients with diseases causing growth disorders (data not shown).

 
Growth disorders

The mean %20K in basal samples from provocative tests in children with GHD, non-GHD short stature, and Turner’s syndrome were 6.5 ± 2.4%, 6.5 ± 3.8%, and 5.9 ± 3.2%, respectively. The basal and peak concentrations of 22K and 20K and the %20K in provocative tests are shown in Table 2Go. There was no significant difference in the %20K in normal children, in basal samples from children with growth disorders, or in peak samples among growth disorders in provocative tests. There was also no significant difference in the %20K between mild and severe GHD, as there was a significant positive correlation in peak GH between 20K and 22K (n = 13; r = 0.994; P < 0.0001).


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  		Table 2. Results for the children with growth disorders

 
Figure 4AGo shows the mean %20K during ATT in non-GHD. There was no significant change in %20K throughout ATT and other provocative tests (data not shown) in GHD, non-GHD, and Turner’s syndrome children. Figure 4BGo shows the mean %20K during 3-h nocturnal sleep in non-GHD subjects. There was also no significant difference in %20K throughout 3-h nocturnal sleep. In neither growth disorders nor normal children did the %20K correlate with the height SD score or BMI.\.



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  		Figure 4. The %20K during the arginine tolerance test (A) and during 3-h nocturnal sleep (B) in non-GHD short stature (mean ± SD). The %20K did not change during the arginine tolerance test or during nocturnal sleep in non-GHD short stature patients.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Although several attempts have been made to measure serum 20K directly, either it has been difficult to obtain specific antibody for 20K or the assay sensitivity of antibody was insufficient to assay serum 20K (14). Indirect estimation has been carried out by calculating the difference between total hGH and 22K (15, 16, 17). Other methods employed to estimate 20K include immunoaffinity chromatography (18), electrophoretic systems (19), and gel-electrophoretic-immunostaining-autoradiographic assay (20). By these methods, 20K has been estimated to range from 5–15% of total hGH. It has also been reported that the quantity of 20K increases after exercise or food intake (16, 17), but the percentage of 20K in hGH does not vary due to these factors.

Boguszewski et al. (10) reported that the percentage of non-20K GH isoforms was higher in short children born small for gestational age and in Turner’s syndrome patients than in normal children and suggested a possible cause for growth failure by non-22K GH isoforms. Boguszewski et al. (15) showed a significant positive correlation between the percentage of non-22K GH isoforms and BMI, weight SD score, or weight for height SD score in prepubertal children. This fact suggested that non-22K GH isoforms may be regulated by nutritional state. In the present study, however, we have demonstrated by direct 20K measurement that the %20K does not vary according to age, puberty, or sex in normal children, and that it does not correlate with the height SD score in normal children or in children with growth disorders. The somatogenic activity of 20K is reported to be almost the same as that of 22K (6). From our study, 20K does not seem to be related to any mechanism causing growth disorder. It remains possible that GH isoforms other than 20K might contribute to the higher percentage of non-22K GH isoforms and be related to short stature. Although 20K has shown a higher lipolytic activity than 22K in vitro (21), we could not find any correlation between %20K and BMI.

In addition, we have examined %20K in nocturnal spontaneous secretion and in response to several provocative tests. In each situation, serum 20K changed in a manner parallel to 22K, and the %20K remained constant regardless of the secretory phases of GH. 20K has been reported to be produced through pre-mRNA alternative splicing (4, 5). On the other hand, clearance of 20K is not known in the human, although it has been reported to be slower than 22K clearance in the rat (22). Our study suggests that the secretion of 20K and 22K from the pituitary is not under different control from that of 22K, suggesting the absence of specific control mechanisms in GH mRNA splicing. These results are comparable to previous findings (18, 20).

As the difference in function between 20K and 22K has been variously reported as diabetogenic action (23), insulin-like action (6, 24, 25), antiinsulin action (24), and affinity for GH receptor or GH-related peptide (8, 9) in vitro, further study is necessary to investigate these functional differences in vivo. Our newly developed assay will be useful to clarify the pathophysiological functions of 20K.

Received July 28, 1998.

Revised October 5, 1998.

Accepted October 13, 1998.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Baumann G. 1991 Growth hormone heterogeneity: genes, isohormones, variants, and binding proteins. Endcr Rev. 12:424–449.
  2. Lewis UJ, Dunn JT, Bonewald LF, Seavey BK, Vanderlaan WP. 1978 A naturally occurring structural variant of human growth hormone. J Biol Chem. 253:2679–2687.[Free Full Text]
  3. Lewis UJ, Bonewald LF, Lewis LJ. 1980 The 20,000-dalton variant of human growth hormone: location of the amino acid deletions. Biochem Biophys Res Commun. 92:512–516.
  4. DeNoto FM, Moore DD, Goodman HM. 1981 Human growth hormone DNA sequence and mRNA structure: possible alternative splicing. Nucleic Acids Res. 9:3719–3730.[Abstract/Free Full Text]
  5. Cooke NE, Ray J, Watson MA, Estes PA, Kuo BA, Liebhaber SA. 1988 Human growth hormone gene and the highly homologous growth hormone variant gene display different splicing patterns. J Clin Invest. 82:270–275.
  6. Lewis UJ, Markoff E, Culler FL, Hayek A, Vanderlaan WP. 1987 Biologic properties of the 20K-dalton variant of human growth hormone: a review. Endocrinol Jpn. 34(Suppl 1):73–85.
  7. McCarter J, Shaw MA, Winer LA, Baumann G. 1990 The 20,000 Da variant of human growth hormone does not bind to growth hormone receptor in human liver. Mol Cell Endocrinol. 73:11–14.[CrossRef][Medline]
  8. Daughaday WH, Trivedi B, Winn HN, Yan H. 1990 Hypersomatotropirsm in pregnant women, as measured by a human liver radioreceptor assay. J clin Endocrinol Metab. 70:215–221.[Abstract]
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  10. Boguszewski CL, Jansson C, Boguszewski MCS, et al. 1997 Increased proportion of circulating non-22-kilodalton growth hormone isoforms in short children: a possible mechanism for growth failure. J Clin Endocrinol Metab. 82:2944–2949.[Abstract/Free Full Text]
  11. Suwa S, Tachibana K. 1993 Standard growth chart for height and weight of Japanese children from birth to 17 years based on a cross-sectional survey of national data. Clin Pediatr Endocrinol. 2:87–97.
  12. Tsushima T, Katoh Y, Miyachi Y, et al. Serum concentration of 20K human growth hormone (20K hGH) measured by a specific ELISA. J Clin Endocrinol Metab. In press.
  13. Uchida H, Naito N, Asada N, et al. 1997 Secretion of authentic twenty kilodalton human growth hormone (20K hGH) in Escherichia coli and properties of the purified product. J Biotechnol. 55:101–112.[CrossRef][Medline]
  14. Mellado M, Rodriguez-Frade JM, Kremer L, Martinez-Alonso C. 1996 Characterization of monoclonal antibodies specific for the human growth hormone 22K and 20K isoforms. J Clin Endocrinol Metab. 81:1613–1618.[Abstract]
  15. Boguszewski CL, Jansson C, Boguszewski MCS, et al. 1997 Circulating non-22 kDa growth hormone isoforms in healthy children of normal stature: relation to height, body mass and pubertal development. Eur J Endcrinol. 137:246–253.[CrossRef]
  16. Dore S, Brisson GR, Fournier A, Massicotte D, Peronner F, Gareau R. 1991 HGH 20K species and variability of GH responses to long-duration exercise in male cyclists fed different food supplements. Horm Metab Res. 23:431–434.[Medline]
  17. Dor{theta} S, Brisson GR, Fournier A, Montpetit R, Perrault H, Boisvert D. Contribution of hGH 20K variant to blood hGH response in sauna and exercise. Eur J Appl Physiol. 62:130–134.
  18. Baumann G, Stolar MW. 1986 Molecular forms of human growth hormone secreted in vivo: non-specificity of secretory stimuli. J Clin Endocrinol Metab. 62:789–790.[Abstract]
  19. BaumannG, MacCart JG, Amburn K. 1983 The molecular nature of circulating growth hormone in normal and acromegaric man: evidence for a principal and minor monomeric forms. J Clin Endocrinol Metab. 56:946–952.[Abstract]
  20. Sinha YN, Gilligan TA, Lee DW, Baxi SC, VanderLaan WP. 1986 Demonstration of 20K growth hormone in human plasma by gel electrophoretic immunostaining autoradiographic assay (GEISAA). Horm Metab Res. 18:402–406.[Medline]
  21. Wada M, Uchida H, Ikeda M, et al. 1997 The 20-kilodalton (kDa) human growth hormone (hGH) differs from the 22-kDa hGH in the complex formation with cell surface hGH receptor and hGH-binding protein circulating in human plasma. Mol Endcrinol. 12:146–156.[Abstract/Free Full Text]
  22. Baumann G, Stolar MW, Buchanan TA. 1985 Slow metabolic clearance rate of the 20,000-dalton variant of human growth hormone: Implications for biological activity. Endocrinology. 117:1309–1313.[Abstract]
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