0021-972X/91/7201-0236$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society
Vol. 72, No. 1 Printed in U.S.A.
Twenty-Four-Hour Profile of Plasma Growth Hormone-Binding Protein ZEEV HOCHBERG, TAMAR AMIT, AND ZVI ZADIK Department of Pharmacology, Faculty of Medicine, and Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology (Z.H., T.A.), Haifa; the Pediatric Endocrine Unit, Kaplan Hospital (Z.Z.), Rehovot; and the Department of Pediatrics, Rambam Medical Center (Z.H.), Haifa, Israel
ABSTRACT. In experimental animals each burst of GH pulse is followed by a wave of receptor turnover and an increase in serum GH-binding protein (GH-BP), which occurs 60 min after the GH peak. The present report describes the 24-h profile of plasma GH-BP and its correlation to GH pulsatility in normal individuals. Four normally growing children in early puberty were the subjects of this study. Blood was withdrawn continuously for 24 h in 30-min fractions. Pulse analysis of both GH and GH-BP was performed by the Pulsar program. The vast
majority of the GH pulses were accompanied by GH-BP pulses within 30 min. Correlation of plasma GH levels to GH-BP levels on the residual series above the smoothed baseline of all 172 individual samples was r = 0.447 (P < 0.001). Thus, plasma GHBP levels fluctuate rapidly in relation to the pulsatility of plasma GH levels. This may influence the GH disappearance rate and brings into question some of the deconvolution calculations of GH secretory impulses. (J Clin Endocrinol Metab 72: 236-239, 1991)
T
HE PITUITARY signals its target organs by intermittent bursts of GH secretion that result from a complex interaction of hypothalamic GH-releasing and inhibiting hormones (1-3). This pattern of GH secretion is an important determinant of the target organs' responses. Thus, impaired pulsatility results in growth retardation, a condition that is now recognized as neurosecretory dysfunction (4, 5), and the growth of normal children has been found to bear a close relationship to this 24-h GH-secretion profile (6). In experimental animals the advantage to the growth process of multiple daily bursts of GH has been documented (7). It has been shown in the rat that this may be mediated by a harmoniously timed turnover cycle of the GH receptors, which down-regulate and up-regulate in concert with the pulsatility of serum GH (8). The immediate decrease in the plasma membrane receptors, induced by the binding of the hormones, is followed by internalization of the hormone-receptor complex to the Golgi apparatus and degradative lysosomes (9, 10). From early reports on serum GH-binding protein (GHBP), a relation to the GH receptor was suspected (11). Indeed, the amino-terminal amino acid sequences of both the GH receptor and the serum GH-BP in the rabbit are identical, the latter comprising the extracellalar domain of the receptor (12). In a variety of clinical conditions
the GH-BP correlates closely with GH receptor-binding capacity (13,14). It has been proposed that upon binding of GH to its receptor, either before or after internalization of the complex, the GH-BP is cleaved off of the receptor and shed into the circulation (15). In experimental animals each wave of GH pulse and receptor turnover is followed by an increase in serum GH-BP, which occurs 60 min after the GH peak (16, 17). The present report describes the 24-h profile of plasma GH-BP and its correlation to GH pulsatility in normal individuals.
Materials and Methods Subjects Four normally growing children, two males and two females, in early puberty (Tanner stage 2) were the subjects of this study (Table 1). Blood was withdrawn continuously for 24 h through a venous catheter and a peristatic pump in 30-min fractions, starting at 0800-0900 h, into heparinized test tubes.
GHRIA Plasma GH was assayed in duplicate by an immunofluorometric assay (18). The sensitivity of the assay was 0.1 ng/mL, and the intra- and interassay coefficients of variation were less than 6% and 10%, respectively. GH-BP
Received April 4,1990. Address requests for reprints to: Zeev Hochberg, M.D., D.Sc, Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, P.O.B. 9697, 31096 Haifa, Israel.
GH-BP was analyzed for binding assay on unprocessed plasma as previously described (19). Plasma and serum were 236
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24-h PROFILE OF GH-BP found to yield identical GH binding. Briefly, 50 /xh plasma were incubated for 24 h with 1 ng [125I]human GH (hGH) with or without 1 ng unlabeled hGH. Bound hormone was separated from free hormone by adding 0.2% dextran-coated 2% charcoal. The intra- and interassay coefficients of variation were 1% and 4.5%, respectively. The assay sensitivity was 2% binding in 20 fiL plasma. Binding results were corrected for endogenous GH bound to plasma GH-BP, based on the plasma GH level at each time point and its capability to displace [125I]hGH from control normal human plasma (13, 19). Figure 1 presents the displacement curve of [125I]hGH binding by unlabeled hGH in normal human serum. To correct the GH-BP levels, the measured binding was divided by the fraction of displacement. With 10 ng/mL (0.5 ng/0.05 mL plasma in the assay tube), [125I]hGH binding was displaced to 0.88. A GH-BP noted at 10%/0.05 mL plasma would be corrected to 11.36%/0.05 mL. Pulse analysis Pulse analysis of both GH and GH-BP was performed by the Pulsar program (20), using 12 h as the smoothing time. A point in the scaled residual series was accepted as forming a peak if it was higher than the cut-off value (GJ of 3.98 or if it belonged to a string of adjacent points higher than the following cut-off parameters: G2, 2.4; G3, 1.7; G4, 1.2; and G5, 0.9. For each pulse, the height above the zero line, the residual amplitude above the calculated smoothed area baseline, and the area under the curve were calculated. Results
The individual results of plasma GH and GH-BP 24h profiles are illustrated in Fig. 2. The vast majority of the GH pulses were accompanied by GH-BP pulses. A GH-BP peak occurred within 30 min of a GH peak in 6 of 12, 6 of 13, 8 of 11, and 9 of 11 peaks in patients AD, respectively. When only GH peak amplitudes greater than 5 ng/mL were examined, a GH-BP peak followed within 30 min in 5 of 7, 6 of 7, 7 of 8, and 6 of 7 peaks, respectively. The pulse analysis is summarized in Table 1. The number of GH pulses in 24 h ranged from 11-13, and that of GH-BP pulses ranged from 9-12 (P > 0.1), with respective pulse frequencies of 0.38-0.55 and 0.460.52 pulses/h (P > 0.1). Next we correlated all plasma GH levels to GH-BP
237
levels. To minimize the noise of pulsatility, calculations were made on the residual series above the smoothed baseline of both GH and GH-BP. The correlation coefficient of all 172 individual samples was r = 0.447 (P < 0.001). Discussion The pulsatile nature of GH secretion has been shown to be advantageous for growth (7) as well as for maximal stimulation of insulin-like growth factor-I secretion (21). In the rat model, GH pulsatility has been shown to be followed by harmonious cycles of GH receptor turnover (8-10). In the rat, each wave of GH surge and receptor internalization has been shown to be accompanied by a wave of GH-BP increase as well as insulin-like growth factor-I secretion (16, 17), and in the mouse GH-BP has been shown to be regulated by GH (22). It has been suggested that in the course of the GH receptor internalization, the GH-BP is cleaved from the receptor and shed into the circulation (15). This, however, has not been conclusively proven. We now show that in healthy human subjects, GH-BP in plasma pulsates in close correlation with the pulsation of plasma GH levels. The pulsatile nature of serum GHBP requires an appropriate correction of serum GH levels to account for the displacement of binding by the endogenous ligand (1). As a result of the small plasma volume in our assay, the extent of this correction was minimal. Whether these pulses of GH-BP resulted from interaction of GH with its plasma membrane receptors cannot be conclusively deduced from these preliminary data. In a study of GH receptor internalization in rat liver, the process was shown to occur within several minutes (23). Our sampling procedure with 30-min intervals did not allow us to identify this timing gap. If indeed GH-BP is shed by cleavage from the receptor (15), the increase in serum GH-BP would be instantaneous and synchronous with the GH peak levels. Whereas the pulsatility of GH-BP and serum GH levels are in significant synchrony, the harmony is imperfect. Indeed, some turnover of the GH receptor does occur in the absence of GH, although at a slower pace
TABLE 1. Pulse analysis of GH and GH-BP in four normally growing children Subject 2
Subject 1 No. of peaks Mean amplitude Mean ht Pulse frequency Area under curve Mean area under pulse Integrated cone.
GH
GH-BP
GH
GH-BP
11 8.6 8.9
12 2.7
12 5.4 5.9
11 1.3 9.2
0.48 193
13.3 0.52
14.0
562 8.6
4.4
12.2
0.51 130 8.0 2.7
Subject 4
Subject 3
0.46 409 2.9 8.7
GH 11
10.9 12.8 0.38 304
23.8 6.7
GH-BP
GH
GH-BP
9 1.6 9.4
13 8.9 9.5
12 2.7
0.47 400
10.5 8.5
0.55 214
13.2 4.5
GH units are nanograms per mL or nanograms per h/mL (for area under curve or pulse). BP units are percentage per 50 percentage per h/50 fiL plasma (for area under curve or pulse).
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11.7 0.51 483 4.2 9.1
plasma or
HOCHBERG, AMIT, AND ZADIK
238
Q
m2 o 5 C8 o. z to o
x S
O < JC u.
0.01
10
100
hGH ng/tube 125
FIG. 1. Displacement curve of [ I]hGH binding by unlabeled hGH in 0.1 mL normal human serum (mean ± SEM; n = 3). The measured hGH levels in the untreated serum were less than 0.2 ng/tube.
(10). These results are in disagreement with a recent study that failed to detect GH-BP pulsatility (24). This study used an incomplete separation method of GH-BP determination, obtained under nonequilibrium conditions, which was chosen to compromise precision over ease of
JCE&MM991 Vol 72 • No 1
performance. The resultant wide range of the assay confidence limits might have masked GH-BP pulsatility. Moreover, these results were not compared to the pulsatility of GH. When studying young healthy individuals, one would expect several GH pulses over the 24-h period, yet the researchers deliberately selected patients with relatively low GH levels. Finally, it is to be expected that hourly measurements will miss pulses that can be detected by half-hourly sampling as in our study. The binding assay employed here for measurement of GH-BP in human plasma has been shown to reach half its maximal specific binding within 100 min at 4 C (19). The in vivo binding at 37 C is much faster, with a ti /2 of 6 min and maximal binding that is only about half of that at the lower temperatures (19), presumably as a result of degradation of the BP or dissociation of the complex at the physiological temperature. In the present study each time point was the result of continuous sampling of blood over 30 min. During these 30 min, the BP and the endogenous GH-BP complex were under conditions of relatively rapid association-dissociation at 37 C in vivo. This was followed by 1-2 h of binding to the endogenous hormone in the test tube at room tempera-
1
s
• fi
T YYft
l » B H K I » 8 i t 2 l ( l
CLOCK TIME FIG.
k
i1 % f
! « l 2 H « l l » 2 » !
CLOCK TIME
2. Twenty-four-hour profiles of GH (shaded area) and GH-BP (solid line) of four healthy children, 9.5-11.5 yr of age.
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24-h PROFILE OF GH-BP ture before plasma separation and frozen storage. We previously reported a dissociation t1/2 of 31 min under these conditions (19). The plasma was then frozen, and the binding assay was later run at 4 C. In calculating BP results, the endogenously bound GH was accounted for by correcting the measured BP for its displacement capability. Thus, the results shown here indicate both freeBP and GH-bound BP. When GH-BP was linked to a stable complex with hGH, it was found to slow down the hGH disappearance rate 10-fold compared to that of free hGH (25). We now show rapid fluctuations of the GHBP. This complex situation, in the absence of further knowledge of the GH-BP disappearance rate, does not permit deconvolution of the data for calculation of discrete pulse bursts of BP. Future deconvolution calculations will enable understanding of the chronological and causal relationships of hGH and GH-BP. Previously reported calculations of discrete GH secretory bursts by simultaneous multiple parameter deconvolution failed to include the BP association-dissociation with GH and its differential t1/2 in vivo at 37 C, at room temperature, and in the assay test tube at 4 C (26). The relationship between GH-BP and the GH receptor has been only indirectly suggested. To reveal the actual relevance of BP pulsatility to GH receptor turnover, this relationship will have to be evaluated in other physiological and pathological states of the GH receptor. The synchronized increase in GH-BP may buffer GH pulsatility by way of its interference with the rapid GH clearance (25). GH-BP back-regulates GH binding to its receptor, thus moderating the biological effect of the GH pulse (27). Acknowledgment We thank Miss Ruth Singer for expert secretarial assistance.
References 1. Finkelstein JW, Roffwarg HP, Boyar RM, Kream J, Hellman L. Age-related changes in the twenty-four-hour spontaneous secretion of growth hormone. J Clin Endocrinol Metab. 1972;35:665-70. 2. Plotnick LP, Thompson RG, Kowarski A, de Lacerda L, Migeon CJ, Blizzard RM. Circadian variation of integrated concentration of growth hormone in children and adults. J Clin Endocrinol Metab. 1975;40:240-7. 3. Miller JD, Tannenbaum GS, Colle E, Guyda HJ. Daytime pulsatile growth hormone secretion during childhood and adolescence. J Clin Endocrinol Metab. 1982;55:989-94. 4. Spiliotis BE, August GP, Hung W, Sonis W, Mendelson W, Bercu BB. Growth hormone neuroscretory dysfunction: a treatable cause of short stature. JAMA. 1984;251:2223-30. 5. Zadik Z, Chalew SA, Raiti S, Kowarski AA. Do short children secrete insufficient growth hormone? Pediatrics. 1985;76:355-6O. 6. Albertsson-Wikland K, Rosberg S. Analyses of 24-hour growth hormone profiles in children: relation to growth. J Clin Endocrinol Metab. 1988;67:493-500.
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7. Clark RG, Jansson FO, Isaksson OGP, Robinson ICAF. Intravenous growth hormone response to patterned infusions in hypophysectomized rats. J Endocrinol. 1985;2O4:53-61. 8. Bick T, Youdim MBH, Hochberg Z. Adaptation of liver membrane somatogenic and lactogenic growth hormone (GH) binding to the spontaneous pulsation of GH secretion. Endocrinology. 1989;125:1711-7. 9. Bick T, Youdim MBH, Hochberg Z. The dynamics of somatogenic and lactogenic growth hormone binding: internalization to Golgi fractions in the male rat. Endocrinology. 1989;125:1718-23. 10. Roupas P, Herington AC. Cellular mechanisms in the processing of growth hormone and its receptor. Mol Cell Endocrinol. 1989;61:1-12. 11. Ymer SI, Herington AC. Evidence for the specific binding of growth hormone to a receptor-like protein in rabbit serum. Mol Cell Endocrinol. 1985;41:153-61. 12. Leung DW, Spencer SA, Cachianes G, et al. Growth hormone receptor and serum-binding protein: purification, cloning, and expression. Nature. 1987;330:537-43. 13. Daughaday WH, Trivedi B. Absence of serum growth hormonebinding protein in patients with growth hormone receptor deficiency (Laron dwarfism). Proc Natl Acad Sci USA. 1987;84:463640. 14. Baumann G, Melissa A, Shaw BS, Merimee TJ. Low levels of highaffinity growth hormone-binding protein in African pygmies. N Engl J Med. 1989;320:1705-9. 15. Trivedi B, Daughaday WH. Release of growth hormone-binding protein from IM-9 lymphocytes by endopeptidase is dependent on sulfydryl group inactivation. Endocrinology. 1988;123:2201-6. 16. Bick T, Amit T, Barkey RJ, Hertz P, Youdim MBH, Hochberg Z. The interrelationship of growth hormone (GH), liver membrane GH receptor, serum GH-binding protein activity, and insulin-like growth factor I (IGF-I) in the male rat. Endocrinology. 1990;126:1914-20. 17. Hochberg Z, Bick T, Amit T, Barkey RJ, Youdim MBH. Regulation by growth hormone (GH) of the GH-receptor turnover. Acta Paediatr Scand. 1990;(Suppl)367:148-52. 18. Strasburger C, Barnard G, Toldo L, Zarmi B, Zadik Z, Kohen F. Somatotropin as measured by a two-site time-resolved immunofluorometric assay. Clin Chem. l989;35:913-7. 19. Amit T, Barkey RJ, Youdim MBH, Hochberg Z. A new and convenient assay of growth hormone-binding protein in human serum. J Clin Endocrinol Metab. 1990;71:474-9. 20. Merriam GR, Wachter KW. Algorithms for the study of episodic hormone secretion. Am J Physiol. 1982;243:E310-8. 21. Maiter D, Underwood LE, Maes M, Davenport ML, Ketelslegers JM. Different effects of intermittent and continuous growth hormone (GH) administration on serum somatomedin C/insulin-like growth factor I and liver GH receptors in hypophysectomized mice. Endocrinology. 1988;123:1053-9. 22. Sanchez-Jimenez F, Fielder PJ, Martinez RR, Smith WC, Talamantes F. Hypophysectomy eliminates and growth hormone (GH) maintains the midpregnancy elevation in GH receptor and serum binding protein in the mouse. Endocrinology. 1990;126:1270-5. 23. Postel-Vinay MC, Kayser C, Desbuquois B. Fate of injected human growth hormone in the female liver in vivo. Endocrinology. 1982;111:244-51. 24. Snow KJ, Shaw MA, Winer LM, Baumann G. Diurnal pattern of plasma growth hormone-binding protein in man. J Clin Endocrinol Metab. 1990;70:417-20. 25. Baumann G, Shaw MA, Buchanan TA. In vivo kinetics of a covalent growth hormone-binding protein complex. Metabolism. 1989;38:330-3. 26. Veldhuis JD, Carlson ML, Johnson ML. The pituitary gland secretes in bursts: appraising the nature of glandular secretory impulses by simultaneous multiple-parameter deconvolution of plasma hormone concentrations. Proc Natl Acad Sci USA. 1987;84:7686-90. 27. Herington AC, Ymer S, Stevenson J. Identification and characterization of specific binding proteins for growth hormone in normal human sera. J Clin Invest. 1986;77:1817-23.
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Vol. 72, No. 1 Printed in U.S.A.
Twenty-Four-Hour Profile of Plasma Growth Hormone-Binding Protein ZEEV HOCHBERG, TAMAR AMIT, AND ZVI ZADIK Department of Pharmacology, Faculty of Medicine, and Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology (Z.H., T.A.), Haifa; the Pediatric Endocrine Unit, Kaplan Hospital (Z.Z.), Rehovot; and the Department of Pediatrics, Rambam Medical Center (Z.H.), Haifa, Israel
ABSTRACT. In experimental animals each burst of GH pulse is followed by a wave of receptor turnover and an increase in serum GH-binding protein (GH-BP), which occurs 60 min after the GH peak. The present report describes the 24-h profile of plasma GH-BP and its correlation to GH pulsatility in normal individuals. Four normally growing children in early puberty were the subjects of this study. Blood was withdrawn continuously for 24 h in 30-min fractions. Pulse analysis of both GH and GH-BP was performed by the Pulsar program. The vast
majority of the GH pulses were accompanied by GH-BP pulses within 30 min. Correlation of plasma GH levels to GH-BP levels on the residual series above the smoothed baseline of all 172 individual samples was r = 0.447 (P < 0.001). Thus, plasma GHBP levels fluctuate rapidly in relation to the pulsatility of plasma GH levels. This may influence the GH disappearance rate and brings into question some of the deconvolution calculations of GH secretory impulses. (J Clin Endocrinol Metab 72: 236-239, 1991)
T
HE PITUITARY signals its target organs by intermittent bursts of GH secretion that result from a complex interaction of hypothalamic GH-releasing and inhibiting hormones (1-3). This pattern of GH secretion is an important determinant of the target organs' responses. Thus, impaired pulsatility results in growth retardation, a condition that is now recognized as neurosecretory dysfunction (4, 5), and the growth of normal children has been found to bear a close relationship to this 24-h GH-secretion profile (6). In experimental animals the advantage to the growth process of multiple daily bursts of GH has been documented (7). It has been shown in the rat that this may be mediated by a harmoniously timed turnover cycle of the GH receptors, which down-regulate and up-regulate in concert with the pulsatility of serum GH (8). The immediate decrease in the plasma membrane receptors, induced by the binding of the hormones, is followed by internalization of the hormone-receptor complex to the Golgi apparatus and degradative lysosomes (9, 10). From early reports on serum GH-binding protein (GHBP), a relation to the GH receptor was suspected (11). Indeed, the amino-terminal amino acid sequences of both the GH receptor and the serum GH-BP in the rabbit are identical, the latter comprising the extracellalar domain of the receptor (12). In a variety of clinical conditions
the GH-BP correlates closely with GH receptor-binding capacity (13,14). It has been proposed that upon binding of GH to its receptor, either before or after internalization of the complex, the GH-BP is cleaved off of the receptor and shed into the circulation (15). In experimental animals each wave of GH pulse and receptor turnover is followed by an increase in serum GH-BP, which occurs 60 min after the GH peak (16, 17). The present report describes the 24-h profile of plasma GH-BP and its correlation to GH pulsatility in normal individuals.
Materials and Methods Subjects Four normally growing children, two males and two females, in early puberty (Tanner stage 2) were the subjects of this study (Table 1). Blood was withdrawn continuously for 24 h through a venous catheter and a peristatic pump in 30-min fractions, starting at 0800-0900 h, into heparinized test tubes.
GHRIA Plasma GH was assayed in duplicate by an immunofluorometric assay (18). The sensitivity of the assay was 0.1 ng/mL, and the intra- and interassay coefficients of variation were less than 6% and 10%, respectively. GH-BP
Received April 4,1990. Address requests for reprints to: Zeev Hochberg, M.D., D.Sc, Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, P.O.B. 9697, 31096 Haifa, Israel.
GH-BP was analyzed for binding assay on unprocessed plasma as previously described (19). Plasma and serum were 236
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24-h PROFILE OF GH-BP found to yield identical GH binding. Briefly, 50 /xh plasma were incubated for 24 h with 1 ng [125I]human GH (hGH) with or without 1 ng unlabeled hGH. Bound hormone was separated from free hormone by adding 0.2% dextran-coated 2% charcoal. The intra- and interassay coefficients of variation were 1% and 4.5%, respectively. The assay sensitivity was 2% binding in 20 fiL plasma. Binding results were corrected for endogenous GH bound to plasma GH-BP, based on the plasma GH level at each time point and its capability to displace [125I]hGH from control normal human plasma (13, 19). Figure 1 presents the displacement curve of [125I]hGH binding by unlabeled hGH in normal human serum. To correct the GH-BP levels, the measured binding was divided by the fraction of displacement. With 10 ng/mL (0.5 ng/0.05 mL plasma in the assay tube), [125I]hGH binding was displaced to 0.88. A GH-BP noted at 10%/0.05 mL plasma would be corrected to 11.36%/0.05 mL. Pulse analysis Pulse analysis of both GH and GH-BP was performed by the Pulsar program (20), using 12 h as the smoothing time. A point in the scaled residual series was accepted as forming a peak if it was higher than the cut-off value (GJ of 3.98 or if it belonged to a string of adjacent points higher than the following cut-off parameters: G2, 2.4; G3, 1.7; G4, 1.2; and G5, 0.9. For each pulse, the height above the zero line, the residual amplitude above the calculated smoothed area baseline, and the area under the curve were calculated. Results
The individual results of plasma GH and GH-BP 24h profiles are illustrated in Fig. 2. The vast majority of the GH pulses were accompanied by GH-BP pulses. A GH-BP peak occurred within 30 min of a GH peak in 6 of 12, 6 of 13, 8 of 11, and 9 of 11 peaks in patients AD, respectively. When only GH peak amplitudes greater than 5 ng/mL were examined, a GH-BP peak followed within 30 min in 5 of 7, 6 of 7, 7 of 8, and 6 of 7 peaks, respectively. The pulse analysis is summarized in Table 1. The number of GH pulses in 24 h ranged from 11-13, and that of GH-BP pulses ranged from 9-12 (P > 0.1), with respective pulse frequencies of 0.38-0.55 and 0.460.52 pulses/h (P > 0.1). Next we correlated all plasma GH levels to GH-BP
237
levels. To minimize the noise of pulsatility, calculations were made on the residual series above the smoothed baseline of both GH and GH-BP. The correlation coefficient of all 172 individual samples was r = 0.447 (P < 0.001). Discussion The pulsatile nature of GH secretion has been shown to be advantageous for growth (7) as well as for maximal stimulation of insulin-like growth factor-I secretion (21). In the rat model, GH pulsatility has been shown to be followed by harmonious cycles of GH receptor turnover (8-10). In the rat, each wave of GH surge and receptor internalization has been shown to be accompanied by a wave of GH-BP increase as well as insulin-like growth factor-I secretion (16, 17), and in the mouse GH-BP has been shown to be regulated by GH (22). It has been suggested that in the course of the GH receptor internalization, the GH-BP is cleaved from the receptor and shed into the circulation (15). This, however, has not been conclusively proven. We now show that in healthy human subjects, GH-BP in plasma pulsates in close correlation with the pulsation of plasma GH levels. The pulsatile nature of serum GHBP requires an appropriate correction of serum GH levels to account for the displacement of binding by the endogenous ligand (1). As a result of the small plasma volume in our assay, the extent of this correction was minimal. Whether these pulses of GH-BP resulted from interaction of GH with its plasma membrane receptors cannot be conclusively deduced from these preliminary data. In a study of GH receptor internalization in rat liver, the process was shown to occur within several minutes (23). Our sampling procedure with 30-min intervals did not allow us to identify this timing gap. If indeed GH-BP is shed by cleavage from the receptor (15), the increase in serum GH-BP would be instantaneous and synchronous with the GH peak levels. Whereas the pulsatility of GH-BP and serum GH levels are in significant synchrony, the harmony is imperfect. Indeed, some turnover of the GH receptor does occur in the absence of GH, although at a slower pace
TABLE 1. Pulse analysis of GH and GH-BP in four normally growing children Subject 2
Subject 1 No. of peaks Mean amplitude Mean ht Pulse frequency Area under curve Mean area under pulse Integrated cone.
GH
GH-BP
GH
GH-BP
11 8.6 8.9
12 2.7
12 5.4 5.9
11 1.3 9.2
0.48 193
13.3 0.52
14.0
562 8.6
4.4
12.2
0.51 130 8.0 2.7
Subject 4
Subject 3
0.46 409 2.9 8.7
GH 11
10.9 12.8 0.38 304
23.8 6.7
GH-BP
GH
GH-BP
9 1.6 9.4
13 8.9 9.5
12 2.7
0.47 400
10.5 8.5
0.55 214
13.2 4.5
GH units are nanograms per mL or nanograms per h/mL (for area under curve or pulse). BP units are percentage per 50 percentage per h/50 fiL plasma (for area under curve or pulse).
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11.7 0.51 483 4.2 9.1
plasma or
HOCHBERG, AMIT, AND ZADIK
238
Q
m2 o 5 C8 o. z to o
x S
O < JC u.
0.01
10
100
hGH ng/tube 125
FIG. 1. Displacement curve of [ I]hGH binding by unlabeled hGH in 0.1 mL normal human serum (mean ± SEM; n = 3). The measured hGH levels in the untreated serum were less than 0.2 ng/tube.
(10). These results are in disagreement with a recent study that failed to detect GH-BP pulsatility (24). This study used an incomplete separation method of GH-BP determination, obtained under nonequilibrium conditions, which was chosen to compromise precision over ease of
JCE&MM991 Vol 72 • No 1
performance. The resultant wide range of the assay confidence limits might have masked GH-BP pulsatility. Moreover, these results were not compared to the pulsatility of GH. When studying young healthy individuals, one would expect several GH pulses over the 24-h period, yet the researchers deliberately selected patients with relatively low GH levels. Finally, it is to be expected that hourly measurements will miss pulses that can be detected by half-hourly sampling as in our study. The binding assay employed here for measurement of GH-BP in human plasma has been shown to reach half its maximal specific binding within 100 min at 4 C (19). The in vivo binding at 37 C is much faster, with a ti /2 of 6 min and maximal binding that is only about half of that at the lower temperatures (19), presumably as a result of degradation of the BP or dissociation of the complex at the physiological temperature. In the present study each time point was the result of continuous sampling of blood over 30 min. During these 30 min, the BP and the endogenous GH-BP complex were under conditions of relatively rapid association-dissociation at 37 C in vivo. This was followed by 1-2 h of binding to the endogenous hormone in the test tube at room tempera-
1
s
• fi
T YYft
l » B H K I » 8 i t 2 l ( l
CLOCK TIME FIG.
k
i1 % f
! « l 2 H « l l » 2 » !
CLOCK TIME
2. Twenty-four-hour profiles of GH (shaded area) and GH-BP (solid line) of four healthy children, 9.5-11.5 yr of age.
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24-h PROFILE OF GH-BP ture before plasma separation and frozen storage. We previously reported a dissociation t1/2 of 31 min under these conditions (19). The plasma was then frozen, and the binding assay was later run at 4 C. In calculating BP results, the endogenously bound GH was accounted for by correcting the measured BP for its displacement capability. Thus, the results shown here indicate both freeBP and GH-bound BP. When GH-BP was linked to a stable complex with hGH, it was found to slow down the hGH disappearance rate 10-fold compared to that of free hGH (25). We now show rapid fluctuations of the GHBP. This complex situation, in the absence of further knowledge of the GH-BP disappearance rate, does not permit deconvolution of the data for calculation of discrete pulse bursts of BP. Future deconvolution calculations will enable understanding of the chronological and causal relationships of hGH and GH-BP. Previously reported calculations of discrete GH secretory bursts by simultaneous multiple parameter deconvolution failed to include the BP association-dissociation with GH and its differential t1/2 in vivo at 37 C, at room temperature, and in the assay test tube at 4 C (26). The relationship between GH-BP and the GH receptor has been only indirectly suggested. To reveal the actual relevance of BP pulsatility to GH receptor turnover, this relationship will have to be evaluated in other physiological and pathological states of the GH receptor. The synchronized increase in GH-BP may buffer GH pulsatility by way of its interference with the rapid GH clearance (25). GH-BP back-regulates GH binding to its receptor, thus moderating the biological effect of the GH pulse (27). Acknowledgment We thank Miss Ruth Singer for expert secretarial assistance.
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