0021-972X/90/7002-0417$2.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 70, No. 2 Printed in U.S.A.
KENNETH J. SNOW, MELISSA A. SHAW, LORI M. WINER, AND -GERHARD BAUMANN Center for Endocrinology, Metabolism, and Nutrition, Department of Medicine, Northwestern University Medical School, Chicago, Illinois 60611
over a 24-h period in six normal adults (three men and three women). A standardized GH binding assay, employing incubation of plasma with [125I]GH and separation of bound from free GH by gel filtration, was used. GH-BP activity showed no significant diurnal variation in any subject. The average GHBP activity was similar in all subjects, although statistically significant differences were found between some subjects. No age- or sex-related differences were identified. We conclude that in normal man plasma GH-BP activity is constant throughout the day, thereby implying 1) constancy of binding protein (and possibly GH receptor) concentration, and 2) absence of significant fluctuations in potential binding inhibitors/enhancers in plasma. (J Clin Endocrinol Metab 70: 417, 1990)
ABSTRACT. The identification of specific GH-binding proteins (GH-BP) in human plasma, one of which is a fragment of the GH receptor, has added new complexity to the state of GH in the circulation. A major proportion of GH circulates in complexed form, which differs in kinetics and possibly bioactivity from free GH. Little is known about the regulation of the GH-BP, their constancy or variation in plasma, or plasma factors affecting GH binding. Consequently, the temporal pattern of bound and free GH in plasma is also unknown. Knowledge about possible spontaneous variability in GH-BP levels/ activity is required for physiological investigations and comparative studies among different populations. To address these issues, we measured GH-binding activity in plasma every hour
A
SPECIFIC, high affinity GH-binding protein (GHBP) in human plasma has recently been discovered (1, 2). This binding protein (BP) is a fragment of the GH receptor (3, 4) and complexes a substantial portion of circulating GH (5). A second, low affinity BP has also been identified (6); it complexes a small minority of GH (1, 7). The BPs act to prolong the biological half-life of GH, dampen the oscillations of plasma GH levels caused by episodic pituitary secretion (8, 9), and may have other, as yet unknown, effects on GH action (10). Little is presently known about the the factors regulating the generation of BP or about the respective roles of bound and free GH in growth promotion. Cross-sectional studies of plasma GH-binding activity, performed on samples collected at random times, have identified marked variability among normal subjects (7, 11). Several factors could contribute to changes in GHbinding activity, such as diurnal variation in BP levels, fluctuating plasma constituents that inhibit or enhance binding (e.g. alimentary or metabolic factors), and intermittent high GH levels resulting in partial saturation of Received July 24, 1989. Address requests for reprints to: Dr. G. Baumann, 303 East Chicago Avenue, Chicago, Illinois 60611. * This work was supported by NIH Grants DK-38128, DK-07169, RR-05370, and RR-48.
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the BP (the latter was corrected for in the studies cited). Although large plasma proteins, such as the GH-BP, are not generally known to vary over short periods of time (minutes to hours), the derivation of the high affinity BP from the GH receptor places it in a special category. Moreover, marked diurnal variation has been demonstrated for at least one peptide hormone-BP, the small insulin-like growth factor BP (12). To develop a normative data base on BP levels, bound and free GH concentrations, and appropriate conditions for blood sampling, these issues need to be addressed. In the present study we have examined the circadian variation of plasma GH-binding activity in normal subjects under physiological conditions. The results indicate that GH-BP activity does not vary over 24 h. Materials and Methods Subjects Heparinized blood (3 mL) was obtained from three normal lean men (aged 26-46 yr) and three normal lean women (aged 22-26 yr) every 20 min during a 24-h sojourn at the Clinical Research Center. The study had been approved by the Northwestern University Review Board for human investigation, and all subjects gave informed consent. Regular meals, sleep, and ambulatory activity were maintained. Blood was collected through an indwelling forearm catheter kept open with a slow
Downloaded from https://academic.oup.com/jcem/article-abstract/70/2/417/2280819 by UMass Amherst Libraries user on 10 February 2019
Diurnal Pattern of Plasma Growth Hormone-Binding Protein in Man*
SNOW ET AL.
418
saline infusion; it was centrifuged immediately after collection, and plasma was stored at -20 C until assayed. Hourly plasma samples were selected for assay. Endogenous GH was determined by RIA to allow for effects of BP saturation. The six subjects were selected for inclusion in this study based on their relatively low GH levels, thereby minimizing the need for correction.
Human (h) GH was obtained and radiolabeled as previously described (1). The purity of monomeric [125I]hGH was assured by two sequential gel filtration steps (5) and by using only material within 4 days of repurification. Deiodination and spontaneous aggregation are minimal under these conditions (
Vol. 70, No. 2 Printed in U.S.A.
KENNETH J. SNOW, MELISSA A. SHAW, LORI M. WINER, AND -GERHARD BAUMANN Center for Endocrinology, Metabolism, and Nutrition, Department of Medicine, Northwestern University Medical School, Chicago, Illinois 60611
over a 24-h period in six normal adults (three men and three women). A standardized GH binding assay, employing incubation of plasma with [125I]GH and separation of bound from free GH by gel filtration, was used. GH-BP activity showed no significant diurnal variation in any subject. The average GHBP activity was similar in all subjects, although statistically significant differences were found between some subjects. No age- or sex-related differences were identified. We conclude that in normal man plasma GH-BP activity is constant throughout the day, thereby implying 1) constancy of binding protein (and possibly GH receptor) concentration, and 2) absence of significant fluctuations in potential binding inhibitors/enhancers in plasma. (J Clin Endocrinol Metab 70: 417, 1990)
ABSTRACT. The identification of specific GH-binding proteins (GH-BP) in human plasma, one of which is a fragment of the GH receptor, has added new complexity to the state of GH in the circulation. A major proportion of GH circulates in complexed form, which differs in kinetics and possibly bioactivity from free GH. Little is known about the regulation of the GH-BP, their constancy or variation in plasma, or plasma factors affecting GH binding. Consequently, the temporal pattern of bound and free GH in plasma is also unknown. Knowledge about possible spontaneous variability in GH-BP levels/ activity is required for physiological investigations and comparative studies among different populations. To address these issues, we measured GH-binding activity in plasma every hour
A
SPECIFIC, high affinity GH-binding protein (GHBP) in human plasma has recently been discovered (1, 2). This binding protein (BP) is a fragment of the GH receptor (3, 4) and complexes a substantial portion of circulating GH (5). A second, low affinity BP has also been identified (6); it complexes a small minority of GH (1, 7). The BPs act to prolong the biological half-life of GH, dampen the oscillations of plasma GH levels caused by episodic pituitary secretion (8, 9), and may have other, as yet unknown, effects on GH action (10). Little is presently known about the the factors regulating the generation of BP or about the respective roles of bound and free GH in growth promotion. Cross-sectional studies of plasma GH-binding activity, performed on samples collected at random times, have identified marked variability among normal subjects (7, 11). Several factors could contribute to changes in GHbinding activity, such as diurnal variation in BP levels, fluctuating plasma constituents that inhibit or enhance binding (e.g. alimentary or metabolic factors), and intermittent high GH levels resulting in partial saturation of Received July 24, 1989. Address requests for reprints to: Dr. G. Baumann, 303 East Chicago Avenue, Chicago, Illinois 60611. * This work was supported by NIH Grants DK-38128, DK-07169, RR-05370, and RR-48.
417
the BP (the latter was corrected for in the studies cited). Although large plasma proteins, such as the GH-BP, are not generally known to vary over short periods of time (minutes to hours), the derivation of the high affinity BP from the GH receptor places it in a special category. Moreover, marked diurnal variation has been demonstrated for at least one peptide hormone-BP, the small insulin-like growth factor BP (12). To develop a normative data base on BP levels, bound and free GH concentrations, and appropriate conditions for blood sampling, these issues need to be addressed. In the present study we have examined the circadian variation of plasma GH-binding activity in normal subjects under physiological conditions. The results indicate that GH-BP activity does not vary over 24 h. Materials and Methods Subjects Heparinized blood (3 mL) was obtained from three normal lean men (aged 26-46 yr) and three normal lean women (aged 22-26 yr) every 20 min during a 24-h sojourn at the Clinical Research Center. The study had been approved by the Northwestern University Review Board for human investigation, and all subjects gave informed consent. Regular meals, sleep, and ambulatory activity were maintained. Blood was collected through an indwelling forearm catheter kept open with a slow
Downloaded from https://academic.oup.com/jcem/article-abstract/70/2/417/2280819 by UMass Amherst Libraries user on 10 February 2019
Diurnal Pattern of Plasma Growth Hormone-Binding Protein in Man*
SNOW ET AL.
418
saline infusion; it was centrifuged immediately after collection, and plasma was stored at -20 C until assayed. Hourly plasma samples were selected for assay. Endogenous GH was determined by RIA to allow for effects of BP saturation. The six subjects were selected for inclusion in this study based on their relatively low GH levels, thereby minimizing the need for correction.
Human (h) GH was obtained and radiolabeled as previously described (1). The purity of monomeric [125I]hGH was assured by two sequential gel filtration steps (5) and by using only material within 4 days of repurification. Deiodination and spontaneous aggregation are minimal under these conditions (
