Heterogeneity of circulating growth hormone.

Human growth hormone (GH) represents a family of related proteins arising from two genes, alternative mRNA splicing, and several post-translational modifications. In addition, post-secretory events occur when GH enters the circulation. The full scale of GH heterogeneity is only beginning to be appreciated, and new GH forms or related proteins may be discovered in the future. GH measurements are affected by GH heterogeneity. Immunoassays are influenced by the mixture of GH variants, but are not sensitive to GH binding proteins (GHBPs). In contrast, radioreceptor assays are sensitive to both GH variant mixtures and to the high affinity GHBP. It is hoped that in the future, these problems can be minimized by rigorous characterization of existing antibodies with respect to epitope recognition on various GH forms, and ultimately, by production of GH variant-specific antibodies that permit direct and individual assessment of the circulating members of the GH family.

Regulation of plasma growth hormone-binding proteins in health and disease.

Binding proteins (BP) for growth hormone (GH) have recently been discovered in human plasma. The main BP is related to the GH receptor and probably corresponds to the extracellular portion of the receptor. The BP influence several aspects of GH homeostasis and action. Their level and activity in blood, therefore, become important variables in overall GH physiology. However, to date little is known about the regulation of GH-BP in health and disease. To gain initial information about this point, GH-BP activity was examined in the plasma of 124 subjects with various physiologic and pathologic conditions. The conditions were selected to provide basic physiologic data (men, women, children, age, pregnancy and to investigate key disease states attended by abnormal GH physiology (liver cirrhosis, uremia, infection, acromegaly). A standardized GH binding assay was used to measure BP activity as an index of BP levels. Both the principal, high affinity BP (peak II) and the minor, low affinity BP (peak I) showed considerable individual variation in all groups. Neonates had the lowest levels of both BPs, but by the age of 1 year the levels had increased and remained fairly stable through the seventh decade. In males but not females between the ages of 1 and 20 years, the main (peak II) BP showed a slight upward trend, whereas the minor (peak I) BP declined moderately. Patients with cirrhosis showed the most variation in both BP, and uremic patients demonstrated decreased peak II, but not peak I, binding. Neither BP was affected in acromegaly. We conclude that BP activity in plasma is well conserved in most conditions, but substantial individual variability exists. BP activity increases dramatically during the first year of life.(ABSTRACT TRUNCATED AT 250 WORDS)

A rapid and simple assay for growth hormone-binding protein activity in human plasma.

The newly discovered circulating growth hormone binding proteins dictate a re-evaluation of the state of GH in plasma in health and disease as the binding proteins are known to affect GH metabolism and action. We describe a rapid and simple GH-binding assay that allows determination of free and complexed plasma GH, as well as GH-binding protein activity as an index of GH-binding protein levels, with relative ease. The method is based on incubation of plasma with 125I-GH and separation of bound from free GH on small DEAE-cellulose columns; it can be used on a large scale for routine determinations. The results obtained by this method are comparable to those obtained with the previously used slow and more cumbersome gel filtration technique. Initial data obtained in normal subjects and certain disease states show that the bound fraction of plasma GH is similar in men, women and children, is unaffected by pregnancy or acute infection, but is marginally decreased in liver cirrhosis. In acromegaly, binding protein activity also appears normal when allowance is made for partial saturation of the binding proteins by the high prevailing GH levels. The technique we describe should facilitate investigations of normal and abnormal regulation of the GH binding proteins.

The circulating growth hormone (GH)-binding protein complex: a major constituent of plasma GH in man.

The recent discovery of a specific binding protein for human GH (hGH) in human plasma suggests that hGH circulates in part as a complex in association with the binding protein(s). However, the magnitude of the complexed fraction prevailing under physiological conditions is unknown because of 1) dissociation of the complex during analysis and 2) potential differences in the binding characteristics of radiolabeled and native hGH. We conducted experiments designed to minimize dissociation during analysis (gel filtration in prelabeled columns, frontal analysis, and batch molecular sieving) with both native and radioiodinated hGH. All three methods yielded similar estimates for the complexed fraction. In normal plasma the bound fraction for 22 K hGH averaged 50.1% (range, 39-59%), that for 20 K hGH averaged 28.5% (range, 26-31%). Above a hGH level of about 20 ng/ml the bound fraction declines in concentration-dependent manner due to saturation of the binding protein. We conclude that a substantial part of circulating hGH is complexed with carrier proteins. This concept has important implications for the metabolism, distribution, and biological activity of hGH.

The effect of circulating growth hormone-binding protein on metabolic clearance, distribution, and degradation of human growth hormone.

We recently described a specific binding protein for human GH (hGH) in human plasma, with which a substantial portion of circulating hGH is complexed. The biological function of the complexed fraction is unknown. To test the hypothesis that complexed hGH may have different in vivo kinetics than free hGH, we compared the MCRs, distribution volumes (Vd), and degradation rates of complexed and free [125I] hGH in the rat. A partially purified GH-binding protein preparation, generated by affinity chromatography on a hGH column, was used for this purpose. A mixture of hGH with binding protein (equivalent to the amount contained in 0.9 mL human plasma) was injected iv as a single dose. Parallel experiments were conducted with hGH in the absence of binding protein. Disappearance of total, immunoprecipitable, and trichloroacetic acid-precipitable radioactivity from rat plasma was followed, and MCR, Vd, and degradation rates were derived by standard mathematical techniques. The MCR was 6-fold slower for complexed than for free hGH (2.3 vs. 14 mL/min X kg), Vd was 4-fold smaller for complexed hGH than for free hGH (71 vs. 256 mL/kg), and initial degradation rate was 4.5-fold lower for complexed than for free hGH (13.2% vs. 59.9%/15 min). The Vd of complexed hGH was close to the intravascular volume, while the Vd for free hGH corresponded to the extracellular volume. We conclude that one function of the hGH-binding protein is relative confinement of hGH to the vascular compartment, thereby protecting it from degradation and prolonging its biological half-life.

The autodecomposition of radiolabeled human growth hormone.

Human growth hormone (hGH) was radiolabeled with 125I, using a gentle lactoperoxidase technique. The stability and decomposition products of this tracer were studied by frequent periodic analysis by Sephadex G-100 chromatography on a long column. Monomeric 125I-hGH showed an exponential decline, with a half-life of 61 days. The main radioactive degradation product was iodide, which appeared with a fractional appearance rate of 0.01136 per day. Secondary degradation products were a series of radioactive oligomers of hGH, which appeared with an overall fractional rate of 0.00525 per day. The kinetic data obtained should provide guidelines for the shelf-life and repurification schedule of radioiodinated polypeptides.

A specific growth hormone-binding protein in human plasma: initial characterization.

Human (h) GH in plasma exists as a series of size isomers, which are in part explained by the presence of hGH oligomers. However, certain aspects of circulating large mol wt hGH, such as its high relative proportion compared to that in the pituitary, are poorly understood. To explore whether binding of hGH to plasma protein(s) could contribute to the phenomenon of large mol wt hGH, we incubated freshly prepared monomeric [125I]hGH or biosynthesized [3H]hGH with normal human plasma or serum at pH 7.4 for various time periods at 22 and 37 C. Plasma radioactive hGH patterns were then analyzed simultaneously with unincubated tracer hGH by Sephadex G-100 and G-200 chromatography. We found that part of the radioactivity was converted to a component with an apparent mol wt of 85,000, suggesting binding to a plasma protein(s). This phenomenon was inhibited in a dose-dependent fashion by unlabeled hGH. Saturation/Scatchard analysis indicated an association constant (Ka) of 2-3 X 10(8) M-1 and a maximum binding capacity of 20 ng hGH/ml plasma. Binding was rapid, reversible, and specific. A number of polypeptide hormones, including human placental lactogen, hPRL and rat GH, did not inhibit hGH binding. However, the 20K variant of hGH interacted weakly with the plasma binding component (Ka, 1.2 X 10(7) M-1; maximum binding capacity, 450 ng/ml). The binding component was heat labile and could be partially purified by gel permeation chromatography and affinity chromatography on a hGH-Sepharose column. Its estimated mol wt is 60,000-65,000, and it appears to bind one molecule of hGH to form a complex of 80,000-85,000 mol wt. The binding component is neither albumin nor an immunoglobulin. Under physiological conditions, a minimum of 15-18% of circulating hGH is presumably bound to this plasma component. We conclude that a specific, high affinity, low capacity binding protein for hGH with mol wt of 60,000-65,000 exists in normal and hypopituitary human plasma. hGH complexed with this protein forms part of big-big hGH. The biological significance of this binding protein remains to be investigated.

Molecular forms of circulating growth hormone during spontaneous secretory episodes and in the basal state.

After pharmacological stimulation of the pituitary gland, human GH (hGH) in plasma consists of three or more monomeric molecular forms and several corresponding oligomers. However, the chemical nature of hGH circulating under physiological (stimulated or basal) conditions is not known. In particular, the molecular basis for the GH-like bioactivity of plasma is poorly understood. To gain information on the type(s) of hGH normally found in blood, we extracted hGH from plasma obtained at the time of spontaneous secretory episodes (nocturnal and random release) and during basal periods in 15 normal subjects. Appropriate plasma volumes (30 or 300 ml) were extracted by immunoadsorbent chromatography, and the extracts were analyzed by native polyacrylamide gel electrophoresis at pH 7.5 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at pH 10. The plasma hGH pattern at the time of spontaneous secretion was similar to that after pharmacological stimulation and consisted of 22K (principal), 20K, and acidic hGH as well as hGH dimer. In contrast, plasma hGH patterns during basal periods were highly variable and included immunoreactive hGH fragments in addition to the known hGH forms. Components with mol wt of 30K, 16K, and 12K were consistently identified. We conclude that 1) endogenously stimulated hGH secretion results in the same plasma hormone patterns as pharmacological stimuli; 2) several immunoreactive hGH fragments contribute to the heterogeneity of plasma hGH; and 3) hGH fragments may become a dominant part of total immunoreactivity in the basal state.

Plasma "big" and "big-big" growth hormone (GH) in man: an oligomeric series composed of structurally diverse GH monomers.

Human GH (hGH) immunoreactivity in plasma can be separated into three species of different molecular size by gel filtration (little, big, and big-big hGH). In contrast to pituitary high mol wt GH forms, the molecular nature of the big hGH forms in blood is not known. Therefore, we purified these hGH size isomers from the plasma of L-dopa-stimulated normal subjects and acromegalic patients. Plasma was chromatographed on Sephadex G-100, and fractions containing big-big, big, and little hGH were generated. hGH was extracted and concentrated from these fractions by immunoadsorbent chromatography and analyzed by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-PAGE, and isoelectric focusing. The resulting gel profiles indicated that the majority (70%) of big and big-big hGH was converted to little hGH during extraction and storage. The remainder migrated as distinct species with mol wt of approximately 45, 62, 80, and 110 K in sodium dodecyl sulfate-PAGE. These forms could be converted almost completely to little hGH by sulfhydryl reduction. Little hGH (both native and converted from big forms) was composed of several monomeric hGH species, namely the 22 K form (principal), the 20 K variant, and at least one acidic form. All hGH size isomers contained the same monomeric building blocks, although in somewhat different proportions. Big hGH, e.g. was particularly rich in 20K. No abnormal or previously unrecognized hGH forms were identified as components of big or big-big hGH. Binding of hGH to plasma proteins could not be demonstrated. We conclude that 1) plasma big and big-big hGH represent an oligomeric series composed of 22K (major), 20K, and one or more acidic hGH monomers, 2) the majority of these oligomers are noncovalently associated, with a smaller fraction consisting of monomers linked by disulfide bridges.

Circadian variation of basal plasma growth hormone in man.

Human GH (hGH) circulating during periods of basal pituitary secretion is frequently undetectable in human plasma by RIA. Such basal periods predominate throughout most of the day. Knowledge of the concentration of basal hGH is important, since hGH plays a role in the homeostasis of several metabolic fuels and ions. We have used an immunoadsorbent technique to extract and concentrate hGH from large plasma samples before RIA in order to precisely measure basal hGH levels [immunoextracted RIA (IERIA)]. Twenty milliliters of blood were drawn from five normal volunteers every 2 h over a 24-h period. The obtained plasma (10-13 ml) was passed over an anti-hGH antibody Sepharose column. The column-bound hGH was eluted and measured by RIA. Recovery of hGH in the plasma extracts was measured using [131I]hGH as an internal standard. Recovery averaged 54 +/- 12% (mean +/- SD). Theoretically, the extraction procedure expanded the useful range of the RIA about 50-fold. In practice, the detection limit was 40 pg/ml plasma. Basal hGH (defined as less than 1 ng/ml by conventional RIA) was found to range from less than 40 to 746 pg/ml by IERIA. Above 2 ng/ml, conventional RIA and IERIA yielded comparable results (r = 0.92; P less than 0.001), but below 2 ng/ml, the two assays did not correlate (r = 0.01; P greater than 0.5). In general, conventional RIA overestimated plasma hGH in that range. As a result, the ratio between nocturnal and daytime hGH secretion, as assessed by IERIA, is higher than previously appreciated. We conclude that basal plasma hGH is highly variable below 1 ng/ml, and may on occasion be as low as less than 40 pg/ml. The episodic nature of hGH release appears to persist in the basal state. The conventional definition for basal hGH (less than 5 ng/ml) should probably be revised downward to less than 0.8 ng/ml.

The molecular nature of circulating growth hormone in normal and acromegalic man: evidence for a principal and minor monomeric forms.

Human GH (hGH) extracted from pituitary glands consists of several molecular forms. Monomeric pituitary forms include the single chain 22,000-dalton polypeptide (22K; hGH-B), a 20,000-dalton variant with a 15-amino acid deletion (20K), 3 proteolytically cleaved 2-chain forms (hGH-C, -D, and -E), 2 deamidated forms, an acetylated form (fast hGH), and other, only partially characterized forms. It is not known which of these forms is secreted, nor what the precise nature of circulating hGH is. To answer these questions, we have extracted hGH from human plasma obtained by plasmapheresis from normal volunteers after L-dopa stimulation of hGH secretion and from acromegalic patients. We extracted and concentrated hGH by immunoadsorbent chromatography and examined its chemical nature by polyacrylamide gel electrophoresis under native and denaturing (sodium dodecyl sulfate and urea), nonreducing and reducing (dithiothreitol) conditions as well as by isoelectric focusing. In all cases, the predominant form of hGH present in plasma was 22K, which accounted for approximately 85% of all immunoreactive hGH. In addition, we found evidence for the presence of 20K as a minor form (approximately 7% of all hGH) and of one or more acidic forms (N-acetylated, deamidated, or cleaved hGH; 5-10% of all hGH). Exact identification of the acidic form(s) was not possible. However, the highly bioactive cleaved forms hGH-D and -E were judged to be undetectable (less than 5% of all hGH). We conclude that 1) several monomeric molecular forms of hGH circulate in normal and acromegalic man; 2) the pattern of circulating hGH forms reflects in part their relative prevalence in the pituitary gland; 3) proteolytically cleaved 2-chain hGH forms with enhanced bioactivity are not detectable in blood; and 4) monomeric hGH circulating in acromegaly is qualitatively indistinguishable from normal hGH.