The immuassay handbook parte80

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The immuassay handbook parte80

  1. 1. 777© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/B978-0-08-097037-0.00063-4 Growth and Growth Hormone Deficiency Gill Rumsby1 (gill.rumsby@uclh.nhs.uk) Peter Hindmarsh1 (p.hindmarsh@ucl.ac.uk) Michael Preece2 Jane Pringle2 1 This edition. 2 Previous editions. Normal Childhood Growth The human growth process is complex and represents the integration of at least three distinct phases of growth spread over a time period of 15–20 years. There are three main phases of growth: infancy, which is predominantly dependent on nutrition and is essentially a continuation of intrauterine growth; childhood, which is dominated by growth hormone (GH); and puberty, where the gonado- tropin and sex steroids interact with GH to generate the pubertal growth spurt. During this process, the progress of growth is influenced by a multitude of factors including genetics, nutrition, the general environment, and internal hormone secretion. These factors bear upon the timing and magnitude of various parts of the growth process, such as the interrelationship between prepubertal growth and the adolescent growth spurt. Thyroid hormones, the gonadotropins, and the sex and adrenal steroids are dealt with elsewhere (see THYROID, INFERTILITY, and ADRENAL CORTEX). This chapter is restricted to GH, its binding protein and the dependent insulin-like growth factors (IGFs) and their binding proteins. The pituitary gland secretes GH under the joint control of the hypothalamic peptides: GH-releasing hormone (GHRH) and GH-release-inhibiting hormone or soma- tostatin. In addition, a third GH secretagogue (GHrelin) has been identified although the physiological role for this substance remains unclear. GHRH and somatostatin inte- grate numerous inputs into the hypothalamus and trans- late a signal to the pituitary to release and synthesize GH. The exact mechanism(s) by which GHRH and somatosta- tin interact in man is unclear, but evidence suggests that GHRH and somatostatin cycle out of phase with each other so that GH secretion arises as a result of rising portal concentrations of GHRH and declining somatostatin con- centrations. What is clear is that GH secretion occurs in a pulsatile manner with intermittent, but well regulated, pulsed release of GH at about 200min intervals, mostly at night. There is a weak association between the first attain- ment of deep sleep and the major GH peak of the night. In serum, GH is associated with specific binding pro- teins. A high-affinity GH-binding protein (GHBP) is pres- ent in quantities approximately equimolar with interpulse levels of GH and may bind about 30% of serum GH at these concentrations. It is homologous to the extracellular domain of the membrane-bound GH receptor and may be derived by proteolytic cleavage during cellular processing of the receptor. Circulating concentrations of the binding protein might therefore reflect expression of the GH receptor and the biological activity of GH. There is a strik- ing age-related increase in GHBP, low in the neonatal period and rising through childhood. GH exerts its further effects through two different path- ways. It has potent metabolic effects that appear to act directly through specific GH receptors, particularly in adipocytes and muscle. These effects are directly opposed to those of insulin and form one of the major counter-regulatory mechanisms to balance insulin action. In contrast, the growth-promoting actions of GH are mediated through the insulin-like growth factors (IGFs). These are two in number (IGF1 and IGF2), and they act in a paracrine/autocrine fashion, locally pro- duced in many tissues. There is, however, a considerable quantity of both IGF1 and IGF2 in the circulation bound to a variety of binding proteins. It is not clear whether these circulating forms have a more general endocrine function or act as a reservoir for IGF or perhaps a buffer of IGF action. IGF1 is thought to be an important signal for many cell types integrating information on GH secretion and nutri- tional status. Appropriate concentrations of IGF1 allow proliferating cells to proceed through the cell cycle, to dif- ferentiate, and to perform specific functions such as pro- duction of extracellular matrix compounds (e.g., the proteoglycans in cartilage). These effects are particularly important in the growing organism. The physiological importance of IGF2 is far less clear. The IGFs in the circulation are tightly bound to one of a number of specific binding proteins such that only about 1% of circulating IGF is in the free form. At least six classes of IGF-binding protein (IGFBP) can be distinguished on the basis of their amino acid structure and are designated IGF-BP1 to IGF-BP6. Clinical Disorders GH DEFICIENCY OR INSUFFICIENCY Severe or total GH deficiency (GHD) occurs relatively rarely after major structural lesions of the hypothalamus and pituitary gland, following surgery for their treatment, or as a result of deletions/mutations within the structural gene for GH or its receptor. A series of GH gene abnor- malities have been described along with gene abnormali- ties in a number of pituitary and forebrain developmental genes that produce either isolated GHD or multiple pituitary hormone deficiencies. C H A P T E R 9.9
  2. 2. 778 The Immunoassay Handbook More commonly, there is a variable severity of GH insufficiency that is clinically almost indistinguishable from the severe forms at one end and normality at the other. Diagnosis of these conditions is largely based on clinical assessment of growth. As GH secretion is pulsatile, unstimulated concentrations are useless and assessment requires measurements of GH concentrations coupled with some form of provocation test of GH secretion, e.g., response to hypoglycemia. Frequent sampling throughout 24h periods has been used but is essentially a research tool because of the resource implications. As indicated above, hypopituitarism or multiple pitu- itary hormone deficiencies may be idiopathic, genetic or secondary to structural lesions. GH is by far the most com- monly affected hormone, with the gonadotropins, thyro- tropin (TSH), and adrenocorticotropin (ACTH) affected in turn. The GH aspects of diagnosis are unaltered except that the clinical picture is usually more severe. GHD is treated by replacement with recombinant human GH, given subcutaneously. Over recent years, vari- ous regimens have been explored, but it is now more or less universally accepted that daily injections are required at doses between 5 and 10mg/m2 body surface area (pure somatropin has a bioactivity of 3IU/mg). GH RESISTANCE GH receptor deficiency resembles clinically severe GHD but is usually characterized by normal or excessively high serum concentrations of GH and very low concentrations of IGF1. There is, usually, a very low concentration of GHBP and absent cell membrane binding of GH leading to the assumption that there is a deficiency of the GH receptor. The condition follows an autosomal recessive pattern of inheritance. A nearly indistinguishable variant has been described where normal circulating GHBP is found; it is believed that the GH receptor is present but that post-ligand-binding events are abnormal. Treatment utilizes recombinant IGF1. Growth acceleration is observed in the short term although the increase in final height is perhaps not as great as in GHD. This may reflect the role that GH plays in the differentiation of pre-chondrocytes. GH antibodies are a rare cause of GH resistance. They do not occur spontaneously but may develop rarely while a patient is on GH therapy. This is probably confined to the few patients with severe gene-deletion GHD where the subject’s immune system has never been exposed to GH and fails to recognize it as self when administered thera- peutically. When antibodies do occur they effectively block the effects of the GH. There is no known treatment for GH antibodies, but recombinant IGF1 therapy may overcome in part the attenuation of GH action. EXCESSIVE GH SECRETION Pituitary gigantism occurs with pituitary adenomas that produce excessive GH secretion during childhood. There is excessive skeletal and visceral growth often to a marked degree; it is extremely rare. Circulating GH concentra- tions may not be strikingly elevated in the unstimulated state. GH concentrations usually fail to suppress after a glucose load, and there is a “paradoxical” rise in GH to stimulation with thyrotropin-releasing hormone. Care must be exercised, however, in evaluating GH secretion in tall adolescents as the pubertal changes in GH secretion can produce findings similar to those observed in pituitary gigantism. In this situation, 24h GH profiles can be helpful in distinguishing the two conditions. Acromegaly is a far commoner condition that follows the development of a pituitary adenoma in adults when growth has ceased. There is still bony overgrowth, but it mostly affects membranous bones because of the fusion of the growth plates in the long bones of the skeleton. As with pituitary gigantism, there are a number of important metabolic sequelae of which the most important is diabetes mellitus and also an increased risk of colonic carcinoma in a number of acromegalic patients. Pituitary surgery to remove the adenoma is the mainstay of therapy in both conditions. Treatment with agents such as somatostatin may be required along with radiotherapy in some cases. More specific GH receptor therapies such as the antagonist Pegvisomant are also of value in long-term management. Analytes GROWTH HORMONE GH (somatotropin) is a heterogeneous protein secreted pre- dominantly as its 191 amino acid form, often referred to as 22 kDa GH. There are several other variants of GH found in the circulation, produced as a result of post-transcriptional, post-translational and post-secretory events. This processing results in variants of GH of differing length such as the 20kDa form (178 amino acids) or with structural changes caused by, for example, deamidation or glycosylation. Aggre- gation of the monomeric variants can result in dimeric and oligomeric forms, all of which can interact in an assay to a greater or lesser degree. There is little evidence to date on the role of these vari- ants and whether the proportion is consistent or alters under particular circumstances. This has implications for the measurement of GH by immunoassay (see ASSAY TECHNOLOGY). It should also be noted that there is a second GH gene, which is only expressed in the placenta. This particular GH is found in late pregnancy serum and may also cross- react in certain GH assays. There is considerable structural variability between different species. Assay Technology There are seven different methods for the measurement of GH registered in the UK NEQAS scheme, most of which are immunometric in type. A single manufacturer domi- nates the field. The assays vary in the combination of mono- clonal or polyclonal antibodies used and in the type of solid-phase element. Most use non-radioisotopic tracers, and many assays are now performed on automated systems. The heterogeneity of GH means that for any two anti- bodies used in combination, there will be differences in the recognition of the GH variants. Some assays only recognize the 22kDa variant, while others will cross-react to varying
  3. 3. 779CHAPTER 9.9 Growth and Growth Hormone Deficiency degrees with other GH variants and their aggregates. If the proportion of variants changes after stimulation then, potentially, the amount of GH recognized will change as well. This leads to huge differences in the absolute number obtained for analysis on any sample. The situation is com- pounded by the different calibrants that are available. The current reference material for GH, 98/574, is a recombi- nant protein (rhGH) consisting of 100% 22kDa GH. The former pituitary-derived standard, IS 80/505, is being phased out and is now only used by a single manufacturer. The relative purity of IS 80/505 for monomeric 22kDa GH and the absolute purity of IRP 98/574 have led to some problems as the 20kDa GH molecule may have in vivo bio- activity, and its presence will lead to assay bias depending on the specificity of the antibodies used. The monomeric nature of the standard can lead to incomplete recognition of the circulating GH components and, consequently, to lower readings. The heterogeneity of hGH contributes to the range of product bias against the “All-Lab Trimmed Mean” (ALTM) seen in the UK National External Quality Assurance Scheme (NEQAS), which can be anything from −30% to +10%. The availability of the traceable standard, 98/574, has led to a move to standardize reporting of GH in terms of mass units (µg/L) (1mg corresponding to 3IU units somatotropin). It is recommended that assays should achieve a lower limit of quantitation of 0.05µg/L with a coefficient of variation of <20%. There are methods available to measure variants of GH other than 22kDa, either directly or indirectly. One is a non-22kDa GH exclusion assay, which is an indirect mea- sure of GH variants other than 22kDa: the 22kDa GH is removed by a specific antibody, and the remaining GH is measured by an immunoassay that recognizes all GH forms. Direct methods for measuring 20kDa GH are also available. Both these assays are mainly for research at pres- ent but may have a role to play when the function of these variants is better understood. Mass spectrometry methods are also entering the field particularly for use in detection of exogenous GH in sport. Conventional immunoassays only provide information related to the structure of the analyte and provide no insight into functional activity. The immunofunctional GH assay was constructed to look at the structural integrity of the GH molecule, and therefore, its potential to be function- ally active. It is not a bioassay, but a sophisticated immuno- assay that relies on the ability of the GH to dimerize the GH receptor, a process required for GH action. This method is available commercially, but it remains to be seen whether it is more useful than a conventional immunoassay. Note that a significant proportion of GH is bound to GHBP in the circulation. For the most part, GHBP does not cause interference, probably because the antibodies used have a higher affinity for GH. However, some assays, particularly those with short incubation times, may be affected by the amount of GHBP present. Reference Interval Because of the pulsatile nature of GH secretion, reference intervals for basal values are without meaning. The het- erogeneous nature of GH described above also makes the assignment of expected values, other than for the specific assay being used, unhelpful. Laboratories offering GH assays, particularly for the diagnosis of GH insufficiency states, need to define their own reference intervals for the assay method in use. There is considerable overlap between the concentrations seen in normal individuals and in those with hypo- or hypersecretion of GH. As a result of the huge differences between assays described above, it is impossible to establish a common reference value following an appropriate provocation test (see DYNAMIC TESTS) above which GH insufficiency can be excluded. Particular care is required around the time of onset of puberty, particularly when delayed, as artificially low responses may be seen. Diagnosis of excess GH secre- tion depends upon the failure to suppress circulating levels following a glucose load, and for this reason, it is impor- tant that assays are able to measure reliably at low levels (see ASSAY TECHNOLOGY). Clinical Applications Measurements of basal levels of GH have no clinical use because of the pulsatile nature of GH secretion. Frequent sampling throughout a 24h period is seldom used because of the time taken and the expense. Diagnostic information is normally sought by using dynamic tests that stimulate or suppress GH secretion. Dynamic Tests for GH Insufficiency Provocation tests for GH insufficiency have been legion, indicating the lack of a clearly ideal test. The following are the most important at this time. The normal responses should be assessed in each laboratory. Insulin tolerance or stress test (ITT or IST) Soluble insulin (0.15U/kg) is given intravenously follow- ing an overnight fast. The induced hypoglycemia acts as a stimulus to GH release, which is measured at 30min intervals for 120min. This is the test with which the great- est experience has been gained, and it has become some- thing of a “gold standard” with an adequate response >6.0µg/L and deficiency defined as <2.7µg/L (both values assay dependent). However, it has a significant mortality if used in an inappropriate way, and it is doubtful whether it should be considered as the first choice test any longer. The risks lie in the development of severe hypoglycemia and then the overzealous correction with hyperosmolar glucose solutions leading to hyperosmolar coma and death. Glucagon tolerance test Intramuscular glucagon (standard adult dose 1mg) is administered after an overnight fast and serum GH mea- sured at 30min intervals for 180min. The reliability of the test is about the same as the insulin tolerance test, but it does take considerably longer. It is important that a meal is given and retained after completion of the test as prolonged hypoglycemia may occur otherwise. Clonidine test Clonidine is a selective α-receptor agonist, stimulating GH release through GHRH secretion; it is administered orally in a dose of 0.15mg/m2. Blood is taken at 30min
  4. 4. 780 The Immunoassay Handbook intervals for 150min. It seems to be an entirely safe test although unpleasant postural hypotension and drowsiness may occur. Arginine test Arginine is one of the basic amino acids that, when admin- istered intravenously, stimulates the release of GH; the mechanism is unclear. The dose is 500mg/kg, and blood is taken at 30min intervals for 150min. It is slightly less reli- able than the previous tests in the sense that there is a greater frequency of false-positive results. GH-releasing hormone The use of GHRH as a diagnostic test for GH insuffi- ciency is still somewhat controversial as the discriminatory power is disputed. It is probably best considered as a research tool at the present time. Dynamic Tests for GH Excess Glucose tolerance test Following an overnight fast, oral glucose (equivalent to 75g anhydrous glucose for adults, proportionally less for children) is ingested. Serum GH concentrations are measured at 30min intervals for 120min. The failure of GH levels to suppress, typically below 6.7µg/L, within 60–120min suggests excess GH secretion. Dynamic Tests for GH Resistance See IGF1. Limitations G ITT and glucagon tests both depend on manipulation of glucose homeostasis and can be dangerous. G All tests have a relatively high rate of false positives (apparently low GH response in an individual who is subsequently seen to be normal) ranging between 15% and 25%. GH-BINDING PROTEIN There is an approximately equimolar relationship between GHBP and interpulse levels of GH. Levels of the binding protein may reflect expression of the GH receptor and the biological activity of GH. There is a striking age-related increase in GHBP, being very low in the neonatal period and rising through childhood. This remains predominantly a research tool and is only available in a number of specialist laboratories. Measure- ment still depends upon the quantitative separation of bound and free radiolabeled GH following incubation with an aliquot of the serum sample of interest. There are no international standards. INSULIN-LIKE GROWTH FACTORS: IGF1 (SOMATOMEDIN C) AND IGF2 IGF1 and IGF2 are peptides of 70 and 67 amino acids, respectively, and share considerable structural homology with each other and proinsulin. Function IGFs are the mediators of the growth-promoting activity of GH. The mechanism of action of IGF1 is much better understood than that of IGF2, which has an uncertain role at present. Approximately 99% of the IGFs in the circulation are bound to one of a group of IGFBPs. Assay Technology IGF1 assays are mainly automated and dominated by a single manufacturer. The number of immunometric meth- ods is increasing and both monoclonal and polyclonal anti- sera exist; the most useful have a low cross-reactivity with IGF2 (less than 1%). The presence of a number of IGFBPs interferes with the assay and some form of serum extrac- tion procedure is desirable. The most widely used involves the extraction of the IGFs into acid–ethanol, sometimes followed by chromatography. It is also possible to mop up the binding proteins, after acidification of the serum, using excess IGF2 and then measuring the IGF1 using a highly specific antibody (no cross-reactivity with IGF2). These procedures give a measure of the total IGF1. All commer- cial assays have been calibrated against an International Reference Preparation for IGF1 (87/518). As stocks of this are now exhausted it will be replaced by a recombinant preparation, 02/254. Results obtained from samples spiked with the recombinant protein show a nonquantitative recovery of peptide, presumably because the 87/518 cali- brator is only 44% pure. The use of the defined reference preparation should lead to less variation in the future. The interest in hGH doping in sport has led to the development of liquid chromatography–tandem mass spectrometry methods for IGF1. Free IGF1 assays also exist. These are mainly a research tool at present although they may prove to be more useful than total IGF1. There are fewer assays available for the measurement of IGF2, although the necessary reagents are available com- mercially. Again IGFBPs can cause interference, and extraction procedures comparable to those performed for IGF1 are required. There is no international standard. At present it must be considered as a research tool only. Reference Interval Reference intervals for IGF1 are age and assay dependent, increasing up to sevenfold from infancy to 15 years. After puberty, there is a gradual decline to the seventh decade. IGF2 is less age dependent with an increase from 132 to 430ng/mL at birth to 330–767ng/mL at 5 years with no significant change thereafter. Clinical Applications IGF1 assays are available commercially and are part of the repertoire of some automated analyzers. Their clinical use is restricted mainly because of difficulties of interpretation. There is considerable within-individual variation of IGF1, which makes interpretation of an isolated result of limited value. Low IGF1 levels (with or without IGFBP-3 levels) are not helpful in determining if a child is GH deficient although normal levels suggest other causes of short stat- ure. There is, however, a value in the use of serum IGF1
  5. 5. 781CHAPTER 9.9 Growth and Growth Hormone Deficiency measurements in the diagnosis and management of condi- tions of GH excess, particularly acromegaly. More recently, IGF1 measurements have been used to titrate and monitor recombinant hGH replacement therapy particularly in adults but increasingly in children. The clinical value of IGF2 is unclear at present, except possibly in the investigation of hypoglycemia, particularly if thought to be tumor related. This situation may be sus- pected as a rare cause of hypoglycemia in the presence of suppressed GH and IGF1 concentration and a raised IGF2:IGF1 ratio. Frequently, the form of IGF2 produced by these tumors is “big-IGF2” in which partial cleavage of the prohormone occurs. In practice, as IGF2 assays are of limited availability, diagnosis is often based on relief of symptoms following removal of the tumor. IGF1 Generation Test (Dynamic Test for GH Resistance) The IGF1 generation test depends upon the measurement of IGF1 and IGFBP-3 concentrations in response to a dose of GH, usually given intramuscularly over a period of 3–5days. The protocols are variable, and this should be considered as a research tool for the time being. Limitations The value of IGF1 measurement in the diagnosis of GH insufficiency is limited because of the considerable overlap between the reference interval and values seen in GH- insufficient states, especially in infancy and young child- hood. Other problems are the very low values found in some normal young children and the need for age- and puberty-matched reference intervals. IGF2 has a very unclear clinical significance. IGF-BINDING PROTEINS There are currently six IGFBPs identified although other putative binding proteins have been reported. They all have different affinities for IGF1 and IGF2, and the role of many of these binding proteins has yet to be fully elucidated. IGF-BP1 is a single-chain polypeptide with a molecular mass of 25.7kDa. Serum levels are high in newborns and decline through childhood. It has a marked circadian rhythm, with high levels at night, and has an inverse rela- tionship with food intake and insulin. Phosphorylated and lesser phosphorylated forms of IGF-BP1 have been reported: the lesser phosphorylated forms appear to have a reduced affinity for IGF1, which may increase IGF1 bioavailability. The most abundant IGFBP in the circulation after the neonatal period is IGFBP-3, binding both IGF1 and IGF2 with similar affinities. It is substantially glycosylated with a total, estimated, molecular mass of 42kDa. IGF1 or IGF2 forms a high molecular mass ternary (three component) complex (120–150kDa) with IGFBP-3 and an acid-labile subunit (ALS). ALS may be involved in transporting of IGFs to the binding protein. There is no international reference preparation of IGFBP-3; most assays being calibrated against a non- glycosylated NIBSC reagent 93/560 although a recombinant (glycosylated) preparation is now available. Differences in calibration will obviously contribute to different reference ranges. Immunoassays, including some commercial kits, exist for IGFBP-1 (non-phosphorylated and phosphorylated), BP-3, and ALS but their significance in clinical endocri- nology is controversial. They should be used with cau- tion due to difficulty in interpreting results. Assays also exist for BP-2 and BPs 4–6, but these are purely research tools. General Strategy The principal tool for the diagnosis of growth disorders remains the careful clinical assessment of the growth pat- tern. A low growth velocity is always suspicious and justi- fies further investigation, which should start with the exclusion of general medical problems that may influence growth. Once this is done, specific endocrine tests are appropriate. In a child who is growing at a normal rate, but below the third centile for height, it is important to take into account parental heights and a measure of skeletal maturity, normally bone age. The commonest cause is simple constitutional growth delay. The biochemical tests must only be considered as adjuncts, except in some highly specific situations. Disor- ders of GH excess are probably an exception, where the measurement of circulating GH levels (and possibly IGF1) in carefully controlled circumstances is of primary importance. Further Reading Preece, M.A. Principles of normal growth: auxology and endocrinology. In: Clinical Endocrinology, 2nd edn (ed Grossman, A.), (Blackwell Scientific Publishers, Oxford, 1998). Clemmons, D.R. Consensus statement on the standardization and evaluation of growth hormone and insulin-like growth factor assays. Clinical Chemistry 57, 555–559 (2011). Cohen, P., Rogol, A.D., Deal, C.L., et al. Consensus statement on the diagnosis and treatment of children with idiopathic short stature: A summary of the Growth Hormone Research Society, the Lawson Wilkins Pediatric Endocrine Society, and the European Society for Paediatric Endocrinology workshop. J. Clin. Endocrinol. Metab. 93, 4210–4217 (2008). Hindmarsh, P.C. Current Indications for Growth Hormone Therapy, 2nd edn (Karger, Basle, 2010). Dattani, M.T., Hindmarsh, P.C. Growth Hormone Deficiency in Children. In: Endocrinology, 6th edn (eds De Groot, L.J., and Jameson, J.L.), (Saunders Elsevier, Philadelphia, 2011).

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