3 Diagnosis of growth hormone deficiency D. SCHONBERG

The diagnosis of growth hormone deficiency relies on direct or indirect evaluation of growth hormone secretion by basal or stimulated levels of human growth hormone (hGH) in plasma or by measuring the concentration of GH-dependent hormone levels such as IGF-1. The investigation of hGH secretion is an important tool for the paediatrician in the differential diagnosis of growth retardation. Awareness of the role of hGH in growth processes dates back to observations of pituitary tumours and acromegaly by Marie (1886), in dwarfism by Hutchinson (1900), and in animal experiments of growth arrest after hypophysectomy in young dogs by Aschner (1912). In young rats the growth promoting capacity of crude pituitary extracts was shown by Evans and Long (1921) but highly purified extracts containing growth hormone were achieved only after many fruitless attempts by Li and Evans (1944). Since then, the elucidation of the molecular genetics, production, regulation and clinical function of growth hormone has been very rapid, hGH belongs to a family of structurally closely linked hormones, which includes prolactin and choriosomatomammotrophin (placental lactogen). Their genes on chromosome 17 are very closely related in structure (up to 90%) and they may have had a common ancestor gene (for details see Thorner et al, 1992). Tests for the secretory capacity of hGH date back 30 years, when reliable and sensitive assays became available (Hunter and Greenwood, 1962). To date there has been no unequivocal agreement on the interpretation of measurements of hGH in serum, let alone in urine. On the other hand there is an increasing need and interest in the diagnostic evaluation of hGH secretion in short children since abundant amounts of biosynthetic hGH are now available. Furthermore there is a relationship between the degree of GH insufficiency and the growth response to GH therapy. The old stimulation tests have been claimed to be too insensitive to evaluate varying degrees of defective hGH secretion and for a more refined testing of hGH secretory capacity, study of spontaneous GH secretion has been proposed. These methods may then be used to re-evaluate the role of pharmacological confirmatory tests. Secretion of hGH may be evaluated directly by measuring hGH in serum or urine under certain conditions, or indirectly by estimating hormones or carrier proteins dependent on hGH, like insulin-like growth factor (IGF)-I and IGF-I binding proteins (Table 1). Baillidre's Clinical Endocrinology and Metabolism-527 Vol. 6, No. 3, July 1992 Copyright © 1992, by Bailli~re Tindall ISBN 0-7020-1620--9 All rights of reproduction in any form reserved

528

D. SCHtJNBER6 Table 1. Direct and indirect evaluation of physiological secretion of hGH.

Direct evaluation

Indirect evaluation

Plasma

Immunoassay, (receptor assay): (basal levels), physiological stimuli: sleep, circadian rhythms, energy expenditure

hGH binding proteins IGF-I basal and stimulated IGF-I binding proteins

Urine

24 h collection hGH (or IGF-I)

Repeated morning collections of hGH (or IGF-I)

Tissue

(Metabolism on fibroblasts, leukocytes) Gene analysis hGH, receptor

Receptor binding on fibroblasts, leukocytes Metabolism on fibroblasts, leukocytes

GENERAL ASPECTS Immunoassay of hGH in serum

The WHO Expert Committee on Biological Standardization has recommended that results of immunoassays of hGH be expressed in units of the First International Reference Preparation (former 1. IRP MRC 66/217, now 80/505 calibrated against the latter (Bangham et al, 1985), or US NIH reference preparation HS 2243E, unfortunately not calibrated against the MRC standards) with the qualification 'by immunoassay' (WHO Report, 1969, 1970). According t o S I regulations, values should be given in microunits per litre, where 1 I~U = 0.4-0.5 ~g, depending on the standard. Using the well-defined, authentic and pure biosynthetic hGH preparations available today a standard with a valid relation of weight/volume will be available soon to provide calculation on this more reliable basis. In addition, modern immunoassays using monoclonal antibodies of high specificity and modern techniques like immunoradiometric assays have been said to hold the promise of unequivocal results and hence good comparability of laboratory results (Dinesen, 1991). The general picture has not changed much between the first comparisons of available tests (Frasier, 1974) and today (Reiter and Martha, 1990). Especially in pharmacological testing the stimuli used and the interpretation of the resulting level of stimulated hGH in serum cannot be interpreted unequivocally. External quality control has given and still gives disquietingly different results (Breuer et al, 1978; relative scatter 37-64%; Bidlingmayer et al, 1991: CV 20-50%). The reason may lie in the many variants of hGH in serum (more than ten epitopes), in its different sizes and states of conglomeration, binding to different proteins and varying clearance from the circulation (Baumann, 1990). Some of these differences are not dependent on the secretory state of the pituitary, some obviously are (Baumann, 1991). Taking these facts into account, more research in the specific binding properties of mono or oligoclonal antibodies and their reaction in assays for hGH has to be undertaken for the construction of reliable assays for hGH in serum (and urine) (Dinesen, 1991). It is to be

DIAGNOSIS OF GHD

529

hoped that in the future more consistent physiological and pathological data can be gathered by modern assays and that principles for the diagnosis of alterations in the secretory states of hGH can be established on an internationally unequivocal basis. In industrial kits, clearly defined data on properties of hormones employed as standard and of the antibodies used in the kits have to be investigated and given. In contrast to the very rigid regulations for drugs in human use no regulations on quality exist for laboratory kits for the assay of hormones. Certainly it is not sufficient to state an adaptation to an international standard (Sch6nberg, 1992). Receptorassay of hGH in serum

Binding of hGH to cell surfaces has been assumed to represent the true biological activity of the circulating hGH in serum (Tsushima and Friesen, 1973; for review see Friesen; 1990) in contrast to the immunoassay. Differences in children with normal variant short stature between normal levels of hGH to routine stimuli but low levels of hGH by receptor assay and low somatomedin C (IGF-I) in serum, as well as good responses to exogenous growth hormone, led to the assumption of bioinactive but immunoassayable normal h G H in these patients (Kowarski et al, 1978; Rudman et al, 1980). These tempting speculations have not been confirmed by Garnier and Job (1977) or recently Ilondo et al (1990). Obvious non-specific effects of serum led to the discovery of binding proteins in plasma of high capacity, which were then identified as the extracellular domain of the cell receptor for hGH (Baumann et al, 1986). Assays using receptors for hGH are considered inferior to immunoassays for routine clinical use due to their lesser sensitivity, limited stability and cost. They are still of interest for research~ and new methods may improve their use. Immunoassay of hGH in urine

Measurement of hGH in urine appears a valuable means of evaluating integrated h G H secretion over the time of urine collection, e.g. overnight, and it underwent a renaissance lately through the introduction of highly sensitive immunometric assays (Baumann and Abramson, 1983; Girard et al, 1987; Hashida et al, 1987). The amount of hGH appearing in urine is calculated as less than 0.01% of daily h G H production with fewer different forms. The detection limits for h G H in urine have to be considerably lower compared with original competitive radioimmunoassays, and ranged between 0.25 and 4 pg hGH/ml urine in a recent comparison of six assays (Girard et al, 1990). Related to time, e.g. 24 h, or overnight, normal values still show a considerable intraindividual and interindividual scatter but clear differences before and during puberty (Table 2) and a correlation between integrated 24 h plasma values and urine. In hGH deficiency a good correlation was shown between peak levels of hGH in serum after stimulatory tests and overnight urinary h G H (Walker et al, 1990). The physiological and pathological differences do not seem so clear when urinary hGH is related to

530

D. SCHONBERG Table 2. Preliminary biological data for urinary hGH (ng/24 h).

Prepubertal Pubertal

Girard et al*

Tanaka et al*

Welling et air

3.03 (2.29) 6.09 (6.07)

4,72 (3.28) 11.42 (7.65)

1.5 (0.2-3.3) 3.8 (1.1-9.6)

* Mean (SD). ? Median (95% confidence limits). From Girard et al (1990).

excretion of creatinine. Reabsorption and excretory influences on circulating hGH in its many forms in the kidney are not yet clearly defined. In renal failure, estimating urinary hGH is unreliable. TESTING hGH SECRETION Factors interfering with interpretation Apart from the differences related to assay methodology and the many variants of hGH and its binding proteins in biological fluids, several biological and pharmacological influences have to be considered in the evaluation of hGH secretion (Table 3). The most prominent factors in clinical diagnostic procedures are obesity, stress and puberty (Sch6nberg, 1983, 1986, 1988). After the first observation of a diminished response to hypoglycaemia in obese subjects (Roth et al, 1963) many authors confirmed this blunted GH response to fasting, exercise, insulin and arginine, etc. Fear of venepuncture, even in the outpatient department, results in a rise of plasma levels of hGH (Frohman et al, 1967). Subsequent stimulation, e.g. in exercise tests, may be blunted thereafter for a longer period of time. Usually, therefore, venepuncture is undertaken 30min before the test proper. Although an elevated level of hGH over 10 txg/1 (20 mU/l) excludes Table 3. Stimulating and depressing influences on secretion of hGH. hGH stimulated

hGH depressed

Natural

Hunger Malnutrition Stress Protein deficit Oestrogens, androgens ACTH Renal dysfunction

Emotional deprivation Hyperglycaemia (postprandial) Refractory period after stress Obesity Lack of sex hormones Hypo/hyperthyroidism Cortisol excess

Pharmacological

Piperidine, L-dopa, diazepam, propranolol, clonidine, amphetamine, metoclopramide

Corticosteroids, aminophylline, theophylline, phenoxybenzamine, ergotamine, reserpin, chlorpromazine, phentolamine, tolazoline, morphine, apomorphine

Adapted from Sch6nberg (1988),

DIAGNOSIS OF GHD

531

an absolute lack of hGH production, more subtle conclusions about partial GH insufficiency are not possible. In untreated hypothyroid children diminished responses to stimuli have been reported, but there is no direct relation to the functional state of the thyroid (Root et al, 1967). After appropriate thyroxine substitution the tests returned to normal. Congenital hypothyroidism should be detected by neonatal screening but juvenile hypothyroidism has to be considered. Thyroid function should always be considered in children presenting with stunted growth. The growth of children undergoing treatment with corticosteroids is poor. Many authors have reported both subnormal (Frantz and Rabkin, 1964) and absolutely normal responses of hGH to stimuli (Root et al, 1967) in such patients. Different treatment regimens or differences in the pharmacodynamics of modern drugs may account for these variations. During puberty the hGH response increases in boys and girls (Shizume et al, 1969), and in delayed puberty these results may be low. Priming with sex steroids has been advocated for the testing of these children (Deller et al, 1970; Illig and Prader, 1970). Poor growth, retarded bone age and impaired response to hGH are frequently observed in emotionally deprived children (Patton and Gardner, 1961). The differentiation from hypopituitarism proper can be difficult. In endogenous depression hGH stimulation is lower than in exogenous depression (Sachar et al, 1971). Treatment of psychiatric disturbance may influence hGH secretion (Matussek, 1980). Pharmacological influences may be summarized according to modern concepts of the neurosecretory regulation of hGH secretion, in that oL-adrenergic, dopamine and acetylcholine agonists and [3-adrenergic antagonists stimulate, and [3-adrenergic agonists and a-adrenergic, dopamine and acetylcholine antagonists depress secretory impulses (Thorner et al, 1992). Preparation for tests

Biological variation lies not only in interindividual differences but also in the circadian secretion of hormones, influences of metabolism, influences of pathological conditions such as inflammation and many drugs. Such possible influences have to be taken into account by the doctor when obtaining the material to be assessed. The susceptibility of the material in biological fluid to destruction or alteration by enzymes has to be known and taken into account in choosing the packing material and conditions for transporting the sample to the laboratory. Last, but certainly not least, a short but informative description of the doctor's reasons for submitting the material should be included. Without this information the clinical chemist will not be able to draw the necessary conclusions from the results obtained and thus cannot interpret the result properly and in the context of the intended diagnostic process. Testing the secretion of hGH is painful for the patient, time consuming for the family and the doctor, and expensive. Apart from clear indications, false

532

D. SC~ONB~RG

positive or negative results should be avoided by careful preparation of the patient, keeping the above-mentioned principles in mind. The administration of the drugs mentioned should be stopped at least 3-4 days before the test. The patient should be in a psychologically and metabolically stable state, having receiving adequate information, and being without food for 10-12h (preferably overnight); glucose containing drinks should also be avoided. The tests should start in the morning on an empty stomach, preferably after a sound sleep on the ward. For lengthy tests the bladder and bowel should be emptied. In all tests requiring several samples of blood to be taken, an indwelling needle or preferably a plastic catheter should be inserted 30 min before the start of the test to avoid stress or pain-induced changes in h G H in serum. Individual reactions to stress and pain, or even their anticipation, is very variable; a reaction to a stimulus may be totally suppressed by a previous spontaneous G H pulse before the test, e.g. at time 0. (Spitz et al, 1972).

SECRETION OF hGH: BASAL STATE, INDIRECT EVALUATION AND PHYSIOLOGICAL STIMULI Direct assay in plasma or urine

In contrast to many other hormones, the basal levels of hGH in plasma determined with the assays currently available have no clinical significance because they do not distinguish pituitary insufficiency from normal, mainly because of the intermittent nature of secretion. Elevated levels may exclude gross insufficiencies but are influenced strongly by the metabolic state, psychological disturbance, etc. of the patient (Table 3). Determination of hGH in urine reflects the integrated secretion over time, e.g. overnight. The restrictions of this method have been discussed above (see Table 2). Direct assay in tissue

It may be assumed that cells of patients with hGH deficiency show deficient numbers of receptors and metabolic signs of subnormal supply of hGH. This concept has been tested in a cellular nutritional assay of leukocytes isolated from short and normal children and differences in several parameters such as intracellular glutamine glycine and adenylate kinase have been found (Metcoff et al, 1989; Mardo, 1990). The isolation and testing of individual peripheral leukocytes and their differentiation are still very tedious procedures prone to false results. A new model of growth regulation and its tests has been proposed by Kiess and Butenandt (1987) for differences in receptors of hGH in peripheral mononuclear cells. Other groups have been unable to confirm these findings. Individual cultures of fibroblasts constitute an in vitro model for a similar approach. These investigations remain unconfirmed and await further refinement in methodology.

DIAGNOSIS OF GHD

533

Direct assay by molecular genetics The elucidation of the h G H gene locus by Hirt et al (1987) opened new vistas on the differential diagnosis of short stature, e.g. in familial cases. In severe and/or isolated hGH deficiency deletions of the hGh-N gene have been found in several families in Israel and Switzerland and this intricate method is becoming increasingly important (Parks, 1989); in the great majority of patients presenting with isolated h G H deficiency no structural abnormalities of the h G H gene cluster are reported (Mullis et al, 1990). Deletions in the hGH receptor gene are accompanied by changes in hGH binding protein in plasma (see below) (Baumann et al, 1986).

Indirect evaluation of secretion in plasma by hGH binding proteins The specific binding proteins for hGH (GH-BP) have been discovered during investigations of the higher molecular weight forms of hGH and of unspecific binding of hGH in radioimmunoassays (Baumann et al, 1986). Since the high affinity GH-BP in plasma was shown to represent the extracellular portion of the h G H receptor by Leung et al (1987), its clinical relevance has been tested (Holl et al, 1991; Baumann, 1992). The high affinity GH-BP may be estimated in GH receptor deficiency, e.g. in Laron dwarfs or pygmies, where it is absent or found in low concentration. Less prominent cases of receptor dysfunction with highly reacting hGH to stimuli and low somatomedin may be a domain of GH-BP determinations in future. On the other hand the high specificity of GH-BP may be a tool for discerning alterations in hGH secreted as a cause of hGH deficiency. If the report of Martha et al (1991) that GH-BP can serve as a measure for the therapeutic response to exogenous h G H can be confirmed, GH-BP-I may be the long awaited indicator for short children in this important respect.

Indirect evaluation of secretion in plasma by IGF-I IGF-I is the most important growth factor for the clinician concerned with growth disorders, and since it is directly dependent on the secretory pattern of hGH its measurement indirectly reflects hGH secretion. It also plays a negative role in feedback of hGH, and so reflects the regulatory situation as well. The absolute levels for age and sex (Rosenfeld et al, 1986; Hintz et al, 1988; Heinrich et al, 1987), the stimulation by exogenous hGH for 4 days or special combinations of clinical facts and IGF-I (Lee et al, 1990) may then be used to try to circumvent more painful and blood consuming larger tests (Table 4). Difficulties in interpretation are obvious between normal levels of IGF-I in 10-15% h G H deficient children in which hGH and IGF-1 levels are inconsistent (Rosenfeld et al, 1986; Ranke et al, 1988) and other states, e.g. in fasting (Silink, 1992).

By IGF-I binding protein (IGFBP-3) IGF-I binding proteins in human serum are present in varying sizes; the large complex has a distinct pattern over age with a moderate peak at

534

D. SCHt3NBERG Table 4. Plasma levels of IGF-I by age and sex in normal controls.

Male

Female

Age (years)

Mean

Range

n

Mean

Range

n

0-2 2--4 4-6 6-8 8-10 10-12 12-14 14-16

42.1 (30) 50.2 (26.1) 87.5 (42.6) 101.3 (31.2) 164.5 (70.8) 190.9(87.2) 275.9 (127) 475 (139)

0-109 21-821 33-172 60-182 77-303 71-351 106--492 318-822

13 14 11 9 15 14 10 9

30.4(22.2) 62 (31.5) 119.7(52.7) 174.4(56.1) 220.3(67.3) 281.7(142) 395.2(80.3) 527.4(142)

0-80 18--120 58-194 113-275 81-349 123-561 292-511 319-756

13 14 11 9 15 14 10 9

Values in parentheses are standard deviations. From Heinrich et al (1987) and recent unpublished data.

puberty. It reflects IGF-I and hence h G H . For its assay and use in the diagnosis of growth disorders a high sensitivity and specificity resulting in very good accuracy have been reported (Blum and Ranke, 1990). Its role in the concert with other tests is now under investigation since a commercial kit became available. Exercise test

Principle. Physiological stimuli are reactions of h G H to metabolic demand in physical exercise and in hunger, to shifts in brain function in deep sleep and circadian rhythms. R e b o u n d of h G H after a glucose load and after strenuous exercise were used early as physiological stimuli (Roth et al, 1963). After work on a bicycle ergometer or stair climbing, h G H rises above 6 Ixg/l 25-30min later in about 70% of normal or short for age children (Buckler, 1972; Stahnke et al, 1975; Sch6nberg et al, 1975; Seip et al, 1990). Since this stimulation of h G H is mediated by catecholamines, improved consistency of positive results up to 80-100% can be achieved by combination with L-dopa (Amendt and Rhode, 1979) at the expense of frequent side-effects. Blunted responses to exercise in children with constitutional delay of growth and adolescence (Sch6nberg et al, 1975) corresponded to the observation of relative insufficiency in spontaneous secretion over 5 h in sleep in many of them (Bierich and Potthoff, 1979).

Procedure. Suitable screening test for outpatient service. Blood drawn for basal h G H in serum early in the morning after overnight fast with empty stomach. After a warm-up phase (energetic exercise to the point of exhaustion for 10-15 min by ergometer, stair climbing, etc. to a heart rate of approximately 170-185 beats/min) 20min rest, thereafter second blood drawn for h G H in serum.

Specialproblems..Positive response to over 5-7 txg/1 h G H in about 70-90% of normals. In constitutional delay of growth and adolescence a negative

DIAGNOSIS OF GHD

535

response is more common, and frank pituitary insufficiency has to be excluded by a confirmatory test.

Deep sleep

Principle. Hunter and Rigal (1966) and Quabbe et al (1966) observed intermittent bursts of hGH in deep sleep. The most consistent finding at night is the rise in hGH 30-60min after the onset of sleep in electroencephalographic (EEG) sleep stage IV (Sch6nberg et al, 1991). Many authors have used this test for screening hGH secretion (Underwood et al, 1971). Levels of hGH above 5 Ixg/l have been reported in 70-100% of the controls. Disadvantages of this method are the uncertain timing of deep sleep if not controlled by electroencephalography and the necessity of admitting the patient to the hospital.

Procedure. Bed in quiet surroundings; cannula in cubital vein 30 min before bed; blood drawn just before usual bedtime; cannula filled with heparin or covalently bound heparin to wall of catheter; drip infusion not recommended. Blood taken at 20-30 minutes after onset of sleep, sampling continued for 5 h at 30 min intervals.

Special problems. Only possible in hospital; problems with undisturbed sleep without 'test' night; reliable only with evaluation and staging of sleep by EEG.

Circadian rhythm

Principle. Physiological secretion of hGH into serum, like other pituitary hormones, is governed by an intricate interplay between stimulatory (growth hormone releasing factor) and inhibitory (somatotrophin release inhibitory factor) hormones resulting in a pulsatile pattern most prominent at night and depending on nutritional and psychological states, sex and sexual development and age (for review see Tannenbaum, 1989). To study more subtle relationships between the magnitude of hGH secretion and growth in children, there is a renaissance in the analyses of circadian or overnight rhythms (Bierich and Potthoff, 1979; Albertsson-Wikland et al, 1983; Hindmarsh et al, 1987; Albertsson-Wikland and Rosberg, 1988; Ho and Weissberger, 1990).

Procedure. Patient in bed, best results with pretest night in quiet surroundings and continuous recording of EEG to assess sleep stages. Indwelling catheter in cubital vein with continuous slow drip of saline, preferably catheter with covalently bound heparin to the inside to prevent clotting. Blood drawn at 20min intervals, or continuous pump withdrawal over periods of 20 min.

Special problems. Applicable in special cases and for scientific research in well-trained groups.

536

D. SCHONBERG

SECRETION OF hGH: PHARMACOLOGICAL

STIMULI

M a n y reports on stimulatory agents have b e e n published since the first papers on h G H physiology ( R o t h et al, 1963; H u n t e r et al, 1965). Frazier (1974) reviewed ten stimulation tests for clinical use, n o n e of t h e m sufficiently safe for the diagnosis of pituitary failure, but s o m e of t h e m widely used either singly or in combination. With growing u n d e r s t a n d i n g o f h G H regulation, substances active in the brain and playing a role in stimulation o f h G H have b e e n a d d e d (Table 5).

Table 5. Stimulation of hGH in serum: physiological and pharmacological stimuli (for description see text). Stimulus

Timing/dose

Blood drawn

Result hGH

0, 30 min 0, 30, 40, 60 min

Basal < 4 ~g/1 Max > 10 Ixg/l 5-10 Ixg/1

Night secretion Circadian rhythms

10 min 45-90 min after sleep onset Overnight 24 h

Every 20 min Every 20 min

5 ~Lg1-1 rain -1. 6--8p,g/l 1.363-1.810 Ixgt

Pharmacological stimuli L-Dopa/carbidopa + propranolol Clonidine

250/25 mg oral 1 mg/kg oral O.15 mg/kg oral

O, 45, 90 min

Max > 10 ixg/l

3 h, every 30 rain

Max > 10 ~g/l Max > 10 Ixg/l

Physiological stimuli

Exercise First deep sleep

Glucagon-propranolol Arginine chloride Insulin (regular) Sequential arginine/insulin GHRH 1-44, 1-40, 1-29 Priming with sex steroids e.g. in pubertal delay

0.1 mg/kg i.m. 1 mg/kg oral 0.5 g/kg 30 min 0.1 units/kg i.v. 0.5 g/kg + 0.1 units/kg i.v. 1 (-3) ~g/kg i.v.

3 h, every 30 min 0, 30, 60, 90 rain 0, 30, 60, 90 rain 0, 15, 30, 45, 60rain 20, 30, 45, 60rain 0, 15, 30, 60 min

Boys: Testosterone 100 mg i.m. daily for 3 days before test

Max > 10 Ixg/1 Max > 10 ~Lg/1 Max > 10 ~Lg/1 Max > 10 ixg/1

Girls: Ethinyloestradiol 0.1 mg oral daily for 3 days before test

* Mean integrated concentration hGH (Rochiccioli et al, 1991). t Integrated concentration sleep, plus area under sleep curve (Garnier et al, 1988). Tests in italics are tests for screening; others are confirmatory.

L-dopa, L-dopa-carbidopa, L-dopa/propranolol Principle. E l e v a t e d levels of h G H had b e e n o b s e r v e d during t r e a t m e n t of

P a r k i n s o n ' s disease with the c a t e c h o l a m i n e r g i c n e u r o t r a n s m i t t e r L-dopa ( B o y d et al, 1970). I n t r o d u c e d for h G H testing by H a y e k and C r a w f o r d (1972) and used by m a n y groups. False-negative results of 5 - 4 0 % ; improvem e n t of sensitivity with [3-adrenergic b l o c k a d e by p r o p r a n o l o l (Collu et al, 1975), or with carboxylase inhibitor L-carbidopa or b o t h ( S c h 6 n b e r g e r et al, 1979) to false-negatives of only 9 . 5 % and less sampling time.

DIAGNOSIS OF GHD

537

Procedure. Suitable screening test for outpatient service. For doses, see Table 5, propranolol orally not more than 40 mg. Blood drawn at 0, 15, 45, 60, 75, 90 min, in combinations at 0, 45, 90 min sufficient.

Special problems. Large interindividual variability of response without combination; nausea and vomiting relatively frequent.

Cionidine

Principle. Catecholaminergic properties used in antihypertensive therapy, based on assumptions of regulation of hGH in brain employed as stimulant. Effectiveness comparable to insulin-induced hypoglycaemia.

Procedure. Suitable screening test for outpatient service. For dose of clonidine, see Table 5. Blood drawn for 3h at 30min intervals since individual response time is very variable.

Special problems. Patient in sitting position for the whole time; frequent effect on blood pressure, since orthostatic dysregulation and vomiting often occur. Drawback: long duration for outpatient service.

Glucagon-propranolol

Principle. Glucagon elicits a rise and subsequent fall of blood sugar. In reaction to this mild relative hypoglycaemia secretion of hGH is stimulated. To augment this reaction propranolol is added.

Performance. Propranolol has to be administered orally 2 h before the test; for dose see Table 5, maximum dose 40 mg. After injection of glucagon intramuscularly blood drawn for 3 h in 10 min intervals.

Special problems. Very potent test. Stimulation of hGH possible even in cases with no reaction to arginine or insulin tests.

Arginine hydrochloride

Principle. Stimulation of hGH by amino acids, e.g. ornithine, glycine, mostly used arginine hydrochloride (Parker et al, 1967), may be mediated by dopaminergic pathways. It is a safe and agreeable way of testing h G H secretory capacity; false-negative tests in normals are between 15 and 25% and can be improved by the additive effect of e-dopa (Weldon et'al, 1975).

Procedure. Arginine hydrochloride is diluted 1 : 10 in physiological saline and infused intravenously over 30min. Blood drawn at start of infusion (time 0), at 30 min intervals to 90 min after start of infusion. In the rare case of side-effects metabolic alkalosis should be considered.

Special problems. None.

538

D. SCHONBERG

Insulin-induced hypoglycaemia

Principle. Hypoglycaemia evoked by intravenous regular insulin is counteracted by secretion of hGH and cortisol for the mobilization of energy. Apart from hGH the function of the hypothalamus-pituitaryadrenal axis can be investigated by determination of cortisol and/or adrenocorticotrophic hormone (ACTH) at the same points of blood drawn. Used by all groups after report from Roth et al (1963). Considered the most effective test for hGH secretion, for a long time the gold standard. Falsenegative results of 0-26% reported, e.g. by Job et al (1971) and Frasier (1974).

Procedure. Former rule of lower dose of 0.05 units regular insulin/kg body weight if growth hormone deficiency is suspected and all other cases 0.1 units insulin/kg body weight no longer strictly applied; 0.1 units/kg body weight gives more consistent results and is usually well tolerated. Test valid only if blood sugar falls by - 50% of level at start of the test, or if absolute level of blood sugar below 40 mg% (< 2.22 mmol/1). O n the spot measuring device for blood sugar, 20% glucose solution for intravenous and glucagon for intramuscular injection must be at hand at the patient's bed. Test to be supervised by experienced nurse and doctor. Blood drawn after intravenous insulin at 0, 30, 60, 90 and 120 min. False-negative results in normal controls 26% or less. Combined tests for adrenal function: blood drawn at 20, 30, 45 and 60 min after insulin for cortisol determination.

Special problems. Frequent side-effects of pallor, sweating, nausea and dizziness about 5-30 min after insulin injection, especially to be anticipated in pituitary insufficiency.

Sequential arginine-insulin test

Principle. Since both arginine and insulin-induced hypoglycaemia have a restricted use in excluding growth hormone deficiency in normals, both tests have been sequentially combined by Penny et al (1969). In context with others the sensitivity of this test is reported at near 100%.

Procedure. Doses of arginine and insulin are comparable with the single tests: arginine hydrochloride 0.5 g/kg body weight to a maximum of 40 g/test diluted 1 : 10 in physiological saline and infused over 30 min. Insulin in the original paper 0.075 units/kg body weight intravenously injected 60min after start of the arginine infusion. Blood drawn (-30), 0, 15, 30, 45 and 60 min after start of arginine; 20, 30, 45 and 60 min after insulin. Combined test for adrenal function: blood drawn at 20, 30, 45 and 60 min after insulin for cortisol determination. For safety measures concerning insulin-induced hypoglycaemia and parameters for sufficient hypoglycaemia, see above.

Special problems. Same as above in single tests. Drawback of increased blood loss during shorter time.

DIAGNOSIS OF GHD

539

hGH-releasing hormone (GHRH)-test

Principle. A bolus intravenous injection of synthetic G H R H releases the secretory reserve of hGH quickly (Borges et al, 1983). The response to this hypothalamic hormone has been used extensively in the differential diagnosis of a secretory insufficiency at the hypothalamic and/or pituitary level. Clinical differentiation in the magnitude and relation of hGH secretion in different tests has shown a high rate of positive responses to G H R H , even in contrast to all other diagnostic measures, resulting in the diagnosis of hGH deficiency. Apart from therapeutic implications the relative value for this test is considered low nowadays (Ranke et al, 1986; Gelato et al, 1986; Cavallo et al, 1990). It can be anticipated that an otherwise intact pituitary gland quiescent without G H R H stimulation for a long time will not readily respond to one bolus of G H R H . Therefore a priming for several days before the test, e.g. 5 days 1 jxg GHRH/kg body weight subcutaneously daily, has been proposed (Butenandt et al, 1986). Potentiating G H R H by the cholinergic drug pyridostigmine may improve the diagnostic reliability of this test (Ghigo et al, 1990).

Procedure. There are no differences in response between the various forms of G H R H 1-44, 1-40 or 1-29 at doses of 1-2 txg/kg body weight as an intravenous bolus. Blood drawn at 0, 15, 30, 45, 60 and 90 min after G H R H . Side-effects, especially in older children, are flushes in about 20%. The stimulated levels of hGH in plasma are higher than with the other tests (see validation of results).

VALIDATION OF RESULTS The responsibility for the validation and control of assays themselves is almost completely in the hands of clinical chemists in the laboratory and they are usually well aware of the problems and possible errors. In contrast, the doctor diagnosing and treating the patients often relies heavily on the data reported and takes them for granted. The interpretation and the conclusions drawn from laboratory results are intricate and complicated processes, which can be analysed to their philosophical and semantic bases. Since laboratory assays contribute to the rising cost of general health care especially if misordered and misinterpretation of data plays an important role in misdiagnosing and mistreating the patients efforts in the improvement of the data retrieval, their transmission to the users, and help for their interpretation are of rising concern to the medical community in quality assurance programmes for medical laboratories (for details see Sch6nberg, 1992). Levels of h G H to diagnose or exclude growth hormone deficiency are given in Table 5 and in the text. Normal ranges for stimulation tests have been reported by Josefsberg et al (1983). It must be borne in mind that until now the absolute measurements vary considerably from kit to kit (see above) and recommendations of one group can only serve as guidelines.

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Many new tools for the evaluation of hGH insufficiency have been added and are now under investigation on a broad clinical basis. In general, levels of hGH above 10 Ixg/1 exclude pituitary insufficiency, levels between 5 and 10 Ixg/1 have been termed subnormal. Results of some investigations like G H R H tests may well exceed these levels. For more refined studies in partial insufficiency of hGH secretion combinations of pharmacological and physiological tests are necessary, e.g. reported by Rochiccioli et al (1991). It may well be that IGF-BP-3, GH-BP-I and refined techniques in the molecular biology of hormones and receptors involved in growth will play an important role in the future. RECOMMENDATIONS OF TESTS FOR hGH DEFICIENCY FOR CLINICAL USE

The main objective for investigating growth hormone secretion is growth in children. Since normal growth in height and weight represents good health, deviations thereof have to be followed. In statistical terms we recommend following children presenting with a height attained between mean for age and sex and 2 standard deviation scores (SDS) below the mean and, if calculated from reliable data, a growth velocity of above the 25th percentile for age and sex. These children should be seen after 6-12 months to ascertain growth velocity. Only basal investigations and laboratory tests are necessary (Table 6, first group). Since growth velocity is a much more discriminant observation the investigation should be extended (Table 6, middle group). Data on renal function and karyotype in girls should be included since both may detect frequent, non-hormonal causes of stunted growth. A radiograph of the hand allows for determination of the skeletal development and the approximate developmental age of the patient. From the relation of chronological age (CA) to bone age (BA) to age for height attained (HA) clinical features of intrauterine growth retardation or familial short stature (CA = BA, H A diminished), constitutional delay of growth and adolescence ( B A = H A diminished), hypothyroidism ( B A ~ H A ~ C A ) and growth hormone deficiency ( H A < B A < C A ) may be deduced. Since many children with defective h G H secretion show small sellae, skull radiography is justified if the clinical findings support this diagnosis. To test the hormonal status, screening tests of hGH are recommended, e.g. basal level of IGF-I, exercise test, L-dopa in combination with clonidine. For these tests the patients have to follow special guidelines (see above). Thyroid function tests should be included, since, although congenital hypothyroidism can now safely be excluded where screening is employed, acquired hypothyroidism has to be regarded as a possibility since slowing of growth may be the first clinical sign. Finally, if the patient is very small and has, at two visits, a growth velocity below the 25th percentile or even below the 3rd percentile a full investigation into growth hormone insufficiency must be undertaken. This includes the above and pharmacological and physiological tests and can only be done reliably in a specialist's department and with the patient on the ward for a

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541

Table 6. Recommendations for stepwise investigation of short stature and growth hormone deficiency. Growth

Recall

Investigation

Height between mean and - 2 SDS; growth velocity above 25th percentile Height between - 2 and -3 SDS; growth velocity below 25th percentile Height below 3 SDS; growth velocity at second visit below 25th or below 3rd percentile

Outpatient control 12 months Laboratory normal: 3-6 months Laboratory normal: 3 months

Outpatient;basal laboratory Outpatient;extended laboratory Ward;full laboratory

Outpatient and basal laboratory investigations: Detailed history especiallygrowth data including development of parents and siblings (height for age of patient, growthspurt, puberty); careful clinical examination; growth data and if reliable, growth velocity/yearcompared with appropriate standard for region, race and sex. Analysis of blood, urine, sedimentation rate. Outpatient and extended laboratory investigations: Same as basal + creatinine, urea nitrogen, phosphate, acid-base state, alkaline phosphatase; karyotype (exclusion Turner's syndrome). X-rays: hand for determination of bone age; skull for size of sella turcica, prediction of final height compared with target height. Hormones: screening for hGH; exercise test or L-dopa/L-carbidopa or clonidine, IGF-I basal, thyrotropin, triodothyronine, thyroxine, (GH-BH-I), IGFBP3. Ward and extended laboratory investigations: Same as basal and extended. Hormones: two confirmatory tests of hGH secretion combined with other tests of pituitary function, e.g. arginine + TRF + CRF/ACTH + LHRH or sequential arginine/insulin, (circadian hGH secretion), IGF-I stimulation test. Adapted from Underwood and van Wyk (1985), Schfnberg (1986) and Brook and Hindmarsh (1991).

few days. The plan of investigations will have to be explained to the patient and his or her parents, and the ward should be visited in advance to prevent arousal and stress, with their effects on stress-dependent hormones like h G H ( U n d e r w o o d and van Wyk, 1992).

SUMMARY Many ways of evaluating the physiological state of h G H secretion exist, some of which have been touched upon and none of which has as yet proven infallible. A p a r t from important clinical features like history, physical data and growth rate, the diagnosis of altered pituitary function is based on tests and their interpretation. The physician responsible has to be informed on their effectiveness and pitfalls. Results should be interpreted in relation to developmental age (bone age) rather than chronological age. Research is under way to try to facilitate the diagnosis of varying degrees of alterations of h G H secretion. Reliability in predicting the effect of therapy with h G H is the ultimate aim in order to prevent unnecessary cost and disappointment for the patients. With the help of doctors involved in child care, such as physicians at kindergarten or school, it should be possible to start the slow process of investigating growth disorders at an early age.

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