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October 1987, Volume 37, Issue 10

Original Article

PROVOCATIVE TESTING OF HUMAN GROWTH HORMONE RELEASE IN SHORT STATURED CHILDREN

Qaisar Hussain Siraj  ( Nuclear Medical Centre, Armed Forces Institute of Pathology, Rawalpindi. )
Asma Inam-ur-Rehman  ( Nuclear Medical Centre, Armed Forces Institute of Pathology, Rawalpindi. )
Amin Waqar  ( Nuclear Medical Centre, Armed Forces Institute of Pathology, Rawalpindi. )
Syed Azhar Ahmed  ( Nuclear Medical Centre, Armed Forces Institute of Pathology, Rawalpindi. )

Abstract

We have studied the combined use of two procedures, i.e, L-Dopa stimulation and exercise stimula­tion as provocative tests of hGH release in establishing the diagnosis of hGH deficiency in 30 short statured children. This combination was found to be a simple, reliable and practical regimen for routine use as an outdoor diagnostic procedure. Our initial experience with the “Cold pressor test”, a new test of hGH stimulation is also reported (JPMA 37:256 ,1987).

INTRODUCTION

When a child falls repeatedly below the third percentile of height standard in relation to its age, his growth may be considered to be abnormal. Children wjth growth failure and dwarfism without a clearly defmed abnormality should be studied for hGH deficiency.
The growth hormone content of human serum may be measured by bioassay, radiore­ceptor assay, or radioimmunoassay (RIA). RIA was first applied to hGH by Greenwood and his colleagues1. Prior to the introduction of RIA techniques for measuring hGH levels in plasma, the definite diagnosis of pituitary dwarfism presented great difficulties. The use of RIA for the measurement of hGH levels in the serum has brought about major impact on physiological studies and clinical practice. In most normal subjects, the basal secretion of hGH is low and it is not possible to differentiate with confidence the normal from hypopituitarism by a single determination of plasma hGH. To evaluate the adequacy of hGH secretion, dynamic testing of hGH release in response to suitable provocative tests has now become established as an effective means to assess the hGH secretory capacity of anterior pituitary or the presence of an abnor­mality of hGH control mechanism.
A large number of stimulatory tests have been employed to aid in the assessment of pos­sible hGH deficiency in the short statured (Table 1).

For our study, we selected the L-Dopa Stimulation and the exercise stimulation tests, from among the range of established stiniulatory tests, on account of their ease of performance, simplicity, reliability and lack of necessity of continuous medical surveillance.
This study was aimed at evaluating, in routine outpatient clinic environment the role of the above tests in investigating hGH deficiency in short statured children. Furthermore,we report our initial experience with the “Cold pressor test”, a new test for hGH stimulation.

MATERIALS AND METHODS

Thirty short statured children ranging in age from 3.5 to 18 years, with a mean age of 10.41 ± 3.80 years referred to the Nuclear Medical Centre, A.F.I.P, Rawalpincli, for investigations of growth hormone deficiency were studied. Serum samples for the estimation of basal resting hGH levels were obtained from all patients in the morning after an overnight fast. Growth hormone stimulation tests were then performed in these patients. L—Dopa stimulation test was performed in 29 patients, exercise stimulation in 25 patients and 10 patients were subjected to the “Cold pressor” stimulation test. 26 children received a combination of two provocative tests and 4 children underwent all three provocative procedures.
STIMULATION TESTS
1. Exercise stimulation test
The patients were exercised maximally on a tread­mill at a speed and gradient varying with their physical size and condition and blood samples obtained at the end of the exercise period.
2. L-Dopa stimulation test
An oral dose of L-Dopa was administered according to the weight of the patient as follows:
Lessthan20Kg-125mg.
From 20 to 35 Kg — 250 mg.
Over3SKg-5OOmg.
Blood samples were taken 90 min.later.
3. Cold pressor test
The patients were made to place one hand in crushed ice for a period of 4-5 min. and blood samples for hGH estimation were obtained 5 min. later.
IMMUNOASSAY FOR HUMAN GROWTH HORMONE
Dac-Cel-hGH-MCA by Weilcome, an inimunora­diometric assay kit utilising antibody to human growth hormone was used to quantify serum hGH.
The kit utilises the two-site immunoradio­metric assay(sandwich) principle in which two different antibodies, combining with distinct antigenic determinants on the growth hormone molecule, are used2. The various stages of the assay take place sequentially. hGH in the sample is first incubated with an excess of labelled antibody to hGH and the labelled antibody-hGH complex is then reacted with a second (unlabelled) antibody to hGH. The labelled antibody-hGH-second anti­body complex is then separated from free labelled antibody using an anti-rabbit serum covalently coupled to cellulose. After solid-phase separation of this “sandwich”, the distribution of radioac tivity is determined. The amount of radioactivity increases proportionately with the concentration of hGH in the sample, which is determined by reference to a standard curve set up at the same time. The labelled antibody to hGH is of mono­clonal origin conferring high specificity on the assay . The intra-assay precision (agreement between replicate measurements) was about 3.9%, and the interassay precision (repeatability of assayed values) was about 6.1%.

RESULTS

Based on th results of the stimulation tests for hGH, the patients were divided into 3 groups. Group 1 included patients who showed normal response to a stimulation test in that their serum hGH level increased to 15 mIU/L or more as a result of the stimulus; Group 2 included patients who showed a response of 10 mJU/L or more but less than 15 mIU/L and these were classified as partially deficient; Group 3 included those patients who failed to achieve a level of lOmIU/L in response to any of the stimulation tests.

Table II lists the relevant clinical details, the basal hGH values, and the results of the stimulation tests for the patients in group 1,

table III for the patients in group 2, and table IV for the patients in group 3.


GROUP 1 PATIENTS
40% (n12) of the patients tested showed a normal response to at least one provocative test. The mean basal hGH value in this group was 5.14 ± 3.6 mIU/L. The mean hGH level in response to L-Dopa (n12) was 213 ± 15.4 mIU/L, with a mean rise of 810% over the basal level; the mean hGH value in response to exercise stimulation (n10) was 17.1 ± 10.7 mIU/L with a mean rise of 355% over the basal value; and the mean hGH value in response to cold pressor (n=4) was 18.7 ± 5.8 mIU/L with a mean rise of 794% over the basal value.
GROUP 2 PATIENTS
16.7% (n5) of the patients showed partial hGH deficiency and the mean basal hGH level in this group was 3.2±1.4 mIU/L. After L-Dopa stimulation the mean hGH value increased to 11.2 ± 2.6 mIU/L, showing a mean increase of 321% over the basal value. In response to exercise sti­ mulation the mean hGH value rose to 8.9 ± 2 mIU/L with a mean increase of 256% over the basal value, and in response to cold pressor the percentag~ increase was only 42% with a mean hGH value of 5.8 mIU/L.
GROUP 3 PATIENTS
43.3% (n=13) of the patients tested proved hGH deficient with a mean basal hGH level of 2 ± 0.7 mIU/L. After L-Dopa stimulation, the mean hGH value rose to 4.4± 1.6 mIU/L, showing a mean percentage rise of 118% over the basal value. After exercise stimulation, the hGH value rose to 4.4 ± 2.2 mIU/L showing a mean percentage rise of 121% over the basal value and after cold pressor stimulation the mean hGH value was found to be 3.7 ± 2.33 mIU/L with a mean percentage rise of 29% over the basal value.
75% of the patients in groups 1 and 2 as compared to 30% of the patients in group 3 had basal levels of serum hGH greater than 2.0 mlU/L.
17% (n=5) of the patients tested with L-Dopa failed to respond to this stimulus, but showed a normal response to other provocative tests. Similarly, 20% (n=5) of the exercise test results also showed a failure to respond, as did 33% of those subjected to cold pressor test.

DISCUSSION

Impairment of hGH secretion either due to abnormal function or destruction of somato­trophic cells of the anterior pituitary, or due to lack of hypothalamic stimulation in childhood leads to the impaired growth called pituitary dwarfism. However, in only a minority of these patients there is evidence of pituitary destruction; 10% are familial with transmission as a recessive gene, most cases have no familial basis and are not the result of any disease process affecting the pituitary; in about one third of the cases isolated hGH deficiency is present; and in the remaining, other pituitary deficiencies exist3. Thus the estimation of plasma hGH appears to have a decisive role to play in establishing the diagnosis of pituitary dwarfism.
The characteristic secretory pattern of hGH is one of low basal levels with secretory episodes associated with sleep, exercise, stress, and high protein meals3. In our study the basal level of serum hGH was seen to be less than 2 mIU/L in 70% of the patients in group 3 as compared to 25% of the patients in groups 1 and 2. Thus although the basal levels of hGH tend to be lower in patients with hGH deficiency than in the normal patients, simple hGH levels alone are not sufficient to arbitrarily decide the issue and the diagnosis of hGH deficiency must be considered in the light of dynamic testing of hGH release and the clinical picture.
L-Dopa, a precursor of catecholamines, induces a significant rise in the peripheral hGH levels and is thought to act via its conversion to dopamine and the subsequent perturbation of the hypothalamic catecholamine4s. The manner in which exercise stimulates hGH release is not known, although it has been observed that for a reliable response the subject should be exercised to exhaustion. 5
The criteria applied to the assessment of hGH stimulation tests are largely empirical. Most workers would accept the failure to achieve a peak level of 10.0 mIU/L as evidence of hGH deficiency. A further category of partial hGH deficiency has been accepted by consensus, although the cut.off for this group varies from 14 to 20 mIU/L in different centres. The normal response of normal subjects to hGH stimulation tests ranges from 50-90%, with most of the tests recording a positive response in approximately 75% of normal subjects if a peak response of 15 mIU/L is to be achieved. This means that one in four of the normal subjects will have partial hGH deficiency on the basis of one test. For this reason it is customary to perform a series of tests to reduce the probability of a false interpretation.6
Our results of the three provocative tests show that the mean response to L-Dopa in the non-deficient and partially deficient patients is higher than that obtained with the other two procedures, and that the mean percentage rise over the basal value by L-Dopa is also greater in these patients. The results of the exercise stimula­tion in the partially hGH deficient patients were not satisfactory in that 50% of these patients failed to respond adequately to the stimulus (see figure).


The superiority of L-Dopa is also reported by other workers. In a comparative study of 5 provocative test procedures conducted in normal volunteers to ascertain the relative effectiveness of each stimulus, Eddy et al found that the most consistent stimulus for producing a positive hGH response was L-Dopa (95%), followed by insulin (90%), arginine (80%), vasopressin (60%), and glucagon (55%). They further reported that the side effects encountered with L-Dopa stimula­tion were considerably less frequent and less consequential than those with insulin induced hypoglycaemia7
None of the three stimulation tests in this study produced a positive response in 17-33% of the hGH non-deficient patients (see table II). However the patients in this group, who failed to respond to L-Dopa, responded adequately to exercise and vice versa, and those who did not respond to cold pressor responded well to exercise and/or L-Dopa. These results agree with the reports in the literature and confirm that failure to responc to one stimulus does not necessarily imply a lack of responsiveness to another, nor does it unequivocally establish the diagnosis of hypopi­tuitarism.
Faraser and Fass et al have reported that of normal children tested with L-Dopa stimulation, 10% showed a response of less than 15 mIU/L8,9
Weldon et al in studying the response of short statured children to various provocative tests found a false negative response to L.Dopa in 13% and to insulin in 15% of those proved to be hGH non-deficient10 Similarly Lacey et al and Buckleret al have reported hGH levels of less than 10 mIU/L after exercise in 20-30% of the normal children tested11,12
Our study has highlighted the effectiveness of the combined use of two simple tests of hGH stimulation, i.e., L-Dopa and exercise stimulation, for evaluating hGH deficiency and established their suitability for routine use as an outdoor diagnostic procedure. Although insulin-induced hypoglycaemia has been regarded as the standard hGH stimulation test, the incidence and severity of side effects and the need for constant medical survefflance often curtail its usefulness. L-Dopa has proved to be a safe and reliable stimulus of hGH release, with few side effects, and the additional use of exercise testing has effectively reduced false interpretation.
The number of patients on whom cold pressor test was performed is as yet too small to merit definite conclusions but the initial results appear promising. It evoked a higher response than L-Dopa in S out of 9 patients who under­went both tests, and also a greater response than to exercise in 3 out of 4 patients subjected to both exercise and cold stimulation. Four patients in group 1 were subjected to cold stimulation and the diagnostic response of greater than 15 mIU/L was elicited by this test in two patients who had failed to respond adequately to L-Dopa and exercise stimulation. These results indicate that this simple, safe, and less time consuming test may be valuable in discriminating patients with hGH deficiency from the non-deficient patients.

REFERENCES

1. Greenwood, F.C., Hunter, W.M., and Glover, J.S. The preparation of 131 I4abelled human growth hormone of high specific activity. Biochem. J.,1963;89: 114.
2. Peake, G.T., Morris, J. and Buckman, M.T. Growth Hormone. in methods of hormone radioimmuno­ assay. Edited by Jaffe, B.M., Behrman, H.R. New York, Academic Press, 1979, p. 223.
3. williams, R.H. Textbook of endocrinology. Edited by Robert H. williams 6th ed. Philadelphia, Saunders, l98l,p.97.
4. Eddy, R.L., Jones, A.L., Chakmakjian, Z.H. and Silverthorne, M.G. Effect of levodopa (L-Dopa) on human hypophyseal trophic hormone release. J. Clin. Endocrinol. Metab., 1971; 33:709.
5. Richard, A.D. Endocrine disorders; a guide to diagnosis. Edited by Richard, A.D. London, Butterworths, 1984, p. 639.
6. Richard, A.D. Endocrine disorders; a guide to diagnosis. Edited by Richard, A.D. London, Butterworths, 1984, p. 292.
7. Eddy, R.L., Gilliland, P.F., Ibarra, J. D. Jr., McMurry, J. F. Jr., and Thompson, J.Q. Human growth  hormone release; comparison of provocative test procedures. Am. J. Med., 1974; 56: 179.
8. Fraser, S.D. A review of growth hormone stimula­tion tests in children. Paediatrics, 1974; 53: 929.
9. Fass,’ B., Lippe, B.M. and Kaplan, S.A. Relative usefulness of three growth hormone stimulation screening tests. Am. J. Dis. Child., 1979; 133:931.
10. weldon, V.V., Gupta, 5K., Haymond, M3V., Pagliara, AS., Jacobs, L.S. and Daughaday, wit The use of L.dopa in the diagnosis of hyposoma­totropism in children. J. Clin. Endocrinol. Metab., 1973;   36:42.
11. Lacey, K.A., Hewison, A. and Parkin, J.M. Exercise as a screening test for growth hormone deficiency in children. Arch. Dis. Child., 1973; 48:508.
12. Buckler, J.M.H Plasma growth hormone response to exercise as a diagnostic aid. Arch. Dis. Child., 1973; 48:565.

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