J. Endocrinol. Invest. 13: 307-316, 1990
A new test for the diagnosis of growth hormone deficiency due to primary pituitary impairment: combined administration of pyridostigmine and growth hormone-releasing hormone E. Ghigo*, E. Imperiale*, G, M. Boffano*, E. Mazza*, J, Bellone*, E. Arvat*, M, Procopio*, S. Goffi*, A. Barreca**, P Chiabotto***, R. ~ala***, C. Oe Sanctis***, M. F. Boghen*, E. E. Müller****, and F. Camanni* *Divisione di Endocrinologia, Dipartimento di Fisiopatologia Clinica, Universita di Torino, **Divisione di Endocrinologia, ISMI, Universita di Genova, ***Divisione di Endocrinologia Pediatrica, Ospedale Regina Margherita, Torino, ****Dipartimento di Farmacologia, Universita di Milano, Italy tively. In contrast even assuming a minimum normal GH peak as high as 20 pg/I, PD + GHRH induced a positive response in 27/27 NS all having a .noct4rnal GH mean concentration (MG) ~ 3 pg/I. Therefore PD + GHRH test gave no false negative responses and this was true not only in NS but even in all FSS and CGD having a GH MC ~ 3 pg/I. On the other hand, PD + GHRH induced a negative GH response in all oGHD and in 8/10 iGHD patients. In the remaining two iGHD patients, PD + GHRH demonstrated anormal pituitary GH reserve in spite of a GH MC < 3 pg/I and low IGF-Ilevel, thus pointing to a hypothalamic pathogenesis for the GHD. Considering FSS and CGD children having a GH MC < 3 pg/I, PD + GHRH showed a primary pituitary GH deficiency in 3/12 CGD with low plasma IGF-I levels. In conclusion, in slowly growing children PD + GHRH test is the most reliable provocative test for the diagnosis of primary pituitary GH deficiency being capable to discriminate between an unequivocally normal and impaired somatotropic function. The study of spontaneous GH secretion is mandatory only for those patients having low height velocity but anormal GH response to PD + GHRH in order to look for the existence of a GHD due to hypothalamic dysfunction.
ABSTRACT. The diagnosis of growth hormone (GH) deficiency (GHD) is currently based on failure to increase plasma GH levels to an arbitrary cutoff point of 7 or 10 pg/I in response to two provocative stimuli. False negative responses to these tests, however, frequently occur thus reducing their diagnostic reliability. Theaim of this study was to assess a combination of pyridostigmine (PD) and GH-releasing hormone (GHRH) (60 mg oral PD 60 min before 1 pg/Kg GHRH iv) as a reliable test probing pituitary somatotropic function. In fact PD, an acetylcholinesterase inhibitor, strikingly potentiates GH response to GHRH likely by inhibiting somatostatin release. The combination PD + GHRH was tested in normal children and adolescents (NS, n = 27) and in a large group of short children classified as having familial short stature (FSS, n = 24), constitutional growth delay (CGD, n = 34) and GH deficiency (organic, oGHD, n = 6; idiopathic, iGHD, n = 10). In all groups results obtained by PD + GHRH were compared with those obtained by testing with GHRH, clonidine (CLON) and PD alone and by studying spontaneous nocturnal GH secretion over 8 hours. Assuming 7 pg/I as minimum normal GH peak, a positive response occurred in only 18/24,11/12 and 12/13 NS for GHRH, CLON, and PD, respec-
INTRODUCTION The criterion for the diagnosis of growth hormone (GH) deficiency (GHD) is based on failure to increase plasma GH levels to an arbitrary cut-off point of 7 or 10 PQ / I in response to at least two provocative stimuli such as arginine, insulin-induced
Key-words: Growth hormone, growth hormone-releasing hormone, pyridostigmine, short stature. Correspondence: Prof. Franeo Camanni, Divisione di Endocrinologia, Ospedale Molinette. Corso Polonia 14, 10126 Torino, Italy.
Received September 29, 1989; accepted February 5, 1990.
307
E. Ghigo, E. Imperiale, G.M. Boffano, et al.
into 4 groups as folIows: 1) Familial short stature (FSS) (16 boys and 8 girls; age 6.9-14.5 yr): a) height below the third percentile; b) predicted adult height within parental target zone (i.e. midparental height ± 8.5 cm) and, in children under 9 yrs, height equal to or above the corrected third percentile; c) bone age corresponding to chronological age ± 24 months. 2) Constitutionalgrowth delay (CGD) (29 boys and 5 girls; age 5.4-16.0 yr): a) height below the third percentile; b) predicted adult height within the parental target zone and, in children under 9 yr, height equal to or above the corrected third percenHle; c) bone age retardation more than 24 months. 3) Idiopathic GH deficiency (lGHD) (8 boys and 2 girls; age 6.5-15.9 yr): a) height below the third percentile; b) predicted adult height below parental target zone and, in children under 9 yr, height below the corrected third percentile; c) height velocity lower than the third percentile curve; d) bone age retardation more than 12 months; e) plasma GH peak< 7 pg/l after insulin 0.1 U/Kg iv. 4) organic GH deficiency (oGHD) (4 boys and 2 girls; age 3.4-14.7 yr): height above or below the third percentile. These patients have been examined 36 months after surgery for a craniopharyngioma. On the basis of the hormonal response to combined iv administration of insulin (0.1 U I kg), TRH (200 pg) and GnRH (100 pg), these subjects were shown having a pituitary deficiency of GH, ACTH, LH, FSH and TSH secretion. Basal plasma PRL levels were normal in all subjects. Diabetes insipidus was shown in one (case 5). All patients were on replacement therapy with cortisone and I-thyroxine at the time of the study. Individual clinical details concerning age, pubertal stage, bone age, height velocity as weil as hormonal levels including IGF-I are reported in tables 1-4. All subjects had normal body weight (± 10% of ideal body weight). None had evidence of nutritional deficiency, systemic disease, dysmorphology or psychological deprivation. Thyroid and adrenal function were normal in all subjects except for organic GH deficient ones (see above). Two out of 10 iGHD (cases 8,9) were previously treated with exogenous GH. Both patients discontinued therapy at least one month before starting the study. The children were studied as in-patients, after informed consent was obtained from parents.
hypoglycemia, clonidine, I-dopa and glucagon (13). Propranolol is sometimes given with the latter two drugs (1 -3). False negative responses to these tests frequently occur also in normal subjects, thus reducing their diagnostic reliability (1 -3). It was been clearly shown that even the GH-releasing hormone (GHRH) test is not a discriminator between normal and GHD children (4-6). In fact great inter- and intraindividual variability and false negative somatotroph responses to the neuropeptide frequently occur, also in normal subjects (7, 8). The measurement of insulin-like growth factor I (IGF-I) can be helpful in confirming the diagnosis, though it cannot be used as a sole test (3, 9, 10). Based on the foregoing, it is now widely accepted that in children with low height velocity the study of spontaneous GH secretion is the only method that can identify subjects with deficient GH secretion (4,11-14). More recently, however, Rose et al. raised criticism against this diagnostic approach, mainly focusing on the great overlap existing between spontaneous GH secretion in normal and GH deficient children, if they are matched for age and pubertal development (15). Therefore, these Authors oppose abandoning classical provocative tests. Taking all into account, it can be stated that what constitutes a correct diagnostic approach of GH deficiency remains matter of debate. We have previously shown that the enhancement of cholinergic activity by pyridostigmine (PD) stimulates GH release and clearly potentiates GH response to GHRH, both in adults and in children with short stature (16-18). The aim of our study was to verify the diagnostic reliability of PD + GHRH as a functional test probing the pituitary somatotropic function. Thus, we have: 1) expanded our previous observation (18) to include normal children and a larger number of short children; 2) compared the GH response to PD + GHRH with those occurring after GHRH, clonidine and PD alone, and with nocturnal spontaneous GH secretion.
MATERIALS AND METHODS Subjects
Twenty-seven normal children and adolescents (NS) (20 boys and 7 girls; aged 5.9-15.5 yr; height ranging from 10th to 50th centile) and 74 subjects with short stature were studied. These were divided
Pharmacological tests After an overnight fast, GH tests were carried out
308
A new test to diagnose GH deficiency
starting at 09:00 h, 30 min after an indwelling catheter was inserted into an antecubital vein. Four tests were performed at least 3 days apart and in random order: 1) GHRH (GHRH 29, Kabivitrum), 1 pg/Kg iv as bolus; 2) clonidine (CLON, Catapresan, Boehringer Ingelheim), 150 pg/m 2 orally; 3) pyridostigmine (PD, Mestinon, Hoffmann-LaRoche), 60 mg orally; 4) PD + GHRH, PD administered 60 min before GHRH. Blood sampies were taken every 15 min, from -15 to 120 min for GHRH and PD GHRH tests, and from 0 to 180 min for both CLON and PD test.
eter had been inserted into an antecubital vein. During nocturnal test all subjects slept. Lights were off at 23:00 h. EEG recording was not performed.
Procedures Height was measured using an Harpenden stadiometer. Height, heigth velocity, corrected third percentile, predicted adult height, bone age (RUS) and pubertal staging were defined according to Tanner et al. (19, 20). Growth measurement were expressed as standard deviation scores for either chronological age or skeletal age. Plasma GH levels were measured in duplicate by RIA using SORIN reagents (Saluggia, Italy). All sampies from an individual subject were analyzed together. The sensitivity of the assay was 0.2 pg/I.
+
Spontaneous nocturnal GH secretion The study of nocturnal GH secretion was performed by taking blood sam pies every 30 min from 23:00 to 07:00 h, starting 30 min after an indwelling cath-
Table 1 - Normal children and adolescents: clinical details and results o( provocative tests and spontaneous noeturnal GH seeretion.
Ca se (no.) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Sex F M M F F
F F M
F F M M M M M M M M M M M M M M M M M
Age (yr) 5.9 8.7 8.9 9.3 9.5 9.5 9.8 10.3 10.5 10.7 11.0 12.9 13.1 13.1 13.5 13.9 14.1 12.5 12.9 130 13.9 14.0 14.2 13.8 15.5 15.0 15.1
Pubertal Stage PH B/G
Bone Age (yr) 5.0 8.8
1 1 1 1 1 1 1 2 2 2 2 2 2 3 3 4 4
14.6
12.9
13.5
Nocfurnal Mean GH Concentration (pg/I)
Highest Nocturnal GH Peak (pg/I)
GHRH
CLON
PO
PO+GHRH
IGF-I (pg/I)
22.2 43.5
13.0 6.3
7.0 18.0
15.0 16.5 21.8 12.7
50.0 50.0 23.8 37.0 43.0 50.0 40.4 40.0 38.0 44.0 80.0 34.0 38.0 43.7 25.0 26.0 26.0 45.0 34.0 50.0 65.0 24.4 35.0 24.0 47.5 50.0 36.0
212.4 99.9 102.1 106.0 199.9 120.0 312.9 104.0 84.0 317.1 140.0 170.5 295.6 76.0 304.0 304.3 298.5 275.3 277.0 306.0 325.0 319.0 323.0 542.0 206.5 492.0 442.6
Height SOS
Height Velocity SOS
+0.4 -1.1 -1.0 -1.0 -0.9 -1.0 -1.0 -0.5 -1.0 - 0.6 -0.7 -1.1 - 0.9 - 0.9 -1.0 -1.0 - 0.5 - 0.8 -1.2 -1.0 - 0.7 - 0.6 -0.8 -0.8 -1.0 -0.4 0
- 0.6 0 +1.1 +1.0 +1.4 +0.4 0 +0.3 - 0.1 - 0.7 +1.2 -0.8 0 -0.7 - 0.9 -1.2 -1.0 + 1.4 + 1.4 - 0.7 -1.1 -1.6 +1.4 -0.5 0 +1.3 +0.1
6.0 13.0
26.0 42.0
7.7
38.0
4.5 3.6 3.0
18.6 11.8 8.6
3.7
9.7
3.0 3.0
8.9 16.2
3.0
11.3
4.0 5.1 5.4
17.0 15.4 18.0
11.5
50.0
6.8
30.5
GH Peak (pg/I)
14.0 26.0 8.5 15.3 5.8 8.5 15.0 25.0 19.3 6.0 23.0 20.8 15.0 11.2 16.5 18.5 44.0 4.0
15.0
14.0 8.2 13.5 16.0 8.0
12.0 12.5
3.2 5.7 5.6 15.0
18.0 12.0
13.0 15.0 12.0 8.2 16.0 7.8 13.3 12.5 6.8 9.3 8.4 10.8 8.4 13.0 11.6
Mean ±SE
12.0 0.5
- 0.8 0.1
0 0.2
5.6 0.8
21.5 3.4
16.3 2.2
12.4 1.0
12.2 0.9
40.7 2.5
250.1 24.0
Range
5.9 15.5
-1.3 +0.4
-0.6 +1.4
3.0 13.0
8.6 50.0
3.2 44.0
6.3 18.0
6.8 21.8
23.8 80.0
76.0 542.0
309
E. Ghigo, E. Imperiale, G.M. Boftano, et al.
RESULTS
The intra- and interassay coefficients of variation were 4.5 and 7.9%, respectively. The GH secretory responses to pharmacological tests were expressed as absolute peak values (pg/l) irrespective of time of occurrence. The spontaneous GH secretion was expressed as mean concentration (MC) and as highest nocturnal peak (HNP). IGF-I was measured by radioimmunoassay, using the immunochemicals and trace provided by Nichols Institute (San Juan Capistrano, CA, USA). IGF-I concentrations are expressed as pg/l with reference to a pure recombinant IGF-I preparation (a generous gift from Ors. L. Fryklund and A. Skottner, Kabivitrum AG, Stockholm, Sweden). Sensitivity of this assay is 9 pg 1I. Interassay coefficient of variation is 14% at a concentration of 364 pg/tube. In order to avoid interference by binding proteins, all plasma sampies were treated with acidethanol, according to Oaughaday et al. (21). The recovery by this acid extraction procedure, determined after overnight incubation of pool sam pies with labelled IGF-I, was 89.85% (SO 2.54, n = 12). Test sampies were diluted appropriately to give a concentration from 5 to 1300 pg/tube. In our laboratory, normal ranges are: for pubertal stage 1 74342 pg/l, for stage 2 75-398 pg/l, for stage 3 and 4 189-592 pg 1I. Statistical analysis of the data was performed by using the two way analysis of variance with Scheffe's f test and unpaired Student's ttest, where appropriate. FAMILI AL SHORT STATURE
NORMAL CHI LOREN
GH OEFICIENCY
0
2-
0
0 80
0
Ir
+
0
0
~o
o~o
60
0
o§
0
J: Cl 0..
±
0.3pg/l. A GH MC;::: 3 pg/l was present in 83% (20/24) of children. There were no significant differences between mean HNP and mean peaks after GHRH,
0
c,
c
Familial short stature (Table 2, Fig. 1) Basal plasma GH levels (mean ± SE) were 1.8
CONSTITUTIONAL GROWTH OELAY
100
J:
Normal subjects (Table 1, Fig. 1) Basal plasma GH levels (mean ± SE) were 1.6 ± 0.3 pg/l. All subjects had a GH MC;::: 3 pg/l. T{1ere were no significant differences between mean HNP and mean peaks after GHRH, CLON and PD. PD + GHRH test induced a mean GH peak clearly higher (p< 0.0001 ) than HNP, GHRH, CLON and PD. Assuming 7 pg/l as minimum normal GH peak, a positive hormonal increase occurred in 75% (18/24),92% (11/12), 92% (12/13) and 100% (27/27) of subjects, in response to GHRH, CLON, PD and PD + GHRH test, respectively. Assuming 10 pg/l as minimum normal GH peak, a positive hormonal increase occurred in 67% (16/24), 75% (9/12),62% (8/13) and 100% (27/27) of subjects, in response to GHRH, CLON, PD and PD + GHRH test, respectively. PD + GHRH test induced GH peak ranging from 23.8 to 80.0 pg/l. Since the mean - 2 SO of these data (after logarithmic transformation) is 20 pg/l, this value was assumed as minimum normal GH peak. All subjects had normal IGF-I levels.
~
GI
0000 0 0 00 00 0
ca
"'ca"
40
GI
a.
J: Cl
.
ca
8000 0 00
00 0 00 0
00 0
0
20
8
8
0
-------
E
0
0
---------------
ca
00
0
0
0
0 0 00 0
0 -----------ö--8
ii: 0
I
,.3
I
I
,.3
A new test for the diagnosis of growth hormone deficiency due to primary pituitary impairment: combined administration of pyridostigmine and growth hormone-releasing hormone E. Ghigo*, E. Imperiale*, G, M. Boffano*, E. Mazza*, J, Bellone*, E. Arvat*, M, Procopio*, S. Goffi*, A. Barreca**, P Chiabotto***, R. ~ala***, C. Oe Sanctis***, M. F. Boghen*, E. E. Müller****, and F. Camanni* *Divisione di Endocrinologia, Dipartimento di Fisiopatologia Clinica, Universita di Torino, **Divisione di Endocrinologia, ISMI, Universita di Genova, ***Divisione di Endocrinologia Pediatrica, Ospedale Regina Margherita, Torino, ****Dipartimento di Farmacologia, Universita di Milano, Italy tively. In contrast even assuming a minimum normal GH peak as high as 20 pg/I, PD + GHRH induced a positive response in 27/27 NS all having a .noct4rnal GH mean concentration (MG) ~ 3 pg/I. Therefore PD + GHRH test gave no false negative responses and this was true not only in NS but even in all FSS and CGD having a GH MC ~ 3 pg/I. On the other hand, PD + GHRH induced a negative GH response in all oGHD and in 8/10 iGHD patients. In the remaining two iGHD patients, PD + GHRH demonstrated anormal pituitary GH reserve in spite of a GH MC < 3 pg/I and low IGF-Ilevel, thus pointing to a hypothalamic pathogenesis for the GHD. Considering FSS and CGD children having a GH MC < 3 pg/I, PD + GHRH showed a primary pituitary GH deficiency in 3/12 CGD with low plasma IGF-I levels. In conclusion, in slowly growing children PD + GHRH test is the most reliable provocative test for the diagnosis of primary pituitary GH deficiency being capable to discriminate between an unequivocally normal and impaired somatotropic function. The study of spontaneous GH secretion is mandatory only for those patients having low height velocity but anormal GH response to PD + GHRH in order to look for the existence of a GHD due to hypothalamic dysfunction.
ABSTRACT. The diagnosis of growth hormone (GH) deficiency (GHD) is currently based on failure to increase plasma GH levels to an arbitrary cutoff point of 7 or 10 pg/I in response to two provocative stimuli. False negative responses to these tests, however, frequently occur thus reducing their diagnostic reliability. Theaim of this study was to assess a combination of pyridostigmine (PD) and GH-releasing hormone (GHRH) (60 mg oral PD 60 min before 1 pg/Kg GHRH iv) as a reliable test probing pituitary somatotropic function. In fact PD, an acetylcholinesterase inhibitor, strikingly potentiates GH response to GHRH likely by inhibiting somatostatin release. The combination PD + GHRH was tested in normal children and adolescents (NS, n = 27) and in a large group of short children classified as having familial short stature (FSS, n = 24), constitutional growth delay (CGD, n = 34) and GH deficiency (organic, oGHD, n = 6; idiopathic, iGHD, n = 10). In all groups results obtained by PD + GHRH were compared with those obtained by testing with GHRH, clonidine (CLON) and PD alone and by studying spontaneous nocturnal GH secretion over 8 hours. Assuming 7 pg/I as minimum normal GH peak, a positive response occurred in only 18/24,11/12 and 12/13 NS for GHRH, CLON, and PD, respec-
INTRODUCTION The criterion for the diagnosis of growth hormone (GH) deficiency (GHD) is based on failure to increase plasma GH levels to an arbitrary cut-off point of 7 or 10 PQ / I in response to at least two provocative stimuli such as arginine, insulin-induced
Key-words: Growth hormone, growth hormone-releasing hormone, pyridostigmine, short stature. Correspondence: Prof. Franeo Camanni, Divisione di Endocrinologia, Ospedale Molinette. Corso Polonia 14, 10126 Torino, Italy.
Received September 29, 1989; accepted February 5, 1990.
307
E. Ghigo, E. Imperiale, G.M. Boffano, et al.
into 4 groups as folIows: 1) Familial short stature (FSS) (16 boys and 8 girls; age 6.9-14.5 yr): a) height below the third percentile; b) predicted adult height within parental target zone (i.e. midparental height ± 8.5 cm) and, in children under 9 yrs, height equal to or above the corrected third percentile; c) bone age corresponding to chronological age ± 24 months. 2) Constitutionalgrowth delay (CGD) (29 boys and 5 girls; age 5.4-16.0 yr): a) height below the third percentile; b) predicted adult height within the parental target zone and, in children under 9 yr, height equal to or above the corrected third percenHle; c) bone age retardation more than 24 months. 3) Idiopathic GH deficiency (lGHD) (8 boys and 2 girls; age 6.5-15.9 yr): a) height below the third percentile; b) predicted adult height below parental target zone and, in children under 9 yr, height below the corrected third percentile; c) height velocity lower than the third percentile curve; d) bone age retardation more than 12 months; e) plasma GH peak< 7 pg/l after insulin 0.1 U/Kg iv. 4) organic GH deficiency (oGHD) (4 boys and 2 girls; age 3.4-14.7 yr): height above or below the third percentile. These patients have been examined 36 months after surgery for a craniopharyngioma. On the basis of the hormonal response to combined iv administration of insulin (0.1 U I kg), TRH (200 pg) and GnRH (100 pg), these subjects were shown having a pituitary deficiency of GH, ACTH, LH, FSH and TSH secretion. Basal plasma PRL levels were normal in all subjects. Diabetes insipidus was shown in one (case 5). All patients were on replacement therapy with cortisone and I-thyroxine at the time of the study. Individual clinical details concerning age, pubertal stage, bone age, height velocity as weil as hormonal levels including IGF-I are reported in tables 1-4. All subjects had normal body weight (± 10% of ideal body weight). None had evidence of nutritional deficiency, systemic disease, dysmorphology or psychological deprivation. Thyroid and adrenal function were normal in all subjects except for organic GH deficient ones (see above). Two out of 10 iGHD (cases 8,9) were previously treated with exogenous GH. Both patients discontinued therapy at least one month before starting the study. The children were studied as in-patients, after informed consent was obtained from parents.
hypoglycemia, clonidine, I-dopa and glucagon (13). Propranolol is sometimes given with the latter two drugs (1 -3). False negative responses to these tests frequently occur also in normal subjects, thus reducing their diagnostic reliability (1 -3). It was been clearly shown that even the GH-releasing hormone (GHRH) test is not a discriminator between normal and GHD children (4-6). In fact great inter- and intraindividual variability and false negative somatotroph responses to the neuropeptide frequently occur, also in normal subjects (7, 8). The measurement of insulin-like growth factor I (IGF-I) can be helpful in confirming the diagnosis, though it cannot be used as a sole test (3, 9, 10). Based on the foregoing, it is now widely accepted that in children with low height velocity the study of spontaneous GH secretion is the only method that can identify subjects with deficient GH secretion (4,11-14). More recently, however, Rose et al. raised criticism against this diagnostic approach, mainly focusing on the great overlap existing between spontaneous GH secretion in normal and GH deficient children, if they are matched for age and pubertal development (15). Therefore, these Authors oppose abandoning classical provocative tests. Taking all into account, it can be stated that what constitutes a correct diagnostic approach of GH deficiency remains matter of debate. We have previously shown that the enhancement of cholinergic activity by pyridostigmine (PD) stimulates GH release and clearly potentiates GH response to GHRH, both in adults and in children with short stature (16-18). The aim of our study was to verify the diagnostic reliability of PD + GHRH as a functional test probing the pituitary somatotropic function. Thus, we have: 1) expanded our previous observation (18) to include normal children and a larger number of short children; 2) compared the GH response to PD + GHRH with those occurring after GHRH, clonidine and PD alone, and with nocturnal spontaneous GH secretion.
MATERIALS AND METHODS Subjects
Twenty-seven normal children and adolescents (NS) (20 boys and 7 girls; aged 5.9-15.5 yr; height ranging from 10th to 50th centile) and 74 subjects with short stature were studied. These were divided
Pharmacological tests After an overnight fast, GH tests were carried out
308
A new test to diagnose GH deficiency
starting at 09:00 h, 30 min after an indwelling catheter was inserted into an antecubital vein. Four tests were performed at least 3 days apart and in random order: 1) GHRH (GHRH 29, Kabivitrum), 1 pg/Kg iv as bolus; 2) clonidine (CLON, Catapresan, Boehringer Ingelheim), 150 pg/m 2 orally; 3) pyridostigmine (PD, Mestinon, Hoffmann-LaRoche), 60 mg orally; 4) PD + GHRH, PD administered 60 min before GHRH. Blood sampies were taken every 15 min, from -15 to 120 min for GHRH and PD GHRH tests, and from 0 to 180 min for both CLON and PD test.
eter had been inserted into an antecubital vein. During nocturnal test all subjects slept. Lights were off at 23:00 h. EEG recording was not performed.
Procedures Height was measured using an Harpenden stadiometer. Height, heigth velocity, corrected third percentile, predicted adult height, bone age (RUS) and pubertal staging were defined according to Tanner et al. (19, 20). Growth measurement were expressed as standard deviation scores for either chronological age or skeletal age. Plasma GH levels were measured in duplicate by RIA using SORIN reagents (Saluggia, Italy). All sampies from an individual subject were analyzed together. The sensitivity of the assay was 0.2 pg/I.
+
Spontaneous nocturnal GH secretion The study of nocturnal GH secretion was performed by taking blood sam pies every 30 min from 23:00 to 07:00 h, starting 30 min after an indwelling cath-
Table 1 - Normal children and adolescents: clinical details and results o( provocative tests and spontaneous noeturnal GH seeretion.
Ca se (no.) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Sex F M M F F
F F M
F F M M M M M M M M M M M M M M M M M
Age (yr) 5.9 8.7 8.9 9.3 9.5 9.5 9.8 10.3 10.5 10.7 11.0 12.9 13.1 13.1 13.5 13.9 14.1 12.5 12.9 130 13.9 14.0 14.2 13.8 15.5 15.0 15.1
Pubertal Stage PH B/G
Bone Age (yr) 5.0 8.8
1 1 1 1 1 1 1 2 2 2 2 2 2 3 3 4 4
14.6
12.9
13.5
Nocfurnal Mean GH Concentration (pg/I)
Highest Nocturnal GH Peak (pg/I)
GHRH
CLON
PO
PO+GHRH
IGF-I (pg/I)
22.2 43.5
13.0 6.3
7.0 18.0
15.0 16.5 21.8 12.7
50.0 50.0 23.8 37.0 43.0 50.0 40.4 40.0 38.0 44.0 80.0 34.0 38.0 43.7 25.0 26.0 26.0 45.0 34.0 50.0 65.0 24.4 35.0 24.0 47.5 50.0 36.0
212.4 99.9 102.1 106.0 199.9 120.0 312.9 104.0 84.0 317.1 140.0 170.5 295.6 76.0 304.0 304.3 298.5 275.3 277.0 306.0 325.0 319.0 323.0 542.0 206.5 492.0 442.6
Height SOS
Height Velocity SOS
+0.4 -1.1 -1.0 -1.0 -0.9 -1.0 -1.0 -0.5 -1.0 - 0.6 -0.7 -1.1 - 0.9 - 0.9 -1.0 -1.0 - 0.5 - 0.8 -1.2 -1.0 - 0.7 - 0.6 -0.8 -0.8 -1.0 -0.4 0
- 0.6 0 +1.1 +1.0 +1.4 +0.4 0 +0.3 - 0.1 - 0.7 +1.2 -0.8 0 -0.7 - 0.9 -1.2 -1.0 + 1.4 + 1.4 - 0.7 -1.1 -1.6 +1.4 -0.5 0 +1.3 +0.1
6.0 13.0
26.0 42.0
7.7
38.0
4.5 3.6 3.0
18.6 11.8 8.6
3.7
9.7
3.0 3.0
8.9 16.2
3.0
11.3
4.0 5.1 5.4
17.0 15.4 18.0
11.5
50.0
6.8
30.5
GH Peak (pg/I)
14.0 26.0 8.5 15.3 5.8 8.5 15.0 25.0 19.3 6.0 23.0 20.8 15.0 11.2 16.5 18.5 44.0 4.0
15.0
14.0 8.2 13.5 16.0 8.0
12.0 12.5
3.2 5.7 5.6 15.0
18.0 12.0
13.0 15.0 12.0 8.2 16.0 7.8 13.3 12.5 6.8 9.3 8.4 10.8 8.4 13.0 11.6
Mean ±SE
12.0 0.5
- 0.8 0.1
0 0.2
5.6 0.8
21.5 3.4
16.3 2.2
12.4 1.0
12.2 0.9
40.7 2.5
250.1 24.0
Range
5.9 15.5
-1.3 +0.4
-0.6 +1.4
3.0 13.0
8.6 50.0
3.2 44.0
6.3 18.0
6.8 21.8
23.8 80.0
76.0 542.0
309
E. Ghigo, E. Imperiale, G.M. Boftano, et al.
RESULTS
The intra- and interassay coefficients of variation were 4.5 and 7.9%, respectively. The GH secretory responses to pharmacological tests were expressed as absolute peak values (pg/l) irrespective of time of occurrence. The spontaneous GH secretion was expressed as mean concentration (MC) and as highest nocturnal peak (HNP). IGF-I was measured by radioimmunoassay, using the immunochemicals and trace provided by Nichols Institute (San Juan Capistrano, CA, USA). IGF-I concentrations are expressed as pg/l with reference to a pure recombinant IGF-I preparation (a generous gift from Ors. L. Fryklund and A. Skottner, Kabivitrum AG, Stockholm, Sweden). Sensitivity of this assay is 9 pg 1I. Interassay coefficient of variation is 14% at a concentration of 364 pg/tube. In order to avoid interference by binding proteins, all plasma sampies were treated with acidethanol, according to Oaughaday et al. (21). The recovery by this acid extraction procedure, determined after overnight incubation of pool sam pies with labelled IGF-I, was 89.85% (SO 2.54, n = 12). Test sampies were diluted appropriately to give a concentration from 5 to 1300 pg/tube. In our laboratory, normal ranges are: for pubertal stage 1 74342 pg/l, for stage 2 75-398 pg/l, for stage 3 and 4 189-592 pg 1I. Statistical analysis of the data was performed by using the two way analysis of variance with Scheffe's f test and unpaired Student's ttest, where appropriate. FAMILI AL SHORT STATURE
NORMAL CHI LOREN
GH OEFICIENCY
0
2-
0
0 80
0
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+
0
0
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60
0
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0.3pg/l. A GH MC;::: 3 pg/l was present in 83% (20/24) of children. There were no significant differences between mean HNP and mean peaks after GHRH,
0
c,
c
Familial short stature (Table 2, Fig. 1) Basal plasma GH levels (mean ± SE) were 1.8
CONSTITUTIONAL GROWTH OELAY
100
J:
Normal subjects (Table 1, Fig. 1) Basal plasma GH levels (mean ± SE) were 1.6 ± 0.3 pg/l. All subjects had a GH MC;::: 3 pg/l. T{1ere were no significant differences between mean HNP and mean peaks after GHRH, CLON and PD. PD + GHRH test induced a mean GH peak clearly higher (p< 0.0001 ) than HNP, GHRH, CLON and PD. Assuming 7 pg/l as minimum normal GH peak, a positive hormonal increase occurred in 75% (18/24),92% (11/12), 92% (12/13) and 100% (27/27) of subjects, in response to GHRH, CLON, PD and PD + GHRH test, respectively. Assuming 10 pg/l as minimum normal GH peak, a positive hormonal increase occurred in 67% (16/24), 75% (9/12),62% (8/13) and 100% (27/27) of subjects, in response to GHRH, CLON, PD and PD + GHRH test, respectively. PD + GHRH test induced GH peak ranging from 23.8 to 80.0 pg/l. Since the mean - 2 SO of these data (after logarithmic transformation) is 20 pg/l, this value was assumed as minimum normal GH peak. All subjects had normal IGF-I levels.
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