Europ. J. Pediat. 122, 85--92 (1976) O by Springer-Verlag 1976

Mean 24-Hour Growth Hormone and Testosterone Concentrations in Relation to Pubertal Growth Spurt in Boys with Normal or Delayed Puberty* O. Butenandt, R. Eder, K. Wohlfarth, F. Bidlingmaier, and D. Knorr Universit~ts-Kinderklinik im Dr. yon tIaunerschen Kinderspital Miinchen (Direktor: Prof. Dr. K. Betke), Abteilung fiir p/~diatrische Endokrinologie (Prof. Dr. D. Knorr) Received January 17, 1976 Abstract. The mean growth hormone concentration during a 24-hour period in 7 boys of short familial stature and a growth rate of 3.2--5.4 era/year was between 1.0 and 4.6 ng/ml serum. In 7 boys with pubertal growth spurt and familial tallness (growth rate 7.2--11.0 cm/ year) it varied from 0.97 to 4.4 ng/ml and in 6 boys with constitutional delay of puberty (a growth rate of 4.2--5.2 era/year prior to puberty) from 1.3 to 4.3 ng/ml. No correlation was found between the 24-hour mean growth hormone concentration and the mean 24-hour testosterone concentration in serum or the growth rate, but a correlation was found between testosterone and the growth rate. It is concluded that the growth spurt in puberty is not due to a change in growth hormone concentration but rather to the increase of androgen production in puberty. Key words: Delayed puberty - - Growth rate - - Growth hormone concentration (mean daily growth hormone concentration) - - Testosterone concentration (mean daily testosterone concentration). The growth rate of boys with constitutional delay of adolescence is lower than that of healthy, normally developing boys of the same age. This is especially visible of the point when normal adolescents reach the pubertal growth spurt and the delayed patients have not yet entered puberty. I t is not clear whether an altered growth hormone secretion rate is responsible for this difference in the growth velocity in the same age group. Okada et al. [16] found a smaller increase of serum growth hormone in patients with delayed puberty following exercise despite a good response to insulin-induced hypoglycemia. Healthy boys had good responses to both tests. Treatment with testosterone provokes a potentiated increase of growth hormone in plasma following hypoglycemia [4, 8, 9]. Since insulin-induced hypoglycemia is an unphysiologie stimulus as well as other pharmacologic stimuli, we have measured the spontaneous growth hormone concentration and their variations in four 6-hour periods over a whole day. The patients continued their life ~s normally as possible without being disturbed too much. This investigation was performed to decide whether the mean daily growth hormone concentration * Supported by Deutsche Forschungsgemeinschaft, SFB 51.

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O. Butenandt et al.

i n boys w i t h c o n s t i t u t i o n a l delay of p u b e r t y a n d low growth rate is different from t h a t in p u b e r t a l boys with high growth velocity (pubertal growth spurt). As a p a r a m e t e r of p u b e r t y testosterone was m e a s u r e d i n the same p l a s m a samples collected over 24 h. Methods Blood was taken according to the methods by Finkelstein et al. [6] or Kowarski et al. [12]. It was collected in regular short intervals (ca. 1 drop per 5 rain) or using a constant withdrawal pump from a small venous catheter inserted into the forearm 1 h prior to the start of collection. Samples from 6 h were pooled, centrifuged, and the supernatant frozen until the determination of growth hormone or testosterone. Determinations of growth hormone were performed radioimmunologieallyusing a double antibody method [3]. Each value is the mean of a triplicate determination. All different samples from one patient were assayed at the same time in the same assay. Testosterone assay: Plasma testosterone was measured by means of a sensitive radioimmunoassay using a highly specific antiserum which was produced by immunizationof rabbits with testosterone-3-(0-carboxymethyl)oxime [5] linked covalently to bovine serum albumin. ~'or duplicate determination 0.05--2.0 ml aliquots of the plasma samples were incubated with small amounts of tritiated testosterone for control of procedural losses and extracted with ether. Purification of the dried extracts and separation of testosterone from cross-reacting steroids was performed by Sephadex LH20 chromatography with benzene/ethanol (95:5) as

Table 1. Clinical data of patients Patient

Age (years; months)

Height (cm)

Growth rate Height for age (cm/year) (em)

a) Familial shortness (prior to puberty) W. tt. 6;8 110 Sp. J. 8;7 119 B. St. 8;8 119 Sch. R. 9;4 123 K.P. i0;10 129 W.W. 12;2 134

5.4 4.6 4.3 5.2 4.7 3.2

K.W.

3.5

119~:11 129~12 130:L12 132:~12 140~13 148~:15 brother ofW.~I. 150=j=15

4.2 4.8 4.5 5.2 9.7 4.5

128• 150~:15 151i15 154=}=16 162=t=17 165=t=17

12;6

136

b) Constitutional delay of development S.G. 8;5 110 G. tt. 12;7 130 M. Ch. 12;10 131.5 R.R. 13;3 140 A.H. 14;5 137 C.W. 14;8 140 c) Familial tallness and puberty St. P. 12;2 160 O.J. 12;5 158 K.R. 13;0 168 W.D. 13;9 168 R.W. 13;10 168 M.K. 15;4 177 M. tt. 15;9 178

8.1 ca. 10 7.2 7.9 10.1 7.8 8,5

148=]=15 149~15 153~:15 158=}=16 159116 168J:16 170=f=16

Growth Hormone and Testosterone in Pubertal Growth Spurt

87

the solvent. The radioimmunologic procedure was similar to that described previously for the measurement of estrogens in childrens plasma [2]. An 8-point standard curve ranging from 10 to 240 pg was prepared for each assay with authentic testosterone. In this way 2 ng/100 ml testosterone could be clearly distinguished :from zero. The interassay coefficient of variation was 15.1~ in prepuberty and early puberty and 10.7~ in advanced puberty and in adult men.

Patients

We measured the mean growth hormone concentration of 7 boys of familial shortness prior to puberty and of 7 boys of familial tallness during puberty as well as of 6 male patients with constitutional delay of development prior to puberty (Table 1). The diagnosis "constitutional delay of development" was made if the following conditions were observed: 1. A retardation of bone age of more than 2 years. 2. A delay of puberty and/or a short stature. 3. At least one parent has a history of delayed puberty with late onset of pubertal growth spurt.

Table 2. Integrated growth hormone concentrations in serum (ng/ml) Patient

10:00 a.m. to 4:00p.m.

4:00 p.m. to 10:00p.m.

10:00p.m. to 4:00 a.m.

4:00 a.m. to 10:00a.m.

24 h (10:00 a.m. to 10:00 a.m.)

4.0 3.5 2.2 6.0 1.5 8.6 4.d 4.31

5.0 1.4 1.4 0.65 1.0 4.8 1.5 2.25

3.6 2.1 1.5 2.2 1.0 4.6 3.5 2.64

5.4 5.0 2.8 4.4 2.1 3.2 3.81

4.0 1.2 1.4 4.2 1.0 1.0 3.13

3.9 3.4 3.0 4.3 1.4 1.3 2.88

4.2 1.5 4.7 4.6 7.8 7.2 8.9 5.56

1.0 1.2 1.5 1.4 2.2 1.6 2.8 1.67

1.9 1.0 2.0 2.7 4.4 2.9 3.7 2.66

a) Familial shortness (prior to puberty) W.H. Sp.J. B. St. Sch. R. K.P. W.W. K.W. Mean(~)

2.8 1.6 0.8 1.6 0.4 3.5 2.0 1.81

2.6 2.0 1.5 0.7 1.1 1.4 6.2 2.21

b) Constitutionel delay of puberty (prior to puberty) S.G. G.H. M. Ch. R.R. A.H. C.W. Mean(2)

3.4 2.8 2.0 3.0 1.0 0.6 2.13

2.8 4.6 5.6 5.6 1.5 0.5 3.43

c) Familial tallness (during puberty) St.P. O.J. K.R. W.D. R.W. ~.K. ~.H. ~ean(~)

0.8 0.4 0.4 2.4 4.6 1.2 1.3 1.58

1.6 0.8 1.2 2.4 3.0 1.6 1.9 1.79

Highest value of each individual is marked.

88

0. Butenandt et al. Results

The results are summarized in Table 2. During daytime (10 : 00 a . m . - - 4 : 00 p.m.) the mean individual growth hormone concentrations in all subjects tested were below 5 ng/ml serum, often even below 2 ng/ml. The mean growth hormone concentrations were often higher during the evening hours (2nd collection period: 4:00 p . m . - - 1 0 : 0 0 p.m.) but reached the m a x i m u m only in the third collection period (10:00 p , m . - - 4 : 0 0 a.m.) in 15 of the 20 tested boys. At this time the mean growth hormone concentration of all 7 boys with tall stature and pubertal growth spurt was higher t h a n the means of the two other groups. However, the individual values were variable in a wide range and there were no statistically significant differences between the three groups. Growth hormone concentrations in the last collection period (4:00 a.m.--10:00 a.m.) were similar to the vMues in the daytime period. The individual mean growth hormone concentrations during the 24-hour periods are illustrated in Figure 1. They v a r y from 1.0 to 4.6 ng/ml serum in all groups. The statistical evaluation of all values using the Wilcoxon test does not show any significant difference between the three groups in any collection period.

Mean

24 hrs Growth Hormone Level

6

-;

4 3 2

o

I Prepubertal

=ubertal

Boys

Boys

Delayed Puberty

rig. 1. Mean 24-hour growth hormone levels in individual patients. No difference has been found between boys prior to puberty, during puberty, or with delayed puberty

+ + o

+

++

+

g:7 : 6~

[ I

0 e

ee I 2

9 0

.~

0

0 O

l 4

J

Mean 24 hP3 growth Hormone Concentra tion

Fig. 2. Correlation between growth rate and mean 24-hour growth hormone concentration. 9 Boys prior to puberty with familial shortness, q- Boys in puberty with familial tallness. 0 Boys with delayed puberty

Growth Hormone and Testosterone in Pubertal Growth Spurt Mean

24 hrs

89

Hormone Concentrot/ons

ng//ml [

." 10

20 30 50 100 200 400 Plasma Testosterone ng~o0 ml

Fig. 3. Mean 24-hour growth hormone concentration in correlation to mean 24-hour testosterone concentration

~11

8

9

9

0:6 ~5

9

~4 3

9

9

9149

9

9 ,

.

.

.

.

.

.

.

i

7o 20 30 50 700 200 Mean 24.hrs Plasma Testosterone

~ ' 400 ng/lO0 ml

Fig. 4. Correlation between mean 24-hour testosterone concentration and growth rate

The growth rate of all patients with constitutional delay of puberty was similar to boys prior to puberty with the exception of one case (patient A. H.) who started his pubertal growth spurt during the months following growth hormone investigation. This growth spurt (9.7 cm/year) was similar to the growth velocity of the tall pubertal boys. However, no correlation was found between the mean individual daily growth hormone concentration and the growth rate (Fig. 2). Also, no correlation was found between the mean growth hormone concentration during the 24-hour period and the mean testosterone content in the same serum specimen (Fig. 3). But a clear increase of the growth rate could be observed when the testosterone content was elevated to pubertal levels (Fig. 4).

Discussion To test the hypothalamo-hypophyseal function in regard to the ability to produce and secrete growth hormone concentrations of this hormone were measured by several authors under different physiologic conditions (i.e., sleep or exercise) and under pharmacologic stimuli (insulin-induced hypoglycemia or arginine infusion)[7, 10, 14, 15, 17, 18]. The former tests giveonlyamomentaneous

90

O. Bu~enandtet al.

result in evaluation of the circulating hormone, the latter ones give the information of pharmacologic regulation and hypophyseal reserve of the hormone. However, nothing can be said about physiologic production and secretion of growth hormone during a whole day and variations dependant on daytime, exercise, or rest. The determination of the mean individual growth hormone concentration permits evaluation of the available circulating hormone. According to Kowarski et al. [12], this method can be used for the determination of the growth hormone production rate. Therefore, the influence of growth hormone upon the pubertal growth spurt and its correlation to testosterone was evaluated by this method. The mean daily growth hormone concentrations either measured in 6-hour periods or determined over the whole 24-hour period did not show significant differences between preadolescent boys with low growth rate, fast growing boys during puberty, and boys with delayed puberty and slow growth velocity. With other words : no correlation was found between the 24-hour mean growth hormone concentration and the growth rate. During sleep, all boys had their highest growth hormone concentrations, again without significant differences between the three mentioned groups. Also, there was no increment of the mean growth hormone concentration with advancing puberty since no correlation existed between the mean growth hormone concentration and the mean testosterone concentration of the same individual. However, a correlation was observed between the testosterone values and the growth velocity. Our results are in accordance with a report by Thompson et al. [19, 20] who did not find differences in the integrated growth hormone concentration in boys of different pubertal stages. Also, Wieland et al. [21] did not find statistical significant changes of growth hormone levels 1--2 h following lunch in boys with advancing puberty despite increasing testosterone levels. The increase of the mean growth hormone concentration during the evening or even more increase during the night is due to the physiologic secretion of this hormone following onset of sleep [1, 7, 10, 17]. tPinkelstein's group [6] stated that children secrete growth hormone physiologically only during sleep but not in daytime while awake. This seems not to be true for all children since we have found clearly measurable hormone concentrations also during wake periods. Also, determinations of growth hormone in a single specimen during daytime sometimes allow the exclusion of hyposomatotropism in short children when the hormonal content is high. Growth spurt in puberty depends on the amount of circulating androgens but not on an increased secretion of growth hormone [11, 19, 21]. Zachmann and Prader [22] reported a normal pubertal growth spurt initiated by a long-acting testosterone preparation in sexually immature boys without growth hormone deficiency. The higher response of growth hormone secretion following different stimuli after testosterone treatment [4, 8, 9, 13] seems to be evoked by extremely high levels of testosterone in serum not reached during normal puberty [18].

Growth Hormone and Testosterone in Pubertal Growth Spurt

91

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21. Wieland, 1% G., Chen, J. C., Zorn, E. iVf.,Hallberg, M. C. : Correlation of growth, pubertal staging, growth hormone, gonadotropins, and testosterone levels during the pubertal growth spurt in males. J. Pediat. 79, 999 (1971) 22. Zachmann, M., Prader, A.: Anabolic and androgenic effect of testosterone in sexually immature boys and its dependance on growth hormone. J. clin. Endocr. 89, 85 (1970) Priv.-Doz. Dr. O. Butenandt UniversitEts-Kinderklinik LindwurmstraBe 4 D-8000 Miknchen 2 Federal Republic of Germany