LECTURE Assessment

of Growth

Hormone Milo

Secretion

in Children

Zachmann

Department of Pediatrics, University of Zurich, Switzerland

(Received for publication

on December 2, 1989)

Abstract. Growth hormone (GH) is secreted in a pulsatile way during the whole life under the reciprocal influence of somatostatin and GH-releasing hormone (GHRH). It mediates many effects by stimulating production of insuline like growth factor I (IGF I) in liver and other tissues, but IGF I is also regulated by the nutritional state. Women secrete more GH than men, and older men and women less than young women. This suggests importance of estradiol in regulating secretion. Sex hormone effects are also demonstrated by the increment of GH and IGF I at puberty, which is an amplitude-modulated phenomenon. Classic metabolic studies have shown that patients with GH-deficiency retain more nitrogen in response to a given dose of exogenous hGH than normal subjects. The use of the stable isotope 15Nhas simplified such studies. In GH-deficient patients, there was with this technique a marked positive hGH-induced balance change. In girls with Turner syndrome (as example of subjects with normal GH-secretion), balance change was less marked with the same dose. Girls with Turner syndrome, who were given a double hGH-dose showed a response in the same range as that in the GH-deficient patients with the lower dose. A conclusion from this is that patients with normal GH-secretion need higher doses to obtain a similar response, than patients with GH-deficiency. The dosage in such patients will have to be selected individually, and needs to be about twice or three times as high as in GH-deficient patients. (Keio J Med 39 (3): 173-186, September, 1990) Key words:

growth

hormone,

hypopituitarism,

Turner

I have suggested to give you a conference on the assessment of growth hormone (GH) secretion in children. Only few years ago, such a task would have been very simple and straightforward: in most centers, two provocation tests (most frequently an insulin toler ance test and an arginine infusion) were carried out, and the GH results were compared with established normal values from the respective laboratory. At present, even though and also because much has been recently learned concerning the dynamics of GH secretion, it is much less clear, when GH secretion has to be regarded as normal in a child with short stature, and when not, and as I will try to show you in the following, it did even become quite meaningless to try a separation between GH de ficiency and normal GH secretion. GH is secreted in an intermittent and pulsatile way during childhood and adulthood. It is therefore clear that GH secretion is important not only for linear growth, but also for its metabolic effects during the whole life. There is strong evidence to suggest that GH is an important regulator of intermediary metabolism, and it plays a role in maintain Reprint Zurich,

requests to: Dr. Switzerland

Milo

Zachmann,

Department

of Pediatrics,

syndrome,

metabolic

response

ing positive nitrogen balance, muscle mass, and bone density, and in avoiding excessive formation of adipose tissue. In this conference, I would like to briefly review first the regulation of GH secretion as a physiological basis for understanding, and then the tests for the evalu ation of GH secretion. The complex nature of regulation of GH secretion is presented in a very simplistic way in Fig. 1. GH is secreted by the somatotrope cells of the anterior pituitary gland under the influence of stimulatory and inhibitory factors. The somatotrope cells are regu lated by two hypothalamic hormones, somatostatin or SRIF and GH-releasing hormone or GHRH (Fig. 2). In addition, insulin like growth factor I or IGF I feeds back at both the pituitary and hypothalamic levels to inhibit GH secretion. IGF I, whose structure was clarified in Zurich by my colleagues Humbel and Rinderknecht (Fig. 3), and whose effects were among others studied by the group of Froesch, has many similarities with insulin both, with respect to its structure as you can see here , and its action. The structure is very similar to that of insulin, particularly with respect to its disulfide bridges . Kinderspital

173

Zurich,

Universitats-Kinderklinik.

Steinwiesstrasse

75 , 8032

174

M . Zachmann:

Table

1

Assessment

Stimuli:

(Reproduction Dekker Inc.) Fig. 1

Fig. 2

Hypothalamo-hypophyseal

Regulation

of Growth

Increasing

permission

GH

Hormone

Secretion

in Children

Secretion.

is granted

by A.D.

Rogol and Marcel

axis.

of growth hormone

and somatomedin

hypothalamus. These neurons are regulated by mono aminergic and cholinergic neurons from other areas of the brain. They act as transducers for environmental factors such as somatic or psychological stress to regulate GH secretion. Following the discovery and biochemical characterization of natural somatostatin, its action profile has been thoroughly investigated. Although the name somatostatin was coined in virtue of its GH release inhibiting properties, a number of central and peripheral endocrine and paracrine actions have been ascribed to this peptide. Its inhibitory effect on a series of pituitary and gastro-intestinal hormones has characterized it as a brain-gut hormone. Physiological factors that stimulate GH secretion thus include deep sleep, notably the REM or rapid eye movement sleep, stress, and physical exercise (Table 1). In addition, nutrient intake modulates GH secretion in humans, and as we will see later has even more important influences on the production of IGF I. Oral glucose loading suppresses GH secretion as blood glucose concentrations increase, but later on, about 120 minutes after glucose administration, GH secretion is stimulated when blood glucose concentration again declines. Glucose tolerance tests therefore can also be used to see, whether high GH levels as e.g. in acromegaly, are suppressible or not. The change or rate of change in blood glucose therefore may be more important than the absolute concentration. Similarly, amino acids, particularly arginine and ornithine, but also e.g. oral intake of a mixture of amino acids, as in the old so-called Borvil test, stimulate GH secretion. The effects of food on GH secretion are variable and unpredictable.

(IGF I)

secretion.

In

phylogenetically

secreted

lower

rhythmically

with

animals a fixed

like

rats,

ultradian

GH

period

is at a

periodicity of approximately 3 hours. Although it has been thought that there is no such ultradian rhythm in Fig. 3

Structure

of IGF

I.

humans,

such

imposed

by

certain It is therefore causes

GHRH tostatin

not

surprising

that

its acute

administration

hypoglycemia. to which are

I will

synthesized

come by

back neurons

later

and within

soma the

rhythms other

situations.

may

influences, During

only but puberty

be

hidden

they

and

may

super

emerge

approximately

in eight

pulses of GH occur every 24 hours. who are fasted for 12-36 hours

Normal young men or longer show an

increased

pulses

basal

and

frequency mean

of GH secretory GH

concentrations,

and increased

as can

be seen

on

Keio J Med 39 (3): 173-186,

1990

175

Fig. 5

hGH secretion pattern during 24 hours and during life.

(Reproduction Dekker Inc.)

Fig.

4

Serum

normal 5 (C)

of a 5-day

above

each

minute/ml)

levels male

fast.

measured prior

The of

permission

the

every

to (A),

Computer

profile. of each

(Reproduction Dekker

GH

37-year-old

20

during

minutes day

identification

shaded

bars

3 days.

(from

is

granted

for

1 (B),

of GH indicate ref.

by

24 hours and

pulses

integrated

GH

and

Rogol and Marcel

day

(ƒÊg

Fig. 6 Rogol

by A.D.

shown

1) A. D.

is granted

in a

during are

permission

Biological effects of IGF I and II.

Marcel

Inc.)

Fig. 4 from Thorner and coworkers.1 Spectral analysis of these data demonstrates several dominant frequencies, the predominant occurring at one episode per 90 minutes. Thus a basic ultradian rhythm together with the effects of nutrients, exercise, sleep, and stress also seems to exist. Besides the short-term rhythms during 24 hour, there is changing secretion during life, as can be seen on this slide. In adults, the 24 hour GH secretion decreases slowly with age (Fig. 5). But even so, the pituitary glands of old people still contain considerable quantities of GH. As you know, before recombinant hGH was available, it was extracted from pituitaries of old people. GH me diates many of its effects by stimulating the production of IGF I in the liver and many other tissues (Fig. 6). Many, but not all effects of GH on adipocytes, muscle, and chondrocytes thus are indirect through IGF I. Circu lating IGF I levels feed back at the hypothalamus and pituitary to inhibit GH secretion. However, IGF I pro duction is regulated not only by the circulating concen

(Reproduction Dekker Inc.)

permission

is granted

by A.D.

Rogol and Marcel

Fig. 7 Fasting plasma growth hormone levels in kwashiorkor and during nutritional recovery) and in marasmus .

(before

176

tration of GH but also by the nutritional state of the individual. For example, in protein-calorie malnutrition, IGF I concentrations are low whereas as can be seen in Fig. 7 from Godard from children with kwashiorkor , GH concentrations are high and normalize with recovery. Nutritional rehabilitation of children results in a rapid increase in IGF I levels, suppression of GH concen trations, and the initiation of accelerated growth . It is possible therefore that the secretion of GH is regulated predominantly by the metabolic requirements of the individual, which in turn depend on the subject's nu tritional state. In malnourished children, normal growth is generally maintained as long as possible at the expense of fat and muscle tissue. As Gloria Tannenbaum2 has shown, somatostatin or SRIF plays an important role in the regulation of the episodic and ultradian rhythm of GH secretion (Fig. 8). Passive immunization of rats with specific antibodies to the 14 and 28 amino acid sequences caused a significant GH elevation. The fact that soma tostatin antiserum was unable to block episodic GH surges indicates that this hormone's release must be regulated by a dual mechanism. Indeed, GH-releasing hormone (GHRH or GRF) seems to be instrumental in the maintenance of pulsatile GH secretion. More over, exogenous GHRH induced a further GH increase predominantly during the period of active secretion. Neutralization of endogenous somatostatin eliminated this time-dependent effect, indicating that this peptide blocks periodical spontaneous GH release. Food depri vation and changes in glucose homeostasis virtually

Fig. 8 Schematic representation of the postulated rhythmic secretion of somatostatin (SRIF) and GH-releasing factor (GRF) into hypo physeal portal blood, with the net result of GH secretion, as observed in plasma. (from ref. 2)

M.

Zachmann:

Assessment

obliterate

the

of Growth

ultradian

Hormone

GH

Secretion

rhythm.

in Children

In this context,

peripheral somatostatin seems to play an important role. Also the central GHRH/somatostatin interplay is re sponsible for a shortloop feedback control on pituitary somatotropes. The

pulsatile

reciprocal schematically result

secretion

secretion shown

in the

of the different

and

GHRH.

normal

during

direct

evidence,

since

effects

the

permitting

animal,

GHRH

and

model

it is probably nutrients,

not

appear

Our

to have

from

a-adrenergic adrenergic

and

stimulation stimulation

cholinergic

the

rat may of GH the

stress effects

mechanisms man,

of of

secretion.

In

there

inhibition

of somatostatin

data

secretion, effects man.

regulating is far

a role

secretion In

stimulate

of

these

secretion

somatostatin. is evidence

of

be a good

in rat and

support

GHRH

may

GHRH is more

because

somatostatin

Experimental

in and

of replacing

of the neurotransmitters

GHRH

complete.

the

somatostatin

effects

nature and

different

from

immunization

for understanding

steroids,

understanding

hypothalamic

While

useful

gonadal

factors

of the

is the

secretion

with elevated In rats there

of endogenous

the pulsatile

are

of GHRH,

by selective

somatostatin.

for studying

GH

infusions

study

As

peaks

of somatostatin

pulsatile

in patients secretion.

can be blocked

by the

this is derived

continuous

and GHRH

GH

secretions

favoring

pulsatile GH secretion levels due to ectopic

is regulated somatostatin.

figure,

of augmented

men

thus and

rhythmic

Evidence

demonstration

of GH

of GHRH

and

for b

addition,

somatostatin of

dopaminergic

secretion.

Let me now turn to some aspects of GH in humans. GH is secreted in bursts at all stages of life. Thorner and Rogol3 carried out a study to analyze the pattern of GH secretion in young and older men and women. Blood samples were withdrawn every 20 minutes for 24 hours for the determination of GH concentrations. The pulses of GH secretion were analyzed using a computer algor ithm. They observed that women secrete more GH than men, and older men and women secrete less GH than young women. The so-called PULSAR computer program is frequently used to identify hGH pulses. It is important that a pulse is defined in terms of the per formance of the assay, but the SD of an assay varies at different concentrations. It would be too complicated to discuss these aspects of pulse analysis, but it is sufficient to mention that the analysis is complicated, requires computer assistance, and that its inaccuracy increases, the smaller the absolute quantities of GH get. In the figure from a paper of Ho and coworkers4, representative profiles of GH secretion over 24 hours are shown from a young woman, young man, older woman, and older man (Fig. 9). In the young woman it is clear that the area under the curve is greater than in the older woman and older man. In addition, the number of pulses tends to be higher. The same is true for the difference between

Keio J Med 39 (3): 173-186,

1990

177

Fig. 9 Representative GH profiles from a young female, young male, older female, and older male sampled every 20 minutes for 24 hours. Pulses were categorized as large (L) or small (S) depending on whether the rise was greater or less than three times the threshold criterion for a pulse. (from ref. 4)

young and older males, but the difference is less marked (Table 2). These data are summarized for the entire group of volunteers on this table. Note that the integrated mean GH concentration is greater in young women than in young men and that young men, older women, and older men secrete roughly the same quantity of GH per day. The integrated GH concentrations correlate with the plasma estradiol concentrations, as do the amplitude of the pulses and the fraction of GH secreted as pulses. These data suggest that in addition to the mentioned factors, estradiol may be an important factor regulating GH secretion in humans. In contrast there was in these studies no correlation of GH with serum testosterone concentrations. The IGF I level also correlated with the amount of GH secreted in pulses. From these obser vations, however, it should not be concluded that tes tosterone has no effect at all on GH-secretion. (Fig. 10)

Table

2

Effects

of Sex and Age

on Pulsatile

GH Secretion

in Man.

For a long time, the so-called androgen-priming has been known. It means that the GH-response to stimu lation tests can be enhanced in prepubertal boys, in boys with constitutional delay of growth and adolescence, or in boys who lack androgens for pathological reasons such as primary hypogonadism or gonadotropin deficiency. This slide from Ruth Illig at our department shows that the maximum GH-response to insulin-induced hypo glycemia can be increased in boys with anorchia or gonadotrophin deficiency. The columns a represent the peak GH levels after insulin without any androgen re placement, the columns b and c the levels after 3 days and 1 week of priming with testosterone. The group of columns at the left are patients with anorchia, then with gonadotropin deficiency, then with constitutional delay, and those who do not respond at all are patients with complete GH-deficiency. (Fig. 11) The effects of sex hormones are also demonstrated by the fact that GH secretion and serum IGF I concentrations increase at puberty in girls and boys. Although there is considerable individual variation, this is clearly shown on this slide . We have prepared this slide, when we studied patients with craniopharyngioma. So please disregard the data on the slide, but look only at the bars, which show , how IGF I normally increases during puberty . Dr. Rosenfeld has presented similar data. His results agree well with the values obtained by Froesch and ourselves using the somewhat different assay of Zapf. However , this phenomenon does not seem to be an exclusive effect of increasing gonadal hormones, because GH and IGF I

178

M.

Fig. 10

Effect

of exogenous

growth and puberty. administration.

Fig.

11

with

excessive

the

concentrations

normally

biochemical GH to

and the

therefore

of

androgens

at the time already

priming

Although

the

is possible

that

for instance The

increase

or

childhood

before

increase

also

be

estrogens

and

the first

The

possibly

testosterone

in boys

mechanism androgen

can

in

related

from

treatment

increase

GH

for this is not

exactly

may be peripherally

in adipose

tissue

in GH

secretion

amplitudemodulated

during

even

I during

decreased •›

the

and converted

phenomenon

at puberty that

or tes secretion. known,

it

aromatized to estrogens. represents

is relatively

an in

1st

Assessment

in 8 patients

testosterone,

IGF

normal •¢,

values

on GH release

before

of adrenarche.

mentioned,

tosterone

+,

of puberty.

I could

secretion

adrenal As

IGF

normal

signs

testosterone column:

Craniopharyngioma:

rise slightly

or clinical

Left

Zachmann:

with constitutional

right column:

year

linear

after

growth,

after

surgery the

of Growth

bars

in

Hormone

delay

Secretion

in Children

of

testosterone

patients

represent

adolescence.

dependent of changes in pulse frequency. Such changes may be secondary to the action of adrenal and/or gonadal sex steroid hormones modulating the responsivity of the somatotrope cells to endogenous GHRH, the amount of GHRH secreted, or the inhibitory tonus of somatostatin. After these physiological considerations, which were based on animal experiments and studies in the human, let me now turn to some comments on tests for growth hormone secretion. Unfortunately, there are no abso lutely reliable tests of GH reserve, and several funda mentally different approaches have been taken. (Fig. 12) The oldest test to diagnose GH-deficiency, which was

Keio J Med 39 (3): 173-186,

1990

179

Fig. 13

15N-balance

studies,

test design.

Table 3 15N-balance (mg/kg) in GH-deficient Patients and Patients with Turner Syndrome before and on Recombinant hGH.

a)-b):

before

and

after

2•~3

IU/m2

on

consecutive

c):

before

and

after

2•~6

IU/m2

on

consecutive

d):

before

and

after

3•~2

IU/m2

on

consecutive

days days days

(lower

15N

- dose)

Fig. 12

hGH-induced

urinary nitrogen retention

(from ref. 5).

developed in Zurich by my teacher Prof. Prader already back in the fifties, long before radioimmunoassays for estimation of GH were available, is testing the metabolic response to exogenous hGH by analyzing the change of nitrogen balance.5 In view of the difficulties with the stimulation tests, which I will briefly discuss later, these tests, which are biologically sound and reliable, but which were mostly abandoned in the sixties and seventies because of their practical difficulties, have recently been refined, and have made quite a comeback. The classic metabolic studies with constant protein intake and more recent techniques using the stable isotope 15N have provided a variety of information concerning protein metabolism in children with growth disorders. They have shown that patients with GH-deficiency retain more nitrogen in response to a given dose of acutely admin istered exogenous hGH than normal subjects, and that short children with normal GH-secretion respond like normal and less than GH-deficient children. This is shown on this old slide and we could entirely confirm these former results using more sophisticated modern techniques. (Fig. 13) The use of the stable isotope 15N,6 which has been introduced for this purpose by our group has greatly simplified such studies, because constant nitrogen intake is not required, and the response can be studied on an outpatient basis in 3 days rather than

during 2-3 weeks in hospital. (Table 3) The data on this slide are from patients with proven idiopathic GH - deficiency. Urinary 15N-balancestudies were performed once before and once on the second day of treatment with hGH. On each test day, the patients were given orally 0.05g per kg of 15N-labelledammonium chloride. As you can see, in the GH-deficient patients, there was a very marked positive balance change. The results in patients with Turner syndrome by contrast were quite different: It can be seen that the basal balance is much higher, and the positive balance change much less marked with the same dose of hGH in these girls. Girls with Turner syndrome, who were given a double hGH - dose of twice 6IU showed that the response is more marked and in the same range as that in the GH -deficient patients with the lower dose. One conclusion from this is that patients without GH-deficiency need higher doses to obtain a similar response. The advent of radioimmunoassays for estimation of GH-concentrations in blood in the early sixties led to the development of many stimulation tests for testing the reserve of GH -secretion. Such tests that utilize what were considered to be more reliable methods for stimulating GH secretion are still today widely employed. They do , however, as we will see, also have their drawbacks . These tests are well-known to everybody and I will not discuss them in detail. They include the induction of hypoglycemia with insulin, the infusion of arginine , and the oral adminis

180

tration of L-dopa. Other pharmacological stimuli such as clonidine have also been used with some success. How ever, most investigators agree that none of these tests is entirely satisfactory since none is physiological. For all of these tests, a GH response above about 7-10ng/ml depending on the laboratory has traditionally been con sidered to exclude GH deficiency. However, all limits which have been suggested to separate GH-deficiency from normal GH-secretion, are not biologically valu able and are in reality completely arbitrary. The most frequently used test is insulin-induced hypoglycemia. If performed under strict and well-supervised conditions, it is relatively safe. It acts on the hypothalamus, probably by suppressing somatostatin secretion. The infusion of arginine has few side effects. Arginine acts through the hypothalamus, although whether it acts by stimulating GHRH or by inhibiting somatostatin or both was un known until very recently. Just a few months ago, Alba Roth and coworkers from Munich could show that arginine stimulates GH-secretion by suppressing endo genous somatostatin secretion rather than by stimulating GHRH. To determine how arginine stimulates GH secretion, they investigated its interaction with GHRH in vivo and in vitro. Their results indicated that arginine administered with GHRH led to higher serum GH levels than did a maximally stimulatory dose of GHRH or arginine alone. The serum TSH response to arginine - TRH also was greater than that to TRH alone. They concluded from this that the stimulatory effects of argi nine are mediated by suppression of endogenous soma tostatin secretion. Arginine also stimulates insulin secretion, but does not cause hypoglycemia during the test, and this mechanism is still many years after using the test poorly understood. The oral administration of clonidine, an a-adrenergic agonist, probably acts by stimulating hypothalamic GHRH secretion. Its disad vantage is that the time course of stimulation of GH is variable, L-Dopa also has a variable time course and often causes unpleasant nausea. Its mechanism of action is unknown, but it appears to act at the hypothalamic level to both stimulate GHRH and inhibit somatostatin secretion. Propranolol, the b-adrenergic blocking drug, cannot be used alone but may act by suppressing hypo thalamic somatostatin release e.g. in combination with glucagon. Although the mentioned classic stimulation tests of GH-reserve, which have been used for 25 years have serious drawbacks, they may after all not be that bad. Rose and coworkers from the NIH recently con cluded from a study that compared patients with GH deficiency with patients with idiopathic short stature that the measurement of the spontaneous GH-secretion in prepubertal short children had lower sensitivity and offered no diagnostic advantage over the classic stimu lation tests. Since the isolation of GHRH by Guillemin and Rivier in 1982, there has been a large number of

M. Zachmann:

Assessment

of Growth

Hormone

Secretion

in Children

publications on the effects of acute administration of GHRH to GH deficient subjects. Brook8 and coworkers have not found that the response to an intravenous bolus of GHRH is of any predictive value in the long term response to treatment with GHRH. It seems therefore not to be a very helpful test in the diagnosis of GH deficiency. Nevertheless, GHRH is now widely available on an experimental basis. It was hoped that this agent would test GH reserve. However, there are several factors causing confusion. First, in normal adults GH responses to GHRH are extremely variable both among subjects and in the same subject on different occasions. This is most likely due to the fact that the response of the pituitary depends on the momentaneous hypothalamic somatostatin secretion. Thus if GHRH is administered at a time when somatostatin secretion is low, there will be a large response. If it is administered at a time when somatostatin levels are high, there will be a very poor GH response. Results of GHRH testing of children with short stature of varying etiologies can be summarized as follows. (Fig. 14) Children with idiopathic short stature who do not meet the classic criteria for GH deficiency

Fig.

14

GH

stature.

Each

uterine

growth

adolescence panel L-dopa

release

in

symbol

response

retardation and/or

to

represents

familial

and

hGRF-40

an (B)

short

in

individual

constitutional stature.

are the mean •}SEM of the peak and hGRF-40. (from ref. 9)

delay

Bars GH

children

with

patients,

at

responses

(A) of growth

the

right to

the

short Intra

of

and each

arginine/

Keio J Med 39 (3): 173-186,

1990

181

what is really happening under physiological conditions demonstrate an increase in GH in response to GHRH. On this slide from Gelato and coworkers9, A represents and 2) they are often less unpleasant for the children. the results from children with intrauterine growth retar Because exercise and sleep are known physiological dation and B those of children with constitutional delay stimuli of GH secretion, two simple tests taking advan or familial short stature. The responses are variable as in tage of this phenomenon are possible. (Fig. 16) One is to normal young adults and in children at various stages of have the patient exercise vigorously (e.g. on a bicycle). puberty. (Fig. 15) On this slide, A represents idiopathic Dr. Greene at our department has carried out a study on GH-deficiency, and B organic hypopituitarism. Most GH-response to exercise. While his results are of physio children with idiopathic GH deficiency thus respond to logical interest, individual variation of the response is GHRH, although in a few the responses are absent. great, and such tests have little diagnostic importance. Many children with organic hypopituitarism fail to re The other such test is to measure serum GH after the spond to GHRH. If the GHRH test causes stimulation patient has been asleep for at least 30-60 minutes or of GH secretion, it indicates that pituitary somatotrophic even better to record an EEG and wait until the patient cells are present and responding to stimulation. Failure obtains a REM sleep, since it has been found that this to respond could either be due to a defect at the level of sleep stage of rapid eye movements is associated with the pituitary somatotrophic cells or to the cells not being the most pronounced GH-peaks. (Fig. 17) Dr. Ruth Illig previously exposed to GHRH. In the latter situation at our department has studied such EEG-related GH repeated injections of GHRH may be useful to demon -secretion, and an example of her results is shown on this strate that there is no pituitary defect. Failure to respond slide. On the top, the sleep stages are classified, on the to GHRH could also result from excessive hypothalamic bottom, the GH-levels. As you can see, GH increases somatostatin secretion. with some delay after a period of deep sleep. Both tests While the mentioned classic tests and the GHRH-test have the advantage that they measure GH in response have their place in the assessment of GH-secretion, to physiological stimuli, and that no unphysiological more physiological tests have recently gained interest for compounds whatsoever have to be injected to the two reasons: 1) they may deliver more information on patients. An blood 24

Fig.

15

stature.

GH

release

Each

symbol

deficiency panel L-dopa

and

are

the

and

(B)

in

response

represents organic

mean •}SEM hGRF-40.

to

hypopituitarism. of the

(from

hGRF-40

an individual

ref.

peak 9)

in

Bars GH

children

patient.

(A) at

responses

the

with

to the

to provocative at intervals

hours.

Data

from

workers10

make

the

testing

of every the

studies

important

also

is to sample

10 or 20 minutes of

point

Spiliotis that

most

over

and

co

normal

short

isolated right

alternative for GH

GH

of each arginine/

Fig.

16

column:

GH

response

basal

value,

to exercise right

column:

in various value

after

groups

of children

exercise.

. Left

182

Fig.

M.

17

bottom:

GH GH

secretion

during

sleep

in one

patient.

Top:

sleep

Zachmann:

Assessment

of Growth

Hormone

Secretion

in Children

stages,

values. Fig. 18

Urinary

growth

hormone

in healthy

children.

children have approximately eight pulses of GH secretion every 24 hours. Kerstin Albertsson Wikland has shown morning urine samples as presented on this slide with a that even in a group of normal children, the taller ones log scale indicating the 3rd, 50th and 97th percentile. secrete more GH and have larger amplitudes than the The values expressed in this way are not significantly shorter ones. The correlation between physiological GH age-related. (Fig. 19) In children with proven GH secretion and the results of dynamic function tests for - deficiency, all values are below these normal limits, as GH reserve is far from satisfactory. Thus, children could you can see here. Our number of analyses is still quite be defined as growth-hormone deficient by pharmaco small, but we are now gathering normal values from a logical testing and yet have apparently normal patterns larger number of normal children as fast as possible, of GH release as shown by frequent sampling methods since the ethical problems, which arise with pharmaco or, probably more commonly, can be considered to logical testing in completely normal children do not arise have impaired spontaneous GH secretion with normal with the simple collection of morning urine. (Fig. 20) responses to dynamic function tests. It is not possible to Interestingly, also some of the prepubertal untreated delineate all of the factors that must be considered when girls with Turner syndrome excreted subnormal quanti evaluating such children, but obviously parental height, ties of GH in urine. From just a few cases we could so far study in such girls before and after small dose estrogen absence of disease, adequate nutrition, skeletal matu ration and the degree of its retardation, and lack of replacement, we do, however, not believe that girls with Turner syndrome really have reduced GH-secretion, but psychosocial problems should be considered. The inter pretation of GH values in the absence of such information that this low excretion is a consequence of their lack of is not very useful. A single basal IGF I level is useful estrogens. Just like prepubertal boys with anorchia, pre information if it is normal. However, as mentioned pubertal girls with Turner syndrome already have in before, nutritional factors play an important role in regulation of IGF I secretion. Many of the IGF I con centrations in mildly growth-retarded children are intermediate between those found in normal and hypo pituitary children. None of the tests of GH reserve have been sufficiently standardized against responses in normal children. Most are standardized against responses of normal young adults, of siblings of growth-hormone - deficient children, or even of children with short stature who have arbitrarily been regarded as having "normal" responses. A recent technique of assessment of GH -secretion, which seems to be quite promising and is even more physiological and less disturbing to the children, is the estimation of GH in urine by an enzyme immuno assay. Dr. Torresani from our laboratory has recently obtained quite interesting results with this method. (Fig. 18) He obtained normal values in ng/g creatinine from Fig. 19 Urinary growth hormone in children with GH-deficiency.

Keio J Med 39 (3): 173-186,

Fig. 20

Urinary

growth

1990

183

hormone

in girls with Turner

syndrome.

creased gonadotrophins, which shows that their estrogen secretion must be lower than that of normal prepubertal girls. (Fig. 21) Androgen priming in boys with consti tutional delay as I have mentioned before also causes a marked rise in GH excretion, as you can see from these few examples we have studied so far. It is still too early to recommend estimations of urinary GH as a screening procedure on a large scale, but we believe that this method has an interesting future and might one day replace the unpleasant pharmacological stimulation tests. Towards

the

end

of

my

conference,

questions arise in this context. of GH secretion are necessary the

amount

pattern

or

pulses related

of GH

secreted

number

to admit

over

of pulses,

that

many

following

What amount and pattern for normal growth? Is it

or

that is most important? to these questions have

have

the

studies of CLark and coworkers11 in rats demonstrate that if GH is administered continuously there is less effect on growth compared to the same amount given intermittently. This seems to indicate that not only the overall produced quantity of GH is important for normal growth, but also the pattern and pulsatility of release. Similar observations have been made with gonadotropin releasing hormone, where accurate pulsatile administra tion seems to be even more important. (Fig. 22) Experi ments in this respect are difficult also for ethical reasons, and have not been reported in humans. However, it could be clearly shown that administrations only once or twice a week are insufficient in GH-deficient children and that the growth velocity increases, when hGH is administered more frequently, but still much less than it is secreted under physiological conditions. This slide from Milner shows that frequency of administration is more important than total dose. (Fig. 23) Furthermore, if hGH is administered daily subcutaneously, the effects of the same overall doses are slightly, but still signifi

a 24-hour the

period,

amplitude

the of the

Although many details been recently learned, I

aspects

are

still

unclear.

The

Fig. 22 Relationship between administration in GH-deficient

Fig.

21

Comparison

androgen priming development.

of in boys

serum with

and

urinary

constitutional

GH delay

before of

and

after

growth

and

Fig.

23

Height

administration

velocity of hGH.

height velocity children.

in GH-deficient

and

children

frequency

with

of hGH

LM . and

S. C.

184

M. Zachmann:

cantly better, as has been shown by Kastrup and co workers. What is normal GH secretion and what criteria define GH deficiency? At the extremes of the spectrum, these answers are quite easy. The child who grows normally, has parents of normal stature, attains a normal adult height, and who has a normal IGF I value certainly has sufficient GH, irrespective of the pattern of GH secretion. (Fig. 24) In contrast, the child who is growing very slowly, has fallen much below the third percentile on the growth chart, has low IGF I, and has no GH response to dynamic function tests clearly has almost complete GH deficiency. This patient of many years ago, who is now an adult and whom we have observed for many years, was given hGH quite late and therefore shows the natural course of the growth disorder in GH - deficiency quite well. (Fig. 25) The problem shows even better on the growth velocity chart. The velocity was very low before treatment, increased somewhat with thyroxine alone, and more with the addition of GH, which at the time was a pituitary extract, and of testosterone. The these may

problem two

also

extreme benefit

lies with the children categories, from

hGH

but

who do not fall into in between,

and

who

treatment.

Ladies and gentlemen. The following conclusions may be drawn from what I have sad. It is clear that there are today in the field of assessment of GH-secretion just as

Fig.

24

Long

term

course

in

a patient

Assessment

of Growth

Hormone

Secretion

in Children

many questions as answers in spite of many years of research and in spite of the fact that much and substantial progress has been made. Certainly, more results in normal children would be required. However, for ethical reasons, it is not easily possible to obtain a large number of such data. Therefore most so-called normal values in stimulation tests and in the analysis of physiological secretion had to be obtained in short, but otherwise normal children. The optimal frequency and duration of sampling for a better evaluation of pulsatility need to be determined. At present the growth velocity of the child, the response to standard dynamic tests, the basal serum IGF I concentration, the pattern of GH secretion over a 24-hour period, metabolic nitrogen retention tests, and possibly the estimation of urinary GH appear to be the most useful. The GHRH test offers the possibility to exclude a primary pituitary defect, but is otherwise not very important. Disturbances of GH secretion may result from neurosecretory disturbances that may be transient, or may represent part of the spectrum between normal variation and pathological GH secretion. Criteria to distinguish these possibilities are lacking at present. As Brook states correctly, there is a continuum in endo genous GH secretion ranging from those children with poor growth velocities who secrete very little called GH deficient, to those with normal growth velocities who secrete a much greater amount. The relationship between

with

combined

pituitary

insufficiency.

Keio J Med 39 (3): 173-186,

1990

Fig.

185

25

Height

velocity

curve

in a patient

growth velocity and GH secretion in short children is described by an asymptotic regression. Further, the re lationship between growth and GH is modulated by GH pulse amplitude. The therapeutic implications of this relationship are that any short child given hGH will grow more rapidly, and that those children growing most slowly with the least endogenous hGH secretion will have the greatest increment in height velocity for a standard dose of exogenous hGH. (Fig. 26) On this slide, which we have made from the results of a multi

with

combined

center

study

children,

treatment.

least

would or

one

when

the

tests

dose,

when

the

possible

were

in the

be year

tests

serious trial

a regular deficiency,

are normal.

there

those

grow

best

on hGH obstacles,

conclusion with

that

hGH

replacement and

Hopefully,

is

children

economic

say as an overall with

GH-deficient

although

in general

a treatment

suggest tests

not

in

clearly,

to stimulation

probably

test

hGH

this quite

variation,

If there

one would

insufficiency.

recombinant

individual

who respond

months

with

you can see

a large

best

pituitary

with

for

the six

dose, a higher

this will

be

future. References

Fig.

26

-induced

Relationship hypoglycemia

between and height

maximum

GH-response

velocity on subsequent

to

insulin

hGH treatment.

1. Thorner MO, Vance ML, Evans WS, Blizzard RM , Rogol AD, Ho K, Leong DA, Borges JLC , Cronin MJ, MacLeod RM, Kovaks K, Asa S, Horvath E, Frohman L, Furlanetto R , Klingensmith GJ, Brook C, Smith P, Reichlin S , Rivier J, Vale W: Physiological and clinical studies of GRF and GH . Recent Prog Horm Res 42: 589-640, 1986 2. Tannenbaum GS, Ling N: The interrelationship of growth hormone(GH)-releasing factor and somatostatin in generation of the ultradian rhythm of GH secretion . Endocrinology 115: 1952-1957, 1984 3. Thorner MO, Rogol AD: Neuroendocrine control of growth hormone secretion. In: Human Growth Hormone , Underwood L E, ed., Marcel Dekker, Inc., New York , Basel, 1988, 113-130 4. Ho KY, Evans WS, Blizzard RM , Veldhuis JD, Merriam GR, Samojlik E, Furlanetto R, Rogol AD, Kaiser DL , Thorner MO: Effects of sex and age on the 24 hour secretory profile of GH

186

5.

6.

7.

M. Zachmann:

secretion in man: Importance of endogenous estradiol concen trations. J Clin Endocrinol Metab 64: 51-58, 1987 Prader A, Zachmann M, Poley JR, Illig R: The metabolic effect of a small uniform dose of human growth hormone in hypopituitary dwarfs and in control children. I. Nitrogen, alpha-amino-N, creative-creatinine and calcium excretion and serum urea-N, alpha-amino-N, inorganic phosphorus and alkaline phosphatase. Acta Endocrinol 57: 115-128, 1968. Zachmann M, Zagalak M, Gitzelmann RP, Prader A: Modifi cation of 15N balance by growth hormone, testosterone, and thyroxine in patients with growth hormone deficiency and hypo thyroidism. In: Stable Isotopes, Klein ER, Klein PD, eds., Academic Press, New York, 1979, 619-622 Rose SR, Ross JL, Uriarte M, Barnes KM, Cassorla FG, Cutler GB: The advantage of measuring stimulated as compared with spontaneous growth hormone levels in the diagnosis of growth

8.

Assessment

of Growth

Secretion

in Children

hormone deficiency. N Engl J Med 319: 201-207, 1988 Brook CGD, Hindmarsh PC, Smith PJ, Stanhope R: features and investigation Clinics in Endocrinology RA, eds., WB Saunders,

9.

Hormone

Clinical

of growth hormone deficiency. In: and Metabolism, Savage MO, Randall Philadelphia, 1986, 479-493

Gelato M, Malozowski S. Nicoletti MC, Ross JL, Pescovitz OH, Rose S, Loriaux DL, Cassorla F, Merriam G: Growth hormone responses to GH-releasing hormone during pubertal development

10.

in normal boys and girls: comparison to idiopathic short stature and GH deficiency. J Clin Endocrinol Metab 63: 174-179, 1986 Spiliotis BE, August GP, Hung W, Sonis W, Mendelson W,

11.

Bercu BB: Growth hormone neurosecretory dysfunction: treatable cause of short stature. JAMA 152: 2223-2251, 1984 Clark RG, Jansson JO, Isaksson O, Robinson ICAF: Intravenous growth hormone: hypophysectomized

growth responses rats. J Endocrinol

to patterned 104: 53-61,

infusions 1985

a

in