The serum growth-hormone response to hypoglycemia in dwarfism

November, 1967 T h e Journal of P E D I A T R I C S

625

The serum growth-hormone response to hypoglycemia in cl arfism The serum growth-hormone response to insulin-induced hypoglycemia has been measured in control subjects and patients with dwarfism o[ various causes. The normal response has been defined and applied to the assessment o[ growth-hormone function in patients with growth retardation. Definitive evaluation of growth-hormone responsiveness was possible in 58 of 59 patients with dwarfism.

S. Douglas Frasier, M.D. "~ WITK

THE

TECIclNICAL

ASSISTANCE

OF

Jean M. Hilburn, B.S., and Nancy L. Matthews, B.A. LOS

ANGELES

AND

POMONA~

CALIF.

etiology o f dwarfism in children remains a perplexing clinical problem. The differential diagnosis of hypopituitarism, constitutional delay in growth and adolescence, and primordial dwarfism is frequently difficult? The possible role of growth-hormone deficiency in specific syndromes associated with dwarfism has not been extensively investigated. The development of radioimmunoassay techniques for the measurement of humanD E T E R M I N A T I O N

of

the

From the Department o[ Pediatrics, University of Cali[ornia at Los Angeles School of Medicine, and Pacific State Hospital, Pomona, Call[. Supported by Grants 62-I4-38; 63-14-38; 66-14-38, #om the State of Cali[ornia, Department of Mental Hygiene. Presented in part to the Western Society for Pediatric Research, Portland, Oregon, Nov. I, I965. *:'Address, Children's Division Building (Room CD 4 E 6), Los Angeles County General Hospital, 1200 N. State St., Los Angeles, Call[. 90033.

growth hormone2-4 provides a method for determining the blood concentration of growth hormone in children with growth retardation. The demonstration of a rapid rise in the plasma concentration of growth hormone following the induction of hypoglycemias suggested a technique for assessing the dynamic aspects of growth-hormone secretion and a possible diagnostic procedure for detecting growth-hormone deficiency. An increase in the blood concentration of growth hormone in rr to hypoglycemia has been widely confirmed in normal adults2 -1~ The response of growth hormone to hypoglycemia as a test of pituitary function has been investigated in adults, and has been shown to provide a means of detecting a deficiency of growth hormone in patients with hypopituitarism?~, 12 There have been preliminary reports of the response of growth Vol. 71, No. 5, pp. 625-638

626

Frasier

hormone to hypoglycemia in dwarfism, ~31~ but no details have been published. This study was designed to determine the usefulness of the response of growth hormone to insulin-induced hypoglycemia in the differential diagnosis of dwarfism, and to assess this response in patients with specific clinical syndromes associated with growth retardation. METHODS

Growth-hormone assay. The concentration of human growth hormone ( H G H ) in serum was measured by the radioimmunoassay technique of Glick and associatesY The hormone (Raben No. 16") was iodinated with carrier-free Naa~I (Iso/Serve) by the method of Greenwood and co-workers ~6 with modifications previously described. 17 Reaction mixtures consisted of HGH~3~I in tracer amounts (0.025 to 0.05 m/sg per milliliter, guinea pig a n t i - H G H serum (R. S. Yalow No. 29: 12-62t), in a final dilution of 1:1,500,000 and either a known quantity of highly purified human-growth hormone (Wilhelmi HS 503A$) or unknown serum. Each tube contained 0.1 ml. HGH~a~I (0.25 to 0.5 m/xg per milliliter), 0.1 ml. a n t i - H G H serum (1:150,000), and either 0.01 to 0.32 ml. standard H G H (5 m/~g per milliliter) or 0.01 to 0.1 ml. unknown serum. The usual volume of unknown serum was 0.05 or 0.1 ml. Final volume of the reaction mixtures was t.0 ml. with 0.05 M barbital buffer (pH 8.6) containing human serum albumin 2.5 mg. per milliliter used as diluent. Reaction mixtures were allowed to equilibrate 4 to 7 days at 4 ~ C. Free and bound HGH131I were separated by ehromatoelectrophoresis 17 on W h a t m a n 3 MC filter paper strips at 600 v. for 60 minutes at 4 ~ C. in 0.1 ]~I barbital buffer ( p H 8.6). A 0.2 ml. aliquot of the reaction mixture was applied to the paper after the r by Dr. M. Raben, "~Provided by Dr. R. S. Yalow through the NationaI Pituitary Agency~ National Institutes of Health. +Supplied by Dr, A. E. Wilhelmi,

The Journal o/ Pediatrics November 1967

addition of 0.02 ml. normal h u m a n plasma stained with bromphenol blue. The added plasma enhanced the separation of free and bound radioactivity and served as a marker for the migration of serum proteins. Free HGH131I was not eluted from the origin under these conditions. Radioactivity was quantitated by scanning chromatoelectrophoretograms in a 4~- radiochromatogram scanner (Packard) equipped with an integrating recorder (Honeywell). Counting error was less than 5 per cent. Standards and unknown sera were assayed in duplicate or triplicate and the mean per cent H G H l a l I bound to antibody utilized in calculating the concentration of H G H . Duplicate or triplicate control mixtures were included with each set of standards and each set of unknown sera to correct for nonspecific protein binding of HGH~I.I~ Insulin induced hypoglycemia. After an overnight fast, hypoglycemia was induced by the intravenous administration of crystalline insulin, 0.05 to 0.i unit per kilogram body weight. The higher dose was generally used except in patients in whom insulin sensitivity was suspected. An intravenous infusion of 0.9 per cent saline was begun in any patient suspected of being insulin sensitive or when there was not easy access to a large superficial vein. A physician was in attendance throughout the test and 50 per cent glucose and glucagon were available to terminate symptomatic hypoglycemia. Blood was obtained for the determination of glucose concentration and for radioimmunoassay of H G H at 0, 15, 30, 60, 90, and 120 minutes after insulin administration. An aliquot of each sample was assayed for glucose concentration by either the glucose oxidase method (Glucostat), or on an autoanalyzer by the ferri-ferrocyanide method. The remainder of each sample was allowed to clot at room temperature for 1 to 2 hours, and the serum was separated and frozen at -20 ~ C. until assayed for growth-hormone concentration. Preliminary observations had shown no difference in the concentration of

Volume 71 .Number 5

Growth-hormone response to hypoglycemia

growth hormone in paired samples of plasma separated and frozen immediately and serum separated after clotting. Ancillary studies. Height age and the deviation from mean height for age were determined from standards developed by the Iowa Child Welfare Research Station. 2~ Bone age was determined by comparison with the standards of Greulich and Pyle. 21 Pituitary function was evaluated in patients with pituitary or central nervous system lesions and in patients with suspected idiopathic hypopituitarism. Thyrotropic hormone (TSH) function was measured by determining protein-bound iodine (PBI) and thyroid radioiodine uptake. A PBI of less than 4 ~g per cent a n d / o r a radioiodine uptake of less than 15 per cent at 24 hours was interpreted as being compatible with T S H deficiency. Adrenocorticotropic hormone (ACTH) function was measured by determining 24 hour urinary 17-hydroxysteroids (Porter-Silber Chromagens) 22 and by assessing the response to mepyrapone (500 mg. per square meter every 4 hours for 24 hours). A baseline 17-hydroxysteroid excretion of less than 2 mg. per square meter per 24 hours 23 a n d / o r failure to double the urinary excretion of 17-hydroxysteroids in response to mepyrapone24, 2~ was interpreted as evidence of A C T H deficiency. Urinary gonadotropins were measured in patients 12 years of age or older by the mouse uterine weight method 26 after extraction by kaolin absorption. 27 Diabetes insipidus was documented by demonstrating polyuria and polydipsia and a response to the administration of Pitressin. Study patients. Control subjects (Group I). The 10 control children studied were aged from 5 months to 13 years (Table I). Heights were +2 standard deviation (S.D.) from the mean for age. Subjects were either normal children studied as outpatients or children hospitalized for the investigation of disorders other than short stature.

Pituitary or central nervous system lesions and growth retardation (Group 1I). Nineteen patients aged 2 years and 7 months

627

to 16 years and 9 months with a lesion of the pituitary or of the central nervous system were investigated (Table III). All patients except 1 showed a subnormal rate of growth (less than 3 cm. per year) and increasing deviation from the mean height for age over a period of observation of 6 months to 7 years. Only 1 patient was observed for less than 1 year. Patient 12 was less than 2 S.D. below the mean height for age at the time of decompression of a craniopharyngioma and grew 7.6 em. over the ensuing 16 months (5.8 cm. per year). Data regarding blood-sugar values are not available on Patient 29. She became mildly symptomatic after the administration of insulin and therefore an adequate fall in concentration of blood sugar has been assumed. All patients except 12 and 15 demonstrated at least 1 associated tropic hormone deficiency and/or diabetes insipidus. Patients who had demonstrable thyrotropin and/or A C T H deficiency were receiving replacement thyroid and glucocorticoid therapy at the time of study. In those with diabetes insipidus, the condition was controlled with vasopressin.

Suspected growth hormone deficiency (Group IH). Twelve patients with clinically suspected deficiency of growth hormone were studied (Table IV). All were 3 S.D. or more below the mean height for age. Patients showed increasing deviation from the mean height for age, and the rate of growth was less than 5 cm. per year over a period of observation of 9 months to 7 years. Only 1 patient was observed for less than 1 year. The ratio of bone age to chronological age varied from 0.29 to 0.80 with a mean of 0.53 and the ratio of height age to chronological age varied from 0,25 to 0.74 with a mean of 0.53. Associated tropic hormone deficiencies were demonstrated in Patients 30 (ACTH, T S H ) , 33 ( A C T H ) , 34 ( A C T H ) , 37 ( T S H ) , and 40 (TSH, gonadotropin). Patient 30 had episodes of spontaneous hypoglycemia and no patient in this group had diabetes insipidus. The protein-bound iodine value

628

Frasier

The Journal of Pediatrics November 1967

was greater than 4 ~g per cent in all patients at the time hypoglycemia was induced. Constitutional delay in growth (Group I V ). Ten patients with constitutional delay in growth were investigated (Table V). All were 3 S.D. or more below the mean height for age. Growth rate varied from 5 to 8.2 cm. per year over a period of observation of 1 to 3 89 years. Height maintained a constant relation to the mean height for age with advancing chronological age. The ratio of bone age to chronological age varied from 0.26 to 0.85 with a mean of 0.59 and a ratio of height age to chronological age varied from 0.23 to 0.69 with a mean of 0.56. Syndromes associated with growth retardation (Group V). Seventeen patients with a variety of recognized nonpituitary disorders associated with growth retardation were studied (Table V I ) . This group included 4 patients with gonadal dysgenesis confirmed by elevated urinary gonadotropins (greater than 140 mouse uterine units per 24 hours); 2 patients with chondrodysplasia; monovular twins with asthma, dwarfism, and elevated serum concentrations of immunoglobulin A28; 1 patient with Rubinstein-Taybi syndrome29; and one patient with dwarfism and sclerodermatous skin changes, s~ Also included are 7 patients in 6 families with dwarfism associated with craniofacial disproportion, mandibulofacial dysostosis and

minor anomalies of the extremities such as hemiatrophy and short incurved fifth fingers; 6 of this group had a history of intrauterine growth retardation, TM 32 with birth weights at term varying from 1,000 to 2,500 Gm. Patient 66 had episodes of symptomatic hypoglycemia. Patients 62 to 65 most closely resembled the dwarf described by RusselP ~ and Patients 66 to 68 most closely resembled the bird-headed dwarf described by Seckel. a~ RESULTS Assay of human growth hormone. Fig. 1 shows a typical standard curve. When a logarithmic scale is employed for the known amounts of H G H added, a log-dose response, which is linear over the range 0.1 to 1.6 m#g per milliliter, is obtained. Plotting the data in this way allows analysis of the stand a r d curve by bioassay statistics, a~ The p value is < 0.001 and the standard curve has an index of precision (X) of 0.065. The sensitivity of the assay varies from 0.05 to 0.2 m/xg per milliliter and thus 0.5 to 2 m~g per milliliter can be measured in a 1:10 dilution of serum. The slope obtained when serial dilutions of plasma from an acromegalic patient are assayed is parallel to the standard curve, indicating that the same substance ( H G H ) is being measured. Plasma from hypophysec-

Table I. Control subjects

Patient

Sex

Age (yr.--too.)

1

F

0--5

2 3 4 5 6 7 8 9 10

F M M F M F M F M

4--2 4--6 8--0 8--10 9--4 10--6 11--3 11--7 13--2

Mean • S.D. Range

Minimum blood sugar Concentration (my.%) Per cent of fasting 38 39 55 33 32 60 27 64 30 38 31 40 23 26 39 42 32 30 32 37 33.9 • 8.7 23-55

40.9 • 10.4 27-64

*Difference between lowest growth-hormone concentrations (0 or 15 minute sample) and peak growth-hormone concentration.

Volume 71 Number 5

Growth-hormone

tomized patients does not inhibit the b i n d i n g of HGH131I, i n d i c a t i n g that the assay is specific for H G H . T h e m e a n difference between duplicate determinations of 30 sera c o n t a i n i n g less t h a n 10 m/xg per milliliter was 25 per cent of the m e a n serum concentration. T h e m e a n difference between duplicate determinations of 22 sera c o n t a i n i n g 10 to 35 m/~g per milliliter was 19 per cent of the m e a n serum concentration. T h e concentration of H G H in serum from a n acromegalic patient assayed in duplicate in 7 different assays was 21.7 _+

. 351 ~, 3o1 ~2s

1I/1o0 I/I40 1/20 I/to [ o

,,,%

629

3.5 m/xg per milliliter. Recovery of H G H added to serum from hypophysectomized patients varied from 77 to 87 per cent over the range 15 to 60 m/xg per milliliter; the m e a n recovery was 82 per cent. Control subjects. T h e response of serum growth h o r m o n e to insulin i n d u c e d hypoglycemia in control subjects is shown in T a b l e I a n d Fig. 2. G r o w t h h o r m o n e was detectable after a n overnight fast in 5 of 10 subjects. T h e m a x i m u m fasting concent r a t i o n was 9.6 m/xg per milliliter. Following i n d u c t i o n of hypoglycemia, a rise was evi-

50

# 400

response to hypoglycemia

.

ra

_~ 30 :z. ::= 20

%

w u ~o

0

o&

",

22

o4~

o!8

,

an acromegalic patient (x x) and the lack of effect of plasma from hypophysectomized patients (black circles) is also shown. HGH added (millimicrogram per milliliter), o o.

< < < < <

I 15 minutes

5.2 4 4 2.8 2 3.0 1 9.6 4.0 2

8.6 -7.7 8.8 2.0 < 2 1.7 -15 23

2.5+-3.2 < 1-96

8.4+-7.7 1.7-23

15.8+-7.5 8-30

60

90

120

{minules)

Fig. 2. Serum growth-hormone response to insulininduced hypoglycemia in control subjects. Individual values are shown by open circles. The mean is shown by closed circles and +- 1 S.D. is enclosed in brackets. The composite response is indicated by the solid line connecting mean values.

Growth hormone (m#g/ml.) I 30 minutes I 60 minutes ] 90 minutes 22 8.3 17 13 7.6 12 8.0 23 17 30

30 TIME

Fig. I. Growth-hormone assay standard curve. Each point represents the m e a n of duplicate determinations of H G H 1 3 1 1 bound to antibody. The parallel effects of serial dilutions of plasma from

0

t5

20 3.5 < 4 7.5 18 32 4.6 25 5.5 20

-< 4 < 4 12 7.4 13 < 1 -6.8 14

13.8-+ 1 0 . 9 < 4-32

6.7+.6.1 < 1-14

I 120 minutes t 2.4 4.0 -4.4 7.8 10 < 1 13 5.8 6 5.9+.4.0 < 1-13

Increase* 16.8 8.3 17 10.2 18 32 8 15.4 13 30 16.9+-8.3 8 32

630

Frasier

The Journal o[ Pediatrics November 1967

T a b l e I I . C o m p a r i s o n of r e s p o n s e to h y p o g l y c e m i a w i t h r e s p o n s e t o s a l i n e

,

15 minutes

30 minutes

60 minutes

< 2

< 2

23

24

78

67

39

23

0

90 minutes

120 minutes

Increase

< 2

24

Patient A M Insulin 0,1 U n i t / K g . I V

HGH (m#g/ml.) Sugar (mg.%)

0.9% Saline 1 c.c. I V

HGH (m~g/ml.) Sugar (mg.%)

<2

<2

84

<2

84

93

<2

5.1 88

96

<2

99

0

<2

88

85

Patient LC Insulin 0.1 U n i t / K g . IV

HGH (m~g/ml.) Sugar (rag.%)

0.9% Saline 1 c.c. I V

HGH ( m~tg/ml. ) Sugar (rag.%)

5.5 72 <2 78

7.3

15

37 2.5

44 <2

74

72

7.2

9.4

2.6

46

57

6:

< 2

< 2

< 2

73

75

70

9.5

2.5

T a b l e I I I . L e s i o n s of t h e p i t u i t a r y o r c e n t r a l n e r v o u s s y s t e m

Minimum blood sugar Patient ] Sex 11 12 13 14 15 16 17 18 19 20t 21 22t 23 24 25 26 27 28 29

M F (1) (2) F F M M M M F M M M F F F M M M F

Age (yr.--too 0 6--1 9--4 10--8 9---6 9--6 9--8 12--5 13--8 13--9 14 15--5 16 16--7 16--9 2--7 12--10 13--8 7--1 9--2 15--3

Diagnosis Craniopharyngioma s Craniopharyngioma ~ Craniopharyngtoma e Craniopharyngmrna ~ Craniopharyngmma Craniopharyngloma ~ Craniopharyngloma Craniopharyngmma ~ Craniopharyngloma Craniopharyngloma Craniopharyngloma Craniopharyngmma Chromophobe adenoma *~ Suprasellar astrocytoma ~ Astrocytoma floor of third ventricle ~ Astrocytoma floor of third ventricle s Reticuloendotheliosis (Hand-Sehfiller-Christian) Reticuloendotheliosis ( Hand-S chillier-Christian ) Basilar skull fracture; cerebrospinal fluid rhinorrhea Mean + S.D. Range

*Postoperative. tTest discontinued because of symptoms.

Concentration (rag. %)

Per cent o[ lasting

58 61 45 56 44 58 34 56 16 25 56 61 26 43 40 41 46 70 50 61 55 55 50 61 47 50 27 36 23 39 32 59 53 72 37 40 41 50 (Symptomatic) 41.1+12.1 16-58

52.3-+12.2 25-72

Volume 71 Number 5

Growth-hormone response to hypoglycemia

dent in the 15 minute sample in 7 of 8 subjects. I n 1, there was a fall between the fasting and 15 minute samples. The peak concentration of growth hormone was detectable in the 30 or 60 minute sample and varied from 8 to 32 m~g per milliliter (mean 19 _+ 8.4 S.D. m/xg per milliliter). The increase in serum concentration of growth hormone, when the peak is compared with the lowest value (fasting or 15 minute sample), varied from 8 to 32 m~g per milliliter (mean 16.9 _+ 8.3 S.D. m/~g per milliliter). An undetectable concentration of growth hormone is arbitrarily designated as zero in this calculation. A fall in the serum concentration of growth hormone was detectable in the 60 or 90 minute sample, and in general, continued through the 120 minute sample. The fall was not as sharp as the rise and in 5 subjects there was a plateau or slight rise between the 90 and I20 minute samples. There was wide variability in the

0 2.4 <4 2.6 <4 1.6 <1 <4 <1 <1
I 15 minutes < 2 -<2 <4 1.2 <1 <4 <1 <1
I

30 minutes 3.5 <4 7.4 <4 <1 <1 <4 <1 <1 2.6

<1

<1

<1

<1

<1

<1

<1

<1

<1

<2 <2 <2
-<2 -<1 <2 <1 <2

<2 <2 <2 <1 <2 <1 <2

63 1

magnitude and shape of the response between patients. In Table I I the response to insulin-induced hypoglycemia is compared with the response to the intravenous administration of 0.9 per cent saline in 2 control patients. A rise in the serum concentration of growth hormone was associated with hypoglycemia, while the administration of saline resulted in no change in the concentrations of growth hormone or blood sugar. Patients with hypothalamic or pituitary lesions. Table I I I shows the serum growth hormone response to insulin-induced hypoglycemia in patients with a lesion of the pituitary gland or central nervous system. Growth hormone was detectable in 3 of 19 patients after an overnight fast. The maximum fasting concentration was 2.6 m/xg per milliliter. There was a fall between the fasting and 15 minute samples in these 3 patients. A definite rise in serum concentra-

Growth hormone (m#g/ml.) t 60 minutes I 90 minutes 2 2.0 10 -5.6 <2 <4 <4 2.9 1.1
112ominutes -<4 -<4 0 <1 <4 <1 <1 <1

I

Increase 3.5 16 7.4 0 2.9 0 0 0 0 2.6

--

--

--

<1

<1

<1

--

--

--

0

-<2 <2
<2 <2 -<1 <2 <1 <2

0 0 0 0 0 0 0

<2 <2 <2 <1 <2 <1 <2

0

0

6 32

Frasier

The Journal o[ Pediatrics November 1967

tion of g r o w t h h o r m o n e was observed only in p at i en t 12. I n this p a ti e n t the response to h y p o g l y c e m i a was tested before and after a 16 m o n t h period, during w h ic h t i m e she g r e w 7.6 cm. T h e increase in serum growthh o r m o n e c o n c e n t r a t i o n was 16 a n d 7.4 mt~g pe r milliliter, respectively. Patients 1I, 14, and 19 showed slight increases in serum g r o w t h - h o r m o n e c o n c e n t r a t i o n of 3.5, 2.9, a n d 2.6 m/~g per milliliter, respectively.

S e r u m g r o w t h h o r m o n e was undetectable after an overnight fast or in response to hypoglycemia in the r em ai n i n g 15 patients. Suspected g r o w t h h o r m o n e deficiency. T a b l e I V shows the serum g r o w t h - h o r m o n e response to insulin-induced h y p o g l y c e m i a in patients with suspected idiopathic hypopituitarism. G r o w t h h o r m o n e was detectable after an overnight fast in 5 of 12 patients, with

T a b l e IV. Suspected g r o w t h - h o r m o n e deficiency

Growth hormone (m#g/ml.)

Minimum blood sugar Patient i Sex

30 31 32 33 34 35 36 37 38 39 40 41 42

F ( 1) (2) F M F M M M F M M F F M

Age (yr.--

15

Conce?~-

too.)

tration (rag. %)

Per cent o[ lasting

1--11 2--8 4--tl 4~11 5--5 6--7 6--10 7--4 7--8 8--5 10--3 12--t 12--8 12--11

50 22 40 39 51 55 48 20 52 30 26 47 37 41

66 29 5I 58 61 63 67 25 54 36 35 70 42 56

39.9-+11.6 22-55

50.9 + 14.9 29-70

Mean -+ S.D. Range

minutes

3.6 1.2 4.8 <2 < 2 < 1 < 2 -<2 < 2 1.4 <2 18 < 1

-1.1 7.6 <2 < 2 < 1 2.9 3.1 <2 < 4 3.8 -19 < 1

30 minutes 3.0 4.2 24 3.2 <~ 2 < 1 < 2 4.2 < 2 17 3.4 <2 80 < 1

60 rainutes 4.9 < I 7.0 <2 < 2 < i < 2 4.3 <2 17 3.9 <2 22 < 1

90 1 2o rain- tl -mmutes r utes 3.8 5.6 2.4 2.2 3.9 3.1 <2 <2 3.0 4.5 < 1 < 1 < 2 < 2 4.8 4.7 <2 <2 7.8 < 4 3.2 3.7 -<2 9.6 4.4 < 1 2.0

Increase

2.0 3.1 19.2 3.2 4.5 0 2.9 1.7 0 17 2.5 0 62 2.0

T a b l e V. Constitutional short stature

Minimum blood sugar Patient

Sex

43 44 45 46 47 48 49 50 51 52

F F M M M M M F M F Mean + S.D, Range

Age (yr.--too 0 1--7 4--2 4--11 5--3 6--4 6--4 9--7 13--5 13--8 13--10

Concentration (rag. % ) 52 48 38 52 33 34 51 47 50 51

Per cent o[ lasting 70 48 44 67 42 45 62 60 60 39

~ 2 12 13 %2 < 2 2.4 1.4 6.0 2.1 3.2

45 6 -+ 7.6 33-52

53.7 + 11.3 39-70

4.0 + 4.8 < 2-13

Volume 71 Number 5

Growth-hormone response to hypoglycemia

a maximum level of 18 m~g per milliliter. There was a clear rise in serum growth hormone detected in the 30 or 60 minute sample in Patients 31, 38, and 41, with a maximal increase of 19.2, 17, and 62 m~g per milliliter, respectively. In 8 tests involving 7 patients a slight rise of 1.7 to 4.5 m~g per milliliter was demonstrated. The peak was detectable in the 15 minute sample of 1 test, in the 30 or 60 minute sample of 3 tests, and was delayed to the 90 or 120 minute sample of 4 tests. There was no detectable serum growth hormone in the fasting state or in response to hypoglycemia in 3 patients. Constitutional delay in growth. The serum growth-hormone response to hypoglycemia in 10 patients with constitutional delay in growth is shown in Table V. Serum growth hormone was detectable in 7 children after an overnight fast with a maximum concentration of 13 m#g per milliliter. There was a fall between the fasting and the 15 minute samples in 3 patients. There was a rise in all patients to a peak of 9 to 39 m/xg per milliliter (mean 18.8 __ 9.2-S.D. m/xg per milliliter). The peak was detected in the 30 or 60 minute sample in 9 patients and at 15 minutes in the tenth. The maximum increase in growth hormone concentration varied from 7.2 to 31.3 mt~g per milliliter (mean 16.6 _+ 8.6 S.D. mt~g per milliliter). A fall

15minutes 3.0 2.1 6.7 --

18 < 2 2.6 8.0 --

8.4 6.1 -+3.8 < 2-18

[

30minutes 25 6.6 39 26 12 3.1 4.1 16

]

was noted in the 60 or 90 minute sample followed by a plateau or slight rise in serum concentration of growth hormone between the 90 and 120 minute sample. The pattern of response in this group is indistinguishabIe from that of control subjects and there is no significant difference between the peak growth-hormone concentration and maximum increase in growthhormone concentration in this group and control subjects. Syndromes associated with growth retardation. Table VI shows the serum growth hormone response to hypoglycemia in a variety of clinical syndromes associated with dwarfism. Growth hormone was detected after an overnight fast in 8 of 16 patients from whom a fasting sample was available. Three patients showed a fall between the fasting and 15 minute samples. A peak serum concentration of growth hormone of 7.8 to 85 m~g per milliliter was reached in 16 of these 17 patients in the 30 or 60 minute sample. In Patient 58 a peak of 11 m/zg per milliliter was not reached until the 120 minute sample. The maximum increase in this group varied from 7.8 to 85 m#g per milliliter. The data has not been analyzed statistically because of the clinical heterogeneity of this group. The pattern of response is similar to that observed in control children and in patients with constitutional short stature.

Growth hormone (m#g/ml.) 60mlnutes [ 90minutes 7.4 19 11 22 9.1 16 9.6 11

9.0

2.9

10.4

6.5

15.1 + 11.5 3.1-39

11.5 + 5.9 6.5-22

63 3

1120minutes

3.5 5.4 6.8

6.4 6.3 4.3 24 12 2.6 3.4 3.4

--

9.5 -4.7 4.2 <

2

9.1 5.4 + 3.1 < 2-9.5

<

2

5.8 6.8 + 6.8 < 2-24

I

Increase 25 16.9 31.3 26 18 16 8.2 10 69 7.2 16.6 + 8.6 7.2-31.3

6 34

Frasier

The ]ournaI of Pediatrics November 1967

Table VI. Syndromes associated with growth retardation Minimum blood sugar

Age

P~z-

tient

(yr.--moO

53 54 55 56 57 58 59r

Sex F F F F F F F

60~ 61 62 63 64t 65t 66 67 68 69

F

F F M M M M M F M

8--11 5--11 10--2 8--6 8 13~ 16--4 2--9 3 7

1~r e a n

+

13--5 14--6 15--8 16--8 4--9 4--3 8--11

Diagnosis

Gonadal dysgenesis Gonadal dysgenesis Gonadal dysgenesis Gonadal dysgenesis Chondrodystrophy Achondroplasia Asthma Dwarfism Elevated immunoglobulin A Rubinstein-Taybi syndrome Dwarfism; sclerodenaaatous skin changes Intrauterine growth retardation, craniofacial disproportion, mandibulo.facial dysostosis, and minor anomalies of extremities

S.D.

Range

Concentration (mg. %) 48 53 38 40 52 33 37

Per cent of fasting 59 61 54 44 69 48 51

29 48 39 40 47 51 64 31 48 50

33 62 42 57 60 66 65 34 62 56

44• 29-64

54.3• 33-69

~'Monovular twins. tSiblings.

DISCUSSION The clinical classification of dwarfism is based on descriptive a n d / o r etiological considerations. When a lesion of the pituitary or hypothalamus is associated with growth failure, growth hormone deficiency is generally assumed. In the absence of such a lesion, the clinical diagnosis of growthhormone deficiency presents a more difficult problem, particularly in the prepubertal child. In this study, patients without lesions have been grouped according to clinical criteria suggested by Wilkins. 1 When no specific clinical syndrome associated with dwarfism was present, patients were divided into 2 groups based on a retarded rate of growth (less than 5 cm. per year) or a normal rate of growth (greater than 5 cm. per year). These groups were designated suspected growth-hormone deficiency and constitutional delay in growth, respectively. They were comparable in the degree of dwarfism (height age/chronological age ratio) and the degree of retardation in bone

maturation (bone age/chronological age ratio). The induction of hypoglycemia was well tolerated by 67 of the 69 children tested. Although transient symptoms of sweating, pallor, and drowsiness were frequently noted, the test was discontinued because of symptomatic hypoglycemia in only 2 patients. Following the administration of insulin, the minimum blood-sugar value varied from 14 to 64 mg. per 100 ml. per cent (mean 41.5 _+ 10.3 S.D. rag. per 100 ml.) and reached 25 to 72 per cent of the fasting concentration (mean 51.5 _+ 12.2 S.D. per cent). In all patients the minimum blood-sugar concentration was 55 mg. per 100 ml. or less a n d / o r the blood-sugar concentration fell to below 66 per cent of the fasting concentration. Since this degree of hypoglycemia resulted in an adequate response in control subjects and patients with constitutional delay in growth, we do not feel that it is necessary to obtain a 50 per cent fall in the concentration of blood sugar to evaluate the

Volume 71 Number 5

0

Growth-hormone response to hypoglycemia

Growth hormone (m#g/ml.) t 15 minutes t 30 minutes I 60 minutes I 90 minutes { 120 minutes [

6.8 2 2.3 < 4 4.0 < 2

8.2 3.4 3.1 < 4 -19

17 30 21 15 22 3.8 49

5.4 < 2 8.4 3.7 2 2 < 4 2.5 23 < 2

10 -52 < 2 24 < 2 7.4 < 1 4.4 < 2

17 15 33 13 85 ~ 2 30 9.6 3.6 12

--

<

2

Increase

19 < 2 19 9.1 28 4.5 10

7.0 < 2 6.3 4.2 7.0 5.7 4.4

2.0 < 2 < 2 < 2 < 4 11 2.3

19 23.2 21 14.7 28 7 49

9.7 20 32 17 33 20 23 < 1 11.4 19

15 26 34 10 21 12 < 4 < 1 5.4 16

14 16 30 14 3.8 3.2 ~ 4 18 3.4 --

11.6 26 43.6 17 85 20 30 18 7.8 19

growth-hormone response. There was no significant difference in the degree of hypoglycemia between the groups studied, and there was no correlation between the degree of hypoglycemia and the magnitude of the serum growth-hormone response in any group. The response of growth hormone to hypoglycemia is blunted in hypothyroidism, ss Since all patients included in this study were clinically euthyroid and the PBI was greater than 4 /xg per cent at the time of testing, this factor has not influenced the responses observed. The normal response of serum growth hormone to hypoglycemia may be defined by a comparison of the response in control children with that in patients with growth failure and a lesion of the pituitary gland or central nervous system. The minimum increase in serum growth-hormone concentration was 8 m/xg per milliliter in control patients. In contrast, only 3 of 18 patients with growth retardation in Group I I r e -

63 5

sponded to hypoglycemia with a rise in serum concentration of growth hormone, and the maximum increase observed was 3.5 m~g per milliliter. The remaining 15 patients had no detectable serum growth hormone after an overnight fast or in response to hypoglycemia. The minimal or absent response in this group is comparable to that seen in control patients after the intravenous administration of 0.9 per cent saline. Based on this comparison the following criteria were defined for evaluating response: (1) complete absence of response indicates deficiency of growth hormone, (2) an increase in serum concentration of growth hormone of greater than 5 mt~g per milliliter indicates an intact growth hormone respons% (3) an increase of less than 5 m/~g per milliliter strongly suggests an impaired growth hormone response to hypoglycemia and the presence of a deficiency of growth hormone. No response was detected in 3 patients with suspected growth hormone deficiency and an increase of 1.7 to 4.5 m/~g per milli-

636

Frasier

liter, which frequently occurred in the 90 or 120 minute sample was demonstrated in an additional 7 patients in this group. A similar subnormal response of 3.1 rn/~g per milliliter was seen in Patient 33 on repeat testing. A diagnosis of growth-hormone deficiency was apparently confirmed in these 10 patients and 9 have continued to show retarded growth rates (less than 5 cm. per year). However, Patient 39 has grown 6 cm. in the year since testing. Thus patients with a response of less than 5 m/~g per milliliter may require further clinical evaluation of growth and repeat testing before a definitive diagnosis is made. Three patients with suspected growthhormone deficiency showed responses comparable to or in excess of control subjects, apparently eliminating hypopituitarism as the cause of dwarfism. Patient 31 has shown an increased rate of growth to greater than 10 cm. per year in the period of observation since the test was performed and is felt to represent dwarfism associated with environmental deprivation. Patient 41 represents delayed growth and adolescence and is now showing pubertal changes and an increased growth rate. The etiology of growth retardation remains obscure in Patient 38 and he continues to grow at a subnormal rate. Patient 42 had normal 24 hour urinary excretion of 17-hydroxycorticosteroids, normal levels of PBI and normal thyroid radioiodine uptake. In the last 6 months, he has shown pubertal development and is felt to have isolated growth-hormone deficiency? 7 The failure to demonstrate an associated tropic hormone deficiency does not eliminate deficiency of growth hormone as the etiology in a child with growth retardation. When patients with pituitary or central nervous system lesions, suspected idiopathic hypopituitarism, and constitutional delay in growth are compared, growth-hormone responsiveness is seen to correlate with growth rate. All patients with growth rates in excess of 5 cm. per year demonstrated a response of greater than 5 m,ag per milliliter. Included in this group is the 1 patient with the craniopharyngioma who is growing

The ]ournaI o[ Pediatrics November 1967

normally. An absent response or an increase of less than 5 mp,g per milliliter was observed in 27 of 30 patients with growth rates below 5 cm. per year. This suggests that a normal growth rate provides clinical evidence against growth-hormone deficiency. Though a subnormal growth rate is suggestive of growthhormone deficiency, it is not diagnostic. However, it is an indication for the evaluation of growth-hormone responsiveness. All patients with a recognizable clinical syndrome associated with dwarfism had a serum growth-hormone response to hypoglycemia within or in excess of the range seen in control subjects. This indicates that growth-hormone deficiency does not play a role in the dwarfism noted in these conditions. A possible exception is the delayed response in Patient 58, who has achondroplasia. Although an increase of 7 mug per milliliter was observed, the peak hormone concentration was in the 120 minute sample. A larger number of patients with achon.droplasia must be studied before the typical response of this group can be defined. The normal responses observed in the dwarf described by Russell (Patients 62 to 65) corroborate the finding previously reported in 1 patient by Snow and colleagues? s Three patients demonstrated an excessive response. In Patient 62 this was sustained throughout the test period. The significance of this type of response is unclear. All subjects studied can be divided into responders and nonresponders based on the standards previously described; 41 patients showed an increase in growth-hormone concentration of 6.9 to 85 m~g per milliliter, while 28 patients showed a response of less than 5 m#g per milliliter with no detectable growth hormone in 19. Growth hormone was detectable after an overnight fast in 23 responders (56 per cent) with a range of 1.4 to 23 mp~g per milliliter and in 13 the fasting growth-~clormone concentration was greater than 5 m/sg per milliliter. These results are similar to those previously published? ~ 4o Serum growth hormone was detectable in 5 nonresponders (17 per cent) after an overnight fast and in none was the concentration

Volume 71 Number 5

Growth-hormone response to hypoglycemia

above 5 m/zg per milliliter (range 1.1 to 3.6 mug per milliliter). This suggests that the fasting concentration of serum growth hormone might be used as a screening test for growth-hormone deficiency. Concentrations above 5 m~g per milliliter suggest intact growth hormone function, while concentrations below this level would be an indication to test the response to insulin-induced hypoglycemia. T h e peak concentration of serum growth hormone was reached in either the 30 or 60 minute sample in 41 of 42 tests in responsive patients and the increase from the fasting sample to the peak varied from 6.9 to 85 mbtg per milliliter. Growth hormone was detectable in 8 of 28 tests at 30 or 60 minutes in nonresponsive patients and the increase from fasting sample to the 30 or 60 minute sample varied from 1.t to 3.5 m~g per milliliter. These data suggest that sampling can be: limited to fasting 30 and 60 minutes without lessening the discriminating ability of the response. An additional sample for the determination of blood-sugar concentration should be obtained at 15 minutes to insure that adequate hypoglycemia has been produced. SUMMARY

The serum growth-hormone response to insulin induced hypoglycemia was evaluated in 10 control subjects, 19 patients with lesions of the pituitary or of the central nervous system, 13 patients with suspected idiopathic growth-hormone deficiency, 10 patients with constitutional delay in growth, and 17 patients with various clinical syndromes associated with dwarfism. The normal response is defined as an increase in concentration of serum growth hormone exceeding 5 m,ag per milliliter detected at 30 or 60 minutes in response to a fail in blood-sugar concentration to 55 mg. per cent or less, a n d / o r to 66 per cent or less of fasting concentrations. A normal response indicates unimpaired growth-hormone function, and no detectable response indicates growth-hormone deficiency. Detectable responses of less than

63 7

5 m#g per milliliter strongly suggest growthhormone deficiency but are not necessarily definitive. Growth-hormone responsiveness was norreal in gonadal dysgenesis, in dwarfism associated with asthma and elevated immunoglobulin A concentrations, and in low-birth-weight dwarfism with multiple anomalies. The response in achondroplasia was incompletely defined. The author wishes to express his appreciation to Drs. Harvey Klevit (University ef Oregon School of Medicine), Henry Sauls (University of Minnesota School of Medicine), Stanton Shuler and Robert ten Bensel (U. S. Naval Hospital, San Diego), Lyman Page (Stanford University School of Medicine), and Thomas Nelson (California College of Medicine), for providing serum samples and case summaries on their patients. The Pediatric House Staff of the UCLA Hospital and the Los Angeles County Harbor General Hospital performed many of the insulin tolerance tests. REFERENCES

I. Wilkins, L.: The diagnosis and treatment of endocrine disorders in childhood and adolescence, ed. 3, Springfield, Ill., 1965, Charles C Thomas, Publisher. 2. Glick, S. M., Roth, J., YaIow, R. S., and Berson, S. A.: Immunoassay of human growth hormone in plasma, Nature 199: 784, 1963. 3. Hunter, W. M., and Greenwood, F. C.: A radioimmunoeleetrophoretic assay for human growth hormone, Biochem. J. 91: 43, 1964. 4. Schalch, D. S., and Parke~, M. L.: A sensitive double antibody immunoassay for human growth hormone in plasma, Nature 203: 1141, 1964. 5. Roth, J., Gliek, S. M., Yalow, R. S., and Berson, S. A.: Hypoglycemia: A potent stimulus to secretion of growth hormone, Science 140: 987, 1963. 6. Hunter, W. M., and Greenwood, F. C.: Studies on the secretion of human pituitary growth hormone, Brit. Med. J. 1: 804, 1964. 7. Hartog, M., Gaafar, M. A., and Fraser, R.: Effect of corticosteroids on serum growth hormone, Lancet 2" 376, i964. 8. Beck, P., Koumans, J. H. T., Winterling, C. A., Stein, M. D., Daughaday, W. H., and Kipnis, D. N.: Studies of insulin and growth hormone secretion in human obesity, J. Lab. & Clin. Med. 64: 654, 1964. 9. Fantz, A. G., and Rabkin~ M. T.: Human growth hormone: Clinical measurement, response to hypoglycemia and suppression by

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i1.

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corticosteroids, New England J. Med. 271: 1375, 1964. Greenwood, F. C., Landon, J., and Sta_rnp, T. C. B.: The plasma sugar, free fatty-acid, cortisol, and growth hormone response to insuIin. I. In control subjects, J. Clin. Invest. 45: 429, 1966. Landon, J., Greenwood, F. C., Stamp, T, C. B., and Wynn, V.: The plasma sugar, free fatty-acid, cortisol, and growth-hormone response to insulin and the comparison of this procedure with other tests of pituitary and adrenal function. II. In patients with hypothalamie or pituitary dysfunction or anorexia nervosa, J. Clin. Invest. 45: 437, I966. Rabkin, M. T., and Frantz, A. G.: Hypopituitarlsm: A study of growth hormone and other endocrine functions, Ann. Int. Med. 64: 1197, 1966. Kaplan, S. L., Abrams, C. A. L., Belt, J. J., Conte, F. A., and Grumbach, M. M.: Serum growth hormone response to insulin-lnduced hypoglycemia in disorders of growth, J. P~mAT. 67: 956, 1965 (Abst.). Laron, Z., and Mannheimer, S.: Measurement of human growth hormone: Description of the method and its clinical applications, Israel J. M. Sc. 2: 115, 1966. Root, A. W., Rosenfield, R. L., and Bongiovanni, A. M., and Eberlein, W. A.: Plasma growth hormone in disturbances of growth, J. PEDIAT. 69: 880, 1966 (Abst,). Greenwood, F. C., Hunter, W. M., and Glover, J. S.: The preparation of lSq-labeled human growth hormone of high specific radioactivity, Biochem. J. 89: 114, 1963. Frasier, S. D., and Smith, F. G,, Jr.: Antibodies to human growth hormone: Development during therapy with human growth hormone, Am. J. Dis. Child. 112: 383, 1966. Berson, S. A., Yalow, R. S., Bauman, A., Rothschild, M. A., and Newerly, K.: InsulinI131. metabolism in human subjects: Demonstration of insulln-blnding globulin in the circulation of insulin treated subjects, J. Clin. Invest. 35: 170, 1956. Yalow, R. S., and Berson, S. A.: Immunoassay of endogenous plasma insulin in man, J. Clin. Invest. 39: II57, 1960. Jackson, R. L., and Kelly, H. G.: Growth charts for use in pediatric practice, J. PEDIAT. 27: 215, 1945. Greulich, W. W., and Pyle, S. I.: Radiographic atlas of skeletal development of the hand and wrist, ed. 2, Stanford, Calif., 1959, Stanford University Press. Glenn, E. M., and Nelson, D, A.: Chemical method for the determination of 17-hydroxycorticosteroids and 17-ketosteroids in urine following hydrolysis with fl-glucuronldase, J. Clin, Endocrinol. 13: 911, 1953. Migeon, C. J., Green, O. C., and Eckert, J. P.: Study of adrenocortical function in obesity, Metabolism t2: 718, 1963.

The ]ournal o[ Pediatrics November 1967

24. Steiker, D. D., Bongiovanni, A. M., Eberlein, W. R., and Leboeuf, G.: Adrenocortical and adrenocorticotropic function in children. J. P~DIAT. 59: 885, i961. 25. Richmond, L., Cbapell, J., and Cleveland, W. W.: Response of children to methopyrapone (Metapirone), J. PEDIAT. 64: 381, 1964. 26. Levin, L., and Tyndale, H. H.: The quantitative assay of "follicle stimulating" substances, Endocrinology 21: 619, 1937. 27. Albert, A.: Human urinary gonadotropins, In Pincus, G.: Recent progress in hormone research, New York, 1957, Academic Press, p. 227. 28. Huntley, C. C., Johnson, H. H., and Lyerly, A . D . : Asthma, short stature, and elevated 7 1A-Globulins, Am. J. Dis. Child. 109: 353, 1965. 29. Rubinstein, J. H., and Taybi, I-I.: Broad thumbs and toes and facial abnormalities: A possible mental retardation syndrome, Am. J. Dis. Child. 105: 588, I963. 30. Thannhauser, J. J.: Werner's syndrome (progeria of the adult) and Rothmund's syndrome: Two types of closely related heredofamilial atrophic dermatoses with juvenile cataracts and endocrine features. A critical study with 5 new cases, Anr~. Intern. Med. 23: 559, 1945. 31. Black, J.: Low-birth-weight dwarfism, Arch. Dis. Childhood 36: 633, 1961. 32. Warkany, J., Monroe, B. B., and Sutherland, B. S.: Intrauterine growth retardation, Am. J. Dis. Child. 102: 249, 1962. 33~ Russell, A.: A syndrome of intrauterine dwarfism recognizable at birth with craniofacial dysostosis, disproportionately short arms, and other anomalies (5 examples), Proc. Roy. Soc. Med, 47: 1040, 1954. 34. Seckel, H. P. G.: Bird-headed dwarfs: Studied in developmental anthropology including human proportions, Springfield, Ill., 1960, Charles C Thomas, Publisher. 35. Bliss, C. I.: The statistics of bioassay with special reference to the vitamins, New York, 1952, Academic Press, pp. 452-474. 36. Sheikholislam, B. M., Lebovitz, H. E., and Stempfel, R. S.: Growth-hormone secretion in hypothyroidism. 43rd meeting of the Endocrine Society, 1966 (Abst. 61). 37. Rimoln, D. L., Merimee, T, J., and McKusick. V. A.: Growth-hormone deficiency in man: An isolated recessively inherited defect, Science 152: 1635, 1966. 38. Snow, R., Sacks, M. O., and Cornblath, M.: Ketotic hypoglycemia in a Russell dwarf, J. PEDIAT. 69: 121, 1966. 39. Greenwood, F. C., Hunter, W, M., and Merrian, V. J.: Growth-hormone levels in children and adolescents, Brk. Med. J. h 25, 1964. 40. Hunter, W, M., and Rigal, W. M.: The diurnal pattern of plasma growth hormone concentration in children and adolescents, J. Endoerinot. 34: 147, 1966.