Twenty-four Hour Plasma GH, FSH and LH Profiles in Patients with Turner's Syndrome MARIA CORAZON R. VILLADOLID, KAZUE TAKANO, NAOMI HIZUKA, KUMIKO ASAKAWA, IZUMI SUKEGAWA, REIKO HORIKAWA AND KAZUO SHIZUME Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women's Medical College, Tokyo 162, Japan and Research Laboratory, the Foundation for Growth Science in Japan Abstract We studied the plasma GH profiles in 6 patients with Turner's syndrome and 6 normal girls of short stature by sampling every 20 min for 24 hours. We observed episodic secretion of GH in these subjects. The mean plasma 24 h GH level in patients with Turner's syndrome was 3.6 }1.4 (SD) ng/ml which was significantly lower than that of normal short girls (7.1 }2.2ng/ml, p<0.01). The GH secretion during both nighttime and daytime was decreased in the patients with Turner's syndrome, however the number of pulses did not differ significantly. There were no correlations between the mean plasma 24 h GH level on one hand and peak GH level obtained after GH provocative test and plasma somatomedin C on the other. Plasma FSH and LH levels were also measured in 4 patients with Turner's syndrome. Both levels were elevated and there observed no clear pulsatile secretion of FSH, but, some pulsatile secretion of LH was observed in two patients. These data indicate that patients with Turner's syndrome have decreased endogenous GH secretion, even though they show normal GH responses to GH provocative tests. Received June 16, 1987 Address all correspondence to: Dr. KAZUE TA- KANO, Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women's Medical College, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162, Japan Turner's syndrome is a genetic disorder with many physical abnormalities including short stature and gonadal dysgenesis. For treatment of short stature, anabolic steroid, low doses of estrogen, and/or hgh have been used (Almqvist et al., 1964; Hutchings et al., 1965; Tanner et al., 1971; Ross et al., 1983; Stahnke 1984). Recently, we reported the effective treatment of short stature in these patients with methionyl hgh (Takano et al., 1986). The mechanism of the effect of hgh treatment in these patients is not clear at this moment. Therefore, in this study, we investigated the 24 h plasma GH profile together with FSH and LH in patients with Turner's syndrome and compared them to those in normal children of short stature.
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VILLADOLID et al. Materials and Methods Subjects Six children with Turner's syndrome and six normal girls of short stature, aged 8 to 14, who came to our hospital for the evaluation of their short stature, were investigated. Tables 1 and 2 list the individual data from clinical and laboratory examinations. They were all prepubertal, in Tanner stage I. The diagnosis of Turner's syndrome was established by sex chromosome analysis. Two patients (Nos. 3 and 6) had previously been treated with methionyl human growth hormone for 6-12 months, but the treatment had been switched to anabolic steroid. Five patients were under treatment with small amounts of anabolic steroid (Stanozolol: Winstrol(R) 0.03 mg/kg) for 3 to 20 months, but this treatment was discontinued one week prior to the study. The height of the normal six girls was below 2SD of the mean height of Japanese girls in the same age group. They showed normal plasma GH responses to GH provocative tests and no physical or laboratory abnormalities other than short stature. We temporarily classified these children as normal short children. All twelve children had normal thyroid function and showed normal GH responses to GH provocative tests (Table 2). Test protocol Assay methods Twenty-four hour plasma GH levels were determined using a double-antibody RIA kindly provided by the National Hormone and Pituitary Program of the NIADDK, NIH, (hgh: AFP-4793B;anti-hGH antiserum: AFP-97720133). The minimal detectable level was 0.5ng/ml. Intra- and inter-assay coefficients of variation for GH were 6.6% and 8.0%, respectively. Twentyfour hour secretory FSH and LH levels were also measured in four of the Turner's patients with commercially available RIA kits. Minimal detectable levels were both 1.9mU/ml. Intraand inter-assay coefficients of variation for FSH were 6.3 and 3.9% and those for LH were 6.7% and 4.5%, respectively. Plasma somatomedin C was measured with a RIA kit (Nichols Institute, San Juan Capistrano, CA). The intra-assay coefficient of variation was 4.1%. All 72 samples from each child obtained over a 24h period were assayed at one time. The following criteria were used in calculating GH, FSH and LH secretion: a) the mean plasma 24 h hormonal value was the mean value for all 72 samples collected over a period of 24 h. Data obtained during the day (8:00 AM to 8:00 PM) and night (8:00PM to 8:00AM) were analyzed separately. b) The criteria for the pulse secretion were defined if the plasma hormonal level was twice as high as the nadir value and, in the case of GH secretion, with an absolute value greater than 2ng/ml. Bone age was estimated according to the standards of Greulich and Pyle (1959). Student's Proper consent was obtained from both patients and their parents. The experimental protocol was approved by the Human Subject Investigations Committee of our department. The patients were assigned to a quiet room with no restrictions on food, activity or leisure. A gauge 19 indwelling butterfly needle was inserted in a forearm vein connected to a non-thrombogenic, heparinized catheter which, in turn, was attached to a small portable withdrawal pump (Cormed Model ML-6-5H) (Kowarski et al., 1971). This pump was adjusted by a flowmeter to extract blood at a constant rate of 6ml/hour. About 2ml of heparinized blood was taken every 20 minutes for a period of 24 h and, at the end, 2ml EDTA plasma was taken to measure plasma somatomedin C. The blood samples were immediately chilled on ice and centrifuged. Plasma was then separated and stored at-20 Ž until the time of the assay. Results Figs. 1 and 2 show plasma GH profiles in patients with Turner's syndrome and normal short children, respectively. Table 3 shows the analyzed data from these GH secretory profiles. The mean plasma GH levels in the six patients with Turner's syndrome ranged from 1.2 to 4.6 ng/ml with a mean of 3.6 }1.4 (SD)ng/ml, which was significantly lower than that of normal short children (7.1 }2.2ng/ml, p<0.01). Mean plasma GH levels during nighttime (2000-0800) were greater than those observed dur-
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VILLADOLID et al. No4. 13.6Y No2. 11.7Y No5. 13.9Y No3. 12.8Y No6. 14.4Y CLOCK TIME Fig. 1. Plasma GH secretory profiles of 6 patients with Turner's syndrome sampled every 20 min for 24 h. Numbers on the figure are the same as those in Table 1. Hatched columns during sleep indicate time of drowsiness.
24H GH, FSH, LH PROFILES IN TURNER GIRL No 7, 8.8Y No 10. 11,5Y No 8, 9.8Y No 11, 12.9Y No 9, 11.0Y No 12, 13.5Y CLOCK TIME Fig. 2. Plasma GH secretory profiles of 6 normal girls of short stature sampled every 20 min for 24 h. Numbers on the figure are the same as those in Table 1. Hatched columns during sleep indicate time of drowsiness.
VILLADOLID et al. Endocrinol. Japon. February 1988
Vol. 35, No. 1 24H GH, FSH, LH PROFILES IN TURNER GIRL ing daytime (0800-2000), but these values were also significantly lower in patients with Turner's syndrome than those in normal short children (p <0.05). The number of pulses of GH secretion during both daytime and nighttime did not differ between these two groups of children. The maximum peak GH level in daytime was significantly decreased in patients with Turner's syndrome (p < 0.01). There were no correlations between mean plasma 24h GH levels and peak plasma GH levels obtained after insulin induced hypoglycemia. We studied 24 h plasma FSH and LH profiles in 4 of the patients with Turner's syndrome (Fig. 3, Table 4). The mean 24 h plasma FSH levels in 4 patients ranged from 64.7 to 144.8 miu/ml with a mean of 89.1 miu/ml. There were no clearly pulsatile increases, although there were small levels during daytime and nighttime did not differ significantly in any of these patients. The mean 24 h plasma LH levels ranged from 56.9 to 84.3 miu/ml with a mean of 67.1 }6.2 miu/ml. The mean plasma LH levels during daytime and nighttime did not differ significanly in three of them, but a significantly increased LH secretion during nigthttime was observed in one patient (No. 2). Pulsatile LH secretion was observed three times in one patient (No. 2) and once in another (No. 5) during nighttime. Plasma somatomedin C in patients with Turner's syndrome varied between 0.65 and 1.60 U/ml with a mean of 1.17 } 0.40 U/ml, which did not differ from that obtained from normal short children. There were no correlations between plasma somatomedin C and mean plasma 24 h GH levels. Discussion We investigated daily profiles of plasma GH, FSH and LH in patients with Turner's syndrome. We observed episodic secretion
VILLADOLID et al. of GH in these patients, but the total amount of GH secreted throughout both day and night was decreased below that in normal short children. The pulse frequencies in patients with Turner's syndrome during 24h did not differ from those of normal short girls, but the peak amplitude was lower. There was no correlation between the mean 24h plasma GH level and peak plasma GH levels obtained after GH provocative tests. Ross et al. (1985) also reported decreased endogenous GH secretion in patients with Turner's syndrome as compared with that in normal subjects. The GH responsiveness to GH provocative tests in patients with Turner's syndrome varied. Some had normal and others subnormal responses to GH provocative tests (Donaldson 1968, Laczi et al., 1979, Takano et al., 1987). However, the GH responsiveness to provocative tests did not reflect the amount of endogenous GH secretion (Bercu et al., 1986). As shown in this study, all patients showed normal GH responses to GH provocative tests, but the total 24h endogenous GH secretion was decreased. The cause of decreased GH secretion may be due to numerous factors. The absence of ovarian steroid together with increased plasma FSH and LH may play a role in this phenomenon. The importance of estrogen in GH secretion, both in 24h GH profiles and GH responsiveness to provocative tests has been reported (Sperling et al., 1970, Gourmelen et al., 1979, Ho et al., 1987). GH neurosecretory dysfunctions, as proposed by Spiliotis et al. (1984) also participate in this interaction. The genetic abnormality of the second sex chromosome might help to explain this phenomenon. Further study will be required to find the reason for the decreased endogenous GH secretion in Turner's syndrome. Two patients with Turner's syndrome (Nos. 3 and 6) had been previously treated with methionyl human growth hormone (mhgh) for 6 and 12 months, respectively. Their height increased from 5.1 to 5.8cm/ year and 2.0 to 3.7cm/year, respectively, in spite of the low doses of m-hgh administered (0.3 IU/kg/week and 0.4 IU/kg/week, respectively). We have reported that 80% of patients with Turner's syndrome showed growth rate increases with hgh treatment, but the rest did not (Takano et al., 1986). The patients with decreased endogenous GH secretion might be those which show good growth response to hgh treatment. Usually there is a pulsatile FSH and LH secretion in children of pubertal age (Marshall and Kelch, 1986). It is well known that plasma FSH and LH levels are elevated in patients with primary hypogonadism due to a feedback mechanism. In this study, we also observed increased plasma FSH and LH concentrations in our patients with Turner's syndrome (normal value:fsh; 2-12 miu/ml, LH;2-20 miu/ml). There was no pulsatile plasma FSH secretion, but some pulsatile plasma LH secretion was observed. The physiological importance of this pulsatile pattern is not yet clear. Acknowledgements This work was partially supported by Grants in Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (No. 61440052, No. 61570566 and No. 61770872), a Research Grant from the Intractable Disease Division, Public Health Bureau, Ministry of Health and Welfare, and a Research Grant from the Foundation for Growth Science in Japan. References Almqvist, S., K. Hall, S. Lindstedt, J. Lindsten, R. Luft and H. Sjoberg (1964). Effects of short term administration of physiological doses of human growth hormone in three patients with Turner's syndrome. Acta Endocrinol. (Copenh) 46, 451-464. Bercu, B. B., D. Shulman, A. W. Root and B. E. Spiliotis (1986). Growth hormone (GH)
24H GH, FSH, LH PROFILES IN TURNER GIRL provocative testing frequently does not reflect endogenous GH secretion. J. Clin. Endocrinol. Metab. 63, 709-716. Donaldson, C. L., L. C. Wegienka, D. Miller and P. H. Forsham (1968). Growth hormone studies in Turner's syndrome. J. Clin. Endocrinol. Metab. 28, 383-386. Gourmelen, M., M. T. Pham-Huu-Trung and F. Girard (1979). Transient partial hgh deficiency in prepubertal children with delay of growth. Pediatr. Res. 13, 221-224. Greulich, W. W. and Pyle, I. S. (1959). Radiographic Atlas of Skeletal Development of the Hand and Wrist, 2ed. Stanford University Press, Stanford. Ho, K. Y., W. S. Evans, R. M. Blizzard et al. (1987). Effects of sex and age on the 24-hour profile of growth hormone secretion in man: Importance of endogenous estradiol concentrations. J. Clin. Endocrinol. Metab. 64, 51-58. Hutchings, J., R. Escamilla, C. Li and P. Forsham (1965). Li human growth hormone administration in gonadal dysgenesis. Amer. J. Dis Child 109, 318-321. Kowarski, A., R. G. Thompson, C. J. Migeon and R. M. Blizzard (1971). Determination of integrated plasma concentrations and true secretion rates of human growth hormone. J. Clin. Endocrinol. Metab. 32, 356-360. Laczi, F., J. Julesz, T. Janaky and F. A. Laszlo (1979). Growth hormone reserve capacity in Turner's syndrome. Horm. Metab. Res. 11, 664-666. Marshall. J. C. and R. P. Kelch (1986). Gonadotropin-releasing hormone: role of pulsatile secretion in the regulation of reproduction. N. Engl. J. Med. 315, 1459-1468. Sperling, M. A., F. M. Kenny and A. L. Drash (1970). Arginine-induced growth hormone responses in children: Effect of age and puberty. J. Pediatr. 77, 462-465. Spiliotis, B. E., G. P. August, W. Hung, W. Sonis, W. Mendelson and B. B. Bercu (1984). Growth hormone neurosecretory dysfunctions. J. A. M. A. 251, 2223-2230. Stahnke, N.(1984). Human growth hormone treatment in short children without growth hormone deficiency. N. Engl. J. Med. 310, 925-926. Takano, K., N. Hizuka and K. Shizume (1986). Treatment of Turner's syndrome with methionyl human growth hormone for six months. Acta Endocrinol. (Copenh) 112, 130-137. Takano, K., K. Shizume, H. Imura, M. Irie, Y. Kumahara, N. Shimizu and the members of study committee for hgrf-44 (SM-8144) (1987). Plasma growth hormone (GH) response to GH-releasing factor (SM-8144) in children of short stature and patients with GH deficiency. Endocrinol. Japon 34, 117-128. Tanner, J., R. Whitehouse, P. Hughes and F. Vince (1971). Effect of human growth hormone treatment for 1 to 7 years on growth of 100 children with growth hormone deficiency, low birth weight, inherited smallness, Turner's syndrome and other complaints. Arch. Dis. Child 46, 745-782. Ross, J. L., F. G. Cassorla, M. C. Skerda, I. M. Valk, D. L. Loriaux and G. B. Cutler (1983). A preliminary study of the effect of estrogen dose on growth in Turner's syndrome. N. Engl. J. Med. 309, 1104-1106. Ross, J. L., L. M. Long, L. D. Loriaux and G. B. Cutler (1985). Growth hormone secretory dynamics in Turner syndrhme. J. Pediatr. 106, 202-206.