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Luteinizing Hormone (LH), Follicle-Stimulating Hormone, and Testosterone Responses to Consecutive Injections of D-Leucine-6-LH-Releasing Hormone Ethylamide in Normal Men
Vol. 28, No.4, April 1977 Printed in U.S.A.
FERTILITY AND STERILITY Copyright © 1977 The American Fertility Society
LUTEINIZING HORMONE (LH), FOLLICLE-STIMULATING HORMONE, AND TESTOSTERONE RESPONSES TO CONSECUTIVE INJECTIONS OF D-LEUCINE-6-LHRELEASING HORMONE ETHYLAMIDE IN NORMAL MEN
LUIS SCHWARZSTEIN, M.D.* NESTOR J. APARICIO, M.D.*t DIEGO TURNER, PH.D.* ELDA A. DE TURNER, PH.D.* DAVID H. COY, PH.D.:!: ANDREW V. SCHALLY, PH.D.:j:
Grupo de Estudio en Fertilidad y Endocrinologia de Rosario, Rosario, Argentina, and Veterans Administration Hospital and Tulane University School of Medicine, New Orleans, Louisiana 70112
Luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T) responses to three consecutive intravenous injections of D-Leu-6-LH-releasing hormone ethylamide (D-Leu-6-LH-RH-EA) at 3-hour intervals were studied in six healthy, fertile, male volunteers 34.2 ± 1.6 years ofage. Each man received three injections of 20 M ofD-Leu-6-LH-RH-EA at 6:00 A.M., 9:00 A.M., and 12:00 noon, respectively. Blood samples were obtained before the first injection and at 1, 2, 3 (before the second injection), 4,-5, 6 (before the third injection), 7,8, and 9 hours after the beginning of the test. Serum levels ofLH, FSH, and T were determined by radioimmunoassay with the double-antibody technique. The response to the first injection ofD-Leu-6-LH-RH-EA confirmed the longer duration of the stimulation ofLH and FSH release caused by this compound as compared with that caused by LH-RH. SErum T levels rose significantly, almost paralleling the variations experienced with gonadotropins. The second injection caused a slight increase in LH and T responses in relation to the first injection. Two and three hours after administration, the third stimulus resulted in hormone levels lower than those obtained with the first two injections. Possible explanations for this finding might be a reduction of pituitary responsiveness as a result of multiple stimulation with D-Leu-6-LH-RH-EA, or spontaneous circadian variation of the pituitary response, or a combination of factors.
After synthetic luteinizing hormone-releasing hormone (LH-RH) became available for clinical use,l several questions arose which are still unanswered. Two of them are focused upon in this paper, namely whether there is or is not a gonadal steroid response concomitant with the pituitary response to LH-RH and whether repeated acute or
chronic stimulation exhausts pituitary responsiveness. With regard to the first problem, some authors have failed to disclose a steroid response or have found a highly variable, erratic response,2 while others have found gonadal steroid responses almost parallel with pituitary responses in both normal men and normal women. 3 · 5 Concerning the possibility that repeated LH-RH stimulation might result in pituitary exhaustion, various studies have indicated that if such an effect existed, it would last less than 90 minutes,6 and that, moreover, consecutive LH-RH injections at 90-minute intervals would result in a progressive and significant increase in the pituitary re-
Accepted November 26, 1976. * Grupo de Estudio en Fertilidad y Endocrinologia de Rosario. tReprint requests: Dr. Nestor J. Aparicio, Manuel Ugarte 2170, BUenos Aires, Argentina. :j:Veterans Administration Hospital and Tulane University School of Medicine.
451
452
SCHWARZSTEIN ET AL.
sponse. 7 In some cases of hypogonadotropic hypogonadism, daily administration of LH-RH for a long period would restore impaired capacity of the pituitary gonadotropin response. 8 Various analogs of LH-RH have been developed recently.9 One of them, D-Leu-6-LH-RH-ethylamide (D-Leu6-LH-RH-EA), causes greater and more sustained LH and follicle-stimulating hormone (FSH) release than does LH-RH.9 Since this compound is now available for clinical trials, an approach to the problems mentioned above was considered worth attempting. On this basis, this study was programmed in order to investigate LH, FSH, and testosterone (T) responses to three consecutive injections of D-Leu-6-LH-RH-EA given at 1BOminute intervals. MATERIALS AND METHODS
The subjects were six normal male volunteers ages 34.2 years ± 1.6 SE, weighing 76.9 kg ± 5.2 SE, and measuring 175.0 cm ± 6.4 SE in height. All had a negative history of endocrine disease and none was under drug therapy of any kind. Preliminary sperm counts were above 40 x 106 sperm/ml with normal sperm vitality and motility parameters. Each man had fathered at least one child. Each subject received three intravenous injections of 20 ILg of D-Leu-6-LH-RH-EA dissolved in 2 ml of 0.9% saline at lBO-minute intervals, starting at 6:00 A.M. All six subjects went through the study simultaneously, following the
April 1977
usual overnight rest. Physical activity was kept to a moderate degree and food intake remained as usual. Blood samples were obtained from an antecubital vein before the first injection of DLeu-6-LH-RH-EA (basal sample) and 1, 2, 3, 4, 5, 6, 7, B, and 9 hours later. The second and third injections of the analog were given after the 3- and 6-hour blood samples were obtained. Serum was extracted from each sample after clot retraction and kept frozen at - 20" C until hormones were assayed. LH, FSH, and testosterone levels were determined in triplicate by the double-antibody radioimmunoassay method, the specifications of which are reported elsewhere. 4 LH and FSH results were expressed in milli-international units per milliliter of serum in terms of Second International Reference Preparation of Human Menopausal Gonadotropin (2nd IRPHMG); testosterone levels were expressed in nanograms per milliliter. The results of comparing hormone levels after each stimulus with the respective basal values and with one another were analyzed with the ttest for paired samples. Regression analysis was used to correlate LH to testosterone and LH to FSH levels. RESULTS
Table 1 shows the serum levels ofLH, FSH, and testosterone obtained in the six volunteers under basal conditions and after each of the three injections of D-Leu-6-LH-RH-EA. Figure 1 shows
TABLE 1. Individual Levels of LB, FSB, and Testosterone in Six Normal Men under Basal Conditions and after Administration of Three Intravenous Injections ofD-Leu-6-LH-RB-EA at 6:00 A.M., 9:00 A.M., and 12:00 Noon Hormone and subject
LH (mIU/ml) Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 Subject 6 FSH (mlU/ml) Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 Subject 6 Testosterone (ng/ml) Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 Subject 6
Time after first injection
Basal (6 A.M.)
1hr (7 A.M.)
2hr (SA.M.)
3hr (9 A.M.)
4hr (10 A.M.)
5hr (11 A.M.)
6hr (12 noon)
7hr (1 P.M.)
8hr (2 P.M.)
9hr (3 P.M.)
5.5 6.2 5.9 5.4 9.1 4.8
32.0 34.0 22.4 39.0 38.0 21.0
87.0 74.0 61.0 52.0 37.0 46.0
56.0 81.0 54.0 56.0 54.0 72.0
69.0 89.0 95.0 89.0 86.0 63.0
84.0 64.0 83.0 91.0 89.0 69.0
80.0 56.0 42.0 82.0 61.0 61.0
95.0 68.0 59.0 87.0 67.0 76.0
37.0 40.0 36.0 86.0 46.0 72.0
26.0 25.0 38.0 43.0 44.0 60.0
6.8 4.9 7.2 9.4 6.2 5.1
24.0 39.0 15.9 21.0 21.0 11.9
77.0 57.0 31.0 28.0 29.0 28.0
41.0 42.0 32.0 27.0 18.0 25.0
29.0 64.0 48.0 29.0 24.0 43.0
58.0 56.0 45.0 36.0 32.0 49.0
47.0 49.0 40.0 37.0 39.0 29.0
64.0 61.0 39.0 30.0 48.0 36.0
39.0 39.0 30.0 36.0 31.0 18.0
28.0 38.0 31.0 30.0 18.0 15.0
6.5 5.8 7.4 5.2 6.7 5.9
14.0 15.0 15.9 15.1 12.8 9.9
16.0 17.0 21.0 14.9 14.1 10.9
14.0 14.9 22.0 16.5 9.4 11.8
18.0 21.0 27.0 17.2 17.2 13.5
15.0 20.0 20.0 13.0 18.0 15.2
14.0 18.0 14.5 12.0 21.0 14.1
25.0 24.0 17.0 19.0 27.0 14.7
17.0 19.0 13.0 14.0 15.0 14.9
16.0 14.0 14.5 8.0 14.0 15.8
LH, FSH, AND T RESPONSES TO D-LEU-6-LH-RH-EA
Vol. 28, No.4
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FIG. 1. Serum levels (means ± standard error) ofLH, FSH, and testosterone obtained in six normal men under basal conditions and after administration of three intravenous injections of20 ILg ofn-Leu-6-LH-RH-EA at 3-hour intervals. Injection times are shown.
the profiles of the three hormones. There was a sustained increase in LH after the first injection, reaching maximal average levels at 3 hours. The second injections resulted in an even greater and more significant increase (P < 0.05 for 3- versus 4-hour levels) 1 hour after administration, with a subsequent decrease until the 6th hour of the test. The values found 1 hour after the first injection did not reach the level attained after the second injection, nor did they differ significantly from the peak values that had followed the first injection. Serum LH levels decreased substantially at 8 and 9 hours (i.e., 2 and 3 hours after the third injection of D-Leu-6-LH-RH-EA, respectively). The average LH level at 9 hours was significantly lower than that observed at 3 hours (P <
453
0.05). Poststimulation LH levels at all times of the test were significantly higher than the basal values (P < 0.05). The levels attained at 4 and 7 hours (i.e., 1 hour after the second and third injections of the analog, respectively) were significantly higher (P < 0.01) than those found 1 hour after the first injection. There was also a progressive increase in FSH after the first injection, with maximal levels at 2 hours followed by a moderate decrease. A new rise was the result of the second injection: FSH levels at 5 hours equaled those observed after the first injection; a slight decrease in FSH levels occurred at 6 hours. One hour after administration of the third injection (i.e., at 7 hours), FSH values showed another slight rise followed by a marked drop. All poststimulation FSH values were significantly higher than basal levels (P < 0.05). The figures obtained 1 hour after the first injection were significantly lower than those observed 1 hour after the second and third injections of DLeu-6-LH-RH-EA (P < 0.02 andP < 0.01, respectively). The FSH levels at 6 hours (i.e., 3 hours after the second injection) were statistically higher than those obtained at 3 and 9 hours (i.e., 3 hours after the first and third injections, respectively [P < 0.02 and P < 0.01]). The testosterone results showed a profile quite similar to that of LH. Following the first injection of D-Leu-6-LH-RH-EA, testosterone levels rose to peak average values between the 2nd and 3rd hours of the test. The second injection caused an even greater and more significant elevation (P < 0.005, 2-hour versus 4-hour values) 1 hour after administration, followed by a drop until 6 hours. A new rise unsued 1 hour after the third injection (P < 0.005 in relation to the 2nd hour), with a marked fall toward 8 and 9 hours. Poststimulation testosterone levels were significantly above base line values throughout the full test period (P < 0.05). LH versus testosterone and LH versus FSH correlated positively and significantly (r = 0.64 and r = 0.67, respectively; difference from 0: P < 0.01 in both cases). No untoward effects were observed in any of the volunteers. DISCUSSION
With the methods applied, the basal LH, FSH, and testosterone levels observed in this study fell within the normal range for males (LH, 3.5 to 15.4 mIU/ml; FSH, 3.5 to 12.5 mIU/ml; testoster-
454
SCHWARZSTEIN ET AL.
one, 3.7 to 8.0 ng/ml). Intra- and interassay variations were as follows: LH, 5.7% and 13.7%; FSH, 7.9% and 16.3%; testosterone, 5.3% and 14.8%. The first injection of n-Leu-6-LH-RH-EA elicited a progressive LH elevation which became maximal at 3 hours, together with a slightly smaller rise in FSH and a frank rise in testosterone levels, which were highest at 2 hours and fell slightly at 3 hours. These results confirm the longer duration of the effect ofn-Leu-6-LH-RH-EA as compared with that of LH-RH9; moreover, they suggest that, even with this more potent analog, stimulation of LH release is greater than that of FSH release. The systematic rise in testosterone levels in all patients (well above variations attributable to hazard or to eventual variations in the radioimmunoassay method) confirms that the analog under study is capable of causing significant and prompt stimulation of gonadal steroid secretion almost paralleling LH rises, by way of pituitary stimuli, as was previously shown for LH-RH.3-S The significant correlation observed between LH and testosterone levels throughout the whole test might be considered a factor in support of a cause-effect relationship between gonadotropic stimulation and gonadal steroid response. The second injection of n-Leu-6-LH-RH-EA resulted in a new rise in LH values to levels even higher than those reached after the first injection, with concomitant rises in testosterone levels and less marked FSH rises. This pattern following the second injection of the analog is similar to that obtained with consecutive LH-RH injections every 90 minutes, 7 except for the differences in duration of action already discussed. 9 The response that followed the third injection of n-Leu-6-LH-RH-EA was characterized by an increase in hormone levels which, 1 hour after administration, did not exceed the levels obtained after the second injection and which was followed by a decrease in all three hormone levels to values significantly lower than those obtained 3 hours after the first and second stimuli were applied. These data suggest that pituitary responsiveness may remain unimpaired after twice-repeated stimulation with n-Leu-6-LH-RH-EA, at least at the doses and intervals used herein. The second injection, on the contrary, might determine a higher hormone response. The values obtained after the third injection of the analog, instead, would point to a reduced response, particularly regarding duration. An explanation for this finding might lie in pituitary exhaustion by consecutive
April 1977
stimuli. However, the period of action of the third injection of the analog extended from 12:00 noon to 3:00 P.M., at which time there seemed to be a physiologic reduction of the pituitary gonadotropic response to LH-RH.4 The lower hormone levels found 2 and 3 hours after administration of the third stimulus, in relation to the first two injections, might be explained as a circadian variation in pituitary response, rather than as a result of exhausted pituitary responsiveness caused by n-Leu-6-LH-RH-EA. In conclusion: (1) The response to the first injection ofn-Leu-6-LH-RH-EA confirmed the longer duration of gonadotropic stimulation determined by this analog as compared with that of LH-RH. (2) The possibility was confirmed that n-Leu-6-LH-RH-EA may stimulate testosterone secretion in a. way almost parallel with variations in serum gonadotropin levels. (3) A second injection of the analog 180 minutes later was not attended by reduced pituitary LH and FSH responses; rather, an increasing trend was seemingly the case. (4) Two and three hours after the third injection of the analog, hormone levels were lower than those obtained after the first and, particularly, the second stimulus. (5) The last finding might result from exhausted pituitary responsiveness subsequent to multiple stimuli, from spontaneous circadian variation in the pituitary response, or from a combinatio~ of factors. Acknowledgments. We gratefully acknowledge the provision of LH, FSH, and testosterone standards and antisera by Serono Immunochemicals (Rome, Italy) and the secretarial aid of Mrs. Ana Maria Mastrimgelo.
REFERENCES 1. Schally AV, Kastin AJ, Arimura A: The hypothalamus and reproduction. Am J Obstet GynecoI1l4:423, 1972
2. Jewelewicz R, Dyrenfurth I, Warren M, Vande Wiele RL: Clinical studies with gonadotropin-releasing hormone. Bull NY Acad Med 50:1097,1974 3. Arai K, Yanaihara T, Okinaga S: Response of ovarian steroid secretion to the intrinsic gonadotropin release caused by the administration of synthetic luteinizing hormone-releasing hormone. Am J Obstet Gynecol 123: 804, 1975 4. Schwarzstein L, de Laborde NP, Aparicio NJ, Turner D, Mirkin A, Rodriguez A, Rodriguez Lhullier F, Rosner JM: Daily variations of FSH, LH and testosterone response to intravenous luteinizing hormone-releasing factor (LRF) in normal men. J Clin Endocrinol Metab 40:313, 1975 5. Figueroa Casas PR, de Laborde NP, Badano A, Pedroza Garcia E, Aparicio N, Miechi H, Mirkin A, Arcangeli 0, Rosner JM: Ovarian response to luteinizing hormonereleasing hormone (LH-RH) in normal women and amenorrheic patients. Reproduction 2:1,1975
Vol. 28, No.4
LH, FSH, AND T RESPONSES TO D-LEU-6-LH-RH-EA
6. ROmmler A, Baumgarten S, Hammerstein J: Hormonal response to multiple stimulation with synthetic luteinizing hormone-releasing hormone (LH-RH) in amenorrheic women (abstr). Acta Endocrinol [Suppl] (Kbh) 173: 87, 1973 7. Figueroa Casas PR, Badano A, Mirkin A, Miechi H, Aparicio NJ: Gonadotropin response and induction of ovulation in long-standing secondary amenorrhea treated with clomiphene citrate and repeated injection of hypothalamic gonadotrophin-releasing hormone (LH-RH). Reproduccion. In press
455
8. Yoshimoto Y, Moridera K, Imura H: Restoration of normal pituitary gonadotropin reserve by administration of luteinizing hormone-releasing hormone in patients with hypogonadotropic hypogonadism. N Engl J Med 292:242, 1975 9. Vilchez-Martinez JA, Coy DH, Arimura A, Coy EJ, Hirotsu Y, Schally AV: Synthesis and biological properties of (Leu-6)-LH-RH and (D-Leu-6, desGly-NH210-LHRH-ethylamide). Biochem Biophys Res Commun 59: 1226, 1974
FERTILITY AND STERILITY Copyright © 1977 The American Fertility Society
LUTEINIZING HORMONE (LH), FOLLICLE-STIMULATING HORMONE, AND TESTOSTERONE RESPONSES TO CONSECUTIVE INJECTIONS OF D-LEUCINE-6-LHRELEASING HORMONE ETHYLAMIDE IN NORMAL MEN
LUIS SCHWARZSTEIN, M.D.* NESTOR J. APARICIO, M.D.*t DIEGO TURNER, PH.D.* ELDA A. DE TURNER, PH.D.* DAVID H. COY, PH.D.:!: ANDREW V. SCHALLY, PH.D.:j:
Grupo de Estudio en Fertilidad y Endocrinologia de Rosario, Rosario, Argentina, and Veterans Administration Hospital and Tulane University School of Medicine, New Orleans, Louisiana 70112
Luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T) responses to three consecutive intravenous injections of D-Leu-6-LH-releasing hormone ethylamide (D-Leu-6-LH-RH-EA) at 3-hour intervals were studied in six healthy, fertile, male volunteers 34.2 ± 1.6 years ofage. Each man received three injections of 20 M ofD-Leu-6-LH-RH-EA at 6:00 A.M., 9:00 A.M., and 12:00 noon, respectively. Blood samples were obtained before the first injection and at 1, 2, 3 (before the second injection), 4,-5, 6 (before the third injection), 7,8, and 9 hours after the beginning of the test. Serum levels ofLH, FSH, and T were determined by radioimmunoassay with the double-antibody technique. The response to the first injection ofD-Leu-6-LH-RH-EA confirmed the longer duration of the stimulation ofLH and FSH release caused by this compound as compared with that caused by LH-RH. SErum T levels rose significantly, almost paralleling the variations experienced with gonadotropins. The second injection caused a slight increase in LH and T responses in relation to the first injection. Two and three hours after administration, the third stimulus resulted in hormone levels lower than those obtained with the first two injections. Possible explanations for this finding might be a reduction of pituitary responsiveness as a result of multiple stimulation with D-Leu-6-LH-RH-EA, or spontaneous circadian variation of the pituitary response, or a combination of factors.
After synthetic luteinizing hormone-releasing hormone (LH-RH) became available for clinical use,l several questions arose which are still unanswered. Two of them are focused upon in this paper, namely whether there is or is not a gonadal steroid response concomitant with the pituitary response to LH-RH and whether repeated acute or
chronic stimulation exhausts pituitary responsiveness. With regard to the first problem, some authors have failed to disclose a steroid response or have found a highly variable, erratic response,2 while others have found gonadal steroid responses almost parallel with pituitary responses in both normal men and normal women. 3 · 5 Concerning the possibility that repeated LH-RH stimulation might result in pituitary exhaustion, various studies have indicated that if such an effect existed, it would last less than 90 minutes,6 and that, moreover, consecutive LH-RH injections at 90-minute intervals would result in a progressive and significant increase in the pituitary re-
Accepted November 26, 1976. * Grupo de Estudio en Fertilidad y Endocrinologia de Rosario. tReprint requests: Dr. Nestor J. Aparicio, Manuel Ugarte 2170, BUenos Aires, Argentina. :j:Veterans Administration Hospital and Tulane University School of Medicine.
451
452
SCHWARZSTEIN ET AL.
sponse. 7 In some cases of hypogonadotropic hypogonadism, daily administration of LH-RH for a long period would restore impaired capacity of the pituitary gonadotropin response. 8 Various analogs of LH-RH have been developed recently.9 One of them, D-Leu-6-LH-RH-ethylamide (D-Leu6-LH-RH-EA), causes greater and more sustained LH and follicle-stimulating hormone (FSH) release than does LH-RH.9 Since this compound is now available for clinical trials, an approach to the problems mentioned above was considered worth attempting. On this basis, this study was programmed in order to investigate LH, FSH, and testosterone (T) responses to three consecutive injections of D-Leu-6-LH-RH-EA given at 1BOminute intervals. MATERIALS AND METHODS
The subjects were six normal male volunteers ages 34.2 years ± 1.6 SE, weighing 76.9 kg ± 5.2 SE, and measuring 175.0 cm ± 6.4 SE in height. All had a negative history of endocrine disease and none was under drug therapy of any kind. Preliminary sperm counts were above 40 x 106 sperm/ml with normal sperm vitality and motility parameters. Each man had fathered at least one child. Each subject received three intravenous injections of 20 ILg of D-Leu-6-LH-RH-EA dissolved in 2 ml of 0.9% saline at lBO-minute intervals, starting at 6:00 A.M. All six subjects went through the study simultaneously, following the
April 1977
usual overnight rest. Physical activity was kept to a moderate degree and food intake remained as usual. Blood samples were obtained from an antecubital vein before the first injection of DLeu-6-LH-RH-EA (basal sample) and 1, 2, 3, 4, 5, 6, 7, B, and 9 hours later. The second and third injections of the analog were given after the 3- and 6-hour blood samples were obtained. Serum was extracted from each sample after clot retraction and kept frozen at - 20" C until hormones were assayed. LH, FSH, and testosterone levels were determined in triplicate by the double-antibody radioimmunoassay method, the specifications of which are reported elsewhere. 4 LH and FSH results were expressed in milli-international units per milliliter of serum in terms of Second International Reference Preparation of Human Menopausal Gonadotropin (2nd IRPHMG); testosterone levels were expressed in nanograms per milliliter. The results of comparing hormone levels after each stimulus with the respective basal values and with one another were analyzed with the ttest for paired samples. Regression analysis was used to correlate LH to testosterone and LH to FSH levels. RESULTS
Table 1 shows the serum levels ofLH, FSH, and testosterone obtained in the six volunteers under basal conditions and after each of the three injections of D-Leu-6-LH-RH-EA. Figure 1 shows
TABLE 1. Individual Levels of LB, FSB, and Testosterone in Six Normal Men under Basal Conditions and after Administration of Three Intravenous Injections ofD-Leu-6-LH-RB-EA at 6:00 A.M., 9:00 A.M., and 12:00 Noon Hormone and subject
LH (mIU/ml) Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 Subject 6 FSH (mlU/ml) Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 Subject 6 Testosterone (ng/ml) Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 Subject 6
Time after first injection
Basal (6 A.M.)
1hr (7 A.M.)
2hr (SA.M.)
3hr (9 A.M.)
4hr (10 A.M.)
5hr (11 A.M.)
6hr (12 noon)
7hr (1 P.M.)
8hr (2 P.M.)
9hr (3 P.M.)
5.5 6.2 5.9 5.4 9.1 4.8
32.0 34.0 22.4 39.0 38.0 21.0
87.0 74.0 61.0 52.0 37.0 46.0
56.0 81.0 54.0 56.0 54.0 72.0
69.0 89.0 95.0 89.0 86.0 63.0
84.0 64.0 83.0 91.0 89.0 69.0
80.0 56.0 42.0 82.0 61.0 61.0
95.0 68.0 59.0 87.0 67.0 76.0
37.0 40.0 36.0 86.0 46.0 72.0
26.0 25.0 38.0 43.0 44.0 60.0
6.8 4.9 7.2 9.4 6.2 5.1
24.0 39.0 15.9 21.0 21.0 11.9
77.0 57.0 31.0 28.0 29.0 28.0
41.0 42.0 32.0 27.0 18.0 25.0
29.0 64.0 48.0 29.0 24.0 43.0
58.0 56.0 45.0 36.0 32.0 49.0
47.0 49.0 40.0 37.0 39.0 29.0
64.0 61.0 39.0 30.0 48.0 36.0
39.0 39.0 30.0 36.0 31.0 18.0
28.0 38.0 31.0 30.0 18.0 15.0
6.5 5.8 7.4 5.2 6.7 5.9
14.0 15.0 15.9 15.1 12.8 9.9
16.0 17.0 21.0 14.9 14.1 10.9
14.0 14.9 22.0 16.5 9.4 11.8
18.0 21.0 27.0 17.2 17.2 13.5
15.0 20.0 20.0 13.0 18.0 15.2
14.0 18.0 14.5 12.0 21.0 14.1
25.0 24.0 17.0 19.0 27.0 14.7
17.0 19.0 13.0 14.0 15.0 14.9
16.0 14.0 14.5 8.0 14.0 15.8
LH, FSH, AND T RESPONSES TO D-LEU-6-LH-RH-EA
Vol. 28, No.4
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FIG. 1. Serum levels (means ± standard error) ofLH, FSH, and testosterone obtained in six normal men under basal conditions and after administration of three intravenous injections of20 ILg ofn-Leu-6-LH-RH-EA at 3-hour intervals. Injection times are shown.
the profiles of the three hormones. There was a sustained increase in LH after the first injection, reaching maximal average levels at 3 hours. The second injections resulted in an even greater and more significant increase (P < 0.05 for 3- versus 4-hour levels) 1 hour after administration, with a subsequent decrease until the 6th hour of the test. The values found 1 hour after the first injection did not reach the level attained after the second injection, nor did they differ significantly from the peak values that had followed the first injection. Serum LH levels decreased substantially at 8 and 9 hours (i.e., 2 and 3 hours after the third injection of D-Leu-6-LH-RH-EA, respectively). The average LH level at 9 hours was significantly lower than that observed at 3 hours (P <
453
0.05). Poststimulation LH levels at all times of the test were significantly higher than the basal values (P < 0.05). The levels attained at 4 and 7 hours (i.e., 1 hour after the second and third injections of the analog, respectively) were significantly higher (P < 0.01) than those found 1 hour after the first injection. There was also a progressive increase in FSH after the first injection, with maximal levels at 2 hours followed by a moderate decrease. A new rise was the result of the second injection: FSH levels at 5 hours equaled those observed after the first injection; a slight decrease in FSH levels occurred at 6 hours. One hour after administration of the third injection (i.e., at 7 hours), FSH values showed another slight rise followed by a marked drop. All poststimulation FSH values were significantly higher than basal levels (P < 0.05). The figures obtained 1 hour after the first injection were significantly lower than those observed 1 hour after the second and third injections of DLeu-6-LH-RH-EA (P < 0.02 andP < 0.01, respectively). The FSH levels at 6 hours (i.e., 3 hours after the second injection) were statistically higher than those obtained at 3 and 9 hours (i.e., 3 hours after the first and third injections, respectively [P < 0.02 and P < 0.01]). The testosterone results showed a profile quite similar to that of LH. Following the first injection of D-Leu-6-LH-RH-EA, testosterone levels rose to peak average values between the 2nd and 3rd hours of the test. The second injection caused an even greater and more significant elevation (P < 0.005, 2-hour versus 4-hour values) 1 hour after administration, followed by a drop until 6 hours. A new rise unsued 1 hour after the third injection (P < 0.005 in relation to the 2nd hour), with a marked fall toward 8 and 9 hours. Poststimulation testosterone levels were significantly above base line values throughout the full test period (P < 0.05). LH versus testosterone and LH versus FSH correlated positively and significantly (r = 0.64 and r = 0.67, respectively; difference from 0: P < 0.01 in both cases). No untoward effects were observed in any of the volunteers. DISCUSSION
With the methods applied, the basal LH, FSH, and testosterone levels observed in this study fell within the normal range for males (LH, 3.5 to 15.4 mIU/ml; FSH, 3.5 to 12.5 mIU/ml; testoster-
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SCHWARZSTEIN ET AL.
one, 3.7 to 8.0 ng/ml). Intra- and interassay variations were as follows: LH, 5.7% and 13.7%; FSH, 7.9% and 16.3%; testosterone, 5.3% and 14.8%. The first injection of n-Leu-6-LH-RH-EA elicited a progressive LH elevation which became maximal at 3 hours, together with a slightly smaller rise in FSH and a frank rise in testosterone levels, which were highest at 2 hours and fell slightly at 3 hours. These results confirm the longer duration of the effect ofn-Leu-6-LH-RH-EA as compared with that of LH-RH9; moreover, they suggest that, even with this more potent analog, stimulation of LH release is greater than that of FSH release. The systematic rise in testosterone levels in all patients (well above variations attributable to hazard or to eventual variations in the radioimmunoassay method) confirms that the analog under study is capable of causing significant and prompt stimulation of gonadal steroid secretion almost paralleling LH rises, by way of pituitary stimuli, as was previously shown for LH-RH.3-S The significant correlation observed between LH and testosterone levels throughout the whole test might be considered a factor in support of a cause-effect relationship between gonadotropic stimulation and gonadal steroid response. The second injection of n-Leu-6-LH-RH-EA resulted in a new rise in LH values to levels even higher than those reached after the first injection, with concomitant rises in testosterone levels and less marked FSH rises. This pattern following the second injection of the analog is similar to that obtained with consecutive LH-RH injections every 90 minutes, 7 except for the differences in duration of action already discussed. 9 The response that followed the third injection of n-Leu-6-LH-RH-EA was characterized by an increase in hormone levels which, 1 hour after administration, did not exceed the levels obtained after the second injection and which was followed by a decrease in all three hormone levels to values significantly lower than those obtained 3 hours after the first and second stimuli were applied. These data suggest that pituitary responsiveness may remain unimpaired after twice-repeated stimulation with n-Leu-6-LH-RH-EA, at least at the doses and intervals used herein. The second injection, on the contrary, might determine a higher hormone response. The values obtained after the third injection of the analog, instead, would point to a reduced response, particularly regarding duration. An explanation for this finding might lie in pituitary exhaustion by consecutive
April 1977
stimuli. However, the period of action of the third injection of the analog extended from 12:00 noon to 3:00 P.M., at which time there seemed to be a physiologic reduction of the pituitary gonadotropic response to LH-RH.4 The lower hormone levels found 2 and 3 hours after administration of the third stimulus, in relation to the first two injections, might be explained as a circadian variation in pituitary response, rather than as a result of exhausted pituitary responsiveness caused by n-Leu-6-LH-RH-EA. In conclusion: (1) The response to the first injection ofn-Leu-6-LH-RH-EA confirmed the longer duration of gonadotropic stimulation determined by this analog as compared with that of LH-RH. (2) The possibility was confirmed that n-Leu-6-LH-RH-EA may stimulate testosterone secretion in a. way almost parallel with variations in serum gonadotropin levels. (3) A second injection of the analog 180 minutes later was not attended by reduced pituitary LH and FSH responses; rather, an increasing trend was seemingly the case. (4) Two and three hours after the third injection of the analog, hormone levels were lower than those obtained after the first and, particularly, the second stimulus. (5) The last finding might result from exhausted pituitary responsiveness subsequent to multiple stimuli, from spontaneous circadian variation in the pituitary response, or from a combinatio~ of factors. Acknowledgments. We gratefully acknowledge the provision of LH, FSH, and testosterone standards and antisera by Serono Immunochemicals (Rome, Italy) and the secretarial aid of Mrs. Ana Maria Mastrimgelo.
REFERENCES 1. Schally AV, Kastin AJ, Arimura A: The hypothalamus and reproduction. Am J Obstet GynecoI1l4:423, 1972
2. Jewelewicz R, Dyrenfurth I, Warren M, Vande Wiele RL: Clinical studies with gonadotropin-releasing hormone. Bull NY Acad Med 50:1097,1974 3. Arai K, Yanaihara T, Okinaga S: Response of ovarian steroid secretion to the intrinsic gonadotropin release caused by the administration of synthetic luteinizing hormone-releasing hormone. Am J Obstet Gynecol 123: 804, 1975 4. Schwarzstein L, de Laborde NP, Aparicio NJ, Turner D, Mirkin A, Rodriguez A, Rodriguez Lhullier F, Rosner JM: Daily variations of FSH, LH and testosterone response to intravenous luteinizing hormone-releasing factor (LRF) in normal men. J Clin Endocrinol Metab 40:313, 1975 5. Figueroa Casas PR, de Laborde NP, Badano A, Pedroza Garcia E, Aparicio N, Miechi H, Mirkin A, Arcangeli 0, Rosner JM: Ovarian response to luteinizing hormonereleasing hormone (LH-RH) in normal women and amenorrheic patients. Reproduction 2:1,1975
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LH, FSH, AND T RESPONSES TO D-LEU-6-LH-RH-EA
6. ROmmler A, Baumgarten S, Hammerstein J: Hormonal response to multiple stimulation with synthetic luteinizing hormone-releasing hormone (LH-RH) in amenorrheic women (abstr). Acta Endocrinol [Suppl] (Kbh) 173: 87, 1973 7. Figueroa Casas PR, Badano A, Mirkin A, Miechi H, Aparicio NJ: Gonadotropin response and induction of ovulation in long-standing secondary amenorrhea treated with clomiphene citrate and repeated injection of hypothalamic gonadotrophin-releasing hormone (LH-RH). Reproduccion. In press
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8. Yoshimoto Y, Moridera K, Imura H: Restoration of normal pituitary gonadotropin reserve by administration of luteinizing hormone-releasing hormone in patients with hypogonadotropic hypogonadism. N Engl J Med 292:242, 1975 9. Vilchez-Martinez JA, Coy DH, Arimura A, Coy EJ, Hirotsu Y, Schally AV: Synthesis and biological properties of (Leu-6)-LH-RH and (D-Leu-6, desGly-NH210-LHRH-ethylamide). Biochem Biophys Res Commun 59: 1226, 1974