Postgraduate Medical Journal (February 1975) 51, 59-64.
The response to luteinizing hormone-releasing hormone in normal men A. R. BoyNs* M. SHAHMANESH M.R.C.P.
Ph.D., B.Sc., M.B.
M. ELLWOOD
G. V. GROOM*
M.R.C.P.
B.Sc.
EVA NELSON
M. HARTOG
A.I.M.L.T.
D.M., F.R.C.P.
University Department of Medicine, Bristol Royal Infirmary and *Tenovus Institute for Cancer Research, Cardiff
Summary Synthetic luteinizing hormone-releasing hormone (LH-RH) was given in doses of 11-100 Vg to normal men. Doses of 50 and 100 t±g caused a two- to ten-fold rise in serum LH in all subjects, but in only 40 % was the serum follicle-stimulating hormone (FSH) elevated. A significantly higher LH response to LHRH was seen in subjects in whom a rise in serum FSH could be detected. Although there was a wide variation in the serum gonadotrophic hormone response, the test was reproducible in individual subjects. A significant rise of serum LH was seen following the injection of 1 jig LH-RH and a linear log-dose response occurred over the range 0-25 to 4 ,ug. There was no rise in serum FSH with the lower doses of LH-RH. Two injections of LH-RH 90 min apart produced comparable rises of serum LH and FSH. The rise of serum LH following a second injection of LH-RH was blunted when the two injections were given 30 or 45 min apart. Introduction Luteinizing hormone releasing hormone (LH-RH) was first isolated by Schally and this colleagues (Schally et al., 1971a). It has subsequently been synthesized and has been shown to be active in man. A rise of serum LH regularly occurs following the injection of doses of 50-100 [Lg (Schally et al., 1971b); Besser et al., 1972; Yen et al., 1972; Nillius and Wide, 1972) and in a proportion of subjects folliclestimulating hormone (FSH) is also released. The hormone has been evaluated in hypogonadal patients but, in the doses used, does not seem to be able to distinguish pituitary from hypothalamic disease (Mortimer et al., 1973). In the present study, we have investigated the reproducibility of the changes in serum gonadotrophin following the administration of LH--RH in
the same subjects, and determined the lowest dose to produce a response. We have also given consecutive injections of the hormone in the hope that the relative changes in serum LH and FSH might thereby be altered (Schally, Arimura and Kastin, 1973).
Subjects and methods The subjects were normal male volunteers aged 19-42 whose consent was given after a full explanation of the nature of the project. LH-RH was synthesized by Dr Derek Schafer of Reckitt and Coleman (Schafer and Black, 1973). The tests were all begun between 9 a.m. and 10 a.m. An indwelling catheter was inserted into the forearm vein and two or three basal blood samples were withdrawn over the next 15-30 min. The LH-RH was then injected rapidly through the catheter and further blood samples withdrawn at frequent intervals. Nine men were given doses of 50 and 100 ,ug at least 1 week apart. In a second experiment, five men received three injections of the same dose of hormone at weekly intervals, two of 50 ,ug and three of 100 ,±g. A third experiment consisted of the administration of two successive injections of 100 jig LH-RH, 90 min apart, to six men, three of whom were later re-tested with doses 45 min apart and another two with doses 30 min apart. Finally, five men received doses of 0 25, 1 and 4 ptg at hourly intervals. Measurements of LH and FSH were undertaken in all serum samples. Serum LH was measured with a double-antibody radioimmunoassay. Purified human pituitary (Fraction IRC 2, kindly donated by by Dr A. Stockell Hartree, Cambridge) was used for labelling with 1"-I. Anti-human pituitary LH (70/229) was supplied by the Medical Research Council. Results were expressed in terms of a human pituitary LH standard (MRC 68/40) ascribed an arbitrary value of 20 u/ampoule. The smallest
M. Shahmanesh et al.
60
amount of LH which could be measured was approximately 0-2 mu/ml serum. Serum FSH was assayed by a double-antibody radioimmunoassay as described previously (Groom et al., 1971). The second International Reference Standard Human Menopausal Gonadotrophin (2nd IRP-HMG) (potency 40 iu/ampoule) was used as a standard. In the assay, the MRC-FSH standard 69/104 has a potency of 16-8 iu FSH/ampoule in terms of the 2nd IRP-HMG. The lower limit of sensitivity of the assay was 1 mu/ml serum and intraassay variation was 10%. Results Serum LH There was a two- to ten-fold rise of serum LH following doses of 50 and 100 ig of LH-RH (Fig. 1). The response to the two doses was similar and the maximum rise in serum LH in individuals showed no significant difference using the paired t test (P > 0-5). The response in individuals tested on three occasions proved to be reproducible (Fig. 2). The maximum rise of serum LH after 100 tig LH-RH showed a variance ten times higher between subjects than within the same subject (P < 0.01). The mean rise of serum LH following a second injection of LH-RH, 90 min after the initial injection was 2-15 mu/ml
3/
2
i I
3
I 2
-15
0
20
40
60 80 100 130 140 Time (min) FIG 2. Individual changes of serum LH levels (mean and s.e. mean) in five normal men receiving 50 (broken line) or 100 (solid line) jg on three separate occasions.
(9±0-34 s.e.m.) and was similar to the rise of 2-49 mu/ml (±0-45) seen after the first injection (Fig. 3). When two doses of LH-RH were given 45 min apart, the maximum rise in serum LH after the two injections was 2-95 mu/ml (±0-62) and 0-28 mu/ml (±0-13). Injections of the hormone at 0 and 30 min produced rises in serum LH of 1-3 mu/ml (±0-45) and 0-12 mu/ml (±0-03) respectively. Only two of the five subjects given 0-25 tig LH-RH showed more than a doubling of their basal LH levels though a small response was seen in the other three (Fig. 4). Following 1 and 4 txg LH-RH, the serum LH was significantly elevated above the levels seen immediately before each injection (P < 0-005 and 0-025 respectively). The maximum rise of serum LH levels after 0-25, 1 and 4 (tg of the hormone showed a linear log-dose response (P< 0-01) (Fig. 5).
4
0
4
I
I 20
I
60 80 100 120 Time (min) FIG. 1. Rise of serum LH (mean and s.e. mean) in nine normal men after intravenous injection of 50 (broken line) and 100 (solid line) ,ug LH-RH. 40
Serum FSH A rise in serum FSH was seen in only eight of the nineteen subjects to whom 50 or 100 tgg LH-RH were given and the range of response was wider than with LH (Fig. 6). The maximum rise in serum FSH showed no significant difference in individuals receiving both doses of the hormone (P > 0-5). Serum FSH levels reached their height later than LH and were maximal 45 min after the injection. The response in the individual was reproducible but not to the extent found with serum LH (Fig. 7) but the range
61
Luteinizing hormone-releasing hormone in men 2 3 E
I I I I I I 20 40 60 80 100 120 140 160
I
)
0
LA-
E.
-t
'
E
.
0 20 40 60 80 100120 140 160 180 L
I
I
I
I 4
0.25
1,
0
Dose of LH-RH (MLg) FIG. 5. The maximum rise of serum LH and FSH (mean and s.e. mean) from pre-inection levels in five normal men given 0-25, 1 and 4 jxg LH-RH at hourly intervals.
, , II I, 20 40 60 80 100 120 140 160 180200220 Time (min)
I0
FIG. 3. Changes of serum LH (mean and s.e. mean) in six normal men given 100 [ag LH-RH 90 min apart (c). In three, the test was repeated with injections 45 min apart (b) and in another two, 30 min apart (a). The arrows show the time of injections.
8
/6\
0 Dose of LH-RH
0
25,9g
1|g
2r5
4g9
T
-
E 20:3
61- /"
2 10
-
---4
0.5 -20
I
0
20
40
60 80 100 120 140 160 180 Time (min)
II
FIG. 4. Serum LH levels (mean and s.e. mean) in five normal men injected with 0-25, 1 and 4 ,[g LH-RH at hourly intervals. The arrows show the time of injections.
was too wide to permit statistical analysis. Paired injections of LH-RH at 90-min intervals elevated serum FSH in two of the five subjects tested (Fig. 8). The rise in serum FSH which followed the second injection in these two subjects was of the same order as that produced by the initial dose. There was no rise in serum FSH with doses of 0-25, 1 and 4 ,tg of LH-RH in the five subjects tested. One of these subjects had previously shown a rise of serum FSH with 100 ,ug of the hormone.
5
_
I I
0
40
80
120
Time (min)
FIG. 6. Serum FSH levels (mean and s.e. mean) in nine normal men after injections of 50 (broken line) and 100 (solid line) tLg LH-RH. Subjects who responded and showed no response are shown separately and the figures indicate the number of subjects in each group.
M. Shahmanesh et al.
62
Discussion The two- to ten-fold rise in serum LH observed in the present study in response to 50 and 100 s±g is similar to previous reports (Schally et al., 1971b; Besser et al., 1972; Yen et al., 1972). Although the response in individuals proved reproducible, there was considerable variation among different subjects which must be taken into consideration when using LH-RH for clinical purposes. Injections of LH-RH have been shown to produce a less consistent and less marked rise of serum FSH (Kastin et al., 1972; Yen et al., 1972; Besser et al., 1972; Nillius and Wide, 1972; Rebar et al., 1973), and in the present study, only 40% of normal men showed a measurable rise of serum FSH following doses of 50 and 100 ag. In another series, 75% of men similarly tested failed to show a rise of serum FSH beyond the normal range (Besser et al., 1972). We were interested to find that the subjects showing a rise of serum FSH tended to be those who attained higher levels of serum LH. The explanation for this is uncertain, but may reflect variations in the endogenous LH-RH secretion. Thus, post-menopausal women (Siler and Yen, 1973) and patients with primary hypogonadism (Mortimer et al., 1973; Siler and Yen, 1973) show an exaggerated response to LH-RH. A significant elevation of serum LH was seen in all the subjects given 1 tag LH-RH (Fig. 4), and a linear log dose-response relationship over the range 0-25-4 jag was observed. There was, however, no rise of serum FSH with these doses of LH-RH. There is conflicting evidence regarding the minimum dose of the hormone required to produce a rise of serum LH. Thus, 1 5 jig (Schally et al., 1971b), 10 Iig (Rebar et al., 1973) and 25 tag (Haug and Torjesen, 1973) have previously been reported to cause a significant release of LH. Such differences could perhaps be due
16-
12
12
j
-
T
4-
Oo
o4A--2V--i-I I
0
40
80 120 Time (min) FIG. 7. Individual changes of serum FSH levels (mean and s.e. mean) in five normal men receiving 50 (broken line) or 100 (solid line) ptg LH-RH on three separate occasions.
The maximum increment of serum LH in subjects who also showed a FSH response to 50 and 100 (ig LH-RH was 3-85 (±1-33 s.d.) mu/ml which was significantly higher (P < 0001) than the mean maximum increment of 2-2 (±0-87) mu/ml in serum LH levels in those who showed no FSH response
(Fig. 9).
4
32 -F
*--x
\
*
,
0
20
40
_
I 60
80
100
120
140
160
180
200
Time (min)
FIG. 8. Changes of serum FSH in two normal men given 100 xAg LH-RH 90 min apart. (Four others were tested but showed no FSH response and are not shown.) The arrows show the time Afif
t
r
220
Luteinizing hormone-releasing hormone in men 9
-
9~~~~~~~~
8_
7
*
0
6 E
63
Two successive injections of LH-RH at 90-min intervals produced similar elevations in serum LH. However, when the interval between the injections was reduced to 45 and 30 min, a much reduced LH response to the second injection was observed. This apparent 'refractory period' has been noted by Schneider and Dahlen (1973). It has been a consistent finding that the increase of serum LH caused by a single injection of LH-RH is greater than that of serum FSH (Kastin et al., 1972; Yen et al., 1972, Besser et al., 1972; Nillius and Wide, 1972; Rebar et al., 1973). This has also been found of biologically active analogues of the molecule (Schally et al., 1973). An enhanced rise of serum FSH has been reported with more prolonged stimulation of the pituitary by LH-RH (Schally et al., 1973). We were, however, unable to show a larger rise of serum FSH with paired injections at intervals of 90 min.
I
-J5
E:3
0
*
) (I1)
We wish to thank Dr D. Shafer of Reckitt and Coleman for the supply of synthetic LH-RH, and R. P. Brown for his helpful advice. The assistance of Miss J. Blatchford is gratefully acknowledged. M.S. is in receipt of a grant from the Medicine Research Council. A.R.B. and G.V.G. are in receipt of a grant from the Tenovus Organization.
C,)
0)*0
:3
E~~~~~~~~~~ 0
* ~~~~~0
*: 0@
A
0
4 S 0
0~~~~~
I~ ~ ~
Acknowledgments
0_ 0 0
No FSH FSH response response FIG. 9. The maximum rise of serum LH levels in subjects who did and did not show an FSH response to 50 and 100 jig LH-RH. P < 0-001.
to variations in the sensitivities of the radioimmunoassays or to the differences in the hormone used. The response to these lower doses of LH-RH may prove more discriminating for testing hypothalamicpituitary function than the larger doses that have been used to date.
References BESSER, G.M., McNEILLY, A.S., ANDERSON, D.C., MARSHALL J.C., HARSOULIS, P.. HALL, R., ORMSTON, B.J., ALEXANDER, L. & COLLINS, W.P. (1972) Hormonal responses to synthetic luteinizing hormone and follicular stimulating hormone-releasing hormone in man. British Medical Journal, Mii, 267. GROOM, G.V., GROOM, M.A., COOKE, I.D. & BOYNES, A.R. (1971) The secretion of immuno-reactive luteinizing hormone and follicle stimulating hormone by the human foetal pituitary in organ culture. Journal of Endocrinology, 49, 335. HAUG, E. & TORJESEN, P. (1973) Effect ofsynthetic luteinizing hormone and follicle stimulating hormone-releasing hormone (LH/FSH-RH) on serum levels of LH, FSH, thyrotrophin (TSH) and growth hormone (HGH) in normal male subjects. Acta endocrinologica, 73, 465. KASTIN, A.J., SCHALLY, A.V., GUAL, C. & ARIMURA, A. (1972) Release of LH and FSH after administration of synthetic LH-releasing hormone. Journal of Clinical Endocrinology and Metabolism, 34, 753. MORTIMER, C.G., BESSER, G.M., McNEILLY, A.S., MARSHALL, J.C., HARSOULIS, P., TUNBRIDGE, W.M.G., GOMEZ PAN, A. & HALL, R. (1973) Luteinizing hormone and follicular stimulating hormone-releasing hormone test in patients with hypothalamic-pituitary-gonadal dysfunction. British Medical.fournal, iv, 73. NILLIUS, S.J. & WIDE, L. (1972) Variation in LH and FSH response to LH-releasing hormone during the menstrual cycle. The Journal of Obstetrics and Gynaecology of the British Commonwealth, 79, 874. REBAR, S.S., YEN, S.S.C., VANDERBERG, G., NAFTOLIN, F., Ehara, Y., ENGBLOM, S., RYAN, K.J., RIVIER, J., AMoss, M. & GUILLEMIN, R. (1973) Gonadotrophic responses to synthetic LRF: Dose-response relationship in men. Journal of Clinical Endocrinology and Metabolism, 36, 10.
64
M. Shahmanesh et al.
SCHAFER, D.J. & BLACK, A.D. (1973) Synthesis of luteinizing hormone releasing hormone (LH-RH): A simplified approach to the synthesis of arginine peptides. Tetrahedron Letters, 41, 4071. SCHALLY, A.V., ARIMURA, A. & KASTIN, A.J. (1973) Hypothalamic regulatory hormones. Science, 179, 341. SCHALLY, A.V., BABA, Y., ARIMURA, A., REDDING, T.W. & WHITE, W.F. (1971a) Evidence for the peptide nature of LH and FSH-releasing hormone Biochemical and Biophysical Research Communications, 42, 50. SCHALLY, A.V., KASTIN, A.J. & ARIMURA, A. (1971b) Hypothalamic follicle-stimulating hormone (FSH) and luteinizing hormone (LH) regulating hormone: Structure,
physiology and clinical studies. Fertility and Sterility, 22, 703. SCHNEIDER, H.P.G. & DAHLEN, H.G. (1973) Reserve capacity of the human pituitary gonadotroph. Acta endocrinologica Suppl., 117, 294. SILER, T.M. & YEN, S.S.C. (1973) Augmented gonadotrophin response to synthetic LRF in hypogonadal state. Journal of Clinical Endocrinology and Metabolism, 37, 491. YEN, S.S.C., REBAR, R., VANDERBERG, G., NAFTOLIN, F., EHARA, Y., ENGBLOM, S., RYAN, K.J., BENIRSCHKE, K., RIVIER, J., AMOS, M. & GUILLEMIN, R. (1972) Synthetic luteinizing hormone-releasing factor: a potent stimulator of gonadotrophin release in man. Journal of Clinical Endocrinology and Metabolism, 34 1108.
The response to luteinizing hormone-releasing hormone in normal men A. R. BoyNs* M. SHAHMANESH M.R.C.P.
Ph.D., B.Sc., M.B.
M. ELLWOOD
G. V. GROOM*
M.R.C.P.
B.Sc.
EVA NELSON
M. HARTOG
A.I.M.L.T.
D.M., F.R.C.P.
University Department of Medicine, Bristol Royal Infirmary and *Tenovus Institute for Cancer Research, Cardiff
Summary Synthetic luteinizing hormone-releasing hormone (LH-RH) was given in doses of 11-100 Vg to normal men. Doses of 50 and 100 t±g caused a two- to ten-fold rise in serum LH in all subjects, but in only 40 % was the serum follicle-stimulating hormone (FSH) elevated. A significantly higher LH response to LHRH was seen in subjects in whom a rise in serum FSH could be detected. Although there was a wide variation in the serum gonadotrophic hormone response, the test was reproducible in individual subjects. A significant rise of serum LH was seen following the injection of 1 jig LH-RH and a linear log-dose response occurred over the range 0-25 to 4 ,ug. There was no rise in serum FSH with the lower doses of LH-RH. Two injections of LH-RH 90 min apart produced comparable rises of serum LH and FSH. The rise of serum LH following a second injection of LH-RH was blunted when the two injections were given 30 or 45 min apart. Introduction Luteinizing hormone releasing hormone (LH-RH) was first isolated by Schally and this colleagues (Schally et al., 1971a). It has subsequently been synthesized and has been shown to be active in man. A rise of serum LH regularly occurs following the injection of doses of 50-100 [Lg (Schally et al., 1971b); Besser et al., 1972; Yen et al., 1972; Nillius and Wide, 1972) and in a proportion of subjects folliclestimulating hormone (FSH) is also released. The hormone has been evaluated in hypogonadal patients but, in the doses used, does not seem to be able to distinguish pituitary from hypothalamic disease (Mortimer et al., 1973). In the present study, we have investigated the reproducibility of the changes in serum gonadotrophin following the administration of LH--RH in
the same subjects, and determined the lowest dose to produce a response. We have also given consecutive injections of the hormone in the hope that the relative changes in serum LH and FSH might thereby be altered (Schally, Arimura and Kastin, 1973).
Subjects and methods The subjects were normal male volunteers aged 19-42 whose consent was given after a full explanation of the nature of the project. LH-RH was synthesized by Dr Derek Schafer of Reckitt and Coleman (Schafer and Black, 1973). The tests were all begun between 9 a.m. and 10 a.m. An indwelling catheter was inserted into the forearm vein and two or three basal blood samples were withdrawn over the next 15-30 min. The LH-RH was then injected rapidly through the catheter and further blood samples withdrawn at frequent intervals. Nine men were given doses of 50 and 100 ,ug at least 1 week apart. In a second experiment, five men received three injections of the same dose of hormone at weekly intervals, two of 50 ,ug and three of 100 ,±g. A third experiment consisted of the administration of two successive injections of 100 jig LH-RH, 90 min apart, to six men, three of whom were later re-tested with doses 45 min apart and another two with doses 30 min apart. Finally, five men received doses of 0 25, 1 and 4 ptg at hourly intervals. Measurements of LH and FSH were undertaken in all serum samples. Serum LH was measured with a double-antibody radioimmunoassay. Purified human pituitary (Fraction IRC 2, kindly donated by by Dr A. Stockell Hartree, Cambridge) was used for labelling with 1"-I. Anti-human pituitary LH (70/229) was supplied by the Medical Research Council. Results were expressed in terms of a human pituitary LH standard (MRC 68/40) ascribed an arbitrary value of 20 u/ampoule. The smallest
M. Shahmanesh et al.
60
amount of LH which could be measured was approximately 0-2 mu/ml serum. Serum FSH was assayed by a double-antibody radioimmunoassay as described previously (Groom et al., 1971). The second International Reference Standard Human Menopausal Gonadotrophin (2nd IRP-HMG) (potency 40 iu/ampoule) was used as a standard. In the assay, the MRC-FSH standard 69/104 has a potency of 16-8 iu FSH/ampoule in terms of the 2nd IRP-HMG. The lower limit of sensitivity of the assay was 1 mu/ml serum and intraassay variation was 10%. Results Serum LH There was a two- to ten-fold rise of serum LH following doses of 50 and 100 ig of LH-RH (Fig. 1). The response to the two doses was similar and the maximum rise in serum LH in individuals showed no significant difference using the paired t test (P > 0-5). The response in individuals tested on three occasions proved to be reproducible (Fig. 2). The maximum rise of serum LH after 100 tig LH-RH showed a variance ten times higher between subjects than within the same subject (P < 0.01). The mean rise of serum LH following a second injection of LH-RH, 90 min after the initial injection was 2-15 mu/ml
3/
2
i I
3
I 2
-15
0
20
40
60 80 100 130 140 Time (min) FIG 2. Individual changes of serum LH levels (mean and s.e. mean) in five normal men receiving 50 (broken line) or 100 (solid line) jg on three separate occasions.
(9±0-34 s.e.m.) and was similar to the rise of 2-49 mu/ml (±0-45) seen after the first injection (Fig. 3). When two doses of LH-RH were given 45 min apart, the maximum rise in serum LH after the two injections was 2-95 mu/ml (±0-62) and 0-28 mu/ml (±0-13). Injections of the hormone at 0 and 30 min produced rises in serum LH of 1-3 mu/ml (±0-45) and 0-12 mu/ml (±0-03) respectively. Only two of the five subjects given 0-25 tig LH-RH showed more than a doubling of their basal LH levels though a small response was seen in the other three (Fig. 4). Following 1 and 4 txg LH-RH, the serum LH was significantly elevated above the levels seen immediately before each injection (P < 0-005 and 0-025 respectively). The maximum rise of serum LH levels after 0-25, 1 and 4 (tg of the hormone showed a linear log-dose response (P< 0-01) (Fig. 5).
4
0
4
I
I 20
I
60 80 100 120 Time (min) FIG. 1. Rise of serum LH (mean and s.e. mean) in nine normal men after intravenous injection of 50 (broken line) and 100 (solid line) ,ug LH-RH. 40
Serum FSH A rise in serum FSH was seen in only eight of the nineteen subjects to whom 50 or 100 tgg LH-RH were given and the range of response was wider than with LH (Fig. 6). The maximum rise in serum FSH showed no significant difference in individuals receiving both doses of the hormone (P > 0-5). Serum FSH levels reached their height later than LH and were maximal 45 min after the injection. The response in the individual was reproducible but not to the extent found with serum LH (Fig. 7) but the range
61
Luteinizing hormone-releasing hormone in men 2 3 E
I I I I I I 20 40 60 80 100 120 140 160
I
)
0
LA-
E.
-t
'
E
.
0 20 40 60 80 100120 140 160 180 L
I
I
I
I 4
0.25
1,
0
Dose of LH-RH (MLg) FIG. 5. The maximum rise of serum LH and FSH (mean and s.e. mean) from pre-inection levels in five normal men given 0-25, 1 and 4 jxg LH-RH at hourly intervals.
, , II I, 20 40 60 80 100 120 140 160 180200220 Time (min)
I0
FIG. 3. Changes of serum LH (mean and s.e. mean) in six normal men given 100 [ag LH-RH 90 min apart (c). In three, the test was repeated with injections 45 min apart (b) and in another two, 30 min apart (a). The arrows show the time of injections.
8
/6\
0 Dose of LH-RH
0
25,9g
1|g
2r5
4g9
T
-
E 20:3
61- /"
2 10
-
---4
0.5 -20
I
0
20
40
60 80 100 120 140 160 180 Time (min)
II
FIG. 4. Serum LH levels (mean and s.e. mean) in five normal men injected with 0-25, 1 and 4 ,[g LH-RH at hourly intervals. The arrows show the time of injections.
was too wide to permit statistical analysis. Paired injections of LH-RH at 90-min intervals elevated serum FSH in two of the five subjects tested (Fig. 8). The rise in serum FSH which followed the second injection in these two subjects was of the same order as that produced by the initial dose. There was no rise in serum FSH with doses of 0-25, 1 and 4 ,tg of LH-RH in the five subjects tested. One of these subjects had previously shown a rise of serum FSH with 100 ,ug of the hormone.
5
_
I I
0
40
80
120
Time (min)
FIG. 6. Serum FSH levels (mean and s.e. mean) in nine normal men after injections of 50 (broken line) and 100 (solid line) tLg LH-RH. Subjects who responded and showed no response are shown separately and the figures indicate the number of subjects in each group.
M. Shahmanesh et al.
62
Discussion The two- to ten-fold rise in serum LH observed in the present study in response to 50 and 100 s±g is similar to previous reports (Schally et al., 1971b; Besser et al., 1972; Yen et al., 1972). Although the response in individuals proved reproducible, there was considerable variation among different subjects which must be taken into consideration when using LH-RH for clinical purposes. Injections of LH-RH have been shown to produce a less consistent and less marked rise of serum FSH (Kastin et al., 1972; Yen et al., 1972; Besser et al., 1972; Nillius and Wide, 1972; Rebar et al., 1973), and in the present study, only 40% of normal men showed a measurable rise of serum FSH following doses of 50 and 100 ag. In another series, 75% of men similarly tested failed to show a rise of serum FSH beyond the normal range (Besser et al., 1972). We were interested to find that the subjects showing a rise of serum FSH tended to be those who attained higher levels of serum LH. The explanation for this is uncertain, but may reflect variations in the endogenous LH-RH secretion. Thus, post-menopausal women (Siler and Yen, 1973) and patients with primary hypogonadism (Mortimer et al., 1973; Siler and Yen, 1973) show an exaggerated response to LH-RH. A significant elevation of serum LH was seen in all the subjects given 1 tag LH-RH (Fig. 4), and a linear log dose-response relationship over the range 0-25-4 jag was observed. There was, however, no rise of serum FSH with these doses of LH-RH. There is conflicting evidence regarding the minimum dose of the hormone required to produce a rise of serum LH. Thus, 1 5 jig (Schally et al., 1971b), 10 Iig (Rebar et al., 1973) and 25 tag (Haug and Torjesen, 1973) have previously been reported to cause a significant release of LH. Such differences could perhaps be due
16-
12
12
j
-
T
4-
Oo
o4A--2V--i-I I
0
40
80 120 Time (min) FIG. 7. Individual changes of serum FSH levels (mean and s.e. mean) in five normal men receiving 50 (broken line) or 100 (solid line) ptg LH-RH on three separate occasions.
The maximum increment of serum LH in subjects who also showed a FSH response to 50 and 100 (ig LH-RH was 3-85 (±1-33 s.d.) mu/ml which was significantly higher (P < 0001) than the mean maximum increment of 2-2 (±0-87) mu/ml in serum LH levels in those who showed no FSH response
(Fig. 9).
4
32 -F
*--x
\
*
,
0
20
40
_
I 60
80
100
120
140
160
180
200
Time (min)
FIG. 8. Changes of serum FSH in two normal men given 100 xAg LH-RH 90 min apart. (Four others were tested but showed no FSH response and are not shown.) The arrows show the time Afif
t
r
220
Luteinizing hormone-releasing hormone in men 9
-
9~~~~~~~~
8_
7
*
0
6 E
63
Two successive injections of LH-RH at 90-min intervals produced similar elevations in serum LH. However, when the interval between the injections was reduced to 45 and 30 min, a much reduced LH response to the second injection was observed. This apparent 'refractory period' has been noted by Schneider and Dahlen (1973). It has been a consistent finding that the increase of serum LH caused by a single injection of LH-RH is greater than that of serum FSH (Kastin et al., 1972; Yen et al., 1972, Besser et al., 1972; Nillius and Wide, 1972; Rebar et al., 1973). This has also been found of biologically active analogues of the molecule (Schally et al., 1973). An enhanced rise of serum FSH has been reported with more prolonged stimulation of the pituitary by LH-RH (Schally et al., 1973). We were, however, unable to show a larger rise of serum FSH with paired injections at intervals of 90 min.
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We wish to thank Dr D. Shafer of Reckitt and Coleman for the supply of synthetic LH-RH, and R. P. Brown for his helpful advice. The assistance of Miss J. Blatchford is gratefully acknowledged. M.S. is in receipt of a grant from the Medicine Research Council. A.R.B. and G.V.G. are in receipt of a grant from the Tenovus Organization.
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No FSH FSH response response FIG. 9. The maximum rise of serum LH levels in subjects who did and did not show an FSH response to 50 and 100 jig LH-RH. P < 0-001.
to variations in the sensitivities of the radioimmunoassays or to the differences in the hormone used. The response to these lower doses of LH-RH may prove more discriminating for testing hypothalamicpituitary function than the larger doses that have been used to date.
References BESSER, G.M., McNEILLY, A.S., ANDERSON, D.C., MARSHALL J.C., HARSOULIS, P.. HALL, R., ORMSTON, B.J., ALEXANDER, L. & COLLINS, W.P. (1972) Hormonal responses to synthetic luteinizing hormone and follicular stimulating hormone-releasing hormone in man. British Medical Journal, Mii, 267. GROOM, G.V., GROOM, M.A., COOKE, I.D. & BOYNES, A.R. (1971) The secretion of immuno-reactive luteinizing hormone and follicle stimulating hormone by the human foetal pituitary in organ culture. Journal of Endocrinology, 49, 335. HAUG, E. & TORJESEN, P. (1973) Effect ofsynthetic luteinizing hormone and follicle stimulating hormone-releasing hormone (LH/FSH-RH) on serum levels of LH, FSH, thyrotrophin (TSH) and growth hormone (HGH) in normal male subjects. Acta endocrinologica, 73, 465. KASTIN, A.J., SCHALLY, A.V., GUAL, C. & ARIMURA, A. (1972) Release of LH and FSH after administration of synthetic LH-releasing hormone. Journal of Clinical Endocrinology and Metabolism, 34, 753. MORTIMER, C.G., BESSER, G.M., McNEILLY, A.S., MARSHALL, J.C., HARSOULIS, P., TUNBRIDGE, W.M.G., GOMEZ PAN, A. & HALL, R. (1973) Luteinizing hormone and follicular stimulating hormone-releasing hormone test in patients with hypothalamic-pituitary-gonadal dysfunction. British Medical.fournal, iv, 73. NILLIUS, S.J. & WIDE, L. (1972) Variation in LH and FSH response to LH-releasing hormone during the menstrual cycle. The Journal of Obstetrics and Gynaecology of the British Commonwealth, 79, 874. REBAR, S.S., YEN, S.S.C., VANDERBERG, G., NAFTOLIN, F., Ehara, Y., ENGBLOM, S., RYAN, K.J., RIVIER, J., AMoss, M. & GUILLEMIN, R. (1973) Gonadotrophic responses to synthetic LRF: Dose-response relationship in men. Journal of Clinical Endocrinology and Metabolism, 36, 10.
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SCHAFER, D.J. & BLACK, A.D. (1973) Synthesis of luteinizing hormone releasing hormone (LH-RH): A simplified approach to the synthesis of arginine peptides. Tetrahedron Letters, 41, 4071. SCHALLY, A.V., ARIMURA, A. & KASTIN, A.J. (1973) Hypothalamic regulatory hormones. Science, 179, 341. SCHALLY, A.V., BABA, Y., ARIMURA, A., REDDING, T.W. & WHITE, W.F. (1971a) Evidence for the peptide nature of LH and FSH-releasing hormone Biochemical and Biophysical Research Communications, 42, 50. SCHALLY, A.V., KASTIN, A.J. & ARIMURA, A. (1971b) Hypothalamic follicle-stimulating hormone (FSH) and luteinizing hormone (LH) regulating hormone: Structure,
physiology and clinical studies. Fertility and Sterility, 22, 703. SCHNEIDER, H.P.G. & DAHLEN, H.G. (1973) Reserve capacity of the human pituitary gonadotroph. Acta endocrinologica Suppl., 117, 294. SILER, T.M. & YEN, S.S.C. (1973) Augmented gonadotrophin response to synthetic LRF in hypogonadal state. Journal of Clinical Endocrinology and Metabolism, 37, 491. YEN, S.S.C., REBAR, R., VANDERBERG, G., NAFTOLIN, F., EHARA, Y., ENGBLOM, S., RYAN, K.J., BENIRSCHKE, K., RIVIER, J., AMOS, M. & GUILLEMIN, R. (1972) Synthetic luteinizing hormone-releasing factor: a potent stimulator of gonadotrophin release in man. Journal of Clinical Endocrinology and Metabolism, 34 1108.
