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Measurement of immunologically reactive follicle stimulating hormone in serum of normal male children and adults
Measurement of Immunologically
Reactive Follicle
Stimulating Hormone in Serum of Normal Male Children By S.RAITI, A.
and Adults
JOHANSON,C. LIGHT, C. J. MIGEON AND R. M. BLIZZARD
Using a radio immunoassay serum, FSH concentrations have been determined in 97 normal male children between the ages of 5 and 18 years, in 30 normal adult males between 25 and 45 years, and in 9 adult patients who were sexually infantile, presumably because of deficient gonadotrop in secretion by the pituitary. FSH reactive material was found in all serums. In normal children serum FSH concentrations begin increasing shortly after 9.0 years of age from the low levels found in early childhood. By the age of 13 years the mean FSH determination was comparable to that of normal adults. When correlated with various stages of sexual development (1 through S), the mean FSH concentrations found in the serums of individuals in stages 1, 2, and 3 differed significantly from each other.
However, mean levels for the individuals in groups 3, 4, and 5 did not differ significantly. There was a low degree of correlation between serum FSH concentration and the excretion of urinary 17ketosteroids. The values of immunologic FSH reported may have no specific correlation with biologically active FSH in serum, and all results are interpretable only in comparison of one group of values against another. The determination of absolute values of FSH in serum will be possible only when there is an adequately defined standard for serum and commonly available antigen and antiserum. Only then will it be possible to compare biologic and immunologically reactive FSH in sera. (Metabolism 18: No. 3, March, 234-240, 1969)
INTERESTED IN MATURATION OF THE CHILD into the adult have been interested in the role that pituitary follicle stimulating hormone (FSH) and luteinizing hormone (LH) play. Concentrations of these hormones at various ages must be known before physiological mechanisms for maturation can be postulated. Therefore, we previously determined and reported1 the LH concentration in the serums of males during childhood and
T
HOSE
From The Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Md. Received for publication September 17, 1968. This work was supported by USPHS Research Grants HD-01852 and AM-OOlBO-16. Institute of Child Health, S. RAITI, M.B.B.S. (Q’L’D), M.R.C.P.: Senior Lecturer, London; Consultant Endocrinologist, The Queen Elizabeth Hospital for Children, London; Honorary Consultant Endocrinologist, The Hospital for Sick Children, London; supported by USPHS Traineeship Grant Tl-AM-5219-08. A. JOHANSON, M.D.: Assistant Professor, supported by USPHS Department of Pediatrics, University of Virginia, Charlottesville; The Johns Traineeship Grant TI-AM-521948. C. LIGHT, M.D.: Consultant Pediatrician, Hopkins Hospitd, Baltimore, Md. C. J. MIGEON, M.D.: Associate Professor, Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Md. R. M. of Pediatrics, The Johns Hopkins University BLIZZARD, M.D.: Professor, Department School of Medicine, Baltimore, Md.
234
IMMUNOLOGICALLY
235
REACTIVE FSH
adolescence. We now are reporting the same individuals.
the FSH concentrations
in the serums of
METHODS
Radioimmunoassay preparation of The technique of Midgley has been applied .e An immunochemical human pituitary FSH (LER 780), with a biologic potency of 50.0 NIH-FSH-SI units* and with an immunologic potency of 86.6 NIH-FSH-SI units2 or 2293 International Units was labeled with laliodine (I) which was at a concentration of (IU)s (2nd IRP-HMG),* 300350 mc./ml. (Cambridge Nuclear Corp.) according to the method of Greenwood, Hunter, and Glover.4 The lsrI-FSH was separated from free 1311 using a 25-cm. column of G-75 sephadex. Elution was carried out using 0.1 M sodium barbital buffer at pH 8.6. Fractions of 0.5 ml. each were collected in tubes containing 0.1 ml. of 30 per cent bovine serum albumin. Four fractions from the upper portion of the descending limb of the protein peak were pooled and passed through a second sephadex column. Fractions from the upper portion of the descending limb of the second protein peak were pooled and used for the immunoassay. The fractions of rsrI-FSH from this area of the peak had the least damage and the greatest binding capacity. In addition this product compared favorably with that separated on disc electrophoresis when LER 780 was the hormone varied between 165 uC/ugm. used for iodination. The specific activity of the IsiI-FSH and 260 uC/ugm. The antiserum to FSH and the volumes and types of reagents were those described by Midgley.2 Two IU of HCG, which previously had been demonstrated to be adequate to neutralize all antibodies cross reacting with LH in the volumes of antiserum used, were added to each tube before addition of any FSH as larI-FSH, as FSH in serum, or as FSH in the form of the 2nd IRP-HMG. Following adsorption with HCG, this antiserum did not cross react in any significant amount with TSH, HGH or HCG.5 Sheep anti-rabbit serum was used in excess and in an amount which produced maximum precipitation of the islI-FSH-anti FSH complex. A Nuclear of Chicago automatic gamma counter with a 2.0” scintillation crystal was used to count both the precipitate and supernate. The bound to free ratio of 1alI-FSH, when no standard (2nd IRP-HMG) was added, varied from 0.31 to 0.86 for 12 assays. In 10 of the 12 assays it was 0.51-0.86. Maximum displacement of bound 1slI-FSH from the antibody with addition of excess standard (2nd IRP-HMG) varied between 50 and 70 per cent of that initially bound. Logit plots were utilized to determine the limits of sensitivity for each assay. The lower limit of sensitivity was usually between 1 and 2 mIU/ml. and never greater than 3.9 mIU/ml. The upper limit of sensitivity varied between 15.6 and 62.4 mIU/ml., but usually was 31.2 mIU/ml. To determine intra-assay variability one serum specimen was run in 9 tubes. The results for single tubes varied between 4.0 and 8.0 mIU/ml. Since triplicate specimens are used in the usual assay, the intra-assay variability was determined by calculating the mean value for tubes No. 1 $ 2 + 3, 2 + 3 + 4, etc. The mean of the resultant determinations was 6.2 mIU/ml. with a SD of 0.6 mIU/ml. To determine the inter-assay variability, 14 separate assays for a single serum specimen were done in triplicate. The mean and SD were 7.1 ‘_ 1.1 mIU/ml. with a range of 5.5 to 9.0.
Patient Material Ninety-seven normal boys whose heights were between the 25th and 75th percentiles for their ages (5 to 18 years) were bled between 7:OO and 8:00 a.m. A physical examination was done, and a stage of sexual development was assigned according to the classification of * Second International Reference Preparation which was kindly supplied by Dr. D. Bangham, London.
of Human Menopausal Gonadotropin National Institute for Medical Research,
236
MIT1
16-
x
tiYPOtONADOTROPlC
o
~IVPOPHYSECTOM~ZED
. .
14-
ET AL.
.
.
. .
.
.
Fig. l.-Serum FSH concentrations in males compared with chronological age.
MEAN 4.2 ~IS.0. to.7 5-8
,
5.2 t1.2
'
9-1,
5.8 8.0 1.5 t 2.0,?3.6, 2 1.91
8.5 +3.6
' 12 ' 13 ' 14-15 ' 16-18
1.4 r1.9 'ADULTS'
YEARS
Tanner.6 The sera were stored at -20” C until thawed for assay purposes. Thirty male physicians between the ages of 25 and 45 years also donated sera. The sera of five adult patients between the ages 20 and 32 years, and who had an isolated gonadotropin deficiency as determined by absence of sexual development, normal growth, and normal ACTH, HGH and TSH function also were tested, as were the serums of 4 adult patients with panhypopituitarism secondary to hypophysectomy for craniopharyngiomas. RESULTS
The mean FSH levels and their standard deviations for different age groups are presented in Fig. 1. FSH was found in all serums. There was a definite trend for the mean FSH level to increase with age, until at 13 years of age the mean FSH value (8.0 mIU/ml.) was approximately the same as that found for the older age groups. Statistical analysis using the “T” test revealed that the 5.0-8.0 age group differed significantly from all others (p < .Ol). Data from individuals in the 9.0-12 year age groups in Fig. 1 were pooled for purposes of comparison with the other groups. This Q.O-12-year group differed significantly (p < .Ol) from the younger and older age groups. After 13 years, there was no significant difference between groups. There was a great deal of scatter in all age groups after the age of 11. It was not determined whether this variability was attributable to a day to day variation or some other factor. Surprisingly, 4 of the 30 adult male subjects had values in the same low range as normal young male children. In Fig. 2, the stages of sexual development are correlated with the chronological ages of these male subjects. By the age of 13 years (Fig. 2)) all of the boys had developed sexually and 6 of the 9 in this age group were either in stages 3 or 4 of their sexual development. In Fig. 3, the FSH levels in serum are correlated with the stages of sexual development. The extreme variability of
IMMIJNOLOGICALLY
REACTIVE
.
...
I
.~e.
I
... .......
’ 5-6
... ’ 7-8
0.0 ‘..’
t:
... ..*.
.
.... .... .....
t:
A: ...
..
’ 9-m
237
FSH
::: . ..
. .
Fig. 2.-Stages of sexual development in normal males compared with chronological age.
I II-12 ’ 13-14 ’ 15-M ’ 17-19 ’ YEARS
serum concentrations noted previously for children after the age of 11 is apparent for individuals in stages 3, 4, and 5 of their sexual development. Stage 1 and 2 differed significantly from all other stages ( p < .Ol). There was no statistically significant difference in the mean values among stages 3, 4, and 5, or between them and those of normal adult males. Six of the 22 adolescent males in stage 5 had values within the range of 1 SD from the mean value for sexually infantile subjects. The correlation coefficient between serum FSH levels and urinary 17ketosteroid excretion is 0.49. This is statistically significant (p < 0.001) but is of low degree. The correlation coefficient between the serum FSH and serum LH levels1 on the same patients is 0.50. DIS~~JSSIOA-
Several authors2,5~7-10 have applied immunoassay procedures for determining small quantities of FSH in human serum or plasma. Different antiserum and different standards have been used, and values that have been reported consequently have not always been comparable. For example, the values obtained for normal males by Ode11 and Parlow were significantly higher than those found by Cargille and Rodbard. The utilization of a urinary standard (2nd IRP-HMG) which may not be ideal for measuring FSH of pituitary origin or in serum, and the possibility that FSH determined by immunologic techniques may not correlate with biologically active FSH, contribute to the difficulty in assessing or interpreting the values of serum FSH concentrations obtained by radioimmunoassay techniques. Those publishing FSH concentrations, and those reading such publications, must recognize
238
RAITI ET AL.
16 !
X HY POGONADOTAOPIC 0
HYPOPHYSECTOHIZED
.
14-
12-
IO-
Fig. 3.-Serum
FSH concen-
trafions in males compared with chronological age.
,g
.
6_
STAGE
- SEXUAL
DEVELOPMENT
that improvement and standardization of techniques will permit reporting more accurate actual determinations. Regardless, the utilization of FSH radioimmunoassays, which have been standardized within an individual laboratory and the limitations of which are recognized by the investigators, permits a comparative study of groups of subjects or various stimuli upon one individual. The current study was devised specifically to compare FSH levels in the sera of children of various ages and at various stages of sexual deveIopment, in order to better understand adolescent development. The study serves this purpose, and inspection of the data permits one to draw several conclusions. First, mean FSH levels for groups of normal boys begin to increase over the values of early childhood, between the ages of 9 and 11 years. They further increase until the children reach approximately 13 years of age and/or reach the third stage of sexual development, at which time the mean levels are comparable to those of the normal adult male. Secondly, our values in young adult males are consistent with those reported by Midgley2 for six similar subjects (3.5-8.6 mIU 2nd IRP-HMG), and may be consistent with values of biologically active FSH, since KelleP reported a value of 8.8 mIU/ml., when he assayed a pooled serum from adult males, and Kulin et al.l* reported a mean assay value of 11.5 mIU/ml. on a separate pool of adult male serum. However, our values should not be regarded as necessarily reflecting those of biologically active FSH in serum. Demura et al.,a and Midgley,2 using immunologic assays, have noted some FSH or FSH immunoreactive material in the sera of hypophysectomized and hypopituitary patients, and Kuhn et a1.13 using a bioassay, intermittently found detectable gonadotropin in pooled urine of preadolescent children. The latter group believed that small amounts of biologically active gonadotropin were
IMMUNOLOGICALLY
239
REACTIVE FSH
excreted during childhood. Therefore, it is probable that the small amount of immunologically reactive material which we detected in the sera of young children and hypopituitary adults is immunologically reactive FSH. When this study was initiated it was hoped that differentiation of hypogonadotropic from normal subjects could be made by assaying a single serum specimen. Unfortunately, the determination of FSH on a random specimen of serum will not always permit this differentiation. Sequential specimens may permit such differentiation, but this remains to be determined, since adequate sequential studies were not carried out in this project. Johanson et al.’ from this clinic have recently reported serum LH values for the same groups of boys and adult males. There is poor correlation between LH and FSH values in individual patients. Further comparison of the LH and FSH data, when expressed in mIU/ml. of 2nd IRP-HMG permits the deduction that mean LH values rise from childhood to adulthood (3.4 to 10.9) proportionately greater than do mean FSH values (4.2 to 7.4). Because we are measuring immunologic and not biologic activity one cannot expressly state, however, that there is an actual greater increase in biologically active LH than FSH. By comparing these data further, it is concluded that the urinary excretion of 17-ketosteroids correlates closely (Cor. Coef. = 0.73) with serum LH concentrations,l but to a much lesser extent: with the FSH concentrations (Cor. Coef. = 0.49). Since ?i or more of the 17-ketosteroid excretion of males is of adrenal origin, one must consider the possibility that LH can in some direct or indirect way affect the development of the zona reticularis of the adrenal, in addition to affecting the maturation of the mature leydig cells in the testes. There is no evidence from these data to suggest that FSH might have such an effect. ACKNOWLEDGMENTS Mr. Walter Davis and Mrs. M. Westervelt assisted in the technical and secretarial work, respectively. Dr. Leo Reichert prepared the FSH. Dr. A. R. Midgley Jr., prepared the FSH antiserum. The National Pituitary Agency, the Endocrinology Study Section, and the National Institutes of Arthritis and Metabolic Diseases made this work possible.
REFERENCES 1. Johanson,
A. J., Migeon,
C. J., Light,
C., Guyda, H., and Blizzard, R. M.: Serum luteinizing hormone ( LH ) by radioimmunoassay in normal children. J. Pediatrics (in press). 2. Midgley, A. R., Jr.: Radioimmunoassay for human follicle stimulating hormone. J, Clin. Endocr. 27:295, 1967. 3. Donini, P., Puzzoli, D., D’Alessio, I., Lunenfeld, B., Eshkol, A., and Parlow, A. F.: Purification and separation of follicle stimulating hormone (FSH) and luteinizing hormone (LH) from human post-menopausal gonadotrophin ( HMG ) . I. Separation of FSH and LH by electrophoresis,
chromatography and gel filtration dures. Acta Endocr. 52:169, 1966. 4. Greenwood, F. C., Hunter, and Glover, J. S.: The preparation labelled human growth hormone specific radioactivity. Biochem. J.
proceW. M., of IsrIof high 89: 114,
1963. 5. Cargille, C. M., and Rodbard, D.: Methodological studies of a 24 hour radioimmunoassay for human follicle stimulating hormone. Abstracts Endocrine Sot., 1967 ( Abstract 165 ). 6. Tanner, J. M.: Growth at Adolescence fed. 2). Oxford, Blackwell, 1962, p. 32. 7. Taymor, M. D., Aono, T., and Gold-
240 stein, D. P.: Radioimmunoassay of FSH utilizing 1251. Program of Endocrine Society, 1967 (Abstract 164). 8. Demura, H., Saxena, B. B., Gandy, H. M., and Peterson, R. E.: Radioimmunoassay for FSH and LH in human plasma. Program Endocrine Society, 1967 (Abstract 63). 9. Odell, W. D., and Parlow, A. F.: Some physiological studies of human FSH using radioimmunoassay. Program of Endocrine Society (Abstract 65). 10. Faiman, C., and Ryan, R. J.: Radioimmunoassay for human FSH. J. Clin. Endocr. 27:444, 1967.
RAITI ET AL.
11. Keller, P. J.: Studies on pituitary gonadotropins in human plasma. II. Follicle stimulating and luteinizing hormone in male and post-menopausal plasma. Acta Endocr. (Kobenhavn) 52:348, 1966. 12. Kulin, H. E., Rifkind, A. B., and Ross, G. T.: Human LH activity in processed and unprocessed urine measured by radioimmunoassay and bioassay. J. Clin. Endocr. 28: 100, 1968. 13. -, -, Ross, G. T., and Odell, W. D.: Total gonadotropin activity in the urine of prepubertal children. J. Clin. Endocr. 27: 1123, 1967.
Reactive Follicle
Stimulating Hormone in Serum of Normal Male Children By S.RAITI, A.
and Adults
JOHANSON,C. LIGHT, C. J. MIGEON AND R. M. BLIZZARD
Using a radio immunoassay serum, FSH concentrations have been determined in 97 normal male children between the ages of 5 and 18 years, in 30 normal adult males between 25 and 45 years, and in 9 adult patients who were sexually infantile, presumably because of deficient gonadotrop in secretion by the pituitary. FSH reactive material was found in all serums. In normal children serum FSH concentrations begin increasing shortly after 9.0 years of age from the low levels found in early childhood. By the age of 13 years the mean FSH determination was comparable to that of normal adults. When correlated with various stages of sexual development (1 through S), the mean FSH concentrations found in the serums of individuals in stages 1, 2, and 3 differed significantly from each other.
However, mean levels for the individuals in groups 3, 4, and 5 did not differ significantly. There was a low degree of correlation between serum FSH concentration and the excretion of urinary 17ketosteroids. The values of immunologic FSH reported may have no specific correlation with biologically active FSH in serum, and all results are interpretable only in comparison of one group of values against another. The determination of absolute values of FSH in serum will be possible only when there is an adequately defined standard for serum and commonly available antigen and antiserum. Only then will it be possible to compare biologic and immunologically reactive FSH in sera. (Metabolism 18: No. 3, March, 234-240, 1969)
INTERESTED IN MATURATION OF THE CHILD into the adult have been interested in the role that pituitary follicle stimulating hormone (FSH) and luteinizing hormone (LH) play. Concentrations of these hormones at various ages must be known before physiological mechanisms for maturation can be postulated. Therefore, we previously determined and reported1 the LH concentration in the serums of males during childhood and
T
HOSE
From The Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Md. Received for publication September 17, 1968. This work was supported by USPHS Research Grants HD-01852 and AM-OOlBO-16. Institute of Child Health, S. RAITI, M.B.B.S. (Q’L’D), M.R.C.P.: Senior Lecturer, London; Consultant Endocrinologist, The Queen Elizabeth Hospital for Children, London; Honorary Consultant Endocrinologist, The Hospital for Sick Children, London; supported by USPHS Traineeship Grant Tl-AM-5219-08. A. JOHANSON, M.D.: Assistant Professor, supported by USPHS Department of Pediatrics, University of Virginia, Charlottesville; The Johns Traineeship Grant TI-AM-521948. C. LIGHT, M.D.: Consultant Pediatrician, Hopkins Hospitd, Baltimore, Md. C. J. MIGEON, M.D.: Associate Professor, Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Md. R. M. of Pediatrics, The Johns Hopkins University BLIZZARD, M.D.: Professor, Department School of Medicine, Baltimore, Md.
234
IMMUNOLOGICALLY
235
REACTIVE FSH
adolescence. We now are reporting the same individuals.
the FSH concentrations
in the serums of
METHODS
Radioimmunoassay preparation of The technique of Midgley has been applied .e An immunochemical human pituitary FSH (LER 780), with a biologic potency of 50.0 NIH-FSH-SI units* and with an immunologic potency of 86.6 NIH-FSH-SI units2 or 2293 International Units was labeled with laliodine (I) which was at a concentration of (IU)s (2nd IRP-HMG),* 300350 mc./ml. (Cambridge Nuclear Corp.) according to the method of Greenwood, Hunter, and Glover.4 The lsrI-FSH was separated from free 1311 using a 25-cm. column of G-75 sephadex. Elution was carried out using 0.1 M sodium barbital buffer at pH 8.6. Fractions of 0.5 ml. each were collected in tubes containing 0.1 ml. of 30 per cent bovine serum albumin. Four fractions from the upper portion of the descending limb of the protein peak were pooled and passed through a second sephadex column. Fractions from the upper portion of the descending limb of the second protein peak were pooled and used for the immunoassay. The fractions of rsrI-FSH from this area of the peak had the least damage and the greatest binding capacity. In addition this product compared favorably with that separated on disc electrophoresis when LER 780 was the hormone varied between 165 uC/ugm. used for iodination. The specific activity of the IsiI-FSH and 260 uC/ugm. The antiserum to FSH and the volumes and types of reagents were those described by Midgley.2 Two IU of HCG, which previously had been demonstrated to be adequate to neutralize all antibodies cross reacting with LH in the volumes of antiserum used, were added to each tube before addition of any FSH as larI-FSH, as FSH in serum, or as FSH in the form of the 2nd IRP-HMG. Following adsorption with HCG, this antiserum did not cross react in any significant amount with TSH, HGH or HCG.5 Sheep anti-rabbit serum was used in excess and in an amount which produced maximum precipitation of the islI-FSH-anti FSH complex. A Nuclear of Chicago automatic gamma counter with a 2.0” scintillation crystal was used to count both the precipitate and supernate. The bound to free ratio of 1alI-FSH, when no standard (2nd IRP-HMG) was added, varied from 0.31 to 0.86 for 12 assays. In 10 of the 12 assays it was 0.51-0.86. Maximum displacement of bound 1slI-FSH from the antibody with addition of excess standard (2nd IRP-HMG) varied between 50 and 70 per cent of that initially bound. Logit plots were utilized to determine the limits of sensitivity for each assay. The lower limit of sensitivity was usually between 1 and 2 mIU/ml. and never greater than 3.9 mIU/ml. The upper limit of sensitivity varied between 15.6 and 62.4 mIU/ml., but usually was 31.2 mIU/ml. To determine intra-assay variability one serum specimen was run in 9 tubes. The results for single tubes varied between 4.0 and 8.0 mIU/ml. Since triplicate specimens are used in the usual assay, the intra-assay variability was determined by calculating the mean value for tubes No. 1 $ 2 + 3, 2 + 3 + 4, etc. The mean of the resultant determinations was 6.2 mIU/ml. with a SD of 0.6 mIU/ml. To determine the inter-assay variability, 14 separate assays for a single serum specimen were done in triplicate. The mean and SD were 7.1 ‘_ 1.1 mIU/ml. with a range of 5.5 to 9.0.
Patient Material Ninety-seven normal boys whose heights were between the 25th and 75th percentiles for their ages (5 to 18 years) were bled between 7:OO and 8:00 a.m. A physical examination was done, and a stage of sexual development was assigned according to the classification of * Second International Reference Preparation which was kindly supplied by Dr. D. Bangham, London.
of Human Menopausal Gonadotropin National Institute for Medical Research,
236
MIT1
16-
x
tiYPOtONADOTROPlC
o
~IVPOPHYSECTOM~ZED
. .
14-
ET AL.
.
.
. .
.
.
Fig. l.-Serum FSH concentrations in males compared with chronological age.
MEAN 4.2 ~IS.0. to.7 5-8
,
5.2 t1.2
'
9-1,
5.8 8.0 1.5 t 2.0,?3.6, 2 1.91
8.5 +3.6
' 12 ' 13 ' 14-15 ' 16-18
1.4 r1.9 'ADULTS'
YEARS
Tanner.6 The sera were stored at -20” C until thawed for assay purposes. Thirty male physicians between the ages of 25 and 45 years also donated sera. The sera of five adult patients between the ages 20 and 32 years, and who had an isolated gonadotropin deficiency as determined by absence of sexual development, normal growth, and normal ACTH, HGH and TSH function also were tested, as were the serums of 4 adult patients with panhypopituitarism secondary to hypophysectomy for craniopharyngiomas. RESULTS
The mean FSH levels and their standard deviations for different age groups are presented in Fig. 1. FSH was found in all serums. There was a definite trend for the mean FSH level to increase with age, until at 13 years of age the mean FSH value (8.0 mIU/ml.) was approximately the same as that found for the older age groups. Statistical analysis using the “T” test revealed that the 5.0-8.0 age group differed significantly from all others (p < .Ol). Data from individuals in the 9.0-12 year age groups in Fig. 1 were pooled for purposes of comparison with the other groups. This Q.O-12-year group differed significantly (p < .Ol) from the younger and older age groups. After 13 years, there was no significant difference between groups. There was a great deal of scatter in all age groups after the age of 11. It was not determined whether this variability was attributable to a day to day variation or some other factor. Surprisingly, 4 of the 30 adult male subjects had values in the same low range as normal young male children. In Fig. 2, the stages of sexual development are correlated with the chronological ages of these male subjects. By the age of 13 years (Fig. 2)) all of the boys had developed sexually and 6 of the 9 in this age group were either in stages 3 or 4 of their sexual development. In Fig. 3, the FSH levels in serum are correlated with the stages of sexual development. The extreme variability of
IMMIJNOLOGICALLY
REACTIVE
.
...
I
.~e.
I
... .......
’ 5-6
... ’ 7-8
0.0 ‘..’
t:
... ..*.
.
.... .... .....
t:
A: ...
..
’ 9-m
237
FSH
::: . ..
. .
Fig. 2.-Stages of sexual development in normal males compared with chronological age.
I II-12 ’ 13-14 ’ 15-M ’ 17-19 ’ YEARS
serum concentrations noted previously for children after the age of 11 is apparent for individuals in stages 3, 4, and 5 of their sexual development. Stage 1 and 2 differed significantly from all other stages ( p < .Ol). There was no statistically significant difference in the mean values among stages 3, 4, and 5, or between them and those of normal adult males. Six of the 22 adolescent males in stage 5 had values within the range of 1 SD from the mean value for sexually infantile subjects. The correlation coefficient between serum FSH levels and urinary 17ketosteroid excretion is 0.49. This is statistically significant (p < 0.001) but is of low degree. The correlation coefficient between the serum FSH and serum LH levels1 on the same patients is 0.50. DIS~~JSSIOA-
Several authors2,5~7-10 have applied immunoassay procedures for determining small quantities of FSH in human serum or plasma. Different antiserum and different standards have been used, and values that have been reported consequently have not always been comparable. For example, the values obtained for normal males by Ode11 and Parlow were significantly higher than those found by Cargille and Rodbard. The utilization of a urinary standard (2nd IRP-HMG) which may not be ideal for measuring FSH of pituitary origin or in serum, and the possibility that FSH determined by immunologic techniques may not correlate with biologically active FSH, contribute to the difficulty in assessing or interpreting the values of serum FSH concentrations obtained by radioimmunoassay techniques. Those publishing FSH concentrations, and those reading such publications, must recognize
238
RAITI ET AL.
16 !
X HY POGONADOTAOPIC 0
HYPOPHYSECTOHIZED
.
14-
12-
IO-
Fig. 3.-Serum
FSH concen-
trafions in males compared with chronological age.
,g
.
6_
STAGE
- SEXUAL
DEVELOPMENT
that improvement and standardization of techniques will permit reporting more accurate actual determinations. Regardless, the utilization of FSH radioimmunoassays, which have been standardized within an individual laboratory and the limitations of which are recognized by the investigators, permits a comparative study of groups of subjects or various stimuli upon one individual. The current study was devised specifically to compare FSH levels in the sera of children of various ages and at various stages of sexual deveIopment, in order to better understand adolescent development. The study serves this purpose, and inspection of the data permits one to draw several conclusions. First, mean FSH levels for groups of normal boys begin to increase over the values of early childhood, between the ages of 9 and 11 years. They further increase until the children reach approximately 13 years of age and/or reach the third stage of sexual development, at which time the mean levels are comparable to those of the normal adult male. Secondly, our values in young adult males are consistent with those reported by Midgley2 for six similar subjects (3.5-8.6 mIU 2nd IRP-HMG), and may be consistent with values of biologically active FSH, since KelleP reported a value of 8.8 mIU/ml., when he assayed a pooled serum from adult males, and Kulin et al.l* reported a mean assay value of 11.5 mIU/ml. on a separate pool of adult male serum. However, our values should not be regarded as necessarily reflecting those of biologically active FSH in serum. Demura et al.,a and Midgley,2 using immunologic assays, have noted some FSH or FSH immunoreactive material in the sera of hypophysectomized and hypopituitary patients, and Kuhn et a1.13 using a bioassay, intermittently found detectable gonadotropin in pooled urine of preadolescent children. The latter group believed that small amounts of biologically active gonadotropin were
IMMUNOLOGICALLY
239
REACTIVE FSH
excreted during childhood. Therefore, it is probable that the small amount of immunologically reactive material which we detected in the sera of young children and hypopituitary adults is immunologically reactive FSH. When this study was initiated it was hoped that differentiation of hypogonadotropic from normal subjects could be made by assaying a single serum specimen. Unfortunately, the determination of FSH on a random specimen of serum will not always permit this differentiation. Sequential specimens may permit such differentiation, but this remains to be determined, since adequate sequential studies were not carried out in this project. Johanson et al.’ from this clinic have recently reported serum LH values for the same groups of boys and adult males. There is poor correlation between LH and FSH values in individual patients. Further comparison of the LH and FSH data, when expressed in mIU/ml. of 2nd IRP-HMG permits the deduction that mean LH values rise from childhood to adulthood (3.4 to 10.9) proportionately greater than do mean FSH values (4.2 to 7.4). Because we are measuring immunologic and not biologic activity one cannot expressly state, however, that there is an actual greater increase in biologically active LH than FSH. By comparing these data further, it is concluded that the urinary excretion of 17-ketosteroids correlates closely (Cor. Coef. = 0.73) with serum LH concentrations,l but to a much lesser extent: with the FSH concentrations (Cor. Coef. = 0.49). Since ?i or more of the 17-ketosteroid excretion of males is of adrenal origin, one must consider the possibility that LH can in some direct or indirect way affect the development of the zona reticularis of the adrenal, in addition to affecting the maturation of the mature leydig cells in the testes. There is no evidence from these data to suggest that FSH might have such an effect. ACKNOWLEDGMENTS Mr. Walter Davis and Mrs. M. Westervelt assisted in the technical and secretarial work, respectively. Dr. Leo Reichert prepared the FSH. Dr. A. R. Midgley Jr., prepared the FSH antiserum. The National Pituitary Agency, the Endocrinology Study Section, and the National Institutes of Arthritis and Metabolic Diseases made this work possible.
REFERENCES 1. Johanson,
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C., Guyda, H., and Blizzard, R. M.: Serum luteinizing hormone ( LH ) by radioimmunoassay in normal children. J. Pediatrics (in press). 2. Midgley, A. R., Jr.: Radioimmunoassay for human follicle stimulating hormone. J, Clin. Endocr. 27:295, 1967. 3. Donini, P., Puzzoli, D., D’Alessio, I., Lunenfeld, B., Eshkol, A., and Parlow, A. F.: Purification and separation of follicle stimulating hormone (FSH) and luteinizing hormone (LH) from human post-menopausal gonadotrophin ( HMG ) . I. Separation of FSH and LH by electrophoresis,
chromatography and gel filtration dures. Acta Endocr. 52:169, 1966. 4. Greenwood, F. C., Hunter, and Glover, J. S.: The preparation labelled human growth hormone specific radioactivity. Biochem. J.
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240 stein, D. P.: Radioimmunoassay of FSH utilizing 1251. Program of Endocrine Society, 1967 (Abstract 164). 8. Demura, H., Saxena, B. B., Gandy, H. M., and Peterson, R. E.: Radioimmunoassay for FSH and LH in human plasma. Program Endocrine Society, 1967 (Abstract 63). 9. Odell, W. D., and Parlow, A. F.: Some physiological studies of human FSH using radioimmunoassay. Program of Endocrine Society (Abstract 65). 10. Faiman, C., and Ryan, R. J.: Radioimmunoassay for human FSH. J. Clin. Endocr. 27:444, 1967.
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11. Keller, P. J.: Studies on pituitary gonadotropins in human plasma. II. Follicle stimulating and luteinizing hormone in male and post-menopausal plasma. Acta Endocr. (Kobenhavn) 52:348, 1966. 12. Kulin, H. E., Rifkind, A. B., and Ross, G. T.: Human LH activity in processed and unprocessed urine measured by radioimmunoassay and bioassay. J. Clin. Endocr. 28: 100, 1968. 13. -, -, Ross, G. T., and Odell, W. D.: Total gonadotropin activity in the urine of prepubertal children. J. Clin. Endocr. 27: 1123, 1967.