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Original Studies |
Department of Growth and Reproduction, Copenhagen University Hospital, DK-2100 Copenhagen Ø; and the Department of Biostatistics, University of Copenhagen (J.H.P.), DK-2200 Copenhagen N, Denmark
Address all correspondence and requests for reprints to: Elisabeth Carlsen, M.D., Department of Growth and Reproduction, GR 5064, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Serum inhibin B was measured in 13 healthy normal male volunteers (median age, 30 yr) by continuous blood drawing, with sampling every 30 min for 24 h. Blood samples were also analyzed for FSH, LH, testosterone, estradiol, and sex hormone-binding globulin.
We found a significant diurnal variation in inhibin B, with peak values in the early morning and nadirs in the late afternoon, followed by gradual increasing nocturnal values. An average decline of 3%/h from 0900 until 1700 h was calculated. Significant cross-correlation was found between inhibin B and testosterone as well as estradiol, whereas no cross-correlation was found between inhibin B and FSH. Two-dimensional time-series analyses revealed a statistically significant influence of testosterone on inhibin B. In addition, estradiol and inhibin B had a significant influence on one another.
In conclusion, we found a significant diurnal variation in inhibin B levels in normal men, with a pattern of higher values in the early morning hours and lower values in the late afternoon and evening. We did not find evidence for a role of FSH in this diurnal variation of inhibin B. However, covariation with serum levels of testosterone and estradiol suggested that these hormones might play a role in the diurnal rhythm of inhibin B, although some other common influence could not be excluded.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit and a
ßB-subunit, may be the only physiologically important form in the
male (1, 2). Inhibin B is of testicular origin and is believed to be
secreted primarily from the Sertoli cells. Recently, a negative
relationship between circulating inhibin B and FSH in normal adult
males has been demonstrated (2, 3).
The release of FSH and LH from the pituitary gland is episodic,
occurring in response to pulsatile GnRH release from the hypothalamus.
Testosterone and estradiol, in turn, also exhibit a pulsatile pattern
that seems to coincide with the gonadotropic stimulus, although the
response is somewhat attenuated. In addition to this pulsatile release,
an overall diurnal rhythm seems to exist for these hormones (4, 5).
Earlier reports have described a diurnal rhythm in the secretion of
inhibin (6, 7, 8). However, as these studies only determined the
-subunit of inhibin, they were unable to discriminate among the
various forms of inhibin present in plasma, i.e. the dimeric
inhibin A and inhibin B and the monomeric precursor forms. The present
study describes for the first time a diurnal pattern of inhibin B as
well as the relationship to gonadotropins and other gonadal
steroids.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Thirteen healthy male volunteers, aged 21–36 yr (median, 30 yr), were studied. They had no history of genital disorders, infertility, or chronic diseases and received no medication. They were all within 10% of ideal body weight. Furthermore, they had a blood sample drawn before entering the study to ensure an inhibin B level within the normal range, as established previously (9). All men agreed to participate by signing a consent form approved by the local human subject review committee.
Protocol
The subjects were admitted in the morning to our in-patient unit. A physical examination was performed, and a heparinized catheter was inserted into a forearm vein. Blood samples were withdrawn continuously (6 mL/h) for 24 h using a portable pump (Swemed lab pump, Carmeda, Sweden) into vacuumed vials containing gel. The vials were changed every 30 min. During the investigation, no restrictions regarding meals and sleep were made, but the subjects recorded their meals, and the period of sleep was registered. Blood sampling was discontinued in subject 3 after 11 h due to clotting of the catheter.
Hormone analyses
Blood was centrifuged, and serum was separated and stored at -20 C until assay. All samples were analyzed in duplicate for inhibin B, FSH, LH, testosterone, sex hormone-binding globulin (SHBG), and estradiol in our department.
Serum inhibin B was measured in a two-site enzyme immunometric assay
using a monoclonal antibody raised against the inhibin ßB-subunit in
combination with a labeled antibody raised against the inhibin
-subunit, as previously described (10). The detection limit was 18
pg/mL, and the intra- and interassay coefficients of variation were
15% and 18%, respectively.
Serum levels of FSH, LH, and SHBG were measured by time-resolved immunofluorometric assay (DELFIA, Wallac, Turku, Finland), with detection limits of 0.06 U/L, 0.05 U/L, and 0.5 nmol/L, respectively. Intra- and interassay coefficients of variation were below 8% for both FSH and LH and were below 6% for SHBG.
Serum levels of testosterone and estradiol were measured by RIA (Coat-a Count, Diagnostic Products, Los Angeles, CA; and Immunodiagnostic Systems, Boldon, UK, respectively). The detection limit for testosterone measurements was 0.23 nmol/L, and the intra- and interassay coefficients of variation were both less than 10%. For the estradiol assay, the detection limit was 18 pmol/L, the intraassay coefficient of variation was less than 8%, and the interassay coefficient of variation was less than 13%.
Statistics
The population median values of inhibin B over the hours of the day was estimated using a standard local linear regression technique (loess) (11), which assumes a Gaussian error distribution for the logarithm of inhibin B.
The possible temporal coupling within and between the hormone measurements for each individual was estimated by auto- and cross-correlations. Thereby, the correlation between individual hormone values within one time series and between two distinct hormone time series a distance (lag) k time units apart could be measured. Because of sustained high auto- and cross-correlations, in part due to diurnal patterns, a two-dimensional time series model including autoregressive terms for both hormones and a term accounting for a possible time trend (nonstationary) was introduced to model the dynamics of the hormones and to reflect synchronous endogenous time dependency (12, 13). Lagged measurements of one hormone were allowed to enter the model for the other hormone to account for the temporal coupling between the two hormones.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Figure 1
shows the diurnal profiles
of serum inhibin B in all subjects. A general trend, with highest
levels in the early morning and declining values toward the evening
followed by gradual increasing values overnight, was observed. The
value on the first morning, when the subjects arrived, was generally
lower than that on the following morning when the sample was taken with
the subject still resting. These diurnal variations, however, were not
present in subject 11, who had relatively constant inhibin B
values.
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. A more conventional cosinor analysis
with the determination of acrophase and amplitude was tried, but did
not describe the data as adequate, probably in part due to the
difference in the levels of inhibin B in the 2 consecutive morning
values for each individual. Eleven of the 12 subjects showed a diurnal
rhythm with a statistically significant nadir in the evening at
1907 h (range, 1553–2114 h) and a median peak on the following
morning at 0800 h (range, 0775–0875 h). The median decrease from
the highest to the lowest level of inhibin B was 37.2% (range,
17.9–49.5%). As expected from the profile of the untransformed values
of inhibin B, subject 11 did not show any statistically significant
variations.
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). The calculation was restricted to the
daytime hours, as in a clinical setting most patients will have blood
samples drawn during the day. An average decline in inhibin B of 21%
(range, 40.2 to -5.3%) from 0900–1700 h was calculated. This
decline, which was highly significant (P = 0.001),
corresponds to an approximate decline of 3%/h.
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Temporal coupling of inhibin B and other hormones
Figure 4 illustrates the
relationship among inhibin B, testosterone, estradiol, and FSH for one
of the subjects. Testosterone and to some extent estradiol seem to
follow approximately the same diurnal pattern as inhibin B, whereas the
diurnal pattern of FSH is different. Cross-correlations were calculated
in 11 of the 13 subjects (excluding subjects 3 and 11) to investigate
the possible temporal coupling between inhibin B and the other measured
hormones. As illustrated in Fig. 5, a
positive correlation was found between inhibin B and testosterone and
between inhibin B and estradiol; thus, high values of inhibin B were
preceded and followed by high values of testosterone and estradiol.
However, no cross-correlation was found between inhibin B and FSH. As
auto- and cross-correlations are very sensitive to time trends, a
two-dimensional time series model was applied. For each time point the
preceding measurements (lagged measurements) of inhibin B and the other
hormones were entered as covariates in a regression. Using this model,
the statistical influence of an individual hormone on other hormones
and the effect of lagged times could be assessed. Because the way
hormones interact may change during sleep, this analysis was performed
only for the wake hours. Table 1 shows
the results of this analysis. Serum testosterone had a significant
influence on the level of inhibin B, with a lag effect of 0.5–1 h,
whereas inhibin B did not influence the level of testosterone. In
addition, estradiol and inhibin B had a significant influence on one
another. No significant lagged effect was demonstrated for FSH and
inhibin B.
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For each subject (except no. 3), mean values of the 24-h measurements of log-transformed FSH, LH, testosterone, estradiol, and SHBG were calculated. These subject-specific mean values for each hormone were correlated to the corresponding mean values of inhibin B. In accordance with previous studies, serum inhibin B and FSH seemed to be negatively correlated (-0.54; P = 0.07). However, no significant correlations were obtained for any of the hormones. Similar results were obtained when only the measurements from the wake hours were included.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit. As in our study, they found a peak
level in the morning between 0700–0900 h, but Tenover et
al. (7) did not find a decline in inhibin until the late
afternoon. Furthermore, in all three studies, the nadir was found
during the night (6, 7, 8). This discrepancy with our results may be
explained by the use of different assays. We found a lower level of
serum inhibin B in the morning on the day of admission to the hospital
compared with that on the following morning. A possible explanation for
the lower level of serum inhibin B could be that the subjects on the
first morning of the study had to get up and come to the hospital,
whereas the following morning they usually had the last blood sample
drawn when they were still in bed. Whether the earlier hour of waking
up, the physical activity involved in transportation to the hospital,
or both played a role is not known.
Significant episodic peaks in inhibin -subunit have been described
previously by Brennemann (6). We could not exclude that episodic peaks
occurred in some of our subjects; however, the continuous
blood-sampling protocol used did not allow us to evaluate episodic
variation in inhibin B levels.
As expected (2, 3, 14), we found a negative correlation between mean values of FSH and inhibin B, although it was not statistically significant, probably due to the relatively small number of men studied. However, to our surprise we found no correlation between the diurnal changes in FSH and inhibin B levels within an individual. Thus, although FSH has an effect on the mean levels of inhibin B, the diurnal changes in inhibin B might be regulated by other factors, i.e. the intrinsic levels of other gonadal hormones and probably also interaction with germ cells (15, 16). In accord with this, it has been suggested that only about 70% of serum inhibin B is gonadotropin dependent (1, 14).
No correlation was found when mean values of inhibin B were compared
with the corresponding mean values of testosterone and estradiol.
However, in support of an influence of intrinsic testicular factors on
inhibin B, we found a positive correlation between inhibin B and
testosterone and between inhibin B and estradiol over the 24-h period.
Our data are in line with results obtained in
previous studies using assays, which were unable to discriminate
between the bioactive dimeric forms and the monomeric -subunit of
inhibin. Thus, Brennemann et al. (6) and Yamaguchi et
al. (8) found a significant correlation between testosterone and
inhibin in peripheral blood, and after catheterization of the
spermatic veins, studies have demonstrated episodic cosecretions of
testosterone and estradiol (17) and of testosterone and inhibin
(18).
Our findings of significant intraindividual diurnal variation in serum inhibin B levels have important implications for the use of inhibin B as a clinical tool. In most of the individuals we found that the late afternoon values were 30–40% lower than those during the early morning hours. Even during the daytime hours, when most blood samples are drawn in the out-patient clinics, we found a quite pronounced decline in serum levels of inhibin B of about 3%/h. Thus, caution should be taken when longitudinal values of an individual are compared unless the blood samples were taken at the same time of the day. One should, however, bear in mind that this estimate is based on only 12 subjects and should, therefore, be taken merely as an indicator of the magnitude of the decline rather than a measure by which values from individual subjects can be accurately adjusted. Furthermore, one individual showed no significant variation in inhibin B during the 24-h period, indicating that individual differences in the diurnal pattern occur.
In conclusion, we found a significant diurnal variation in inhibin B levels in normal men, with a pattern of higher values in the early morning hours and lower values in the late afternoon and evening. We did not find evidence for a role of FSH in this diurnal variation in inhibin B. However, covariation with serum levels of testosterone and estradiol suggested that these hormones might play a role in the diurnal rhythm of inhibin B, although some other common influence could not be excluded.
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Footnotes |
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Received September 3, 1998.
Revised October 29, 1998.
Revised February 8, 1999.
Accepted February 17, 1999.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit mRNA levels by specific germ cell types. Mol Cell
Endocrinol. 72:13–22.[CrossRef][Medline]
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