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Original Studies |
Department of Pediatrics, University of Parma (L.G., M.E.S., A.V., G.M., M.V.), 43100 Parma, Italy; and the Department of Pediatrics, University of Modena (S.B.), 41100 Modena, Italy; Evgenidion Hospital, Endocrine Unit, Athens University Medical School (G.M.), 11528 Athens, Greece; and the Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health (G.P.C.), Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Lucia Ghizzoni, M.D., Department of Pediatrics, University of Parma, Via Gramsci 14, 43100 Parma, Italy. E-mail: lughizzo{at}IPRUNIV.CCE.UNIPR.IT
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In man, endogenous hypercortisolism (Cushing’s syndrome) is associated with a decrease in mean 24-h plasma TSH caused by a decrease in TSH pulse amplitude (10). Pharmacological doses of dexamethasone rapidly suppressed basal serum TSH levels and completely inhibited the pulsatile secretion of this hormone, but left the TSH response to exogenous TRH unaffected (11). Activation of the stress system is associated with decreased production of TSH, which may be caused by the increased levels of glucocorticoids (9, 12). CRH-stimulated increases in SRIH might also participate in the central component of thyroid axis suppression during stress, which is counteracted by the stimulatory effects of the activated locus ceruleus-norepinephrine system (13). On the other hand, in patients with primary adrenal insufficiency, plasma TSH levels are increased and return to normal after corticosteroid replacement (14, 15, 16). Such findings suggest a direct suprapituitary and/or pituitary effect of glucocorticoids on TSH secretion. In normal subjects, Re et al. (1) found an increase in TSH within 24 h in response to metyrapone, suggesting that physiological plasma cortisol levels may have a suppressive effect on TSH secretion. However, abolition of the circadian cortisol rhythm by metyrapone administration did not lead to disruption of the TSH circadian rhythm (17).
In normal men and women it appears that the diurnal rhythms of cortisol and TSH are in reverse phase (18), suggesting that the hypothalamic-pituitary-adrenal and thyroid axes may have reciprocal interactions. As most of the studies on the interactions between these axes have been performed in disease states or under experimental conditions using pharmacological doses of glucocorticoids, we investigated the relation between these two axes under physiological or slightly disturbed conditions. To do this, we evaluated the spontaneous cortisol and TSH secretion in short normal children and in children with nonclassical congenital adrenal hyperplasia (NCCAH) due to 21-hydroxylase deficiency (19), who had mild nocturnal cortisol insufficiency (20, 21).
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Subjects and Methods |
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This study was approved by the Clinical Research Committee of
the Department of Pediatrics at the University of Parma (Parma, Italy).
Eight patients with NCCAH and eight normal children were studied. The
clinical characteristics of all subjects are summarized in Table 1
. All patients were clinically and
biologically euthyroid. Integrated concentrations of T4,
T3, free T4 (FT4), and free
T3 (FT3) from the 12-h daytime and nighttime
periods of control and NCCAH children are shown in Table 2. The diagnosis of NCCAH was based on a
serum 17-hydroxyprogesterone (17-OHP) level exceeding 45 nmol/L 60
min after an iv bolus dose of 250 µg corticotropin (Synacthen,
Ciba-Geigy, Basel, Switzerland). Patients 3 and 7 had no symptoms
and were identified while testing family members of affected patients.
All other patients presented with or had a history of premature
pubarche. Control children were chosen among subjects with familial
short stature whose hypothalamic-pituitary function was normal and were
matched to NCCAH patients according to baseline adrenal steroid levels,
which were all within the normal range for Tanner stage I or II for
breast in girls and for testicular size in boys (22) and were similar
in the two groups.
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At 1000 h, after an overnight fast, an indwelling nonthrombogenic catheter was inserted into an antecubital vein and connected to a portable constant withdrawal pump, according to the method of Kowarski et al. (23). The rate of withdrawal was 4 mL/h, and blood collection tubes were changed every 30 min for 24 h. During this time, children were encouraged to continue normal activity and had a standard hospital diet. We arbitrarily chose the period from 2200 h in the evening to 1000 h in the morning and named it nighttime, because we expected the main circadian peaks of cortisol and TSH during this period. We started the pulsatility study at 1000 h to avoid dividing the morning cortisol surge.
Blood samples for measurements of cortisol, TSH, T4, T3, FT4, and FT3 concentrations were kept at room temperature and centrifuged within 24 h. After centrifugation, serum was stored at -20 C until assayed. T4, T3, FT4, and FT3 levels were measured in pooled samples collected during the 12-h daytime and nighttime periods.
Bone age was determined by the method of Greulich and Pyle (24).
Hormone assays
Commercial kits were used for the estimation of serum cortisol (RIA, Radim, Pomezia, Italy), TSH (immunoradiometric assay, Nichols Institute, San Juan Capistrano, CA), T3, T4, FT3, and FT4 (enzyme-linked immunosorbent assay, Boehringer Mannheim, Mannheim, Germany), and 17-OHP (RIA, Diagnostic Products Corp., Los Angeles, CA) concentrations. The sensitivities of the assays were 2.48 nmol/L for cortisol, 0.04 mU/L for TSH, 0.46 nmol/L for T3, 7.7 nmol/L for T4, 0.46 pmol/L for FT3, 1.3 pmol/L for FT4, and 0.2 nmol/L for 17-OHP. Mean intra- and interassay coefficients of variation were 4.1% and 6.5% for cortisol, 4.4% and 6.8% for TSH, 5.2% and 9.8% for T3, 4.9% and 8% for T4, 4.2% and 5% for FT3, 5.6% and 9.1 for FT4, and 4.6% and 5.1% for 17-OHP, respectively.
Pulse analysis
The Pulsar program was used to quantitate the pulse properties of cortisol and TSH time series objectively (25). Samples were analyzed for mean 24- and 12-h serum hormone concentrations, area under the curve above baseline (AUCb), area under the curve above zero line (AUCo), number of significant pulses, mean pulse height, mean pulse amplitude, mean pulse area, mean pulse length, and mean interpulse interval. The cut-off parameters G1–5 were set at 5.78, 2.89, 1.84, 1.27, and 0.89 times the intraassay SD as criteria for accepting peaks 1, 2, 3, 4, and 5 points wide, respectively. The smoothing time was set at half the total profile time, that is 12 h (24 points) and 24 h (48 points) for the 12- and 24-h profiles, respectively.
Statistical analysis
Values are reported as the mean ± SEM unless otherwise stated. A test for normality was performed on all data. Statistical significance was determined by the Wilcoxon signed rank test or the Wilcoxon rank sum test, as appropriate. P < 0.05 was considered significant.
Correlation analysis
To search for a time-ordered relation between TSH and cortisol,
we staggered and correlated the arithmetic or log-transformed values of
the concentration-time series of TSH with those of cortisol. As
circadian periodicity in the TSH and cortisol series might result in
significant correlation between them, reflecting the relative phases of
the two circadian rhythms, we looked for the presence of positive or
negative correlations on a finer time scale between the detrended
series (using a five-point moving average) and the first difference
series (
series: second value minus first, third minus second,
etc.). These transformations mitigate the effect of baseline
shifts and the circadian component. All of the above correlations were
performed using Statistica software for IBM computers (26).
Cross-correlation analysis between cortisol and TSH was computed at various time lags covering the 24-h study period, as previously described (21).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Daytime (1000–2200 h) and nighttime (2200–1000 h) mean, mean
AUCb, AUCo, peak characteristics (height, amplitude, area, and length),
interpeak interval, and number of peak TSH values (±SEM)
in the control and NCCAH groups of children are reported in Table 3. In both groups, mean and mean AUCo
values were higher at night than during the day (P <
0.05). In control children, the nighttime mean pulse height and number
of pulses were also higher than those during the day (P
< 0.05). The rest of the peak characteristics did not show a day/night
difference in this group. In NCCAH children, nighttime mean AUCb, pulse
height, area, and length were higher than those during the day
(P < 0.05).
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The mean TSH values and the secretory characteristics analyzed over the
24 h did not differ between the two groups (data not shown). No
difference was observed in the 12-h pooled (daytime and nighttime)
T4,T3, FT4, and FT3
values (Table 2
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Cortisol 24-h profile and pulsatility in control and NCCAH children
Daytime (1000–2200 h) and nighttime (2200–1000 h) mean, mean
AUCb, AUCo, peak characteristics (height, amplitude, area, and length),
interpeak interval, and number of peak cortisol values
(±SEM) in the control and NCCAH groups of children are
reported in Table 4. In the control
group, mean, mean AUCb, AUCo, and peak height, amplitude, and area
values were higher at night than during the day (P <
0.05). In the NCCAH group, most of these values were lower at night
than during the day; however, none of them was significantly
different.
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The mean cortisol values and the secretory characteristics analyzed over 24 h did not differ between the two groups (data not shown). Data for pulsatile cortisol secretion were concordant with those reported previously (23).
Correlation analyses
The correlation analysis at lag 0 of raw and values, after
logarithmic transformation and detrending, showed a positive, but not
significant, correlation between cortisol and TSH in both control and
NCCAH groups of children (data not shown).
The graphs depicting the mean coefficients of correlation from the
cross-correlation analyses over the 24 h between the cortisol and
TSH raw values of both groups are shown in Fig. 2. A strongly significant negative
correlation over time (Fig. 2a) was observed between cortisol and TSH
concentrations, peaking at lag time 2.5 h for both the control and
NCCAH groups of children (P < 0.05), with cortisol
leading TSH by this time interval. Similar coefficients of correlation
were observed from 2–2.5 h and from 2–4 h lag times for control and
NCCAH children, respectively. In addition, a slightly significant
positive correlation was observed over time between cortisol and TSH
concentrations, peaking at lag time 13 and 14 h for the control
and NCCAH groups of children (P < 0.05), respectively,
with cortisol leading TSH by these time intervals (Fig. 2b).
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addition, a significant negative correlation was observed over time
between cortisol and TSH concentrations, peaking at lag time -16.5 and
-17.5 h for the control and NCCAH groups of children, respectively,
with TSH leading cortisol by these time intervals (Fig. 2d).
When the cross-correlation analysis was performed between the cortisol
and TSH and detrended values, no significant correlation was
found.
Comparison of TSH secretory profiles in control and NCCAH children after 3-h lagging of NCCAH TSH time series
To verify whether the daytime differences in TSH secretory parameters between the two groups were due to the distinct secretory profile of TSH in the NCCAH group, we analyzed the TSH daytime and nighttime time series of the NCCAH children after shifting them backward by 3 h with respect to control children. We did so, based on the results of the cross-correlation analysis showing an absolute 3-h difference between the lag time of the stronger positive correlation of TSH over cortisol time series in both the control (8.5 h) and NCCAH (5.5 h) groups. Specifically, the 1000–2200 and 2200–1000 h TSH values of the control children were compared with the 1300–0100 and 0100–1300 h values of the patients. In contrast to the previous analysis, there was no difference in the daytime secretory characteristics of TSH between the two groups. Nighttime TSH secretory parameters remained similar in the two groups.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The relation between the 24-h concentration-time series of plasma cortisol and TSH raw values, as demonstrated by cross-correlation analyses, was almost similar in the two groups. The highest negative correlation was observed when cortisol preceded TSH values by a 2.5-h lag in both groups of children, suggesting that cortisol might negatively regulate TSH secretion even under physiological conditions. This reciprocal relation might reflect a direct negative effect of glucocorticoids on TRH and TSH. Alternatively, glucocorticoids might exert their negative regulation indirectly through inhibition of the noradrenergically mediated stimulation of TRH (27). Furthermore, CRH-activated SRIH release also might be responsible for TRH and TSH inhibition. It has been shown that glucocorticoid secretion during stress is associated with TSH inhibition due to CRH-induced SRIH increase (13). It should be pointed out that this negative correlation of cortisol over TSH was sustained over a longer period of time in NCCAH than in control children. This probably reflects the greater variability in cortisol secretion in this group due to different levels of 21-hydroxylase deficiency (28).
A strong positive correlation between the two hormones was observed at
-8.5 and -5.5 h lag times for the control and NCCAH groups of
children, respectively, with TSH leading cortisol, indicating that
sustained secretion of TSH precedes a cortisol surge by these time
intervals. The shorter interval between the secretory phases of these
two hormones observed in the group of NCCAH children suggests that in
these patients the nocturnal TSH peak occurs temporally closer to the
morning cortisol peak than in controls. This is also evident with
simple inspection of the secretory hormone profiles (Fig. 3). This temporally delayed TSH secretion
in NCCAH children might be due either to the lower nighttime cortisol
levels and/or to a glucocorticoid-unopposed norepinephrine-induced
TRH stimulation. Furthermore, the nighttime low cortisol secretion in
the NCCAH children might also be responsible for the lower daytime TSH
release via mild nocturnal cortisol deficiency-induced CRH
hypersecretion and stimulation of SRIH resulting in rearrangement of
the TSH circadian rhythmicity. In fact, the quantitative daytime
difference in TSH secretion disappeared when the TSH levels of the
control group were compared with those of the NCCAH group shifted
backward by 3 h. The latter is the absolute temporal difference
between the lag time of the positive correlation of TSH over cortisol
in the two groups. The slightly higher levels of cortisol secretory
parameters during the day in NCCAH children than in control children
might also reflect and/or explain the difference in daytime TSH
secretion in the patient group.
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Footnotes |
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Received March 3, 1997.
Revised July 8, 1997.
Accepted July 17, 1997.
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for all children at the lag time 