European Journal of Endocrinology (1997) 137 172–175
`
`ISSN 0804-4643
`
`SHORT COMMUNICATION
`
`One microgram is the lowest ACTH dose to cause a maximal
`cortisol response. There is no diurnal variation of cortisol
`response to submaximal ACTH stimulation
`
`Gabriel Dickstein, Doron Spigel, Eldad Arad and Carmela Shechner
`
`Division of Endocrinology, Bnai Zion Medical Center, Haifa 31048, Israel
`
`(Correspondence should be addressed to G Dickstein, Division of Endocrinology, Oregon Health Sciences University, L607, 3181 SW Sam Jackson Park
`Road, Portland, Oregon 97201–3098, USA)
`
`Abstract
`
`There are many suggestions in the literature that the adrenal gland is more sensitive to ACTH in the
`evening than in the morning. However, all these studies in humans were conducted when the basal
`cortisol level was not suppressed, and were based on the observation that, after stimulation, the
`increases in cortisol differed, though the peak values were the same. To examine this, we established
`the lowest ACTH dose that caused a maximal cortisol stimulation even when the basal cortisol was
`suppressed, and used a smaller dose of ACTH for morning and evening stimulation. The lowest ACTH
`dose to achieve maximal stimulation was found to be 1.0 mg, with which dose cortisol concentration
`increased to 607.2 ⫾ 182 nmol/l, compared with 612.7 ⫾ 140.8 nmol/l with the 250 mg test
`(P > 0.3). The use of smaller doses of ACTH (0.8 and 0.6 mg) achieved significantly lower cortisol
`responses ( 312 ⫾ 179.4 and 323 ⫾ 157.3 nmol/l respectively; both P < 0.01 compared with the 1 mg
`test). When a submaximal ACTH dose (0.6 mg) was used to stimulate the adrenal at 0800 and 1600 h,
`after pretreatment with dexamethasone, no difference in response was noted at either 15 min
`(372.6 ⫾ 116 compared with 394.7 ⫾ 129.7 nmol/l) or 30 min (397.4 ⫾ 176.6 compared with
`403 ⫾ 226.3 nmol/l; P > 0.3 for both times). These results show that 1.0 mg ACTH, used latterly as
`a low-dose test, is very potent in stimulating the adrenal, even when baseline cortisol is suppressed;
`smaller doses cause reduction of this potency. Our data show that there is probably no diurnal
`variation in the response of the adrenal to ACTH, if one eliminates the influence of the basal cortisol
`level and uses physiologic rather than superphysiologic stimuli.
`
`European Journal of Endocrinology 137 172–175
`
`Introduction
`
`It was previously suggested (1–4) that the adrenal
`gland is more sensitive to stimuli in the evening than
`in the morning. These studies
`showed that,
`in
`humans, the increase in cortisol in the evening was
`higher than that in the morning,
`in response to
`adrenocorticotropic hormone (ACTH)
`(1),
`insulin-
`induced hypoglycemia (2) or corticotropin-releasing
`hormone (CRH) (3). However, in all these studies the
`peak cortisol concentration was similar, and unrelated
`to the time of day. The studies therefore do not
`necessarily indicate a greater response, but may reflect
`only the maximal ability of the adrenal to respond to
`ACTH (4). In rats, however, it was shown that, not
`only was the increase in corticosterone greater in the
`evening, but the peak cortisol value was also higher,
`thus truly demonstrating a greater evening response
`(5). In a previous study (4) we tried to eliminate the
`effect of the basal cortisol level on the final result, by
`
`䉷 1997 Society of the European Journal of Endocrinology
`
`pretreatment with dexamethasone. We found that,
`under these conditions, while the 30-min response to
`ACTH was the same, there seemed to be an earlier
`increase in cortisol in the evening. We concluded that
`this might indeed indicate a greater sensitivity of the
`adrenal to ACTH in the evening.
`Latterly, after we introduced the 1.0 mg ACTH test
`(4), we became interested in identifying the lowest
`ACTH dose that would still cause a maximal adrenal
`response, unrelated to the basal cortisol
`level. We
`hypothesized that, by using a smaller dose than that at
`different times of the day, we might disclose a different
`adrenal response, representing a difference in adrenal
`sensitivity to ACTH at different times of day.
`
`Subjects and methods
`
`Ten normal volunteers (five women, five men, aged
`20–56 years) were tested with the low-dose (1.0 mg)
`
`Amerigen Exhibit 1175
`Amerigen v. Janssen IPR2016-00286
`
`

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`EUROPEAN JOURNAL OF ENDOCRINOLOGY (1997) 137
`
`Adrenal response to low-dose ACTH
`
`173
`
`*
`
`*
`
`1000
`
`750
`
`500
`
`250
`
`0
`
`Cortisol (nmol/l)
`
`0.6
`
`0.8
`
`1.0
`
`250
`
`ACTH dose used (mg)
`
`Figure 1 Plasma cortisol concentrations 30 min after i.v. injection of
`different doses of ACTH(1–24) to ten normal volunteers pretreated
`with 1 mg dexamethasone 8 h before the test. Vertical bars
`represent means. *P < 0.01 for responses to 1.0 and 250 mg ACTH
`compared with those to 0.8 and 0.6 mg ACTH.
`
`dexamethasone (Fig. 2), no difference was noted
`between basal
`levels (41.4 ⫾ 35.9 compared with
`38 ⫾ 28.5 nmol/l) or at 15 min (372.6 ⫾ 116 com-
`pared with 394.7 ⫾ 129.7 nmol/l) or 30 min (397.4 ⫾
`176.6 compared with 403 ⫾ 226.3 nmol/l; P > 0.3 for
`both times).
`
`Discussion
`
`It has been claimed that there is circadian regulation of
`the sensitivity of the response of the adrenal cortex to
`ACTH (1–5, 7). The absolute increment in cortisol
`response to an ACTH stimulation test is greater when
`the test is performed at circadian nadir than at the peak
`
`and with the conventional (250 mg) ACTH tests, 8 h
`after pretreatment with 1.0 mg dexamethasone. Seven
`of the ten volunteers participated in both studies; the
`remaining three differed in the two studies. All tests
`were conducted between 0800 and 0900 h.
`All ten normal volunteers from the previous 1.0 mg
`test group (five women, five men, aged 20–26 years),
`were then given 0.8 and 0.6 mg ACTH i.v. between 0800
`and 0900 h, after pretreatment with 1.0 mg dexa-
`methasone at midnight. At least 48 h were allowed to
`pass between the different tests, and the order of the
`tests was changed between participants.
`In the third part of the study, 0.6 mg ACTH was
`injected i.v. to ten volunteers (five women, five men,
`aged 18–28 years) at 0800 h and at 1600 h, 8 h after
`pretreatment with 1.0 mg dexamethasone. At least 48 h
`were allowed between the two tests, and their order was
`reversed in 50% of the subjects.
`Cortisol was measured using a solid phase RIA as
`previously described (4). ACTH solutions for i.v. injec-
`tion were kept diluted to 5 mg/ml and refrigerated for up
`to 4 months. Further dilution was made shortly before
`the test. This procedure was proven to be satisfactory in
`earlier studies (4).
`
`Analysis of data
`
`Results are presented as means ⫾ S.D. For multiple
`comparisons, ANOVA was performed. This test was
`followed by the Mann–Whitney test for the 1.0 and
`0.8 mg tests. Student’s t-test was performed for paired
`comparison of the morning and evening tests.
`
`Results
`
`Response to low ACTH concentrations
`
`Figure 1 compares the cortisol response to stimula-
`tion with 0.6, 0.8, 1.0 and 250 mg ACTH after
`suppression of basal cortisol. No difference was
`found at 30 min between the 1.0 and 250 mg doses
`(607.2 ⫾ 182 compared with 612.7 ⫾ 140.8 nmol/l
`respectively, P = 0.86).
`At 30 min, there was a significantly lower response to
`0.8 mg than to 1.0 mg (312 ⫾ 179.4 compared with
`607.2 ⫾ 182 nmol/l, P < 0.01). No further reduction in
`response to 0.6 mg was noted (323 ⫾ 157.3 nmol/l,
`P = 0.95).
`Only three of the volunteers in the 0.6 mg test,
`and three in the 0.8 mg test had a 30-min cortisol level
`of 500 nmol/l or greater, which would be considered
`a normal stimulation (6). In contrast, all volunteers
`showed a 30-min cortisol level of more than 500 nmol/l
`in response to the 1.0 mg test.
`
`Effect of time of day
`
`When the cortisol responses to 0.6 mg ACTH at 0800 h
`and 1600 h were compared after pretreatment with
`
`Figure 2 Plasma cortisol concentrations in response to 0.6 mg
`ACTH i.v. at 0800 and 1600 h, after pretreatment with 1.0 mg
`dexamethasone 8 h before the test.
`
`

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`174
`
`G Dickstein and others
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`EUROPEAN JOURNAL OF ENDOCRINOLOGY (1997) 137
`
`(1, 8). This is true also for the cortisol response to
`insulin-induced hypoglycemia (2) and to CRH (3).
`It is known that injection of very low doses of ACTH is
`enough to evoke some adrenal production of cortisol. It
`has been shown that 400 ng b-ACTH(1–24) caused an
`increment of about 190 nmol/l cortisol (9). DeBold
`(personal communication) found that i.v. injection of
`2.0 ng b-ACTH(1–24) caused a mean increase in
`plasma ACTH of 5 pmol/l within 5 min, with a
`subsequent mean increase in plasma cortisol of
`165 nmol/l 10 min later. However, these low doses of
`ACTH are effective only when introduced i.v.; s.c.
`injection with 2.5 mg (10) or even 4.0 mg (11) ACTH
`will cause no increase in plasma cortisol. Daidoh et al.
`(12) found that 0.5 mg ACTH i.v. was the smallest dose
`that caused maximal stimulation of cortisol under
`normal conditions, starting with normal basal cortisol
`levels of about 200 nmol/l.
`We tried to identify the lowest dose of ACTH that will
`cause normal adrenal stimulation, even when basal
`cortisol level is suppressed. One microgram ACTH, the
`dose used for the low-dose ACTH test, was found to be
`that dose. This probably shows that the basal cortisol
`level is not important for interpretation of the low-dose
`ACTH test, and that this test can be performed at any
`time of the day.
`A trial
`to reduce further the ACTH dose used
`produced a significant
`reduction in the adrenal
`response. Moreover, the response range widened con-
`siderably; some individuals showed a normal increase in
`cortisol, but most did not. This is further proof that the
`1.0 mg dose used for the low-dose test is indeed the most
`appropriate dose.
`It should be emphasized again,
`however,
`that
`these studies were conducted after
`baseline suppression of cortisol. It might be, therefore,
`that many patients will continue to respond normally to
`these very low doses of ACTH (0.8 or 0.6 mg) if the
`baseline cortisol level is normal, as suggested by Daidoh
`et al. (12); others, however, will probably not, so that the
`specificity of the test will deteriorate.
`We then used the sub-maximal dose of 0.6 mg ACTH
`to examine if there is truly a diurnal change in the
`adrenal response to ACTH (1–3, 6, 7). We did not
`consider an absolute increment in cortisol response to
`be good enough a criterion to prove such greater
`adrenal sensitivity, and believed that starting the test
`from the same basal cortisol
`levels is crucial
`for
`interpretation of the results. In our previous paper (4)
`we achieved that by pretreatment with dexamethasone,
`as did Kaneko et al. (5) in their study on rats, in which
`they indeed found a greater sensitivity of the adrenal in
`the evening. One must remember, though, that in the
`rat, a nocturnal animal, corticosterone levels are higher
`in the evening than in the morning, and a higher
`adrenal sensitivity in the evening should therefore
`not be surprising, unlike in humans. However, in our
`earlier study we used a pharmacologic rather than a
`physiologic dose of ACTH (250 mg), and under those
`
`conditions we found that the 30-min cortisol response
`was equal in the morning and in the evening, though at
`15 min the evening response was significantly higher
`(4). We interpreted these results as proof of a greater
`adrenal sensitivity in the evening. However, from the
`present study, using a sub-maximal dose of ACTH, one
`can see that when the cortisol increase is less than
`maximal, no difference in the response can be noted
`between morning and afternoon results. In our opinion,
`this proves that there is no real diurnal difference in
`response, and that all the data previously presented that
`claimed such a difference in man merely represented
`achievement of
`the same maximal-possible cortisol
`level, starting from a different baseline and therefore
`representing a different increment.
`We conclude that 1.0 mg ACTH is sensitive for
`measuring adrenal responses, even when the basal
`cortisol level is suppressed, and that this is probably the
`lowest ACTH dose that will produce a maximal
`response. Smaller doses of ACTH give variable responses
`when baseline cortisol is low. Use of such lower doses
`reveals no diurnal variation in adrenal cortisol response
`to a physiologic ACTH stimulus.
`
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`Received 6 January 1997
`Accepted 29 April 1997