Clinical Endocrinology (2000) 53, 199±204
`
`Establishment of reference values for standard dose
`short synacthen test (250 mg), low dose short
`synacthen test (1 mg) and insulin tolerance test for
`assessment of the hypothalamo±pituitary±adrenal axis
`in normal subjects
`
`J. Gonza lbez*, C. Villabona*, J. Ramo n², M. A.
`Navarro³, O. Gime nez*, W. Ricart§ and J. Soler*
`*Departments of Endocrinology and ²Public Health and
`³Hormone Unit, Ciutat SanitaÁ ria i UniversitaÁ ria de
`Bellvitge, Universitat de Barcelona, Barcelona and
`§Department of Endocrinology, Hospital Dr Josep
`Trueta, Girona, Spain
`
`(Received 15 November 1999; returned for revision 12 January
`2000; ®nally revised 14 February 2000; accepted 5 April 2000)
`
`5th percentile in each test were: at ‡ 30 minutes:
`(HDT: 537, LDT: 489 nmol/l), peak: (HDT 649, LDT
`498, ITT: 539 nmol/l).
`CONCLUSIONS Comparison of the plasma cortisol
`response at ‡ 30 minutes with both short ACTH tests
`and the peak in the insulin tolerance test did not
`reveal differences. Each test, for each time point
`and for each biochemical method, requires its own
`minimum threshold of normality to assess the
`hypothalamo±pituitary±adrenal axis.
`
`Summary
`
`OBJECTIVE To assess the integrity of the hypotha-
`lamo±pituitary±adrenal(HPA) axis, many authors
`have proposed the short synacthen test (ACTH1±24,
`Tetracosactrin) as a replacement for the insulin toler-
`ance test (ITT). The aim of this study was to compare
`the plasma cortisol response obtained with both short
`synacthen tests (high dose (HDT, 250 mg) and low
`dose (LDT, 1 mg)) with the peak reached during the
`ITT in healthy volunteers, and to establish the plasma
`cortisol cut-off level in each test.
`SUBJECTS AND METHODS Thirty healthy subjects
`(16 F, 14 M), mean age 34 years, underwent both
`short synacthen tests. Twenty healthy subjects, 15
`of whom (11 F, nine M) belonged to the above
`group, mean age 30 years, underwent an ITT. Plasma
`cortisol was measured using a chemiluminiscence
`immunoassay.
`RESULTS There were no differences between plasma
`cortisol 30 minutes after both short synacthen tests
`(HDT: 684 6 123, LDT: 669 6 119 nmol/l) and the peaks
`reached with the LDT (691 6 123 nmol/l) and the ITT
`(673 6 99 nmol/l).The only difference (P < 0´001) was
`found in the comparison of plasma cortisol peak
`reached with the HDT (802 6 142 nmol/l) with the
`other tests. Plasma cortisol levels obtained in the
`
`Correspondence: J. GonzaÂlbez MorgaÂez (c) Esteve Pila 48±54 B
`8221, (08190) Sant Cugat del ValleÁs, Barcelona, Spain. E-mail:
`josegonzalbez@arrakis.es
`
`The insulin tolerance test (ITT) is considered the standard
`reference test for assessing the integrity of the hypothalamo±
`pituitary±adrenal (HPA) axis (Plumpton & Besser, 1969;
`Grinspoon & Biller, 1994). Plasma cortisol responses to hypo-
`glycaemia correlate well with response to surgical stress
`(Plumpton & Besser, 1969). However, it is well known that
`this test is unpleasant for the patient, potentially hazardous in
`patients with coronary heart disease or with a history of
`seizures, and is not advisable in children and the elderly. In
`addition, close medical supervision is required, and the test is
`relatively expensive.
`As a result, many authors have examined other tests,
`including short synacthen (ACTH1±24)
`tests standard,
`conventional or high dose (HDT, 250 mg) or low dose (LDT,
`1 mg) metyrapone, naloxone, and the CRH test (Lindholm et
`al., 1978; Lindhom & Kehlet, 1987; Crowley et al., 1991;
`Broide et al., 1995; Kane et al., 1995; Tordjman et al., 1995;
`Ammari et al., 1996; Hurel et al., 1996; Orme et al., 1996;
`Wang et al., 1996; Mukherjee et al., 1997). In most of these
`tests adequate maximum plasma cortisol response usually
`varied between 500 to 600 nmol/l for each test, though many of
`the studies did not include a control group.
`Since Lindhom & Kehlet (1987) found a highly signi®cant
`correlation between the plasma cortisol reached at ‡ 30 minutes
`during the HDT and the plasma cortisol peak reached during the
`ITT, the HDT has increasingly gained acceptance as a good
`substitute for the ITT. However, the test result only re¯ects the
`presence or absence of adrenocortical atrophy secondary to
`corticotrophin insuf®ciency (Hjortrup et al., 1983). Furthermore,
`
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`
`199
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`
`200 J. Gonza lbez et al.
`
`the HDT uses a
`several researchers have considered that
`supraphysiological dose and is not sensitive enough to detect
`mild degrees of secondary adrenal insuf®ciency. They report
`that
`the HDT underdiagnoses clinically signi®cant adrenal
`insuf®ciency, with potentially serious consequences (Borst et
`al., 1982; Cunningham et al., 1983; Tsatsoulis et al., 1988;
`Ammari et al., 1996; Soule et al., 1996; Streeten et al., 1996).
`In recent years several authors have reduced the dose,
`demonstrating that the plasma cortisol response to 1 mg is
`equivalent to that obtained with 250 mg in normal subjects
`(Oelkers et al., 1988; Crowley et al., 1991; Daidoh et al., 1995;
`Rasmuson et al., 1996; Talwar et al., 1998) and that the test is
`thus able to detect mild adrenal
`insuf®ciency. Without
`modifying the reference test's threshold of stimulated plasma
`cortisol, Tordjman et al. (1995) and Dickstein et al. (1991)
`found that the LDT increased sensitivity for assessment of
`secondary adrenal insuf®ciency. However, Mayenknecht et al.
`(1998) established that the diagnostic sensitivity of the tests is
`the same, provided different thresholds of normality are applied
`for each test. The lower normal
`limit of plasma cortisol
`response varies widely from study to study and this is one cause
`of discrepancies in sensitivity and speci®city. To address this
`issue we attempted to compare the response of plasma cortisol
`achieved with both short synacthen tests (HDT and LDT) and
`with the ITT in healthy volunteers and for each test, to establish
`the normal cut-off level at which the stimulated plasma cortisol
`excludes impairment of the HPA axis.
`
`Subjects and methods
`
`Control subjects
`
`Thirty-®ve healthy subjects mostly Hospital personnel or
`patients' family members were recruited to establish the
`normal range of plasma cortisol responses to HDT, LDT and
`ITT. None were taking any medication known to affect the
`HPA axis. Thirty of them (16 females, 14 males, aged 21±
`64 years, mean 34 years, body mass index (BMI): 24´8 6 4´8 kg/
`m2), underwent both short synacthen tests in random order.
`Twenty healthy volunteers, 15 of whom belonged to the above
`group (11 females, nine males, aged 21±56 years, mean
`30 years, BMI 23´1 6 3´7 kg/m2) underwent an ITT. All tests
`were performed in the morning between 0800 and 0900 hours
`after a fast. The minimum and maximum intervals between
`tests were 1 week and 1 month, respectively. The study was
`approved by the hospital's ethical committee; each subject was
`fully informed and gave written consent.
`
`Test protocol
`
`The synacthen tests were performed in subjects who had been
`quietly seated in armchairs for 30 minutes An indwelling
`
`venous cannula was placed in one forearm and 250 mg
`Tetracosactrin (ACTH1±24) (Synacthen, Novartis, Basel, Swit-
`zerland) was injected as a bolus after a basal blood sample had
`been taken to measure plasma cortisol. Additional blood
`samples were obtained 30 and 60 minutes after injection. The
`LDT was also performed in these subjects, at a dose of 1 mg.
`One ml of the content of the Tetracosactrin ampule (250 mg)
`was diluted in 49 ml of saline serum; later, 1 ml of this solution
`was diluted again with 49 ml more of saline serum. Ten ml of
`the solution thus contained 1 mg of Tetracosactrin. This freshly
`prepared solution was also injected as a bolus, and blood
`samples for plasma cortisol measurement were withdrawn at
`baseline and 30 and 60 minutes postinjection. No adverse
`effects were reported with either the HDT or the LDT.
`The insulin tolerance test was performed in the morning
`between 0800 and 0900 h. The insulin dose was 0´1±0´15 IU/kg
`body weight, with sampling at 15, 0, ‡ 30, 45, 60 and
`90 minutes The results were accepted if blood glucose
`levels had dropped below 2´2 mmol/l (40 mg/dl). During the
`test the capillary glucose was monitored with the glucose±
`oxidase method (glucocard, Menarini Diagnostics, Kyoto,
`Japan). All subjects recovered normoglycaemia quickly and
`spontaneously.
`
`Laboratory methods
`
`For all tests blood samples were immediately centrifuged at
`2000 g at 4 8C for 15 minutes and the resulting plasma was
`stored at 20 8C until plasma cortisol measurement.
`Plasma cortisol was measured using a direct chemiluminis-
`cence immunoassay (ACS 180, Chiron Diagnostics, Halstead,
`UK). The intra-assay and interassay coef®cients of variation
`were 5´1±7´0% and 6´4±9´7%, respectively.
`
`Statistical methods
`
`the distribution of
`Using the Kolmogorov±Smirnov test,
`plasma cortisol results in the healthy volunteers for each time
`point was found to be normal. The results are expressed as mean
`6 standard deviation (m 6 SD). However, as their distribution
`was non-Gaussian, plasma cortisol data were also expressed
`as medians and percentiles. Statistical analysis was performed
`by one-way ANOVA. Differences between two tests in subjects
`were analysed with the paired two-tailed Students'
`test.
`Correlations were examined by computing Pearson's correla-
`tion coef®cients. The highest value of plasma cortisol at ‡ 30
`or ‡ 60 minutes in each test was regarded as peak. The plasma
`cortisol increment was obtained by determining the difference
`between the peak and the basal plasma cortisol concentration. A
`P-value less than 0´05 was considered signi®cant.
`
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`
`

`
`Reference values for HPA junction 201
`
`Table 1 Plasma cortisol response at
`‡ 30 minutes and peak cortisol with low dose
`test (LDT), standard dose test (HDT) and peak
`cortisol with insulin tolerance test (ITT).
`Results are expressed as 5th, 10th and 50th
`percentiles
`
`Percentile
`
`Peak ITT
`
`‡ 30 minutes HDT
`
`‡ 30 minutes LDT
`
`Peak HDT
`
`Peak LDT
`
`5th
`10th
`50th
`
`539
`562
`654
`
`537
`564
`632
`
`489
`534
`649
`
`649
`654
`806
`
`498
`534
`657
`
`Results
`
`There were no differences between the mean basal plasma
`cortisol
`levels on any of the three tests. There were no
`differences between the mean plasma cortisol level reached at
`‡ 30 minutes after both short synachten tests and the peaks
`reached with the LDT and the ITT (at ‡ 30 minutes with HDT:
`684 6 123 nmol/l and LDT: 669 6 119 nmol/l; peak with LDT:
`691 6 123 nmol/l and ITT: 673 6 99 nmol/l. P > 0´87). The
`only difference (P < 0´001) found was between the peak plasma
`cortisol with the HDT (802 6 142 nmol/l) and the other tests.
`Likewise, the plasma cortisol mean increment after the HDT
`(439 6 127 nmol/l) was signi®cantly higher (P < 0´001) than the
`increment after the other two tests (LDT: 311 6 117; ITT:
`316 6 131 nmol/l).
`The plasma cortisol peak with the HDT was obtained in
`100% of cases at ‡ 60 minutes In contrast, with the LDT the
`peak was reached at ‡ 30 minutes in 23 subjects, and at
`‡ 60 minutes in only seven.
`The correlation coef®cients between plasma cortisol levels
`were as follows: between ‡ 30 minutes with HDT and the peak
`with ITT r ˆ 0´51 P < 0´004; between ‡ 30 minutes with LDT
`and the peak with ITT r ˆ 0´11 P ˆ 0´65; between ‡ 30 minutes
`with both short synachten tests r ˆ 0´49 P < 0´006.
`There was no statistically signi®cant correlation regarding
`sex and age at any point in any of the tests (data not shown). The
`only signi®cant correlation was between plasma cortisol
`response and the weight of healthy volunteers at certain
`points with the LDT (at ‡ 30 minutes, r ˆ 0´37 P ˆ 0´039; at
`60 min, r ˆ 0´53, P ˆ 0´002; peak, r ˆ 0´42, P ˆ 0´02).
`There were no correlations between the other tests at any point
`and the basal plasma cortisol with the LDT.
`The plasma cortisol responses, expressed as median and 10th
`and 5th percentiles, are shown in Table 1. The plasma cortisol
`level values in the 10th percentile with LDT at ‡ 30 minutes
`
`Fig. 1 Box plots showing median values, together with the 5th, 25th,
`50th, 75th and 95th percentiles of plasma cortisol level for peak with
`insulin tolerance test (PEAK ITT), at ‡ 30 minutes with standard and
`low dose synacthen test (‡ 30 MIN HDT, ‡ 30 MIN LDT), and peak
`with standard and low dose synacthen test (PEAK HDT and PEAK
`LDT).
`
`and the values in the 5th percentile at the peak during ITT and at
`‡ 30 minutes in HDT were very similar. Figure 1 shows the
`distribution of plasma cortisol results in our healthy subjects
`expressed as median and different percentiles.
`Table 2 shows that the proportion of falsely subnormal HPA
`responses varies according to the test and time point used. The
`percentage of falsely subnormal responses at the cut off level of
`500 nmol/l with the LDT at both times (peak, ‡ 30 minutes),
`was very similar to the percentage of falsely subnormal
`responses using a cut off of 550 nmol/l with the ITT and at
`‡ 30 minutes in the HDT.
`
`Discussion
`
`The comparisons of the mean plasma cortisol levels obtained by
`each test in our healthy volunteers are in agreement with those
`of other researchers in patients with pituitary disease without
`HPA impairment. Lindholm & Kehlet (1987) carried out a
`
`Table 2 Percentage of falsely subnormal
`responses calculated for our series of healthy
`subjects at the different cut-off levels (500,
`550, 600 nmol/l) habitually used in the
`literature at ‡ 30 minutes and peak with low
`dose test (LDT), standard dose test (HDT) and
`peak to insulin tolerance test (ITT)
`
`Cortisol
`
`Peak ITT
`
`‡ 30 minutes HDT
`
`‡ 30 minutes LDT
`
`Peak HDT
`
`Peak LDT
`
`$ 500
`$ 550
`$ 600
`
`0%
`5%
`10%
`
`0%
`7%
`17%
`
`7%
`13%
`30%
`
`0%
`0%
`0%
`
`7%
`13%
`23%
`
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`

`
`202 J. Gonza lbez et al.
`
`study in a group of patients with pituitary diseases in whom, in
`most cases, the HPA axis was preserved. In those patients, the
`authors found no differences between the mean plasma cortisol
`level at ‡ 30 minutes with the HDT and peak plasma cortisol
`with the ITT. In another study carried out in children with
`profound adrenal insuf®ciency, Weintrob et al. (1998) found no
`differences between the mean plasma cortisol
`level at
`‡ 30 minutes with both short synacthen tests and the plasma
`cortisol peak during the ITT.
`The large differences between the short synacthen tests and
`the ITT reported in the literature are probably due to the fact
`that the three tests were compared in patients with mild adrenal
`gland atrophy (Borst et al., 1982; Cunningham et al., 1983;
`Jones et al., 1994; Kane et al., 1995; Tordjman et al., 1995;
`Ammari et al., 1996; Hurel et al., 1996; Orme et al., 1996;
`Rasmuson et al., 1996; Soule et al., 1996; Streeten et al.,
`1996; Mukherjee et al., 1997; Bangar & Clayton, 1998
`Mayenknecht et al., 1998). Mayenknecht et al. (1998) consider
`that this state of partial or mild secondary adrenal insuf®ciency
`occurs when the peak plasma cortisol during the ITT is between
`500 and 550 nmol/l.
`In order to reduce the problems associated with the reported
`lack of sensitivity of the short synacthen test, the LDT has been
`proposed as a substitute for the ITT. Tordjman et al. (1995)
`evaluated only the plasma cortisol peak, and arbitrarily applied
`the same normality threshold (500 nmol/l) for three different
`ACTH1±24 stimuli (1, 5 and 250 mg). Other authors showed that
`the LDT (1 mg) is the most sensitive test for detecting states of
`partial adrenal insuf®ciency in patients with pituitary disease
`(Dickstein et al., 1991; Broide et al., 1995; Rasmuson et al.,
`1996; Dickstein et al., 1997; Talwar et al., 1998; Weintrob et
`al., 1998), though the increased sensitivity of the test is only
`due to the use of the same threshold for this dose.
`However, Mayenknecht et al. (1998) found no differences in
`the plasma cortisol stimulated by the two short synacthen tests.
`They found the two tests to be equally valid if different
`normality thresholds were established for each, and observed
`that both tests identify patients with moderate or severe adrenal
`insuf®ciency but not mild degrees of insuf®ciency.
`In our study, peak and incremental cortisol were higher with
`the HDT than with the other tests (LDT, ITT). Our ®ndings
`suggest that plasma cortisol at 60 minutes with the HDT should
`not be used or, at
`least, should be evaluated with another
`threshold. This conclusion would invalidate the many studies
`which consider the same threshold for peak and increment in all
`tests (Brost et al., 1982; Cunningham et al., 1983; Tsatsoulis et al.,
`1988; Jackson et al., 1994; Jones et al., 1994; Kane et al., 1995;
`Tordjman et al., 1995; Soule et al., 1996; Streeten et al., 1996;
`Ambrosi et al., 1998; Talwar et al., 1998; Weintrob et al., 1998).
`In our study, the LDT and the ITT were not well correlated.
`This may be because the low dose was not adjusted for body
`
`weight, or because there may have been a large variability in the
`dose during handling. The insulin dose in all subjects was
`adjusted to reach the necessary glycaemic level; the standard
`dose was supraphysiological and did not require adjustment for
`body weight. In fact, we only found a relationship between
`plasma cortisol response and weight in LDT; there was no
`correlation with weight in the other tests.
`In a large group of healthy volunteers, Clark et al. (1998)
`showed that with plasma cortisol stimulated with ACTH1±24
`administered via intramuscular injection the results varied
`widely according to the biochemical method used. They found
`that the value of plasma cortisol at ‡ 30 minutes varied between
`510 and 626 nmol/l. For their part, Mayenknecht et al. (1998)
`used the DPC (Diagnostic Products Corp DPC, Llanberis, UK),
`one of the methods that obtains the highest values of plasma
`cortisol. Applying the result of mean 2SD as the normality
`threshold (although it presented a high standard deviation) they
`obtained a value around 535 nmol/l at ‡ 30 minutes with the
`LDT and 618 and 725 nmol/l at minutes 30 and 60 with the
`HDT. In that study, reference levels were obtained in a group of
`healthy subjects and a group of hospitalized patients without
`adrenal disorders. For the ITT, a plasma cortisol peak of
`550 nmol/l was applied as a cut-off point. However, the results
`of Oelkers (1998) for ITT in healthy volunteers showed a
`mean 2SD value of 569 nmol/l, below the minimum normal
`level (599 nmol/l). As in our study, Oelkers obtained very similar
`minimum values at ‡ 30 minutes with the HDT (596 nmol/l) and
`with the peak on the ITT. In addition, the lowest range at
`‡ 30 minutes with the LDT was lower (483 nmol/l) than with the
`other two tests. In that study the low dose was adjusted for body
`surface area. Using the same biochemical method, Ambrosi et al.
`(1998) would not have obtained a similar result with the LDT;
`their normality threshold (336 nmol/l) would be far below the
`lowest value obtained in the control group. This result would
`give an unacceptably low sensitivity. Hurel et al. (1996) used a
`different biochemical method, but in that study the normality
`threshold can be deduced to be around 390 nmol/l for the HDT.
`They considered a value of around 520 nmol/l as normal during
`the ITT when applying con®dence intervals, and 600 nmol/l at
`‡ 30 minute with HDT, because with this value they obtained a
`false negative rate of only 10%.
`Two recent studies (Bangar & Clayton, 1998; Abdu et al.,
`1999) propose raising the cut-off to 600 nmol/l in order to
`increase sensitivity with both synacthen tests. We found that this
`arbitrary value gives rise to a high proportion of falsely
`subnormal response (up to 30%) with both synacthen tests and
`therefore many patients with pituitary disease would be
`administered unnecessary chronic treatment with hydrocortisone.
`We agree with Clark et al. (1998) that the plasma cortisol
`results obtained in healthy subjects with both short synacthen
`tests and with ITT do not have a Gaussian distribution.
`
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`

`
`Reference values for HPA junction 203
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`
`Therefore, if we consider the mean 2SD result as threshold,
`the plasma cortisol value considered as normal would be far
`below the lowest value obtained in our group of healthy
`volunteers. For this reason, we prefer to study the distribution of
`plasma cortisol results in percentiles, and consider the result
`obtained in the 5th percentile in each of the three tests and in
`each of the times as the plasma cortisol cut-off. The cut-off at
`‡ 30 minutes with the HDT and at the peak with the ITT would
`thus be the same (around 540 nmol/l). The cut-off at ‡ 30
`minute and at the peak on the LDT would be lower, around
`500 nmol/l. The cut-off at the peak on the HDT would be
`around 650 nmol/l.
`We conclude that different minimum normality thresholds
`should be established for each test, for each point, and for each
`biochemical method. These data should be applied to a group of
`patients with pituitary disease to establish the sensitivity and
`speci®city of each test.
`
`References
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`the hypothalamo±pituitary±adrenalaxis in patients with pituitary
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