Clinical Endocrinology (1998) 49, 287–292
`
`Defining the normal cortisol response to the short
`Synacthen test: implications for the investigation of
`hypothalamic-pituitary disorders
`
`P. M. Clark*, I. Neylon†, P. R. Raggatt‡,
`M. C. Sheppard† and P. M. Stewart†
`*The RegionalEndocrineLaboratory,UniversityHospital
`BirminghamNHSTrust, Birmingham, †Departmentof
`Medicine,Universityof Birmingham,QueenElizabeth
`Hospital,Birminghamand ‡Departmentof Clinical
`Biochemistry,AddenbrookesHospital,Cambridge,UK
`
`(Received8January1998;returnedfor revision24February
`1998;finallyrevised19March1998;accepted23March1998)
`
`Summary
`
`OBJECTIVE To define the normal cortisol response to
`the Short Synacthen Test using four different cortisol
`immunoassays and to assess the implications for the
`investigation of hypothalamic-pituitary disorders.
`DESIGN AND PATIENTS The cortisol response to 250
`mg im ACTH1–24 (Synacthen, Ciba Geigy)
`in 100
`healthy volunteers using four different cortisol immu-
`noassays has been measured. In 44 newly diagnosed
`and untreated patients with pituitary disease, basal
`and 30 minute post-ACTH cortisol results were also
`determined using the four immunoassays.
`RESULTS The distribution of cortisol results at all
`time points and for all methods were non-Gaussian
`and significant differences in the absolute values of
`the 5th – 95th percentiles were found between meth-
`ods (P< 0·01). At 30 min post-Synacthen in normals
`the 5th percentile of the cortisol response ranged
`from 510 to 626 nmol/l with the different methods.
`Similarly the relationship between assay results dif-
`fered at different time points. No effect of age on the
`cortisol response was found but for stimulated corti-
`sol values and the incremental responses females
`showed significantly higher responses than males
`(P< 0·05) for most methods. Although there was a
`significant positive linear correlation (P< 0·001)
`between stimulated and basal cortisol values for all
`methods, no significant
`relationship was found
`
`Correspondence: Dr PM Clark, Regional Endocrine Laboratory,
`University Hospital Birmingham NHS Trust, Selly Oak Hospital,
`Raddlebarn Road, Birmingham B29 6JD, UK. Fax: þ44 (0)121 414
`0078.
`
`between the incremental response and basal cortisol
`values. In the pituitary disease patients basal and 30
`minute post-ACTH cortisol results were significantly
`lower (P< 0·05 and < 0·001) than the control group
`using the same cortisol assay. When the results
`were compared to the 5th percentile of the gender
`and assay specific control group 33·3% of male and
`17·4% of female patients failed the Synacthen test at
`30 min.
`‘normal’
`the
`of
`definition
`CONCLUSIONS The
`response to Synacthen should be both gender and
`method related at all time points. The data suggest
`that up to one-third of untreated patients with pituitary
`disease may have subtle defects in the hypothalamic-
`pituitary-adrenal axis.
`
`The short Synacthen test (SST) was introduced first in the 1960s
`as a means of rapidly assessing adrenal function (Wood et al.,
`1965). Based on the premise that the adrenal gland will respond
`to an exogenous bolus of synthetic ACTH when there is
`endogenous ACTH reserve, the SST has also been used to
`assess the integrity of the hypothalamo-pituitary-adrenal (HPA)
`axis in patients with suspected or established pituitary disease
`(Lindholm & Kehlet, 1987; Stewart et al., 1988) and in patients
`treated chronically with corticosteroids (Kehlet & Binder,
`1973; Kane et al., 1995). In both instances the cortisol response
`to the SST compares favourably with the response to the ‘gold
`standard’ of the insulin tolerance test (ITT). As a result many
`endocrinologists now use the SST as the first line test in
`documenting function of the HPA axis (Stewart et al., 1988;
`Grinspoon & Biller, 1994; Clayton, 1996), reserving the ITT for
`patients who fail the SST, or for those who also require an
`assessment of growth hormone reserve. However, based on the
`use of pass/fail cut-off values for the cortisol response to the
`SST and ITT, discrepancies have been reported between the
`two tests (Borst et al., 1982; Ammari et al., 1996; Soule et al.,
`1996; Streeton et al., 1996), leading some endocrinologists to
`question the predictive value of the SST. The definition of a
`‘pass’ for the SST is not consistent in clinical practice, at least
`amongst British endocrinologists (Stewart et al., 1988). This
`may reflect the fact that the definition of a ‘pass’ response in the
`literature is derived from studies using the fluorimetric methods
`for the measurement of free 11-hydroxycorticoids in human
`
`䉷 1998 Blackwell Science Ltd
`
`287
`
`Amerigen Exhibit 1176
`Amerigen v. Janssen IPR2016-00286
`
`

`
`288 P.M. Clarket al.
`
`Table 1 Serum cortisol response to Synacthen in healthy volunteers. Results are expressed as median [5th-95th percentile] in nmol/l
`
`Time
`
`0 min
`
`30 mins
`
`60 mins
`
`30–0 min
`
`60–0 min
`
`Method
`TDX
`ACS
`Delfia
`DPC
`
`349 [164–870]
`352 [195–650]
`309 [163–620]
`391 [200–904]
`
`811 [626–1431]
`741 [569–1078]
`707 [510–1088]
`866 [590–1548]
`
`972 [766–1655]
`885 [661–1263]
`849 [619–1291]
`1047 [722–1830]
`
`488 [289–776]
`399 [208–593]
`409 [222–641]
`494 [222–762]
`
`645 [433–1036]
`520 [307–724]
`544 [329–810]
`650 [344–1037]
`
`plasma (Mattingly, 1962; Wood et al., 1965) which is likely to
`give significantly higher results than immunoassays because of
`the measurement of both cortisol and corticosterone in the
`fluorimetric assays particularly at high concentrations. Further-
`more there has been a proliferation of cortisol immunoassays
`and significant deviations from isotope dilution gas chromato-
`graphy-mass
`spectrometry (GC-MS)
`results have been
`described for some methods, differences which are not reflected
`in different quoted reference ranges (De Brabandere et al.,
`1995). These deviations may reflect differences in assay
`specificity and calibration.
`The aim of this study was to define the normal cortisol
`response to the SST using four widely available immunoas-
`says. Using the generated ‘normal’ reference range, the use of
`the SST in the investigation of the hypothalamic-pituitary-
`adrenal axis in newly diagnosed pituitary patients was also
`studied.
`
`Materials and methods
`
`Patients andcontrols
`
`One hundred normal healthy volunteers on no therapy (67
`females, aged 19–63 years, median 25 years) were studied. All
`subjects rested for 30 min prior to the test. A short Synacthen
`test was performed between 0900 and 1200 hours by the
`administration of ACTH1–24 im (250 mg, Ciba Geigy). Blood
`
`was collected basally and at 30 and 60 min, into plain tubes. The
`samples were allowed to clot and serum separated and stored at
`¹ 20⬚C, prior to analysis.
`In addition, 44 patients (23 females, aged 21–77 years,
`median 43 years) with newly diagnosed pituitary disease were
`investigated prior to any form of treatment. Thirteen patients
`had acromegaly, 18 prolactinomas, 9 nonfunctioning tumours
`and 4 idiopathic hypopituitarism. Twenty-eight patients had
`macroadenomas. A short Synacthen test was performed as
`described above (0900–1200 h) with blood samples collected
`basally and at 30 min.
`The study had the approval of the local ethical committee and
`all patients and controls gave informed written consent.
`
`Cortisolassays
`
`Serum cortisol was measured by four different commercially
`available immunoassays. The assays used were chosen to
`include those widely used by laboratories, both automated and
`manual assays and those with isotopic and nonisotopic labels.
`The assays used were: TDX (Abbott Diagnostics, Maidenhead,
`UK), ACS 180 (Chiron Diagnostics, Halstead, UK), Delfia
`(Pharmacia Wallac, Milton Keynes, UK), Coat-a-Count
`(Diagnostic Products Corp DPC, Llanberis, UK). The following
`interassay imprecision was achieved, given as the coefficient of
`variation over the stated concentration range:
`TDX: less than 8% (106–1099 nmol/l), ACS less than 10%
`
`Table 2 Differences in the distribution of cortisol results and the incremental values for different methods
`
`Methods
`
`Time
`
`P
`
`Time
`
`P
`
`Time
`
`P
`
`Time
`
`P
`
`TDX vs DPC
`TDX vs Delfia
`TDX vs ACS
`DPC vs Delfia
`DPC vs ACS
`Delfia vs ACS
`
`0
`0
`0
`0
`0
`0
`
`NS
`NS
`NS
`0·05
`NS
`NS
`
`30 mins
`30 mins
`30 mins
`30 mins
`30 mins
`30 mins
`
`NS
`< 0·001
`NS
`< 0·001
`< 0·01
`NS
`
`60 mins
`60 mins
`60 mins
`60 mins
`60 mins
`60 mins
`
`NS
`< 0·001
`< 0·05
`< 0·001
`< 0·001
`NS
`
`30–0
`30–0
`30–0
`30–0
`30–0
`30–0
`
`NS
`< 0·01
`< 0·01
`< 0·001
`< 0·001
`NS
`
`Time
`
`60–0
`60–0
`60–0
`60–0
`60–0
`60–0
`
`P
`
`NS
`< 0·01
`< 0·001
`< 0·01
`< 0·001
`NS
`
`Kolmogorov Smirnov test, NS, not significant.
`
`䉷 1998 Blackwell Science Ltd, Clinical Endocrinology, 49, 287–292
`
`

`
`The short Synacthentest 289
`
`Table 3 Bias ratio of the cortisol assays compared to the ACS method
`
`Method
`
`Bias ratio
`(mean)
`
`0 vs 30 min
`
`0 vs 60 min
`
`30 vs 60 min
`
`Significance of difference (P)
`
`TDX vs ACS
`0 min
`30 min
`60 min
`DPC vs ACS
`0 min
`30 min
`60 min
`Delfia vs ACS
`0 min
`30 min
`60 min
`
`1·053
`1·152
`1·185
`
`1·181
`1·207
`1·244
`
`0·899
`0·964
`0·983
`
`(NS, not significant).
`
`< 0·01
`
`NS
`
`< 0·05
`
`< 0·01
`
`NS
`
`< 0·05
`
`NS
`
`NS
`
`NS
`
`(53–993 nmol/l), Delfia less than 8% (76–925 nmol/l), DPC
`less than 10% (80–1033 nmol/l), respectively. The quoted
`cross-reactivity of these assays with corticosterone is 6·3, 2·8,
`27·7 and 0·9%, respectively. The manufacturers recommended
`procedures were followed. The TDX assay was used to assay
`the samples from the pituitary patients.
`
`Statisticalmethods
`
`The distribution of cortisol results in the healthy volunteers for
`each time point, analysed by each method showed a non-
`Gaussian distribution. As a result, cortisol data were expressed
`as medians and the 5th and 95th percentiles.
`
`The distribution of cortisol results at each time point and the
`distribution of the differences in cortisol results between time
`points, i.e. 30 min minus basal and 60 min minus basal, were
`compared for each assay using the Kolmogorov-Smirnov test
`with a Bonferroni correction for multiple comparisons.
`The relative bias of the methods was studied by arbitrarily
`choosing one method (ACS) against which to compare the
`others, thus for each sample a result by a given method was
`expressed as a ratio to the matched ACS result (bias ratio) and
`this was calculated for each time point. Comparison of the bias
`ratio at different time points was performed using the Student’s
`t-test. The Mann–Whitney U-test with correction for ties was
`used to investigate the effect of age and gender on the cortisol
`response and to compare the cortisol responses of the newly
`diagnosed pituitary patients and normal volunteers. The
`relationship between the 30 and 60 minute responses to the
`basal value were examined by least squares linear regression
`analysis.
`
`Results
`
`The distribution of serum cortisol results and the cortisol
`increments in the control subjects obtained with the four
`different
`immunoassays are shown (Table 1). Significant
`differences in these distributions (P < 0·01) were found at
`each time point between methods (Table 2).
`The bias ratios were found to be significantly different
`between methods and within a method at different time points
`(Table 3).
`Comparing the results for each time point and increment for
`each method from the healthy volunteers aged less than
`40 years with those aged greater than 40 years, revealed no
`significant differences. Significant differences, however, were
`
`Table 4 Serum cortisol response to Synacthen in healthy volunteers according to gender
`
`Method
`
`Time
`
`0 min
`
`30 min
`
`60 min
`
`30–0 min
`
`60–0 min
`
`Gender
`
`TDX
`
`ACS
`
`Delfia
`
`DPC
`
`M
`F
`M
`F
`M
`F
`M
`F
`M
`F
`
`313 [166–527]
`368 [150–884]
`750 [585–909]
`871*** [629–1456]
`888 [676–1077]
`1066*** [800–1879]
`404 [238–552]
`509*** [295–808]
`540 [344–716]
`681*** [488–1037]
`
`326 [186–578]
`352 [194–691]
`705 [554–876]
`786** [543–1193]
`830 [624–998]
`927** [662–1354]
`350 [120–509]
`406* [193–607]
`468 [254–660]
`539** [351–742]
`
`309 [164–475]
`309 [162–632]
`689 [501–900]
`729 [510–1383]
`814 [553–956]
`897** [631–1317]
`376 [218–533]
`417 [216–673]
`481 [221–657]
`582** [337–830]
`
`356 [203–573]
`424 [200–935]
`786 [605–1040]
`920** [586–1571]
`990 [632–1282]
`1057** [778–1834]
`423 [219–698]
`511 [206–773]
`547 [255–864]
`676* [375–1037]
`
`Values are given as median, [5th – 95th percentiles], nmol/l. *P < 0·05, ** P < 0·01, *** P < 0·001 vs males.
`
`䉷 1998 Blackwell Science Ltd, Clinical Endocrinology, 49, 287–292
`
`

`
`290 P.M. Clarket al.
`
`Fourteen of the 44 patients (32%) had a 30 minute serum
`cortisol concentration less than the 5th percentile of the control
`group with the appropriate assay (< 626 nmol/l). When the
`results were compared to the 5th percentile of the gender
`specific control group, 7 of the male patients (33·3%) and 4 of
`the female patients (17·4%) had a 30 minute cortisol less than
`the gender and method specific ranges.
`
`Discussion
`
`The use of the short Synacthen test for the investigation of
`disorders of the hypothalamic-pituitary-adrenal axis has come
`under increasing scrutiny as comparisons of its diagnostic
`efficiency with the insulin tolerance test have shown a number
`of discrepancies (Borst et al., 1982; Ammari et al., 1996; Soule
`et al., 1996; Streeton et al., 1996). It has been recognized that a
`number of factors may contribute to this situation. Thus, for
`example, there has been much debate as to whether the peak
`cortisol value or the cortisol increment should be used, whether
`the route of administration should be iv or im, the dose of
`ACTH that should be used and whether the diurnal variation in
`ACTH/cortisol secretion is of importance. Whilst these issues
`have been addressed and can be taken into consideration
`(Grinspoon & Biller, 1994), discrepancies between the SST and
`ITT may still be found, in patients with Cushing’s disease and
`immediately following a pituitary insult such as surgery or
`apoplexy when the adrenal glands can still respond to ACTH
`(Hjortrup et al., 1983). This aside, the greatest clinical concern
`relates to patients who ‘pass’ the SST but who ‘fail’ the ITT
`(Borst et al., 1982; Ammari et al., 1996; Soule et al., 1996;
`Streeton et al., 1996). Central to these studies is the definition of
`a ‘normal’ response to Synacthen (and also to the ITT). Early
`studies using a fluorimetric assay for 11-hydroxycorticoids
`(Mattingly, 1962) are likely to have overestimated the cortisol
`
`*
`
`**
`
`**
`
`2000(cid:127)
`
`1500(cid:127)
`
`1000(cid:127)
`
`500(cid:127)
`
`0
`
`Pituitary Control Pituitary Control Pituitary Control
`0 min
`30 – 0 min
`30 min
`
`Cortisol (nmol/l)
`
`Fig. 1 Distribution of cortisol results in patients with pituitary
`disease at diagnosis and controls. Results are expressed as median,
`[5th – 95th percentile] in nmol/l and were obtained using the TDX
`assay. *P < 0·05, **P < 0·01 compared to the control group.
`
`found for stimulated and incremental values (but not basal
`values) for all assays between male and female volunteers
`(Table 4).
`A significant positive linear relationship (P < 0·001) was
`obtained between the results at 30 and 60 min and with the basal
`cortisol concentrations for all methods (30 mins vs 0 min, range
`for all cortisol assays: slope ¼ 0·836–1·049, intercept ¼ 407–
`485 nmol/l, r ¼ 0·7113–0·8396, 60 minute vs 0 minute, range
`for all cortisol assays: slope ¼ 0·777–1·162, intercept ¼ 524–
`662 nmol/l, r ¼ 0·6473–0·8050). No significant correlation was
`obtained between the incremental values (30–0 min and 60–
`0 min and the basal cortisol concentration.
`In the newly
`diagnosed pituitary patients both basal cortisol (290 [113–
`650] nmol/l) (median [5th-95th percentile]) and 30 minute
`cortisol (748 [302–1000] nmol/l) were significantly lower than
`the control group (P < 0·05 and P < 0·001, respectively, Mann
`Whitney U-test) and this was independent of tumour type and
`size (Fig. 1). Indeed, compared to their gender matched controls
`(Table 5) the pituitary patients gave significantly lower results
`at all time points for females and for the 30 minute post-
`Synacthen value for males.
`
`Table 5 Range of cortisol results in patient with pituitary disease at diagnosis
`
`Group
`
`Pituitary
`Control
`Pituitary
`Control
`Pituitary
`Control
`Pituitary
`Micro adenoma
`Macro adenoma
`
`Gender
`
`M
`M
`F
`F
`M & F
`M & F
`
`M & F
`M & F
`
`0 min
`
`30 min
`
`30–0 min
`
`290 [110–511]
`313 [166–527]
`290* [69·5–688]
`368 [150–884]
`290* [113–650]
`349 [164–870]
`
`274 [108–715]
`290 [85–572]
`
`660* [300–995]
`750 [585–909]
`780** [219–995]
`871 [629–1456]
`748** [302–1000]
`811 [626–1431]
`
`720 [291–1073]
`760 [224–963]
`
`420 [114–720]
`404 [238–552]
`364* [110–608]
`509 [295–808]
`373** [112–667]
`488 [289–776]
`
`400 [112–575]
`370 [105–713]
`
`Results are expressed as median [5th – 95th percentile] in nmol/l and were obtained using the TDX assay. *P < 0·05, ** P < 0·01 compared with control
`group.
`
`䉷 1998 Blackwell Science Ltd, Clinical Endocrinology, 49, 287–292
`
`

`
`The short Synacthentest 291
`
`Some indication of the relative bias of cortisol assays is given by
`the six month cumulative bias for the period of the study reported
`by UK NEQAS (Dr J Middle, personal communication): for
`TDX ¼ 9·80%, DPC ¼ 5·30%, ACS ¼ – 1·75%, Delfia ¼
`¹ 11·0% which approximates the ranking of the methods as
`shown in Table 1. It should be emphasized that all analytical
`methods are subject to variation over a period of time, for example
`the subsequent recalibration of the DPC method and this should
`be taken account of when determining reference ranges.
`There was no effect of age on the response to Synacthen in
`this adult population, but the effect of gender on the response to
`cortisol was significant with some variation between the
`methods. Gender differences in the cortisol response to the
`SST have not been noted in earlier studies using fluorimetric
`assays (Wood et al., 1965) though analysis of the data using
`parametric statistics combined with the imprecision and
`nonspecificity of the assay may have obscured any differences.
`If steroids other than cortisol do cross-react in the assays, it is
`possible these are released in response to Synacthen in higher
`concentrations in females vs males. Alternatively, studies of
`ACTH and cortisol pulsatility (Horrocks et al., 1990; Roelf-
`sema et al., 1993) suggest that there may be a greater sensitivity
`of the adult female adrenal cortex to ACTH. The dose of ACTH
`used in this study, however, is supraphysiological (Oelkers,
`1996) suggesting an analytical explanation for the gender
`differences may be the most likely.
`Several studies have demonstrated a negative correlation
`between the incremental cortisol response and basal concentra-
`tions (Leisti & Perheentupa, 1978; Kukreja & Williams, 1981;
`May & Carey, 1985; Dickstein et al., 1991) though these studies
`have been limited by small numbers, the use of nonspecific
`fluorimetric cortisol assays or have studied an unselected,
`hospital population.Within the tightly defined conditions of this
`study no such relationship was demonstrated for either
`incremental value (30–0 min or 60–0 min).
`Defining a ‘pass’ for the SST as a 30 minute cortisol response
`greater than the 5th percentile value for the assay used, we have
`demonstrated that 32% of newly diagnosed and untreated
`pituitary patients had some dysfunction of the HPA axis. When
`the appropriate gender-related 5th percentile value at þ 30 min
`was used, this value was 33·3% for male patients and 17·4% for
`females. Rather surprisingly this was unrelated to the under-
`lying endocrine status or size of the pituitary lesion, suggesting
`that any deficiency of the HPA axis may be secondary to
`neuroendocrine abnormalities rather than structural
`loss of
`pituitary corticotrophs.
`We conclude that the definition of the ‘normal’ response to
`Synacthen should be both method and gender related at all time
`points. The statistical techniques used to compare data from
`different groups should also take into account
`that
`the
`distributions are non-Gaussian and that the relationships of the
`
`response to Synacthen because of the nonspecificity of the
`assays. Despite the development of immunoassays for the
`measurement of serum cortisol significant method-related
`variations in the measurement of serum cortisol have been
`reported. Indeed during 1984, 65% of all laboratories would
`have more than a 20% positive bias from GC-MS targets, the
`then reference method (Moore et al., 1985). There remain
`significant method-related differences in measured serum
`cortisol concentrations despite improvements in calibration
`and specificity of immunoassays (De Brabandere et al., 1995).
`These differences might well be reflected in different reference
`ranges for basal cortisol and for the cortisol response to
`Synacthen and also the ITT.
`In this study we have demonstrated that the response of serum
`cortisol to the SST in healthy volunteers shows a non-Gaussian
`distribution and significant method related differences. This has
`been shown for both the basal, stimulated values at both 30 and
`60 min post-Synacthen and for the incremental values. The nature
`of the distribution of cortisol results may not have been noted in
`earlier studies either because of the small number of subjects
`studied or the imprecision of the assays used (Stewart et al., 1988;
`Hurel et al., 1996) but because of this inappropriate statistical
`techniques may have led to an incorrect definition of the ‘normal’
`response. The data presented here shows significant differences in
`the cortisol distributions between methods, particularly at 30 and
`60 min post-Synacthen.
`In addition to the variation in the distribution of the results
`with different cortisol assays,
`the significant differences
`between the methods shown by the bias ratio do not show
`consistency at the different time points. This greater method-
`related difference in specimens taken after Synacthen compared
`to basal specimens might be explained by the release of steroids
`other than cortisol in response to Synacthen which affect some
`of the immunoassays more than others either by direct cross-
`reactivity or by their effect on the displacement reaction of
`cortisol from cortisol binding globulin. Synacthen is known to
`cause the release of other adrenal steroids including 17 a–
`hydroxyprogesterone, 17 a–hydroxy-pregnenolone, dehydroe-
`piandrosterone, androstenedione, androstenediol and aldoster-
`one (Grunwald et al., 1990; Lashansky et al., 1991). Whether
`such substances contribute significantly to the ‘cortisol’ result
`will depend on individual assay specificity and the absolute
`concentration of cortisol released. This difference in bias has
`important implications for the comparison of methods. It is
`clear that where a study has involved the use of more than one
`cortisol immunoassay comparison must be made on both basal
`and stimulated samples and full details published (Hurel et al.,
`1996; Orme et al., 1996). The use of quality control materials
`alone to compare assays is not likely to give data applicable to
`patient samples and comparison should be based on analysis of
`samples collected from patients basally and post-stimulation.
`
`䉷 1998 Blackwell Science Ltd, Clinical Endocrinology, 49, 287–292
`
`

`
`292 P.M. Clarket al.
`
`distributions differ with time. Controversy in the literature
`surrounding the use of the SST in the assessment of the HPA axis
`seems
`likely to have been complicated by the use of
`inappropriate ‘cut-off’values. Whilst further appraisals of the
`SST in the assessment of endogenous HPA function are required,
`our data suggests that up to one-third of untreated patients with
`pituitary disease may have subtle deficits in pituitary-adrenal
`reserve, irrespective of the underlying diagnosis.
`
`Acknowledgements
`
`The staff of the Regional Endocrine Laboratory, Birmingham
`and Clinical Biochemistry, Addenbrookes Hospital are thanked
`for their technical support. The statistical advice of Drs R
`Holder and IR Stevens, University of Birmingham is gratefully
`acknowledged. Dr J Middle, UK NEQAS, Birmingham kindly
`supplied data on the cumulative bias of the cortisol assays. This
`study was supported in part by Ciba Geigy, Abbott Diagnostics,
`Chiron Diagnostics, Pharmacia Wallac and Diagnostic Pro-
`ducts. PMS is an MRC Senior Clinical Fellow. We thank the
`volunteers who took part in this study and Professor J Franklyn
`and Dr D Heath for allowing us to study their patients.
`
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`䉷 1998 Blackwell Science Ltd, Clinical Endocrinology, 49, 287–292