About the Use and Misuse of an ACTH 1-39 Radioimmunoassay to Measure ACTH 1-24

M. Grino, P. Darmon, F. Dadoun, C. Frachebois, J.-G. Velut, S. Boullu, A. Dutour and C. Oliver

Laboratoire de Neuroendocrinologie Expérimentale, INSERM U 501 (M.G., F.D., C.F., J.-G.V., S.B., A.D., C.O.) Laboratoire de Biochimie (M.G.) Service d’Endocrinologie, Maladies Métaboliques et de la Nutrition (P.D., F.D., C.F., S.B., A.D., C.O.) Hôpital Nord, Marseille Cedex 20, France

In a recent paper Mayenknecht et al. (1) have shown that the diagnostic sensitivities of the low (LDT) and high (HDT) dose tetracosactin (ACTH 1-24) stimulation tests are almost identical. Their conclusions are supported in part by the measurements of plasma immunoreactive tetracosactin, which show that "In the HDT, plasma tetracosactin rose to more than 60,000 pg/mL shortly after injection. In the LDT, it rose to 1900 pg/mL." Based on these results and on the observation that the response of the normal human adrenal to an increment in plasma corticotropin to 70–80 pg/mL is almost maximal (2), the authors assumed that the LDT cannot be more sensitive than the HDT. However, we feel that these tetracosactin measurements were largely overestimated. Indeed, assuming a distribution space of 10 L, injection of 1 µg (i.e. the dose used in the LDT) or 250 µg (i.e. the dose used in the HDT) tetracosactin would give maximal plasma concentration of 100 and 25,000 pg/mL, respectively. This discrepancy is most probably related to the methodology used to measure plasma tetracosactin. Indeed, the authors have used a radioimmunoassay (distributed by CIS) designed to measure ACTH 1-39. They specify that the antiserum used in the assay recognizes the N-terminal sequence of ACTH. However, this does not necessarily imply that it recognizes equally ACTH 1-24 and ACTH 1-39. Our findings indicate that the antiserum used in this radioimmunoassay crossreacts about 5 times more (on a mass basis) with ACTH 1-24 than with ACTH 1-39. In addition, standard curves obtained with ACTH 1-24 or ACTH 1-39 are not strictly parallel [this can be explained by the fact that the antiserum may contain several species of anti-ACTH immunoglobulins having different affinity for ACTH 1-24 or ACTH 1-39]. As a consequence, plasma tetracosactin levels measured using the CIS antiserum and plotted against a standard curve generated with ACTH 1-39 will be overestimated by at least 5 times (see Fig. 1Go).



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Figure 1. Displacement curves obtained in ACTH radioimmunoassay with ACTH 1-24 and ACTH 1-39.

 
We have studied, in eight normal subjects, the kinetic profile of ACTH 1-24 in plasma from 2 to 30 min after i.v. injection of 1, 5, or 250 µg tetracosactin as a bolus. Immunoreactive ACTH 1-24 was measured using the CIS antiserum and plotted against a standard curve generated with ACTH 1-24. Our results show that injection of various amounts of tetracosactin induced a short-lasting (t1/2 = 4.91 ± 0.23 min) linear dose-related increase in plasma immunoreactive ACTH 1-24 (area under the curve: 21.4 ± 2.0, 112.0 ± 19.4, and 5,215 ± 369 pg/mL/min for the 1, 5, or 250 µg dose, respectively), while the ACTH 1-24 peak value (determined 2 min after injection) was 38 ± 9, 255 ± 55, and 17,000 ± 2,060 pg/mL for the 1, 5, or 250 µg dose, respectively (3).

These observations indicate that one should be very cautious when using a radioimmunoassay designed for a given peptide to measure another highly structurally related peptide; also, the plasma levels of tetracosactin reached after injection of 1 µg of this substance lie within a physiological range.

Footnotes

Received October 16, 1998. Address correspondence to: Michel Grino, M.D., Ph.D., Laboratoire de Neuroendocrinologie Expérimentale, INSERM U501, Faculté de Médecine Secteur Nord, Boulevard Pierre Dramard, 13916, Marseille Cedex 20, France.

References

  1. Mayenknecht J, Diederich S, Bähr V, Plöckinger U, Oelkers W. 1998 Comparison of low and high dose corticotropin stimulation test in patients with pituitary disease. J Clin Endocrinol Metab. 83:1558–1562.[Abstract/Free Full Text]
  2. Oelkers W. 1996 Dose-response aspects in the clinical assessment of the hypothalamopituitary adrenal axis, and the low-dose adrenocorticotropin test. Eur J Endocrinol. 135:27–33.[Medline]
  3. Darmon P, Dadoun F, Frachebois C, et al. On the meaning of the Low-Dose ACTH 1-24 tests to assess functionality of the hypothalamic-pituitary-adrenal axis. Eur J Endocrinol. In press




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