Analytical and clinical aspects of adrenocorticotrophin determination

Abstract

Adrenocorticotrophin (ACTH) is formed from the cleavage of pro-opiomelanocortin. Measurement of plasma ACTH is a key step in the differential diagnosis of hypothalamic-pituitary-adrenal disorders.Prior to the development of radioimmunoassay, the bioassays employed for the determination of ACTH were highly complex, time-consuming and costly in terms of number of animals used. Their sensitivity was such that the normal early morning peak of ACTH could not be determined. The introduction of immunoassay methodology enabled the measurement of low normal ACTH concentrations. Immunological recognition of ACTH by the antibodies employed offered improvements with regard to speci city. The development of two-site immunometric assays further improved speci city and the ability to measure low normal ACTH concentrations without the need to extract large volumes of plasma. The quanti cation of ACTH is now routinely performed in clinical laboratories, with nonradioisotopic methods becoming increasingly popular.ACTH measurement is of limited value in distinguishing between the causes of Cushing's syndrome, as there is considerable overlap in circulating ACTH concentrations in subjects with either a pituitary or an ectopic tumour. The role of ACTH precursor and related peptides in normal and pathological states and the clinical utility of their measurement remain to be fully elucidated. However, there is evidence that measurement of ACTH precursors can be a useful diagnostic tool in identifying the aetiology of Cushing's syndrome. This review will primarily address methodological aspects of ACTH measurement in the diagnosis of clinical conditions. Ann Clin Biochem 2003; 40: 453-471 Structure and nomenclature of ACTH and its related peptidesAdrenocorticotrophin (ACTH) and related peptides are derived by selective proteolytic cleavage of a common glycoprotein called pro-opiomelanocortin (POMC; MW 32 000) (see Fig. 1). Within human pituitary corticotrophs, POMC is cleaved by the enzyme prohormone convertase 1 (PC1) into an N-terminal terminal glycopeptide (N-POC, 76 aa), joining peptide (JP, 30 aa), ACTH (39 aa) and a C-terminal fragment called b-lipotrophin (bLPH, 89 aa). There is no further processing in the adult human pituitary and therefore the ACTH-related peptides in the circulation are N-POC, JP, ACTH and bLPH. However, in addition, the ACTH precursors POMC and pro-ACTH are present in the human circulation at concentrations of 5-40 pmol/L, which is ¢ve-fold greater than that of ACTH (54.1-51.4 ng/L, 50.9-11.3 pmol/L) (see Fig. 2). 1 ACTH concentrations are lower than those of N-POC (5.6-16.8 pmol/ L) and approximately equimolar with those of bLPH (2.5-6.7 pmol/L). However, b-endorphin concentrations (41.7 pmol/ L) are consistently lower than those of the other peptides. Further processing can occur in animals that have neurointermediate lobes in the pituitary. These cells contain prohormone convertase 2 (PC2), which can cleave ACTH to a-melanocyte-stimulating hormone (aMSH, 13 aa) and corticotrophin-like intermediat...

The Effect of Temperature and Aprotinin on the preanalytical stability of adrenocorticotrophic hormone

“…The ACTH assay we used in our study employs two monoclonal antibodies specific for ACTH [9][10][11][12] and for the C-terminal region (ACTH 36-39). Due to common antigenic structure, the antibodies recognize intact biologically active ACTH 1-39 and the ACTH precursors POMC and pro-ACTH [1]. Enzymatic cleavage or modification of ACTH may have occurred, even in the presence of aprotinin, at other sites in the mol- As expected, for each of the three study conditions we observed a decreasing trend of measured ACTH concentrations with time.…”

“…Analysis of plasma adrenocorticotropin (ACTH) is a key step in differentiating hypothalamic-pituitary-adrenal disorder diagnosis and of great clinical significance for both Cushing's syndrome and adrenal insufficiency [1]. ACTH in the blood is considered highly unstable because of proteolytic degradation [2][3][4][5].…”

“…In particular, it is recommended that the blood sample is collected into chilled tubes, stored in ice immediately after collection, centrifuged in a cooled centrifuge (4°C) and analyzed within 1 h of collection, and stored at -20°C. Moreover, it is recommended that blood is collected into plastic tubes with the tri-potassium (K3) salt of ethylenediaminetetraacetic acid (K3EDTA) as anticoagulant or siliconized glass tubes as ACTH sticks to the glass [1]. Furthermore, it is usually recommended that plasma for endogenous ACTH analysis remain frozen during shipment to the referral laboratory, requiring dry ice and overnight mail service [6,7].…”

The diagnosis and investigation of adrenal insufficiency in adults

“…Two of these preparations, from the National Hormone and Pituitary Program and the National Institute for Biological Standards and Control (code 74/ 555), have similar immunoreactivity but differ significantly in biological potency. 121 In the absence of an agreed international standard for ACTH, it is difficult to draw conclusions regarding assay bias. The predominant assay method in use in the UK is the Siemens Immulite.…”

Multiplexed Ultrasensitive Determination of Adrenocorticotropin and Cortisol Hormones at a Dual Electrochemical Immunosensor

“…In fact, various immunoassays were proposed for the determination of ACTH based on different antibodies and using radioactivity, chemiluminiscence or fluorescence as detection techniques. These methodologies allowed ranges between a few units up to various hundreds of ng/L ACTH to be covered [19][20][21], concentrations that are much larger than those appearing in the linear range obtained for this hormone with the developed Strept-AP/ Biotin-ACTH/anti-ACTH/APBA/SPCE1 immunosensor. On the other hand, the electrochemical immunosensor for ACTH previously described by our group provided a detection limit of 18 pg/L [15].…”