Long-acting porcine sequence ACTH in the diagnosis of adrenal insufficiency: a cost-effective alternative to the ACTH1-24 test.

While the ACTH1-24 test has some well-documented shortcomings, it is the most widely used test to diagnose primary and secondary adrenal insufficiency. However, this synthetic ACTH preparation is not readily available in some countries. Research from India has demonstrated that using a long-acting porcine sequence ACTH has similar diagnostic performance to ACTH1-24 at around 25% of the cost. This may allow access to a robust test for adrenal insufficiency to developing countries and potentially allow thousands of patients to be identified and appropriately treated.

Identification of metabolites of peptide-derived drugs using an isotope-labeled reporter ion screening strategy.

Background Peptide-derived drugs represent an emerging class of prohibited substances in professional sports and, thus, in modern doping controls. After parental administration (e.g. subcutaneous, intravenous), these drugs undergo various metabolic processes, which degrade them to biologically active or inactive peptides. Knowledge about these metabolic processes and the hereby produced metabolites plays a key role in successful doping controls due to the effective design of analytical assays under consideration of optimal analytical targets. Unfortunately, the complexity of biological matrix (e.g. blood or urine) complicates the immediate identification of relevant metabolites due to the enormous excess of naturally occurring peptides and their degradation products. Methods In this study, a strategy employing in-vitro metabolism of stable isotope-labeled peptides producing characteristic reporter ions derived from labeled immonium ions is shown. The in-vitro experiments were performed with human skin tissue microsomes (S9), and model drugs representing prohibited peptide hormones were synacthen, insulin, and corticorelin (respectively, their stable isotope-labeled analogs). After generic sample preparation, the metabolites were identified by means of liquid chromatography (LC) coupled to high-resolution mass spectrometry (MS) in an untargeted approach. Results and conclusions For all three model peptides, several metabolic products were readily identified. While insulin and corticorelin were found to be comparably stable, synacthen was fully degraded, yielding a plethora of metabolic products. A proof of concept concerning the transferability of the obtained data was accomplished by analyzing plasma samples collected post-administration of recombinant human insulin, corroborating the presence of a skin protease-indicative insulin metabolite in vivo.

Cosyntropin testing does not predict response to glucocorticoids in community-acquired pneumonia in a randomized controlled trial.

OBJECTIVE: Glucocorticoids have been shown to improve outcome in community-acquired pneumonia (CAP). However, glucocorticoids have potential side-effects, and treatment response may vary. It is thus crucial to select patients with high likelihood to respond favourably. In critical illness, cosyntropin testing is recommended to identify patients in need for glucocorticoids. We investigated whether cosyntropin testing predicts treatment response to glucocorticoids in CAP. DESIGN: Predefined secondary analysis of a randomized controlled trial. PATIENTS: Hospitalized patients with CAP. MEASUREMENTS: We performed 1 µg cosyntropin tests in a randomized trial comparing prednisone 50 mg for 7 days to placebo. We investigated whether subgroups based on baseline and stimulated cortisol levels responded differently to glucocorticoids with regard to time to clinical stability (TTCS) and other outcomes by inclusion of interaction terms into statistical models. RESULTS: A total of 326 patients in the prednisone and 309 patients in the placebo group were evaluated. Neither basal cortisol nor a Δcortisol <250 nmol/L after stimulation nor the combination of basal cortisol and Δcortisol predicted treatment response as measured by TTCS (all P for interaction >0.05). Similarly, we found no effect modification with respect to mortality, rehospitalization, antibiotic treatment duration or CAP-related complications (all P for interaction >0.05). However, glucocorticoids had a stronger effect on shortening length of hospital stay in patients with a baseline cortisol of ≥938 nmol/L (P for interaction = 0.015). CONCLUSIONS: Neither baseline nor stimulated cortisol after low-dose cosyntropin testing at a dose of 1 µg predicted glucocorticoid responsiveness in mild to moderate CAP. A treatment decision for or against adjunct glucocorticoids in CAP should not be made depending on cortisol values or cosyntropin testing results.

International survey on high- and low-dose synacthen test and assessment of accuracy in preparing low-dose synacthen.

OBJECTIVE: The short synacthen test (SST) is widely used to assess patients for adrenal insufficiency, but the frequency and protocols used across different centres for the low-dose test (LDT) are unknown. This study aimed to survey centres and test the accuracy of ten different synacthen preparation strategies used for the LDT. METHODS: Members of 6 international endocrine societies were surveyed regarding diagnostic tests used for adrenal insufficiency, and in particular the SST. Synacthen was diluted for the LDT and concentrations measured using a synacthen ELISA. RESULTS: Survey responses were received from 766 individuals across 60 countries (52% adult, 45% paediatric endocrinologists). The SST is used by 98% of centres: 92% using high-dose (250 µg), 43% low-dose and 37% both. Ten low-dose dilution methods were assessed and variation in synacthen concentration was demonstrated with intramethod coefficients of variation (CV) ranging from 2.1% to 109%. The method using 5% dextrose as a diluent was the least variable (CV of 2.1%). The variation in dilution methods means that the dose of synacthen administered in a LDT may vary between 0.16 and 0.81 µg. CONCLUSIONS: The high-dose SST is the most popular diagnostic test of adrenal insufficiency, but up to 72% of paediatric endocrinologists use a LDT. There is considerable variation observed both within and between low-dose synacthen dilution methods creating considerable risk of inaccurate dosing and thereby invalid results.

Detection of tetracosactide in plasma by enzyme-linked immunosorbent assay (ELISA).

As a synthetic analogue of adrenocorticotropic hormone (ACTH), tetracosactide is prohibited in sport by the World Anti-Doping Agency (WADA). An enzyme-linked immunosorbent assay (ELISA) method is proposed for detection of this drug in plasma. Since its structure corresponds to the 24 N-terminal of the 39 amino acids of the natural endogenous peptide ACTH, tetracosactide can be detected with a commercial ELISA kit for ACTH that uses antibodies, the epitopes of which are located in the 1-24 part of ACTH. However, an essential condition for detection specificity is the preliminary total clearance of endogenous ACTH in the plasma samples. This is achieved by a preparative step based on cation-exchange chromatography before ELISA. The method is specific and sensitive (LOD: 30 pg/mL) and may be used as a screening analysis in anti-doping control. The pre-analytical conditions are shown to be of the upmost importance and recommendations for blood collection (EDTA tubes), sample transport (4 °C) and plasma sample storage (-20 °C) are presented.

Different cut-off values of the insulin tolerance test, the high-dose short Synacthen test (250 µg) and the low-dose short Synacthen test (1 µg) in assessing central adrenal insufficiency.

OBJECTIVE: The short Synacthen test (SST) is widely used as alternative test to the insulin tolerance test (ITT) to investigate central adrenal insufficiency (CAI), but the methodology and cut-off values of the SST are controversial. Our aim was to evaluate the cut-off value of the ITT in normal subjects and to assess the different cut-off values of the high-dose SST (250 µg, HDT) and the low-dose SST (1 µg, LDT) in subjects with suspected CAI. SUBJECTS AND METHODS: We conducted ITTs in 208 normal subjects to establish the cut-off value for the ITT, and 28 of those subjects underwent the HDT and LDT. From 1999 to 2007, 182 patients with suspected CAI were recruited and underwent ITTs, LDTs and HDTs to establish cut-off values and compare the diagnostic accuracy between the LDT and HDT. RESULTS: The 95th percentile of the peak cortisol level during the ITT in the normal control subjects was 14·8 µg/dl. Receiver operator characteristics (ROC) analysis revealed that the optimal cut-off values of peak cortisol in the LDT and HDT in patients with suspected CAI were 15·8 and 17·4 µg/dl, respectively. However, the cut-off values from normative data (mean - 2 SD) were 18·3 µg/dl for the LDT and 20·5 µg/dl for the HDT in normal control. CONCLUSIONS: The optimal cut-off values of SSTs needed to be individualized according to the type of SST and tested patient population.

Expression of leukemia inhibitory factor and leukemia inhibitory factor receptor in the canine pituitary gland and corticotrope adenomas.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine of the IL-6 family that activates the hypothalamic-pituitary-adrenal axis and promotes corticotrope cell differentiation during development. The aim of this study was to investigate the expression of LIF and its receptor (LIFR) in the canine pituitary gland and in corticotrope adenomas, and to perform a mutation analysis of LIFR. Using immunohistochemistry, immunofluorescence, and quantitative expression analysis, LIF and LIFR expression were studied in pituitary glands of control dogs and in specimens of corticotrope adenoma tissue collected through hypophysectomy in dogs with pituitary-dependent hypercortisolism (PDH, Cushing's disease). Using sequence analysis, cDNA was screened for mutations in the LIFR. In the control pituitary tissues and corticotrope adenomas, there was a low magnitude of LIF expression. The LIFR, however, was highly expressed and co-localized with ACTH(1-24) expression. Cytoplasmatic immunoreactivity of LIFR was preserved in corticotrope adenomas and adjacent nontumorous cells of pars intermedia. No mutation was found on mutation analysis of the complete LIFR cDNA. Surprisingly, nuclear to perinuclear immunoreactivity for LIFR was present in nontumorous pituitary cells of the pars distalis in 10 of 12 tissue specimens from PDH dogs. These data show that LIFR is highly co-expressed with adrenocorticotropic hormone (ACTH) and alpha-melanocyte-stimulating hormone (alpha-MSH) in the canine pituitary gland and in corticotrope adenomas. Nuclear immunoreactivity for LIFR in nontumorous cells of the pars distalis may indicate the presence of a corticotrope adenoma.

Reversed-phase capillary high-performance liquid chromatography with on-line UV, fluorescence and electrospray ionization mass spectrometric detection in the analysis of peptides and proteins.

Analysis of peptide mixtures by reversed-phase capillary HPLC with gradient elution using three detectors in series: UV (214 nm), fluorescence (lambda exc. = 280 nm, lambda emiss. = 356 nm), and electrospray ionization mass spectrometry (ES-MS) is reported. The chromatographic integrity of the system and the detection limits were evaluated. The effect of the mass spectrometer's acquisition rate on the total ion current (TIC) profile was also examined. The utility of fluorescence monitoring with UV and ES-MS detection was demonstrated in the analysis of proteolytic digests of proteins. The native fluorescence character of tryptophan-containing peptides provides selectivity in peptide mapping, while monitoring UV absorption at 214 nm affords detection of the peptide bond. Three tryptophan-containing tryptic peptides of bovine serum albumin were immediately located by fluorescence among many UV peaks and ES-MS provided molecular masses allowing the peptides to be identified.

Adrenocorticotropin(1-10) and -(11-24) promote adrenal steroidogenesis by different mechanisms.

ACTH1-10 and ACTH11-24 each elicit cortisol secretion submaximally in freshly dispersed or cultured beef adrenal cortical cells. The combination of ACTH1-10 and ACTH11-24 promotes cortisol release to the maximal level elicited by ACTH1-24. Maximal cortisol release by ACTH11-24, but not by ACTH1-24 or ACTH1-10, was enhanced by forskolin. The calcium channel blockers nifedipine and verapamil inhibited cortisol release by ACTH1-10, ACTH1-24 or ACTH11-24, suggesting calcium influx to be essential for steroid secretion regardless of the secretagogue. Vanadium, in a dose-dependent manner, inhibited cortisol secretion elicited by ACTH1-24 and ACTH1-10 but not that caused by ACTH11-24. These results suggest that there are at least two receptors mediating ACTH1-24-dependent steroid secretion. One class of receptor recognizes ACTH1-10 but not ACTH11-24 and is linked to the cAMP messenger pathway.

Immunohistological study of Crooke's cells.

By immunohistochemical methods, 28 postmortem pituitary glands with unequivocal Crooke's hyaline change were examined to identify the hyaline material. Crooke's hyaline was positive for 55-57 kilodalton (KD) cytokeratin (CK) but negative for 68 KD CK, vimentin, desmin, neurofilament and glial fibrillary acidic protein. Involucrin, a new marker for keratinocyte differentiation, could also not be demonstrated. It was concluded that Crooke's hyaline was an abnormally accumulated CK which is a normal constituent of the ACTH cell.

[1H-NMR study of the peptide corresponding to the ACTH-like sequence of the variable region of human G1 Eu heavy chain immunoglobin]. / 1H-IaMR-issledovanie peptida, sootvetstvuiushchego AKTG-podobnoi posledovatel'nosti variabel'noi chasti tiazheloi tsepi immunoglobulina G1 Eu cheloveka.

Synthetic decapeptide corresponding to ACTH-like sequence of the variable part of the heavy chain of immunoglobulin G1 Eu was studied by two-dimensional 1H-NMR spectroscopy (400 MHz). A complete assignment of signals in the peptide spectrum was made. The decapeptide was shown not to have any ordered spatial structure, and was characterized by a high extent of flexibility of the oligopeptide chain, except for the peptide bond with an N-terminal residue.

[Analysis of protected peptides--ACTH(1-24) fragments using high performance liquid chromatography]. / Analiz zashchishchennykh peptidov--fragmentov ACTH-(1-24) metodom vysokoéffektivnoi zhidkostnoi khromatografii.

The applicability of reversed-phase ion-pair HPLC for separating the ACTH-(1-24) fragments obtained at the final stage of the synthesis has been examined. Optimization of chromatographic conditions (selection of proper packing material, eluent and concentration of ion-pairing reagent) for separation of protected peptides has been performed. A mixture of the ACTH fragments 1-4, 5-12, 13-24, 5-24, and 1-24 has been resolved on a Zorbax C8 column using methanol - 0,01 M tetrabutylammonium bromide (94:6) as mobile phase.

A radioassay for nonoxidized methionine in peptides. A method for identifying (after isoelectric focusing) and for estimating biologically active forms of corticotropin and other hormones.

A radioassay for nonoxidized methionine in peptides is described; it has advantages over other methods currently used because of its simplicity, sensitivity, accuracy, and applicability to individual peptide components in mixtures and to many samples at a time. Methionyl residues were S-carboxymethylated with iodo[2-14C]acetic acid; iodo[2-3H]acetic acid did not provide a stable radioactive tracer. The labeled peptide was isolated by carboxymethylcellulose chromatography or by isoelectric focusing (IEF) or electrophoresis in polyacrylamide gel, and its radioactivity measured. The assay was applied to corticotropins, alpha-melanotropin, bombesin, glucagon, substance P, parathormone, and calcitonin. Twenty-four to thirty samples were conveniently analyzed at a time with a lower detection limit of less than 1 nmol of methionine per sample. The accuracy of the assay, assessed also by reverse-phase high-performance liquid chromatography, is a consequence of its precision, the specificity of the reaction with iodoacetic acid, and the use of an appropriate standard of the peptide being assayed. Methionine was identified, and could be estimated, in individual peptide components of a mixture by using IEF to separate simultaneously the labeled peptide from iodo[2-14C]acetic acid and from other peptide and protein components. This was facilitated by a convenient method for detecting and quantifying these peptides after IEF. The assay is particularly useful for several peptide hormones whose biological activity depends on their sole methionine residue being in a nonoxidized state. It can be used for monitoring their isolation or synthesis and their stability during processing and storage, as well as for evaluating differences in biological potency between preparations and analogues.

The International Reference Preparation of Tetracosactide for Bioassay: characterization and estimation of its (1-24)corticotrophin-tetracosapeptide content by physicochemical and biological methods.

The preparation and nature of the International Reference Preparation of Tetracosactide for Bioassay (IRP; in ampoules coded 80/590) are described. The IRP was studied by six laboratories in five countries using in-vivo and in-vitro bioassays and various physicochemical methods. The bulk (1-24)corticotrophin-tetracosapeptide (batch 000179) from which the IRP was prepared contained 10.4% (w/w) acetic acid and 8.3% (w/w) water; its (1-24)corticotrophin-tetracosapeptide content was estimated to be 71.7% (w/w) by amino acid analysis, 74.2% (w/w) by high performance liquid chromatography (HPLC) and 77.5% (w/w) by spectrophotometry. (1-24)Corticotrophin-tetracosapeptide accounted for more than 90% (w/w) of the total peptide in the IRP as judged by HPLC, thin-layer chromatography, carboxymethyl-cellulose chromatography, isoelectric focusing (IEF) and electrophoresis. The homogeneity of the peptide in the IRP was similar by all methods to that in batch 000179 from which it was prepared. The (1-24)corticotrophin-tetracosapeptide content of the IRP (with 95% confidence limits), in terms of batch 000179, was found to be 491 micrograms/ampoule by HPLC and spectrophotometry, 473 (433-513) micrograms/ampoule by IEF and 505 (473-539) micrograms/ampoule by the in-vitro rat adrenocortical cell assay. A comparison in the same bioassay system of the IRP with a laboratory house standard of (1-24)corticotrophin-tetracosapeptide, which originated from a different manufacturer, gave similar results. Accelerated thermal degradation studies of the IRP by adrenocortical cell assay, HPLC and IEF suggested that more than 99.9% of its original content of (1-24)corticotrophin-tetracosapeptide would remain after 10 years under normal storage conditions of -20 degrees C in the dark. Bioassay estimates of samples of the IRP which had undergone significant degradation were higher than estimates by HPLC, indicating that molecular species other than (1-24)corticotrophin-tetracosapeptide contributed to their corticotrophic activity. The corticotrophic activity of the IRP was demonstrated by cytochemical bioassay and by in-vivo bioassays as well as by the adrenocortical cell assay. After consideration of these data, the Expert Committee on Biological Standardization of the World Health Organization established the ampoule d preparation, coded 80/590, as the International Reference Preparation of Tetracosactide for Bioassay and assigned to it a potency of 490 i.u./ampoule; thus the i.u. is represented by 1 microgram (1-24)corticotrophin-tetracosapeptide.

[Possibility of using the larvae of anuran amphibians as a test model for the quantitative determination of hypophyseal hormones]. / O vozmozhnosti primeneniia lichinok beskhvostykh amfibii v kachestve test-modeli dlia kolichestvennogo opredeleniia nekotorykh gipofizarnykh gormonov.

A possibility to use the larvae of ecaudate amphibia as a test model for the quantitative determination of certain hypophyseal hormones has been shown. The physiological reactions of the pigmentary cells in tadpoles of Rana temporaria can be used for a quick and exact testing of the adrenocorticotropic hormone preparations (ACTH): corticotropin and Zn-corticotropin. Evidently the method elaborated for the biological testing of hypophyseal hormones may be used in the production of the endocrinous preparations as a quick assessment of melanocyte-stimulating hormones in integral raw and sublimated hypophyses.

Immunocytochemistry of pituitary corticotroph cells in terrestrial turtle (Testudo mauritanica). / Etude immunocytochimique des cellules corticotropes de l'hypophyse de la Tortue terrestre (Testudo mauritanica)

The immunocytochemical and histochemical characters of the corticotroph cells of the Turtle adenohypophysis have been studied. These cells are localised in the rostral part of the gland and are revealed by Is anti ACTH (1-24) and (17-39). They are also colored with lead hematoxyline and PAS-Orange G. The corticotroph nature of these cells is confirmed by the study of their modifications after treatment with amphenone and ACTH. The Is anti ACTH also reveal most of the cells of the pars intermedia; while the Is anti beta-MSH reveals only these cells and some scatter cells of the pars distalis.