A septic shock is considered a systemic stressor eliciting a prolonged activation of the hypothalamic-pituitary-adrenal (HPA) axis by release of the corticotropin-releasing hormone (CRH) from cells of the paraventricular nucleus into the hypophyseal blood supply. This, in turn, stimulates the anterior pituitary gland to release corticotropin (ACTH) and further proopiomelanocortin (POMC) derivatives, for example, β-endorphin (β-END) (1), into the cardiovascular compartment.
In the pituitary of lower species such as the rat, POMC is processed by two enzyme systems present in different types of cells: corticotroph-type and melanotroph-type cells. In corticotroph-type cells of the pituitary, the enzymatic cleavage of POMC by the prohormone convertase PC-1 leads to the release of ACTH, β-lipotropin (β-LPH), and β-END; in melanotroph-type cells, these fragments are additionally processed by the prohormone convertase PC-2 to release smaller fragments. Furthermore, the smaller fragments are modified, for example, by N-terminal acetylation or C-terminal amidation, which results in the release of derivatives such as α-melanotropin (α-melanocyte-stimulating hormone [α-MSH]) or N-acetyl-β-END (Nac-β-END) (2, 3). In the human pituitary, unlike in the rat, corticotroph- and melanotroph-type enzymatic cleavage of POMC apparently occurs in all POMC processing cells, which are dispersed throughout the anterior lobe of the pituitary (4).
Much data have been published concerning the release of the ACTH and the consecutive glucocorticoid response during septic shock, and there is evidence that an intact HPA axis and resultant glucocorticoid release are critical for the host survival (5, 6). As yet, there is no information about the physiological significance of further POMC derivatives after activation of the HPA axis by septic shock as a systemic stressor, although further POMC derivatives such as the melanotroph-type α-MSH are known to be released under stress into the cardiovascular compartment (7-10), whereby α-MSH is known to be a modulator of inflammatory processes by impairing important functions of antigen-presenting cells and T cells (11). Moreover, several anti-inflammatory effects have been described for α-MSH, such as suppression of fever induced by IL-1 or IL-6, induction of the anti-inflammatory mediator IL-10, and inhibition of macrophage function and leukocyte migration (11, 12). Therefore, we hypothesized that further POMC derivatives such as α-MSH may play a critical role at the early stage of septic shock.
Thus, in 17 patients with septic shock without adrenocortical insufficiency, and in 16 healthy volunteers, we studied the response of corticotroph-type and melanotroph-type pituitary POMC systems after i.v. administration of 100 μg human CRH; 60 min after CRH administration, 250 mg dexamethasone was given intravenously. We excluded patients with adrenal insufficiency because we wanted to study the significance of pituitary functions for the HPA control circuit and thus tried to exclude an influence of adrenal failure on the HPA control circuit.
MATERIALS AND METHODS
This prospective study in patients was performed between June 2004 and July 2006 in an intensive care unit (ICU) of a university hospital. Patients of the ICU as far as observed to be at the onset of severe sepsis were subjected to an ACTH stimulation test using an i.v. bolus of 250 μg tetracosactin (Synacthene; Novartis Pharma Ltd., Nürnberg, Germany). The ACTH stimulation tests were performed between 8:00 am and 4:00 pm. Blood samples were taken immediately before and 30 and 60 min after the test. Cortisol response was defined as the difference between the highest of the concentrations taken after tetracosactin administration and those taken before the test. Adrenocortical insufficiency was defined by a response cortisol concentration of less than 248 nmol/L (9 μg/dL) (13, 14). Only responders were included in the study, and of those, only the patients who developed septic shock were included.
In the current study, patients during the acute phase of septic shock (between 4 and 15 h after the need for vasopressors to maintain a systolic blood pressure >90 mmHg or a mean arterial pressure >60 mmHg; Table 2) showing no symptoms of adrenocortical insufficiency (i.e., patients with an intact HPA control circuit) were enrolled if they met the criteria of septic shock proposed by the American College of Chest Physicians and the Society of Critical Care Medicine Consensus Conference Committee of 1992 (15). Exclusion criteria were age less than 18 years, pregnancy, known endocrine disorders, immunosuppressive medication, chemotherapy, radiation therapy or cardiopulmonary resuscitation within 1 month before the study, and life expectancy of less than 2 months as well as administration of corticosteroids, opiates, or etomidate within 48 h before study commencement. In addition, 16 individuals without clinical signs of HPA axis dysfunction (healthy participants) were included for comparison. Approval of the study was obtained from the ethics committee of the University of Giessen (Medical Faculty); written consent to participate in the study was given by 17 patients or their surrogate (for characteristics, see Table 1). Twenty-eight days after the diagnosis of septic shock, the study was terminated in the event of death (nonsurvivor) or with discharge (survivor) of the patient from the ICU without any signs of sepsis or organ dysfunctions. We followed the hypothalamus-pituitary response to the administration of exogenous CRH within 24 h after diagnosis of septic shock.
The CRH tests (patients and healthy participants) were performed between 4:00 and 5:00 pm and more than 24 h after the ACTH stimulation test, starting with an i.v. bolus injection of 100 μg human CRH (CRH Ferring; Ferring, Kiel, Germany). Before (t0) and 15 (t15), 30 (t30), 45 (t45), and 60 (t60) min after CRH administration, blood was drawn from a central venous catheter (patients) or from a forearm catheter (healthy participants) to determine POMC derivatives such as ACTH, authentic β-END [β-END(1-31)], β-END immunoreactive material (IRM), β-LPH IRM, Nac-β-END IRM, and α-MSH as well as cortisol. Thereafter, 250 mg dexamethasone was intravenously injected; 60 min after dexamethasone administration (tdex+60), blood was drawn from the catheter again.
Plasma sample collection
Twenty-four milliliters of blood was taken from the central venous catheter (patients) or from a forearm catheter (healthy participants), mixed with 300 μL EDTA (0.08 g/mL), immediately placed on ice, and centrifuged at 1,000 g (15 min, 4°C). Two 5-mL aliquots of plasma were acidified, each with 0.5 mL HCl (1 mol/L); approximately 2 mL plasma was used for determination of ACTH and cortisol. All samples were immediately frozen at 80°C until extraction. For standardization of the assays, β-END(1-31), Nac-β-END(1-31), α-MSH, and β-LPH were added to EDTA-plasma samples from three healthy volunteers (consent to participate in the study was given) in concentrations of 0.33, 1, 3.3, 10, 33, 100, and 1,000 pmol/L. These plasma standard samples were treated in exactly the same way as the patients' samples.
The extraction of the patients' and the standard plasma samples was conducted as described previously (16). In brief, the samples were thawed at 4°C, and the 5-mL aliquots of acidified plasma were passed at 4°C through Sep-Pak C18 cartridges (Waters, Milford, Mass), which had been activated before with 5 mL methanol followed by 5 mL urea (8 mol/L) and 10 mL water (bidistilled, 4°C). Then the cartridges were washed with 10 mL acetic acid (4% in water) and 10 mL water. Elution of the POMC derivatives from the cartridges was achieved with 10 mL 1-propanol with 4% acetic acid (96/4 = vol/vol). The eluate was dried at room temperature using a SpeedVac Concentrator (Savant, New York, NY). The remaining aqueous phase was lyophilized; the residue was reconstituted on ice in 0.5 mL buffer 1 (see below), and 100-μL aliquots-referred to as "extracts" (see below)-were frozen at −80°C and kept for further analyses.
Determination of POMC fragments and cortisol
Peptides, reagents, and buffer
Synthetic human β-END(1-31), Nac-β-END(1-31), and β-LPH were obtained from Bachem, Heidelberg, Germany; from Nova-Biochem, Bad Soden, Germany; and from Dr A. F. Parlow, Torrance, Calif, respectively. A monoclonal antibody against the N-terminus of β-END, code 3E7, had been obtained from C. Gramsch (deceased), Schwabhausen, Germany. Buffers used were buffer 1 (0.02 mol/L sodium phosphate [pH 7.40] containing 0.15 mol/mL sodium chloride, 0.1% [wt/vol] gelatin, 0.01% [wt/vol] bovine serum albumin, and 0.01% [wt/vol] thimerosal) and buffer 2, which consisted of buffer 1 with an additional 0.1% (vol/vol) Triton X-100.
A fluid-phase, two-site immunoprecipitation radioimmunoassay (RIA), as described previously, was used to determine authentic β-END in plasma extracts (17). In brief, a monoclonal mouse antibody (code 3E7), directed against the N-terminus of β-END(1-31), and a polyclonal rabbit antiserum (code 11P), directed against the C-terminal fragment of β-END(1-31), were used. The monoclonal mouse antibody (3E7) was radioactively labeled with iodine 125 (125I) using published techniques (16, 18); for purification, the iodination product was incubated with charcoal for 15 min on ice, the suspension was centrifuged at 8,000 g (5 min), and the supernatant containing the 125I-labeled antibody (3E7) was stored at 4°C.
For the assay, plasma extracts (in buffer 1) were thawed at 4°C and were incubated together with the labeled antibody (3E7) and the rabbit antiserum (11P). A 125I-3E7/β-END(1-31)/11P complex was formed in the case of samples containing β-END(1-31). The complex was immunoprecipitated by a goat antiserum directed against rabbit immunoglobulin (IgG). The immunoprecipitate was centrifuged; the radioactivity measured in the pellet indicated the amount of precipitated β-END(1-31).
This two-site immunoprecipitation RIA proved highly specific for β-END(1-31). Neither β-LPH, fragments of β-END (e.g., α- or γ-END), β-END(1-27), N-acetylated endorphins, metenkephalin, or any other POMC peptide cross-reacted with this assay. The intra-assay and interassay coefficients of variation were 3.7% or 3.8%, respectively. The detection limit varied between 2 and 5 pmol β-END(1-31)/L plasma and was determined separately for each assay (17, 19-23).
β-Lipotropin (45-54) IRM was determined using a competitive fluid-phase RIA conducted, in principle as described for previously published RIAs (16). In brief, plasma extracts (in buffer 1) were thawed at 4°C and incubated together with a polyclonal rabbit antiserum (code 93E) against the (45-54) fragment of human β-LPH and with 125I-labeled β-LPH(37-58), that is, β-MSH (in buffer 2) at 4°C for 24 h. To separate antibody-bound from free-labeled peptides, the samples were incubated together with charcoal and subsequently centrifuged to remove charcoal-adsorbed peptide. The supernatants were analyzed for radioactivity in a gamma-counter to determine antibody-bound labeled peptides.
Intra-assay and interassay coefficients of variation were 9.6% or 10.2%, respectively. The cross-reactivity with α-MSH was 0.43%; cross-reactivities with γ2-MSH, β-END(1-31), and further peptides tested were less than 0.1%. The detection limit varied between 1.7 and 24.8 pmol β-LPH/L plasma and was determined separately for each assay.
N-acetyl-β-END IRM was determined using a competitive fluid-phase RIA, which was conducted according to previously described assays (16). In brief, plasma extracts (in buffer 1) were thawed at 4°C and were incubated together with a polyclonal rabbit antiserum (code 27P) against the acetylated N-terminus of β-END and with 125I-labeled Nac-β-END(1-27) (in buffer 2) at 4°C for 24 h (for further conduction of the assay, see section β-Lipotropin IRM). The assay picks up all N-acetylated β-END derivatives but does not recognize nonacetylated derivatives. The detection limit was 3.5 pmol/L plasma; the intra-assay coefficient of variation was 3.6%; the interassay coefficient of variation was 4.3%.
β-Endorphin IRM was determined in a one-site fluid-phase RIA, in principle as described previously (24). In brief, plasma extracts (in buffer 1) were thawed at 4°C and were incubated together with a polyclonal rabbit antiserum (code 84E) against the (17-26) fragment of human β-END (diluted 1:10,000 with buffer 1) and with 125I-labelled β-END(1-31) (in buffer 2) at 4°C for 24 h (for further conduction of the assay, see section β-Lipotropin IRM).
α-Melanocyte-stimulating hormone (1-13)
α-Melanocyte-stimulating hormone (1-13) was determined using a competitive fluid-phase RIA, which was conducted according to previously described assays (16). In brief, plasma extracts (in buffer 1) were thawed at 4°C and were incubated together with a polyclonal rabbit antiserum (code 174E) against the amidiated C-terminus of α-MSH and with 125I-labelled α-MSH(1-13) (in buffer 2) at 4°C for 24 h (for further conduction of the assay, see section β-Lipotropin IRM). The assay picks up only α-MSH(1-13) but does not recognize the N-terminus of α-MSH(1-10) and Ac-N(Tyr5)-α-MSH(1-5). Furthermore, the cross-reactivities with ACTH(1-10), as well as β-, γ-, and γ2-MSH, were less than 0.1%. The detection limit varied between 1 and 3 pmol/L plasma; the intra-assay coefficient of variation was 11%; the interassay coefficient of variation was 3.5%.
ACTH(1-39) in plasma was measured using a commercially available two-site chemiluminescence assay (Nichols, San Juan Capistrano, Calif). The intra-assay coefficient of variation at 6.6 ng/L was 3.4%, and the limit of detection was 0.5 ng/L.
Cortisol in plasma was measured using a commercially available enzyme-linked immunosorbent assay (Enzymun-Test Cortisol; Boehringer Mannheim Diagnostica, Mannheim, Germany). The detection limit was 27.6 nmol/L.
For descriptive statistics, minimum and maximum values, as well as first, second (median), and third quartiles, for example, of POMC derivative plasma concentrations, were determined. Analytical statistics were performed using the Wilcoxon signed rank test for nonparametric data. The significance of differences between plasma concentrations of the POMC derivatives or cortisol at different times was examined by calculating Hodges-Lehmann estimators (Symbol) associated with the Wilcoxon signed rank statistics (25). The level of significance was set at the two-sided 95% confidence interval for Symbol. For a concentration difference deviating significantly from zero, both the lower and the upper values of a 95% confidence interval had to be either positive or negative; however, in either case, one of them still was allowed to be zero.
The data determined before the CRH administration at t0 were entered as baseline values. The responses to CRH were calculated as the net areas under the plasma concentration curves (AUCsPOMC derivative). To obtain these values, first, the area under the response curve was calculated by integrating the times on the abscissa and the dependent variables on the ordinate as measured over all times. Then, a second area was calculated by using only the y-value measured at t0 (baseline value before CRH administration) for integration over all measured times. Finally, the second area was subtracted from the first one.
Seventeen patients during the acute phase of septic shock were included in the current study; 7 of those 17 patients survived, and 10 died. The general characteristics of the patients with septic shock enrolled in the current study are given in Table 1. No significant differences were found between survivors (n = 7) and nonsurvivors (n = 10), considering the mean Simplified Acute Physiology Score II (SAPS II) number of sequential organ failure assessment, age, body mass index, sex, pre-existing diseases, infections, and microorganisms. The catecholamine infusions, the times from shock onset to administration of a vasopressor, clinical and biochemical data, and the cortisol response after 0.25 mg i.v. injection of synthetic ACTH(1-24) (Synacthene) in the patients with septic shock are shown in Table 2.
Corticotroph-type POMC systems in patients with septic shock
Intravenous administration of 100 μg CRH induced in all patients and in the controls a significant increase in the plasma concentrations of corticotroph-type POMC derivatives, ACTH and β-END(1-31) (Figs. 1 and 4, A and B), β-END IRM, and β-LPH IRM (data not shown) as observed 15 min after injection of CRH (t15) in comparison to basal levels at t0. Correspondingly, the net AUCs, AUCACTH, AUCβ-END IRM, AUCβ-END(1-31), and AUCβ-LPH IRM did not significantly differ between survivors, nonsurvivors, and controls (Fig. 5). In patients, the release of ACTH and β-END(1-31), as well as for β-END IRM and β-LPH IRM (data not shown), into the cardiovascular compartment was suppressed 60 min after i.v. injection of dexamethasone.
Melanotroph-type POMC systems in patients with septic shock
In survivors, the plasma concentrations of α-MSH were found to be increased 15 min after CRH injection and increased again between 30 and 60 min after treatment with CRH (Fig. 2A), whereas in the nonsurvivors, no significant release of α-MSH after treatment with CRH was observed (Fig. 2B). In the controls, we found an increase of plasma concentrations of α-MSH from t0 to t15 and, further on, from t15 to t30 (Fig. 4C).
In survivors, a permanent increase of Nac-β-END IRM plasma concentrations was found over an observation period of 60 min after injection of CRH (Fig. 3A). In contrast, no significant increase of Nac-β-END IRM plasma concentrations was found after administration of CRH in nonsurvivors (Fig. 3B). In controls, no significant increase of Nac-β-END IRM plasma concentrations was observed within the first 30 min after injection of CRH (Fig. 4D); however, from 30 to 45 min after CRH administration, Nac-β-END IRM plasma concentrations increased significantly.
In survivors but not in nonsurvivors, the release of α-MSH and Nac-β-END IRM into the cardiovascular compartment was found to be suppressed 60 min after injection of dexamethasone (Figs. 2 and 3); however, the plasma concentration of Nac-β-END IRM was found to be increased. In accordance with the plasma concentrations of α-MSH and Nac-β-END IRM after injection of CRH, the net areas under the concentration curves, AUCα-MSH, and AUCNac-β-END IRM in nonsurvivors were significantly lower than in survivors and controls (Fig. 5).
Much data have been published concerning the functional integrity of the HPA axis in patients with septic shock. However, septic shock should be considered a systemic stressor eliciting the release not only of ACTH but also of other corticotroph-type POMC derivatives such as β-END or β-LPH as well as melanotroph-type POMC derivatives such as Nac-β-END or α-MSH, which have not yet been under study during septic shock.
In contrast to all previous studies, we did study not only the release of ACTH as the only parameter for the functional integrity of the HPA axis; in our study, we examined the release of further corticotroph-type and melanotroph-type POMC derivatives in patients under conditions during the acute phase of septic shock. It is well known that HPA axis activation and glucocorticoid response are critical for the survival of the host and that adrenal insufficiency in patients with septic shock carries prognostic implications (5, 6, 26); thus, in the current study, only patients without adrenal insufficiency but with septic shock were included.
In the current study, we examined the function of corticotroph-type and melanotroph-type POMC systems of the pituitary in patients with septic shock to obtain information about their relevance for the patient's survival during septic shock.
Corticotroph-type POMC system in patients with septic shock
In this study on patients during the acute phase of septic shock in comparison to healthy volunteers, the i.v. administration of 100 μg CRH caused increased plasma levels of corticotroph-type POMC derivatives such as ACTH, β-END IRM, β-END(1-31), and β-LPH. In all patients (survivors and nonsurvivors), we found elevated levels of ACTH, β-END IRM, β-END(1-31), and β-LPH after CRH stimulation as expected. Thus, we conclude that, during the acute phase of septic shock, the ability of the pituitary to release corticotroph-type POMC derivatives is not related to severity of septic shock, or in turn, the release of corticotroph POMC derivatives into the cardiovascular compartment does not influence the prognosis of septic shock. Furthermore, in both survivors and nonsurvivors, the release of all corticotroph-type POMC derivatives was suppressed by dexamethasone. Therefore, we conclude that the corticotroph POMC system of the pituitary is not linked to the survival rate of septic shock during its acute phase.
In several studies, authors have demonstrated elevated plasma cortisol concentrations (27) or inadequate increment of cortisol after ACTH stimulation in patients with septic shock (28), but during the acute phase of critical illness, the increased cortisol release is correlated with increased ACTH levels (29). However, during the chronic phase of sepsis, there is a dissociation between ACTH and cortisol levels, ACTH being low while cortisol levels are high [3 to 4 days after administration to ICU (29)]. This suggests that the prolonged disease state results in a non-ACTH-mediated glucocorticoid release (30). The high glucocorticoid level together with the low ACTH level during the prolonged phase of sepsis suggests an immunosuppressive state, which might be the reason for the increased susceptibility to infectious complications during the chronic phase of septic shock. In any case, the glucocorticoid physiology and regulation in endotoxemia and septic shock are far from being completely understood.
Melanotroph-type POMC system in patients with septic shock
Although it was shown that CRH, which has a pivotal role in stress response, is a stimulator of α-MSH (31, 32) and Nac-β-END IRM (7) release from the pituitary into the cardiovascular compartment, melanotroph-type POMC derivatives such as Nac-β-END IRM or α-MSH have not been studied in patients with septic shock as yet.
In our study, the plasma concentrations of melanotroph-type POMC derivatives such as Nac-β-END IRM and α-MSH showed a significant increase in response to CRH administration in survivors, whereas in nonsurvivors, no significant release of melanotroph-type POMC derivatives was observed. Thus, with regard to the prognosis of septic shock, we have to distinguish between corticotroph-type and the melanotroph-type POMC systems of the pituitary. Failure of the melanotroph-type POMC system seems to be associated with a poor prognosis in patients with septic shock. We conclude that not only the release of ACTH and the adequate function of the adrenals, but also the release of melanotroph-type POMC derivatives and their target tissues, are related to the outcome in sepsis or septic shock. However, we do not know whether the melanotroph-type POMC system of the pituitary has any influence on the severity of illness or mortality of patients with septic shock, or vice versa, whether the conditions of septic shock influence the release of the melanotroph-type POMC derivatives from the pituitary into the cardiovascular compartment. In any case, we have to consider the functional significance of melanotroph-type POMC derivatives under the conditions of septic shock.
It is well known that α-MSH, a melanotroph-type POMC derivative, is a significant modulator of inflammatory processes (33) by impairing both antigen-presenting cells and T cells (11); specific receptors for α-MSH have been found on neutrophils and monocytes (34). Several anti-inflammatory effects have been described for α-MSH, such as suppression of fever induced by IL-1 or IL-6, induction of the anti-inflammatory mediator IL-10, and inhibition of macrophage function and leukocyte migration (11, 12). To the best of our knowledge, there is only one clinical study in patients with septic shock that demonstrated that plasma concentrations of α-MSH are reduced in the early phase of septic shock, and addition of α-MSH to LPS-stimulated blood samples reduces production of cytokines involved in the development of septic shock (35).
In our study, nonsurvivors showed no significant increase of α-MSH and Nac-β-END but a significant increase of β-END(1-31) and β-END IRM after treatment with CRH. One of the reasons may be that the enzyme N-acetyltransferase in the pituitary may be decreased in nonsurvivors. Interestingly, previous studies have demonstrated that hepatic N-acetyltransferase, a major enzyme for the conjugation of bile acids, is significantly decreased during sepsis (36), and moreover, augmentation of hepatic N-acetyltransferase improved survival rate in a rodent model of sepsis (37). However, the possibility of a correlation between the survival rate under septic conditions and the activity of the enzyme N-acetyltransferase requires further investigations. Furthermore, exaggerated inflammatory response, resulting in high cytokine levels, might provide an alternative explanation for the melanotroph-type POMC system dysfunction as observed.
In summary, the present study shows that, during the acute phase of septic shock in survivors and nonsurvivors, there is no characteristic difference in the release of corticotroph-type POMC derivatives into the cardiovascular compartment after CRH administration and that there is no significant correlation between the concentrations of corticotroph-type POMC derivatives and the patient's outcome. In contrast to the corticotroph-type POMC derivatives, the plasma concentrations of α-MSH and Nac-β-END IRM after injection of CRH and, thus, the net areas under the concentration curves, AUCα-MSH, and AUCNac-β-END IRM were significantly lower in the nonsurvivors than in the survivors and the controls. Thus, the corticotroph-type and the melanotroph-type pituitary POMC system should be regarded separately. In fact, plasma concentrations of melanotroph-type POMC derivatives such as α-MSH or Nac-β-END but not of corticotroph-type POMC derivatives correlated with survival in septic shock. However, these data are preliminary, and we cannot draw definite conclusions on the prognostic value of melanotroph-type POMC derivatives as yet. Further studies with a larger cohort of patients are needed to verify our results. Nevertheless, understanding the function of melanotroph-type POMC derivatives such as α-MSH and Nac-β-END under septic conditions might provide key information for further development of therapeutic strategies for treatment of patients under septic shock conditions.
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Corticotropin (ACTH); authentic β-endorphin [β-endorphin (1-31)]; β-lipotropin (β-LPH); N-acetyl-β-endorphin (Nac-β-END); α-melanocyte-stimulating hormone (α-MSH); corticotropin releasing hormone (CRH); hypothalamic-pituitary-adrenal (HPA) axis