In this study, we found that the plasma levels of mature and total AM on day 1 of ICU admission in patients with sepsis were significantly higher than in those without sepsis. The good correlation between the plasma levels of both the peptides recognized in the present study demonstrates that both total and mature AM are equally useful in diagnosing sepsis based on the latest Sepsis-3 definitions. On the contrary, with regard to the clinical outcome in patients with sepsis, a significant difference in the level of each AM was recognized between survivors and non-survivors 3 days after the admission. In addition, the levels of both the AM forms could predict the 28-day mortality in patients with sepsis on day 3, but not on day 1. Therefore, unlike in previous studies, we found that it may take several days for these AM peptides to prognosticate the 28-day mortality in patients with sepsis, because AM levels increased even in patients with a good prognosis on day 1.
In some previous studies, the levels of total AM were assessed and their utility in diagnosing sepsis was compared with those of other chemical biomarkers, such as CRP, PCT, lactate, and PSEP [8, 9, 18, 20, 24-26]. We found that among all the clinical parameters examined, the diagnostic accuracy for sepsis on the first day of ICU admission was the highest for total AM, and that of mature AM was the second highest. It is assumed that mature AM is biologically active, and would thus be a more informative biomarker than total AM in patients with sepsis. In our study, the cut-off level of mature AM for the diagnosis of sepsis was approximately 31.2 pg mL-1, and its AUC was 0.848, with a sensitivity of 88.1% and a specificity of 67.9%. Other investigators have also reported that the ability of mature AM to identify patients with sepsis was modest because an AUC of 0.76 and a Youden’s index derived cut-off of 37 pg mL-1 generated a sensitivity of 61% and a specificity of 80% [20]. These cut-off values of mature AM were close, indicating that the values of about 30 pg mL-1 could be applicable to diagnose sepsis, which is a novel finding for mature AM.
The half-life of mature AM in the plasma is approximately 20 min. As a biomarker of acute cardiovascular failure, the levels of mature AM can be significantly changed during the first 24 h after diagnosis [10]. Indeed, in our study, the levels of mature AM in patients with sepsis significantly increased on day 1 but reverted to the same levels as in patients without sepsis, 3 days after admission, indicating that a time frame in which sepsis can be identified by this peptide may be confined to an early stage after the onset.
Accumulating evidence indicates that the clinical outcome in patients with sepsis can be diagnosed based on AM levels [10-12, 14-17]. However, investigators have pointed to the need for more data to evaluate the mortality rate in patients with sepsis prognosticated by the levels of mature AM [8]. Marino et al. [15] demonstrated that the levels of mature AM at ICU admission strongly correlated with the 28-day mortality rate in patients with sepsis. Patients with AM levels > 70 ng L-1 had a 28-day survival rate of 55%. Recent evidence supports this result. Kim et al. [10] reported that the cut-off value of mature AM levels for 28-day mortality was 97.12 pg mL-1. However, in the present study, the levels of total and mature AM on day 1 could not be used to predict the 28-day sepsis-related mortality. It is well known that AM levels increase in patients with heart failure, myocardial infarction, pulmonary hypertension, SIRS, inflammatory bowel diseases, and renal failure, resulting in increased AM levels even in patients with sepsis having a good prognosis [2, 27-30]. This notion can explain our results presented in Figure 3, which indicates that the levels of mature and total AM were not significantly different between non-survivors and survivors on day 1.
We found that the levels of mature and total AM on day 3 in patients with sepsis had a high predictive value for sepsis-related mortality as determined by ROC analysis. Interestingly, Mebazaa et al. [11] reported that in patients with the levels of mature AM > 70 pg mL-1 on admission, a decrease in its levels below 70 pg mL-1 on day 2 was associated with the recovery of organ function on day 7 and a better 28-day outcome (9.5% mortality), whereas persistently elevated levels of mature AM on day 2 were associated with prolonged organ dysfunction and high 28-day mortality (38.1% mortality). These results suggest that the levels of mature AM may remain high if the treatment is unsuccessful, and organ damage continues in sepsis. Indeed, as demonstrated in Figure 3, the sustained increase in the levels of mature AM in patients with sepsis correlated with worsening prognoses.
In the present study, the cut-off levels of mature and total AM on day 3 for the diagnosis of the 28-day sepsis-related mortality were approximately 32.4 and 435.0 pg mL-1, respectively. The cut-off level of mature AM was lower than that in previous studies (30 pg mL-1 versus 70 pg mL-1). This could be explained by the sensitivity of the assay used in each study, indicating that the cut-off values obtained by different assay methods may be different. Recently, Lundberg et al. [20] reported a Yuden’s index cut-off of 45 pg mL-1 for predicting the 28-day sepsis-related mortality in their sepsis cohort, which was as low as in the present study.
It has been presumed that total AM represents the sum of synthesized intermediate AM and mature AM. However, little is known about the ratio of each peptide contained in total AM. We suspected that if the composition ratio would vary for each patient with sepsis, the levels of total AM could not directly reflect the functional status of mature AM [31]. However, as discussed previously, we found a definite correlation between the levels of mature and total AM, both of which were measured from an identical blood sample, with a resultant increase in a similar fashion. Based on our results, it may be sufficient to measure either mature or total AM for the diagnosis of sepsis.
This study has some limitations. First, this study was performed as a small, single-centre study focusing on the diagnostic performance of AM for sepsis and septic shock based on the Sepsis-3 definitions. Given the similarities in responses and outcomes, such a study will likely require a much larger patient sample size and would potentially be best accomplished with a multi-centre design. Second, as discussed previously, the AM-binding protein, complement factor H, hinders the reliable measurement of mature AM [9, 18, 32]; this technical difficulty in the assay precludes its practical use. Recently, Weber et al. [19] reported a new assay to reliably measure the bio-active AM using a double monoclonal sandwich immunoassay that can measure C-terminally amidated biologically active AM. We used our originally developed assay for measuring mature AM and found that the limits of detection and quantitation were 0.133 and 0.085 pmol L-1, respectively, according to the Clinical and Laboratory Standards Institute (CLSI) protocols. The intra- and inter-assay coefficients of variation were 1.8% and 5.1%, respectively [23]. Thus, we believe our assay is as reliable as the one reported by Weber et al. Third, we do not provide any evidence of whether AM might resolve or worsen the septic state itself. Although the effects of AM or AM antibody therapy for sepsis have been evaluated in some preclinical animal studies [33], it is still unclear whether AM will have beneficial therapeutic effects in patients with sepsis. There is an ongoing, multi-centre study evaluating the AM-binding antibody therapy for sepsis (AdrenOSS-2) [34]. The results of this study might eventually be useful in characterizing the efficacy of AM therapy in patients with sepsis.