Relationship between Nonhepatic Serum Ammonia Levels and Sepsis-Associated Encephalopathy: A Retrospective Cohort Study

Objectives Nonhepatic hyperammonemia often occurs in patients with sepsis. Ammonia plays an essential role in the occurrence of hepatic encephalopathy. However, the relationship between nonhepatic serum ammonia levels and sepsis-associated encephalopathy (SAE) remains unclear. Thus, we aimed to evaluate the association between serum ammonia levels and patients with SAE. Methods Data of critically ill adults with sepsis who were admitted to the intensive care unit were retrieved from the Medical Information Mart for Intensive Care IV (MIMIC IV) between 2008 and 2019 and retrospectively analyzed. Data of patients with sepsis patients and serum ammonia not related to acute or chronic liver disease were not included. Results Data from 720 patients with sepsis were included. SAE was found to have a high incidence (64.6%). After adjusting for other risk factors, a serum ammonia level of ≥45 μmol/L (odds ratio (OR): 3.508, 95% confidence interval (CI): 2.336–5.269, p < 0.001) was found to be an independent risk factor for patients with SAE; moreover, as the serum ammonia level increased, the hospital mortality of SAE gradually increased in a certain range (serum ammonia <150 μmol/L). Serum ammonia levels of ≥45 μmol/L were associated with higher Simplified Acute Physiology Score II and Sequential Organ Failure Assessment (SOFA) scores in patients with SAE. Besides, our study found that patients with SAE used opioid analgesics (OR:3.433, 95% CI: 1.360–8.669, p = 0.009) and the SOFA scores of patients with SAE (OR: 1.126, 95% CI: 1.062–1.194, p < 0.001) were significantly higher than those without SAE. Conclusions Nonhepatic serum ammonia levels of ≥45 μmol/L evidently increased the incidence of SAE. Serum ammonia levels should be closely monitored in patients with sepsis.


Introduction
Sepsis, a life-threatening organ dysfunction caused by a dysregulated host response to infection [1], can cause multiple organ dysfunction syndromes (MODS).In sepsis, brain dysfunctions occur easily and early on [2], and brain dysfunction in sepsis without direct infection of the nerve is called sepsisassociated encephalopathy (SAE) [3].Te latest research shows that SAE has a high incidence of 49.6% [4] and a mortality of 50.3% [5].Moreover, 45% of sepsis survivors have long-term cognitive dysfunction [6].Since there is no efective treatment for patients with SAE patients, it is essential to control the modifable factors to prevent SAE.
High serum ammonia levels play an essential role in the pathogenesis of hepatic encephalopathy [7].Ammonia crosses the blood-brain barrier where it is further metabolized to glutamine; consequently, high glutamine levels in the brain induce brain edema and increase cerebral volume, and ammonia decreases excitatory neurotransmission.Patients often show consciousness changes and delirium and even go into coma [8,9].In recent years, nonhepatic hyperammonemia has gradually gained attention, especially in patients with sepsis.In our previous study, we found a 40.3% incidence of nonhepatic hyperammonemia in sepsis.In addition, we found that the incidence of encephalopathy was signifcantly higher in the nonhepatic hyperammonemia group (37.3%) than in the nonhepatic nonhyperammonemia group (19.6%), and the nonhepatic hyperammonemia group had higher short-term (hospital mortality: 59.8%; 30-day mortality: 47.7%) and long-term mortality (90-day mortality: 61.7%; 1-year mortality: 67.7%) than the nonhepatic nonhyperammonemia group [10].Yazan et al. found that elevated ammonia levels can be a novel biomarker for sepsis [11].
Serum ammonia is an important neurotoxic molecule, and elevated nonhepatic serum ammonia levels are commonly seen in patients with sepsis.Our previous research found that the serum ammonia level was associated with higher SOFA scores and lactate levels in patients with SAE [12].However, the relationship between nonhepatic serum ammonia levels and incidence of SAE remains unclear.Terefore, we aimed to evaluate the association of nonhepatic serum ammonia levels with SAE.

Materials and Methods
2.1.Setting.Tis study was a retrospective cohort study based on data from the Medical Information Mart for Intensive Care IV (MIMIC IV 0.4) from 2008 to 2019 [13].MIMIC IV is a publicly available database of patients admitted to the Beth Israel Deaconess Medical Center in Boston, MA, USA.Te use of data from the MIMIC IV database was approved by the Institutional Review Boards of the Massachusetts Institute of Technology and Beth Israel Deaconess Medical Center [13].Te requirement for individual patient consent was waived because the study did not impact clinical care, and all protected health information was deidentifed.We included data from 69,619 ICU patients, which included 19,658 patients with sepsis, among whom 1,377 patients with sepsis had their serum ammonia levels evaluated.

Patients.
First, the database was searched for patients with sepsis.Sepsis was defned as a suspected infection combined with an acute increase in the Sequential Organ Failure Assessment (SOFA) score of ≥2 [1].Patients with an infection site or prescriptions for antibiotics and those who underwent sampling of bodily fuids for a microbiological culture were considered to have a suspected infection.When the antibiotic was administered before sampling, we assumed the microbiological sample must have been collected within 24 h; when microbiological sampling was conducted before administering antibiotics, we assumed the antibiotic must have been administered within 72 h [14].Second, we searched for patients with sepsis who underwent serum ammonia level testing.We excluded patients diagnosed with acute and chronic liver disease and diseases that afect consciousness.We divided the patients into an SAE group and a non-SAE group.SAE in the study was defned as a Glasgow Coma Scale (GCS) score of <15, diagnosed delirium, or usage of haloperidol in patients with sepsis [4,5,15].

Data Collection.
Patients' baseline variables, including age, sex, type of admission, ethnicity, coexisting illness (Supplementary materials 10-14), site of infection, microbiology type, length of hospital stays, and hospital mortality were extracted from the database.Te patient's vital signs, including heart rate, systolic blood pressure, diastolic blood pressure, mean arterial pressure, respiratory rate, temperature, and arterial oxygen saturation (SpO 2 ), were extracted.Te patients' laboratory parameters, including alanine aminotransferase, aspartate aminotransferase, albumin, ammonia, lactate, bilirubin, creatinine, blood urea nitrogen, glucose, and hemoglobin levels along with platelet count, partial thrombin time, international normalized ratio, prothrombin time, white blood cell count, neutrophil count, lymphocyte count, partial pressure of carbon dioxide (PaCO 2 ), and partial pressure of oxygen (PaO 2 ), were extracted.Te patient's disease severity score, including Sequential Organ Failure Assessment (SOFA) score, Simplifed Acute Physiology Score II (SAPS), and Glasgow Coma Scale (GCS) score in the frst 24 h of the patient's admission to the ICU, was extracted.Te analgesic and sedative drugs used, including midazolam, propofol, and/or opioids, were extracted.Te use of vasopressors, mechanical ventilation, and renal replacement therapy was extracted.Te worst laboratory parameters and the mean value of vital signs during the frst 24 h of ICU admission were used in the analysis in this study.R statistical software (R Foundation for Statistical Computing, Vienna, Austria) was used to extract the patient's indexes.Serum ammonia in the SAE group was signifcantly higher than that in the non-SAE group as shown in Table 1.We applied propensity score matching to match the covariates with in-between diferences as shown in Supplementary Material 15.We considered that it may be related to the occurrence of SAE.Our data were all nonnormally distributed.We used generalized additive models [18] to further determine the relationship between serum ammonia levels and SAE.Our cohort study found that serum ammonia levels of ≥45 μmol/L can increase the incidence of SAE.For further verifcation, we used the multivariate logistic regression analysis.After adjusting for other risk factors, serum ammonia levels of ≥45 mmol/l were identifed as an independent risk factor for SAE.We used the survive package to observe the trend of changes between serum ammonia levels and the hospital mortality of SAE.Data were analyzed using R software.

Baseline Characteristics.
As shown in Figure 1, 1,377 patients with sepsis had data on their serum ammonia level in the MIMIC IV database.In total, 393 patients were diagnosed with acute and chronic liver diseases, 264 patients were diagnosed with traumatic brain injury, and other diseases were excluded.Finally, 720 patients were included in the fnal cohort.

Relationship between Serum Ammonia Levels and SAE
Incidence.A generalized linear model was used to explore the relationship between serum ammonia levels and SAE incidence (Figure 2(a)).Serum ammonia ≥45 μmol/L can increase the incidence of SAE.Furthermore, Figure 2(b) shows that as serum ammonia levels increase, the incidence of SAE increases.

Relationship between Serum Ammonia Levels and SAE
Hospital Mortality.Te survival package of R Studio was used to observe the relationship between serum ammonia levels and the hospital mortality of patients with SAE. Figure 3 demonstrates that, when serum ammonia levels were <150 μmol/L, an increase in the serum ammonia level was associated with a gradual increase in the hospital mortality of patients with SAE.

Te Efect of Serum Ammonia Level ≥45 μmol/L on
Prognosis of SAE Patients.Patients with SAE who have serum ammonia levels of ≥45 μmol/L had higher SOFA scores (7 (5-10) vs. 4 (6-9), p � 0.022) than those with serum ammonia levels of <45 μmol/L; however, there was no diference in length of hospital stays and hospital mortality between the two groups (Table 3).

Discussion
In the present cohort study, we demonstrated an association between serum ammonia levels and the incidence of SAE.Te incidence of SAE in this cohort study was 64.4%.Patients with SAE have a worse prognosis in terms of longer hospital stays and higher hospital mortality than those without SAE.Furthermore, among patients with sepsis, those with a serum ammonia level ≥45 μmol/L were 3.508 times more likely to develop SAE than those with serum ammonia levels of <45 μmol/L.Patients with SAE with serum ammonia levels of ≥45 μmol/l had higher SAPS II and SOFA scores than those with serum ammonia levels Emergency Medicine International    Our study found that serum ammonia levels, use of opioids, and high SOFA scores were potentially modifable factors for the incidence of SAE.
Ammonia is a potential neurotoxin, and elevated serum ammonia levels can cause neurological symptoms and changes in the patient's consciousness.Elevated serum ammonia levels can be due to hepatic hyperammonemia [21,22] and nonhepatic hyperammonemia [16,23,24].Elevated nonhepatic serum ammonia levels need attention.Nonhepatic hyperammonemia had a high incidence and was associated with a poor prognosis in critically ill patients.Yao et al. found that the incidence of nonhepatic hyperammonemia was as high as 75.98%.Patients with severe nonhepatic hyperammonemia had longer ICU stays and higher acute physiology assessment, chronic health assessment, and SOFA scores [23].Stergachis et al. [24] reported that 57% of patients with severe nonhepatic hyperammonemia survived after having reversible neurological changes and 4% survived with irreversible neurological changes.Our previous study demonstrated that the incidence of nonhepatic hyperammonemia was 42.2% in critical care patients.Patients with sepsis accounted for 26.2% of the patients with nonhepatic hyperammonemia [25].Among patients with sepsis, those with nonhepatic hyperammonemia had a signifcantly higher incidence of encephalopathy (37.4%) and delirium (15.9%) than those with normal serum ammonia levels [10].Sakusic et al. [16] reported that 71% of patients with nonhepatic  Emergency Medicine International hyperammonemia presented with acute encephalopathy.Our cohort study found that the incidence of SAE was 64.4% and that nonhepatic serum ammonia levels in the SAE group were signifcantly higher than those in the non-SAE group (Table 1); moreover, patients with serum ammonia levels of ≥45 μmol/L had 3.501 times higher incidence of SAE than those with serum ammonia levels of <45 μmol/L (Figure 2, Table 2).Our study results further confrm those of previous research.Te neurotoxicity caused by nonhepatic blood ammonia may be attributed to serum ammonia-induced TNF-α in glial cells; TNFR1 activated by released TNF-α, TNFR1 leads to nuclear translocation of NF-κB and an increase of TNF-α.[26].In addition, the logistic analysis showed that Escherichia coli infection was more frequent in patients with SAE patients than in those without SAE, thereby indicating that the occurrence of nonhepatic hyperammonemia may be related to Escherichia coli infection and increased ammonia production [27].Serum ammonia is a fat-soluble substance, and increased serum ammonia can enter the brain through the blood-brain barrier and afect the activities of glutaminase, glutamine synthetase, and glutamate dehydrogenase, which are important enzymes for nervous system function [28,29].Nonhepatic serum ammonia is a risk factor for SAE, especially serum ammonia levels of ≥45 μmol/L, although these levels do not afect the mortality in patients with SAE (Table 3); as serum ammonia levels increase, the mortality rate (serum ammonia <150 μmol/L) and incidence of SAE increase (Figure 3); patients with serum ammonia levels of ≥45 μmol/L had higher SOFA and SAPS II scores than those with serum ammonia levels of <45 μmol/L (Table 3).We recommend that the serum ammonia levels should be closely monitored in patients with sepsis, and in patients with serum ammonia levels of ≥45 μmol/L, timely and efective interventions should be taken to reduce the incidence and improve the prognosis of SAE.Te mechanism of nonhepatic blood ammonia levels leading to SAE needs to be studied in the future.Sonneville et al. found that the rate of use of opioids in patients with and without SAE was 43.7% and 25.9%, respectively [5].Our results show that the incidence of SAE in patients with sepsis who used opioids was 3.433 times that of those who did not use opioids.Te use of opioids is an independent risk factor for SAE.Our study results are consistent with those of Sonneville et al.Opioid-induced delirium has been confrmed in other patients, except for those with SAE.Pavone et al. found that among critically ill patients, those exposed to opioids were 2.5 times more likely to develop delirium than those not exposed to it [30].Te odds of postoperative day 2 delirium increased signifcantly with the increase in intraoperative opioid use in patients with chronic kidney disease (OR � 1.6) [31].Future research should focus on the optimal dose and timing of administration of the opioid and pain management approaches to prevent delirium.
Leite et al. found that the SOFA score was a risk factor for the incidence of delirium in patients weaned from mechanical ventilation [32].A previous study found that patients with SAE patients had higher SOFA scores [20], and a kidney SOFA score of >2 was associated with a 1.41 times higher incidence of SAE [5].Our study found that the SOFA score was an independent risk factor for SAE (OR: 1.126).Our study results support those of previous studies.Tus, the more severe the organ dysfunction in patients with sepsis, the more likely SAE is to occur.

4.1.
Limitations.Tis study had some limitations.First, our defnition of SAE and nonhepatic serum ammonia were diferent from those used in previous studies.According to the reference literature retrieved from retrospective studies, the defnition of SAE is based on the GCS score, the diagnosis of delirium, or the use of haloperidol drugs, and the exclusion of cerebral hemorrhage, brain trauma, and epilepsy, severe liver and kidney disease caused by consciousness disorder, or mental disorders.Te defnition of nonhepatic serum ammonia is based on the diagnostic code to exclude acute and chronic liver disease.Tey all lack imaging data, such as computed tomography, magnetic resonance imaging, electroencephalogram, and ultrasound data.Hence, there may be information bias in the SAE cohort.Second, the results of this study demonstrated that patients with sepsis who had serum ammonia levels of ≥45 μmol/L had a 3.508 times higher chance of developing SAE than those with serum ammonia levels of <45 μmol/L; however, a causal association with serum ammonia level ≥45 μmol/L and the incidence of SAE could not be proved due to the retrospective nature of our study.Its causality should be assessed in future studies using a prospective study design.Te consciousness of patients with SAE could not be observed through our intervention of serum ammonia levels.Finally, critically ill patients often have various other comorbidities.Due to the interaction between the diseases, some confounding factors still cannot be ruled out and can cover up or exaggerate the relationship between the risk factors and SAE.Te small sample size of this study may lead to bias in the study results and require larger sample sizes to validate in the future.

Conclusions
Serum ammonia levels of ≥45 μmol/L are associated with the incidence of SAE.Tese levels can provide a reference for clinicians to intervene in patients with SAE.Serum ammonia levels of ≥45 μmol/L provide a reference target for future prospective research.

Figure 1 :
Figure 1: Flowchart of the study's selection process.

Figure 2 :
Figure 2: Association between serum ammonia levels and SAE incidence.

Figure 3 :
Figure 3: Relationship between serum ammonia levels and hospital mortality of patients with SAE.
IQR).Categorical variables, including sex, type of admission, ethnicity, coexisting illness, site of infection, microbiology type, length of hospital stays, hospital mortality, use of analgesic and sedative drugs, vasopressors, and mechanical ventilation, were expressed as numbers and proportions.Continuous variables were examined using the Mann-Whitney U test in the SAE group and the non-SAE group; categorical variables were compared using Pearson's exact test.

Table 1 :
Baseline characteristics and outcome.Sonneville et al. conducted a multicenter study and demonstrated that the incidence of SAE was 53% [5].Tomonori Yamamoto et al.'s multicenter, randomized, controlled trial found that patients with SAE had a longer ICU stay than those without SAE [19].Feng et al. found that patients with SAE presented with high APACHE II and SOFA scores and high short-term of <45 μmol/L.Besides, we found that the use of opioids and high SOFA scores were independent risk factors for SAE.SAE has a high incidence and poor outcome.

Table 2 :
Multivariate logistic analysis of risk factors in patients with SAE.

Table 3 :
Te efect of serum ammonia on the prognosis of patients with SAE.