Serum anion gap is associated with mortality in intensive care unit patients with diastolic heart failure

Serum anion gap (AG) is closely related to mortality in critically ill patients with several diseases. We aimed to determine the relationship between serum AG levels and 28-day intensive care unit (ICU) mortality in patients with diastolic heart failure (DHF). This cohort study enrolled critically ill patients with DHF from the Medical Information Mart for Intensive Care IV (MIMIC-IV) database. Serum AG levels were calculated using the traditional and albumin-adjusted methods. Multivariate Cox proportional hazards regression and restricted cubic spline curves were used to determine the correlation between serum AG levels and 28-day ICU mortality. We used receiver operating characteristic (ROC) curves and area under the curve (AUC) to compare the ability of traditional and albumin-adjusted AG to predict mortality. Overall, 3290 patients were included. Multivariate analysis showed an association of high levels of traditional (hazard ratio [HR], 1.48; 95% confidence interval [CI], 1.1–1.98, p = 0.009) and albumin-adjusted AG (HR, 1.36; 95% CI, 1.02–1.79, p = 0.033) with higher risk of 28-day ICU mortality. Restricted cubic spline curves indicated a linear relationship between AG level and 28-day ICU mortality. Comparison of the ROC curves revealed that albumin-adjusted AG had a greater ability to predict 28-day ICU mortality compared with traditional AG (AUCs of 0.569 [95% CI, 0.536–0.601] and 0.619 [95% CI, 0.588–0.649], respectively). In ICU patients with DHF, higher levels of traditional and albumin-adjusted AG were associated with higher 28-day ICU mortality. Albumin-adjusted AG exhibited greater predictive ability for mortality compared with traditional AG.


Baseline characteristics of patients
A total of 3290 patients were included in this retrospective study (Fig. 1).The baseline characteristics of the traditional and albumin-adjusted AG groups are presented in Tables 1 and 2, respectively.Among the patients, there were 1515 men and 1775 women, and the median age was high, 75.5 ± 12.6 years.Of these, 72.3% were white and 7.5% received RRT.
In the traditional AG group, patients with higher AG levels had higher levels of several laboratory parameters, such as albumin, BUN, creatinine, calcium, potassium, lactate, and glucose.They also had more comorbidities, such as hypertension, diabetes, and renal disease, and received more vasopressor treatments with dopamine and dobutamine.These patients received more RRT and had higher SAPS II and APS III and mortality rates.
Similarly, in the albumin-adjusted AG group, patients with higher AG levels had higher WBC, and BUN, creatinine, potassium, and glucose levels.Interestingly, these patients had lower calcium levels.Their heart and respiratory rates were higher.They had a higher incidence of renal disease, lower incidence of chronic

Association between AGs and 28-day ICU mortality
In the univariate analysis, both traditional and albumin-adjusted AG were associated with an increased risk of 28-day ICU mortality in patients with DHF when used as continuous variables, the hazard ratios (HRs) were 1.05 (95% confidence interval [CI], 1.03-1.07;p < 0.001), 1.05 (95% CI, 1.03-1.06;p < 0.001), respectively.As shown in Table 3, AGs measured using the two methods were used as categorical variables, with the group with the lowest AG level as the reference group.The group with high levels of traditional AG showed significantly increased 28-day ICU mortality (HR, 1.68; 95% CI, 1.3-2.19;p < 0.001).The same effect was replicated in the group with high levels of albumin-adjusted AG (HR, 1.57; 95% CI, 1.22-2.02;p < 0.001).In contrast, the groups with moderate levels of traditional and albumin-adjusted AG both showed no significant association with 28-day ICU mortality.The variables of age; WBC count; BUN, creatinine, and lactate levels; pH; PO 2 ; SBP; DBP; MBP; respiratory rate; temperature; SpO 2 ; presence of hypertension, liver disease, and malignant cancer; use of dobutamine, phenylephrine, and norepinephrine; SOFA score; SAPS II; and APS III were associated with 28-day ICU mortality.Table 4 shows the adjusted analysis of AGs with 28-day ICU mortality in patients with DHF using multivariate Cox proportional hazards models.In Model I, when used as continuous variables, traditional (HR, 1.05; 95% CI, 1.03-1.07;p < 0.001) and albumin-adjusted AG (HR, 1.05; 95% CI, 1.03-1.07;p < 0.001) were associated with increased risk of 28-day ICU mortality with adjustments for sex, age, marital status, and ethnicity.When used as categorical variables, the groups with high levels of traditional and albumin-adjusted AG demonstrated a higher risk of 28-day ICU mortality, the adjusted HRs were 1.64 (95% CI, 1.26-2.13;p < 0.001) and 1.61 (95% CI, 1.25-2.07;p < 0.001), respectively.After adjustment in Model II, no significant effects of traditional and albumin-adjusted AG as continuous variables were observed on 28-day ICU mortality.Similar to Model I, groups with high levels of traditional (HR, 1.48; 95% CI, 1.1-1.98;p = 0.009) and albumin-adjusted AG (HR, 1.36; 95% CI, 1.02-1.79;p = 0.033) remained significantly associated with an increased risk of 28-day ICU mortality, wherein the groups with the lowest AG levels were considered the reference groups.However, the groups with moderate levels of traditional (HR, 1.31; 95% CI, 0.98-1.74;p = 0.066) and albumin-adjusted AG (HR, 1.07; 95% CI, 0.8-1.43;p = 0.655) yielded the same results as in Model I, of no significant associations with 28-day ICU mortality.
Restricted cubic spline curves indicated a linear relationship between AGs and 28-day ICU mortality after adjusting for confounders, as shown in Fig. 2. The results are similar to those of P for the trends listed in Table 4.The Kaplan-Meier (K-M) survival curve illustrated that patients with elevated levels of traditional and albuminadjusted AG had decreased survival rates, as shown in Fig. 3 (p < 0.0001).

ROC curve analysis
A comparison of the ROC curves between traditional AG and albumin-adjusted AG revealed a significant difference.The ability of albumin-adjusted AG to predict 28-day ICU mortality was higher than that of traditional AG (AUCs of 0.569 [95% CI, 0.536-0.601]and 0.619 [95% CI: 0.588-0.649],respectively) (Fig. 4).

Subgroup analysis
Subgroup analysis was applied to evaluate the trend of effect sizes between the traditional AG and 28-day ICU mortality in Models I and II (Fig. 5).There were no significant interactions in most strata of the two models, except for the receipt of RRT (P for interaction in model I = 0.017; P for interaction in model II = 0.038) and  the presence of diabetes (P for interaction in model I = 0.008; P for interaction in model II = 0.006) and chronic pulmonary disease (P for interaction in model I = 0.025; P for interaction in model II = 0.002).Additionally, we noted that a few variables such as lactate level (P for interaction = 0.008), phenylephrine use (P for interaction = 0.021), and presence of renal disease (P for interaction = 0.014) showed an interaction between traditional AG and 28-day ICU mortality in Model I.Meanwhile, temperature (P for interaction = 0.009) showed a similar interaction in Model II.Consistent results were observed in Supplementary Tables S4 and S5.Similar results were observed between the albumin-adjusted AG and 28-day ICU mortality, as shown in Fig. 6.Only presence of diabetes showed an interaction in the two models (P for interaction in Model I = 0.008; P for interaction in Model II = 0.01).Variables such as lactate level (P for interaction = 0.004), phenylephrine use (P for interaction = 0.008), receipt of RRT (P for interaction = 0.038), and presence of renal disease (P for interaction = 0.027) demonstrated an interaction between albumin-adjusted AG and 28-day ICU mortality in Model I. Additionally, variables such as temperature (P for interaction = 0.03), APS III (P for interaction = 0.048), and presence of chronic pulmonary disease (P for interaction = 0.002) demonstrated a similar interaction.Consistent results are presented in Supplementary Tables S6 and S7.

Discussion
This study demonstrated the relationship between serum AG levels and 28-day ICU mortality in patients with DHF.Serum AG levels can be calculated using the traditional and albumin-adjusted methods.There was a potential linear trend between AGs measured using the two methods and mortality; higher serum AG levels were associated with an increased risk of 28-day ICU mortality and a shorter survival time.Both traditional and albumin-adjusted AG remained independent predictors of 28-day ICU mortality in critically ill patients with DHF after adjusting for confounders.Moreover, no significant interactions were found between the AGs and 28-day ICU mortality in most subgroups.Specifically, the ROC curves of traditional and albumin-adjusted  AG showed a significant difference in mortality prediction.The predictive ability of albumin-adjusted AG was greater than that of traditional AG, with AUCs of 0.619 and 0.569, respectively.Serum AG is an inexpensive and effective tool that aids in the detection of various acid-base imbalances and differential diagnosis of metabolic acidosis.It can be calculated with serum or plasma electrolytes; serum values are used most frequently, and reflect the differences between the concentrations of unmeasured anions and cations 6 .The average serum AG in healthy individuals measured in mEq/L is 12 ± 4 17 .With the invention of automated analyzers that enabled measurement of electrolytes in large groups of patients, particularly in critically ill patients, the serum AG can be obtained easier and easier.As such, many previous studies have revealed the relationship between serum AG levels and clinical outcomes or predicting prognosis of critically ill patients.One study found that high serum AG levels were a significant risk for ICU and hospital all-cause mortality in patients with non-traumatic SAH 9 .Tang et al. 16 also measured the association between serum AG and the 30-day and 90-day all-cause mortalities of critically ill patients with CHF and suggested that high AG levels were associated  www.nature.com/scientificreports/with increased risk mortality.Another retrospective study of critically ill patients with acute ischemic stroke demonstrated there was a potential linear trend between the plasma AG and in-hospital mortality and revealed that the AG was an independent risk factor for in-hospital mortality 15 .Similarly, our findings were consistent with these studies and showed a positive correlation between serum AG and 28-day ICU mortality in critically ill patients with DHF.Most studies used traditional AG to explore the relationship between serum AG levels and clinical outcomes of critically ill patients, however, this study compared the association between traditional AG and albumin-adjusted AG to mortality in critically ill patients.Most of the serum AG is due to the sum of anionic charges on circulating proteins, of which the most abundant one under normal conditions is albumin 18 .Hence, changes in the concentration of serum albumin can alter serum AG levels, especially in individuals with hypoalbuminemia 6 .Therefore, Serum AG levels should be corrected for serum albumin to obtain an albumin-adjusted AG.Abramowitz et al. 7 reported that higher serum AG levels, calculated using three methods (traditional AG, albumin-adjusted AG and full AG), were associated with an increased risk of mortality independent of eGFR and albuminuria.Kim et al. indicated that corrected AG may be used to predict mortality in children, and that an elevated level of corrected AG at admission was associated with higher mortality in the pediatric ICU 19 .Similarly, albumin-adjusted AG was used to evaluate its relationship with mortality to reduce bias and increase predictive value in our study.We observed the same trend, enhancing the stability and consistency of the results.Furthermore, there was a significant difference between traditional and albumin-adjusted AG in prediction of 28-day ICU mortality.However, forecasting the risk of mortality by using a single measurement of either traditional or albumin-adjusted AG may not be effective.Therefore, a more accurate evaluation strategy for predicting clinical outcomes should be considered in patients with DHF.
However, the specific mechanism underlying the close correlation between serum AG levels and 28-day ICU mortality in patients with DHF remains unclear.DHF can occur when a ventricle with normal or low end-diastolic volume cannot accept a normal venous return 1 .Its pathophysiology mainly includes impaired left ventricular filling, delayed relaxation, and increased stiffness.As a result, an upward displacement of the diastolic pressure-volume relationship with increased end-diastolic, left atrial, and pulmonary wedge pressure may be generated, leading to symptoms of pulmonary congestion 20 .Signs and symptoms such as breathlessness, coughing, tachypnea, dyspnea on exertion, or paroxysmal nocturnal dyspnea may be present 2 , which may cause respiratory acid-base imbalance.Meanwhile, circulatory disturbances are caused by the heart losing its ability to efficiently pump blood, leading to a decreased perfusion of tissue, hypoxia of cells, and metabolic acid-base imbalance 21 .Ongoing acid-base imbalance can aggravate the severity of the disease and is associated with poor prognosis of the patient 22 .Serum AG can reflect various acid-base imbalances; this is the explanation that we propose to clarify the relationship between serum AG levels and 28-day ICU mortality in patients with DHF.
More recently, DHF has been redefined as HF with normal or preserved EF (HFnEF or HFpEF) 1 .Additionally, Bozkurt et al. 23 proposed a new and revised classification of HF according to the LVEF and ended a history of inconsistent definitions and classifications of HF in March 2021.Symptomatic HF with LVEF ≥ 50% is defined as HFnEF or HFpEF.Because the information of patients in the MIMIC-IV database was recorded from 2008 to 2019, the previous standard for diagnosing DHF was used.The patients with DHF in our study, whose EF were ≥ 0.50, were similar to individuals with HFnEF or HFpEF.Therefore, the findings of this retrospective study may be applicable to patients with HFnEF and HFpEF.
To our knowledge, this is the first study to show an independent association of traditional and albuminadjusted AGs with 28-day ICU mortality in patients with DHF admitted to the ICU.However, the present study has some limitations.Firstly, due to the nature of the MIMIC-IV database, the absence of some variables, such as lactate level, pH, PO 2 , and PCO 2 was close to 35%, and multiple imputations based on five replications were employed to reduce bias.Secondly, the diagnosis of DHF was based on administrative codes.Although the first sequence of diagnosis was used, it is possible that some false associations were caused by misclassifications.Thirdly, although we had performed Multivariate Cox proportional hazard regression models to control the confounding, there would exist some potential confounders that would not be measured in our study.Fourth, this was a single-institution retrospective study, and there may have been a selection bias.Therefore, these results may not apply to all patients with DHF and should be regarded only as of reference and must be further verified.Nevertheless, the independent associations of traditional and albumin-adjusted AG with 28-day ICU mortality remain unclear.
In conclusion, this retrospective observational study showed that higher levels of traditional and albuminadjusted AG were associated with higher 28-day ICU mortality in patients with DHF.AG measured using both traditional and albumin-adjusted methods could be used as simple and effective tools to predict the 28-day ICU mortality in these patients.Furthermore, the predictive ability for mortality of albumin-adjusted AG was greater than that of traditional AG.

Study design and data source
This retrospective cohort study adhered to the Strengthening the Reporting of Observational Studies in Epidemiology statement 24 .Data were collected from the Medical Information Mart for Intensive Care IV (MIMIC-IV version 1.0) database (https:// mimic.mit.edu/) 25 , which contains information on patients at a Tertiary Academic Medical Center from 2008 to 2019.The Beth Israel Deaconess Medical Center (BIDMC, Boston, MA, USA) approved the permission to use the database.All researchers who complete the Collaborative Institutional Training Initiative examination can access it for data extraction purposes.One author of this study had completed the examination and received the permission (certification number 40287955 for Hongyu Xu).All data in the database were de-identified to protect patient privacy.Therefore, informed consent was not required.

Table 1 .
Baseline characteristics of the patients with DHF grouped by traditional AG.Data were mean ± SD or median (IQR) for skewed variables or numbers (proportions) for categorical variables.AG anion gap; UO urine output; RRT renal replacement therapy; WBC white blood cells; BNU blood urea nitrogen; PO 2 partial oxygen pressure; PCO 2 partial pressure of carbon dioxide; SBP systolic blood pressure; DBP diastolic blood pressure; MBP mean blood pressure; SpO 2 saturation of peripheral oxygen; SOFA sequential organ failure assessment score; SAPS II simplified acute physiology score II; APS III acute physiology score III; ICU intensive care unit.

Table 2 .
Baseline characteristics of the patients with DHF grouped by albumin-adjusted AG.Data were mean ± SD or median (IQR) for skewed variables or numbers (proportions) for categorical variables.AG anion gap; UO urine output; RRT renal replacement therapy; WBC white blood cells; BNU blood urea nitrogen; PO 2 partial oxygen pressure; PCO 2 partial pressure of carbon dioxide; SBP systolic blood pressure; DBP diastolic blood pressure; MBP mean blood pressure; SpO 2 saturation of peripheral oxygen; SOFA sequential organ failure assessment score; SAPS II simplified acute physiology score II; APS III acute physiology score III; ICU intensive care unit.

Table 3 .
Results of univariate analysis for 28-day ICU mortality.Data presented are HRs and 95% CIs.
ICU intensive care unit; AG anion gap; WBC white blood cells; BNU blood urea nitrogen; PO 2 partial oxygen pressure; SBP systolic blood pressure; DBP diastolic blood pressure; MBP mean blood pressure; SOFA sequential organ failure assessment score; SAPS II simplified acute physiology score II; APS III acute physiology score III.

Table 4 .
Multivariable cox proportional hazard models analysis of AG with 28-day ICU mortality.Data presented are ORs and 95% CIs.AG anion gap.Model I adjusted for gender, age, marital status, ethnicity.Model II adjusted for model I + weight, albumin (for traditional AG), BUN, lactate, PH, PO 2 , PCO 2, HR, SBP, RR, temperature, SpO 2 , chronic pulmonary disease, renal disease, liver disease, diabetes, hypertension, malignant cancer, dopamine, dobutamine, phenylephrine, RRT, SAPS II, APS III.Relationship between serum AGs measured in two methods and the risk of 28-day ICU mortality.(A) Traditional AG, (B) albumin-adjusted AG.The solid pink lines represent the smooth curves fit between variables.Purple bands depict the 95% confidence intervals.Blue areas show sample size distribution.Data were adjusted for gender, age, marital status, ethnicity, weight, albumin (only for traditional AG), BUN, lactate, PH, PO 2 , PCO 2 , heart rate, SBP, respiratory rate, temperature, SpO 2 , chronic pulmonary disease, renal disease, liver disease, diabetes, hypertension, malignant cancer, dopamine, dobutamine, phenylephrine, RRT, SAPS II, APS III.