The Association Between Alkaline Phosphatase and Neurological Outcome in Patients with Post-cardiac Arrest: a Secondary Analysis of Data from Belgium


 Background: Prognostication of the unfavourable neurological outcome(UNO) after Cardiac arrest(CA) is multimodal while blood biomarkers are an attractive option.Serum alkaline phosphatase(ALP) is shown to be associated with ischemic stroke and considered as an independent prognostic factor for long-term functional outcome after acute cerebral infarction.We aimed to study the association between ALP and UNO in 3 months in patients after CA.Methods:Review of consecutive patients admitted to the ICU at Erasme Hospital, Brussels (Belgium) following CA between January 2007 and December 2015. The outcome was the UNO in 3 months. We used multivariable logistic regression models to calculate the adjusted odd ratio (OR) with 95% confidence interval(CI). Interaction and stratified analyses were conducted according to Shock,Dobutamine.used,Hypertension,Cardiac.Etiology and Shockable.Rhythm.Results: We included 374 CA patients. The multivariate logistic regression analyses revealed that the risk of UNO in 3 months after CA was significantly associated with ALP.When ALP was Equal is divided in 3 groups, the risk of ALP>91 level and adverse outcomes was 1.7407 times of ALP<64 level (P for trend=0.19709).In the non-hypertension group, the risk of adverse outcomes increased with increased ALP by 1.018 (1.0041.01,1.032).Conclusions: ALP is an independent risk factor for the UNO in 3 months after CA, especially in non-hypertension. Elevated ALP was significantly associated with increased UNO in 3 months after CA. However, the prediction significance of ALP for long-term neurological outcome in patients after CA is needed further studied.


Introduction
Cardiac arrest (CA) which had an annual incidence of nearly 350,000 out-of-hospital and about 750,000 in-hospital events is common in the United States 1,2 .Even after the rescue, the ischemia-reperfusion injury that occurs after the return of spontaneous circulation (ROSC) contributes to a systemic in ammatory which further increases the likelihood of a poor outcome including hypoxic-ischemic brain injury.Neurological prognostication after CA is challenging due to the lack of early and reliable predictors for clinically relevant brain damage.The prediction of brain injury is multimodal, including repeated clinical neurological examinations, diagnostic imaging, neurophysiological tests, and blood biomarkers 3 .Among blood biomarkers are an attractive choice because they are not affected by sedative drugs and are considered objective measures.Currently, the only recommended biomarkers after cardiac arrest is neuron-speci c enolase evaluated 48-72h after CA 4 .But earlier, simpler and reliable outcome predictors are needed to strati cation and improve care after CA 5 .
Alkaline phosphatase (ALP), an enzyme rst discovered in the 1920s, has been evaluated as a potential biomarker for many diseases 6 .There are four isoenzymes in humans. Intestinal tract (IAP), placenta, germinal tissue (GCAP), and tissue-speci c (TNAP).TNAP can be expressed through a variety of tissues, including multiple cell types in the brain 7 .Ermonval et al and Brun-Heath et al have suggested that ALP could be expressed in cerebral endothelial cells and neurons 8, 9 .Lee et al suggested that the increase of ALP at stroke is related to the degree of cerebral cerebrovascular disease 10 .Ischemic stroke is similar to neurological injury after CA.Brain cells are very sensitive to hypoxia.After Ischemic stroke, ischemic stoves will have ischemic and hypoxic injury, if reperfusion treatment can be achieved, and reperfusion injury.After CA occurs, blood supply to the whole brain stopped, and ischemia and hypoxia injury to the brain, when effective CRP or ROSC, the brain recovered blood supply, followed by reperfusion injury.Therefore, we argue that the brain after CA suffers blows of the same nature as ischemic stroke.Several studies have also noted that ALP is associated with ischemic stroke as an independent prognostic factor for the long-term functional outcome after acute cerebral infarction 11-13 . Thus, the goal of this study is to study the relationship between ALP and post-CA neural results, and also to evaluate the effects of other classical risk factors on associations through interaction and hierarchical analysis.

Data Source
Date were obtained from 'DATADRYAD' database (www.Datadryad.org). This website permitted users to freely download the raw data. According to Dryad Terms of Service, we cited Dryad data package in the present study 14 ."Data from: Acute liver dysfunction after cardiac arrest," Dryad, ataset, https://datadryad.org/stash/dataset/doi:10.5061/dryad.qv6fp83)in the present study. Authors of the original study have waived all copyright and related ownership of these data. Therefore, we could use these data for secondary analysis without infringing on the authors' rights.

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The protocol of post-resuscitation management had been extensively described elsewhere 15 . Advanced hemodynamic monitoring was used (PiCCO, Pulsion, Munich, Germany) and cardiac function was assessed by repeated transesophageal and/or transthoracic echocardiography. Mean arterial pressure (MAP) was maintained at >65-70 mmHg using volume resuscitation, dobutamine, and/or noradrenaline, whenever needed. Intra-aortic balloon counterpulsation (IABP) or extracorporeal membrane oxygenation (ECMO) was also used in cases of severe cardiogenic shock. Ventilation was set to maintain normocapnia and SpO 2 >94%. Blood glucose was kept between 110 and 150 mg/dl using a continuous insulin infusion. Enteral nutrition was initiated during TTM and continued thereafter according to gastric tolerance. Neurological monitoring and prognostication of coma after CA were performed using a multimodal approach, as previously described 16 .

Data Collection and Measurements
We collected data on demographics (including chronic use of any anticoagulant), preexisting chronic diseases, cardiopulmonary resuscitation (CPR) (initial rhythm, bystander CPR, time-to-ROSC, total-adrenaline-dose), and blood test in all patients. Blood samples were analyzed for Glutamic-oxaloacetic mg/dl) and brinogen (normal values > 150 mg/dl) on admission and then once daily until ICU discharge.
As described before 14 , Neurological evaluation was assessed at 3 months after CA using the cerebral performance categories score (CPC; 1 = no or mild neurological disability, 2 = moderate neurological disability, 3 = severe neurological impairment, 4 = vegetative state, 5 = death) 17 .Unfavorable neurological outcome at 3 months after CA(Unfavourite Outcome in 3 months) was considered as a CPC 3-5; favorable neurological outcome as CPC 1-2 18 .The shock was de ned as the need for vasopressor agents for more than 6 hours. Statistical Analysis.
All normally distributed and skewed continuous variables were presented as mean (SD) or median (interquartile range [IQR]). Categorical variables were indicated as frequencies (%). For baseline characteristics analysis, the statistical differences among tertile groups of ALP were tested with one-way ANOVA for continuous variables and chi-square test for categorical variables. The effect of ALP on unfavourable neurological outcome(UNO) in 3 months was evaluated using logistic regression models (odd ratio [OR] and 95% con dence interval [CI]).Both nonadjusted and multivariate-adjusted models were used. To assess confounding, we entered covariates into a binary logistic regression model in the basic model or eliminated the covariates in the complete model one by one and compared the regression coe cients 23 .
Variables thought to be confounders based on existing literature and clinical judgment were included [19][20][21][22] .In this study, the logistic regression models were adjusted for age+gender+Weight+Witnessed.Arrest+Bystander.CPR+time.to.ROSC+ Epinephrine.total.dose+Out.of.Hospital+Cardiac.Etiology+Shockable.Rhythm+Corticoids+Chronic.Anticoagulation+Chron.Heart.Failure+Hypertension+Coron.Ar trends were conducted with linear regression by entering the median value of each ALP tertile as a continuous variable in the models.
Logistic regression model with cubic spline functions and the smooth curve tting were used to identifying linear relationships between ALP and UNO in 3 months. The subgroup analyses were conducted by Dobutamine.used and Shock using strati ed logistic regression model. Interaction across subgroups was tested using the likelihood ratio test. For all statistical analyses, we used statistical packages R version 3.4.3 (The R Foundation, Vienna, Austria) and EmpowerStats (X&Y Solutions, Inc., Boston, MA, USA). P< 0.05 was considered statistically signi cant.
For other continuous variables with missing values less than 6%, the missing values were replaced by the mean values.

Results
Population 435 comatose patients (Glasgow Coma Scale, GCS < 9) admitted after IHCA or OHCA were included.After excluding participants who were died less than 24 hours(n=51) after ICU admission, and missing data on liver function(n=10) because of ALP being a kind of liver enzyme, and we needed to adjust the indexes related to liver.The nal cohort included 374 patients. The ow chart of the study patients selection is presented in Figure 1.

Baseline Characteristics of Study Participants by Categories of Serum ALP Levels
The baseline characteristics of all participants were listed in Table 1.The ALP is evenly divided into 3 groups based on the serum ALP.226 (60.4%) patients had an UNO in 3 months.159(42.5%) patients had hypertension.221(59.1%) patients had cardiac etiology.153(40.9%) patients had shockable rhythm during CA.200(53.5%) participants suffered shock during ICU stayed,and 201 (53.7%) participants used Dobutamine during ICU stayed. The mean ALP level was 93.7 ± 66.7 IU/L. Participants with higher serum ALP levels were more likely to be associate with IHCA, Shockable.Rhythm, Corticoids, Chronic Renal Failure, Shock, Dobutamine.used,VSP, AKI, CRRT.used, higher CRP, higher Cr, higher GOT, higher GGT, and higher BIL  Association between ALP levels and unfavorable neurological outcome in 3 months in the study Subgroup analyses of the association between ALP levels on unfavorable neurological outcome in 3 months in the study.
Shock,Dobutamine.used,Hypertension,Cardiac.Etiology and Shockable.Rhythm are known confounders of ALP and UNO in 3 months association. To see if the association between serum ALP levels and UNO in 3 months is stable in different subgroups, we did strati ed analyses and interactive analyses ( Figure  3). The association between ALP and UNO in 3 months in the strati ed analysis was consistent with that in the multivariable logistic regression analysis.The strati ed analysis demonstrated a statistically signi cant association between ALP levels and UNO in 3 months in non.Dobutamin.used subjects (adjusted OR, 1.0324; 95% CI, 1.009-1.0564), non-Hypertension subjects (adjusted OR, 1.018; 95% CI, 1.0041-1.032). Data showed that Hypertension played an interactive role in the association between ALP and UNO in 3 months (P for interaction 0.004).

Discussion
In this population-based cohort study, ALP was found to be associated with an elevated risk of the UNO in 3 months, independent of age + gender + Weight + Witnessed.Arrest + Bystander.CPR + time.to. ROSC + Epinephrine.total.dose + Out.of.Hospital + Cardiac.Etiology + Shockable.Rhythm + Corticoids + Chronic.Anticoagulation + Chron.Heart.Failure + Hypertension + Coron.Artery.Dis + Diabetes + COPD.Asthma + Previous.Neurol.Disease + Chronic.Renal.Failur + Liver.Cirrhosis + ICU.stay + TTM + IABP + ECMO + Shock + VSP.used + Dobutamine.used + Mechanical.Ventilation.used + CRRT.used + MAP + Lactate + CRP + Creatinine + ScvO2.SvO2 + AST + ALT + LDH + GGT + Total.bilirubin + APTT + PT + INR + Platelets + Proteins + Glucose + pH + PaO2 + PaCO2.We revealed a linear relationship between serum ALP levels and the risk of the UNO in 3 months. The relationship was characterized as follows: A 7.7% higher risk of UNO in 3 months, associated with a 10 U/L increased in serum ALP. In addition, in the multi-factor analysis ( Table 2), we could also nd that the relationship between the ALP and the prognostic becomes more obvious as the ALP increases,especially obvious in the T3 group.After adjustment, ALP increased 1 unit, the prognosis risk increased 74.07% (1.7407 (0.7676,3.9472)), while the corresponding T2 groups increased only 10.29% (1.1029 (0.5364,2.2674)).In the strati ed analysis, we observed signi cant associations in patients with non-hypertension, but not in patients with hypertension.Interestingly, we also observed signi cant associations in the non-Dobutamine.used group, despite no interaction between ALP and the results.We found no interaction in the shock, cardiac.Etiology,shockable.Rhythm groups, indicating that the relationship between ALP and the results was unrelated to the cardiogenic cause, de brillation rhythm, and shock.
ALP, an enzyme rst discovered in the 1920 s, has been evaluated as a potential biomarker for many diseases 6 .The rise in ALP is a common manifestation of ischemic stroke. It has been made clear that ALP is an independent risk factor for the adverse prognosis of ischemic stroke and has a positive correlation 13,23,24 .Neurodysfunction of ischemic stroke mainly comes from ischemic damage of nerve cells.The injury to brain cells in patients after CA also mainly stems from ischemic and hypoxia injury during cardiac arrest and reperfusion injury after recovery.It shows that the two have some similarities, and our results suggest that the positive correlation of ALP with bad prognosis also agrees with previous studies.
The reasons for ALP rise in CA may be the following:First, some basic studies have shown that ALP activity can be expressed in brain endothelial cells and neurons. 8,9 .An association between ALP and minor cerebrovascular disease was found by Lee et al 10 .When blood supply disorder occurs in the arterial artery, the corresponding brain tissue develops ischemia, damage or even necrosis, and blood ALP increases.The greater the infarct range, the higher the ALP, the more obvious the corresponding nerve function damage. Brichacek et al. also found that ALP caused adverse outcomes by playing a role in the broken bloodbrain barrier (BBB), neurological in ammation, and vascular dysfunction in stroke patients 6 .Because small areas of cerebral infarction are often associated with a neurologic prognosis, this explains why OR increases in ALP > 91 and ALP < 64.Second, serum ALP levels are also associated with in ammation or malnutrition. Similar to the C-reactive protein, ALP also re ects the in ammatory state of the body 25 .The reperfusion after prolonged cardiac arrest ischemia leads to increased in ammatory cytokines 26 .Due to the systemic in ammatory response, patients after CA showed a sepsis-like state.In fact, in this study, elevated ALP levels were associated with CRP levels and several nutritional states.However, we found that the elevation of ALP levels and the adverse stroke outcome remained stable after adjusting these indicators.Thus, it is unlikely for the link between ALP and adverse outcomes of stroke to rely solely on malnutrition and in ammation.However, some studies conclude not exactly the same as ours.It was found that ALP was not signi cantly associated with neurofunctional results from a year later 20 .The reason may be the grouping of ALP was different.Their study was based on the ALP grouping of the ending, while our research was based on grouping by the ALP value.
In the subgroup analysis,Our subgroup analysis revealed that the association between ALP and risk of UNO in 3 months existed between the layers except hypertension.It showed that ALP concentration was positively associated with UNO in 3 months in nonhypertension (OR 1.018(1.0041,1.032)), P for trend 0.01083)However, the associations were not observed in hypertensions.The reasons for the unclear relationship between ALP and the adverse outcome in the hypertension group were unclear.It has been proposed that there are various complications in patients with hypertension, so TTM is associated with good neurological recovery in patients without hypertension, but not in patients with hypertension 27,28 .Eujene Jung suggests that hypertension does not have an adverse effect on the prognosis of patients with cardiac arrest 27 .This may be associated with hypertension patients with greater awareness of activating emergency medical services (EMS) with ischemic symptoms.And lead to short cardiac arrest to EMS recovery 29 .Another possibility is that the effects of hypertension drugs can be taken into account.There have been studies that calcium channel blockers can be treated in ischemic pretreatment reserved with cardiac arrest and angiotensin converting enzyme inhibitors to reduce recurrent myocardial infarction and ischemia 30,31 .In addition, most patients with hypertension have cerebrovascular sclerosis, which involves too many diseases 32 .In the future, it is necessary to study the factors related to HTN which improve the favorable neurological outcome of CA patients.
This study has several limitations. First, this is a population-based observational study, not an intervention study. There are likely to be signi cant potential biases that were not considered.Second,because we studied the population of patients who recovered heart breathing after CA, we excluded the early death (n = 51) to reduce bias. Also, the dependent variable we studied ALP is one of the liver enzymes, so we excluded the absence of data on liver transaminases, coagulation, and/or total bilirubin (n = 10). This leads to a reduced number of studies, but after regression analysis and hierarchical analysis,the results remain robust and reliable. Third, compared to previous studies, we lack variate data like hypertension medication, however, our data have 48 covariates that reduce the bias as possible, and our results all show consistency after adjusting the covariates, which also shows that our results are stable.These limitations should be considered in future studies.

Conclusion
ALP is an independent risk factor for the UNO in 3 months after CA, especially in non-hypertension. Elevated ALP was signi cantly associated with increased UNO in 3 months after CA. This is similar to the relationship between ALP and the prognosis of ischemic stroke.However, the prediction signi cance of ALP for long-term neurological outcome in patients after CA is needed.

Declarations
Funding None Availability of data and material Data can be downloaded from the DRYAD database (http:// www.Datadryad.org).

Competing interests
The author declares no con icts of interest in this work.

Consent to publish
Not applicable/all data used for the present study have been anonymized, and the submission does not include information that may identify individual persons.