Mac-2 binding protein glycosylation isomer is a potential biomarker to predict portal hypertension and bacterial infection in cirrhotic patients

Objectives Mac-2-binding protein glycosylation isomer (M2BPGi) is a novel plasma biomarker for liver fibrosis, but less is known about its role in portal hypertension. We aimed to evaluate the association between M2BPGi and hepatic venous pressure gradient (HVPG) and to investigate its predictive value on prognosis of cirrhotic patients. Methods Forty-eight cirrhotic patients who underwent HVPG measurement in Taipei Veterans General hospital were retrospectively enrolled. The Spearman’s correlation test was used to analyze the correlation between plasma M2BPGi levels and HVPG and other parameters. Cox proportional hazards regression models were used to identify predictors for clinical outcomes. Results Plasma M2BPGi levels were higher in cirrhotic patients than healthy subjects and significantly correlated with HVPG levels (rs = 0.45, p = 0.001). On multivariate Cox regression analysis, higher plasma M2BPGi levels [≥ 6 cut-off index (C.O.I)] did not predict mortality within five years for cirrhotic patients and the result was similar in patients without hepatocellular carcinoma. Interestingly, M2BPGi ≥ 6 C.O.I was a potential predictor of bacterial infection within five years [Hazar ratio (HR) = 4.51, p = 0.003]. However, M2BPGi failed to predict occurrence of other cirrhosis-related complications, including variceal bleeding, ascites formation, spontaneous bacterial peritonitis, hepatorenal syndrome and hepatic encephalopathy. Conclusion Plasma M2BPGi levels positively correlated with HVPG and higher serum M2BPGi levels might have a potential role in predicting development of bacterial infection for cirrhotic patients with portal hypertension.

Introduction Portal hypertension is a major and unfavorable consequence of liver cirrhosis. Portal pressure is determined indirectly by measurement of the hepatic venous pressure gradient (HVPG), the difference between the wedged hepatic venous pressure and the free hepatic venous pressure, via hepatic vein catheterization [1]. With the progression of hepatic fibrosis, portal pressure increases and then progresses to clinically significant portal hypertension (HVPG at or above 10mmHg), which is an important cut-off value for developing cirrhosis-related complications, such as ascites formation, variceal bleeding, hepatic encephalopathy, and hepatic decompensation [2,3]. Therefore, evaluation of the presence and degree of portal hypertension in cirrhotic patients is a critical issue to avoid adverse clinical outcomes.
Currently, HVPG measurement has been used clinically for predicting events of clinical decompensation in patients with compensatory cirrhosis, monitoring response of non-selective beta-blockers, and assessing the risk of liver failure after liver resection in patients with chronic liver disease or cirrhosis [4][5][6]. Though HVPG measurement is considered a safe procedure, the invasiveness of this procedure limits its clinical applications for cirrhotic patients. Consequently, non-invasive diagnostic tools have emerged as alternatives to assess liver fibrosis and portal hypertension.
Recently, Mac-2-binding protein glycosylation isomer (M2BPGi) has been introduced as a novel biomarker for liver fibrosis [7,8]. Emerging studies have demonstrated that M2BPGi is a potential predictor for hepatic fibrosis of various etiologies, such as viral hepatitis, nonalcoholic fatty liver disease, autoimmune hepatitis, and primary biliary cirrhosis and biliary atresia [9]. Its plasma concentration reflects the effect of antiviral treatment in both chronic hepatitis B and hepatitis C and plays a role in risk evaluation of hepatocellular carcinoma development in viral hepatitis [9,10]. M2BPGi is also a surrogate marker for postoperative ascites after receiving resection of hepatocellular carcinoma [11]. Higher plasma levels of M2BPGi were also associated with poor prognosis in cirrhotic patients [12]. Although M2BPGi levels have good correlation with the degree of liver fibrosis, there is limited study to investigate the predictive role of M2BPGi in complications and prognosis in cirrhotic patients with portal hypertension.
In this study, we aimed to investigate the association between M2BPGi and HVPG and the role of M2BPGi in predicting cirrhosis-related complications in portal hypertension.

Patients
From April 2000 to July 2020, forty-eight cirrhotic patients who underwent hemodynamic studies for evaluation of the presence and degree of portal hypertension at Taipei Veterans General hospital were retrospective enrolled. Written informed consent was obtained from each patient before hemodynamic study. This study has been approved by the Institutional Review Board of Taipei Veterans General Hospital (IRB No., 2021-02-012CC) and follows the tenants of the Declaration of Helsinki .  The diagnosis of liver cirrhosis was based on clinical, biochemical, and imaging findings for all  patients. Patients were excluded if they have portal vein thrombosis, hepatocellular carcinoma or  other liver tumor, active infection, active variceal bleeding, active alcohol drinking, hepatic  encephalopathy, use of non-selective beta-blockers or vasoactive drugs, history of previous transjugular intrahepatic portosystemic shunt placement or previous operations for portal hypertension. Demographic characteristics, laboratory data, and medical history were collected retrospectively by reviewing patients' medical records. Severity of underlying liver disease was measured using the Child-Pugh score and Model for End-Stage Liver Disease (MELD) score. Patients were followed until death, liver transplant or until five years of follow-up. During the followup period, clinical events were recorded, which included (1) complication of cirrhosis, such as spontaneous bacterial peritonitis (SBP), hepatic encephalopathy, newly developed ascites, hepatorenal syndrome, first or recurrent variceal bleeding, (2) newly diagnosed hepatocellular carcinoma, (3) events of bacterial infection, including SBP, (4) death, and (5) liver transplant.

Hemodynamic measurements
The enrolled patients underwent hemodynamic evaluation after overnight fasting. Under local anesthesia, a 7-Fr Swan-Ganz thermodilution catheter (Viggo-Spectramed, Oxnard, CA, USA) was placed in the right jugular vein or right femoral vein and was then advanced into the hepatic vein using the Seldinger technique as previous described [13]. The free hepatic venous pressure and wedge hepatic venous pressure were measured by inflation and deflation of the balloon with a multichannel recorder (model 78534C, Hewlett Packard, Palo Alto, CA). HVPG was calculated as the difference between wedged hepatic venous pressure and free hepatic venous pressure. After hepatic vein catheterization, the catheter was then advanced into right side of heart and the pulmonary artery to measure systemic hemodynamics, including right arterial pressure (RAP), mean pulmonary arterial pressure (MPAP), and pulmonary capillary wedge pressure (PCWP). Mean arterial pressure (MAP). Cardiac output (CO) was measured by using the thermodilution method. Heart rate and mean artery pressure (MAP) were recorded with an external vital sign monitor (Dinamap 8100, Critikon, Tampa, FL). Systemic vascular resistance (SVR; dynes•s/cm5) was calculated using the following equation: 80 x (MAP-RAP)/CO.

Statistical analysis
Data were expressed as mean ± standard deviation (SD) or as counts, as appropriate. The Mann-Whitney U-test was applied for assessing statistical significance of differences of between groups. Univariate and multivariate Cox regression analysis were performed to identify predictors for clinical outcomes. The results of the Cox regression analysis were reported as p value, hazard ratio (HR), and 95% confidence interval (CI). The cut-off values of MELD score, Child-Pugh score, M2BPGi, Fibrosis-4 (FIB-4) scores, and AST to platelet ratio index (APRI) scores in Cox regression analysis were determined by using median levels and the cut-off value of HVPG was according to the previous study [16]. Correlation between continuous variables was analyzed using Spearman's correlation test and Spearman's correlation coefficient was expressed as r s . Statistical significance was defined as p � 0.05. All statistical analysis were performed using IBM SPSS Statistics for Windows, Version 24.0 (IBM Corp. Armonk, NY, USA).

Patient characteristics
A total of 48 cirrhotic patients were retrospectively enrolled in this study, which included 36 males and 12 females with a mean age of 62.7 years. All the patients have portal hypertension (HVPG at or above 6mmHg) and 46 (95.8%) patients have clinically significant portal hypertension (HVPG at or above 10mmHg), with the mean HVPG of 17.5 mmHg. The demographic, laboratory and clinical characteristics are summarized in Table 1.

Association between M2BPGi and HVPG in liver cirrhosis
Plasma M2BPGi levels were significantly higher in cirrhotic patients compared to healthy subjects (7.19 ± 4.44 vs. 0.34 ± 0.11, p < 0.001) (Fig 1A). Interestingly, M2BPGi levels increased with the progression of portal hypertension (Fig 1B) in cirrhotic patients. Correlation between M2BPGi and HVPG was statistically significant (r s = 0.45, p = 0.001) (Fig 1C). However, correlation between M2BPGi and other hemodynamic parameters, including MAP, CO, and SVR, were not found. The median plasma level of M2BPGi was 6 C.O.I. in our study. Cirrhotic patients were then divided into group with M2BPGi � 6 C.O.I. and group with M2BPGi < 6 C.O.I. The HVPG levels between the two groups were compared (Fig 1D). Patients with plasma M2BPGi � 6 C.O.I. had significantly higher HVPG levels than those with M2BPGi < 6 C.O.I. We also examined the association between HVPG and FIB-4 scores as well as APRI scores and found that HVPG levels did not correlated with both parameters (r s = 0.09, p = 0.556 and r s = 0.12, p = 0.404, respectively).

Role of M2BPGi for predicting clinical outcomes in cirrhotic patients with portal hypertension
The median follow-up time was 20.1 (0.7-60.0) months. During the follow-up period, 32 patients died. Cox regression analysis was used to evaluate potential predictors associated with mortality within five years (Table 2). On univariate analysis, higher Child-Pugh scores (� 7) and higher plasma levels of M2BPGi (� 6 C.O.I) were significant predictors for mortality (Table 2 and S1

Fig).
However, these parameters failed to predict mortality on multivariate analysis. The results were similar after excluding 14 patients with hepatocellular carcinoma (S1 Table). Additionally, FIB-4 and APRI did not show a predictive role for mortality in these patients. We further examined potential parameters to predict cirrhosis-related complications in patients with portal hypertension. Twenty-seven patients developed bacterial infection events, including bacteremia (n = 9), pneumonia (n = 2), urinary tract infection (n = 6), soft tissue infection (n = 3), and SBP (n = 11). Of the 11 patients with SBP, 7 patients suffered from both SBP and other infection events. On univariate analysis, Child-Pugh scores � 7, albumin � 3.5 g/dL and plasma M2BPGi levels � 6 C.O.I were significant predictors for occurrence of bacterial infection within five years. However, on multivariate Cox regression analysis, plasma M2BPGi levels � 6 C.O.I was the only significant predictor for bacterial infection events (HR = 4.51, p = 0.003) ( Table 3 and Fig 3). Nevertheless, none of these parameters showed a predictive value for occurrence of SBP and other cirrhosis-related complications (S2-S6 Tables).

Discussion
In the present study, we demonstrated that plasma levels of M2BPGi correlated with HVPG levels in cirrhotic patients. Furthermore, bacterial infection events occurred at higher rates in those with higher plasma M2BPGi concentrations, suggesting that M2BPGi could be a new prognostic marker for bacterial infection for cirrhotic patients with portal hypertension. The results in our study provide novel information that may help clinical physicians to identify patients who are benefit from aggressive surveillance of cirrhosis-related complications.   It is known that HVPG levels correlate with the degree of fibrosis on liver histology [17][18][19] and has been used to predict clinical outcomes among cirrhotic patients. However, the utility of HVPG measurement is limited in clinical use owing to the invasiveness. Therefore, numerous studies aim to find a non-invasive tool which is highly accurate, repeatable, and convenient in clinical use. Recently, M2BPGi, an altered form of Mac-2 binding protein (M2BP) due to changes in the N-glycosylation during the progression of hepatic fibrosis, has been reported as a novel biomarker for liver fibrosis [7]. Although studies have confirmed the diagnostic accuracy of M2BPGi for liver fibrosis with various etiologies, especially for advanced fibrosis [8,20], no studies have investigated the association of M2BPGi and portal pressure in cirrhotic patients. In the present study, we found that there is a positive correlation between M2BPGi and HVPG in cirrhotic patients. Importantly, we identified cut-off values of M2BPGi to stratify patients with different degree of portal hypertension: 2.33 C.O.I for patients with clinical significantly portal hypertension (HVPG � 10mmHg), 3.89 C.O.I for patients with severe portal hypertension (HVPG � 12mmHg) and 5.93 C.O.I for patients with increased risk of mortality (HVPG � 16mmHg). The findings in our study suggest that M2BPGi might play a role in recognizing cirrhotic patients with different severity of portal hypertension.  Additionally, previous studies have demonstrated that plasma M2BPGi levels were higher in patients with decompensated cirrhosis and higher plasma M2BPGi level was an independent risk factor for liver-related mortality or all-cause mortality in cirrhotic patients [21][22][23][24]. Nevertheless, in our studies, we found that M2BPGi did not show a predictive value for allcause mortality and occurrence of cirrhosis-related complications, including SBP, hepatic encephalopathy, variceal bleeding, hepatorenal syndrome during the follow-up period. One of the reasons for the discrepant results may be attributed to the different enrolled populations between studies. In our study, 95.8% of patientshave clinically significant portal hypertension with the mean HVPG of 17.5 mmHg, which indicated that most patients enrolled in our study had more advanced fibrosis and were prone to have poor outcomes and higher mortality, leading to the different results as compared with the other studies. Furthermore, the different cutoff values of M2BPGi between studies and a small patient population in the present study might also contribute to different results.
An interesting finding in the present study was that M2BPGi was a potential predictor for the occurrence of bacterial infection. Plasma M2BPGi levels were also associated with plasma IL-1α and IL-1Ra levels. Although there was no statistic correlation between M2BPGi and IL-1β, there was a trend between the parameters. Bacterial infection is a troublesome consequence of cirrhosis, because in the course of advanced cirrhosis, patients encounter cirrhosis-associated immune dysfunction, with impaired immune system and dysregulated immune cell activation [25]. Bacterial-derived toxins in cirrhotic patients may trigger the production of proinflammatory cytokines from macrophage or monocytes. In vitro, human M2BP induces production of IL-1and other cytokines by blood monocytes [26]. IL-1, which includes IL-1α and IL-1β, is a central mediator of innate immunity and inflammation. The proinflammatory activities of IL-1 are controlled by several endogenous inhibitors and one of which is IL-1 receptor antagonist (IL-1Ra) [27]. In our previous study, we demonstrated that IL-1α, IL-1β and IL-1Ra were increased in cirrhotic patients and increased IL-1Ra levels predicted bacterial infection events [13]. As elevated IL-1 and IL-1Ra might reflect the immunodeficient status and dysregulated immune cell activation in cirrhotic patients, higher plasma concentration of M2BPGi might also reflect an immunosuppressive condition and these patients are susceptible to bacterial infection. Nevertheless, M2BPGi failed to predict the development of SBP on multivariate analysis (S4 Table), which was an important type of bacterial infection for cirrhotic patients. The possible reasons might be attributed to the small sample size and limited events in our study. High mortality rate in our studies might also contribute a role because some patients might die due to other complications before occurrence of SBP. Further prospective studies with larger sample size and longer follow-up time are needed to elucidate the diagnostic accuracy of M2BPGi in predicting HVPG levels and bacterial infection, especially SBP, in cirrhotic patients.
This study has some limitations. First, this is a retrospective study using data from a single medical center with small sample size, and caution must be taken in interpreting data. Second, patients with hepatic encephalopathy, active infection and active bleeding were excluded at enrollment and information about these patient populations is lacking. Furthermore, most of the patients had clinically significant portal hypertension, which indicates advanced cirrhosis in these patients. Therefore, the findings in our study might only be applied to cirrhotic patients with advanced portal hypertension.
In conclusion, plasma levels of M2BPGi correlated with HVPG levels and might predict severity of portal hypertension in cirrhotic patients. Additionally, higher plasma levels of M2BPGi could help to predict the occurrence of bacterial infection. The results in this study suggest that M2BPGi is a potential biomarker that could provide information to clinical physicians about the presence and severity of portal hypertension in cirrhotic patients and help them to identify patients who are at risk of developing bacterial infection.