Enzymatic liver function measured by LiMAx – a reliable diagnostic and prognostic tool in chronic liver disease

Chronic liver disease (CLD) is a major cause of morbidity and mortality worldwide. Non-invasive assessment of hepatic disease severity represents a relevant issue to further improve clinical management and therapeutic treatment. We retrospectively compared the diagnostic and prognostic performance of different non-invasive tools (LiMAx, transient elastography (TE), and biomarkers) in detecting different severity stages during the course of CLD. Patients were divided into four groups based on clinical parameters: (1) patients without CLD (control group), (2) patients suffering from CLD without having cirrhosis, (3) patients with CLD and compensated cirrhosis, and finally, (4) patients with CLD and decompensated cirrhosis. Patients with acute liver failure were excluded from the analysis. A total of 464 patients who underwent LiMAx measurement at the University Clinic of Essen between 10/2016 and 11/2017 were included in this study. Distribution of the different groups were n = 72 patients for group 1, n = 134 patients for group 2, n = 160 patients for group 3, and n = 98 patients for group 4, respectively. Median LiMAx values significantly declined with respect to increasing degree of CLD: (1) 510 µg/h/kg, (2) 390 µg/h/kg, (3) 264 µg/h/kg, and (4) 151 µg/h/kg (p < 0.001). When comparing the diagnostic accuracy of the LiMAx test in detecting patients with presence of cirrhosis (groups 1 and 2 vs. groups 3 and 4), an AUROC of 0.942 was found (cut-off 322 µg/h/kg, sensitivity 86.1%, specificity 91.3%, p < 0.0001). LiMAx was superior to TE and serum biomarkers in predicting patients’ outcome by 90-day mortality (AUROC 0.811, p < 0.001). Enzymatic liver function measured by LiMAx was closely associated with different severity stages of CLD and was a reliable diagnostic and prognostic tool with an accuracy comparable to current standard methods.


patients and Methods
Study population. The study population consisted of patients with CLD of various etiologies who were treated at the Department of Gastroenterology and Hepatology (University Clinic of Essen, Germany) between October 2016 and December 2017. Patients with acute liver failure were excluded from the analysis. Based on clinical, laboratory, ultrasound, endoscopic, and -if available -histological criteria (n = 184/464, 39.6%), patients were classified into four groups: • group I: patients without (history of) CLD (control group) • group II: patients with CLD without cirrhosis • group III: patients with CLD and compensated cirrhosis (cirrhosis with absence of decompensation signs) • group IV: patients with CLD and decompensated cirrhosis (at least one of the following) • presence of hepatic ascites ± spontaneous bacterial peritonitis • presence of portal hypertension (PH)-induced gastrointestinal/variceal hemorrhage • presence of renal dysfunction/hepatorenal syndrome (HRS) • presence of hepatic encephalopathy (HE) The study was conducted in accordance with the Declaration of Helsinki. The protocol was approved by the ethics committee of the University of Duisburg-Essen. non-invasive tools for the assessment of chronic liver disease. Inquiry of data for non-invasive assessment of CLD as stated below was performed within 24 hours of the LiMAx measurement.
Liver maximum capacity (LiMAx). LiMAx test (Humedics, Berlin, Germany) was performed after a minimum of 3 h fasting. The measurement is based on hepatocellular-specific metabolism of intravenously administered 13 C-labeled methacetin -an exclusive substrate for the hepatic cytochrome P450 1A2 enzyme. 13 C-methacetin in hepatocytes is immediately demethylated into acetaminophen and 13 CO 2 ; the latter is subsequently exhaled, leading to an increase of 13 CO 2 concentration in breath. Prior to substrate injection, the individual baseline ratio 13 CO 2 / 12 CO 2 concentration of a patient is measured and thus liver function capacity can be calculated from the analysis of the 13 CO 2 / 12 CO 2 ratio within 60 minutes after injection. Results are given in µg/h/kg. Serum biomarkers and CLD scoring systems. The following laboratory investigations for calculating different indexes and scoring systems were evaluated: aspartate aminotransferase (AST) to alanine aminotransferase (ALT) ratio (AAR), AST-to-platelet ratio index (APRI), fibrosis-4 (FIB-4) score, Model of End Stage Liver Disease (MELD) Score, and Child-Pugh Score (CPS). These were calculated as listed below:  For receiver operating characteristics (ROC) curves, the ROC-statement of PROC LOGISTICS was used. The model specified for the ROC curve included the classification system for the clinical severity stage as dependent variable and the liver test of interest as independent variable. 95% confidence intervals were obtained by the ROCCONTRAST statement which uses the Wald method by default for calculation. In general, optimal cut-offs were determined by the highest Youden index which corresponds to the sum of sensitivity and specificity minus 1.
ethics approval and consent to participate. The ethics committee of the University of Duisburg-Essen approved the anonymous collection and publication of the data.

Discussion
Non-invasive identification of patients with advanced stages of chronic liver disease (CLD) is clinically important as it serves as a relevant determining factor in the assessment of disease progression and long-term outcome. Today, different diagnostic tools are used for estimation of hepatic disease severity and individual prognosis. Our study focused on the measurement of hepatic function in CLD using the LiMAx test. LiMAx has been previously evaluated in various clinical settings such as liver surgery, liver transplantation, cirrhosis, oncology, acute liver failure, sepsis, and drug management [12][13][14][15][16][17][18][19][20]24,25 . Hence, we applied the test to determine enzymatic liver function in a large cohort of 464 patients with CLD. We hypothesized that the LiMAx test could provide additional information on disease severity, possibly allowing better stratification of these patients. We therefore compared the   Fig. 5. The superiority of LiMAx, however, is not essentially surprising: the applied substrate ( 13 C-methacetin) is metabolized by the cytochrome P450 1A2 enzyme -which is expressed in hepatocytes exclusively. In a recently published work of our group, we could demonstrate that structural changes in the course of CLD (reflected by different fibrosis-stages) were closely associated with changes of enzymatic liver function measured by LiMAx (Spearman's r −0.68) 13 . In contrast, routine laboratory parameters -used for the calculation of above-mentioned serum indices -are not liver-specific and released in various organs of the human body. Furthermore, these parameters are influenced by factors such as iatrogenic substitution, or biological half-life time.
Liver cirrhosis is the clinical endpoint of CLD irrespective of its underlying etiology. Development of portal hypertension (PH), defined by HVPG values ≥6 mmHg, is the key factor in the natural history of CLD and represents the main driver of complications. Ongoing disease progression exceeding the threshold of 10 mmHg ("clinical significant portal hypertension" (CSPH)) is highly associated with risk of clinical decompensation events (ascites, variceal bleeding, jaundice, and hepatic encephalopathy) tremendously increasing morbidity, and mortality [2][3][4] . Acquisition of histology by liver biopsy for the detection of cirrhosis and determination of the HVPG for PH assessment are considered as diagnostic gold-standards among patients with CLD 3,26-28 . However, due to their invasiveness, these methods are not applicable in the daily clinical setting. Thus, non-invasive markers for the  www.nature.com/scientificreports www.nature.com/scientificreports/ determination of hepatic disease severity have been subject of extensive scientific research during the last decades. Nonetheless, diagnostic accuracy of AAR, APRI, and FIB-4 for detecting liver cirrhosis is poor with AUROCs ranging from 0.52-0.72 depending on CLD etiology [29][30][31][32][33] . Liver stiffness measurement by transient elastography (TE) is probably the most validated non-invasive technique for the detection of hepatic fibrosis and cirrhosis. According to current literature, AUROC values for detection of cirrhosis (F4) range between 0.80 and 0.98 10,34,35 . Liver stiffness, however, is affected by various (patho)physiological conditions such as hepatic inflammation, congestion, mechanical cholestasis, and food intake 34 . Furthermore, in a significant number of patients, valid stiffness measurements are not obtainable due to technical reasons and/or presence of ascites (n = 99/464; 21.3% in our patient collective). Our results are consistent with those from current literature: LiMAx ® and TE (both AUROCs 0.94) were superior in detecting cirrhosis when compared to serum biomarkers (AUROCs 0.77-0.89) (Fig. 2). The determined cut off values for LiMAx (322 µg/h/kg) and TE (15 kPa) were comparable to those recently published in a histologically-proven cohort of our group including 102 patients 13 .
Apart from diagnostic purposes, prognostic factors and scoring systems have been investigated and (partly) implemented in clinical guidelines. Among patients with advanced CLD and cirrhosis, PH is the main determinant of complications, increasing the mortality of this patient collective. Measurement of HVPG is the gold standard for its evaluation, and the severity of PH is directly correlating with the long-term prognosis of cirrhosis 36 . However, due its invasiveness and indispensable operators' expertise, its use is restricted mainly to tertiary centers. CPS and MELD score are the most common and widely used non-invasive models for outcome prediction in cirrhotic patients with good prognostic accuracy [37][38][39][40] . Consequently, MELD and CPS showed reliable results in predicting 90-day mortality in our collective (AUROCs of 0.8317 for MELD and 0.8284 for CPS, respectively) and were superior to TE (AUROC 0.7259), and serum biomarkers (AUROCs 0.6362-0.7170). Malinowski et al. already showed strong negative correlation of LiMAx with CPS (r = −0.707, p < 0.001) and MELD score (r = −0.686, p < 0.001) in their cohort of 347 cirrhotic patients 19 . Furthermore, Jara and colleagues demonstrated that LiMAx was, apart from serum creatinine levels, an independent predictor of short-term mortality in patients with liver cirrhosis (n = 268, AUROC 0.75) 14 . Accordingly, in our patient collective, the prognostic accuracy of LiMAx for 90-day mortality was excellent with an AUROC of 0.8115 and comparable to CPS (AUROC 0.8284) and MELD (AUROC 0.8317).
Besides its powerful diagnostic and prognostic validity, LiMAx represents a safe and robust non-invasive examination: no adverse events or test failures were documented during the 464 measurements. It is even possible to perform the test in mechanical ventilated patients or patients with extracorporeal membrane oxygenation devices 16,24,41 .
However, it should be mentioned that our study has various limitations. At first, histology was not available for all patients and exact classification of groups I-IV might be slightly modified. Secondly, TE was not obtainable in all patients and the actual results for liver stiffness measurement might be slightly better. Furthermore, this was a retrospectively performed single-center cohort study. However, our present findings confirm the results of our previously published histology-proven study for LiMAx and TE in large patient cohort. A prospective multicenter study would be desirable to further validate the potential of LiMAx in the assessment of disease severity in patients with CLD.
In conclusion, the present study demonstrates that enzymatic liver function was closely associated with different clinical stages occurring during the course of CLD. Our results indicate that LiMAx is not only an accurate diagnostic, but also a valuable prognostic tool in patients with CLD comparable to current standard methods.

Data Availability
Datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.