Retinol-binding protein-4 expression marks the short-term mortality of critically ill patients with underlying liver disease: Lipid, but not glucose, matters

The implications of retinol-binding protein-4 (RBP4) expression in critically ill patients with underlying liver diseases remain unclear. A prospective cohort study involving 200 liver intensive care unit (ICU) patients was conducted, with 274 blood donors as controls. Patient outcomes were assessed using Cox and Kaplan-Meier analyses. Of the 200 ICU patients (mean age: 56.0 yrs), 79.5% were male, 72.5% were cirrhotic, 62% were septic, 29.5% were diabetic, and 29% expired in the ICU (median admission: 7.5 days). ICU patients had lower baseline RBP4 (25.6+/−18.4 vs. 43.8+/−35.0 mg/L, p < 0.001) and total cholesterol (TC) levels than controls. The surviving ICU patients had lower baseline international normalized ratios (INRs) of prothrombin time, model for end-stage liver disease (MELD) scores and sepsis rates, but higher estimated glomerular filtration rates (eGFRs) and RBP4 levels than non-surviving patients. eGFRs, INRs and TC levels were independently associated with RBP4 levels. Only surviving patients exhibited significantly increased RBP4 levels after ICU discharge. Baseline RBP4 levels and MELD scores predicted 21-day (≤10 mg/L) and 1-year (≥25) mortality, respectively. In critically ill patients with underlying liver disease, with a link to eGFRs, INRs and TC levels, the baseline RBP4 may serve as a marker for short-term mortality.

phospholipids and free fatty acids. HSC activation is crucial to the development of hepatic fibrosis and presents as lipid droplet release 12 . Elevated retinoic acid-responsive gene expression is associated with elevated hepatic triglyceride levels 13 ; thus, lipid metabolism is closely associated with HSC activation and hepatic fibrosis. Serum RBP4 levels are associated with insulin resistance (IR), hypertriglycemia, obesity, metabolic syndrome, diabetes and fatty liver 9,[14][15][16][17][18][19][20] . Although serum RBP4 concentrations have been linked to acute mortality in critically ill patients 6 , whether RBP4 is a suitable marker for prognoses and how lipids affect RBP4 expression in patients with underlying liver disease remain unclear. Thus, we conducted a prospective study involving critically ill patients who were admitted to a liver ICU to determine the implications of RBP4 expression in this patient population by comparing this marker with other known prognostic markers.
Factors associated with baseline RBP4 levels. The factors associated with baseline RBP4 are listed in Table 2. The univariate analysis demonstrated that age, diabetes, hypertension, C-peptide levels, uric acid levels, eGFRs, TC levels, triglyceride (TG) levels, ALT levels, bilirubin (t) levels, high sensitivity C-reactive protein (Hs-CRP) levels and INRs were associated with baseline RBP4 levels, whereas the multivariate analysis confirmed that the baseline eGFRs, TC levels and INRs were independently associated with the baseline RBP4 levels. The correlations between RBP4 levels and eGFRs (person correlation = −0.458, p < 0.001), TC levels (person correlation = 0.371, p < 0.001), and INRs (person correlation = −0.404, p < 0.001) are presented in Fig. 2.
Subgroup analyses according to sepsis, cirrhosis and diabetes. Given that septic and cirrhotic patients accounted for the majority of the patients enrolled in this study and that sepsis, pulmonary disease 7 , cirrhosis 5,6 , and diabetes 21, 22 may profoundly affect the RBP4 levels, we performed subgroup analyses of the RBP4 levels according to the patients' sepsis, pulmonary disease, cirrhosis and diabetes statuses. Diabetic (31.87+/−21.33 vs. 23.25+/−16.50 mg/L, p = 0.015) patients had higher baseline RBP4 levels than non-diabetic patients. However, there was no difference in the baseline RBP4 levels between the septic and non-septic patients [p = 0.161, regardless of adjusting for pulmonary disease (yes: p = 0.337; no: p = 0.221)], between those with or without pulmonary disease (p = 0.879), nor was there a difference in the baseline RBP4 levels between the cirrhotic and non-cirrhotic patients (p = 0.303). Regarding the independent predictors of RBP4 levels, higher TC levels (112.1+/−58.2 vs. 106.3+/−37.2, p = 0.039), but lower eGFRs (41.7+/−62.8 vs. 70.1+/−59.7 mL/min, p = 0.009), were noted in diabetic patients than in non-diabetic patients. However, no differences in the INR were noted between diabetic and non-diabetic patients.
Outcome predictors in critically ill patients with underlying liver disease. The usefulness of baseline RBP4 levels and MELD and APACHE IV scores in predicting liver ICU patient outcomes was investigated. The results are presented in Fig. 3. The baseline RBP4 levels could predict short-term (within 21 days of ICU admission) (Fig. 3A), but not long-term (1-year) mortality ( Fig. 3B) in critically ill patients with underlying liver diseases. The RBP4 cut-off value for 21-day survival was 10 mg/L. However, no definitive cut-off levels for survival outcomes based on follow-up times longer than 21 days could be determined. Surviving patients (in this ICU admission, 27.3+/−19.1 vs. 33.3+/−21.4 mg/L, p = 0.02), but not non-surviving patients (expired during this ICU admission, 20.1+/−17.2 vs. 25.9+/−15.8 mg/L, p = 0.078), exhibited significant increases in their RBP4 levels upon discharge from or resuscitation prior to expiring in the ICU. In contrast, baseline MELD scores could predict short-term (Fig. 3C) and long-term (Fig. 3D) mortality up to 1 year, with a cut-off value of 25, whereas APACHE IV scores exhibited a negligible ability to predict both short-term (7-day, p = 0.061, 14-day, p = 0.186, 21-day, p = 0.455, 30-day, p = 0.154) and long-term mortality (1-year, p = 0.095).

Discussion
To the best of our knowledge, this is the first prospective study to elucidate the clinical implications of serum RBP4 levels in critically ill patients with underlying liver disease. The following are the most compelling results of this study: (1). Liver ICU patients had lower RBP4 and TC levels than normal controls. Among ICU patients, surviving patients and diabetic patients had higher baseline RBP4 levels than their counterparts (2). eGFRs and INRs were negatively associated with RBP4 levels, whereas TC levels were positively associated with RBP4 levels.
(3) Only surviving ICU patients had significantly increased RBP4 levels upon discharge from the ICU. (4) Among liver ICU patients, RBP4 levels could predict short-term mortality (21-day), with a cut-off value of 10 mg/L, whereas baseline MELD scores could predict both short-and long-term (1-year) mortality (cut-off value: 25).
Control subjects had higher baseline RBP4 levels than the liver ICU patients, indicating that stress has a negative impact on RBP4 levels 23 , whereas the surviving liver ICU patients had higher baseline RBP4 levels than the non-surviving patients, suggesting that RBP4 plays a crucial role in restoring vital functions during crises, even in patients with underlying liver disease, which exerts fundamental effects on RBP4 levels 9 . Consistent with this finding, under stress, the body activates an acute phase response to down-regulate negative acute phase proteins including RBP4. Retinoids are RBP4 cargos that regulate cellular proliferation, differentiation, and apoptosis and hence affect immunity maintenance and barrier integrity 24 . The impact of sepsis on the RBP4 levels of critically ill patients may be different in various underlying diseases. In ICU patients with underlying pulmonary disease, lower RBP4 levels were noted in septic patients than in non-septic patients 7 . In contrast, another ICU cohort study demonstrated that sepsis had a negligible impact on RBP4 levels in critically ill patients, regardless of the origin of illness 6 . In our study, no differences in baseline RBP4 levels were noted between the septic and non-septic ICU patients with underlying liver disease, regardless of adjusting for pulmonary disease. Moreover, although cirrhotic critically ill patients had been reported to exhibit lower RBP4 levels than non-cirrhotic critically ill patients 6 , surprisingly, our study did not show such difference. Thus, the impacts of cirrhosis on RBP4 levels may vary between critically ill patients with or without underlying liver diseases. Multivariate analysis demonstrated that both eGFRs and INRs were negatively associated with RBP4 levels, indicating that renal and liver function negatively and positively contribute to serum RBP4 levels, respectively, findings consistent with those of previous studies 9, 25,26 . Furthermore, although diabetic patients had higher RBP4 levels than non-diabetic patients in the current study, in addition to eGFP and INR levels, TC levels rather than any indicators of glucose metabolism were independently associated with RBP4 levels. Additionally, the above differences in RBP4 levels between diabetic and non-diabetic patients occurred subsequent to increases in TC levels and decreases in the eGFR in diabetic patients rather than being directly driven by diabetes itself. Previous studies have consistently demonstrated that the high RBP4 levels noted in diabetic patients are caused by diminished renal function rather than by altered glucose metabolism 25,26 . Some studies concluded that glucose metabolism profoundly affects RBP4 levels 6,21,22 ; however, others have noted a link between lipid metabolism and RBP4 levels [23][24][25][26][27][28][29] . Rat studies demonstrated that acute stress had a direct influence on liver lipid metabolism 30 , and human studies demonstrated that low TC levels are associated with a poor prognosis in patients with prolonged sepsis 31 . Moreover, both HDL-C and low-density lipoprotein (LDL-C) levels decreased because of lecithin cholesterol acyltransferase impairment in critical illness 32 . Consistent with these findings, in liver ICU patients, we also showed that HDL-C levels were lower in non-surviving patients than in surviving patients. Collectively, all of the above findings indicate that lipids play a critical role in acute stress. Given that both liver function and RBP4 are closely linked to lipid metabolism 20, 23-29 and that RBP4 is an acute response protein 6 , we would like to stress that TC levels, but not glucose levels (or glucose metabolism), were independently associated with RBP4 levels in liver ICU patients. Thus, in addition to restoring liver and renal function, supplementing patient diets with adequate TC or related precursors 32 may up-regulate RBP4-associated pathways and improve the immediate outcomes of critically ill patients with underlying liver disease.
The findings that only the surviving patients exhibited significantly increased RBP4 levels compared with the baseline levels after discharge from the ICU and that baseline RBP4 levels were most effective in predicting short-term mortality confirmed that RBP4 plays a negative acute-phase reactant role in critically ill patients. The negligible roles of the APACHE scores 1 and the reliabilities of the baseline MELD scores 33 in predicting both short-term and long-term mortality indicated that only scoring systems focused on liver functional reserve have predictive value with respect to outcomes in critically ill patients with underlying liver diseases.
Because liver and adipose tissue are the major sources of RBP4 9, 10 , the main limitation of this study was its lack of pathological studies of liver and adipose tissue samples. Moreover, the findings documented in the current study should be validated in an independent cohort of liver ICU patients. Future studies of RBP4 incorporating surveys of liver and adipose tissue pathology samples from critically ill patients with underlying liver disease, as well as studies utilizing related cellular or animal models, may be required to elucidate the fundamental molecular mechanisms underlying the findings described herein.
Taken together, critically ill patients with underlying liver disease had lower baseline RBP4 and TC levels than controls. With an association with the eGFR, TC and INR levels, baseline RBP4 levels may serve as a marker for short-term mortality within 21 days of liver ICU admission, with a cut-off level of < or =10 mg/L. In contrast, baseline MELD scores can predict short-term and long-term mortality in liver ICU patients. These findings may facilitate improvements in the outcomes of critically ill patients with underlying liver disease in probing associated metabolic alterations and crucial organ dysfunction.

Research Design and Methods
Patients. The study group comprised critically ill subjects aged 18 years or older who were admitted to the liver ICU with underlying diseases, such as chronic hepatitis B, defined as the presence of hepatitis B surface antigens (HBsAg) for >24 weeks 34 ; chronic hepatitis C, defined as the presence of documented HCV antibody positivity and detectable HCV RNA for >24 weeks 35 ; liver cirrhosis, diagnosed by histologic findings or repeated abdominal ultrasound findings consistent with cirrhosis and supplemented with clinical features such as varices and thrombocytopenia, as described elsewhere 36 ; hepatocellular carcinoma; and hepatic failure (complicated by overt hepatic encephalopathy 37 ) subsequent to alcohol intoxication and shock. The liver ICU admission criteria included unstable hemodynamic status and single or multiple organ failure in the aforementioned patients. Cirrhosis-related complications were defined as overt hepatic encephalopathy, esophageal variceal bleeding and spontaneous bacterial peritonitis. Overt hepatic encephalopathy was defined as a spectrum of global neurologic deficits in patients with liver dysfunction after the exclusion of brain disease 34,38 , and was diagnosed and classified as described previously 39 . Esophageal bleeding was determined using a fiberoptic panendoscope. Spontaneous bacterial peritonitis was defined as an ascitic fluid infection without an evident intra-abdominal surgically treatable source 40 and was diagnosed according to the clinical manifestations and ascitic fluid findings as described previously 41 . Severe acute exacerbation of chronic hepatitis B was defined as an abrupt ALT elevation >5 × the upper limit of the normal range associated with jaundice and a prolonged prothrombin time in patients with chronic hepatitis B 42 . Subjects with human immunodeficiency virus, coronary heart disease, terminal stage of malignancy or solid organ transplant recipients were excluded from the study. Blood donors aged 18 years or older without sepsis, cirrhosis and fatty liver served as the controls.     hemoglobin levels, neutrophil percentages, lymphocyte percentages, NLRs, platelet counts, INRs for prothrombin time and RBP4 levels (R&D Systems, MN, USA), upon their admission to the ICU using fasting serum samples. Illness severity was accessed using the APACHE IV and MELD scores. RBP4 levels were evaluated again in the ICU patients upon discharge from or following expiration in the ICU. Abdominal ultrasound and dynamic computed tomography studies were performed to determine if cirrhosis or hepatocellular carcinoma was present.

Materials.
Biochemical tests were performed in the hospital clinical pathology laboratory using routine automated techniques. Serum hepatitis markers, including HBsAg and HBeAg antibody levels and anti-HDV antibody levels, were assayed using radioimmunoassay kits (Abbott Diagnostics, North Chicago, IL, USA), and anti-HCV antibody levels were assayed using a commercial third-generation enzyme immunoassay (Axsym HCV, version 3 Abbott Diagnostics, North Chicago, IL, USA).
Statistical Analyses. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS package version 21, SPSS Inc., Chicago, USA) software. For the between-group comparisons, continuous variables were analyzed using the Student's t-test or the non-parametric test (Mann-Whitney U), whereas categorical variables were analyzed using the chi-squared test or Fisher's exact test when appropriate.
The continuous variables were expressed as the mean+/− standard deviation (SD) and the median (range), and the categorical variables were expressed as the number (No.) and percentage (%). Correlations between variables were analyzed using Spearman's correlation tests. Kaplan-Meier and Cox regression analyses were performed to assess the relationships between the various variables and patient outcomes. The co-linearity of the various variables was determined via a linear regression. Variables found to be significant in the linear regression analysis were included in the multivariate Cox regression models, and univariate and multivariate linear or logistic regression models were used to assess the relationships between the various dependent and independent variables. Paired t-tests were used to compare the variables assessed upon ICU admission and discharge within the same individual. Based on the results of the uni-and multivariate Cox regression analyses, Kaplan-Meier curves were generated and log-rank test calculations were performed to evaluate the different RBP4 (round figures, 9-19 mg/L) and MELD score cut-off values (round figures, . The optimal cut-off values were selected based on the results of the Kaplan-Meier analyses and log-rank tests. Statistical significance was defined at the 5% level based on the results of two-tailed tests of the null hypothesis.