- Email: [email protected]
Increased serum levels of quinolinic acid indicate enhanced severity of hepatic dysfunction in patients with liver cirrhosis
Human Immunology 74 (2013) 60–66
Contents lists available at SciVerse ScienceDirect
www.ashi-hla.org
journal homepage: www.elsevier.com/locate/humimm
Increased serum levels of quinolinic acid indicate enhanced severity of hepatic dysfunction in patients with liver cirrhosis q Imad Lahdou a,1,⇑, Mahmoud Sadeghi a,1, Hani Oweira b,1, Gerhard Fusch c, Volker Daniel a, Arianeb Mehrabi b, GE. Jung b, Hazem Elhadedy b, Jan Schmidt b, Flavius Sandra-Petrescu a, Mircea Iancu a, Gerhard Opelz a, Peter Terness a, Joerg C. Schefold d a
Department of Transplantation Immunology, University of Heidelberg, Germany Department of Visceral Surgery, University of Heidelberg, Heidelberg, Germany Department of Paediatrics, McMaster University, Hamilton, Canada d Department of Nephrology and Intensive Care, Charité University Medicine, Campus Virchow Clinic, Berlin, Germany b c
a r t i c l e
i n f o
Article history: Received 8 June 2012 Accepted 10 September 2012 Available online 6 October 2012
a b s t r a c t Background: The Model for End-Stage Liver Disease (MELD) score is a tool for assessment of the degree of hepatic insufficiency/failure. Quinolinic acid (QuinA) is a tryptophan metabolite produced by activated macrophages. Here we investigate whether the degree of systemic inflammation (QuinA, neopterin, CRP and IL-6) correlates with clinical liver dysfunction according to the MELD Score. Method: Ninety-four patients with liver cirrhosis were categorized into 2 groups according to baseline MELD score (group I, MELD <20, n = 61, and group II, MELD P20, n = 33). Results: Serum levels of QuinA, neopterin, CRP, and IL-6 significantly correlated with MELD score (r = 0.77, 0.75, 0.57, and 0.50; p < 0.0001, respectively). Patients of group II had significantly higher serum levels of QuinA, neopterin, CRP, and IL-6 than group I (p 6 0.0001). ROC curve analysis showed that QuinA and neopterin are more sensitive markers for severity of liver disease than established markers of inflammation such as CRP and IL-6 (sensitivity = 86% and 79%, respectively) (AUC = 0.89 and 0.89, respectively). QuinA provided the most sensitive index with regard to the identification of patients with hepatic encephalopathy. Conclusion: Serum levels of QuinA reflect the degree of liver dysfunction. Moreover, high levels of QuinA may serve as a sensitive indicator of hepatic encephalopathy. Ó 2012 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
1. Introduction The Model for End-Stage Liver Disease (MELD) score is used as a prognostic measure for hepatic insufficiency/failure and includes assessment of the patients’ serum creatinine, serum bilirubin, and international normalized ratio (INR) for prothrombin time [1,2]. The MELD score was shown to predict both short- and medium-term survival of patients with cirrhosis of the liver [1]. q Imad Lahdou designed the study, prepared the samples, wrote the manuscript, and cooperated in management and performance of the tests and Mahmoud Sadeghi assisted in study design, in writing the manuscript, and analyzed the data. G. Fusch and Joerg C. Schefold measured tryptophan metabolites. Joerg C. Schefold, Volker Daniel, Mircea Iancu, Flavius Sandra-Petrescu, Gerhard Opelz, and Peter Terness assisted in writing the manuscript. Hani Oweira treated the patients and assisted in study design and in writing the manuscript. Jan Schmitt, Hazem Elhadedy and Arianeb Mehrabi treated the patients. ⇑ Corresponding author. Address: Institute of Immunology, Department of Transplantation Immunology, University of Heidelberg, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany. Fax: +49 6221 564049. E-mail address: [email protected] (I. Lahdou). 1 Authors who contributed equally to this work.
Recent studies suggest that the MELD score predicts survival of patients with liver cirrhosis even more accurately than Child-Pugh category [2,3]. Moreover, the MELD score predicts perioperative morbidity and mortality in patients with End-Stage Liver Disease [4]. Importantly, based on Wiesner’s report, the mortality of patients in the ensuing 3 months is significantly increased if the MELD score is over 20 [2]. C-reactive protein (CRP), interleukin (IL)-6 and neopterin are well-known inflammatory markers [5–7]. Assessment of CRP as an acute-phase reactant is widely performed in daily practice. Serum levels of CRP have been found elevated in patients with cirrhotic liver diseases [8]. Bota et al. showed no differences in CRP concentrations among patients with cirrhosis with different disease severity as assessed on the basis of the Child-Pugh category [9]. Other data show that CRP is not significantly correlated with encephalopathy scores in cirrhotic patients [10]. Serum level of IL-6 has been demonstrated to increase during inflammation [11]. IL-6 had significant correlations with psychometric test scores in cirrhotic patients [10,12]. Higher levels of IL-6 are due in part to impaired hepatic removal of IL-6 [13].
0198-8859/$36.00 - see front matter Ó 2012 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics. http://dx.doi.org/10.1016/j.humimm.2012.09.009
I. Lahdou et al. / Human Immunology 74 (2013) 60–66
Lemmers et al. found increased levels of IL-6 with the stage of chronic liver disease and significant correlation with disease severity [14]. Neopterin, a pteridine mainly synthesized by activated macrophages, is a marker of inflammation and immune system activation [15]. It was shown that production and release of neopterin is inducible in human monocytes/macrophages by interferon gamma (IFNc) [6]. Goris considered neopterin concentration along with other parameters as a tool for inflammation monitoring [16]. High serum levels of neopterin were observed in patients with cirrhosis and also in patients with chronic hepatitis (B and C) [17,18]. Increased serum neopterin levels over 19.15 nmol/l are associated with higher mortality rates in patients with alcoholic liver disease [19]. Based on the low sensitivity and specificity of Interleukin-6 and CRP as inflammatory markers in decompensated cirrhotic patients, a more sensitive test would be desirable [20]. Quinolinic acid (QuinA) is an excitotoxic kynurenine pathway metabolite of tryptophan [21–23]. The kynurenine metabolic pathway is catalyzed by two enzymes, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO). QuinA levels in the bloodstream and numerous tissues rise sharply after immune stimulation [24] and monocytic cells are known to be the major producers of QuinA. Macrophages have the ability to produce approximately 20–30-fold more QuinA than microglia [25]. The neurotoxic capacity of QuinA in vivo has implicated it in a variety of neurodegenerative disorders, including Huntington’s disease [23,26,27] and Alzheimer’s disease [28]. Moreover, QuinA is increased in clinical conditions such as renal dysfunction [29] and sepsis [30–32]. We investigated whether QuinA and other serum inflammatory biomarkers are associated with severity of disease in patients with hepatic cirrhosis. The sensitivity and specificity of these markers of immune activation and systemic inflammation was assessed.
61
and 2 patients in group 2 (p = 0.074) obtained immmunosuppressive drugs such as tacrolimus, cyclosporine A and prednisolone. Serum levels of QuinA, neopterin, IL-6 and CRP were similar (p = n.s.) in patients with and without immunosuppressive drugs. Fifty healthy volunteers (26 males, mean ± SD 40.5 ± 11.2 years) served as controls. Controls were free of infectious disease. Demographic details of the study groups are depicted in Table 1a. The local ethics committee approved the study and written informed consent was obtained from all study patients. 3. Methods 3.1. Serum separation Serum was collected after blood clotting in a serum separator tube by centrifugation at 4000 rpm for 15 min at 4 °C. The serum was snap frozen and stored at 20 °C until testing. All serum samples were thawed only once. 3.2. Determination of serum quinolinic acid Assessment of tryptophan catabolite quinolinic acid was performed using tandem mass spectrometry as described elsewhere [29,31]. 3.3. Serum cytokine assay with multiplex analysis system IL-6 (lower detection limit: 3 pg/ml) was determined using a Multiplex assay (R&D system, Wiesbaden, Germany) which uses multi-analyte profiles (MAPs) based on Luminex xMAPÒ (Luminex Corporation, Austin TX) technology to discover biomarker patterns within very small sample volumes. 3.4. Determination of serum CRP
2. Patients and methods 2.1. Patients 2.1.1. Characteristics of study population Concentrations of quinolinic acid (QuinA), neopterin, IL-6 and Creactive protein (CRP) were measured in serum samples of 94 cirrhotic patients (69 males) with different disease severity according to the Model for End-Stage Liver Disease (MELD) score [1,2]. Patients were evaluated as inpatients for potential liver transplantation at the Department of Gastroenterology at the University of Heidelberg, Germany, and all patients received a liver transplant in the years 2008 or 2009. According to the MELD score, patients were grouped into 2 cohorts: patients with MELD score <20 were considered as patients with ‘‘moderate’’ (study group I: n = 61) and those with MELD score P20 as patients with ‘‘severe’’ liver dysfunction (study group II: n = 33) (Table 1a). According to the ChildPugh category, the patients were classified in 3 cohorts (Child A, B and C) (Table 1b). The following etiologies of liver failure applied: 31 patients were diagnosed with hepatitis C and/or B, 35 with alcoholic liver cirrhosis, and 28 showed evidence of other pathologies including congenital and autoimmune liver disease. Forty-three patients were diagnosed with hepatic encephalopathy (termed enceph+ in tables and figures). Eighty patients received 0–8 (median 5 in group 1 and 6 in group 2) different drugs including diuretics, beta blockers, proton pump inhibitors, antihypertensives, ACE inhibitors and various supplements (vitamins and minerals). The remaining 14 patients (3 patients in group 1 and 11 patients in group 2: p = 0.0004) did not receive any drugs. Distribution of drugs was similar in the two groups. Eleven patients in group 1
Serum CRP was assessed in a certified laboratory at the Heidelberg University Hospital. 3.5. Determination of serum neopterin Serum neopterin was measured with the Neopterin ELISA kits (Brahms, Berlin, Germany). Based on control measurements in 70 healthy individuals, >15 nmol/l was considered abnormally high. 3.6. Statistical analysis Mann–Whitney-U test, v2 and Fisher exact test were applied using the Statistical Package for the Social Sciences (SPSS, Chicago, USA). p Values of 60.05 were considered significant and are bold printed in table and figures. Spearman rank correlation test was used to determine associations between variables, receiver operating characteristic (ROC) curve analyses were performed to determine the diagnostic sensitivity and specificity of parameters, whereas multivariate parametric analysis was done in order to assess the relationship between the clinical characteristics and prognostic parameters for patients. 4. Results 4.1. Comparison of liver panel tests using MELD score and Child-Pugh category Common liver panel tests such as Albumine, Bilirubin, International normalized ratio (INR), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Gam-
62
I. Lahdou et al. / Human Immunology 74 (2013) 60–66
Table 1a Demographic and clinical characteristics of the study population (According to MELD score). Parameters (normal range)
All patients (n = 94)
Group I MELD <20 (n = 61)
Group II MELD P20 (n = 33)
p
HCs (n = 50)
Age (mean ± SD years) Male gender (n) Albumin (g/L) (30–50) INR ( 1.2) Bilirubine (mg/dL) ( 1.0) Creatinine (mg/dL) (0.1–1.3) ALT (u/L) ( 50) AST (u/L) ( 50) ALP (u/L) (40–130) GGT (u/L) ( 60) HBVAb+ (n) HCVAb+ (n) Hepatic encephalopathy (n)
52.3 ± 10.5 69 31 ± 7.3 1.4 ± 0.6 8.0 ± 8.7 1.5 ± 0.9 240 ± 481 280 ± 672 181 ± 191 157 ± 11 22 43
52.6 ± 11.0 45 32 ± 8.0 1.2 ± 0.1 3.6 ± 3.9 1.1 ± 0.2 218 ± 517 300 ± 552 196 ± 226 157 ± 130 9 17 22
51.9 ± 9.6 24 29 ± 5.0 1.8 ± 0.9 16.0 ± 9.0 2.0 ± 1.3 281 ± 411 526 ± 841 152 ± 91 155 ± 315 2 5 21
0.53 0.66 0.02 <0.001 <0.001 <0.001 0.24 0.86 0.98 0.03 0.41 0.16 0.01
40.5 ± 11.2 26 – – – – – – – – – – –
Original disease Congenital/autoimmune (n) Alcoholic (n) Viral (n)
28 35 31
18 19 24
10 16 7
0.94 0.50 0.07
– – –
Mann–Whitney test v2 or Fisher exact test were used.
Table 1b Demographic and clinical characteristics of the study population (According to the Child-Pugh category). Parameters (normal range)
All patients (n = 94)
Child A (n = 32)
Child B (n = 27)
Child C (n = 35)
p A vs. B
p A vs. C
p B vs. C
Age (mean ± SD years) Male gender (n) Albumin (g/L) (30–50) INR ( 1.2) Bilirubine (mg/dL) ( 1.0) Creatinine mg/dL) (0.1–1.3) ALT (u/L) ( 50) AST (u/L) ( 50) ATP (u/L) (40–130) GGT (u/L) ( 60) HBVAb+ (n) HCVAb+ (n) Hepatic encephalopathy (n)
52.3 ± 10.5 69 31 ± 7.1 1.4 ± 0.6 8 ± 8.7 1.3 ± 1 240 ± 481 380 ± 672 181 ± 191 157 ± 220 11 22 43
54,3 ± 8.8 25 35.8 ± 7.7 1.2 ± 0.1 1.73 ± 1.5 0.95 ± 0.4 278 ± 678 342 ± 711 193 ± 257 175 ± 150 6 7 4
53 ± 11.6 19 29.9 ± 4.6 1.5 ± 0.5 10.1 ± 9 1.2 ± 0.6 157 ± 186 250 ± 349 178 ± 169 148 ± 182 2 9 11
50 ± 10.3 25 27.5 ± 5.8 1.63 ± 0.8 12 ± 9.1 1.8 ± 1.3 269.4 ± 399 515 ± 804 172 ± 133 146 ± 294 3 6 28
0.9 0.49 0.003 0.001 <0.001 0.2 0.9 0.2 0.7 0.05 0.27 0.32 0.013
0.07 0.53 <0.001 <0.001 <0.001 0.001 0.3 0.01 0.8 0.002 0.29 0.64 <0.001
0.2 0.93 0.09 0.2 0.2 0.07 0.5 0.13 0.5 0.5 1.00 0.14 0.002
Mann–Whitney test, Kruskal–Wallis-test, v2 or Fisher exact test were used.
ma-glutamyl transferase (GGT) were assessed in a certified hospital laboratory in patients’ sera. Analysis was performed to screen the degree of liver dysfunction (Table 1). Liver enzymes such as ALT, AST, ALP, GGT and serum albumin were similar in the two MELD groups (Table 1a). As expected, patients in the Child A category had significantly higher serum albumin and lower INR, bilirubine, and creatinine levels than patients in category C (Table 1b). 4.2. Correlation of liver dysfunction with tryptophan metabolite QuinA and inflammatory markers A pronounced inflammatory response in patients with End-Stage Liver Disease was noted. The tryptophan metabolite QuinA and markers of inflammation (CRP, IL-6, neopterin) were all found to correlate with the established clinical indices MELD score and ChildPugh category. In detail, the MELD score positively correlated with serum levels of QuinA (r = 0.77), neopterin (r = 0.75), CRP (r = 0.57), and IL-6 (r = 0.50) (p < 0.0001 for all investigations) (Fig. 1a–d). Child-Pugh categories positively correlated with serum QuinA, neopterin, CRP, and IL-6 as well (p < 0.0001 for all investigations). 4.3. Elevated inflammatory markers in patients with End-Stage Liver Disease (ESLD) compared to healthy controls Patients with severe liver dysfunction (group II) had significantly higher QuinA levels than healthy controls (p < 0.0001),
whereas in patients with moderate liver dysfunction the QuinA levels were lower than in healthy individuals (p = 0.03) (Fig. 2a). In patients with severe or moderate liver dysfunction, significantly higher serum levels of neopterin, CRP, and IL-6 were noted when compared to controls (all p 6 0.0001) (Fig. 2b–d) indicating a pronounced inflammatory response. With advancing liver dysfunction, the levels were more increased (p 6 0.0001; group I vs. group II) (Fig. 2a–d), showing a strong association of inflammatory parameters with severity of disease.
4.4. Sensitivity and specificity of inflammatory parameters in patients with ESLD A receiver operating curve (ROC) analysis was performed to calculate the sensitivity and specificity of the assessed markers. Although all markers were found rather sensitive, statistical analysis showed that QuinA and neopterin had the highest sensitivity and specificity for identification of patients with advanced liver failure (group II). When discriminating group II from group I patients, the areas under curve (AUC) were 0.89, 0.89, 0.73, and 0.74, for QuinA, neopterin, CRP, and IL-6, respectively (p 6 0.0002 for all investigations, sensitivity = 86%, 79%, 76%, 76%, respectively) (Fig. 3a). When sensitivity and specificity of markers was calculated based on Child-Pugh category C vs A + B, AUC were 0.75, 0.73, 0.68, and 0.68 for QuinA, neopterin, CRP, and IL-6, respectively
I. Lahdou et al. / Human Immunology 74 (2013) 60–66
63
Fig. 1. MELD score correlates with serum levels of QuinA and other inflammatory markers. MELD scores of 94 patients with cirrhosis were correlated with serum concentration of QuinA (a), neopterin (b), CRP (c), and IL-6 (d). Spearman rank correlation test was used for statistical calculation.
(p 6 0.003 for all investigations) (Fig. 3b). These results indicate that the tryptophan metabolite ‘‘QuinA’’ is a sensitive marker of severity of disease in patient with ESLD (Fig. 3a and b). 4.5. Relationship between severity of liver disease and QuinA in multivariate parametric analysis Although CRP (95% CI; 22–38 mg/L: p = 0.02) and IL-6 (95% CI; 33–170 pg/ml/L: p = 0.04) significantly correlated with MELD score, statistical analysis showed that QuinA (95% CI; 1.38–1.87: p < 0.0001) and neopterin (95% CI; 48–70 lmol/L: p < 0.0001) had the highest correlations. The results again confirm that QuinA is a distinguished marker in patients with ESLD. 4.6. Higher inflammatory markers in patients with ESLD and hepatic encephalopathy We also investigated whether the assessed inflammatory markers correlate with the presence of hepatic encephalopathy. Both the patients groups with and without hepatic encephalopathy had significantly higher serum levels of neopterin, CRP, and IL-6 when compared to healthy controls (HCs; p < 0.0001 for all investigations) (Fig. 4b and d). Patients with ESLD who did not show clinical signs of encephalopathy (enceph ) had similar QuinA serum levels when compared to HCs, whereas patients with encephalopathy (enceph+) had significantly higher QuinA levels than patients without encephalopathy (p = 0.003) and HCs
(p 6 0.0001) (Fig. 4a). The levels of all indices were significantly higher in patients with encephalopathy than in patients without encephalopathy (p 6 0.001 for all investigations) (Fig. 4a–d). These results indicate that QuinA also serves as a sensitive marker for hepatic encephalopathy. The potential mechanism of mediation of this disturbance remains speculative and requires further analysis in future studies. 5. Discussion The objective of the present study was to investigate whether increased serum levels of activation and inflammatory markers correlate with the degree of clinical liver failure. Enhanced serum levels of the inflammatory markers CRP and IL-6 and increased activation of cellular immunity as assessed by neopterin were noted in patients with liver dysfunction/failure. Interestingly, QuinA, a downstream breakdown product of tryptophan (Trp) along the kynurenine pathyway, was also increased in patients with severe liver dysfunction. To the best of our knowledge, this is the first description of increased (systemic) serum levels of the kynurenine pathway catabolite QuinA in patients with advanced liver dysfunction. From a clinical perspective, this is of interest because kynurenine pathway catabolites are well-known inducers of neurological dysfunction, neuropathy and encephalopathy [21,27,33]. Here, we demonstrate strongly increased serum levels of QuinA, the most potent inducer of neuropathy. Induction of QuinA in the cohort of patients investigated here is of particular
64
I. Lahdou et al. / Human Immunology 74 (2013) 60–66
Fig. 2. Increased serum QuinA levels in patients with severe liver cirrhosis. Based on MELD score ninety-four patients with cirrhosis were grouped in 2 cohorts: patients with MELD score <20 were considered as moderate liver dysfunction (study group I: n = 61) and MELD score P20 as severe liver dysfunction (study group II: n = 33). Fifty healthy volunteers served as controls (HCs). The difference between the medians of parameters in the two groups and HCs are showed by boxplots; (a) QuinA, (b) neopterin, (c) CRP, and (d) IL-6. The p Values were calculated using the Mann–Whitney U test.
interest since hepatic failure/dysfunction is often accompanied by hepatic encephalopathy (HE), which was shown to have a profound impact on the patients´ prognosis [34]. Evaluation of systemic levels of ammonia, which is often used as a marker to assess the degree of HE [35], was not done in the present study whereas our results suggest that QuinA contributes to the development and progression of HE, this remains to be proven in subsequent studies. Most strikingly, when sensitivity/specificity analyses were performed, QuinA was the marker most closely related with the degree of liver failure (according to the MELD scoring system). This indicates that liver dysfunction or failure is associated with a strongly activated catabolic pathway of Trp degradation, which in turn may enhance and support the inflammatory reaction often observed in this cohort of patients. Degradation of the essential amino acid Trp with resulting increased serum levels of the respective catabolites is a consequence of IDO activation [36]. Macrophages stimulated with IFN-c or lipopolysaccharide (LPS) upregulate IDO and increase their QuinA production [37,38]. While the exact mechanism of IDO upregulation in hepatic dysfunction/ failure remains to be elucidated, one might speculate that mechanisms including LPS activation, possibly via edema-induced altered gut barrier dysfunction, may play a role [39]. An enzyme with identical activity to IDO is tryptophan 2,3dioxygenase (TDO). This is a hepatic enzyme which oxidates L-tryptophan and is induced in clinical situations associated with increased endogenous glucocorticoid levels [40]. Although the role of TDO remains unclear in the clinical setting of liver dysfunction,
it might be theoretically responsible for increased levels of QuinA as described in this study. Another interesting aspect of this study is that besides QuinA, neopterin, which are released by activated macrophages in response to infectious and non-infectious inflammatory stimuli [6], was found to be increased in the patient cohort. QuinA and neopterin exceeded CRP and IL-6 in terms of sensitivity and specificity for hepatic dysfunction/failure. This probably indicates activation of cellular immunity and subsequent systemic release of QuinA and neopterin in patients with severe liver dysfunction. We recognize that our study has limitations. Serum levels of QuinA were shown increased also in other clinical situations such as in chronic renal dysfunction/failure [29]. Although no data exist on whether this may also be the case in acute (ARF) or acute-onchronic renal dysfunction, one might speculate that serum levels of these catabolites are increased in these acute clinical conditions too. Since the patients in MELD group II were found to have significantly higher serum creatinine levels when compared to group I, renal dysfunction might have influenced our data. Although we are unable to definitely rule out an influence of renal dysfunction in our analysis, we found that the data were unchanged after we corrected for systemic creatinine levels (there is no significant correlation between QuinA/Cr ratio and MELD score p = 0.10). We also investigated serum levels of QuinA in patients of Group II who had serum Cr 61.3 mg /dl (n = 12) or Cr >1.3 mg/dl (n = 21) respectively. Cr levels did not significantly correlate with QuinA levels (p = 0.38).
I. Lahdou et al. / Human Immunology 74 (2013) 60–66
65
Fig. 3. (a) Predicting value of QuinA for severity of liver disease according to MELD scores. Receiver operating characteristic (ROC) Curve analysis of MELD score was performed to assess the area under the curve (AUC), sensitivity and specificity for QuinA, neopterin, CRP, and IL-6 between patients with moderate (group I: n = 61) and severe liver dysfunction (group II: n = 33); (b) predicting value of QuinA for severity of liver disease according to Child-Pugh category. Receiver operating characteristic (ROC) Curve analysis of Child-Pugh category was performed to assess area under the curve (AUC), sensitivity and specificity for QuinA, neopterin, CRP, and IL-6 between patients with severe (Child-Pugh category C: n = 35) versus moderate liver dysfunction (Child-Pugh categories A + B: n = 59).
Fig. 4. Elevated serum levels of QuinA as marker of hepatic encephalopathy. Patients with liver cirrhosis were divided in two cohorts: patients who did not show clinical signs of encephalopathy (enceph : n = 51), and patients with encephalopathy (enceph+, n = 43). Fifty healthy volunteers used as controls (HCs). The difference between the medians of parameters in the two groups and HCs showed by boxplots; (a) QuinA, (b) neopterin, (c) CRP, and (d) IL-6. The p Values were calculated using the Mann–Whitney U test.
In summary, the degree of liver dysfunction/failure according to the MELD scoring system correlates with markers of inflammation
and activation of cellular immunity. We found increased serum levels of QuinA in cirrhotic patients with advanced liver dysfunc-
66
I. Lahdou et al. / Human Immunology 74 (2013) 60–66
tion and may reflect the severity of this clinical condition. We hypothesize that QuinA may contribute to the development of HE, which is of major clinical importance in patients with liver dysfunction/failure. We concluded that hepatic dysfunction is associated with pathophysiological reactions including activation of the inflammatory cascade. Our data indicate for the first time that increased serum levels of QuinA and neopterin reflect the degree of hepatic dysfunction/failure as assessed using the established MELD score. We speculate that QuinA induction may be a contributor to the development of hepatic encephalopathy, which is often observed in these patients. Acknowledgements We would like to acknowledge the skillful technical assistance of Martina Kutsche-Bauer and Regina Seemuth. References [1] Kamath PS, Wiesner RH, Malinchoc M, Kremers W, Therneau TM, Kosberg CL, et al. A model to predict survival in patients with End-Stage Liver Disease. Hepatology 2001;33(2):464. [2] Wiesner R, Edwards E, Freeman R, Harper A, Kim R, Kamath P, et al. Model for End-Stage Liver Disease (MELD) and allocation of donor livers. Gastroenterology 2003;124(1):91. [3] Huo TI, Lee SD, Lin HC. Selecting an optimal prognostic system for liver cirrhosis: the Model for End-Stage Liver Disease and beyond. Liver Int 2008;28(5):606. [4] Xia VW, Taniguchi M, Steadman RH. The changing face of patients presenting for liver transplantation. Curr Opin Organ Transplant 2008;13(3):280. [5] Jawa RS, Anillo S, Huntoon K, Baumann H, Kulaylat M. Interleukin-6 in surgery, trauma, and critical care part II: clinical implications. J Intensive Care Med 2011;26(2):73. [6] Fuchs D, Weiss G, Reibnegger G, Wachter H. The role of neopterin as a monitor of cellular immune activation in transplantation, inflammatory, infectious, and malignant diseases. Crit Rev Clin Lab Sci 1992;29(3–4):307. [7] Lavie CJ, Church TS, Milani RV, Earnest CP. Impact of physical activity, cardiorespiratory fitness, and exercise training on markers of inflammation. J Cardiopulm Rehabil Prev 2011;31(3):137. [8] Tilg H, Wilmer A, Vogel W, Herod M, Nölchen B, Judmaier G, et al. Serum levels of cytokines in chronic liver diseases. Gastroenterology 1992;103(1):264. [9] Bota DP, Van Nuffelen M, Zakariah AN, Vincent JL. Serum levels of C-reactive protein and procalcitonin in critically ill patients with cirrhosis of the liver. J Lab Clin Med 2005;146(6):347. [10] Montagnese S, Biancardi A, Schiff S, Carraro P, Carlà V, Mannaioni G, et al. Different biochemical correlates for different neuropsychiatric abnormalities in patients with cirrhosis. Hepatology 2011;53(2):558. [11] Ito H. IL-6 and Crohn’s disease. Curr Drug Targets Inflamm Allergy 2003;2(2):125. [12] Montoliu C, Piedrafita B, Serra MA, del Olmo JA, Urios A, Rodrigo JM, et al. IL-6 and IL-18 in blood may discriminate cirrhotic patients with and without minimal hepatic encephalopathy. J Clin Gastroenterol 2009;43(3):272. [13] Wiest R, Weigert J, Wanninger J, Neumeier M, Bauer S, Schmidhofer S, et al. Impaired hepatic removal of interleukin-6 in patients with liver cirrhosis. Cytokine 2011;53(2):178. [14] Lemmers A, Gustot T, Durnez A, Evrard S, Moreno C, Quertinmont E, et al. An inhibitor of interleukin-6 trans-signalling, sgp130, contributes to impaired acute phase response in human chronic liver disease. Clin Exp Immunol 2009;156(3):518. [15] De Rosa S, Cirillo P, Pacileo M, Petrillo G, D’Ascoli GL, Maresca F, et al. Neopterin: from forgotten biomarker to leading actor in cardiovascular pathophysiology. Curr Vasc Pharmacol 2011;9(2):188. [16] Goris RJ. Multiple organ failure: whole body inflammation? Schweiz Med Wochenschr 1989;119(11):347. [17] Gulcan EM, Tirit I, Anil A, Adal E, Ozbay G. Serum neopterin levels in children with hepatitis-B-related chronic liver disease and its relationship to disease severity. World J Gastroenterol 2008;14(44):6840.
[18] Zwirska-Korczala K, Dziambor AP, Wiczkowski A, Berdowska A, Gajewska K, Stolarz W. Hepatocytes growth factor (HGF), leptin, neopterin serum concentrations in patients with chronic hepatitis C. Przegl Epidemiol 2001;55(Suppl. 3):164. [19] Gonzalez-Reimers E, Santolaria-Fernandez F, Rodriguez-Rodriguez E, Rodríguez-Moreno F, Martínez-Riera A, Milena-Abril A, et al. Serum neopterin levels in alcoholic liver disease. Drug Alcohol Depend 1993;33(2):151. [20] Connert S, Stremmel W, Elsing C. Procalcitonin is a valid marker of infection in decompensated cirrhosis. Z Gastroenterol 2003;41(2):165. [21] Heyes MP. Quinolinic acid and inflammation. Ann N Y Acad Sci 1993;679:211. [22] Sadeghi M, Lahdou I, Daniel V, Schnitzler P, Fusch G, Schefold JC, et al. Strong association of phenylalanine and tryptophan metabolites with activated cytomegalovirus infection in kidney transplant recipients. Hum Immunol 2012;73(2):186–92. [23] Schwarcz R, Foster AC, French ED, Whetsell Jr WO, Kohler C. Excitotoxic models for neurodegenerative disorders. Life Sci 1984;35(1):19. [24] Saito K, Markey SP, Heyes MP. Effects of immune activation on quinolinic acid and neuroactive kynurenines in the mouse. Neuroscience 1992;51(1):25. [25] Guillemin GJ, Smith DG, Smythe GA, Armati PJ, Brew BJ. Expression of the kynurenine pathway enzymes in human microglia and macrophages. Adv Exp Med Biol 2003;527:105. [26] Stone TW. Endogenous neurotoxins from tryptophan. Toxicon 2001;39(1):61. [27] Beal MF, Matson WR, Swartz KJ, Gamache PH, Bird ED. Kynurenine pathway measurements in Huntington’s disease striatum: evidence for reduced formation of kynurenic acid. J Neurochem 1990;55(4):1327. [28] Widner B, Leblhuber F, Walli J, Tilz GP, Demel U, Fuchs D. Tryptophan degradation and immune activation in Alzheimer’s disease. J Neural Transm 2000;107(3):343. [29] Schefold JC, Zeden JP, Fotopoulou C, von Haehling S, Pschowski R, Hasper D, et al. Increased indoleamine 2,3-dioxygenase (IDO) activity and elevated serum levels of tryptophan catabolites in patients with chronic kidney disease: a possible link between chronic inflammation and uraemic symptoms. Nephrol Dial Transplant 2009;24(6):1901. [30] Schefold JC, Bierbrauer J, Weber-Carstens S. Intensive care unit-acquired weakness (ICUAW) and muscle wasting in critically ill patients with severe sepsis and septic shock. J Cachexia Sarcopenia Muscle 2010;1(2):147. [31] Schefold JC, Zeden JP, Pschowski R, Hammoud B, Fotopoulou C, Hasper D, et al. Treatment with granulocyte-macrophage colony-stimulating factor is associated with reduced indoleamine 2,3-dioxygenase activity and kynurenine pathway catabolites in patients with severe sepsis and septic shock. Scand J Infect Dis 2010;42(3):164. [32] Zeden JP, Fusch G, Holtfreter B, Schefold JC, Reinke P, Domanska G, et al. Excessive tryptophan catabolism along the kynurenine pathway precedes ongoing sepsis in critically ill patients. Anaesth Intensive Care 2010;38(2):307. [33] Chiarugi A, Cozzi A, Ballerini C, Massacesi L, Moroni F. Kynurenine 3-monooxygenase activity and neurotoxic kynurenine metabolites increase in the spinal cord of rats with experimental allergic encephalomyelitis. Neuroscience 2001;102(3):687. [34] Moroni F, Lombardi G, Carla V, Lal S, Etienne P, Nair NP. Increase in the content of quinolinic acid in cerebrospinal fluid and frontal cortex of patients with hepatic failure. J Neurochem 1986;47(6):1667. [35] Toris GT, Bikis CN, Tsourouflis GS, Theocharis SE. Hepatic encephalopathy: an updated approach from pathogenesis to treatment. Med Sci Monit 2011;17(2):RA53. [36] Kwidzinski E, Bechmann I. IDO expression in the brain: a double-edged sword. J Mol Med (Berl) 2007;85(12):1351. [37] Guillemin GJ, Smythe GA, Veas LA, Takikawa O, Brew BJ. A beta 1–42 induces production of quinolinic acid by human macrophages and microglia. Neuroreport 2003;14(18):2311. [38] Heyes MP, Saito K, Major EO, Milstien S, Markey SP, Vickers JH. A mechanism of quinolinic acid formation by brain in inflammatory neurological disease. Attenuation of synthesis from L-tryptophan by 6-chlorotryptophan and 4chloro-3-hydroxyanthranilate. Brain 1993;116(Pt 6):1425. [39] Basile AS, Saito K, Li Y, Heyes MP. The relationship between plasma and brain quinolinic acid levels and the severity of hepatic encephalopathy in animal models of fulminant hepatic failure. J Neurochem 1995;64(6):2607. [40] Oxenkrug GF. Tryptophan kynurenine metabolism as a common mediator of genetic and environmental impacts in major depressive disorder: the serotonin hypothesis revisited 40 years later. Isr J Psychiatry Relat Sci 2010;47(1):56.
Contents lists available at SciVerse ScienceDirect
www.ashi-hla.org
journal homepage: www.elsevier.com/locate/humimm
Increased serum levels of quinolinic acid indicate enhanced severity of hepatic dysfunction in patients with liver cirrhosis q Imad Lahdou a,1,⇑, Mahmoud Sadeghi a,1, Hani Oweira b,1, Gerhard Fusch c, Volker Daniel a, Arianeb Mehrabi b, GE. Jung b, Hazem Elhadedy b, Jan Schmidt b, Flavius Sandra-Petrescu a, Mircea Iancu a, Gerhard Opelz a, Peter Terness a, Joerg C. Schefold d a
Department of Transplantation Immunology, University of Heidelberg, Germany Department of Visceral Surgery, University of Heidelberg, Heidelberg, Germany Department of Paediatrics, McMaster University, Hamilton, Canada d Department of Nephrology and Intensive Care, Charité University Medicine, Campus Virchow Clinic, Berlin, Germany b c
a r t i c l e
i n f o
Article history: Received 8 June 2012 Accepted 10 September 2012 Available online 6 October 2012
a b s t r a c t Background: The Model for End-Stage Liver Disease (MELD) score is a tool for assessment of the degree of hepatic insufficiency/failure. Quinolinic acid (QuinA) is a tryptophan metabolite produced by activated macrophages. Here we investigate whether the degree of systemic inflammation (QuinA, neopterin, CRP and IL-6) correlates with clinical liver dysfunction according to the MELD Score. Method: Ninety-four patients with liver cirrhosis were categorized into 2 groups according to baseline MELD score (group I, MELD <20, n = 61, and group II, MELD P20, n = 33). Results: Serum levels of QuinA, neopterin, CRP, and IL-6 significantly correlated with MELD score (r = 0.77, 0.75, 0.57, and 0.50; p < 0.0001, respectively). Patients of group II had significantly higher serum levels of QuinA, neopterin, CRP, and IL-6 than group I (p 6 0.0001). ROC curve analysis showed that QuinA and neopterin are more sensitive markers for severity of liver disease than established markers of inflammation such as CRP and IL-6 (sensitivity = 86% and 79%, respectively) (AUC = 0.89 and 0.89, respectively). QuinA provided the most sensitive index with regard to the identification of patients with hepatic encephalopathy. Conclusion: Serum levels of QuinA reflect the degree of liver dysfunction. Moreover, high levels of QuinA may serve as a sensitive indicator of hepatic encephalopathy. Ó 2012 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
1. Introduction The Model for End-Stage Liver Disease (MELD) score is used as a prognostic measure for hepatic insufficiency/failure and includes assessment of the patients’ serum creatinine, serum bilirubin, and international normalized ratio (INR) for prothrombin time [1,2]. The MELD score was shown to predict both short- and medium-term survival of patients with cirrhosis of the liver [1]. q Imad Lahdou designed the study, prepared the samples, wrote the manuscript, and cooperated in management and performance of the tests and Mahmoud Sadeghi assisted in study design, in writing the manuscript, and analyzed the data. G. Fusch and Joerg C. Schefold measured tryptophan metabolites. Joerg C. Schefold, Volker Daniel, Mircea Iancu, Flavius Sandra-Petrescu, Gerhard Opelz, and Peter Terness assisted in writing the manuscript. Hani Oweira treated the patients and assisted in study design and in writing the manuscript. Jan Schmitt, Hazem Elhadedy and Arianeb Mehrabi treated the patients. ⇑ Corresponding author. Address: Institute of Immunology, Department of Transplantation Immunology, University of Heidelberg, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany. Fax: +49 6221 564049. E-mail address: [email protected] (I. Lahdou). 1 Authors who contributed equally to this work.
Recent studies suggest that the MELD score predicts survival of patients with liver cirrhosis even more accurately than Child-Pugh category [2,3]. Moreover, the MELD score predicts perioperative morbidity and mortality in patients with End-Stage Liver Disease [4]. Importantly, based on Wiesner’s report, the mortality of patients in the ensuing 3 months is significantly increased if the MELD score is over 20 [2]. C-reactive protein (CRP), interleukin (IL)-6 and neopterin are well-known inflammatory markers [5–7]. Assessment of CRP as an acute-phase reactant is widely performed in daily practice. Serum levels of CRP have been found elevated in patients with cirrhotic liver diseases [8]. Bota et al. showed no differences in CRP concentrations among patients with cirrhosis with different disease severity as assessed on the basis of the Child-Pugh category [9]. Other data show that CRP is not significantly correlated with encephalopathy scores in cirrhotic patients [10]. Serum level of IL-6 has been demonstrated to increase during inflammation [11]. IL-6 had significant correlations with psychometric test scores in cirrhotic patients [10,12]. Higher levels of IL-6 are due in part to impaired hepatic removal of IL-6 [13].
0198-8859/$36.00 - see front matter Ó 2012 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics. http://dx.doi.org/10.1016/j.humimm.2012.09.009
I. Lahdou et al. / Human Immunology 74 (2013) 60–66
Lemmers et al. found increased levels of IL-6 with the stage of chronic liver disease and significant correlation with disease severity [14]. Neopterin, a pteridine mainly synthesized by activated macrophages, is a marker of inflammation and immune system activation [15]. It was shown that production and release of neopterin is inducible in human monocytes/macrophages by interferon gamma (IFNc) [6]. Goris considered neopterin concentration along with other parameters as a tool for inflammation monitoring [16]. High serum levels of neopterin were observed in patients with cirrhosis and also in patients with chronic hepatitis (B and C) [17,18]. Increased serum neopterin levels over 19.15 nmol/l are associated with higher mortality rates in patients with alcoholic liver disease [19]. Based on the low sensitivity and specificity of Interleukin-6 and CRP as inflammatory markers in decompensated cirrhotic patients, a more sensitive test would be desirable [20]. Quinolinic acid (QuinA) is an excitotoxic kynurenine pathway metabolite of tryptophan [21–23]. The kynurenine metabolic pathway is catalyzed by two enzymes, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO). QuinA levels in the bloodstream and numerous tissues rise sharply after immune stimulation [24] and monocytic cells are known to be the major producers of QuinA. Macrophages have the ability to produce approximately 20–30-fold more QuinA than microglia [25]. The neurotoxic capacity of QuinA in vivo has implicated it in a variety of neurodegenerative disorders, including Huntington’s disease [23,26,27] and Alzheimer’s disease [28]. Moreover, QuinA is increased in clinical conditions such as renal dysfunction [29] and sepsis [30–32]. We investigated whether QuinA and other serum inflammatory biomarkers are associated with severity of disease in patients with hepatic cirrhosis. The sensitivity and specificity of these markers of immune activation and systemic inflammation was assessed.
61
and 2 patients in group 2 (p = 0.074) obtained immmunosuppressive drugs such as tacrolimus, cyclosporine A and prednisolone. Serum levels of QuinA, neopterin, IL-6 and CRP were similar (p = n.s.) in patients with and without immunosuppressive drugs. Fifty healthy volunteers (26 males, mean ± SD 40.5 ± 11.2 years) served as controls. Controls were free of infectious disease. Demographic details of the study groups are depicted in Table 1a. The local ethics committee approved the study and written informed consent was obtained from all study patients. 3. Methods 3.1. Serum separation Serum was collected after blood clotting in a serum separator tube by centrifugation at 4000 rpm for 15 min at 4 °C. The serum was snap frozen and stored at 20 °C until testing. All serum samples were thawed only once. 3.2. Determination of serum quinolinic acid Assessment of tryptophan catabolite quinolinic acid was performed using tandem mass spectrometry as described elsewhere [29,31]. 3.3. Serum cytokine assay with multiplex analysis system IL-6 (lower detection limit: 3 pg/ml) was determined using a Multiplex assay (R&D system, Wiesbaden, Germany) which uses multi-analyte profiles (MAPs) based on Luminex xMAPÒ (Luminex Corporation, Austin TX) technology to discover biomarker patterns within very small sample volumes. 3.4. Determination of serum CRP
2. Patients and methods 2.1. Patients 2.1.1. Characteristics of study population Concentrations of quinolinic acid (QuinA), neopterin, IL-6 and Creactive protein (CRP) were measured in serum samples of 94 cirrhotic patients (69 males) with different disease severity according to the Model for End-Stage Liver Disease (MELD) score [1,2]. Patients were evaluated as inpatients for potential liver transplantation at the Department of Gastroenterology at the University of Heidelberg, Germany, and all patients received a liver transplant in the years 2008 or 2009. According to the MELD score, patients were grouped into 2 cohorts: patients with MELD score <20 were considered as patients with ‘‘moderate’’ (study group I: n = 61) and those with MELD score P20 as patients with ‘‘severe’’ liver dysfunction (study group II: n = 33) (Table 1a). According to the ChildPugh category, the patients were classified in 3 cohorts (Child A, B and C) (Table 1b). The following etiologies of liver failure applied: 31 patients were diagnosed with hepatitis C and/or B, 35 with alcoholic liver cirrhosis, and 28 showed evidence of other pathologies including congenital and autoimmune liver disease. Forty-three patients were diagnosed with hepatic encephalopathy (termed enceph+ in tables and figures). Eighty patients received 0–8 (median 5 in group 1 and 6 in group 2) different drugs including diuretics, beta blockers, proton pump inhibitors, antihypertensives, ACE inhibitors and various supplements (vitamins and minerals). The remaining 14 patients (3 patients in group 1 and 11 patients in group 2: p = 0.0004) did not receive any drugs. Distribution of drugs was similar in the two groups. Eleven patients in group 1
Serum CRP was assessed in a certified laboratory at the Heidelberg University Hospital. 3.5. Determination of serum neopterin Serum neopterin was measured with the Neopterin ELISA kits (Brahms, Berlin, Germany). Based on control measurements in 70 healthy individuals, >15 nmol/l was considered abnormally high. 3.6. Statistical analysis Mann–Whitney-U test, v2 and Fisher exact test were applied using the Statistical Package for the Social Sciences (SPSS, Chicago, USA). p Values of 60.05 were considered significant and are bold printed in table and figures. Spearman rank correlation test was used to determine associations between variables, receiver operating characteristic (ROC) curve analyses were performed to determine the diagnostic sensitivity and specificity of parameters, whereas multivariate parametric analysis was done in order to assess the relationship between the clinical characteristics and prognostic parameters for patients. 4. Results 4.1. Comparison of liver panel tests using MELD score and Child-Pugh category Common liver panel tests such as Albumine, Bilirubin, International normalized ratio (INR), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Gam-
62
I. Lahdou et al. / Human Immunology 74 (2013) 60–66
Table 1a Demographic and clinical characteristics of the study population (According to MELD score). Parameters (normal range)
All patients (n = 94)
Group I MELD <20 (n = 61)
Group II MELD P20 (n = 33)
p
HCs (n = 50)
Age (mean ± SD years) Male gender (n) Albumin (g/L) (30–50) INR ( 1.2) Bilirubine (mg/dL) ( 1.0) Creatinine (mg/dL) (0.1–1.3) ALT (u/L) ( 50) AST (u/L) ( 50) ALP (u/L) (40–130) GGT (u/L) ( 60) HBVAb+ (n) HCVAb+ (n) Hepatic encephalopathy (n)
52.3 ± 10.5 69 31 ± 7.3 1.4 ± 0.6 8.0 ± 8.7 1.5 ± 0.9 240 ± 481 280 ± 672 181 ± 191 157 ± 11 22 43
52.6 ± 11.0 45 32 ± 8.0 1.2 ± 0.1 3.6 ± 3.9 1.1 ± 0.2 218 ± 517 300 ± 552 196 ± 226 157 ± 130 9 17 22
51.9 ± 9.6 24 29 ± 5.0 1.8 ± 0.9 16.0 ± 9.0 2.0 ± 1.3 281 ± 411 526 ± 841 152 ± 91 155 ± 315 2 5 21
0.53 0.66 0.02 <0.001 <0.001 <0.001 0.24 0.86 0.98 0.03 0.41 0.16 0.01
40.5 ± 11.2 26 – – – – – – – – – – –
Original disease Congenital/autoimmune (n) Alcoholic (n) Viral (n)
28 35 31
18 19 24
10 16 7
0.94 0.50 0.07
– – –
Mann–Whitney test v2 or Fisher exact test were used.
Table 1b Demographic and clinical characteristics of the study population (According to the Child-Pugh category). Parameters (normal range)
All patients (n = 94)
Child A (n = 32)
Child B (n = 27)
Child C (n = 35)
p A vs. B
p A vs. C
p B vs. C
Age (mean ± SD years) Male gender (n) Albumin (g/L) (30–50) INR ( 1.2) Bilirubine (mg/dL) ( 1.0) Creatinine mg/dL) (0.1–1.3) ALT (u/L) ( 50) AST (u/L) ( 50) ATP (u/L) (40–130) GGT (u/L) ( 60) HBVAb+ (n) HCVAb+ (n) Hepatic encephalopathy (n)
52.3 ± 10.5 69 31 ± 7.1 1.4 ± 0.6 8 ± 8.7 1.3 ± 1 240 ± 481 380 ± 672 181 ± 191 157 ± 220 11 22 43
54,3 ± 8.8 25 35.8 ± 7.7 1.2 ± 0.1 1.73 ± 1.5 0.95 ± 0.4 278 ± 678 342 ± 711 193 ± 257 175 ± 150 6 7 4
53 ± 11.6 19 29.9 ± 4.6 1.5 ± 0.5 10.1 ± 9 1.2 ± 0.6 157 ± 186 250 ± 349 178 ± 169 148 ± 182 2 9 11
50 ± 10.3 25 27.5 ± 5.8 1.63 ± 0.8 12 ± 9.1 1.8 ± 1.3 269.4 ± 399 515 ± 804 172 ± 133 146 ± 294 3 6 28
0.9 0.49 0.003 0.001 <0.001 0.2 0.9 0.2 0.7 0.05 0.27 0.32 0.013
0.07 0.53 <0.001 <0.001 <0.001 0.001 0.3 0.01 0.8 0.002 0.29 0.64 <0.001
0.2 0.93 0.09 0.2 0.2 0.07 0.5 0.13 0.5 0.5 1.00 0.14 0.002
Mann–Whitney test, Kruskal–Wallis-test, v2 or Fisher exact test were used.
ma-glutamyl transferase (GGT) were assessed in a certified hospital laboratory in patients’ sera. Analysis was performed to screen the degree of liver dysfunction (Table 1). Liver enzymes such as ALT, AST, ALP, GGT and serum albumin were similar in the two MELD groups (Table 1a). As expected, patients in the Child A category had significantly higher serum albumin and lower INR, bilirubine, and creatinine levels than patients in category C (Table 1b). 4.2. Correlation of liver dysfunction with tryptophan metabolite QuinA and inflammatory markers A pronounced inflammatory response in patients with End-Stage Liver Disease was noted. The tryptophan metabolite QuinA and markers of inflammation (CRP, IL-6, neopterin) were all found to correlate with the established clinical indices MELD score and ChildPugh category. In detail, the MELD score positively correlated with serum levels of QuinA (r = 0.77), neopterin (r = 0.75), CRP (r = 0.57), and IL-6 (r = 0.50) (p < 0.0001 for all investigations) (Fig. 1a–d). Child-Pugh categories positively correlated with serum QuinA, neopterin, CRP, and IL-6 as well (p < 0.0001 for all investigations). 4.3. Elevated inflammatory markers in patients with End-Stage Liver Disease (ESLD) compared to healthy controls Patients with severe liver dysfunction (group II) had significantly higher QuinA levels than healthy controls (p < 0.0001),
whereas in patients with moderate liver dysfunction the QuinA levels were lower than in healthy individuals (p = 0.03) (Fig. 2a). In patients with severe or moderate liver dysfunction, significantly higher serum levels of neopterin, CRP, and IL-6 were noted when compared to controls (all p 6 0.0001) (Fig. 2b–d) indicating a pronounced inflammatory response. With advancing liver dysfunction, the levels were more increased (p 6 0.0001; group I vs. group II) (Fig. 2a–d), showing a strong association of inflammatory parameters with severity of disease.
4.4. Sensitivity and specificity of inflammatory parameters in patients with ESLD A receiver operating curve (ROC) analysis was performed to calculate the sensitivity and specificity of the assessed markers. Although all markers were found rather sensitive, statistical analysis showed that QuinA and neopterin had the highest sensitivity and specificity for identification of patients with advanced liver failure (group II). When discriminating group II from group I patients, the areas under curve (AUC) were 0.89, 0.89, 0.73, and 0.74, for QuinA, neopterin, CRP, and IL-6, respectively (p 6 0.0002 for all investigations, sensitivity = 86%, 79%, 76%, 76%, respectively) (Fig. 3a). When sensitivity and specificity of markers was calculated based on Child-Pugh category C vs A + B, AUC were 0.75, 0.73, 0.68, and 0.68 for QuinA, neopterin, CRP, and IL-6, respectively
I. Lahdou et al. / Human Immunology 74 (2013) 60–66
63
Fig. 1. MELD score correlates with serum levels of QuinA and other inflammatory markers. MELD scores of 94 patients with cirrhosis were correlated with serum concentration of QuinA (a), neopterin (b), CRP (c), and IL-6 (d). Spearman rank correlation test was used for statistical calculation.
(p 6 0.003 for all investigations) (Fig. 3b). These results indicate that the tryptophan metabolite ‘‘QuinA’’ is a sensitive marker of severity of disease in patient with ESLD (Fig. 3a and b). 4.5. Relationship between severity of liver disease and QuinA in multivariate parametric analysis Although CRP (95% CI; 22–38 mg/L: p = 0.02) and IL-6 (95% CI; 33–170 pg/ml/L: p = 0.04) significantly correlated with MELD score, statistical analysis showed that QuinA (95% CI; 1.38–1.87: p < 0.0001) and neopterin (95% CI; 48–70 lmol/L: p < 0.0001) had the highest correlations. The results again confirm that QuinA is a distinguished marker in patients with ESLD. 4.6. Higher inflammatory markers in patients with ESLD and hepatic encephalopathy We also investigated whether the assessed inflammatory markers correlate with the presence of hepatic encephalopathy. Both the patients groups with and without hepatic encephalopathy had significantly higher serum levels of neopterin, CRP, and IL-6 when compared to healthy controls (HCs; p < 0.0001 for all investigations) (Fig. 4b and d). Patients with ESLD who did not show clinical signs of encephalopathy (enceph ) had similar QuinA serum levels when compared to HCs, whereas patients with encephalopathy (enceph+) had significantly higher QuinA levels than patients without encephalopathy (p = 0.003) and HCs
(p 6 0.0001) (Fig. 4a). The levels of all indices were significantly higher in patients with encephalopathy than in patients without encephalopathy (p 6 0.001 for all investigations) (Fig. 4a–d). These results indicate that QuinA also serves as a sensitive marker for hepatic encephalopathy. The potential mechanism of mediation of this disturbance remains speculative and requires further analysis in future studies. 5. Discussion The objective of the present study was to investigate whether increased serum levels of activation and inflammatory markers correlate with the degree of clinical liver failure. Enhanced serum levels of the inflammatory markers CRP and IL-6 and increased activation of cellular immunity as assessed by neopterin were noted in patients with liver dysfunction/failure. Interestingly, QuinA, a downstream breakdown product of tryptophan (Trp) along the kynurenine pathyway, was also increased in patients with severe liver dysfunction. To the best of our knowledge, this is the first description of increased (systemic) serum levels of the kynurenine pathway catabolite QuinA in patients with advanced liver dysfunction. From a clinical perspective, this is of interest because kynurenine pathway catabolites are well-known inducers of neurological dysfunction, neuropathy and encephalopathy [21,27,33]. Here, we demonstrate strongly increased serum levels of QuinA, the most potent inducer of neuropathy. Induction of QuinA in the cohort of patients investigated here is of particular
64
I. Lahdou et al. / Human Immunology 74 (2013) 60–66
Fig. 2. Increased serum QuinA levels in patients with severe liver cirrhosis. Based on MELD score ninety-four patients with cirrhosis were grouped in 2 cohorts: patients with MELD score <20 were considered as moderate liver dysfunction (study group I: n = 61) and MELD score P20 as severe liver dysfunction (study group II: n = 33). Fifty healthy volunteers served as controls (HCs). The difference between the medians of parameters in the two groups and HCs are showed by boxplots; (a) QuinA, (b) neopterin, (c) CRP, and (d) IL-6. The p Values were calculated using the Mann–Whitney U test.
interest since hepatic failure/dysfunction is often accompanied by hepatic encephalopathy (HE), which was shown to have a profound impact on the patients´ prognosis [34]. Evaluation of systemic levels of ammonia, which is often used as a marker to assess the degree of HE [35], was not done in the present study whereas our results suggest that QuinA contributes to the development and progression of HE, this remains to be proven in subsequent studies. Most strikingly, when sensitivity/specificity analyses were performed, QuinA was the marker most closely related with the degree of liver failure (according to the MELD scoring system). This indicates that liver dysfunction or failure is associated with a strongly activated catabolic pathway of Trp degradation, which in turn may enhance and support the inflammatory reaction often observed in this cohort of patients. Degradation of the essential amino acid Trp with resulting increased serum levels of the respective catabolites is a consequence of IDO activation [36]. Macrophages stimulated with IFN-c or lipopolysaccharide (LPS) upregulate IDO and increase their QuinA production [37,38]. While the exact mechanism of IDO upregulation in hepatic dysfunction/ failure remains to be elucidated, one might speculate that mechanisms including LPS activation, possibly via edema-induced altered gut barrier dysfunction, may play a role [39]. An enzyme with identical activity to IDO is tryptophan 2,3dioxygenase (TDO). This is a hepatic enzyme which oxidates L-tryptophan and is induced in clinical situations associated with increased endogenous glucocorticoid levels [40]. Although the role of TDO remains unclear in the clinical setting of liver dysfunction,
it might be theoretically responsible for increased levels of QuinA as described in this study. Another interesting aspect of this study is that besides QuinA, neopterin, which are released by activated macrophages in response to infectious and non-infectious inflammatory stimuli [6], was found to be increased in the patient cohort. QuinA and neopterin exceeded CRP and IL-6 in terms of sensitivity and specificity for hepatic dysfunction/failure. This probably indicates activation of cellular immunity and subsequent systemic release of QuinA and neopterin in patients with severe liver dysfunction. We recognize that our study has limitations. Serum levels of QuinA were shown increased also in other clinical situations such as in chronic renal dysfunction/failure [29]. Although no data exist on whether this may also be the case in acute (ARF) or acute-onchronic renal dysfunction, one might speculate that serum levels of these catabolites are increased in these acute clinical conditions too. Since the patients in MELD group II were found to have significantly higher serum creatinine levels when compared to group I, renal dysfunction might have influenced our data. Although we are unable to definitely rule out an influence of renal dysfunction in our analysis, we found that the data were unchanged after we corrected for systemic creatinine levels (there is no significant correlation between QuinA/Cr ratio and MELD score p = 0.10). We also investigated serum levels of QuinA in patients of Group II who had serum Cr 61.3 mg /dl (n = 12) or Cr >1.3 mg/dl (n = 21) respectively. Cr levels did not significantly correlate with QuinA levels (p = 0.38).
I. Lahdou et al. / Human Immunology 74 (2013) 60–66
65
Fig. 3. (a) Predicting value of QuinA for severity of liver disease according to MELD scores. Receiver operating characteristic (ROC) Curve analysis of MELD score was performed to assess the area under the curve (AUC), sensitivity and specificity for QuinA, neopterin, CRP, and IL-6 between patients with moderate (group I: n = 61) and severe liver dysfunction (group II: n = 33); (b) predicting value of QuinA for severity of liver disease according to Child-Pugh category. Receiver operating characteristic (ROC) Curve analysis of Child-Pugh category was performed to assess area under the curve (AUC), sensitivity and specificity for QuinA, neopterin, CRP, and IL-6 between patients with severe (Child-Pugh category C: n = 35) versus moderate liver dysfunction (Child-Pugh categories A + B: n = 59).
Fig. 4. Elevated serum levels of QuinA as marker of hepatic encephalopathy. Patients with liver cirrhosis were divided in two cohorts: patients who did not show clinical signs of encephalopathy (enceph : n = 51), and patients with encephalopathy (enceph+, n = 43). Fifty healthy volunteers used as controls (HCs). The difference between the medians of parameters in the two groups and HCs showed by boxplots; (a) QuinA, (b) neopterin, (c) CRP, and (d) IL-6. The p Values were calculated using the Mann–Whitney U test.
In summary, the degree of liver dysfunction/failure according to the MELD scoring system correlates with markers of inflammation
and activation of cellular immunity. We found increased serum levels of QuinA in cirrhotic patients with advanced liver dysfunc-
66
I. Lahdou et al. / Human Immunology 74 (2013) 60–66
tion and may reflect the severity of this clinical condition. We hypothesize that QuinA may contribute to the development of HE, which is of major clinical importance in patients with liver dysfunction/failure. We concluded that hepatic dysfunction is associated with pathophysiological reactions including activation of the inflammatory cascade. Our data indicate for the first time that increased serum levels of QuinA and neopterin reflect the degree of hepatic dysfunction/failure as assessed using the established MELD score. We speculate that QuinA induction may be a contributor to the development of hepatic encephalopathy, which is often observed in these patients. Acknowledgements We would like to acknowledge the skillful technical assistance of Martina Kutsche-Bauer and Regina Seemuth. References [1] Kamath PS, Wiesner RH, Malinchoc M, Kremers W, Therneau TM, Kosberg CL, et al. A model to predict survival in patients with End-Stage Liver Disease. Hepatology 2001;33(2):464. [2] Wiesner R, Edwards E, Freeman R, Harper A, Kim R, Kamath P, et al. Model for End-Stage Liver Disease (MELD) and allocation of donor livers. Gastroenterology 2003;124(1):91. [3] Huo TI, Lee SD, Lin HC. Selecting an optimal prognostic system for liver cirrhosis: the Model for End-Stage Liver Disease and beyond. Liver Int 2008;28(5):606. [4] Xia VW, Taniguchi M, Steadman RH. The changing face of patients presenting for liver transplantation. Curr Opin Organ Transplant 2008;13(3):280. [5] Jawa RS, Anillo S, Huntoon K, Baumann H, Kulaylat M. Interleukin-6 in surgery, trauma, and critical care part II: clinical implications. J Intensive Care Med 2011;26(2):73. [6] Fuchs D, Weiss G, Reibnegger G, Wachter H. The role of neopterin as a monitor of cellular immune activation in transplantation, inflammatory, infectious, and malignant diseases. Crit Rev Clin Lab Sci 1992;29(3–4):307. [7] Lavie CJ, Church TS, Milani RV, Earnest CP. Impact of physical activity, cardiorespiratory fitness, and exercise training on markers of inflammation. J Cardiopulm Rehabil Prev 2011;31(3):137. [8] Tilg H, Wilmer A, Vogel W, Herod M, Nölchen B, Judmaier G, et al. Serum levels of cytokines in chronic liver diseases. Gastroenterology 1992;103(1):264. [9] Bota DP, Van Nuffelen M, Zakariah AN, Vincent JL. Serum levels of C-reactive protein and procalcitonin in critically ill patients with cirrhosis of the liver. J Lab Clin Med 2005;146(6):347. [10] Montagnese S, Biancardi A, Schiff S, Carraro P, Carlà V, Mannaioni G, et al. Different biochemical correlates for different neuropsychiatric abnormalities in patients with cirrhosis. Hepatology 2011;53(2):558. [11] Ito H. IL-6 and Crohn’s disease. Curr Drug Targets Inflamm Allergy 2003;2(2):125. [12] Montoliu C, Piedrafita B, Serra MA, del Olmo JA, Urios A, Rodrigo JM, et al. IL-6 and IL-18 in blood may discriminate cirrhotic patients with and without minimal hepatic encephalopathy. J Clin Gastroenterol 2009;43(3):272. [13] Wiest R, Weigert J, Wanninger J, Neumeier M, Bauer S, Schmidhofer S, et al. Impaired hepatic removal of interleukin-6 in patients with liver cirrhosis. Cytokine 2011;53(2):178. [14] Lemmers A, Gustot T, Durnez A, Evrard S, Moreno C, Quertinmont E, et al. An inhibitor of interleukin-6 trans-signalling, sgp130, contributes to impaired acute phase response in human chronic liver disease. Clin Exp Immunol 2009;156(3):518. [15] De Rosa S, Cirillo P, Pacileo M, Petrillo G, D’Ascoli GL, Maresca F, et al. Neopterin: from forgotten biomarker to leading actor in cardiovascular pathophysiology. Curr Vasc Pharmacol 2011;9(2):188. [16] Goris RJ. Multiple organ failure: whole body inflammation? Schweiz Med Wochenschr 1989;119(11):347. [17] Gulcan EM, Tirit I, Anil A, Adal E, Ozbay G. Serum neopterin levels in children with hepatitis-B-related chronic liver disease and its relationship to disease severity. World J Gastroenterol 2008;14(44):6840.
[18] Zwirska-Korczala K, Dziambor AP, Wiczkowski A, Berdowska A, Gajewska K, Stolarz W. Hepatocytes growth factor (HGF), leptin, neopterin serum concentrations in patients with chronic hepatitis C. Przegl Epidemiol 2001;55(Suppl. 3):164. [19] Gonzalez-Reimers E, Santolaria-Fernandez F, Rodriguez-Rodriguez E, Rodríguez-Moreno F, Martínez-Riera A, Milena-Abril A, et al. Serum neopterin levels in alcoholic liver disease. Drug Alcohol Depend 1993;33(2):151. [20] Connert S, Stremmel W, Elsing C. Procalcitonin is a valid marker of infection in decompensated cirrhosis. Z Gastroenterol 2003;41(2):165. [21] Heyes MP. Quinolinic acid and inflammation. Ann N Y Acad Sci 1993;679:211. [22] Sadeghi M, Lahdou I, Daniel V, Schnitzler P, Fusch G, Schefold JC, et al. Strong association of phenylalanine and tryptophan metabolites with activated cytomegalovirus infection in kidney transplant recipients. Hum Immunol 2012;73(2):186–92. [23] Schwarcz R, Foster AC, French ED, Whetsell Jr WO, Kohler C. Excitotoxic models for neurodegenerative disorders. Life Sci 1984;35(1):19. [24] Saito K, Markey SP, Heyes MP. Effects of immune activation on quinolinic acid and neuroactive kynurenines in the mouse. Neuroscience 1992;51(1):25. [25] Guillemin GJ, Smith DG, Smythe GA, Armati PJ, Brew BJ. Expression of the kynurenine pathway enzymes in human microglia and macrophages. Adv Exp Med Biol 2003;527:105. [26] Stone TW. Endogenous neurotoxins from tryptophan. Toxicon 2001;39(1):61. [27] Beal MF, Matson WR, Swartz KJ, Gamache PH, Bird ED. Kynurenine pathway measurements in Huntington’s disease striatum: evidence for reduced formation of kynurenic acid. J Neurochem 1990;55(4):1327. [28] Widner B, Leblhuber F, Walli J, Tilz GP, Demel U, Fuchs D. Tryptophan degradation and immune activation in Alzheimer’s disease. J Neural Transm 2000;107(3):343. [29] Schefold JC, Zeden JP, Fotopoulou C, von Haehling S, Pschowski R, Hasper D, et al. Increased indoleamine 2,3-dioxygenase (IDO) activity and elevated serum levels of tryptophan catabolites in patients with chronic kidney disease: a possible link between chronic inflammation and uraemic symptoms. Nephrol Dial Transplant 2009;24(6):1901. [30] Schefold JC, Bierbrauer J, Weber-Carstens S. Intensive care unit-acquired weakness (ICUAW) and muscle wasting in critically ill patients with severe sepsis and septic shock. J Cachexia Sarcopenia Muscle 2010;1(2):147. [31] Schefold JC, Zeden JP, Pschowski R, Hammoud B, Fotopoulou C, Hasper D, et al. Treatment with granulocyte-macrophage colony-stimulating factor is associated with reduced indoleamine 2,3-dioxygenase activity and kynurenine pathway catabolites in patients with severe sepsis and septic shock. Scand J Infect Dis 2010;42(3):164. [32] Zeden JP, Fusch G, Holtfreter B, Schefold JC, Reinke P, Domanska G, et al. Excessive tryptophan catabolism along the kynurenine pathway precedes ongoing sepsis in critically ill patients. Anaesth Intensive Care 2010;38(2):307. [33] Chiarugi A, Cozzi A, Ballerini C, Massacesi L, Moroni F. Kynurenine 3-monooxygenase activity and neurotoxic kynurenine metabolites increase in the spinal cord of rats with experimental allergic encephalomyelitis. Neuroscience 2001;102(3):687. [34] Moroni F, Lombardi G, Carla V, Lal S, Etienne P, Nair NP. Increase in the content of quinolinic acid in cerebrospinal fluid and frontal cortex of patients with hepatic failure. J Neurochem 1986;47(6):1667. [35] Toris GT, Bikis CN, Tsourouflis GS, Theocharis SE. Hepatic encephalopathy: an updated approach from pathogenesis to treatment. Med Sci Monit 2011;17(2):RA53. [36] Kwidzinski E, Bechmann I. IDO expression in the brain: a double-edged sword. J Mol Med (Berl) 2007;85(12):1351. [37] Guillemin GJ, Smythe GA, Veas LA, Takikawa O, Brew BJ. A beta 1–42 induces production of quinolinic acid by human macrophages and microglia. Neuroreport 2003;14(18):2311. [38] Heyes MP, Saito K, Major EO, Milstien S, Markey SP, Vickers JH. A mechanism of quinolinic acid formation by brain in inflammatory neurological disease. Attenuation of synthesis from L-tryptophan by 6-chlorotryptophan and 4chloro-3-hydroxyanthranilate. Brain 1993;116(Pt 6):1425. [39] Basile AS, Saito K, Li Y, Heyes MP. The relationship between plasma and brain quinolinic acid levels and the severity of hepatic encephalopathy in animal models of fulminant hepatic failure. J Neurochem 1995;64(6):2607. [40] Oxenkrug GF. Tryptophan kynurenine metabolism as a common mediator of genetic and environmental impacts in major depressive disorder: the serotonin hypothesis revisited 40 years later. Isr J Psychiatry Relat Sci 2010;47(1):56.