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ORIGINAL ARTICLE
Year : 2019  |  Volume : 32  |  Issue : 1  |  Page : 363-367

Study of serum endotoxin in patients with liver cirrhosis with and without hepatic encephalopathy


1 Department of Tropical Medicine, Faculty of Medicine, Menoufia University, Shebeen El-Kom, Egypt
2 Department of Medical Biochemistry, Faculty of Medicine, Menoufia University, Shebeen El-Kom, Egypt
3 Tropical Medicine at Ministry of Health, Damanhour Fever Hospital, Damanhour, Egypt

Date of Submission26-Sep-2017
Date of Acceptance03-Dec-2017
Date of Web Publication17-Apr-2019

Correspondence Address:
Enas F El Mezain
Damanhour 17323, Beheira Governorate
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_653_17

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  Abstract 


Objective
To study serum endotoxin in patients with liver cirrhosis with and without hepatic encephalopathy (HE).
Background
HE is a common complication of liver cirrhosis. Studies suggest that endotoxin and ammonia play an important role in its pathogenesis.
Patients and methods
This prospective study was conducted from November 2016 to May 2017 on 30 patients with liver cirrhosis without HE as group I, 30 patients with HE (group II) and 30 age-matched and sex-matched healthy individual as the control group. All were subjected to history taking, complete medical examination, and thorough laboratory and radiological investigations. Serum endotoxin was measured by enzyme-linked immunosorbent assay technique and arterial ammonia was measured by the enzymatic method.
Results
There were significant increase in endotoxin and arterial ammonia in cirrhotic patients than controls, with the highest levels in group II and higher levels in comatosed patients than those in precoma. There was a positive correlation between endotoxin and ammonia in group I (r = 0.62, P < 0.001) and group II (r = 0.92, P < 0.001). To predict cirrhosis, serum endotoxin had a sensitivity of 92%, specificity of 80%, and a cutoff point of 0.28 EU/ml, while at a cutoff point of 38 μmol/l arterial ammonia had a sensitivity of 80 and a, specificity of 77%. To predict HE, serum endotoxin had a sensitivity of 93%, specificity of 77% at a cutoff point of 0.42 EU/ml, while at a cutoff point of 75.5 μmol/l arterial ammonia had a sensitivity of 80% and a specificity of 73%.
Conclusion
Serum endotoxin and arterial ammonia were elevated in patients with liver cirrhosis with higher levels in HE and the highest in hepatic coma.

Keywords: ammonia, endotoxins, hepatic encephalopathy, liver cirrhosis


How to cite this article:
El-Lehleh AM, Algamal AA, El-Shazly RA, El Mezain EF. Study of serum endotoxin in patients with liver cirrhosis with and without hepatic encephalopathy. Menoufia Med J 2019;32:363-7

How to cite this URL:
El-Lehleh AM, Algamal AA, El-Shazly RA, El Mezain EF. Study of serum endotoxin in patients with liver cirrhosis with and without hepatic encephalopathy. Menoufia Med J [serial online] 2019 [cited 2019 May 27];32:363-7. Available from: http://www.mmj.eg.net/text.asp?2019/32/1/363/256136




  Introduction Top


Cirrhosis is a chronic progressive liver disease that results in portal hypertension and liver cell failure [1]. Hepatic encephalopathy (HE) is characterized by neuropsychiatric manifestations in patients with liver dysfunction after exclusion of other brain diseases. It can be manifested by personality changes, intellectual impairment, and a depressed level of consciousness [2]. Multiple pathophysiologic mechanisms can result in HE by inducing functional impairment of the central nervous system [3]. Ammonia dysmetabolism cannot explain all the neurological changes that occur in patients with HE [4]. Endotoxin, the lipopolysaccharide in the outer membrane of Gram-negative bacteria, enters the systemic circulation due to intestinal bacterial translocation and portosystemic shunting results in chronic endotoxemia [5]. Circulating endotoxins from the gut cause sterile inflammation by releasing proinflammatory mediators which directly signal to the brain [6]. In cirrhotic patients with minimal HE inflammation or hyperammonemia alone did not result in cognitive impairment but the synergistic effect of both was involved. This suggests that in liver cirrhosis, inflammation and its mediators modify the cerebral effect of hyperammonemia and there is a synergistic relationship between them in the pathogenesis of HE [7]. In cirrhotic patients without evidence of proven infection, endotoxemia plays an important role in the inflammatory pathogenesis of HE [8]. Patients with HE and in deep coma had higher endotoxin levels than those with light coma. In cirrhosis, endotoxemia without sepsis is a constant finding and higher levels of endotoxins are associated with encephalopathy, hepatic failure, and death [9].

The aim of this work was to study serum endotoxin in patients with liver cirrhosis with and without HE.


  Patients and Methods Top


This prospective study was conducted during the period from November 2016 to May 2017 at Damanhour Fever Hospital (Egypt). The study protocol was approved by the Local Ethics Committee of the Menoufia University and the Hepatology Department, Damanhour Fever Hospital. All persons gave written informed consent before inclusion into the study.

A total of 90 persons, 60 patients with liver cirrhosis and 30 controls were included in the study who were divided into three groups: group I included 30 cirrhotic patients without HE and their ages ranged between 45 and 65 years; group II included 30 cirrhotic patients with HE and their ages ranged between 48 and 60 years and they were attended or admitted to the Hepatology Department, Damanhour Fever Hospital, and group III included 30 age-matched and sex-matched healthy individuals.

Inclusion criteria were age more than or equal to 18 years, liver cirrhosis of any etiology, and HE of any grade.

Exclusion criteria included patients in coma due to causes other than HE, patients with a history of gastrointestinal bleeding in the previous 6 weeks, active ingoing infection, receiving antibiotic therapy in the previous 2 weeks, serum creatinine more than 1.5 mg/dl, electrolyte impairment (serum sodium <130 meq/ml, serum potassium <3.5 meq/ml or >5 meq/ml), use of psychotropic drugs in the previous 6 weeks, recent alcohol use (<6 weeks), previous transjugular intrahepatic shunt placement or shunt surgery, hepatocellular carcinoma, and severe comorbidity such as congestive heart failure, pulmonary disease, neurological, and psychiatric problems impairing the quality of life. Patients and controls were subjected to full history taking, complete clinical examination, through laboratory, and radiological investigations.

Diagnosis of cirrhosis was based on clinical, biochemical, and ultrasonographic findings.

Collection of blood samples: about 5 ml of blood was aseptically withdrawn from all persons. A measure of 3 ml was transferred slowly into a plain tube. The serum was allowed to separate in a serum separator tube (about 4 h) at room temperature, then centrifugation at ∼1000g for 15 min was done. The serum samples were stored at −80°C until further use for endotoxin measurement and repeated freeze–thaw cycles were avoided. In addition, the rest of the blood samples were subsequently used for clinical chemistry.

Laboratory investigations included complete blood picture, liver function tests (total bilirubin, direct bilirubin, serum albumin, aspartate aminotransferase, alanine aminotransferase, international normalized ratio, and prothrombin time), viral hepatitis markers [hepatitis B surface antigen (HBsAg) and hepatitis C virus antibody], kidney function tests (blood urea and serum creatinine), and random blood sugar. Complete blood count was done using Phoenix NCC-3300 (NeoMedica, Nis, Serbia). All parameters of liver function tests (except for prothrombin time), parameters of kidney function, and random blood sugar tests were carried out using Respons 920 chemistry analyzer (DiaSys Diagnostics Systems, Holzheim, Germany). Prothrombin time was done using human thromboplastin, by Seasc Clot 2 (Bio Med Diagnostics, Hannover, Germany). Measurement of arterial blood ammonia was done by the enzymatic method using Cobas c311 analyzer (Roche Diagnostics GmbH, Mannheim, Germany). HBsAg was detected by enzyme-linked immunosorbent assay (ELISA) kit (Murex HBsAg version 3; Diasorin, Dartford, UK). Hepatitis C virus antibodies were detected by ELISA kit (Ortho-Clinical Diagnostics, Raritan, New Jersey, USA). Serum endotoxin levels were done by ELISA kit (Aviva Systems Biology, San Diego, California, USA).


  Statistical Analysis Top


Results were statistically analyzed by SPSS version 22 (SPSS Inc., Chicago, Illinois, USA). Analysis of variance (F-test) was used for parametric data. Mann–Whitney and Kruskal–Wallis tests were used for nonparametric data. χ2-Test and Fisher's exact tests were used for qualitative variables. Spearman's and Pearson's correlation tests were used for detecting the strength and direction of association between variables. P value less than 0.05 is considered significant. Receiver operating characteristic (ROC) curve is a graphical plot of the sensitivity versus false positive rate (1 − specificity). Sensitivity or true positive rate = true positive/(true positive + false negative). Specificity or true negative rate = true negative/(true negative + false positive) = 1 − false positive rate. Accuracy = (true positive) + (true negative)/(positive + negative). Positive predictive value (PPV): true positive/(true positive + false positive). Negative predictive value (NPV): true negative/(true negative + false negative). P value was considered significant if up to 0.05.


  Results Top


There was no significant difference between the studied groups regarding age (P = 0.374) and sex (P = 0.955) distribution. The causes of liver cirrhosis were chronic viral hepatitis C and B. There was a statistically significant difference between the three groups regarding serum endotoxin and blood ammonia levels (P < 0.001) as shown in [Table 1]. There was high statistically significant difference in serum endotoxin and blood ammonia levels among patients in group II regarding the level of consciousness (P < 0.001) as shown in [Table 2]. There was a statistically significant difference with negative correlation between endotoxin level and hemoglobin (r = −0.59, P = 0.001), platelets count (r = −0.64, P < 0.001), and albumin (r = −0.70, P < 0.001), while there was a high statistically significant difference with positive correlation between endotoxin levels and (total bilirubin (r = 0.58, P = 0.001), direct bilirubin (r = 0.58, P = 0.001), international normalized ratio (r = 0.72, P < 0.001), prothrombin time (r = 0.70, P < 0.001), and ammonia (r = 0.92, P < 0.001) in group II. There was a statistically significant difference with negative correlation between endotoxin level and albumin (r = −0.89, P < 0.001), while there was a statistically significant difference and positive correlation between endotoxin levels and [total (r = 0.70, P < 0.001), direct bilirubin (r = 0.68, P < 0.001), and ammonia (r = 0.62, P < 0.001)] in group I as shown in [Table 3]. At a cutoff point of 0.28 EU/ml, the sensitivity of endotoxin test was 92%, the specificity was 80%, PPV was 90%, NPV was 83%, accuracy was 88%, and the area under the curve (AUC) was 0.94 to predict patients with cirrhosis, while at a cutoff point of 38 μmol/l the sensitivity of ammonia test was 80%, the specificity was 77%, PPV was 87%, NPV was 65%, accuracy was 78%, and the AUC was 0.89 to predict patients with cirrhosis as shown in [Table 4] and [Figure 1]. At a cutoff point of 0.42 EU/ml, the AUC for endotoxin was 0.95 with a sensitivity of 93%, specificity of 77%, PPV of 80%, NPV of 92%, and accuracy of 85% to predict HE, while at a cutoff point of 75.5 μmol/l, the AUC for ammonia was 0.80 with the sensitivity of the test being 80%, the specificity 73%, PPV 75%, NPV 78%, and accuracy being 77% to predict patients with HE as shown in [Table 4] and [Figure 2].
Table 1: Serum endotoxin and ammonia levels of the studied groups

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Table 2: Relation between the level of consciousness, endotoxin, and ammonia in the studied group II patients

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Table 3: Correlation between endotoxin level and the studied parameters of the studied groups

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Table 4: Validity of endotoxin and ammonia levels between the studied groups

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Figure 1: Receiver operating characteristic (ROC) curve for endotoxin and ammonia levels between the studied total patients (group I+group II) and group III (controls).

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Figure 2: Receiver operating characteristic (ROC) curve for endotoxin and ammonia levels between the studied patients (group I vs. group II).

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  Discussion Top


The present study shows significant increase in ammonia levels in group II than in group I. This comes in agreement with Iwasa et al. [10] who found statistically significant difference between the HE group and non-HE group in ammonia levels with a mean of 68 ± 46 versus 37 ± 25 μmol/l, respectively. This was in disagreement with Wang and Saab [11] who did not suggest that the measurement of ammonia levels can diagnose HE or assess its severity and its role is limited in the pathogenesis of HE. In the present study, serum endotoxin levels were higher in group II than in groups I and III; these come in agreement with Jain et al. [12] who found the median of serum endotoxin levels were 0.35 (0.25–0.50) in cirrhotic without HE, 0.55 (0.4–0.6) in grade I HE, 0.62 (0.5–0.7) in grade II, 0.74 (0.5–0.8) in grade III, 0.9 (0.8–1.0) in grade IV, and 0.25 (0.25–0.30) in the control group, both arterial ammonia and serum endotoxin levels were significantly higher in patients with HE as compared with patients without HE and healthy controls. These results were in disagreement with Fukui et al. [13] who found no relation between serum endotoxin levels and the severity of liver disease. The present study showed that in group II there was a statistically significant difference in serum endotoxin and blood ammonia levels regarding the level of consciousness (higher levels in hepatic coma than in precoma). This can be explained by Romero-Gómez et al. [14] who suggest that elevated blood ammonia and inflammation are involved in the pathogenesis of HE. Patients in precoma had a mean blood ammonia of 68.70 ± 24.0, while patients in coma had a mean of 100.69 ± 12.09. This was in agreement with Ong et al. [15] who reported that arterial ammonia increased with the severity of HE. There was a statistically significant difference between the different grades of HE (grades I and II were with ammonia level <150 μmol/l, while grades III and IV were with ammonia level >150 μmol/l). These results are in disagreement with Kundra et al. [16] who found no statistically significant correlation in patients with elevated ammonia levels and the presence of HE. This can be explained by Shawcross et al. [17] who found that systemic inflammation and not arterial ammonia is associated with the severity of HE with no statistically significant difference in arterial ammonia between patients in grades 3 and 4 HE. In the current study, there was a statistically significant difference in serum endotoxin regarding the level of consciousness in group II with a mean of 0.60 ± 0.12 in precoma, while patients in coma were with a mean of 0.86 ± 0.02. This comes in agreement with Bigatello et al. [9] who reported that endotoxemia in the HE group was higher than in well-compensated cirrhotic group; it was higher in deep than in light coma.

This was in disagreement with Chen et al. [18] who reported that neutralization of endotoxin by the administration of either low-dose or high-dose polymyxin B did not significantly improve the degree of HE and their findings were against the role of endotoxin in the pathogenesis of HE. In the current study, there was a statistically significant difference with a positive correlation between serum endotoxin and arterial ammonia level in groups I and II. This was in agreement with Jain et al. [8] who found a positive correlation between serum endotoxin and arterial ammonia level (r = 0.502) in the HE group.

Using the ROC analysis, endotoxin curve yielded a better test than arterial ammonia to distinguish cirrhotic patients from healthy control (with 92% sensitivity, 80% specificity, while the ROC curve analysis of ammonia was with 80% sensitivity and 77% specificity. The result of endotoxin was in agreement with Lee et al. [19] who reported that for distinguishing cirrhotic patients from healthy controls the cutoff point of serum endotoxin was 21.5 pg/ml (0.215 EU/ml), with AUC 1.0, 100% sensitivity, and 95.5% specificity. The result of arterial ammonia was in agreement with Jain et al. [8] who reported that arterial ammonia levels were significantly higher in cirrhotic patients compared with healthy controls (62 vs. 23 mcg/dl) respectively. Using the ROC analysis, the endotoxin curve yielded a better test than arterial ammonia for the detection of HE in cirrhotic patients with 93% sensitivity and 77% specificity, while the ROC curve analysis of ammonia was with 80% sensitivity and 73% specificity. This was in agreement with Papadopoulos et al. [20] who diagnosed HE with a cutoff point of 75 μmol/l, with 87.9% sensitivity, 88.6% specificity, 87.9% PPV, 88.6% NPV, and the AUC was 0.95. This was in disagreement with Gundling et al. [21] who reported that the cutoff point of ammonia to predict HE was 65.5 μmol/l with 95.7% specificity, a low sensitivity of 41.7%, 59.3% accuracy, 77.3% PPV, and 48.6% NPV. The result of endotoxin was in agreement with Jain et al. [12] who reported that its levels in cirrhotic with HE were higher than cirrhotic without HE.


  Conclusion Top


Serum endotoxin and arterial ammonia were elevated in patients with liver cirrhosis with higher levels in HE and the highest in hepatic coma.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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