Edited by: Christoph Thiemermann, Queen Mary University of London, United Kingdom
Reviewed by: Frank Tacke, Uniklinik RWTH Aachen, Germany; Lukas Martin, Uniklinik RWTH Aachen, Germany
Specialty section: This article was submitted to Inflammation, a section of the journal Frontiers in Immunology
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Liver dysfunction (LD) and liver failure are associated with poor outcome in critically ill patients. In patients with severe sepsis or septic shock, LD occurred in nearly 19% of patients. An early diagnosis of LD at time of initial damage of the liver can lead to a better prognosis of these patients because an early start of therapy is possible. We performed a second prospective study with septic patients to test a new cell-based cytotoxicity device (biosensor) to evaluate clinical relevance for early diagnosis of LD and prognostic capacity. In the clinical study, 99 intensive care unit patients were included in two groups. From the patients of the septic group (
The development of liver dysfunction (LD) and liver failure in intensive care unit (ICU) patients have a relatively high incidence of 11% in all ICU patients and over 19% in patient with septic shock and is associated with increased in-hospital mortality (
Because physiological and online parameters are unable to diagnose LD early, laboratory parameters, like transaminases, albumin, and coagulation factors are commonly used, however, without convincing clinical data for detection of early LD (
LD, however, occurs as an early organ dysfunction in severe ill patients, e.g., in septic patients (
Hepatotoxicity of inflammatory mediators like nitric oxide, chemokines and cytokines, endogen and exogen toxins like lipopolysaccharides, plasma cascade factors, and hepatic ischemia are the main pathophysiological factors for the development of LD leading to hyperbilirubinemia and intrahepatic cholestasis (
In addition, activated neutrophils in response to infectious stimuli casting out their deoxyribonucleic acid DNA as main part of neutrophil-derived extracellular traps (NETs); so-called circulating-free deoxyribonucleic acid/neutrophil (derived) extracellular traps [cf-DNA/NETs; (
To verify the clinical relevance, especially for (early) diagnosis of LD of a new cell-based test device [biosensor; (
Approval for the study from the responsible ethics committee (University of Rostock; II HV 16/2005) was obtained and for all included patients written informed consent was received. Furthermore, the study was carried out under the principles of the Declaration of Helsinki and good clinical practice.
Between June 2005 and May 2008, 51 septic patients were included in the study after screening in the two-perioperative ICUs of the University Hospital of Rostock for fulfilling the criteria of septic shock or severe sepsis (
From each patient, 20 ml blood was obtained for testing with the biosensor and screening for blood parameters, cytokines, and circulating-free deoxyribonucleic acid/neutrophil-derived extracellular traps (cf-DNA/NETs) at inclusion, after 3, and after 7 days. Patients were followed up to assess hospital survival and organ function; demographic data, illness severity, SOFA–acute physiology and chronic health evaluation (APACHE)-II, cytokines, microbiological results, and pre-morbidity were documented (Table
Laboratory parameters and results of APACHE II-, and SOFA scores at inclusion, after 3, and 7 days in the septic- (SG,
Values | At inclusion |
After 3 days |
After 7 days |
|||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Sepsis group ( |
CG ( |
Sepsis group ( |
CG ( |
Sepsis group ( |
CG ( |
|||||||
SG (all) | SSG | SNSG | SG (all) | SSG | SNSG | SG (all) | SSG | SNSG | ||||
Lactate (mmol/l) | 1.7 (1.5–2.9) |
1.7 (1.5–2.5) |
2.4 (1.4–5.3) |
1.1 (0.9–1.3) | 1.7 (1.2–2.4) |
1.6 (1.2–2.2) |
1.9 (1.8–3.4) |
1.3 (0.9–1.6) | 1.4 (1.2–2.1) | 1.4 (1.1–2.0) | 2.2 (1.4–3.2) | 1.6 (1.3–2.8) |
Bilirubin (μmol/l) | 19.9 (12.1–36.8) |
19.2 (13.9–37.1) |
21.4 (11.7–33) | 15.2 (11.3–18.8) | 15.7 (10.7–30.9) |
15.0 (10.8–30.0) |
21.5 (10.0–35.9) |
11.0 (8.6–15.7) | 13.4 (9.1–24.3) |
11.9 (8.6–23.9) |
16.8 (12.3–27.8) |
9.1 (7.6–10.8) |
Ammonia (mmol/l) | 51.3 (34.5–63.9) |
43.1 (30.9–60.4) |
54.4 (43.0–68.7) |
31.7 (22.4–39.4) | 49.3 (38.4–65.4) |
48.1 (38.7–60.2) |
50.4 (35.5–78.8) |
31.8 (25.5–38.7) | 43.7 (37.3–59.2) |
43.6 (36.8–59.8) |
48.4 (38.3–80.2) |
32.4 (24.0–43.5) |
Creatinine (μmol/l) | 136 (94–202) |
130 (93–193) |
161 (103–245) |
78 (66–94) | 130 (92–215) |
127 (92–190) |
148 (108–232) |
77 (65–88) | 83 (64–165) |
82 (62–155) | 95 (82–249) |
79 (65–89) |
Urea (mmol/l) | 10.5 (7.3–17.4) |
9.4 (7.3–14.5) |
17.3 (7.7–22.7) |
4.4 (3.4–5.8) | 12.2 (7.9–18.1) |
10.8 (7.0–15.1) |
18 (11.4–26.7) |
4.6 (3.3–5.5) | 11.3 (6.3–19.7) |
11 (6.1–16.2) |
18.6 (8.2–35.0) |
4.5 (3.6–5.4) |
PCT (ng/ml) | 12.2 (3.9–32.5) |
10.5 (3.3–28.8) |
18.2 (4.4–47.2) |
0.5 (0.2–0.8) | 5.0 (2.0–15.7) |
4.6 (2.0–12.5) |
6.9 (2.2–24.0) |
0.4 (0.1–0.6) | 0.7 (0.4–3.3) |
0.7 (0.4–2.2) |
4.6 (0.4–6.7) |
0.2 (0.1–0.2) |
Leukocytes (GpT/l) | 14.8 (9.1–23.0) |
14.6 (8.2–22.1) |
20.2 (11.8–51.5) |
9.1 (7.6–11.7) | 12.6 (8.6–16.7) |
11.6 (8.3–15.4) |
14.6 (10.5–24.4) |
8.9 (7.3–11.5) | 13.3 (11.0–18.3) |
13.2 (10.4–17.7) |
15.3 (12.4–25.3) |
9.0 (8.0–10.9) |
Thrombocytes (GpT/l) | 191 (106–254) | 201 (107–254) | 174 (65–248) | 184 (136–238) | 170 (100–244) | 187 (109–256) | 149 (71–189) |
187 (149–252) | 229 (135–350) |
272 (157–369) |
184 (113–236) |
334 (238–431) |
Prothrombin time (%) | 71 (61–82) |
73 (61–86) |
66 (61–75) |
90 (80–95) | 81 (70–98) |
84 (72–102) |
65 (50–80) |
102 (92–112) | 88 (74–99) |
88 (76–101) |
84 (64–96) |
99 (89–113) |
APACHE II | 32 (26–36) |
30 (25–35) |
36 (28–42) |
9 (7–12) | ||||||||
SOFA | 13 (11–15) |
12 (10–14) |
15 (12–16) |
2 (0–4) | 11 (9–14) |
10 (8–13) |
13 (11–17) |
1 (0–2) | 9 (3–12) |
7 (3–11) |
13 (9–17) |
0 (0–0) |
For the hepatocytes-based cytotoxicity assay, the human hepatocyte cell line HepG2/C3A (American Type Culture Collection CRL-10741) was used. The cells were cultivated at 37°C in a 5% CO2 humidified incubator with Dulbecco’s modified Eagle’s medium (GIBCO Life Technologies, Eggenstein, Germany), 10% fetal bovine serum (FBS, PAA Laboratories, Pasching, Germany), 1% antibiotics solution (penicillin G: 10,000 IE/ml/streptomycin: 10 mg/ml; PAA), and 1% 200 mM
For testing the hepatotoxicity of patients’ plasma, the cells were seeded in 24-well microtiter plates in a density of 250,000 cells/well. Then, the cells were incubated for 3 days with 1 ml heparinized plasma from the subjects following a 3-day incubation period with fresh medium (1 ml). Cells and cell culture supernatants were obtained for the measurement of viability (XTT-test: dehydrogenases activity in the mitochondria, trypan blue staining: cell-count and vitality), cytochrome 1A2 activity, and synthesis of albumin. All test batches from test subjects and measurements were taken twice and a medium control was added.
The test of metabolism of ethoxyresorufine (Molecular Probes, Eugene, OR, USA) to resorufine was used for measurement of the activity of cytochrome P450 1A2; following the protocol of Kelly and Sussman (
Albumin was measured from cell culture medium supernatant, carried out by a nephelometrical method (Immage 800, Beckman Coulter GmbH, Krefeld, Germany).
The XTT-test (Roche Diagnostics GmbH, Mannheim, Germany) was used following the describing of Scudiero et al. (
Interleukin (IL)-1 beta, IL-6, IL-10, and tumor necrosis factor (TNF)-alpha were measured in patients’ serum with commercial ELISA kits as described by the supplier (BioSource International, Camarillo, CA, USA).
The quantification of cf-DNA/NETs was performed with a fluorescent assay (Leukocare AG, Munich, Germany). A green fluorescent dye binds DNA and the intensity of fluorescence (emission at 530 nm wavelength; Fusion, PerkinElmer, Monza, Italy) correlates with the amounts of DNA. The measurement range was between 50 and 3,000 ng/ml (
The results are expressed as the median with 0.25–0.75 quartile and are displayed as box plots in the figures. Nonparametric analyses were used after (negative) testing of normal distribution (with the Kolmogorov–Smirnov test; SPSS, Chicago, IL, USA). Statistical significance was analyzed with the Kruskal–Wallis one-way, the two-tailed Mann–Whitney
The in-hospital mortality of the septic group (SG) was 23.5% (between day 3 and 20,
Summaries of laboratory parameters and the results of the APACHE II-, and SOFA scores at inclusion, on days 3, and 7 of the CG, and the SG are displayed in Table
Twenty-five patients of the SG (49%) developed acute kidney injury, and 31.4% (16 patients) needed renal replacement therapy (continuous methods). Criteria for LD (
The sources of primary infection and results of microbiological analysis in the SG are displayed in Table
Source of primary infection and results of microbiological analysis in the septic group (SG,
Source of primary infection | Peritonitis | Wound-infection/abscess | Pneumonia | Urogenital infection |
---|---|---|---|---|
Patients ( |
24 | 7 | 20 | 1 |
Fungi ( |
11 | 1 | 0 | 0 |
Gram-positive bacteria ( |
10 | 3 | 1 | 1 |
Gram-negative bacteria ( |
11 | 3 | 12 | 2 |
All values of IL-1-beta, TNF-alpha, and IL-10 were below 21 pg/ml (as median) in the SG and CG (Table
Cytokines values at inclusion, and after 3 days in the septic- (SG,
Cytokine (pg/ml) | At inclusion |
After 3 days |
||||||
---|---|---|---|---|---|---|---|---|
Sepsis group ( |
CG ( |
Sepsis group ( |
CG ( |
|||||
SG (all) | SSG | SNSG | SG (all) | SSG | SNSG | |||
IL-1 beta | 3.9 (2.4–4.5) | 5.8 (2.5–6.5) | 2.7 (2.5–2.9) | 2.5 (2.4–2.6) | 2.5 (2.3–2.6) | 2.6 (2.4–2.8) | 2.4 (2.2–2.9) | 2.6 (2.5–2.7) |
IL-6 | 268 (106–557.5)+,° | 268 (116.5–557.5)#,° | 226 (98–501.5)§,° | 78 (42.1–143)° | 45.3 (25.9–90.5) | 48.2 (27–89) | 42.3 (25.4–155.9) | 30.5 (22.1–58) |
IL-10 | 11.1 (2.4–25.9)+ | 12.3 (3.8–24.1)#,° | 4.6 (2.5–33.3) | 2.5 (2.3–8.6) | 7.5 (2.4–12)+ | 7.2 (2.3–11.9)# | 7.9 (2.5–17.5)§ | 2.5 (2.2–2.6) |
TNF-alpha | 20.4 (12.9–32.2)+,° | 20.4 (13.9–29.8)#,° | 20.7 (11.2–41.9)§,° | 7.7 (5.7–9.7) | 14.7 (10.2–19.9)+ | 15.5 (11.2–19.9)# | 10.8 (7.1–23.2) | 6.6 (5.6–9.7) |
The values of cf-DNA/NETs were significantly higher in the SG than in the CG at inclusion, after 3, and after 7 days (Figure
The values of circulating-free deoxyribonucleic acid (cf-DNA, median/0.25–0.75 quartile) at inclusion, after 3, and 7 days in the survivors (
At inclusion, after 3, and 7 days the vitality and the cell count (Figure
Cell count and vitality (trypan blue staining, median/0.25–0.75 quartile) of HepG2/C3A cells incubated with plasma from survived and non-survived septic patients and non-septic control patients at inclusion, after 3, and 7 days. *
Results of the XTT-test (dehydrogenases activity in the mitochondria) and albumin synthesis of HepG2/C3A cells incubated with plasma from survived and non-survived septic patients and non-septic control patients at inclusion, after 3, and 7 days displayed as median/0.25–0.75 quartile. *
Metabolism of ethoxyresorufine to resorufine (activity of cytochrome 1A2, median/0.25–0.75 quartile) of HepG2/C3A cells incubated with plasma from survived and non-survived septic patients and non-septic control patients at inclusion, after 3, and 7 days. *
Correlations between the hepatocyte-based cytotoxicity test parameters with bilirubin, alanine aminotransferase (ALAT), asparagine aminotransferase (ASAT), and ammonia were not observed. Significant negative correlations were found between the cytochrome 1A2 activity, the XTT-test, the vitality, and the cell count with the APACHE II-, and SOFA scores, lactate, and PCT (ρ between −0.3 and −0.5,
The values of bilirubin correlated with the APACHE II-, SOFA scores, and lactate (ρ = +0.3,
Significant correlations were found between the values of cf-DNA/NETs with the APACHE II-, and SOFA scores, and PCT (ρ between +0.4 and +0.6,
The measured cytokines did not correlate with ALAT, ASAT, and ammonia; however, the cytokines IL-6, IL-10, and TNF-alpha correlated with bilirubin (ρ between +0.2 and +0.4,
In this study, we included 51 patients in general septic shock and observed a relatively low in-hospital mortality of 23.5% in comparison to other studies (
Liver dysfunction or liver failure occurred in 33.3% (
In the state of severe sepsis or septic shock, the functionality of hepatocytes is partly decreased (
In this study, we saw an impairment of viability and functionality of test cells after incubation with plasma from patients in general septic shock. The cytochrome 1A2 activity, the XTT values (activity of mitochondrial dehydrogenases), the cell count, and the vitality were significantly lower in the SG compared to the CG at inclusion, after 3, and after 7 days. In addition, the plasma of non-survivors in the SG led to a more pronounced impairment of test cells than the plasma of survivors at inclusion. These results support the aim of the study that the cell-based biosensor used may be a tool of early diagnosis for LD and has prognostic value. In addition to this thesis, we found significant negative correlation between the cytochrome 1A2 activity, the XTT-test, the vitality, and the cell count with the APACHE II- and SOFA scores, lactate, and PCT at inclusion. By contrast, the albumin synthesis in test cells seems to be a late changing parameter and is not valuable for early detection of LD.
In this study, correlations between static liver markers like bilirubin with the results of the hepatocyte cytotoxicity test parameters at inclusion were not seen. This may be due to the fact that bilirubin and other classical liver parameters increase late in LD or liver failure, especially in septic patients (
The impairment of cell function and viability seen in HepG2/C3A after incubation with septic plasma can be caused by endogenous and exogenous toxins, drugs, and metabolites (
Due to relative low values of cytokines with exception of IL-6, we observed only a few correlations between cytokines and parameters of the biosensor: between the cytochrome 1A2 activity with TNF-alpha, IL-6, and IL-10 at inclusion and results of the XTT-test, and the vitality with the levels of TNF-alpha also at inclusion.
Many cytokines, mainly the pro-inflammatory cytokines TNF-alpha, IL-1 beta, and IL-6, cause an impairment or dysregulation of the viability, the function, and apoptosis of human hepatocytes and hepatocyte cell lines, e.g., in HepG2/C3A cells [for review, see Ref. (
For our hepatocyte-based cytotoxicity test, we worked with the well-characterized cell line HepG2/C3A (
In a previous work from our research group, the testing of antimycotics (caspofungin, anidulafungin, and fluconazole) with HepG2/C3A cells compared with human primary isolated hepatocytes provided similar results regarding cytochrome 1A2 activity, vitality, and activity of mitochondrial dehydrogenase [for review, see Ref. (
NETs play a diametrical role in sepsis (
In line with results of former studies (
The test time with the hepatocyte-based biosensor of 3–6 days in this study is not suitable for early diagnosis of LD. However, by optimization of the biosensor, the incubation time has meanwhile been able to have reduced to 20 h (unpublished data). By technical improvements of the cell culture system (shaking instead of a resting system) and the increase of the concentration of FBS in the cell culture medium, we achieved comparable viability and functionality of sensor cells in human plasma after incubation times of 6 days and of 20 h. The reduction of test time is an important condition for usability in clinical practice (Appendix).
Plasma of healthy volunteers was not tested in the presented analysis. In our former study, the values of the hepatocyte-based test were comparable and without significant difference between healthy volunteers and the postoperative non-septic CG (
In conclusion, hepatotoxicity of septic plasma was already detected with the liver cell-based cytotoxicity at inclusion and also in the course of disease. The causes of these cellular impairments need further basic science and clinical investigations. The influence of NETs on the development of liver failure in septic patients seems to be an interesting approach. Higher levels of cf-DNA/NETs and impairments in all parameters of the hepatocyte biosensor were associated with a worse outcome in this study. Since bilirubin is a late parameter in LD and only markedly increased in advanced liver damage (
The study received ethics approval from the local research ethics committee (University of Rostock; II HV 16/2005), which include compliance with the principles of good clinical practice. The study abided by the principles of the Declaration of Helsinki. Written informed consent was obtained from all subjects or from the patients’ representatives if direct consent could not be received.
MS and GN-S participated in the design of the study. MS did the regulatory work and coordinated the study; wrote the manuscript; revising work was done from all other authors. MS, GR, CH, and SD did the data analysis. MS, CH, TM, JE, GN-S, and SMitzner were clinical investigators. SMargraf and JA analyzed the clinical probes for cf-DNA/NETs.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors want to thank Heike Potschka and Helga Weiss-Reining for valuable technical support. All the routine measurements were made at Institute of Clinical Chemistry and Laboratory Medicine, University Hospital of Rostock. Special thanks go to Johannes Brenner, Maria Brettschneider, and Swantje Kruspi for their great help in the clinical setting and collection of clinical data.
The Supplementary Material for this article can be found online at
ALAT, alanine aminotransferase; APACHE, acute physiology and chronic health evaluation; ASAT, asparagine aminotransferase; ATCC, American Type Culture Collection; cf-DNA, circulating-free deoxyribonucleic acid; CG, control group; ICU, intensive care unit; IL, interleukin; LPS, lipopolysaccharides; NETs, neutrophil-derived extracellular traps; PCT, procalcitonin; SG, septic group; SIRS, systemic inflammatory response syndrome; SOFA, sepsis-related organ failure assessment; TNF, tumor necrosis factor.