Conceived and designed the experiments: GZL YL. Performed the experiments: EV MK. Analyzed the data: EV MK YL ER IE LB. Contributed reagents/materials/analysis tools: MS SR IE LB. Wrote the paper: GZL.
GZL and MK own patents WO2004081029 and US7741065B2, “A novel non-invasive marker for liver disease”. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
The human asialoglycoprotein receptor is a membrane heterooligomer expressed exclusively in hepatocytes. A soluble secreted form, sH2a, arises, not by shedding at the cell surface, but by intracellular cleavage of its membrane-bound precursor, which is encoded by an alternatively spliced form of the receptor H2 subunit. Here we determined and report that sH2a, present at constant levels in serum from healthy individuals is altered upon liver fibrosis, reflecting the status of hepatocyte function.
We measured sH2a levels in serum using a monoclonal antibody and an ELISA assay that we developed, comparing with routine liver function markers. We compared blindly pretreatment serum samples from a cohort of 44 hepatitis C patients, which had METAVIR-scored biopsies, with 28 healthy individuals.
sH2a levels varied minimally for the healthy individuals (150±21 ng/ml), whereas the levels deviated from this normal range increasingly in correlation with fibrosis stage. A simple algorithm combining sH2a levels with those of alanine aminotransferase allowed prediction of fibrosis stage, with a very high area under the ROC curve of 0.86.
sH2a has the potential to be a uniquely sensitive and specific novel marker for liver fibrosis and function.
A soluble secreted form of the human asialoglycoprotein receptor (ASGPR), termed sH2a, is formed by cleavage in the endoplasmic reticulum of hepatocytes of its precursor
Experimental serum markers that have been proposed for fibrosis include extracellular matrix (ECM) macromolecules and their degradation products. Unfortunately, these markers are not very sensitive and are not liver specific, they can also reflect inflammatory processes in other tissues. The same is true for the levels of normally intracellular enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptidase (GGT), etc.). These markers are indicative of liver damage but do not reflect directly hepatocyte function. Function is diagnosed with classical markers like albumin and prothrombin time (PT), but these are sensitive only to severe disease. Likewise, methods that combine several markers, like Fibrotest
Therefore, it would be highly beneficial to have a non-invasive serum marker that is a specific and sensitive test for hepatocyte function, which would also serve as an early indicator of fibrosis. Here we show for a cohort of HCV patients compared to healthy individuals that ASGPR sH2a may be a valid candidate for such a role.
The study had a priori approval by Tel Aviv Sourasky Medical Center hospital ethical committee according to the Helsinki Declaration and written informed consent was obtained from all participants.
Alkaline phosphatase (ALP) substrate p-nitrophenylphosphate (p-NPP) was from Chemicon International (Temecula, CA). Imperial protein stain was from (Pierce). Common reagents were from Sigma.
The preparation of a monoclonal antibody against sH2a and the development of a competitive ELISA using this antibody were described before
Retrospective samples were from a group of healthy blood donors and a cohort of consecutive pretreatment HCV-infected patients at the Liver Unit, Tel Aviv Sourasky Medical Center. Patients co-infected with HIV, HBV or with additional diseases from other etiologies were excluded. Overweight healthy individuals were also excluded.
Patients had routine laboratory tests performed by a certified central lab, using common commercial methods. These tests included total bilirubin, ALP, ALT, AST, GGT, albumin, serum cholesterol, PT and HCV RNA level (RT-PCR). Similar tests were done on healthy individuals except for PT and HCV RNA.
Percutaneous liver biopsy was performed using a Tru-Core II [R] biopsy instrument under ultrasound guidance. A single pathologist blinded to all clinical and serological results evaluated all slides. Two patients where biopsies were found inadequate were excluded from the study. Biopsies were METAVIR-scored
Comparison between groups of patients with regard to demographic (age, gender) and clinical parameters was performed using Chi-square tests and Kruskal-Wallis non-parametric analysis of variance (ANOVA), as applicable. The association between stage and other parameters was examined using logistic regression. Results are presented as odds ratio (OR), sensitivity and specificity with 95% confidence intervals (CI). The diagnostic value of the combination score of sH2a and ALT with regard to fibrosis stages was evaluated by the area under the receiver operating characteristic curve (AUROC). The statistical significance level was set to 0.05. SPSS for Windows software, version 14.0 (Chicago, IL) was used for the analysis.
We recently showed that sH2a is present at constant levels in healthy individuals and strongly reduced in cirrhotic HCV patients
Characteristic | Median | [IQR] | range |
Male gender (%) | 63.64 | ||
Age | 41.5 | 29–53 | 19–64 |
Weight (kg) | 73 | 66–84 | 56–127 |
total bilirubin (mg/dl) | 2 | 1–3 | 0.3–4 |
bilirubin/normal |
0.29 | 0.14–0.43 | 0.04–0.71 |
ALP (U/l) | 75 | 62–96.5 | 44–194 |
ALP/normal |
0.63 | 0.50–0.77 | 0.36–1.50 |
ALT (U/l) | 75 | 43–103 | 30–458 |
ALT/normal |
1.77 | 1.21–2.66 | 0.70–13.47 |
AST(U/l) | 47 | 33–59 | 21–291 |
AST/normal |
1.31 | 0.96–1.67 | 0.58–8.56 |
GGT (U/l) | 33 | 23–52 | 10–246 |
GGT/normal |
0.56 | 0.37–0.94 | 0.18–5.02 |
albumin (g/l) | 45 | 43–47 | 38–54 |
albumin/normal |
0.92 | 0.88–0.96 | 0.78–1.10 |
serum cholesterol (mg/dl) | 166 | 142–199 | 102–323 |
cholesterol/normal |
0.83 | 0.71–1.00 | 0.51–1.62 |
PT (sec) | 12 | 11.4–12.2 | 10.4–14.8 |
PT/normal |
0.81 | 0.77–0.82 | 0.70–0.99 |
sH2a (ng/ml) | 145 | 118–167 | 31–237 |
Fibrosis stage | 1 | 0–3 | 0–4 |
Inflammation grade | 2 | 1–3 | 0–4 |
Median of absolute values divided by upper limit of normal range.
Characteristic | Median | [IQR] | range |
Male gender (%) | 53.57 | ||
Age | 40.00 | 29–48 | 22–64 |
total bilirubin (mg/dl) | 0.36 | 0.22–0.50 | 0.06–1.05 |
bilirubin/normal | 0.05 | 0.05–0.16 | 0.009–0.42 |
ALP (U/l) | 48.00 | 36–55 | 22–88 |
ALP/normal | 0.35 | 0.33–0.51 | 0.17–0.68 |
ALT (U/l) | 19.00 | 14–27 | 9–38 |
ALT/normal | 0.42 | 0.37–0.65 | 0.26–1.03 |
AST(U/l) | 27.00 | 20–31 | 14–39 |
AST/normal | 0.67 | 0.49–0.89 | 0.35–1.08 |
GGT (U/l) | 12.50 | 3–18 | 1–38 |
GGT/normal | 0.18 | 0.06–0.38 | 0.02–0.62 |
albumin (g/l) | 43.00 | 41–46 | 31–50 |
albumin/normal | 1.24 | 1.20–1.39 | 0.89–1.52 |
serum cholesterol (mg/dl) | 190.00 | 167–219 | 101–274 |
cholesterol/normal | 0.86 | 0.83–1.09 | 0.50–1.37 |
sH2a (ng/ml) | 148 | 136–163 | 105–188 |
We analyzed the relation between levels of the different markers and fibrosis stage (
Characteristic | Healthy | HCV patients | P value | Odds ratio (95% CI) | |
Fibrosis stage | |||||
N (n = 28) | 0–2 (n = 31) | 3–4 (n = 13) | |||
Male gender |
15 (53.6) | 18 (58.1) | 10 (76.9) | 0.235 | 2.41 (0.55–10.52) |
Median age (IQR) | 40 (29–48) | 35 (27–49) | 51 (47–55) | 0.019 | 1.08 (1.01–1.16) |
bilirubin/normal >1 | 0 (0) | 0 (0) | 0 (0) | - | - |
ALP/normal >1 | 0 (0) | 1 (3.23) | 3 (23.1) | 0.027 | 10.00 (0.92–108.33) |
ALT/normal >2 | 0 (0) | 10 (32.3) | 8 (61.5) | 0.047 | 4.20 (1.09–17.32) |
AST/normal >1 | 1 (3.6) | 18 (58.1) | 12 (92.3) | 0.008 | 9.33 (1.81–48.24) |
GGT/normal >1 | 0 (0) | 5 (16.1) | 5 (38.5) | 0.075 | 3.71 (0.83–16.55) |
albumin/normal <0.8 | 0 (0) | 1 (3.23) | 0 (0) | 0.529 | 1.4 (1.16–1.70) |
cholesterol/normal >1.2 | 2 (7.1) | 1 (3.23) | 1 (7.69) | 0.476 | 2.73 (0.16–47.46) |
abnormal sH2a | 6 (21.4) | 11 (35.5) | 9 (69.2) | 0.040 | 4.09 (1.02–16.40) |
(sH2a <125 ng/ml or > = 175 ng/ml) |
Data is presented as n (%) except for age, which is presented as median (IQR); odds ratios are from simple logistic regression, using the forced entry method, on each variable. The dependent variable in the regression is state and the outcome is METAVIR score (1 if state is 3–4); CI, confidence interval presented for each explanatory variable.
Given that sH2a levels would reflect liver function, whereas the levels of ALT are indicative of liver damage, we reasoned that a combination of these values would compensate for individual fluctuations in the overall state of each patient. An algorithm combining the levels of sH2a with those of ALT, gave a score of zero for the healthy individuals and a very good correlation with fibrosis stage for the HCV patients (
Our results reveal ASGPR sH2a as a novel non-invasive marker for liver fibrosis and function. SH2a, the enzymes ALT, AST, GGT and ECM components like collagen and hyaluronic acid (HA) reflect different stages of the fibrogenic process. Hepatocytes are targets for most insults to the liver, including hepatitis viruses and alcohol
The membrane-bound precursor of sH2a is cleaved in the endoplasmic reticulum of healthy hepatocytes, traverses the secretory pathway and is secreted to the plasma (step 1). As a consequence of HCV infection and possibly also upon other insults, hepatocytes are damaged, their overall function is compromised and sH2a secretion is reduced, while intracellular enzymes like ALT are released to the plasma (step 2). This starts a process of inflammation (step 3), activation of hepatic stellate cells leading to fibrosis and then cirrhosis, which involve secretion and progressive accumulation of ECM components (step 4).
HA, as well as other proposed tests, can only differentiate very mild from very severe disease (
A method that is being increasingly used is transient elastography (FibroScan), which measures liver stiffness, which in turn is correlated to fibrosis stage. Using this method the AUROC values obtained for significant fibrosis ranged from 0.76 to 0.83, quite suitable but lower than with the combination of sH2a with ALT
The sensitive assessment of hepatocyte function by sH2a could allow the measuring of success of patient treatment. In a longitudinal study during treatment of HCV patients, the change in sH2a levels correlated, unlike other markers, with the success of the therapy (our unpublished results). Studies of membrane ASGPR levels upon resection and recovery or in liver transplantation suggest that sH2a could also be measured to monitor these interventions
SH2a should be further evaluated as it emerges as a good candidate for a suitable liver fibrosis marker. It has been argued that such a marker should: be specific for the liver, be readily available, not be subject to false positive results, identify the stage of fibrosis
We would like to thank Doron Comaheshter of the Tel Aviv Sourasky Medical Center for his assistance in the statistical analysis.