Plasma and urine biomarkers in acute viral hepatitis E

Abstract

Background: Hepatitis E, caused by the hepatitis E virus (HEV), is endemic to developing countries where it manifests as waterborne outbreaks and sporadic cases. Though generally self-limited with a low mortality rate, some cases progress to fulminant hepatic failure (FHF) with high mortality. With no identified predictive or diagnostic markers, the events leading to disease exacerbation are not known. Our aim is to use proteomic tools to identify biomarkers of acute and fulminant hepatitis E.

Results: We analyzed proteins in the plasma and urine of hepatitis E patients and healthy controls by two-dimensional Differential Imaging Gel Electrophoresis (DIGE) and mass spectrometry, and identified over 30 proteins to be differentially expressed during acute hepatitis E. The levels of one plasma protein, transthyretin, and one urine protein, alpha-1-microglobulin (alpha1m), were then quantitated by enzyme immunoassay (EIA) in clinical samples from a larger group of patients and controls. The results showed decreased plasma transthyretin levels (p < 0.005) and increased urine alpha1m levels (p < 0.001) in acute hepatitis E patients, compared to healthy controls. Preliminary results also showed lower urine zinc alpha glycoprotein levels in fulminant hepatitis E compared to acute disease; this remains to be confirmed with more fulminant cases.

Conclusion: Our results demonstrate the utility of characterizing plasma and urine proteomes for signatures of the host response to HEV infection. We predict that plasma transthyretin and urine alpha1m could be reliable biomarkers of acute hepatitis E. Besides the utility of this approach to biomarker discovery, proteome-level changes in human biofluids would also guide towards a better understanding of host-virus interaction and disease.

Figure 1

Workflow for proteomic analysis. Proteins in the plasma or urine of healthy controls and hepatitis E patients were labelled with Cy2, Cy3 or Cy5 dyes as described in Methods. The labelled samples were mixed and subjected to different fractionation strategies. In workflow A, the labelled samples were first subjected to Multi Lectin Affinity Chromatography (MLAC) to obtain fractions enriched in glycoproteins (G fraction) and non-glycoproteins (NG fraction). These fractions were then separately fractionated by mixed cation-anion exchange chromatography (CAX). The six fractions obtained by step salt elution were then analysed by SDS-PAGE. In workflow B, the labelled samples were subjected to MLAC and the enriched fractions were resolved by two-dimensional gel electrophoresis (2DGE). Workflows A and B were followed for plasma samples. Workflow C was followed for urine samples for which the labelled samples were directly fractionated by 2DGE. The differentially expressed spots were analysed with DeCyder 2D v6.5, the selected spots were cut out and the proteins identified by mass spectrometry.

Figure 2

Analysis of plasma fractions. (A) Workflow A, NG fraction separated by CAX. Lanes: 1, NG fraction; 2, CAX flow-through fraction; 3-8, CAX eluates with 100, 200, 300, 400, 500 or 600 mM NaCl. The boxed bands NG-1, NG-2, NG-3 and NG-4 were identified by mass spectrometry. (B) DIGE images of (1) glycosylated and (2) non-glycosylated protein enriched fractions resolved by 2DGE. The proteins in spots P1 to P14 were identified by mass spectrometry and are shown in Table 1.

Figure 3

Analysis of the urine proteome. (A) In Workflow C, pooled urine samples were labelled and resolved by 2DGE. (1) DIGE image of proteins from pools of healthy normals and hepatitis E patients labelled with Cy3 and Cy5, respectively. (2) DIGE image of proteins from pools of hepatitis E patients and healthy normals labelled with Cy3 and Cy5, respectively. (B) DIGE image of urine proteins from acute mild hepatitis patient (H3) labelled with Cy3 and those from a case of fulminant hepatitis E (H4) labelled with Cy5. Box 1 shows proteins spots that were downregulated in fulminant hepatitis compared to acute mild infection, while Box 2 shows proteins that remained unchanged between the two forms of disease.

Figure 4

Validation of representative biomarkers by EIA. The EIAs were carried out as described in Methods. (A) Plasma levels of transthyretin were estimated for various groups of hepatitis patients and controls. The p values shown are based on the Mann-Whitney test; the significance remains the same with the Student's t-test. (B) The levels of alpha-1-microglobulin in urine were estimated for various groups of hepatitis patients and controls. The p-values shown are based on both parametric (Student's t test) and non-parametric (Mann-Whitney U test) tests. All p values shown are relative to Group 5, except in (B) in which Groups 1 and 3 were also compared.