Serum Sphingolipids Reflect the Severity of Chronic HBV Infection and Predict the Mortality of HBV-Acute-on-Chronic Liver Failure

<div><p>Patients with HBV-acute-on-chronic liver failure (HBV-ACLF) have high mortality and frequently require liver transplantation; few reliable prognostic markers are available. As a class of functional lipids, sphingolipids are extensively involved in the process of HBV infection. However, their role in chronic HBV infection remains unknown. The aim of this study was to determine the serum sphingolipid profile in a population of patients with chronic HBV infection, paying special attention to exploring novel prognostic markers in HBV-ACLF. High performance liquid chromatography tandem mass spectrometry was used to examine the levels of 41 sphingolipids in 156 serum samples prospectively collected from two independent cohorts. The training and validation cohorts comprised 20 and 28 healthy controls (CTRL), 29 and 23 patients with chronic hepatitis B (CHB), and 30 and 26 patients with HBV-ACLF, respectively. Biometric analysis was used to evaluate the association between sphingolipid levels and disease stages. Multivariate analysis revealed difference of sphingolipid profiles between CHB and HBV-ACLF was more drastic than that between CTRL and CHB, which indicated that serum sphingolipid levels were more likely to associate with the progression HBV-ACLF rather than CHB. Furthermore, a 3-month mortality evaluation of HBV-ACLF patients showed that dhCer(d18∶0/24∶0) was significantly higher in survivors than in non-survivors (including deceased patients and those undergoing liver transplantation, <i>p</i><0.05), and showed a prognostic performance similar to that of the MELD score. The serum sphingolipid composition varies between CTRL and chronic HBV infection patients. In addition, dhCer(d18∶0/24∶0) may be a useful prognostic indicator for the early prediction of HBV-ACLF.</p></div

Identification of potential biomarkers through OPLS-DA.

<p>(<b>A</b>),(<b>B</b>). Score plots obtained from the training cohort by OPLS-DA. R2Y(cum) and Q2(cum) values are displayed below each panel; these values have a range of 0 to 1 and represent the stability and predictability of the model, respectively. Dots with different colors representing different groups are well separated based on their sphingolipidome data, indicating serum sphingolipidome varied among groups significantly. (<b>C</b>),(<b>D</b>). T-predicted scatter plot of the OPLS-DA model using sphingolipidome data obtained from the validation cohort. The result shows that the OPLS-DA models correctly predicted 86% of V-CTRL and 83% of V-CHB patients within the 95% CI (panel C, CTRL vs. CHB), and correctly predicted 100% of V-CHB and 100% of V-CTRL subjects within the 95% CI (panel D, CHB vs. ACLF). (<b>E</b>),(<b>F</b>). Validation plot of the PLS-DA models obtained using 100 permutation tests to reveal the risk of overfit from the model. The intercept for the blue Q2 line should be below 0.1. Note: V-: corresponding validation group in the validation cohort.</p

Annual total cost and the composition among CHB patients from 2008 to 2012.

<p>Annual total cost and the composition among CHB patients from 2008 to 2012.</p

Demographic characteristic of CHB patients from 2008 to 2012 after PS matching.

<p>Demographic characteristic of CHB patients from 2008 to 2012 after PS matching.</p

Annual total cost and the composition among CHB patients with different insurance types from 2008 to 2012.

<p>Annual total cost and the composition among CHB patients with different insurance types from 2008 to 2012.</p

Serum sphingolipid levels in the training cohort (healthy controls, chronic hepatitis patients, and HBV-ACLF patients).

<p>Data are expressed as the mean ± SEM. *, significant difference compared with healthy controls (*  = <i>p</i><0.05, **  = <i>p</i><0.01); &, significant difference compared with CHB (&  = <i>p</i><0.05, &&  = <i>p</i><0.01).</p

Patient selection procedure.

<p>Patient selection procedure.</p

Antiviral composition of outpatients prescribed antivirals with different insurance types from 2008–2012.

<p>*Note: some patients treated with more than one antiviral at the same time may increase the sum of antiviral composition to more than 1 in each year.</p

Demographic characteristic of CHB patients from 2008 to 2012.

<p>Demographic characteristic of CHB patients from 2008 to 2012.</p

Baseline characteristics of patients with HBV-ACLF.

a<p>Medicine: Methylprednisolone.</p>b<p>Medicine: Thymic peptide α1 or Thymopentin.</p