4-HNE Immunohistochemistry and Image Analysis for Detection of Lipid Peroxidation in Human Liver Samples Using Vitamin E Treatment in NAFLD as a Proof of Concept

Abstract

Lipid peroxidation is a common feature of liver diseases, especially non-alcoholic fatty liver disease (NAFLD). There are limited validated tools to study intra-hepatic lipid peroxidation, especially for small specimen. We developed a semi-quantitative, fully automated immunohistochemistry assay for the detection of 4-hydroxynoneal (4-HNE) protein adducts, a marker of lipid peroxidation, for adaptation to clinical diagnostics and research. We used Hep G2 cells treated with 4-HNE to validate specificity, sensitivity, and dynamic range of the antibody. Staining and semi-quantitative automated readout were confirmed in human needle-biopsy liver samples from subjects with NAFLD and normal liver histology. The ability to detect changes in lipid peroxidation was tested in paired liver biopsies from NAFLD subjects, obtained before and after 4 weeks of treatment with the antioxidant vitamin E (ClinicalTrials.gov NCT01792115, n=21). The cellular calibrator was linear and NAFLD patients had significantly higher levels of 4-HNE adducts compared to controls (p=0.02). Vitamin E treatment significantly decreased 4-HNE (p=0.0002). Our findings demonstrate that 4-HNE quantification by immunohistochemistry and automated image analysis is feasible and able to detect changes in hepatic lipid peroxidation in clinical trials. This method can be applied to archival and fresh samples and should be considered for use in assessing NAFLD histology.

Keywords: 4-HNE adducts; 4-hydroxynonenal; NAFLD; immunoassay; immunohistochemistry; lipid peroxidation.

Conflict of interest statement

Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.

Calibration curves of cells treated with 4-HNE. Hep G2 cells treated with different doses of 4-HNE (1 = media, 2 = 0, 3 = 25, 4 = 50, 5 = 75, 6 = 100, 7 = 150, 8 = 200 µmol/l) were incubated with mouse monoclonal [HNEJ-2] at either 1:100 (filled circle, 1 µg/ml) or 1:200 (empty circle, 2 µg/ml) dilution for 1 hr at room temperature. (A) Shows three individual arrays for both dilutions. Background staining of the solvent control (0 µmol/l) was subtracted from all points. In the linear regression, 25 µmol/l was excluded for both dilutions as it did not differ from the background staining (B). Slopes were 1.071 (1:100) and 1.262 (1:200) while Y intercepts were -22.84 (1:100) and -65.44 (1:200), respectively. Quantification was performed using Visiopharm software. Data are shown as mean ± SD. Fig. 1C shows a representative image of 4-HNE staining in human liver with the corresponding image analysis (1D). Cytoplasmic 4-HNE was quantified and cells were classified as positive cells (white-colored spots) or negative cells (blue-colored spots). Yellow marks nuclei and green represents the combination of blue and yellow. Data are shown as mean ± SD. Scale bar, 100 µm. Abbreviations: 4-HNE, 4-hydroxynoneal; HNEJ-2, mouse monoclonal anti-4 hydroxynonenal antibody.

Figure 2.

4-HNE staining is increased in NAFLD. Compared to controls with normal liver histology (n=5), patients with NAFLD (n=21) show a significant increase in 4-HNE positive cells. Data is shown as mean + SEM. Abbreviations: 4-HNE, 4-hydroxynoneal; NAFLD, non-alcoholic fatty liver disease.

Figure 3.

4-HNE staining is not correlated with (A) ALT, (B) BMI, or (C) liver fat determined by magnetic resonance spectroscopy. A Spearman’s rank correlation coefficient, B, C Pearson’s correlation coefficient. Abbreviations: ALT, alanine aminotransferase; BMI, body mass index; 4-HNE, 4-hydroxynoneal.

Figure 4.

Four weeks of treatment with RRR-α-tocopherol (200–800 IU/day) decreased 4-HNE positive cells (A), with no differences between doses (B). Data are shown as mean + SEM. Abbreviations: 4-HNE, 4-hydroxynoneal; RRR-α-tocopherol, natural vitamin E.