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. 2005 Jul;54(7):987-93.
doi: 10.1136/gut.2004.057968.

Immune response towards lipid peroxidation products as a predictor of progression of non-alcoholic fatty liver disease to advanced fibrosis

E Albano  1 , E Mottaran, M Vidali, E Reale, S Saksena, G Occhino, A D Burt, C P Day
Affiliations

Immune response towards lipid peroxidation products as a predictor of progression of non-alcoholic fatty liver disease to advanced fibrosis

E Albano et al. Gut. 2005 Jul.

Abstract

Aims: Factors responsible for the progression of non-alcoholic fatty liver disease (NAFLD) to more severe liver injury are poorly understood. In the present study, we investigated the association between immune reactions triggered by oxidative stress and stage of NAFLD.

Methods: Titres of IgG against human serum albumin adducted with malondialdehyde (MDA-HSA) or arachidonic acid hydroperoxide (AAHP) and against oxidised cardiolipin (Ox-CL) were measured in 167 NAFLD patients with steatosis only (n = 79), steatohepatitis (n = 74), or steatosis plus cirrhosis (n = 14), and in 59 age and sex matched controls.

Results: Circulating IgG against lipid peroxidation products was significantly higher (p<0.001) in NAFLD patients than in controls. Oxidative stress dependent immune responses were not associated with obesity, type 2 diabetes, or with serum cholesterol, ferritin, or aminotransferase levels. Titres of lipid peroxidation related antibodies were also independent of the extent of steatosis and were similarly distributed in patients with and without necroinflammation. In contrast, the same antibodies were significantly increased in patients with advanced fibrosis or cirrhosis. Logistic regression analysis confirmed that anti-MDA antibodies were independently associated with progression of NALFD and that NAFLD patients with titres of anti-MDA-HSA antibodies above the control threshold value had a threefold (relative risk 2.82 (95% confidence interval 1.35-5.90); p = 0.007) higher risk of having advanced fibrosis/cirrhosis than patients whose antibody titres were within the control range.

Conclusions: These results indicate that the presence of immune reactions triggered by oxidative stress can be an independent predictor of progression of NAFLD to advanced fibrosis.

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Figures

Figure 1
Figure 1
Titres of IgG against human serum albumin adducted with malondialdehyde (MDA-HSA) and arachidonic acid hydroperoxides (AAHP-HSA) or against oxidised cardiolipin (Ox-CL) in patients with non-alcoholic fatty liver disease (NAFLD) and in healthy controls. Sera were tested at a 1:50 dilution in microplate ELISA plates coated with different antigens and revealed with peroxidase linked goat antihuman IgG antiserum. Results are expressed as optical density (od) at 490 nm after subtracting the background reactivity of each serum. Boxes show values within 25th and 75th percentiles, horizontal bar represents the median, 80% of values are between the extremities of the vertical bars (10th–90th percentiles), and extreme values are represented by individual symbols. Values under the boxes represent the percentage of subjects with IgG titres above the cut off value, calculated on the 97.5th percentile of the control population. Statistical significance, NAFLD versus controls: MDA-HSA p<0.0001 (95% confidence interval (CI) −0.169 to −0.11); AAHP-HSA, p<0.0001 (95% CI −0.042 to −0.018); Ox-CL p<0.0001 (95% CI −0.059 to −0.020).
Figure 2
Figure 2
Distribution of IgG against human serum albumin adducted with malondialdehyde (MDA-HSA) and arachidonic acid hydroperoxides (AAHP-HSA) or against oxidised cardiolipin (Ox-CL) in patients with non-alcoholic fatty liver disease (NAFLD) according to the extent of steatosis (A) or the presence of necroinflammation (B). Steatosis was scored as mild/moderate (grades 1–2) or severe (grade 3), as reported in the methods section. Sera were tested at 1:50 dilution in microplate enzyme linked immunoabsorbent assay plates and antibody binding was revealed with peroxidase linked goat antihuman IgG antiserum. Results are expressed as optical density (od) at 490 nm after subtracting the background reactivity of each serum. Boxes show values within 25th and 75th percentiles, horizontal bar represents the median, 80% of values are between the extremities of the vertical bars (10th–90th percentiles), and extreme values are represented by individual symbols.
Figure 3
Figure 3
Titres of IgG against human serum albumin adducted with malondialdehyde (MDA-HSA) (A) or against oxidised cardiolipin (Ox-CL) (B) in patients with non-alcoholic fatty liver disease according to fibrosis stage. Sera were tested at a 1:50 dilution in microplate enzyme linked immunoabsorbent assay plates coated with different antigens and revealed with peroxidase linked goat antihuman IgG antiserum. Boxes show values within 25th and 75th percentiles, horizontal bar represents the median, 80% of values are between the extremities of the vertical bars (10th–90th percentiles), and extreme values are represented by individual symbols. Statistical significance: *p<0.05 versus no fibrosis (95% confidence interval (CI) 0.01 to 0.15) and fibrosis stages 1–2 (95% CI 0.01 to 0.16); **p<0.05 versus no fibrosis (95% CI 0.01 to 0.12) and fibrosis stages 1–2 (95% CI 0.01–0.11).
Figure 4
Figure 4
Characterisation of the antibody response towards malondialdehyde (MDA) adducts in patients with non-alcoholic fatty liver disease (NAFLD). Sera of 28 NAFLD patients with reactivity against MDA adducts and 13 sera from healthy controls (1:50 dilution) were preincubated overnight at 4°C with 20 μmol/l synthetic hexyl-4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde (MDD) diluted in coating buffer. The same sera were also similarly incubated overnight without additions. Aliquots of the sera were then added in duplicated to enzyme linked immunoabsorbent assay plates coated with MDA-human serum albumin (HSA) and the antibody binding was assayed as described in the methods section. Affinity purified rabbit polyclonal IgG directed against the MDD adduct was used as reference. The insert show the chemical structure of the hexyl-MDD adduct.
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