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. 2001 Dec;49(6):852–859. doi: 10.1136/gut.49.6.852

Antiphospholipid antibodies associated with alcoholic liver disease specifically recognise oxidised phospholipids

R Rolla 1, D Vay 1, E Mottaran 1, M Parodi 1, M Vidali 1, M Sartori 1, C Rigamonti 1, G Bellomo 1, E Albano 1
PMCID: PMC1728559  PMID: 11709522

Abstract

BACKGROUND—Circulating antiphospholipid antibodies (aPL) are often detected in patients with alcoholic liver disease (ALD) but little is known about the causes of their formation.
AIMS—We have evaluated whether ethanol mediated oxidative injury might promote the development of aPL in ALD.
PATIENTS AND METHODS—IgG against β2 glycoprotein 1 (β2-GP1), cardiolipin, and human serum albumin (HSA) complexed with either oxidised arachidonic acid (HSA-APP) or malondialdehyde (HSA-MDA) were assayed by ELISA in heavy drinkers with or without ALD and in healthy subjects.
RESULTS—Circulating IgG recognising cardiolipin were significantly higher in ALD patients than in controls. However, anticardiolipin reactivity of ALD sera was only evident using, as the antigen, oxidised cardiolipin but not oxidation protected cardiolipin. In ALD patients, individual values of IgG antioxidised cardiolipin were associated with the titres of antibodies against HSA-MDA and HSA-APP (r=0.68 and 0.72, respectively; p<0.0001) used as markers of oxidative stress. ALD patients also displayed increased levels of antibodies against phospholipid binding protein β2-GP1, and individual reactivity towards oxidised cardiolipin and β2-GP1 were highly correlated (r=0.85; p<0.0001). IgG binding to oxidised cardiolipin, HSA-MDA, and HSA-APP was also significantly higher in β2-GP1 positive than in β2-GP1 negative sera. However, preadsorption of β2-GP1 positive sera on β2-GP1 coated ELISA plates reduced reactivity to oxidised cardiolipin by 80%, without affecting that to HSA-APP or HSA-MDA.
CONCLUSIONS—Ethanol induced oxidative injury is associated with the development of antibodies targeting complexes between oxidised cardiolipin and β2-GP1. These antibodies might account for high aPL titres observed in patients with severe ALD.


Keywords: oxidative stress; lipid peroxidation; β2 glycoprotein 1; ethanol; autoantibodies

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Figure 1  .

Figure 1  

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Immune reactivity against native or oxidised cardiolipin (CL) of sera from 36 patients with alcoholic cirrhosis with or without alcoholic hepatitis (ALD) and from 42 healthy control subjects. IgG binding to ELISA plates coated with CL allowed to auto-oxidise in air or oxidation protected CL was evaluated, as reported in the patients and methods section. The horizontal bars represent median values in each group. Values in each group were normally distributed, as evaluated by the Komolgorov-Smirnov test.

Figure 2  .

Figure 2  

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Reactivity with antigens derived from the reaction of proteins with lipid peroxidation products of sera from patients with alcoholic liver disease positive (Ox-CL+) or negative (Ox-CL ) for the presence of antibodies against oxidised cardiolipin. Human serum albumin (HSA) modified by the reaction with either malondialdehyde (HSA-MDA) or products from arachidonic acid peroxidation (HSA-APP) were used as antigens, as reported in the patients and methods section. Results are mean (SD) ELISA values. **p<0.01, ***p<0.0001 versus Ox-CL subjects.

Figure 3  .

Figure 3  

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Reactivity with oxidised cardiolipin or with antigens derived from the reaction of proteins with lipid peroxidation products of sera from patients with alcoholic liver disease positive (β2-GP1+; n=23) or negative (β2-GP1 ; n=13) for the presence of antibodies against β2-glycoprotein 1. ELISA plates coated with oxidation protected (CL) or oxidised (Ox-CL) cardiolipin, or with malondialdehyde (HSA-MDA) or products from arachidonic acid peroxidation (HSA-APP) were used to evaluate IgG binding. Results are mean (SD) ELISA values. **p<0.005, ***p<0.001 versus β2-GP1 negative subjects; †††p<0.001 versus native cardiolipin.

Figure 4  .

Figure 4  

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Reactivity with oxidised cardiolipin or human serum albumin (HSA) complexed with lipid peroxidation products following preabsorption with the β2 glycoprotein 1 (β2-GP1) of sera from alcoholic patients. Fifteen sera from patients with alcoholic liver disease displaying high titres of IgG against both cardiolipin and β2-GP1 were incubated overnight at 4°C on irradiated ELISA plates coated with β2-GP1 and tested for IgG binding to oxidised cardiolipin (Ox-CL) or HSA modified by reaction with malondialdehyde (HSA-MDA) or products of arachidonic acid peroxidation (HSA-APP). Results are mean (SD) ELISA values obtained with native sera or β2-GP1 preabsorbed sera. *p<0.0001 versus non-pre-absorbed sera.

Figure 5  .

Figure 5  

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Reactivity against oxidised cardiolipin (Ox-CL) in relation to the severity of alcohol liver injury. The clinical severity of hepatic damage was estimated according to Maddrey's DF classification23 (A) or Child-Turcotte classification (B). Patients with DF values less than 90 (n=26) were considered to have mild or moderate hepatic injury and patients with DF values greater than 90 (n=10) represented the group with severe damage. Child A group consisted of 15 patients, while 21 subjects belonged to Child B-C groups. The optical density (od) values in each group were normally distributed, as evaluated by the Komolgorov-Smirnov test. ***p<0.0002 versus DF <90; **p<0.01 versus Child A group.

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