Protein-bound acrolein: potential markers for oxidative stress

Abstract

Acrolein (CH2==CH---CHO) is known as a ubiquitous pollutant in the environment. Here we show that this notorious aldehyde is not just a pollutant, but also a lipid peroxidation product that could be ubiquitously generated in biological systems. Upon incubation with BSA, acrolein was rapidly incorporated into the protein and generated the protein-linked carbonyl derivative, a putative marker of oxidatively modified proteins under oxidative stress. To verify the presence of protein-bound acrolein in vivo, the mAb (mAb5F6) against the acrolein-modified keyhole limpet hemocyanin was raised. It was found that the acrolein-lysine adduct, Nepsilon-(3-formyl-3, 4-dehydropiperidino)lysine, constitutes an epitope of the antibody. Immunohistochemical analysis of atherosclerotic lesions from a human aorta demonstrated that antigenic materials recognized by mAb5F6 indeed constituted the lesions, in which intense positivity was associated primarily with macrophage-derived foam cells and the thickening neointima of arterial walls. The observations that (i) oxidative modification of low-density lipoprotein with Cu2+ generated the acrolein-low-density lipoprotein adducts and (ii) the iron-catalyzed oxidation of arachidonate in the presence of protein resulted in the formation of antigenic materials suggested that polyunsaturated fatty acids are sources of acrolein that cause the production of protein-bound acrolein. These data suggest that the protein-bound acrolein represents potential markers of oxidative stress and long-term damage to protein in aging, atherosclerosis, and diabetes.

Figure 1

Introduction of carbonyl groups into protein by reaction with acrolein. BSA (1 mg/ml) was incubated with 1 mM acrolein in 50 mM sodium phosphate buffer (pH 7.4) at 37°C. The protein carbonyl content was determined by the procedure using 2,4-dinitrophenylhydrazine.

Figure 2

Structure of N^α-acetyl-N^ɛ-(3-formyl-3, 4-dehydropiperidino)lysine (N^α-acetyl-FDP-lysine) (A) and N^α-acetyl-N^im-propanalhistidine (B).

Figure 3

Specificity of mAb5F6. (A) Immunoreactivity of mAb5F6 to the aldehyde-treated protein. Affinity of mAb5F6 was determined by a direct ELISA using aldehyde-treated BSA as the absorbed antigen. A coating antigen was prepared by incubating 1 mg of BSA with 1 mM aldehyde in 1 ml of 50 mM sodium phosphate buffer, pH 7.4, for 2 h at 37°C. (B) Competitive ELISA with FDP-lysine. Competitors: ○, N^α-acetyllysine; •, N^α-acetyl-FDP-lysine; ▵, N^α-acetylhistidine; ▴, N^α-acetyl-N^im-propanalhistidine

Figure 4

Immunohistochemical detection of protein-bound acrolein in fatty streak lesions of arterial tissue. Arterial tissue specimen from a 69-year-old male with atherosclerosis was immunostained with anti-CD68 antibody (A), mAb5F6 (B and D), and rabbit polyclonal antiserum raised against acrolein-modified KLH (C). For a competitive experiment, mAb5F6 preincubated with an excess of N^α-acetyl-N^ɛ-(3-formyl-3, 4-dehydropiperidino)lysine (N^α-acetyl-FDP-lysine) was used (D). The nuclei were counterstained with Meyer’s hematoxylin. The staining with polyclonal antiserum and mAb5F6 was positive in fatty streak lesions. (×200.)

Figure 5

In vitro formation of protein-bound acrolein via lipid peroxidation. (A) Formation of protein-bound acrolein in the oxidized LDL. LDL (0.5 mg/ml) was incubated in the absence (○) or presence of 5 μM Cu²⁺ (•) in 50 mM sodium phosphate buffer (pH 7.4) at 37°C. (B) Formation of protein-bound acrolein in BSA during the iron-catalyzed oxidation of arachidonate. The iron-catalyzed oxidation of arachidonate in the presence of BSA was performed by incubating BSA (1 mg/ml) with 2 mM arachidonate in the presence of either 10 μM Fe²⁺, 1 mM ascorbate, or 10 μM Fe²⁺ and 1 mM ascorbate in 0.1 ml of 0.1 M sodium phosphate buffer (pH 7.4) at 37°C. ○, Control (without arachidonate); ⋄, arachidonate; □, Fe²⁺ + arachidonate; ▪, ascorbate + arachidonate; •, Fe²⁺/ascorbate + arachidonate.