Smoking Has No Effect on the Amino Acid Composition of Apolipoprotein B100 of LDL While Directly Influencing the Antioxidant Status Ananth Sekher Pannala,* K. Richard Bruckdorfer,† and Catherine A. Rice-Evans* ,1 *Wolfson Centre for Age Related Diseases, GKT School of Biomedical Sciences, King’s College London, St. Thomas’ Street, London SE1 9RT, United Kingdom; and †Department of Biochemistry and Molecular Biology, Royal Free and University College Hospital & Medical School, Rowland Hill Street, London NW3 2PF, United Kingdom Received February 13, 2002 Previous studies have demonstrated increased plasma levels of oxidised low-density lipoprotein (oxLDL) in chronic smokers, which has been associ- ated with the extent of endothelial dysfunction. In this study we examine the relationship between the amino acid composition of apolipoprotein B100 (apo B) of low-density lipoprotein (LDL), by reverse phase HPLC after precolumn derivatisation, between smokers (>40 cigarettes/day) and nonsmokers in relation to their plasma and LDL antioxidant status. While there was a significant difference in the levels of plasma vitamin C and -tocopherol between female smokers and non- smokers, as well as in the levels of LDL -tocopherol, there was no significant difference in the amino acid composition of apo B between the two groups. © 2002 Elsevier Science (USA) Key Words: smoking; vitamin C; -tocopherol; amino acid analysis; protein hydrolysis; precolumn deriva- tisation; o-phthalaldehyde; apolipoprotein B100. There is increasing evidence that increased concen- trations of low-density lipoprotein (LDL) and biologi- cally modified lipoproteins, such as oxidised low-den- sity lipoprotein (oxLDL), are associated in the patho- genesis of atherosclerosis [1– 6]. Apolipoprotein B 100 (apo B) containing 4536 amino acid residues [7] is the major protein moiety of LDL. The altered recognition properties of the oxidised LDL particle, induced by the modification of specific amino acid residues on the apo B, are considered to be responsible for uptake of oxLDL by scavenger receptors on target macrophages, a pro- cess involved in the pathogenesis of the disease [1, 2]. Supplementation with antioxidant nutrients (vita- min E, vitamin C, carotenoids) as well as increased fruit and vegetable intake have been demonstrated to protect LDL from oxidation, when examined ex vivo, in nonsmokers, smokers and subjects exposed to passive smoking [8 –16]. It has been reported that smoking is associated with elevations in plasma LDL levels and a decrease in plasma high-density lipoprotein cholesterol levels [17]. Other indications suggest that smoking cessation leads to elevated levels of HDL cholesterol [18]. It has been proposed that modifications to amino acids such as lysine, which alter the charge and the recognition properties of apo B, increase its recognition and uptake by the macrophage scavenger receptors [2]. To determine the structure of apo B many techniques such as sequencing via mRNA and cDNA [19] and more conventional methods of tryptic digestion [20 –22] and cyanogen bromide [23] cleavage have been utilised. However, because of the relative insolubility of the protein and difficulty in separating the complex pep- tide fractions generated from such cleavage, estima- tion of the accurate composition of the protein has often been difficult. In this study we apply an HPLC analytical approach for the determination of the protein composition of LDL-apo B, by quantitative analysis of the amino acids in protein hydrolysates as their o-phthalaldehyde/ 2-mercaptoethanol derivatives, and the influence of smoking (40 cigarettes/day) on such parameters. The results are compared with the modifications to the amino acid composition after copper-mediated oxida- tion of LDL, conventionally used to study LDL oxidis- ability and uptake. The method described here for amino acid analysis has been standardised through Abbreviations used: LDL, low-density lipoprotein; OPA, o-phthal- aldehyde; RP-HPLC, reversed-phase high-performance liquid chro- matography; apo B, apolipoprotein B100. 1 To whom correspondence and reprint requests should be ad- dressed at Wolfson Centre for Age Related Diseases, GKT School of Biomedical Sciences, King’s College London, St. Thomas’ Street, London SE1 9RT, UK. Fax: +44 207 848 6143. E-mail: catherine. rice-evans@kcl.ac.uk. Biochemical and Biophysical Research Communications 292, 175–183 (2002) doi:10.1006/bbrc.2002.6616, available online at http://www.idealibrary.com on 175 0006-291X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.