Abstract
Advances in analytical methodologies in the isolation, characterization, and quantitation of lipid-soluble antioxidants, such as vitamin E, have evolved since its discovery in 1922 (1). The primary purpose of vitamin E (tocopherol) and its tocopherol homologs is to act as free radical scavengers to control lipid oxidation in the body (2). The physiological and biochemical processes lipid-soluble vitamins possess are beneficial in preventing a number of degenerative diseases and conditions such as Alzheimer’s disease and Parkinson’s disease (3–7), cardiovascular disease (8–10), and cancer (11–13). Pharmaceutical, medical, as well as industrial disciplines have been interested in the clinical outcomes of tocopherol studies. Thus, a critical need exists to measure multiple tocopherols and their oxidation products simultaneously in human tissue (2,14–19). Our laboratory is interested in studying lipid nitration products in biological matrices primarily as biomarkers to determine the level of oxidative stress and tissue damage. 3-Nitrotyrosine has been determined to be a useful protein biomarker employed to measure oxidative stress based in previous publications (20–26). The most commonly measured nitrated tocopherol compound of interest is 5-NO2-γ-tocopherol (2,19,27,28). This and other tocopherol congeners of interest are depicted in Fig. 1. High performance liquid chromatography (HPLC) with combined electrochemical and photodiode array detection (HPLC-ECD-PDA) is a relatively new, versatile and reliable chromatographic technique employed to generate high sensitivity and selectivity, reproducibility, and accuracy in the determination of tocopherol homologs.
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References
Serbinova, E. A. and Packer, L. (1994) Antioxidant properties of α-tocopherol and α-tocotrienol. Methods Enzymol. 234, 354–366.
Hensley, K., Williamson, K. S., and Floyd, R. A. (2000) Measurement of 3-nitrotyrosine and 5-nitro-γ-tocopherol by high-performance liquid chromatography with electrochemical detection. Free Radic. Biol. Med. 28, 520–528.
Sano, M., Ernesto, C., Thomas, R. G., Klauber, M. R., Schafer, K., Grundman, M., et al. (1997) A controlled trial of selegiline, alpha-tocopherol, or both as treatment for Alzheimer’s disease. N. Engl. J. Med. 336, 1216–1222.
Jiménez-Jiménez, F. J., Bustos de, F., Molina, J. A., Benito-León, J., Tallón-Barranco, A., Gasalla, T., et al. (1997) Cerebrospinal fluid levels of alpha-tocopherol (vitamin E) in Alzheimer’s disease. J. Neural Transm. 104, 703–710.
Metcalfe, T., Bowen, D. M., and Muller, D. P. R. (1989) Vitamin E concentrations in human brain of patients with Alzheimer’s disease, fetuses with Down’s syndrome, centenarians, and controls. Neurochem. Res. 14, 1209–1212.
Grundman, M. (2000) Vitamin E and Alzheimer’s disease: the basis for additional clinical trials. Am. J. Clin. Nutr. 71(suppl), 630S–636S.
Adams, Jr., J. D., Klaidman, L. K., Odunze, I. N., Shen, H. C, and Miller, C. A. (1991) Alzheimer’s and Parkinson’s disease: Brain levels of glutathione, glutathione disulfide, and vitamin E. Mol. Chem. Neuropathol. 14, 213–226.
Janero, D. R. (1991) Therapeutic potential of vitamin E in the pathogenesis of spontaneous atherosclerosis. Free Radic. Biol. Med. 11, 129–144.
Rimm, E. B., Stampfer, M. J., Ascherio, A., Giovannucci, E., Colditz, G A., and Willett, W.C. (1993) Vitamin E consumption and the risk of coronary heart disease in men. N. Engl. J. Med. 328, 1450–1456.
Armstrong, N. C, Paganga, G., Brunner, E., Miller, N. J., Nanchahal, K., Shipley, M., et al. (1997) Reference values for α-tocopherol and β-carotene in the Whitehall II Study. Free Rad. Res. 27, 207–219.
Gridley, G., McLaughlin, J. K., Block, G., Blot, W.J., Gluch, M., and Fraumeni, Jr., J. F. (1992) Vitamin supplement use and reduce risk of oral and pharyngeal cancer. Am. J. Epidemiol. 135, 1083–1092.
Wald, N. J., Boreham, J., Hayward, J. L., and Bulbrook, R. D. (1984) Plasma retinol, β-carotene, and vitamin E levels in relation to the future risk of breast cancer. Br. J. Cancer. 49, 321–324.
Bostick, R. M., Potter, J. D., McKenzie, D. R., Sellers, T. A., Kushi, L. H., Steinmetz, K. A., and Folsom, A. R. (1993) Reduced risk of colon cancer with high intake of vitamin E: the Iowa’s women health study. Cancer Res. 53, 4230–4237.
Simon, E., Paul, J. L., Atger, V., Simon, A., and Moatti, N. (2000) Study of vitamin E net mass transfer between α-tocopherol-enriched HDL and erythrocytes: application to asymptomatic hypercholesterolemic men. Free Radic. Biol. Med. 28, 815–823.
Schüep, W. and Rettenmaier, R. (1994) Analysis of vitamin E homologs in plasma and tissue: high-performance liquid chromatography. Methods Enzymol. 234, 294–302.
Zaman, Z., Fielden, P., and Frost, P. G (1993) Simultaneous determination of vitamins A and E and carotenoids in plasma by reverse-phase HPLC in elderly and younger subjects. Clin. Chem. 39, 2229–2234.
Stahl, W., Graf, P., Brigelius-Flohé, R., Wechter, W., and Sies, H. (1999) Quantification of the α-and γ-tocopherol metabolites 2,5,7,8-tetramethyl-2-(2′-carboxyethyl)-6-hydroxychroman and 2,7,8-trimethyl-2-(2′-carboxyethyl)-6-hydroxychroman in human serum. Anal. Biochem. 275, 254–259.
Katsanidis, E. and Addis, P. B. (1999) Novel HPLC analysis of tocopherols, tocotrienols, and cholesterol in tissue. Free Radic. Biol. Med. 27, 1137–1140.
Hensley, K., Williamson, K. S., Maidt, M. L., Gabbita, S. P., Grammas, P., and Floyd, R. A. (1999) Determination of biological oxidative stress using high performance liquid chromatography with electrochemical detection (HPLC-ECD). J. High Res. Chromatogr. 22, 429–437.
Hensley, K., Maidt, M. L., Pye, Q. N., Stewart, C. A., Wack, M., Tabatabaie, T., and Floyd, R. A. (1997) Quantitation of protein-bound 3-nitrotyrosine and 3,4-dihyroxyphenylalanine by high-performance liquid chromatography with electrochemical array detection. Anal. Biochem. 251, 187–195.
Hensley, K., Maidt, M. L., Yu, Z. Q., Sang, H., Markesbery, W. R., and Floyd, R. A. (1998) Electrochemical analysis of protein nitrotyrosine and dityrosine in the Alzheimer brain reveals region-specific accumulation. J. Neurosci. 18, 8126–8132.
Maruyama, W., Hashizume, Y., Matsubara, K., and Naoi, M. (1996) Identification of 3-nitro-l-tyrosine, a product of nitric oxide and superoxide, as an indicator of oxidative stress in the human brain. J. Chromatogr. B Biomed. Appl. 676, 153–158.
Shigenaga, M. K. (1999) Quantitation of protein-bound 3-nitrotyrosine by high-performance liquid chromatography with electrochemical detection. Methods Enzymol. 301, 27–40.
Kaur, H., Lyras, L., Jenner, P., and Halliwell, B. (1998) Artefacts in HPLC detection of 3-nitrotyrosine in human brain tissue. J. Neurochem. 70, 2220–2223.
Evans, P., Kaur, H., Mitchinson, M. J., and Halliwell, B. (1996) Do human atherosclerotic lesions contain nitrotyrosine. Biochem. Biophys. Res. Commun. 226, 346–351.
Herce-Pagliai, C., Kotecha, S., and Shuker, D. E. (1998) Analytical Methods for 3-nitrotyrosine as a marker of exposure to reactive nitrogen species: a review. Nitric Oxide 2, 324–336.
Cooney, R. V., Harwood, P. J., Franke, A. A., Narala, K., Sundström, A. K., Berggren, P. O., and Mordan, L. J. (1995) Products of γ-tocopherol reaction with NO2 and their formation in rat insulinoma (RINm5F) cells. Free Radic. Biol. Med. 19, 259–269.
Williamson, K. S., Gabbita, S. P., Mou, S., West, M., Pye, Q. N., Markesbery, W. R., et al. (2002) The nitration product 5-nitro-γ-tocopherol is increased in the Alzheimer brain. Nitric Oxide 6, 221–227.
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Williamson, K.S., Hensley, K., Floyd, R.A. (2003). HPLC With Electrochemical and Photodiode Array Detection Analysis of Tocopherol Oxidation and Nitration Products in Human Plasma. In: Hensley, K., Floyd, R.A. (eds) Methods in Biological Oxidative Stress. Methods in Pharmacology and Toxicology. Humana Press. https://doi.org/10.1385/1-59259-424-7:67
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DOI: https://doi.org/10.1385/1-59259-424-7:67
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