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Clinical Chemistry, Vol 35, 1921-1927, Copyright © 1989 by American Association for Clinical Chemistry
H Refsum, PM Ueland and AM Svardal
Department of Pharmacology and Toxicology, University of Bergen, Haukeland Hospital, Norway.
Homocysteine exists in human plasma as various (mixed) disulfides. Most plasma homocysteine (about 70%) is protein bound, probably via a disulfide bond to albumin, whereas homocysteine-cysteine mixed disulfide is the predominating form in the free fraction. We here present a method for the determination of total homocysteine, which includes both fractions. Plasma was initially treated with sodium borohydride to reduce the disulfide bonds, and the liberated thiols were derivatized with monobromobimane. The derivatized sample, still containing the plasma proteins, was injected onto a strong cation- exchange column, from which the homocysteine derivative was directed by column switching into a cyclohexyl silica (CH) column. The homocysteine derivative was top-concentrated on the CH column, then rapidly eluted with a steep gradient of methanol. Both the derivatization procedure and chromatography were performed with a combined sample processor and sample injector from Gilson (Model 232-401). Within-run and between-run precision (CV) was less than 4%, and the detection limit of 0.2 pmol was sufficiently low for monitoring homocysteine in plasma. We verified the assay against two established manual methods for the determination of total homocysteine in plasma. This, the first fully automated assay for total plasma homocysteine, allows the unattended analysis of 70 samples per 24 h.
The following articles in journals at HighWire Press have cited this article:
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  E. Nurk, C. A Drevon, H. Refsum, K. Solvoll, S. E Vollset, O. Nygard, H. A Nygaard, K. Engedal, G. S Tell, and A D. Smith Cognitive performance among the elderly and dietary fish intake: the Hordaland Health Study Am. J. Clinical Nutrition, November 1, 2007; 86(5): 1470 - 1478. [Abstract] [Full Text] [PDF]
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P. Berstad, S. V Konstantinova, H. Refsum, E. Nurk, S. E. Vollset, G. S Tell, P. M Ueland, C. A Drevon, and G. Ursin Dietary fat and plasma total homocysteine concentrations in 2 adult age groups: the Hordaland Homocysteine Study Am. J. Clinical Nutrition, June 1, 2007; 85(6): 1598 - 1605. [Abstract] [Full Text] [PDF]
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 H. S. Abu-Lebdeh, R. Barazzoni, S. E. Meek, M. L. Bigelow, X.-M. T. Persson, and K. S. Nair Effects of Insulin Deprivation and Treatment on Homocysteine Metabolism in People with Type 1 Diabetes J. Clin. Endocrinol. Metab., September 1, 2006; 91(9): 3344 - 3348. [Abstract] [Full Text] [PDF]
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 P.H. Whincup, J.A. Gilg, A.E. Donald, M. Katterhorn, C. Oliver, D.G. Cook, and J.E. Deanfield Arterial Distensibility in Adolescents: The Influence of Adiposity, the Metabolic Syndrome, and Classic Risk Factors Circulation, September 20, 2005; 112(12): 1789 - 1797. [Abstract] [Full Text] [PDF]
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A. Brevik, S. E. Vollset, G. S Tell, H. Refsum, P. M. Ueland, E. B. Loeken, C. A Drevon, and L. F. Andersen Plasma concentration of folate as a biomarker for the intake of fruit and vegetables: the Hordaland Homocysteine Study Am. J. Clinical Nutrition, February 1, 2005; 81(2): 434 - 439. [Abstract] [Full Text] [PDF]
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 R. Rej Clinical Chemistry through Clinical Chemistry: A Journal Timeline Clin. Chem., December 1, 2004; 50(12): 2415 - 2458. [Abstract] [Full Text] [PDF]
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E. Nurk, G. S Tell, S. E Vollset, O. Nygard, H. Refsum, R. M Nilsen, and P. M Ueland Changes in lifestyle and plasma total homocysteine: the Hordaland Homocysteine Study Am. J. Clinical Nutrition, May 1, 2004; 79(5): 812 - 819. [Abstract] [Full Text] [PDF]
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 E. Bozkurt, S. Keles, M. Acikel, M. Islek, and S. Atesal Plasma Homocysteine Level and the Angiographic Extent of Coronary Artery Disease Angiology, May 1, 2004; 55(3): 265 - 270. [Abstract] [PDF]
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H. Refsum, A. D. Smith, P. M. Ueland, E. Nexo, R. Clarke, J. McPartlin, C. Johnston, F. Engbaek, J. Schneede, C. McPartlin, et al. Facts and Recommendations about Total Homocysteine Determinations: An Expert Opinion Clin. Chem., January 1, 2004; 50(1): 3 - 32. [Abstract] [Full Text] [PDF]
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L. El-Khairy, S. E. Vollset, H. Refsum, and P. M. Ueland Plasma Total Cysteine, Mortality, and Cardiovascular Disease Hospitalizations: The Hordaland Homocysteine Study Clin. Chem., June 1, 2003; 49(6): 895 - 900. [Abstract] [Full Text] [PDF]
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L. El-Khairy, S. E Vollset, H. Refsum, and P. M Ueland Plasma total cysteine, pregnancy complications, and adverse pregnancy outcomes: the Hordaland Homocysteine Study Am. J. Clinical Nutrition, February 1, 2003; 77(2): 467 - 472. [Abstract] [Full Text] [PDF]
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L. El-Khairy, S. E. Vollset, H. Refsum, and P. M. Ueland Predictors of Change in Plasma Total Cysteine: Longitudinal Findings from the Hordaland Homocysteine Study Clin. Chem., January 1, 2003; 49(1): 113 - 120. [Abstract] [Full Text] [PDF]
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 P.H. Whincup, J. Danesh, M. Walker, L. Lennon, A. Thomson, P. Appleby, A. Rumley, and G.D.O. Lowe von Willebrand factor and coronary heart disease. Prospective study and meta-analysis Eur. Heart J., November 2, 2002; 23(22): 1764 - 1770. [Abstract] [Full Text] [PDF]
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S. Tonstad, K. Smerud, and L. Hoie A comparison of the effects of 2 doses of soy protein or casein on serum lipids, serum lipoproteins, and plasma total homocysteine in hypercholesterolemic subjects Am. J. Clinical Nutrition, July 1, 2002; 76(1): 78 - 84. [Abstract] [Full Text] [PDF]
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 E. Nurk, G. S. Tell, S. E. Vollset, O. Nygard, H. Refsum, and P. M. Ueland Plasma Total Homocysteine and Hospitalizations for Cardiovascular Disease: The Hordaland Homocysteine Study Arch Intern Med, June 24, 2002; 162(12): 1374 - 1381. [Abstract] [Full Text] [PDF]
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 E. Hogervorst, H. M. Ribeiro, A. Molyneux, M. Budge, and A. D. Smith Plasma Homocysteine Levels, Cerebrovascular Risk Factors, and Cerebral White Matter Changes (Leukoaraiosis) in Patients With Alzheimer Disease Arch Neurol, May 1, 2002; 59(5): 787 - 793. [Abstract] [Full Text] [PDF]
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S. E. Vollset, H. Refsum, A. Tverdal, O. Nygard, J. E. Nordrehaug, G. S Tell, and P. M. Ueland Plasma total homocysteine and cardiovascular and noncardiovascular mortality: the Hordaland Homocysteine Study Am. J. Clinical Nutrition, July 1, 2001; 74(1): 130 - 136. [Abstract] [Full Text] [PDF]
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L. El-Khairy, P. M. Ueland, H. Refsum, I. M. Graham, and S. E. Vollset Plasma Total Cysteine as a Risk Factor for Vascular Disease : The European Concerted Action Project Circulation, May 29, 2001; 103(21): 2544 - 2549. [Abstract] [Full Text] [PDF]
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 M K Al-Obaidi, P J Stubbs, R Amersey, and M I M Noble Acute and convalescent changes in plasma homocysteine concentrations in acute coronary syndromes Heart, April 1, 2001; 85(4): 380 - 384. [Abstract] [Full Text]
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 E. Nurk, G. S. Tell, O. Nygård, H. Refsum, P. M. Ueland, and S. E. Vollset Plasma Total Homocysteine Is Influenced by Prandial Status in Humans: The Hordaland Homocysteine Study J. Nutr., April 1, 2001; 131(4): 1214 - 1216. [Abstract] [Full Text]
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 M. K. Al-Obaidi, P. J. Stubbs, P. Collinson, R. Conroy, I. Graham, and M. I. M. Noble Elevated homocysteine levels are associated with increased ischemic myocardial injury in acute coronary syndromes J. Am. Coll. Cardiol., October 1, 2000; 36(4): 1217 - 1222. [Abstract] [Full Text] [PDF]
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P. J. Stubbs, M. K. Al-Obaidi, R. M. Conroy, MusB, P. O. Collinson, MRCPath, I. M. Graham, FRCPI, and M. I. M. Noble Effect of Plasma Homocysteine Concentration on Early and Late Events in Patients With Acute Coronary Syndromes Circulation, August 8, 2000; 102(6): 605 - 610. [Abstract] [Full Text] [PDF]
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S. E. Vollset, H. Refsum, L. M Irgens, B. M. Emblem, A. Tverdal, H. K Gjessing, A. L. B. Monsen, and P. M. Ueland Plasma total homocysteine, pregnancy complications, and adverse pregnancy outcomes: the Hordaland Homocysteine Study Am. J. Clinical Nutrition, April 1, 2000; 71(4): 962 - 968. [Abstract] [Full Text] [PDF]
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C.-L. Chao, T.-L. Kuo, and Y.-T. Lee Effects of Methionine-Induced Hyperhomocysteinemia on Endothelium-Dependent Vasodilation and Oxidative Status in Healthy Adults Circulation, February 8, 2000; 101(5): 485 - 490. [Abstract] [Full Text] [PDF]
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M. K. Al-Obaidi, H. Philippou, P. J. Stubbs, A. Adami, R. Amersey, M. M. Noble, and D. A. Lane Relationships Between Homocysteine, Factor VIIa, and Thrombin Generation in Acute Coronary Syndromes Circulation, February 1, 2000; 101(4): 372 - 377. [Abstract] [Full Text] [PDF]
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K. Gempel, K.-D. Gerbitz, B. Casetta, and M. F. Bauer Rapid Determination of Total Homocysteine in Blood Spots by Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry, Clin. Chem., January 1, 2000; 46(1): 122 - 123. [Full Text] [PDF]
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L. El-Khairy, P. M Ueland, O. Nygard, H. Refsum, and S. E Vollset Lifestyle and cardiovascular disease risk factors as determinants of total cysteine in plasma: the Hordaland Homocysteine Study Am. J. Clinical Nutrition, December 1, 1999; 70(6): 1016 - 1024. [Abstract] [Full Text] [PDF]
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P H Whincup, H Refsum, I J Perry, R Morris, M Walker, L Lennon, A Thomson, P M Ueland, and S B J Ebrahim Serum total homocysteine and coronary heart disease: prospective study in middle aged men Heart, October 1, 1999; 82(4): 448 - 454. [Abstract] [Full Text] [PDF]
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 T. H. ROSENQUIST, A. M. SCHNEIDER, and D. T. MONAGHAN N-methyl-D-aspartate receptor agonists modulate homocysteine-induced developmental abnormalities FASEB J, September 1, 1999; 13(12): 1523 - 1531. [Abstract] [Full Text]
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M. J. Magera, J. M. Lacey, B. Casetta, and P. Rinaldo Method for the Determination of Total Homocysteine in Plasma and Urine by Stable Isotope Dilution and Electrospray Tandem Mass Spectrometry Clin. Chem., September 1, 1999; 45(9): 1517 - 1522. [Abstract] [Full Text] [PDF]
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G. Holleland, J. Schneede, P. M. Ueland, P. K. Lund, H. Refsum, and S. Sandberg Cobalamin Deficiency in General Practice. Assessment of the Diagnostic Utility and Cost-Benefit Analysis of Methylmalonic Acid Determination in Relation to Current Diagnostic Strategies Clin. Chem., February 1, 1999; 45(2): 189 - 198. [Abstract] [Full Text] [PDF]
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J. Pietzsch and A. Pixa Determination of Total Homocysteine Clin. Chem., August 1, 1998; 44(8): 1781 - 1782. [Full Text] [PDF]
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D. W. Jacobsen Homocysteine and vitamins in cardiovascular disease Clin. Chem., August 1, 1998; 44(8): 1833 - 1843. [Abstract] [Full Text] [PDF]
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A. Pastore, R. Massoud, C. Motti, A. L. Russo, G. Fucci, C. Cortese, and G. Federici Fully automated assay for total homocysteine, cysteine, cysteinylglycine, glutathione, cysteamine, and 2-mercaptopropionylglycine in plasma and urine Clin. Chem., April 1, 1998; 44(4): 825 - 832. [Abstract] [Full Text] [PDF]
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F. Frantzen, A. L. Faaren, I. Alfheim, and A. K. Nordhei Enzyme conversion immunoassay for determining total homocysteine in plasma or serum Clin. Chem., February 1, 1998; 44(2): 311 - 316. [Abstract] [Full Text] [PDF]
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S. Tonstad, H. Refsum, and P. M. Ueland Association Between Plasma Total Homocysteine and Parental History of Cardiovascular Disease in Children With Familial Hypercholesterolemia Circulation, September 16, 1997; 96(6): 1803 - 1808. [Abstract] [Full Text]
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 T. Fiskerstrand, P. M. Uel, and a. H. Refsum Folate Depletion Induced by Methotrexate Affects Methionine Synthase Activity and Its Susceptibility to Inactivation by Nitrous Oxide J. Pharmacol. Exp. Ther., September 1, 1997; 282(3): 1305 - 1311. [Abstract] [Full Text]
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 O. Nygard, J. E. Nordrehaug, H. Refsum, P. M. Ueland, M. Farstad, and S. E. Vollset Plasma Homocysteine Levels and Mortality in Patients with Coronary Artery Disease N. Engl. J. Med., July 24, 1997; 337(4): 230 - 237. [Abstract] [Full Text] [PDF]
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 I. M. Graham, L. E. Daly, H. M. Refsum, K. Robinson, L. E. Brattstrom, P. M. Ueland, R. J. Palma-Reis, G. H. J. Boers, R. G. Sheahan, B. Israelsson, et al. Plasma Homocysteine as a Risk Factor for Vascular Disease: The European Concerted Action Project JAMA, June 11, 1997; 277(22): 1775 - 1781. [Abstract] [PDF]
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P. M. Gallagher, R. Meleady, D. C. Shields, K. S. Tan, D. McMaster, R. Rozen, A. Evans, I. M. Graham, and A. S. Whitehead Homocysteine and Risk of Premature Coronary Heart Disease: Evidence for a Common Gene Mutation Circulation, November 1, 1996; 94(9): 2154 - 2158. [Abstract] [Full Text]
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 S. Tonstad, O. Joakimsen, E. Stensland-Bugge, T. P. Leren, L. Ose, D. Russell, and K. H. Bonaa Risk Factors Related to Carotid Intima-Media Thickness and Plaque in Children With Familial Hypercholesterolemia and Control Subjects Arterioscler Thromb Vasc Biol, August 1, 1996; 16(8): 984 - 991. [Abstract] [Full Text]
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O. Nygard, S. E. Vollset, H. Refsum, I. Stensvold, A. Tverdal, J. E. Nordrehaug, P. M. Ueland, and G. Kvale Total Plasma Homocysteine and Cardiovascular Risk Profile: The Hordaland Homocysteine Study JAMA, November 15, 1995; 274(19): 1526 - 1533. [Abstract] [PDF]
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