Clinical Chemistry, Vol 39, 263-271, Copyright © 1993 by American Association for Clinical Chemistry

Homocysteine and other thiols in plasma and urine: automated determination and sample stability

T Fiskerstrand, H Refsum, G Kvalheim and PM Ueland
Department of Pharmacology and Toxicology, University of Bergen, Norway.

We have developed a modified version of our fully automated column- switching HPLC method for determining total plasma homocysteine based on single-column (reversed-phase) separation. Homocysteine, cysteine, and cysteinylglycine in plasma (total concentrations), acid- precipitated plasma (non-protein-bound concentrations), and urine can be determined. The derivatization and chromatography were performed automatically by a sample processor. The successful separation of all thiol species (within 15 min) was accomplished by accurate adjustment of the pH of the mobile phase to 3.65 (plasma) or 3.50 (acid- precipitated plasma, urine). Maximal fluorescence yield of cysteine, cysteinylglycine, and, to a lesser degree, homocysteine was dependent on optimal concentrations of EDTA and dithioerythritol during reduction (with NaBH4) and derivatization (with monobromobimane). The method is sensitive (detection limit approximately 0.05 pmol) and has a high degree of precision (CV < 5%). The sample output is approximately 70 samples in 24 h. Serum and heparin plasma can also be analyzed. Hemolysis up to approximately 2.0 g/L of hemoglobin did not interfere with the analytical recovery of homocysteine or cysteine. Collection of blood, separation of plasma from whole blood, and acid precipitation must be standardized to obtain reproducible thiol results. Our modifications and the standardization of blood-sampling procedures have substantially improved the method and broadened its applications.

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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]

				O. T. Raitakari, N. Lai, K. Griffiths, R. McCredie, D. Sullivan, and D. S. Celermajer Enhanced peripheral vasodilation in humans after a fatty meal J. Am. Coll. Cardiol., August 1, 2000; 36(2): 417 - 422. [Abstract] [Full Text] [PDF]

				S. Hustad, P. M. Ueland, S. E. Vollset, Y. Zhang, A. L. Bjorke-Monsen, and J. Schneede Riboflavin as a Determinant of Plasma Total Homocysteine: Effect Modification by the Methylenetetrahydrofolate Reductase C677T Polymorphism Clin. Chem., August 1, 2000; 46(8): 1065 - 1071. [Abstract] [Full Text] [PDF]

				E. Nexo, F. Engbaek, P. M. Ueland, C. Westby, P. O'Gorman, C. Johnston, B. F. Kase, A. B. Guttormsen, I. Alfheim, J. McPartlin, et al. Evaluation of Novel Assays in Clinical Chemistry: Quantification of Plasma Total Homocysteine Clin. Chem., August 1, 2000; 46(8): 1150 - 1156. [Abstract] [Full Text] [PDF]

				G. G. Klee Cobalamin and Folate Evaluation: Measurement of Methylmalonic Acid and Homocysteine vs Vitamin B12 and Folate Clin. Chem., August 1, 2000; 46(8): 1277 - 1283. [Abstract] [Full Text] [PDF]

				A. Gottsater, I. Anwaar, K.-F. Eriksson, I. Mattiasson, F. Lindgarde, and A. Gottsater Homocysteine Is Related to Neopterin and Endothelin-1 in Plasma of Subjects with Disturbed Glucose Metabolism and Reference Subjects Angiology, June 1, 2000; 51(6): 489 - 497. [Abstract] [PDF]

				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]

				S. E. Brett, J. M. Ritter, and P. J. Chowienczyk Diastolic Blood Pressure Changes During Exercise Positively Correlate With Serum Cholesterol and Insulin Resistance Circulation, February 15, 2000; 101(6): 611 - 615. [Abstract] [Full Text] [PDF]

				M. Cahill, M. Karabatzaki, R. Meleady, H. Refsum, P. Ueland, D. Shields, D. Mooney, and I. Graham Raised plasma homocysteine as a risk factor for retinal vascular occlusive disease Br J Ophthalmol, February 1, 2000; 84(2): 154 - 157. [Abstract] [Full Text]

				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]

				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]

				A. de Graaf-Hess, F. Trijbels, and H. Blom New Method for Determining Cystine in Leukocytes and Fibroblasts Clin. Chem., December 1, 1999; 45(12): 2224 - 2228. [Abstract] [Full Text] [PDF]

				J.-F. Salazar, B. Herbeth, G. Siest, and P. Leroy Stability of Blood Homocysteine and Other Thiols: EDTA or Acidic Citrate? Clin. Chem., November 1, 1999; 45(11): 2016 - 2019. [Full Text] [PDF]

				V. Fonseca, S. C. Guba, and L. M. Fink Hyperhomocysteinemia and the Endocrine System: Implications for Atherosclerosis and Thrombosis Endocr. Rev., October 1, 1999; 20(5): 738 - 759. [Abstract] [Full Text]

				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]

				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]

				C. M. Pfeiffer, D. L. Huff, S. J. Smith, D. T. Miller, and E. W. Gunter Comparison of Plasma Total Homocysteine Measurements in 14 Laboratories: An International Study Clin. Chem., August 1, 1999; 45(8): 1261 - 1268. [Abstract] [Full Text] [PDF]

				R. Brandl, R. Probst, B. Muller, S. Powarzynski, P. C. Maurer, and D. Neumeier Evaluation of the Measurement of Lysate Homocysteine in Patients with Symptomatic Arterial Disease and in Healthy Volunteers Clin. Chem., May 1, 1999; 45(5): 699 - 702. [Full Text] [PDF]

				M. van Dusseldorp, J. Schneede, H. Refsum, P. M Ueland, C. M. Thomas, E. de Boer, and W. A van Staveren Risk of persistent cobalamin deficiency in adolescents fed a macrobiotic diet in early life Am. J. Clinical Nutrition, April 1, 1999; 69(4): 664 - 671. [Abstract] [Full Text] [PDF]

				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]

				G. B. Anker, H. Refsum, P. M. Ueland, D. C. Johannessen, E. A. Lien, and P. E. Lonning Influence of Aromatase Inhibitors on Plasma Total Homocysteine in Postmenopausal Breast Cancer Patients Clin. Chem., February 1, 1999; 45(2): 252 - 256. [Abstract] [Full Text] [PDF]

				P. W. Siri, P. Verhoef, and F. J. Kok Vitamins B6, B12, and Folate: Association with Plasma Total Homocysteine and Risk of Coronary Atherosclerosis J. Am. Coll. Nutr., October 1, 1998; 17(5): 435 - 441. [Abstract] [Full Text] [PDF]

				M. P. Hughes, T. H. Carlson, M. K. McLaughlin, and D. D. Bankson Addition of Sodium Fluoride to Whole Blood Does Not Stabilize Plasma Homocysteine But Produces Dilution Effects on Plasma Constituents and Hematocrit Clin. Chem., October 1, 1998; 44(10): 2204 - 2206. [Full Text] [PDF]

				A. B. Guttormsen, P. M. Ueland, P. E. Lonning, O. Mella, and H. Refsum Kinetics of Plasma Total Homocysteine in Patients Receiving High-Dose Methotrexate Therapy Clin. Chem., September 1, 1998; 44(9): 1987 - 1989. [Full Text] [PDF]

				D. W. Jacobsen Homocysteine and vitamins in cardiovascular disease Clin. Chem., August 1, 1998; 44(8): 1833 - 1843. [Abstract] [Full Text] [PDF]

				R. Probst, R. Brandl, M. Blumke, and D. Neumeier Stabilization of Homocysteine Concentration in Whole Blood Clin. Chem., July 1, 1998; 44(7): 1567 - 1569. [Full Text] [PDF]