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Semin Thromb Hemost 2000; Volume 26(Number 03): 233-242
DOI: 10.1055/s-2000-8468
Copyright © 2000 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel.: +1(212) 584-4662

Assay Methods for the Measurement of Total Homocyst(e)ine in Plasma

JOHAN. B. UBBINK
  • Department of Chemical Pathology, University of Pretoria, South Africa
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Publication History

Publication Date:
31 December 2000 (online)

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ABSTRACT

Analytical methods to measure plasma total homocyst(e)ine (tHcy) concentrations are reviewed. High-pressure liquid chromatography (HPLC) with fluorometric detection is the most widely used method to determine plasma tHcy concentrations. Both monobromobimane and ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F) are popular thiol-specific fluorogenic agents suitable for tHcy analysis. Monobromobimane has the advantage that it is highly reactive towards thiols, but its hydrolysis products are also fluorogenic, thus necessitating complex chromatography to obtain satisfactory separation between the compounds of interest and interferents. SBD-F does not show fluorescence, thus allowing isocratic separation of SBD-F derivatized thiols. SBD-thiol adducts are light sensitive and require protection against light to ensure reliable results.

HPLC with electrochemical detection is also often used and has the advantage that no derivatization of thiols is required prior to detection. A recently reported liquid chromatography electrospray tandem mass spectrometric assay has the potential to become the reference method for plasma tHcy analysis.

Other methods to measure plasma tHcy concentrations include gas-liquid chromatography, capillary electrophoresis, and immunoassays. Fluorescence polarization immunoassay compares well with gas chromatography-mass spectrometry and may become the method of choice in routine diagnostic clinical chemistry laboratories.

Instability of tHcy in whole blood as well as postprandial and orthostatic variation are preanalytical factors that should be accounted for in plasma tHcy analysis. Between-method and between-laboratory variations in serum tHcy analysis are not yet satisfactory; certified reference material and standardization of the plasma tHcy assay will be essential to reduce between-laboratory bias.

KEYWORD

HPLC - gas chromatography - immunoassay - preanalytical variation - homocysteine

REFERENCES

  • 1 Stipanuk M H. Metabolim of sulfur-containing amino acids.  Annu Rev Nutr . 1986;  6 179-209
    CrossrefPubMedGoogle Scholar
  • 2 Christensen B, Refsum H, Vintermyr O, Ueland P M. Homocysteine export from cells cultured in the presence of physiological or superfluous levels of methionine: Methionine loading of non-transformed, transformed, proliferating, and quiescent cells in culture.  J Cell Physiol . 1991;  146 52-62
    CrossrefPubMedGoogle Scholar
  • 3 Brattström L E, Hardebo J E, Hultberg B L. Moderate homocysteinemia-A possible risk factor for arteriosclerotic cerebrovascular disease.  Stroke . 1984;  15 1012-1016
    PubMedGoogle Scholar
  • 4 Wilcken D EL, Gupta V J. Cysteine-homocysteine mixed disulphide: Differing plasma concentrations in normal men and women.  Clin Sci . 1979;  57 211-215
    PubMedGoogle Scholar
  • 5 Boers G H, Smals A G, Trijbels F J, Leermakers A I, Kloppenborg P W. Unique efficiency of methionine metabolism in premenopausal women may protect against vascular disease in the reproductive years.  J Clin Invest . 1983;  72 1971-1976
    PubMedGoogle Scholar
  • 6 Andersson A, Isaksson A, Brattström L, Hultberg B. Homocysteine and other thiols determined in plasma by HPLC and thiol-specific post-column derivatization.  Clin Chem . 1993;  39 1590-1597
    PubMedGoogle Scholar
  • 7 Refsum H, Ueland P M, Svardal A M. Fully automated fluorescence assay for determining total homocysteine in plasma.  Clin Chem . 1989;  35 1921-1927
    PubMedGoogle Scholar
  • 8 Jacobsen D W, Gatautis V J, Green R. Determination of plasma homocysteine by high-performance liquid chromatography with fluorescence detection.  Anal Biochem . 1989;  178 208-214
    CrossrefPubMedGoogle Scholar
  • 9 Baeyens W, Van der Weken G, Lin Ling B M, De Moerlose B. HPLC determination of N-acetylcysteine in pharmaceutical preparations after pre-column derivatization with thiolyte RMB using fluorescence detection.  Anal Lett . 1988;  21 741-757
    PubMedGoogle Scholar
  • 10 Fiskerstrand T, Refsum H, Kvalheim G, Ueland P M. Homocysteine and other thiols in plasma and urine: Automated determination and sample stability.  Clin Chem . 1993;  39 263-271
    PubMedGoogle Scholar
  • 11 Jacobsen D W, Gatautis V J, Green R. Rapid HPLC determination of total homocysteine and other thiols in serum and plasma: Sex differences and correlation with cobalamin and folate concentrations in healthy subjects.  Clin Chem . 1994;  40 873-881
    PubMedGoogle Scholar
  • 12 Arnesen E, Refsum H, Bonaa K H. Serum total homocysteine and coronary heart disease.  Int J Epidemiol . 1995;  24 704-709
    CrossrefPubMedGoogle Scholar
  • 13 Perry I J, Refsum H, Morris R W. Prospective study of serum total homocysteine concentration and risk of stroke in middle-aged British men.  Lancet . 1995;  346 1395-1398
    CrossrefPubMedGoogle Scholar
  • 14 Nygård O, Vollset S E, Refsum H. Total plasma homocysteine and cardiovascular risk profile. The Hordaland homocysteine study.  JAMA . 1995;  274 1526-1533
    CrossrefPubMedGoogle Scholar
  • 15 Amundsen T, Ueland P M, Waage A. Plasma homocysteine levels in patients with deep venous thrombosis.  Arterioscler Thromb Vasc Biol . 1995;  15 1321-1323
    PubMedGoogle Scholar
  • 16 Robinson K, Arheart K, Refsum H. Low circulating folate and vitamin B6 concentrations. Risk factors for stroke, peripheral vascular disease, and coronary artery disease.  Circulation . 1998;  97 437-443
    PubMedGoogle Scholar
  • 17 Nygård O, Nordrehaug J E, Refsum H. Plasma homocysteine levels and mortality in patients with coronary artery disease.  N Engl J Med . 1997;  337 230-236
    CrossrefPubMedGoogle Scholar
  • 18 Carmel R, Green R, Jacobsen D W. Serum cobalamin, homocysteine, and methylmalonic acid concentrations in a multiethnic elderly population: Ethnic and sex differences in cobalamin and metabolite abnormalities.  Am J Clin Nutr . 1999;  70 904-910
    PubMedGoogle Scholar
  • 19 Evans R W, Shaten B J, Hempel J D, Cutler J A, Kuller L H. Homocyst(e)ine and risk of cardiovascular disease in the multiple risk factor intervention trial.  Arterioscler Thromb Vasc Biol . 1997;  17 1947-1953
    PubMedGoogle Scholar
  • 20 Imai K, Toyo'oka T, Watanabe Y. A novel fluorogenic reagent for thiols: Ammonium 7-fluorobenzo-2-oxa-1, 3-diazole-4-sulfonate.  Anal Biochem . 1983;  128 471-473
    CrossrefPubMedGoogle Scholar
  • 21 Araki A, Sako Y. Determination of free and total homocysteine in human plasma by HPLC with fluorescence detection.  J Chromatogr . 1987;  422 43-52
    PubMedGoogle Scholar
  • 22 Ubbink J B, Vermaak W JH, Bissbort S. Rapid high-performance liquid chromatographic assay for total homocysteine levels in human serum.  J Chromatogr . 1991;  565 441-446
    PubMedGoogle Scholar
  • 23 Kuo K, Still R, Cale S, McDowell I. Standardization (external and internal) of HPLC assay for plasma homocysteine.  Clin Chem . 1997;  43 1653-1655
    PubMedGoogle Scholar
  • 24 Pfeiffer C M, Huff D L, Gunter E W. Rapid and accurate HPLC assay for plasma total homocysteine and cysteine in a clinical setting.  Clin Chem . 1999;  45 290-292
    PubMedGoogle Scholar
  • 25 Vester B, Rasmussen K. High performance liquid chromatography method for rapid and accurate determination of homocysteine in plasma and serum.  Eur J Clin Chem Clin Biochem . 1991;  29 549-554
    PubMedGoogle Scholar
  • 26 Fermo I, Arcelloni C, Mazzola G, D'Angelo A, Paroni R. High-performance liquid chromatographic method for measuring total plasma homocysteine levels.  J Chromatogr . 1998;  719 31-36
    PubMedGoogle Scholar
  • 27 Dudman N PB, Guo X W, Crooks R, Xie L, Silberberg J S. Assay of plasma homocysteine: Light sensitivity of the fluorescent 7-benzo-2oxa-1,3-diazole-4-sulfonic acid derivative, and use of appropriate calibrators.  Clin Chem . 1996;  42 2028-2032
    PubMedGoogle Scholar
  • 28 Durand P, Fortin L J, Lucier-Cacan S, Davignon J, Blache D. Hyperhomocysteinaemia induced by folic acid deficiency and methionine load-applications of a modified HPLC method.  Clin Chim Acta . 1996;  252 83-93
    CrossrefPubMedGoogle Scholar
  • 29 Accinni R, Campolo J, Bartesaghi S. High-performance liquid chromatographic determination of total plasma homocysteine with or without internal standards.  J Chromatogr . 1998;  828 397-400
    CrossrefPubMedGoogle Scholar
  • 30 Rizzo V, Montalbetti L, Valli M. Study of factors affecting the determination of total plasma 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD)-thiol derivatives by liquid chromatography.  J Chromatogr . 1998;  706 209-215
    PubMedGoogle Scholar
  • 31 Gilfix B M, Blank D W, Rosenblatt D S. Novel reductant for determination of total plasma homocysteine.  Clin Chem . 1997;  43 687-688
    PubMedGoogle Scholar
  • 32 Pfeiffer C M, Twite D, Shih J, Holets-McCormack S R, Gunter E W. Method comparison for total plasma homocysteine between the Abbott IMX analyzer and an HPLC assay with internal standardization.  Clin Chem . 1999;  45 152-153
    PubMedGoogle Scholar
  • 33 Wald N J, Watt H C, Law M R. Homocysteine and ischemic heart disease. Results of a prospective study with implications regarding prevention.  Arch Intern Med . 1998;  158 862-867
    CrossrefPubMedGoogle Scholar
  • 34 Alfthan G, Pekkanen J, Jauhiainen M. Relation of serum homocysteine and lipoprotein(a) concentrations to atherosclerotic disease in a prospective Finnish population based study.  Atherosclerosis . 1994;  106 9-19
    CrossrefPubMedGoogle Scholar
  • 35 Ubbink J B, Fehily A M, Pickering J, Elwood P C, Vermaak W JH. Homocysteine and ischaemic heart disease in the Caerphilly cohort.  Atherosclerosis . 1998;  140 349-356
    CrossrefPubMedGoogle Scholar
  • 36 Selhub J, Jacques P F, Bostom A G. Association between plasma homocysteine concentrations and extracranial carotid-artery stenosis.  N Engl J Med . 1995;  332 286-291
    CrossrefPubMedGoogle Scholar
  • 37 Bostom A G, Shemin D, Lapane K L. Hyperhomocysteinemia and traditional cardiovascular disease risk factors in end-stage renal disease patients on dialysis: A case-control study.  Atherosclerosis . 1995;  114 93-100
    CrossrefPubMedGoogle Scholar
  • 38 Dalery K, Lussier-Cacan S, Selhub J. Homocysteine and coronary artery disease in French Canadian subjects: Relation with vitamins B-12, B-6, pyridoxal phosphate and folate.  Am J Cardiol . 1995;  75 1107-1111
    CrossrefPubMedGoogle Scholar
  • 39 Bostom A G, Gohh R Y, Beaulieu A. Treatment of hyperhomocyst(e)inemia in renal transplant patients. A randomized, placebo-controlled trial.  Ann Intern Med . 1997;  127 1089-1092
    PubMedGoogle Scholar
  • 40 Lussier-Cacan S, Xhignesse M, Piolot A. Plasma total homocysteine in healthy subjects: Sex-specific relation with biological traits.  Am J Clin Nutr . 1996;  64 587-593
    PubMedGoogle Scholar
  • 41 Dudman N PB, Guo X W, Crooks R, Xie L, Silberberg J S. Assay of plasma homocysteine: Light sensitivity of the fluorescent 7-benzo-2oxa-1,3-diazole-4-sulfonic acid derivative, and use of appropriate calibrators.  Clin Chem . 1996;  42 2028-2032
    PubMedGoogle Scholar
  • 42 Maddocks J L, MacLachlan J. Application of new fluorescent thiol reagent to diagnosis of homocystinuria.  Lancet . 1991;  338 1043-1044
    CrossrefPubMedGoogle Scholar
  • 43 Nakashima K, Umekawa C, Yoshida H, Nakatsuji S, Akiyama S. HPLC for the determination of thiols in biological samples using N-[4-(6-dimethylamino-2-benzofuranyl)-maleimide.  J Chromatogr . 1987;  414 11-17
    PubMedGoogle Scholar
  • 44 Cavrini V, Gatti R, Roveri P, Di Pietra A. Analysis of thiols by HPLC after fluorescent prelabelling with 4-(6-methylnaphthalen-2-yl)-4-oxobuten-2-oic acid.  Chromatographia . 1989;  27 185-190
    PubMedGoogle Scholar
  • 45 Hyland K, Bottiglieri T. Measurement of total plasma and cerebrospinal fluid homocysteine by fluorescence following high-performance liquid chromatography and precolumn derivatization with o-phthaldialdehyde.  J Chromatogr . 1992;  579 55-62
    PubMedGoogle Scholar
  • 46 Carducci C, Birarelli M, Nola M, Antonozzi I. Automated high-performance liquid chromatographic method for the determination of homocysteine in plasma samples.  J Chromatogr . 1999;  846 93-100
    CrossrefPubMedGoogle Scholar
  • 47 Rabenstein D L, Yamashita G T. Determination of homocysteine, penicillamine, and their symmetrical and mixed disulfides by liquid chromatography with electrochemical detection.  Anal Biochem . 1989;  180 259-263
    CrossrefPubMedGoogle Scholar
  • 48 Smolin L A, Schneider J A. Measurement of total plasma cysteamine using high-performance liquid chromatography with electrochemical detection.  Anal Biochem . 1988;  168 374-379
    CrossrefPubMedGoogle Scholar
  • 49 Martin S C, Tsakas-Ampatzis I, Bartlett W A, Jones A F. Measurement of plasma total homocysteine by HPLC with colometric detection.  Clin Chem . 1999;  45 150-152
    PubMedGoogle Scholar
  • 50 Verhoef P, Hennekens C H, Malinow R. A prospective study of plasma homocysteine and risk of ischemic stroke.  Stroke . 1994;  25 1924-1930
    CrossrefPubMedGoogle Scholar
  • 51 Wu L L, Wu J, Hunt S C. Plasma homocyst(e)ine as a risk factor for early familial coronary artery disease.  Clin Chem . 1994;  40 552-561
    PubMedGoogle Scholar
  • 52 Eritsland J, Arnesen H, Seljeflot I. Influence of serum lipoprotein(a) and homocysteine levels on graft patency after coronary artery bypass grafting.  Am J Cardiol . 1994;  74 1099-1102
    CrossrefPubMedGoogle Scholar
  • 53 Hopkins P N, Wu L L, Hunt S C. Higher plasma homocyst(e)ine and increased susceptibility to adverse effects of low folate in early familial coronary artery disease.  Arterioscler Thromb Vasc Biol . 1995;  15 1314-1320
    PubMedGoogle Scholar
  • 54 Malinow M R, Ducimetiere P, Luc G. Plasma homocyst(e)ine levels and graded risk for myocardial infarction: Findings in two populations at contrasting risk for coronary heart disease.  Atherosclerosis . 1996;  126 27-34
    CrossrefPubMedGoogle Scholar
  • 55 De J ong, Stehouwer C DA, Makaay A JC. High prevalence of hyperhomocysteinemia and asymptomatic vascular disease in siblings of young patients with vascular disease and hyperhomocysteinemia.  Arterioscler Thromb Vasc Biol . 1997;  17 2655-2662
    PubMedGoogle Scholar
  • 56 D'Eramo J L, Finkelstein A E, Boccazzi F O, Fridman O. Total homocysteine levels in plasma: High-performance liquid chromatographic determination with electrochemical detection and glassy carbon electrode.  J Chromatogr . 1998;  720 205-210
    PubMedGoogle Scholar
  • 57 Magera M J, Lacey J M, Casetta B, Rinaldo P. Method for the determination of total homocysteine in plasma and urine by stable isotope dilution and electrospray tandem mass spectrometry.  Clin Chem . 1999;  45 1517-1522
    PubMedGoogle Scholar
  • 58 Labadarios D, Moodie I M, Shephard G S. Gas chromatographic analysis of amino acids in physiological fluids: A critique.  J Chromatogr . 1984;  310 223-231
    PubMedGoogle Scholar
  • 59 Kataoka H, Takagi K, Makita M. Determination of total plasma homocysteine and related aminothiols by gas chromatography with flame photometric detection.  J Chromatogr . 1995;  664 421-425
    PubMedGoogle Scholar
  • 60 Johnson J E, Lovelock J E. Electron capture sulfur detector: Reduced sulfur species detection at the femtomole level.  Anal Chem . 1988;  60 812-816
    CrossrefPubMedGoogle Scholar
  • 61 Stabler S P, Marcell P D, Podell E R, Allen R H. Quantitation of total homocysteine, total cysteine, and methionine in normal serum and urine using capillary gas chromatography-mass spectrometry.  Anal Biochem . 1987;  162 185-196
    CrossrefPubMedGoogle Scholar
  • 62 Allen R H, Stabler S P, Savage D G, Lindenbaum J. Metabolic abnormalities in cobalamin (vitamin B-12) and folate deficiency.  FASEB J . 1993;  7 1344-1353
    PubMedGoogle Scholar
  • 63 Savage D G, Lindenbaum J, Stabler S P, Allen R H. Sensitivity of serum methylmalonic acid and total homocysteine determinations for diagnosing cobalamin and folate deficiencies.  Am J Med . 1994;  96 239-246
    CrossrefPubMedGoogle Scholar
  • 64 Vecchione G, Margaglione M, Grandone E. Determining sulfur-containing amino acids by capillary electrophoresis: A fast novel method for total homocyst(e)ine in human plasma.  Electrophoresis . 1999;  20 569-574
    CrossrefPubMedGoogle Scholar
  • 65 Ueland P M, Helland S, Broch O J, Schanche J. Homocysteine in tissues of the mouse and rat.  J Biol Chem . 1984;  259 2360-2364
    PubMedGoogle Scholar
  • 66 Refsum H, Helland S, Ueland P M. Radioenzymatic determination of homocysteine in plasma and urine.  Clin Chem . 1985;  31 624-628
    PubMedGoogle Scholar
  • 67 Frantzen F, Faaren A L, Alfheim I, Nordhei A K. Enzyme conversion immunoassay for determining total homocysteine in plasma or serum.  Clin Chem . 1998;  44 311-316
    PubMedGoogle Scholar
  • 68 Schipchandler M T, Moore E G. Rapid, fully automated measurement of plasma homocysteine with the Abbott IMX analyser.  Clin Chem . 1995;  41 991-994
    PubMedGoogle Scholar
  • 69 Andersson A, Isaksson A, Hultberg B. Homocysteine export from erythrocytes and its implication for plasma sampling.  Clin Chem . 1992;  38 1311-1315
    PubMedGoogle Scholar
  • 70 Ubbink J B, Vermaak W JH, Van der Merwe A, Becker P J. The effect of blood sample aging and food consumption on plasma total homocysteine levels.  Clin Chim Acta . 1992;  207 119-128
    CrossrefPubMedGoogle Scholar
  • 71 Møller J, Rasmussen K. Homocysteine in plasma: Stabilization of blood samples with fluoride.  Clin Chem . 1995;  41 758-759
    PubMedGoogle Scholar
  • 72 Hughes M P, Carlson T H, McLaughlin M K, Bankson D D. Addition of sodium fluoride to whole blood does not stabilize plasma homocysteine but produces dilution effects on plasma constituents and hematocrit.  Clin Chem . 1998;  44 2204-2206
    PubMedGoogle Scholar
  • 73 Probst R, Brandl R, Blumke M, Neumeier D. Stabilization of homocysteine concentration in whole blood.  Clin Chem . 1998;  44 1567-1569
    PubMedGoogle Scholar
  • 74 Guttormsen A B, Schneede J, Fiskerstrand T, Ueland P M, Resfum H M. Plasma concentrations of homocysteine and other aminothiol compounds are related to food intake in healthy human subjects.  J Nutr . 1994;  124 1934-1941
    PubMedGoogle Scholar
  • 75 Thirup P, Ekelund S. Day-to-day, postprandial, and orthostatic variation of total plasma homocysteine.  Clin Chem . 1999;  45 1280-1283
    PubMedGoogle Scholar
  • 76 Ubbink J B, Delport R, Riezler R, Vermaak W JH. Comparison of three different plasma homocysteine assays with gas chromatography-mass spectrometry.  Clin Chem . 1999;  45 670-675
    PubMedGoogle Scholar
  • 77 Eliason S C, Ritter D, Chung H D, Creer M. Interlaboratory variability of total homocysteine analysis in plasma.  Clin Chem . 1999;  45 315-316
    PubMedGoogle Scholar
  • 78 Pfeiffer C M, Huff D L, Smith S J, Miller D T, Gunter E W. Comparison of plasma total homocysteine measurements in 14 laboratories: An international study.  Clin Chem . 1999;  45 1261-1268
    PubMedGoogle Scholar
  • 79 Møller J, Rasmussen K, Christensen L. External quality assessment of methylmalonic acid and total homocysteine.  Clin Chem . 1999;  45 1536-1542
    PubMedGoogle Scholar
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