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A highly sensitive single-enzyme homocysteine assay

Nature Protocols volume 3, pages 1388–1394 (2008)Cite this article

Abstract

A protocol for measuring the total plasma homocysteine (tHCY) concentration in very small samples using a selective, recombinant homocysteine α, γ-lyase (rHCYase) and a small portable fluorescence reader is described. The rHCYase produces hydrogen sulfide (H2S) from tHCY without interference from physiological concentrations of Cys or other plasma components. H2S is determined through reactivity with N,N-dibutyl phenylene diamine (DBPDA), which leads to the formation of a fluorescent chromophore. Only 5 μl of plasma/serum sample is required, which can be obtained from a finger prick, suggesting great potential for mass screening. The assay takes ∼20 min.

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Figure 1
Figure 2
Figure 3: Ten tests for five plasma samples containing different levels of L-HCY (3.2–44.6 μM) were measured by the enzymatic HCY assay on the Opulen reader.
Figure 4: Methods comparison.
Figure 5: Comparison of plasma and serum samples.
Figure 6: Interference of Cys in the portable enzymatic HCY assay.

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References

  1. McCully, K.S. Homocysteine and vascular disease. Nat. Med. 2, 386–389 (1996).

    Article  CAS  Google Scholar 

  2. Nurk, E. et al. Plasma total homocysteine and hospitalizations for cardiovascular disease: the Hordaland Homocysteine Study. Arch. Intern. Med. 162, 1374–1381 (2002).

    Article  Google Scholar 

  3. Selhub, J. et al. Association between plasma homocysteine concentrations and extracranial carotid-artery stenosis. N. Engl. J. Med. 332, 286–291 (1995).

    Article  CAS  Google Scholar 

  4. Nygard, O. et al. Plasma homocysteine levels and mortality in patients with coronary artery disease. N. Engl. J. Med. 337, 230–236 (1997).

    Article  CAS  Google Scholar 

  5. Malinow, M.R. et al. Reduction of plasma homocysteine levels by breakfast cereal fortified with folic acid in patients with coronary heart disease. N. Engl. J. Med. 338, 1009–1015 (1998).

    Article  CAS  Google Scholar 

  6. Lindner, A., Bankson, D.D., Stehman-Breen, C., Mahuren, J.D. & Coburn, S.P. Vitamin B6 metabolism and homocysteine in end-stage renal disease and chronic renal insufficiency. Am. J. Kidney Dis. 39, 134–145 (2002).

    Article  CAS  Google Scholar 

  7. Refsum, H., Wesenberg, F. & Ueland, P.M. Plasma homocysteine in children with acute lymphoblastic leukemia: changes during a chemotherapeutic regimen including methotrexate. Cancer Res. 51, 828–835 (1991).

    CAS  PubMed  Google Scholar 

  8. Geisler, J. et al. Changes in folate status as determined by reduction in total plasma homocysteine levels during leucovorin modulation of 5-fluorouracil therapy in cancer patients. Clin. Cancer Res. 4, 2125–2128 (1998).

    CAS  PubMed  Google Scholar 

  9. Sun, C.F., Haven, T.R., Wu, T.L., Tsao, K.C. & Wu, J.T. Serum total homocysteine increases with the rapid proliferation rate of tumor cells and decline upon cell death: a potential new tumor marker. Clin. Chim. Acta. 321, 55–62 (2002).

    Article  CAS  Google Scholar 

  10. Clarke, R. et al. Folate vitamin B12, and serum total homocysteine levels in confirmed Alzheimer disease. Arch. Neurol. 55, 1449–1455 (1998).

    Article  CAS  Google Scholar 

  11. Vafai, S.B. & Stock, J.B. Protein phosphatase 2A methylation: a link between elevated plasma homocysteine and Alzheimer's disease. FEBS Lett. 518, 1–4 (2002).

    Article  CAS  Google Scholar 

  12. Selhub, J. Homocysteine metabolism. Annu. Rev. Nutr. 19, 217–246 (1999).

    Article  CAS  Google Scholar 

  13. Duan, W. et al. Dietary folate deficiency and elevated homocysteine levels endanger dopaminergic neurons in models of Parkinson's disease. J. Neurochem. 80, 101–110 (2002).

    Article  CAS  Google Scholar 

  14. Allain, P. et al. Sulfate and cysteine levels in the plasma of patients with Parkinson's disease. Neurotoxicology 16, 527–529 (1995).

    CAS  PubMed  Google Scholar 

  15. Wollesen, F. et al. Plasma total homocysteine and cysteine in relation to glomerular filtration rate in diabetes mellitus. Kidney Int. 55, 1028–1035 (1999).

    Article  CAS  Google Scholar 

  16. Stehouwer, C.D., Gall, M.A., Hougaard, P., Jakobs, C. & Parving, H.H. Plasma homocysteine concentration predicts mortality in non-insulin-dependent diabetic patients with and without albuminuria. Kidney Int. 55, 308–314 (1999).

    Article  CAS  Google Scholar 

  17. Vollset, S.E. et al. Plasma total homocysteine, pregnancy complications, and adverse pregnancy outcomes: the Hordaland homocysteine study. Am. J. Clin. Nutr. 71, 962–968 (2000).

    Article  CAS  Google Scholar 

  18. Righetti, M., Serbelloni, P., Milani, S. & Ferrario, G. Homocysteine-lowering vitamin B treatment decreases cardiovascular events in hemodialysis patients. Blood Purif. 24, 379–386 (2006).

    Article  CAS  Google Scholar 

  19. Ubbink, J.B., Hayward Vermaak, W.J. & Bissbort, S. Rapid high-performance liquid chromatographic assay for total homocysteine levels in human serum. J. Chromatogr. 565, 441–446 (1991).

    Article  CAS  Google Scholar 

  20. Ueland, P.M. et al. Total homocysteine in plasma or serum: methods and clinical applications. Clin. Chem. 39, 1764–1779 (1993).

    CAS  PubMed  Google Scholar 

  21. Fiskerstrand, T., Refsum, H., Kvalheim, G. & Ueland, P.M. Homocysteine and other thiols in plasma and urine: automated determination and sample stability. Clin. Chem. 39, 263–271 (1993).

    CAS  PubMed  Google Scholar 

  22. Dudman, N.P.B., Guo, X.W., Crooks, R., Xie, L. & Silberberg, J.S. Assay of plasma homocysteine: light sensitivity of the fluorescent 7-benzo-2-oxa-1, 3-diazole-4-sulfonic acid derivative, and use of appropriate calibrators. Clin. Chem. 42, 2028–2032 (1996).

    CAS  PubMed  Google Scholar 

  23. Araki, A. & Sako, Y. Determination of free and total homocysteine in human plasma by high-performance liquid chromatography with fluorescence detection. J. Chromatogr. 422, 43–52 (1987).

    Article  CAS  Google Scholar 

  24. Shipchandler, M.T. & Moore, E.G. Rapid, fully automated measurement of plasma homocyst(e)ine with the Abbott IMx analyzer. Clin. Chem. 41, 991–994 (1995).

    CAS  PubMed  Google Scholar 

  25. Frantzen, F., Faaren, A.L., Alfheim, I. & Nordhei, A.K. Enzyme conversion immunoassay for determining total homocysteine in plasma or serum. Clin. Chem. 44, 311–316 (1998).

    CAS  PubMed  Google Scholar 

  26. Nexo, E. et al. Evaluation of novel assays in clinical chemistry: quantification of plasma total homocysteine. Clin. Chem. 46, 1150–1156 (2000).

    CAS  PubMed  Google Scholar 

  27. 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. 45, 1261–1268 (1999).

    CAS  PubMed  Google Scholar 

  28. Wang, W., Escobedo, J.O., Lawrence, C.M. & Strongin, R.M. Direct detection of homocysteine. J. Am. Chem. Soc. 126, 3400–3401 (2004).

    Article  CAS  Google Scholar 

  29. Tan, Y. et al. Total-homocysteine enzymatic assay. Clin. Chem. 46, 1686–1688 (2000).

    CAS  PubMed  Google Scholar 

  30. Tripodi, A., Chantarangkul, V., Tan, Y., Hoffman, R.M. & Cattaneo, M. Evaluation of an enzymatic method to measure total homocysteine in plasma. Thromb. Haemost. 87, 172–173 (2002).

    Article  CAS  Google Scholar 

  31. Tan, Y. et al. Automated enzymatic assay for homocysteine. Clin. Chem. 49, 1029–1030 (2003).

    Article  CAS  Google Scholar 

  32. NCCLS. Evaluation of Precision Performance of Clinical Chemistry Devices: Approved Guideline NCCLS document EP5-A (ISBN 1-56238-368-X).

  33. Refsum, H. et al. Facts and recommendations about total homocysteine determinations: an expert opinion. Clin. Chem. 50, 3–32 (2004).

    Article  CAS  Google Scholar 

  34. Bland, J.M. & Altman, D.G. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1, 307–310 (1986).

    Article  CAS  Google Scholar 

  35. Han, Q. et al. High expression, purification, and properties of recombinant homocysteine α, γ-lyase. Protein Expr. Purif. 14, 267–274 (1998).

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. A/C Diagnostics, LLC and AntiCancer, Inc., 7917 Ostrow Street, San Diego, 92111, California, USA

    Yuying Tan & Robert M Hoffman

  2. Department of Surgery, University of California, San Diego, 200 West Arbor Drive, San Diego, 92103-8220, California, USA

    Robert M Hoffman

Authors
  1. Yuying Tan
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Correspondence to Robert M Hoffman.

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Competing interests

R.M.H. is President of AntiCancer Inc. and Chairman of A/C Diagnostics LLC and Y.T. is the Research Manager at AntiCancer Inc. and A/C Diagnostics LLC. AntiCancer Inc. and A/C Diagnostics LLC have commercial activities in the area of diagnostic kits for homocysteine and related analytes, vitamin B6 and cysteine.

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Tan, Y., Hoffman, R. A highly sensitive single-enzyme homocysteine assay. Nat Protoc 3, 1388–1394 (2008). https://doi.org/10.1038/nprot.2008.117

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