FEATURES             

·         Purely enzymatic in vitro diagnostic assay for total homocysteine in plasma.

·         Assay is based on genetically-engineered recombinant homocysteine cleaving enzyme.

·         Excellent correlation with HPLC.

·         High precision.

·         High throughput with a linear calibration curve.

·         No immunochemical reactions.

·         Simplest total homocysteine assay.

·     Patented.

GENERAL DESCRIPTION

High levels of homocysteine have been called "the hidden cause of heart attacks."  In fact, people with homocysteine levels in the top 5% of the normal range have been shown to have three times the risk of a heart attack than those with levels in the lower 90%.  This has created a great demand for a widely-available, highly-specific, accurate assay for homocysteine. AC Diagnostics' development of a highly-specific genetically-engineered homocysteinase rHCYase (1) has made such an assay possible: the total Homocysteine (tHcy) Enzymatic Assay (2).

Cardiovascular disease is the No. 1 killer in the US, causing one million heart attack and stroke deaths each year.  It has long been thought that cardiac disease is related primarily to elevated cholesterol.  Indeed 5% of the adult population, some 9 million people, take cholesterol-lowering drugs.  The total market for such treatment is greater than $10 billion per year.

Unfortunately, a quarter of all heart attacks occur in people without elevated cholesterol or other known risk factors.  New studies now show that it is homocysteine that plays a critical role in heart disease and stroke (3,4).  In fact, it may be that monitoring and reducing homocysteine levels is more important than monitoring and lowering cholesterol for preventing cardiovascular disease (5).  Current research indicates that it is homocysteine in combination with cholesterol which trigger plaque formation and atherosclerosis.

Hyperhomocystenemia is also a risk factor and possible agent for other diseases including diabetes (6) and neurodegenerative diseases (7).  Homocystenemia is also a risk factor for  noncardiovascular mortality (8,9).  Homocysteine seems to have a direct pathological effect on endothelial cells (10,11).

PRINCIPLE OF THE tHOMOCYSTEINE (tHcy) ENZYMATIC ASSAY

In the tHcy Enzymatic Assay, protein-bound Hcy is first reduced to free homocysteine. Genetically-engineered recombinant homocysteinase (rHCYase) then reacts with the free Hcy to form hydrogen sulfide, ammonia, and a-ketobutyrate:

           rHCYase

   tHcy  ® ®  ®  a-ketobutyrate + NH₄ + H₂S

Hydrogen sulfide then reacts with a proprietary fluorophore to give a fluorescence or absorption change.  For fluorescence, the excitation wavelength is 665 nm and the emission is at 690 nm.  Absorbance can be read between 660 and 680 nm.  The test is linear from 1 mM to 50 mM.  The tHcy Enzymatic Assay has been specifically optimized to minimize any cross reactivity with cysteine and methionine.

AVAILABILITY

tHcy testing with the tHcy Enzymatic Assay is available for everyone at the A/C Diagnostics Laboratory.  Contact the Company for small scale and bulk testing for any amount of samples.  The tHcy Enzymatic Assay is now being applied to Hitachi and all major analyzers as well as microplate fluorescence and absorbance readers.  Kits will be available in 2002.

SPECIMEN COLLECTION AND  PREPARATION

EDTA-plasma is recommended for homocysteine determination. Storage should be at 4°C for short periods.  For longer storage, samples should be kept frozen at -20°C.  Overnight fasting is recommended before blood is drawn.  Standardized sampling procedures are crucial.

tHOMOCYSTEINE ENZYMATIC ASSAY PATENTS

                     US Patent 5,985,540

                     US Patent 5,998,191

                     US Patent 6,066,467

                     US Patent 6,140,102

                     US Patent 6,448,446

                     US Patent 6,468,762

                    Japan Patent 3,337,693

                     Worldwide patents pending.

KEY REFERENCES

1. Han,Q.,  Lenz, M., Tan, Y., Xu, M., Sun, X., Tan, X-Z., Tan, X-Y., Hoffman, R.M. High expression, purification and properties of recombinant homocysteine, a, g-lyase. Protein Expression and Purification 14, 267-274, 1998. [PDF] 

2. Tan, Y., Tang, L., Sun, X., Zhang, N., Han, Q., Xu, M., Baranov, E., Tan, X-Z., Tan, X-Y., Rashidi, B., An, Z., Perry, A.W., Hoffman, R.M.   Total-homocysteine enzymatic assay. Clinical Chemistry 46, 1686-1688, 2000. [PDF] 

3. Nygard, O., Nordrehaug, J.E., Refsum, H., Ueland, P.M., Farstad, M., Vollset, S.E. Plasma homocysteine levels and mortality in patients with coronary artery disease. New Engl. J. Med. 337, 230-236, 1997.

4. Schnyder, G., Pin, R., Roffi, M., Flammer, Y., Hess, O.M. Association of plasma homocysteine with the number of major coronary arteries severely narrowed. Am. J. Cardiol. 88, 1027-1030, 2001. 

5. Tice, J.A., Ross, E., Coxson, P.G., Rosenberg, I., Weinstein, M.C., Hunink, M.G., Goldman, P.A., Williams, L., Goldman, L. Cost-effectiveness of vitamin therapy to lower plasma homocysteine levels for the prevention of coronary heart disease: effect of grain fortification and beyond. JAMA 286, 936-943, 2001. 

6. Glowinska, B., Urban, M., Peczynska, J., Florys, B., Szydlowska, E.   Elevated concentrations of homocysteine in children and adolescents with arterial hypertension accompanying Type 1 diabetes. Med. Sci. Monit. 7, 1242-1249, 2001. 

7. Rosenberg IH. B vitamins, homocysteine, and neurocognitive function. Nutr. Rev. 59, S69-73; disc. S73-74, 2001. 

8. Vollset, S.E., Refsum, H., Tverdal, A., Nygard, O., Nordrehaug, J.E., Tel, G.S., Ueland, P.M.  Plasma total homocysteine and cardiovascular and noncardiovascular mortality: the Hordaland Homocysteine Study. Am. J. Clin. Nutr. 74, 130-136, 2001. 

9. Malinow MR. Plasma concentrations of total homocysteine predict mortality risk. Am. J. Clin. Nutr. 74, 3, 2001. 

10. Zhang, C., Cai, Y., Adachi, M.T., Oshiro, S., Aso, T., Kaufman, R.J., Kitajima, S. Homocysteine induces programmed cell death in human vascular endothelial cells through activation of the unfolded protein response. J. Biol. Chem. 276, 35867-35874, 2001. 

11. Thambyrajah, J., Landray, M.J., Jones, H.J., McGlynn, F.J., Wheeler, D.C., Townend, J.N.   A randomized double-blind placebo-controlled trial of the effect of homocysteine-lowering therapy with folic acid on endothelial function in patients with coronary artery disease. J. Am. Coll. Cardiol. 37, 1858-1863, 2002

12. Tripodi, A., Chantarangkul, V., Tan, Y., Hoffman, R.M., and Cattaneo, M. Evaluation of of an Enzymatic Method to Measure Total Homocysteine in Plasma. Thrombosis and Haemostasis 87, 172-173, 2002 [PDF] 

13. Han, Q., Xu, M., Tang, L., Tan, X-Z., Tan, X-Y., Tan, Y., and Hoffman, R.M. Homogeneous nonradioactive enzymatic assay for plasma pyridoxal 5-phosphate. Clinical Chemistry 48, 1560-1564, 2002. [PDF] 

14. Tan, Y., Sun, X., Tang, L., Zhang, N., Han, Q., Xu, M., Tan, X., Tan, X., Hoffman, R.M. Automated enzymatic assay for homocysteine. Clinical Chemistry 49, 1029-1030, 2003. [PDF]

15. Han, Q., Xu, M., Tang, Li., Sun, X., Zhang, N., Tan, X., Tan, Y., Hoffman, R.M. Homogeneous enzymatic colorimetric assay for total cysteine. Clinical Chemistry 50, 1229-1231, 2004

16. Han, Q., Sun, X., Xu, M., Zhang, N., Tang, L., Tan, Y., and Hoffman, R.M. 3-deazaadenosine, a stabilizer of whole-blood homocysteine content, does not interfere with single enzyme homocysteine assay while totally inhibiting the enzyme conversion homocysteine immunoassay. Clinical Chemistry. 50, 1703-1704, 2004. [PDF]