B6 ENZYMATIC ASSAY

B₆ ENZYMATIC ASSAY

NEW PUBLICATIONS FROM A/C DIAGNOSTICS:

Han Q., Xu M., Tang L., Tan X., Tan X., Tan Y., Hoffman R.M. Homogeneous, Nonradioactive, Enzymatic Assay for Plasma Pyridoxal 5-Phosphate. Clinical Chemistry 48:9, 1560-1564, 2002:

Background: Pyridoxal 5'-phosphate (PLP) is the biologically active form of vitamin B6. Clinical studies suggest that low PLP concentrations are an independent risk factor for cardiovascular and other diseases. However, PLP concentrations are not routinely diagnosed because of the lack of a homogeneous, nonradioactive assay. We describe a homogeneous, nonradioactive, enzymatic PLP assay that uses the apo form of the PLPdependent recombinant enzyme, homocysteine-a,g-lyase (rHCYase). Methods: PLP was removed from holoenzyme rHCYase by incubation with hydroxylamine to obtain apo-rHCYase. The restoration of enzymatic activity by reconstitution of the holoenzyme was linearly related to the amount of PLP bound to the enzyme. The amplification principle of the assay allowed nanomolar concentrations of PLP to be measured by the conversion (by reconstituted holorHCYase) of millimolar concentrations of homocysteine **to H2S. N,N-Dibutylphenylenediamine (DBPDA) was used for determination of H2S, the combination of which forms a chromophore with high absorbance. The assay was initiated by incubation of 5 mL of plasma with apo-rHCYase in a binding buffer for 60 min at 37 °C. Homocysteine (2.5 mmol/L) was added to the assay buffer and incubated at 37 °C for 20 min. The DBPDA reaction was allowed to progress for 10 min and then read at 675 nm. Results: The PLP enzymatic assay has a lower limit of detection of 5 nmol/L and is linear to 200 nmol/L. The recovery of PLP was 98%. The mean within- and between-run CVs were 9.6% and 12%, respectively. Correlation of 45 samples in the PLP enzymatic assay and the B6 ³H radioenzymatic assay (American Laboratory Products Co., Ltd.) yielded: y = 0.9367x + 10.569 (R² =0.9201). Conclusions: This new PLP assay is the first homogeneous, nonradioactive, vitamin B6 diagnostic method. The assay is applicable to chemistry automated analyzers and may have wide clinical use. © 2002 American Association for Clinical Chemistry**

TECHNICAL BULLETIN

FEATURES

· Assay is based on genetically-engineered recombinant homocysteine cleaving enzyme, which requires PLP for activity.

· Patent: Homogeneous enzymatic assay for vitamin B₆ and improvements in H₂S detection US 6,426,194

GENERAL DESCRIPTION

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. Unfortunately, a quarter of heart attacks occur in people without elevated cholesterol or other known risk factors. Studies now show that vitamin B₆ plays a critical role in heart disease and stroke (1).

Homocysteine is metabolized by the vitamin B₆-dependent trans-sulfuration pathway to cysteine. When these reactions are impaired, homocysteine accumulates in the cell and is exported to the circulation. High levels of homocysteine have been called "the hidden cause of heart attacks." It is estimated that people with homocysteine levels in the top 5% of the normal range have three times the risk of a heart attack than those with levels in the lower 90% (2). Optimum levels of B₆ are necessary to maintain normal homocysteine levels.

Epidemiological evidence has indicated that populations with higher plasma levels of PLP, the active form of B₆, have lower risk of coronary heart disease (2-4). One study has indicated that for men in the lowest 20% of vitamin B₆ values, the relative risk for myocardial infarction was 1.5 (4). Another study has indicated that increasing vitamin B₆ intake to 4.6 mg per day halved the relative risk for coronary heart disease (1). Recent studies indicate that a low level of B₆ is an independent risk factor for cardiovascular disease (5) and are related to a new marker, C-reactive protein (6). Therefore, it is of critical importance to monitor B₆ levels in the general population.

PRINCIPLE OF THE ASSAY

Pyridoxal 5'-phospate (PLP) is removed from the holoenzyme of recombinant homocysteinase (holo-rHCYase) (7) to obtain apo-rHCYase. The restoration of enzymatic activity by reconstitution of the holoenzyme depends linearly on the amount of PLP bound to the enzyme, which in turn depends on the amount of PLP in the plasma. The B₆ assay is based on the PLP-dependent homocysteine a,g-lyase reaction catalyzed by rHCYase:

Apo-rHCYase

The enzyme activity is measured by the modified methylene blue determination of H₂S. The amplification principle of the assay allows nM levels of B₆ to be measured using the signal of mM conversion of homocysteine to H₂S. The amplification of the B₆ signal is on the order of 10⁶.

Hydrogen sulfide reacts with DBPDA to give an absorbance change. The absorbance is read at 675 nm (8). The B₆ test is linear from 1 nM to 1 mM.

The assay can be performed on microtiter plates or on any manual or automated blood chemistry analyzer.

SPECIMEN COLLECTION AND PREPARATION

EDTA-plasma is recommended for B₆ 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.

We recommend each laboratory use an internal control with known value. Controls can be obtained from A/C Diagnostics. The currently accepted normal reference range is 20-120 nm/L but each laboratory should confirm the characteristics of the population being tested.

PERFORMANCE DATA

Recovery and Linearity: The A/C Diagnostics B₆ assay is linear to at least 1,000 nM. Recovery is approximately 100%.

AVAILABILITY

The Enzymatic B₆ 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 Enzymatic B₆ 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.

A/C Diagnostics has filed a series of worldwide patent applications covering all aspects of genetically-engineered recombinant homocysteinase to measure B₆.

REFERENCES

1. Rimm et al. Folate and vitamin B₆ from diet and supplements in relation to risk of coronary heart disease among women. JAMA 279, 359-364, 1998.

2. Selhub et al. Association between plasma homocysteine concentrations and extracranial carotidartery stenosis. N. Engl. J. Med. 332, 286-291, 1995.

3. Verhoef et al. Homocysteine metabolism and risk of myocardial infarction: relation with vitamins B₆, B₁₂, and folate. Am. J. Epidemiol. 143, 845-859, 1996.

4. Chasan-Taber et al. A prospective study of folate and vitamin B₆ and risk of myocardial infarction in US physicians. J. Am. Coll. Nutr. 15, 136-143, 1996.

5. Cattaneo et al. Low plasma levels of vitamin B₆ are independently associated with a heightened risk of deep-vein thrombosis. Circulation 104, 2442-2446, 2001.

6. Friso et al. Low circulating vitamin B₆ is associated with elevation of the inflammation marker C-reactive protein independently of plasma homocysteine levels. Circulation 103, 2788-2791, 2001.

7. Han et al. High expression, purification and properties of recombinant homocysteine, a, g-lyase. Protein Expression and Purification 14, 267-274, 1998.

8. Tan et al. Total-homocysteine enzymatic assay. Clin. Chem. 46, 1686-1688, 2000.