1. Intended Use

The A/C Enzymatic Cysteine Assay is intended for the quantitative determination of total cysteine in plasma (12).

2. General Description

Elevated plasma total homocysteine (tHcy) is a risk factor for cardiovascular disease. Cysteine is structurally similar and metabolically linked to tHcy. Recent studies have demonstrated that plasma total cysteine (tCys) levels are a risk factor of vascular disease in the coronary, cerebral, and peripheral vessels (1). Overall, a U-shaped relationship was observed between tCys and risk of vascular disease with the middle range of 250 to 275 micromol/L tCys used as the reference category. The adjusted risk of vascular disease at low (</=225 micromol/L) tCys levels was 2.1, and the risk at high (>300 micromol/L) tCys levels was 1.6 (1).

High levels of tCy and tHcy increased the risk of venous thrombosis and myocardial infarction (MI). The data suggested that plasma tCys levels are a risk factor for venous thrombosis and MI independently of tHcy levels and that it may be appropriate to study both analytes simultaneously (2).

Considering that albumin and Cys serve as covalent carriers of most of the Hcy in circulation, these components may affect circulating tHcy and the physiological action of Hcy (3). Cys has the potential for multiple interactions with Hcy, because Cys is not only a covalent carrier but also a competitor for binding sites on proteins, a potential competitor for uptake into cells, and a metabolite of Hcy via the transulfuration pathway (3)

Individuals with homocystinuria attributable to defects in the transulfuration pathway provide further evidence for the importance of the relationship between tCys and tHcy. These individuals have low plasma tCys despite severe hyperhomocysteinemia (3,4). It is estimated that 1% of the population is heterozygous for deficiency of cystathionine-b-synthase (CBS), the enzyme for which homozygous deficiency leads to homocystinuria (4). tHcy is increased and tCys is decreased in heterozygous deficiency of this enzyme, and a low tCys/tHcy ratio assists in identifying heterozygotes (4).

Unusually low tCys/tHcy ratios should trigger a review of whether there is a physiological explanation or a preanalytical problem such as delayed separation of plasma from blood cells (3).

Renal clearance of amino acids may have a role in lowering tCys concentrations because both tCys and tHcy are increased in patients with renal failure (5).

tCys was positively associated with age, total cholesterol concentration, diastolic blood pressure, and coffee consumption. Body mass index was a strong determinant of tCys but was not related to tHcy (6). Several factors known to influence tHcy, including smoking status, folate and vitamin intake, heart rate, and physical activity, were not associated or were only weakly associated with tCys (6).

Thus, plasma tCys is strongly related to several factors that constitute the cardiovascular disease risk profile. This should be an incentive to determine the role of tCys in cardiovascular disease (6).

3. Principle of the Assay

In the A/C Enzymatic Cysteine Assay, two recombinant enzymes are used: S-adenosylhomocysteine hydrolase (rSAHH) cloned from T. vaginalis (7) and L-methionine-a-deamino-g-mercaptomethane-lyases (rMETase) cloned from Pseudomonas putida. rMETase reacts with L-cysteine to form hydrogen sulfide, ammonia, and pyrurate (8,9). Since this enzyme also converts homocysteine to H₂S, homocysteine is first removed by SAHH which combines HCY and adenosine to form s-adenosyl-L-homocysteine (Ado-Hcy) (10).

In the first step, samples are reduced by dithiothreitol (DTT) to generate free reduced Cys and Hcy. Simultaneous use of SAHH with excess adenosine converts the reduced Hcy to SAH. Hcy strongly interferes with the A/C Enzymatic Cysteine Assay.

Enzymatic Reactions in the A/C Enzymatic Cysteine Assay

Step 1

rSAHH

Reduced Hcy + Ado Ado-Hcy + H₂0

Step 2

rMETase

Reduced Cys Pyrurate + H₂S + NH₃

Step 3

H₂S combines with DBPDA to form an absorbant compound. The absorbance is read at 675 nm.

K₃Fe(CN)₆

DBPDA + H₂S ® ® ®

HCl

3,7-Bis(dibutylamino) phenothiazine-5 chloride

The chromophore DBPDA reacts specifically with hydrogen sulfide to give a fluorescence or absorption reading. For fluorescence (11), the excitation wavelength is 665 nm and the emission is at 690 nm. Absorbance can be read between 660 and 680 nm.

4. Reagents as Supplied

(For 50 samples with duplicate tubes)

Assay Buffer: 20 mM potassium phosphate buffer pH 8.3, 150 mM NaCl, 0.2% Triton X-100, 1.0 mmol/L DTT and 100 mmol/L. Before use, 200 ml SAHH (3 mg/L) is added.

Lyophilized rSAHH: 50 mg enzyme powder

Lyophilized rMETase: 50 mg enzyme powder

L-Cysteine Standard: 20 mg

Chromogenic Reagent I: 130 mg DBPDA

Chromogenic Reagent II: 200 mg potassium ferricyanide

5. Preparation of Working Reagents

SAHH Enzyme: 6.0 mg SAHH is dissolved in 2.0 ml 20 mM potassium phosphate buffer pH 7.6 and stored frozen at –20°C.

rMETase Enzyme: 0.375 mg/ml rMETase is stored at –20°C.

L-Cysteine Standard: 400 mmol/L

Chromogenic Reagent I: 32.5 mg DBPDA is dissolved in 1.5 ml 6 M HCl, then 1.5 ml 20 mM potassium phosphate buffer pH 7.6 is added and mixed well.

Chromogenic Reagent II: 50 mg potassium ferricyanide is dissolved in 3.0 ml of potassium phosphate buffer pH 7.6.

6. Assay Procedure

Blank Cysteine Standard Test

Step 1

Assay

Buffer (ml) 980 970 970

Plasma (ml) 20 --- 20

Cysteine

Standards (ml) --- 20 ---

(different concentrations)

Mix well and pre-incubate at 37°C for 30 minutes.

Step 2

(0.375 mg/L rMETase (ml) --- 10 10

Mix well and pre-incubate at 37°C for 10 minutes.

Step 3

Chromogenic

Reagent I (ml) 50 50 50

Chromogenic

Reagent II (ml) 50 50 50

Incubate at 37°C for 10 minutes and read absorbance at 675 nm. All samples are run in duplicate. The total cysteine values are calculated according to the calibration curve.

7. Schematic of Assay Procedure

Assay buffer (with samples and rSAHH enzyme)

Incubate at 37°C for 30 minutes

Add rMETase enzyme

Incubate at 37°C for 10 minutes

Add chromogenic agent I and II (chromophore and oxidant)

Incubate at 37°C for 10 minutes

Read absorbance at 675 nm

8. Specimen Collection and Preparation

EDTA-plasma is recommended for total cysteine determination. If stored for longer periods, samples should be kept frozen at –20°C.

9. Quality Control

We recommend each laboratory use an internal control with known value. Controls can be obtained from A/C Diagnostics.

10. Performance Data

Recovery and Linearity: The A/C Enzymatic Cysteine Assay is linear to at least 400 mmol/L. Recovery is approximately 99.4%.

Standard Curve:

Figure 1. The linearity of the calibration curve for the L-cysteine enzymatic assay.

The five different L-Cysteine concentrations of 10, 62.5, 125, 250 and 500 mmol/L were determined using the present method.

11. Patents for the A/C Enymatic Cysteine

Assay

A/C Diagnostics has filed world-wide patent applications for the A/C Enzymatic Cysteine Assay.

12. References

1. El-Khairy L, Ueland PM, Refsum H, Graham IM, Vollset SE. Plasma total cysteine as a risk factor for vascular disease: The European Concerted Action Project. Circulation 103, 2544-2549, 2001.

2. Marcucci R, Brnelli T, Giusti B, Fedi S, Pepe G, Poli D, Prisco D, Abbate Gensini GF. The role of cysteine and homocysteine in venous and arterial thrombotic disease. Am. J. Clin. Pathol. 116, 56-60, 2001.

3. Hortin GL, Sullivan P, Csako G. Relationships among homocysteine, cysteine, and albumin concentrations: potential utility of assessing the cysteine/homocysteine ratio. Clin. Chem. 47, 1121-1124, 2001.

4. Boddie AM, Steen MT, Sullivan KM, Pasquali M. Dembure PP, Coates RJ. Cystathionine-b-synthase deficiency: detection of heterozygotes by the ratios of homocysteine to cysteine and folate. Metabolism 47, 207-211, 1998.

5. Mansoor MA, Ueland PM, Aarsland A, Svardal AM. Redox status and protein binding of plasma aminothiols during the transient hyperhomocysteinemia that follows homocysteine administration. Clin. Chem. 39, 980-985, 1993.

6. El-Khairy L, Ueland PM, Nygard O, Refsum H, Vollset SE. Lifestyle and cardiovascular disease risk factors as determinants of total cysteine in plasma; the Hordaland Homocysteine Study. Am. J. Clin. Nutr. 70, 1016-1024, 1999.

7. Minotto, L., Ko, GA., Edwards, MR., and Bagnara, AS. Trichomonas vaginalis: Expression and Characterisation of Recombinant S-Adenosylhomocysteinase. Eperimental Parasitology 90, 175-180, 1998.

8. Tan Y, Xu M, Tan X, Tan X, Wang X, Saikwa Y, Nagahama T, Sun X, Lenz M, Hoffman RM. Overexpression and large-scale production of recombinant L-methionine-a-deamino -g-mercaptomethane-lyase for novel anticancer therapy. Protein Purification and Expression 9, 233-245, 1997.

9. Han Q, Lenz M, Tan Y, Xu M, Sun X, Tan X, Tang L, Miljkovic D, Hoffman RM. High expression, purification and properties of recombinant homocysteine, a, g-lyase. Protein Expression and Purification 14, 267-274, 1998.

10. Frantzen, F., Faareen, AL., Alfheim I., and Nordhei, AK. Enzyme conversion immunoassay for determining total homocysteine in plasma or serum. Clin. Chem. 44, 311-316, 1998.

11. Tan Y, Tang L, Sun X, Zhang N, Han Q, Xu M, Baranov E, Tan X, Tan X, Rashi B, An Z, Perry AW, Hoffman RM. Total-homocysteine enzymatic assay. Clin. Chem. 46, 1686-1688, 2000.

12. Han, Q., Xu, M., Tang, L., Sun, X., Zhang, N., Tan, X-H., Tan, X-Y., Tan, Y., and Hoffman, R.M. Homogeneous enzymatic colorimetric assay for total cysteine. Clin. Chem. 50, 1229-1231, 2004.

Enzymatic Reactions in the A/C Enzymatic Cysteine Assay

Step 2

H2S combines with DBPDA to form an absorbant compound. The absorbance is read at 675 nm.

K3Fe(CN)6

HCl

Step 1

Step 2

Step 3

H₂S combines with DBPDA to form an absorbant compound. The absorbance is read at 675 nm.

K₃Fe(CN)₆