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Homogeneous enzymatic colorimetric assay for total cysteine.

Patients with vascular disease have significantly higher concentrations of plasma total cysteine (tCYS) than do healthy individuals (1-7). The present method is a new, rapid, and sensitive enzymatic colorimetric assay for tCYS in plasma samples and is homogeneous in that it avoids separation methods. The tCYS assay uses only the recombinant enzymes methionine [alpha],[gamma]-lyase (rMETase) and S-adenosylhomocysteine hydrolase (rSAHH) cloned from Pseudomonas putida and Trichomonas vaginalis, respectively. We have also developed enzymatic assay methods for total homocysteine (tHCY) (8,9) and vitamin [B.sub.6] (10) in plasma that use the same analyte, [H.sub.2]S, that is used for tCYS in the present report. Simultaneous assay of tHCY, vitamin [B.sub.6], and tCYS may be relevant to the study for the occurrence and prevalence of cardiovascular disease (11).

The instrumentation included a Hitachi U-2000 Spectrophotometer (Hitachi, Ltd.) and F1-1000 fluorescence spectrophotometer and a Hitachi HPLC (L-6200A Intelligent Pump) equipped with a Supelcosil LC-18DB column [25 cm x 4.8 mm (i.d.); particle size, 5 [micro]m; Supelco].

The chemicals used were L-cysteine, D,L-homocysteine, L-methionine, adenosine (ADO), L-dithiothreitol, Triton X-100, pyridoxal 5-phosphate, and potassium ferricyanide and were purchased from Sigma Chemical Co. N,NDibutylphenylenediamine (DBPDA) was synthesized in our laboratory (8, 9,12). rMETase and SAHH were produced in our laboratory (13,14).

Four reagents were used for the assay. Reagent 1 contained 20 mmol/L potassium phosphate (pH 8.3), 150 mmol/L NaCl, 9 mg/L rSAHH, 2 mL/L Triton X-100, 1.0 mmol/L L-dithiothreitol, and 100 [micro]mol/L ADO. Reagent 2 contained rMETase (1.08 g/L protein) in 20 mmol/L potassium phosphate (pH 7.2). Reagent 3 contained 40 mmol/L DBPDA in 3 mol/L HCI. Reagent 4 contained 15 mmol/L potassium ferricyanide in 20 mmol/L potassium phosphate (pH 7.2).

Whole blood was collected in evacuated blood-collection tubes without additive or containing EDTA, heparin, or citrate as anticoagulants. Centrifugation was carried out as soon as possible at 2000g for 10 min. The plasma was collected and stored at -70 [degrees]C until analysis. The principle of the assay is as follows: In step 1, samples are reduced by L-dithiothreitol to generate free reduced CYS and HCY. Simultaneous use of rSAHH with excess ADO converts the reduced HCY to S-adenosylhomocysteine (SAH).


In step 2, rMETase is added to generate HZS from tCYS:


In step 3, HZS combines with DBPDA to form a compound that is fluorescent and has ultraviolet absorbance. For this assay, the absorbance is read at 675 nm.


3,7-bis(dibutylamino)-phenothiazine-5'-ium chloride

The tCYS enzymatic assay protocol was as follows:

Step 1. To remove the tHCY in samples, 20 [micro]L of calibrator or plasma sample and 980 [micro]L of reagent 1 were added. This reaction was carried out at 37 [degrees]C for 30 min. This reaction was essential for release of HCY from disulfide linkages in plasma proteins and its removal to form S-adenosylhomocysteine.

Step 2. For production of [H.sub.2]S from tCYS by rMETase, reagent 2 (10 [micro]L) was added to the tubes, which were then vortex-mixed and incubated at 37 [degrees]C for 10 min.

Step 3. For the chromogenic reaction, the enzymatic reaction was stopped by addition of 50 [micro]L of reagent 3 followed by 50 [micro]L of reagent 4. The chromogenic reaction was carried out at 37 [degrees]C for 10 min. tCYS was measured by its absorbance at 675 mn. Calibration was performed with use of calibrators before the first run every day.

The HPLC assay for tCYS and tHCY used the ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate derivatization method as described by Ubbink et al. (15) and Dudman et al. (16) with some modifications. Plasma samples were reduced with tri-n-butylphosphine and then derivatized with ammonium 7-fluorobenzo-2-oxa1,3-diazole-4-sulfonate. tCYS was measured by HPLC with a reversed-phase Supelcosil LC-18DB column at room temperature.

Within-run imprecision was calculated by measuring tCYS in duplicate 20 times. The imprecision (CV) was 4.3% at a mean tCYS concentration of 189.1 [micro]mol/L and 4.5% at a mean tCYS concentration of 283.4 [micro]mol/L. Between-run imprecision was based on results from 20 successive analyses performed over 20 days. The imprecision (CV) was 4.9% and 4.7% at mean tCYS concentrations of 189.1 and 283.4 /,mol/L, respectively.

To examine the linearity of the calibration curve, we measured five different L-CYS solutions (10, 62.5,125, 250, and 500 [micro]mol/L) with the tCYS enzymatic assay. The calibration curve was linear between 10 and 500 [micro]mol/L, and linear regression analysis gave a relationship of: y = 0.0005x + 0.0008 [micro]mol/L ([r.sup.2] = 0.9973).

The analytical recoveries for crystalline L-CYS added to pooled human plasma were determined. L-CYS (50-200 [micro]mol/L) was added to the plasma samples containing 153 [micro]mol/L endogenous L-CYS. The mean recovery was -99%.

The tCYS concentrations in 40 different human plasma samples were measured with the tCYS enzymatic assay and a conventional HPLC method. As shown in Fig. 1, the methods showed excellent agreement. Deming regression analysis (17) comparing the enzymatic assay (x) and the HPLC method (y) yielded the following: y = 1.0054x - 0.6716 ([r.sup.2] = 0.93; n = 40). The mean difference between the methods, as determined by Bland-Altman difference plot (18) of paired means of the enzymatic and HPLC tCYS assays, was 0.645 [micro]mol/L.

We tested the interference in the enzymatic tCYS assay by adding L-HCY, L-methionine, L-cystathionine, and cysteinylglycine to plasma samples. This experiment tested the cross-reactivity of rMETase to potentially interfering compounds (Table 1). No interference by HCY up to 200 [micro]mol/L, L-methionine up to 100 [micro]mol/L, L-cystathionine up to 130 [micro]mol/L, and cysteinylglycine up to 200 [micro]mol/L was observed. This is expected because of the high specificity of rMETase (19). To test whether the assay was influenced by hemolysis, bilirubin, and triglycerides, we added different concentrations of hemoglobin, bilirubin, and triglycerides to the plasma preparation. The assay was unaffected by hemoglobin up to 1.0 g/L, bilirubin up to 0.82 g/L, and triglycerides up to 5.0 g/L.


DOI: 10.1373/clinchem.2004.032920


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Qinghong Han, Mingxu Xu, Li Tang, Xinghua Sun, Nan Zhang, Xuezhong Tan, Xiuying Tan, Yuying Tan, and Robert M. Hoffman* (A/C Diagnostics LLC and AntiCancer, Inc., 7917 Ostrow St., San Diego, CA 92111;

* author for correspondence: fax 858-268-4175, e-mail
Table 1. Interference in the enzymatic tCYS assay.

Analyte added tCYS, Interference,
 [micro]mol/L (a) %

L-HCY, [micro]mol/L
 0 147.0
 50 139.5 -5.1
 100 153.7 4.6
 200 152.0 3.4
L-Methionine, [micro]mol/L
 0 147.0
 50 149.5 1.7
 100 140.3 -4.6
 200 133.7 -9.0
L-Cystathionine, [micro]mol/L
 0 260.3
 32.5 250.2 -3.8
 65 257.0 -1.3
 130 252.2 -3.8
Cysteinylglycine, [micro]mol/L
 0 203.2

 50 207.4 2.1
 100 201.7 -0.7
 200 206.4 1.6

(a) (Sample with added analyte - sample without added analyte)/sample
without added analyte x 100%.
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Title Annotation:Technical Briefs
Author:Han, Qinghong; Xu, Mingxu; Tang, Li; Sun, Xinghua; Zhang, Nan; Tan, Xuezhong; Tan, Xiuying; Tan, Yuy
Publication:Clinical Chemistry
Date:Jul 1, 2004
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