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)(2)(3)(4)(5)(6)(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 α,γ-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 B6 (10) in plasma that use the same analyte, H2S, that is used for tCYS in the present report. Simultaneous assay of tHCY, vitamin B6, 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 Fl-1000 fluorescence spectrophotometer and a Hitachi HPLC (L-6200A Intelligent Pump) equipped with a Supelcosil LC-18DB column [25 cm × 4.8 mm (i.d.); particle size, 5 μm; 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,N-Dibutylphenylenediamine (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 μmol/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 HCl. 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 °C until analysis. The tCYS enzymatic assay protocol was as follows: Step 1. To remove the tHCY in samples, 20 μL of calibrator or plasma sample and 980 μL of reagent 1 were added. This reaction was carried out at 37 °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 H2S from tCYS by rMETase, reagent 2 (10 μL) was added to the tubes, which were then vortex-mixed and incubated at 37 °C for 10 min. Step 3. For the chromogenic reaction, the enzymatic reaction was stopped by addition of 50 μL of reagent 3 followed by 50 μL of reagent 4. The chromogenic reaction was carried out at 37 °C for 10 min. tCYS was measured by its absorbance at 675 nm. 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-oxa-1,3-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 μmol/L and 4.5% at a mean tCYS concentration of 283.4 μmol/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 μmol/L, respectively. To examine the linearity of the calibration curve, we measured five different l-CYS solutions (10, 62.5, 125, 250, and 500 μmol/L) with the tCYS enzymatic assay. The calibration curve was linear between 10 and 500 μmol/L, and linear regression analysis gave a relationship of: y = 0.0005x + 0.0008 μmol/L (r2 = 0.9973). The analytical recoveries for crystalline L-CYS added to pooled human plasma were determined. L-CYS (50–200 μmol/L) was added to the plasma samples containing 153 μmol/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 (r2 = 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 μmol/L. Comparison of the enzymatic and HPLC tCYS assays. (A), correlation between the enzymatic and HPLC tCYS assays by Deming regression analysis (17). The results are expressed in μmol/L for the Deming regression analysis: y = 1.0054x − 0.6716 μmol/L (r2 = 0.93; n = 40). (B), Bland-Altman plot of the difference in tCYS values between the enzymatic and HPLC tCYS assays as a function of their mean value ± 2 SD (18). The mean difference between the methods was 0.0645 μmol/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 μmol/L, l-methionine up to 100 μmol/L, l-cystathionine up to 130 μmol/L, and cysteinylglycine up to 200 μmol/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. Interference in the enzymatic tCYS assay. [(Sample with added analyte − sample without added analyte)/sample without added analyte] × 100%. Interference in the enzymatic tCYS assay. [(Sample with added analyte − sample without added analyte)/sample without added analyte] × 100%.