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R & D Division, Azwell Inc., 2-24-3 Sho, Ibaraki, Osaka 567-0806, Japan
aauthor for correspondence: fax 81-726-22-4909, e-mail nskrd-06{at}mbd.sphere.ne.jp
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Introduction |
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Plasma or serum total Hcy (tHcy) concentrations are most commonly measured by HPLC (9), which is time- consuming and expensive, and by immunochemical (10)(11)(12) or enzymatic (13) methods, which may not be applicable to all colorimetric-based clinical chemistry analyzers.
The thiol group of Hcy allows it to form a disulfide bond with other thiol-containing molecules, such as Hcy itself, cysteine, and the cysteine residue of plasma proteins. Biologic fluids may often contain both reduced and oxidized species of Hcy, and the sum of all the forms of Hcy is usually called total Hcy (tHcy) (14). Most clinical studies concerning Hcy have relied on the measurement of tHcy. An initial chemical reduction step of the sample is inevitable in the tHcy assay. Because reducing agents can interfere with the oxidation of redox indicators, such as Trinder’s reagents and derivatives of methylene blue generally used in diagnostic reagents, methods for tHcy that use these indicators have not been developed.
The present method is a new enzymatic colorimetric assay for tHcy in biologic samples. The principle is as follows. In the first step, samples are reduced by dithiothreitol to generate free reduced Hcy. Simultaneously, Hcy methyltransferase (EC 2.1.1.10) transfers the methyl group of D-methionine methylsulfonium to Hcy, leading to the generation of L-methionine and D-methionine. In the second step, D-amino acid oxidase (EC 1.4.3.3) oxidizes D-methionine with the simultaneous production of hydrogen peroxide, followed by oxidation of 10-(carboxymethyl-aminocarbonyl)-3,7-bis(dimethylamino) phenothiazine (DA-67) to yield methylene blue with an absorbance maximum at 660 nm. This assay system contains N-ethylmaleimide in the second step to capture the thiol group of the remaining dithiothreitol, enabling the oxidation of the redox indicator and generation of the colored product.
L-Homocystine and D-amino acid oxidase were from Sigma. D-Methionine methylsulfonium bromide was obtained from Acros. DA-67 was obtained from Wako Pure Chemical Industries. Dithiothreitol and N-ethylmaleimide were obtained from Nacalai Tesque. Hcy methyltransferase was prepared from baker’s yeast (15). One unit of Hcy methyltransferase activity was defined as the amount of enzyme that catalyzed the synthesis of 1 µmol of D-methionine per minute. All other materials used were of analytical reagent grade.
The tHcy assay was performed as follows. One hundred microliters of saline (for reagent blank), calibrator, or sample was added to 50 µL of reagent A, containing 9.6 U/L Hcy methyltransferase, 15 mmol/L dithiothreitol, 1.5 mmol/L D-methionine methylsulfonium bromide, 0.5 mmol/L ZnBr2, 35 mmol/L sodium phosphate (pH 7.0), and 0.1 g/L Triton X-100. To serve as the background, 100 µL of the same sample was also added to the reagent that did not contain Hcy methyltransferase. The mixtures were incubated for 90 min at 37 °C, and the reactions were terminated by the addition of 150 µL of reagent B, containing 18 mmol/L N-ethylmaleimide and 0.1 g/L Triton X-100. The generated D-methionine was measured with the Hitachi Model 7170 analyzer with a two-point assay. The reaction mixture (20 µL) was added to 160 µL of reagent 1, containing 0.24 mmol/L DA-67, 0.025 mmol/L dithiothreitol, 0.003 mmol/L potassium ferrocyanide, 97 mmol/L sodium phosphate (pH 7.0), and 0.1 g/L Triton X-100. The mixture was incubated for 5 min at 37 °C, followed by the addition of 100 µL of reagent 2, containing 1.4 kU/L D-amino acid oxidase, 4.4 kU/L peroxidase, 1 mmol/L FAD, 93 mmol/L sodium phosphate (pH 7.0), and 0.1 g/L Triton X-100. The mixture was again incubated for 5 min at 37 °C. Absorbance [660 nm (main) and 750 nm (subsidiary)] was measured at 5 and 10 min after starting the reaction. The net absorbance change for tHcy was calculated by subtracting the absorbance change of the background (Hcy methyltransferase absent in reagent A) from that of the test (Hcy methyltransferase present in reagent A). The concentration of tHcy was calculated with reference to the net absorbance change of saline-based Hcy calibrator (25 µmol/L, i.e., 6.71 mg/L L-homocystine).
The conventional HPLC method (16) for tHcy determination was performed at SRL Inc.
We studied linearity with Hcy solutions of 0, 5, 10, 20, 40, and 80 µmol/L. The results were linear from 5 to 80 µmol/L. We estimated the detection limit of the present method by assaying physiologic saline seven times (obtained mean ± SD value, 0.17 ± 0.55 µmol/L). The detection limit, defined as the mean + 3 SD of measured values, was 1.82 µmol/L. For the within-assay precision study, two different human serum samples were assayed seven times. The mean ± SD and CV values for the concentration of tHcy were as follows: serum 1, 11.0 ± 0.31 µmol/L and 2.8%; and serum 2, 31.9 ± 0.33 µmol/L and 1.0%. The analytical recoveries for tHcy added to human pooled serum are shown in Table 1
. The concentrations of tHcy in 39 human serum samples were determined with the present method (Fig. 1
, y axis) and HPLC (Fig. 1
, x axis). The regression equation was: y = 1.032x - 1.0651; r2=0.9893; Sy|x = 1.220.
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Because plasma is often used as a sample for the determination of tHcy, the influences of anticoagulants on the present method were studied. Although no influences were observed with heparin and citrate, EDTA completely inhibited the assay (data not shown). It is known that Hcy methyltransferase requires the divalent ions Zn2+ or Cd2+ (17). When excess Zn2+ was added to the reagent, the influence of EDTA disappeared.
Hcy methyltransferase [S-adenosyl-L-methionine: L-Hcy S-methyltransferase] catalyzes the direct transfer of the methyl group of either S-adenosyl-L-methionine or S-methyl-L-methionine (L-methionine methylsulfonium) to Hcy. The enzyme has been found in extracts of various microorganisms, higher plant seeds, and rat liver (17). The enzyme may also weakly act on S-adenosyl-D-methionine (18). When these substrates are used as the methyl donor, L-methionine will be formed in all cases, and so Hcy may be determined by detecting the amino acid. However, biologic samples generally contain an interfering concentration of L-methionine. We used D-methionine methylsulfonium in the present method as a substrate for Hcy methyltransferase followed by detection of generated D-methionine by D-amino acid oxidase. Fortunately, D-amino acid oxidase acts minimally on D-methionine methylsulfonium around a neutral pH, despite its structural similarity to D-methionine. The concentration of D-amino acids in the aforementioned biologic samples is low, but might slightly increase in some diseases. For accuracy, we also determined the background concentration of D-amino acid in each sample.
In conclusion, the new assay for tHcy can use redox reagents, such as derivatives of methylene blue or Trinder’s reagents. After the treatment of a biologic sample with Hcy methyltransferase and D-methionine methylsulfonium in the presence of dithiothreitol, the generated D-methionine is determined by D-amino acid oxidase, peroxidase, and the redox indicator in the presence of N-ethylmaleimide. This enzymatic colorimetric assay method for measuring tHcy may be suitable for routine use, although it requires manual pretreatment of samples. To avoid this inconvenience, we are now trying to develop a fully automated method based on this assay system.
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The following articles in journals at HighWire Press have cited this article:
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  E.-C. Chan, P.-Y. Chang, T.-L. Wu, and J. T. Wu Enzymatic Assay of Homocysteine on Microtiter Plates or a TECAN Analyzer Using Crude Lysate Containing Recombinant Methionine {gamma}-Lyase Ann. Clin. Lab. Sci., April 1, 2005; 35(2): 155 - 160. [Abstract] [Full Text] [PDF]
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