Simplified Simultaneous Assay of Total Plasma Homocysteine and Methionine by HPLC and Pulsed Integrated Amperometry

¹ Depts. of Lab. Med. & Pathobiol., Med. and Paediatr. (Genetics), Univ. of TorontoBanting Inst., Rm. 415, 100 College St., Toronto, ON M5G 1L5Canada

To the Editor:

Measurement of total plasma homocysteine (tHcy) can be a useful adjunct in the diagnosis of cobalamin or folate deficiency and is emerging as an independent predictor in many vaso-occlusive diseases (1). As clinical interest in this metabolite grows, the demand for simple and efficient methods of determination has increased. In some situations, a methionine-loading test may be conducted to evaluate homocysteine catabolism, but methionine is rarely measured concomitantly, because it usually requires a different assay methodology altogether. In homocystinuria caused by cystathionine beta-synthase deficiency, circulating methionine is often increased, whereas homocystinuria resulting from a relative deficiency of the remethylation pathway is characterized by hypomethioninemia (2).

In our previously reported serum assay for tHcy (3), we used the DX-500 Ion Chromatograph (Dionex Canada), outfitted with two pumps (in parallel), valves, and two columns (a 4 x 50 mm OmniPac PCX-500 precolumn and a 4 x 250 mm OmniPac PCX-500 analytical column) plumbed in series to permit "heart-cut" trapping of tHcy (4). However, with the ED40 electrochemical detector set for pulsed integrated amperometry (PIA) mode, any compound with a reduced sulfur atom, including methionine, will generate a signal proportional to concentration (5).

In our initial procedure, the disulfide reduction procedure with sodium borohydride (NaBH₄) (6) was the most labor-intensive step and constituted a substantial source of assay error. Here, we report a simplified protocol for the tHcy assay that permits accurate simultaneous quantification of methionine.

As suggested by Gilfix et al. (7), we used tris(2-carboxyethyl)phosphine (TCEP) as a reductant instead of NaBH₄. To 300 µL of plasma we added 30 µL of 100 g/L TCEP (Pierce Chemical Co.) and gently mixed with a rotating stirrer at room temperature for 30 min. Then, we added 1170 µL of mobile phase (150 mmol/L NaClO₄, 100 mmol/L HClO₄, and 50 mL/L CH₃CN) and centrifuged the mix at 10 000g for 5 min. The supernatant was passed through a C₁₈ solid-phase extraction cartridge, as described before (3), and 50 µL of filtrate was injected directly. Altering the valve-switch times to 1 min and 2 min generated a larger "heart-cut" of the eluting peaks, with homocysteine eluting at 7.9 min and methionine at 11.3 min.

With our plasma control, we found that TCEP reduction is complete within a minute or so at room temperature (Fig. 1A ). Reduction of Hcy by borohydride at the same temperature was still incomplete at 30 min, and even at 50 °C required at least 15 min to approach completion. Moreover, use of the TCEP reductant significantly decreased between-run variation (CV = 3.1%, n = 10, P <0.05, F test for comparison of variances) in comparison with reduction of the same sample with NaBH₄ and use of our initial protocol (CV = 7.4%, n = 10). Omission of the urea denaturant resulted in a 4% increase of our target tHcy value for the control sample, but the chromatographic profile without urea was less noisy and the assay variation (within-run CV) was correspondingly decreased from 4.8% (n = 16) to 3.8% (n = 16).

View larger version (22K): [in this window] [in a new window]   Figure 1. (A) Time course of homocysteine reduction with TCEP at room temperature (), borohydride at room temperature (), and borohydride at 50 °C (); (B) comparison of homocysteine (Hcy) assayed after reduction with NaBH4 as described before (3) and reduction with TCEP as described in the text; (C) comparison of methionine (Met) assayed by amino acid analysis (by AAA) with that assayed by HPLC followed by electrochemical detection (by ECD) and pulsed integrated amperometry. Insets show Bland–Altman plots comparing differences between paired values with their mean. Horizontal lines indicate mean ± 2s (standard deviations) of the values for paired differences.

Assay of 58 patients' samples with a wide range of homocysteine values (Fig. 1B ) showed excellent correlation (r = 0.96, S_y|x = 0.80), and the line of best fit was not significantly different from the line of identity: slope 0.947 (95% confidence interval 0.91–1.02); y-intercept 0.23 (0.52– 0.97). Analysis of residuals by a runs test and examination of the Bland–Altman plot (8) revealed no significant nonlinear trends (Fig. 1B , inset).

In evaluating our methionine assay, we found near-quantitative recovery (98.4% ± 3.1%, n = 6) of 6.0 µmol/L reagent-grade L-methionine added to a sample with a nominal methionine concentration of 12.1 µmol/L. For 31 samples (Fig. 1C ), the correlation between our method and conventional amino acid chromatography with ninhydrin detection (Beckman 7300 Amino Acid Analyzer) (9) was excellent (r = 0.96, S_y|x = 0.755). By linear regression analysis, the line of best fit (y = 0.97x - 0.89) passed through the origin (95% confidence interval for y-intercept: -0.71 to 2.5). Runs test analysis of residuals and Bland–Altman plot (Fig. 1C , inset) revealed no significant deviation from linearity. The within-run CV was 3.4% (n = 8) and the between-run CV was 4.3% (n = 6). Although identical control sample aliquots were stored at -74 °C and used only once, the measured methionine concentration showed a noticeable downward drift, equivalent to a decrease of 1.6% per week. Similar changes were observed with the calibrators. The susceptibility of methionine to oxidation (forming methionine sulfoxide) is well-described (10) and should be kept in mind when interpreting plasma methionine data (11).

For our group, we found a mean ± SD plasma methionine concentration of 21.9 ± 2.4 µmol/L (range 18.3–26.5 µmol/L), which is within 3.5% of, and intermediate between, the means reported by Guttormsen et al. (22.7 ± 3.5 µmol/L, n = 12) (12) and Potgieter et al. (21.3 ± 2.1 µmol/L, n = 127) (11), who used phthalic aldehyde derivatization and fluorescence HPLC. Our results were also within 3.5% of the values obtained with the ninhydrin-based amino acid chromatography method (22.7 ± 0.35 µmol/L, n = 10) (13).

Our method simplifies the assay of tHcy and reduces assay time and cost. It may also enhance the assessment of methionine loading as a tool for the investigation of hyperhomocystinemia and the potential role of methionine as an antioxidant (14). It offers a rapid and simple alternative to the separate assay of methionine by conventional amino acid chromatography or tandem mass spectrometry (15) and is an attractive alternative to the simultaneous assay of homocysteine and methionine by GC-MS (16).

Acknowledgments

Supported by a grant from the Heart and Stroke Foundation of Ontario.

Footnotes

* Author and address for correspondence. Fax (416) 978-5650; e-mail davidec.cole{at}utoronto.ca

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