Evaluation and Intermethod Comparison of the Bio-Rad High-Performance Liquid Chromatographic Method for Plasma Total Homocysteine

¹ University of Alberta Hospital, Edmonton, Alberta T6G 2B7, Canada, and
² Foothills Medical Center, Calgary, Alberta T6G 2B7, Canada;
^a author for correspondence: fax 403-492-8599, e-mail vdias{at}gpu.srv.ualberta.ca

Total homocysteine (tHcy) is a major risk factor for venous thromboembolism and atherosclerosis (1); hence, there has been an strong interest in developing the most robust methodology for its quantification (2). Reduction and derivatization followed by HPLC separation and fluorescent detection is the most widely applied technique (3). The most frequently used derivatizing agents are the halogen sulfonylbenzofurans [7-benzo-2-oxa-1,3-diazole-4-sulfonic acid (SBD-F) and 4-aminosulfonyl-7-fluoro-2,1,3-benzoxdiazole (ABD-F)] because of their good tHcy-adduct stability and high HPLC resolution. In this study, we evaluated a new HPLC fluorescence assay (Bio-Rad^® Laboratories) that uses ABD-F as the derivatizing agent for quantification of tHcy. Less sample handling, faster reduction, and derivatization with optimized HPLC separation make this assay less laborious and thus improve specimen throughput. Using patient samples, we compared this ABD-F assay with an HPLC fluorescence assay that uses SBD-F as the derivatizing agent and with an enzyme immunoassay (EIA) method (Axis^® Biochemicals) that requires reduction and conversion of tHcy to S-adenosyl-L-homocysteine (SAH) before solid-phase competitive immunoassay that uses a monoclonal anti-SAH antibody.

All subjects underwent a >11-h overnight fast. Blood was collected in EDTA anticoagulant tubes cooled to <4 °C, and the plasma was removed from the cells within 1 h in a refrigerated centrifuge (<10 °C). Aliquots of plasma were prepared for each subsequent method and were kept at -20 °C until analysis. The Axis EIA assay method was used with no modifications to the recently published protocol (4). For the ABD-F assay, equivalent 50-µL volumes of plasma and internal standard were mixed with 50 µL and 100 µL of trialkylphosphine and ABD-F, the reducing and derivatizing agents, respectively. Samples were then incubated at 60 °C for 7 min and then at 4 °C for 7 min, followed by trichloroacetic acid precipitation of plasma proteins. The supernatant was removed for HPLC analysis after centrifugation for 5 min at 10 000g. The chromatographic conditions were as follows: a Perkin-Elmer Liquid Chromatograph 85, a Bio-Rad analytical reversed-phase C₁₈ column (70 x 3.2 mm i.d.), 38 °C, Bio-Rad mobile phase, isocratic flow rate of 0.7 mL/min, and an HP-1046 fluorescence detector, with the excitation and emission wavelengths of 385 nm and 515 nm, respectively, for detection of ABD-F thiols. Under these chromatographic conditions, internal standard and Hcy peaks were completely resolved from other thiol-containing compounds with respective retention times of 2.90 and 3.67 min. The SBD-F HPLC assay used the method of Fortin et al. (5), with minor modifications. Briefly, 240 µL of plasma, 50 µL of acetylcysteine (internal standard), and 30 µL of tri-n-butylphosphine (reducing agent) were mixed and incubated at 4 °C for 30 min. Plasma proteins were precipitated by the addition of 300 µL of perchloric acid. After centrifugation, 50 µL of the supernatant was derivatized with 50 µL of SBD-F (Sigma Chemical Co.) at 60 °C for 60 min. HPLC separation and fluorescent detection were as described above. For this assay, calibrators (0, 5, 10, 15, 25, and 35 µmol/L) were prepared by addition of crystalline D,L-homocysteine (Sigma) to pooled patient EDTA plasma samples.

The imprecision data for the ABD-F, SBD-F, and EIA assays are shown in Table 1 . For the ABD-F and EIA assays, controls were assayed in duplicate daily for 21 days. For the SBD-F assay, pools of previously analyzed patient plasma specimens were assayed daily for 31 days. We assessed the linearity of the Bio-Rad ABD-F assay by serial dilution with Bio-Rad Assay Reconstitution Buffer of a high tHcy patient pool, which gave a linear relationship between expected (x) and actual (y) tHcy concentrations from 0.5 to 100 µmol/L (r = 0.99; regression line, y = 1.03x - 0.68), with mean recoveries and tHcy concentrations ranging from 83% to 101%, respectively. The split sample comparisons used 95 patient samples submitted from cardiology and nephrology clinics for the evaluation of patients who have greater risk for increased tHcy concentrations. The mean difference between sample duplicates (assayed on 2 separate days) for the ABD-F, SBD-F, and EIA methods were 1.02, 1.16, and 2.12 µmol/L, respectively. As shown in Fig. 1 , A and B, linear regression equations were found for HPLC [ABD-F = 0.89(SBD-F) - 3.1, r = 0.983, S_yx = 1.17, n = 95] and for EIA [ABD-F = 0.88(EIA) - 0.59, r = 0.975, S_yx = 1.01, n = 95]. Fig. 1C shows a plot of the differences between patient tHcy (range, 3.4–55.1 µmol/L) by each method and the all-method mean. The mean bias was -2.5 (P <0.001), -0.1, and 2.6 µmol/L (P <0.001) for the ABD-F, EIA, and SBD-F methods, respectively. To further investigate these intermethod biases, the ABD-F method calibrator was assayed in triplicate on both the SBD-F and EIA methods and vice versa. Assay of the EIA method calibrators was not possible because these calibrators are made with SAH, which was not detectable by the HPLC methods. The ABD-F calibrator assayed with the SBD-F and EIA method yielded higher tHcy (4.5 and 1.1 µmol/L, respectively). Conversely, the SBD-F calibrators assayed with the ABD-F and EIA method yielded lower tHcy (-5.5 and -3.3 µmol/L, respectively). Using the ABD-F assay, we established our tHcy reference interval (5th and 95th centiles) for healthy men (n = 42; age range, 19–61 years) to be 4.5 and 12.0 µmol/L, respectively, with tHcy ranging from 3.4 to 19.4 µmol/L; for healthy women (n = 83; age range, 19–58 years) the tHcy centiles were 3.7 and 10.5 µmol/L, respectively, with a range of 2.8–14.3 µmol/L. In all subjects, vitamin B₁₂ and erythrocyte folate concentrations were found to be within reference limits (data not shown). The men had significantly higher tHcy than the women (P <0.05). In our review of the tHcy reference intervals for various HPLC and gas chromatography–mass spectrometry methods in the literature, we found that our results were closest to the SBD-F method of Araki and Sako (6) and the gas chromatography–mass spectrometry method of Stabler et al. (7).

View larger version (39K): [in this window] [in a new window]   Figure 1. Comparison of the three assays for tHcy. Comparison of plasma tHcy concentrations in patients determined by the ABD-F vs the SBD-F (A) and the ABD-F vs the EIA (B) assay methods. In A and B, the dotted line indicates line of identity (x = y). (C) Difference plot comparing the overall mean tHcy concentration of all three methods vs the SBD-F, EIA, and ABD-F assay methods. (), SBD-F; (), EIA; (), ABD-F.

Our estimates of within-run and between-run reproducibility of the Bio-Rad ABD-F method are comparable to that stated by the manufacturer and are acceptable at both apparently healthy (7.6 µmol/L) and high (23.1 µmol/L) tHcy concentrations. Thus in our hands, assay reproducibility and linearity of the ABD-F method are clinically acceptable. The intermethod bias was consistent with the bias among method calibrators. For the most reliable intermethod comparisons of tHcy, the use of a standardized matrix-matched calibrator prepared with the disulfide form would have been optimal, especially because the protein-bound disulfide form represents the major species of tHcy in plasma (8), and it controls the reduction step in the method. Commercially available preparations of disulfide D,L-homocystine have a higher purity, less lot variability, and better stability in plasma than the reduced form (9)(10). The reduction and thiol derivatization steps, which are cumbersome and laborious among HPLC methods and are sources of intramethod variability, were optimized in the Bio-Rad assay. For example, the use of the more water-soluble reducing agent trialkylphosphine in the Bio-Rad assay instead of the tri-n-butylphosphine used in the SBD-F assay enabled faster reduction, generally within 5–7 min instead of 30 min, respectively (11)(12). Similarly, because ABD-F reacts 30 times faster with thiols than SBD-F (13), a shorter incubation time—7 min instead of 60 min at 60 °C, respectively—was required. In addition, the efficiency of SBD-F thiol derivatization can be matrix-dependent (9) and substantially affected by lot-to-lot variations and by differences among manufacturers (11). The best between-run replicate sample measurements among the three methods were consistent with the the ABD-F method requiring less technologist handling and specimen transfer steps when compared with the other two assays. For example, with the ABD-F assay, reduction and derivatization occur in the same tube progressively with short incubation times. The SBD-F method requires reduction before protein precipitation, the subsequent removal of the plasma proteins, and the transfer of an aliquot of the resulting supernatant to another tube for derivatization. In our hands, the manual EIA assay was the most labor-intensive in reagent preparation and sample treatment, with 9 different reagents, a total of 10 different dilution and incubation steps of various times, and 2 microtiter plate washing procedures (4).

In conclusion, the Bio-Rad method was reproducible, rapid, and easy to use for the quantification of plasma tHcy, with the advantages of commercially available reagents and calibrators and reduced preparation time. Cross-method standardization remains problematic for this assay, especially because it markedly impacts the interpretation of many epidemiological studies.

Acknowledgments

We wish to thank Linda Forward and Carol Shalapay for technical assistance.

References

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