Plasma homocysteine determined by capillary electrophoresis with laser-induced fluorescence detection.
A widely used technique for measuring total plasma Hcy is reversed-phase HPLC with fluorescence detection after derivatization of plasma thiols (4-6). Some studies use gas chromatography-mass spectrometry techniques (7) with various derivatization protocols (8).
The increasing demand for the determination of plasma total Hcy prompted us to develop a rapid, automated method based on capillary electrophoresis (CE) and laser-induced fluorescence detection (LIF), in which all specific thiols are detected selectively.
Blood was collected by venipuncture into evacuated tubes containing EDTA from patients with episodes of deep-vein thrombosis or vascular disease. The plasma was separated by centrifugation and was stored at -20[degrees]C until analysis.
Plasma samples (100 [micro]L) were treated for 30 min at room temperature with 10 [micro]L of a novel thiol-reducing agent [35 mmol/L tris(2-carboxyethyl)phosphine; Molecular Probes], as reported recently by Gilfix et al. (9). Compared with the widely employed tributyl phosphine, this new phosphine is nonvolatile, stable, soluble in water, and without disagreeable odor (5,6). When we used this procedure, total Hcy (mixed and symmetric disulfides, including protein-bound Hcy) was analyzed in its reduced form.
All patient samples and plasma-based calibrators were incubated with tris(2-carboxyethyl)phosphine under the same conditions. The internal standard [5 [micro]L of 10 mmol/L D-penicillamine (DP)] was mixed with plasma. The solution was deproteinized with 60 [micro]L of 0.8 mol/L 5-sulfosalicylic acid with vortex-mixing, followed by centrifugation at 10 000g for 15 min.
We mixed 100 [micro]L of the supernatant with 50 [micro]L of 0.3 mol/L carbonate buffer, pH 9.5, and 5 [micro]L of 5 mol/L sodium hydroxide. We then added 50 [micro]L of a 1 g/L 6-iodoacetamidofluorescein solution (6-IAF) in dimethyl sulfoxide (Sigma Chemical Co.), and the mixture was incubated overnight in the dark at room temperature to allow analysis after the reaction has plateaued (>2 h). IAF-labeled samples were stored at -20 [degrees]C until analysis; they could be preserved for up to 3 months. Each derivatized plasma was analyzed directly after being diluted 5000-fold before the CE injection. The analyses were performed on a Zeta CE instrument (Zeta Technology) equipped with a modular LIF detector and an argon ion laser (wavelength, 488 nm). The different thiols were separated on a 75 cm x 50 [micro]m (i.d.) fused-silica capillary (Polymicro Technology) with an effective length of 43 cm and a total length of 75 cm. The separation buffer consisted of 10 mmol/L sodium dodecyl sulfate, 50 mmol/L boric acid, and 20 mmol/L 3-(cyclohexylamino)-1-propanesulfonic acid adjusted to pH 9.5 by addition of a sodium hydroxide solution (10 mol/L). The separation voltage was +25 kV, producing an electrophoretic current of 25 [micro]A.
All samples were assayed in triplicate. Peaks were identified by use of solutions of Hcy, Cys, or glutathione, and the internal standards cysteamine or DP. Fig. 1A shows the thiol solutions prepared in carbonate 0.3 mmol/L buffer, pH 9.5, and then diluted in deionized water. Under the electrophoretic conditions used, the Hcy derivative is well separated with a migration time of 7.2 min.
Electropherograms of a human plasma sample with added DP as internal standard produced several peaks (Fig. 1, B and C). Known quantities of Hcy, Cys, or DP solutions were added to the plasma sample to identify Hcy, Cys, and DP peaks.
Hcy calibrators were prepared from a plasma pool (30, 15, 10, 5, and 2 [micro]mol/L), and the concentrations were calculated according to peak heights. The calibration curves for Hcy quantification were obtained by plotting the peak height ratio of Hcy/DP and Hcy/Cys. The equations, as a function of the Hcy concentration x (expressed in [micro]mol/L) were, respectively, as follows: y = 0.033x + 0.013 (r = 0.9998) for Hcy/DP and y = 0.011x + 0.003 (r = 0.9992) for Hcy/Cys.
The detection response was linear over the concentration range between 2 and 200 [micro]mol/L for all the plasma thiols. The detection limit for Hcy, defined as a signal-to-noise ratio >3, was -0.25 [micro]mol/L in plasma and was quite identical for the other thiols. The detection limit of the diluted sample injected (after the 5000 fold-dilution) was 50 pmol/L, corresponding to a quantity of 1.1 attomoles.
The within-day reproducibility of the assay for Hcy was determined on aliquots (n = 10) prepared independently from the same plasma sample, and the between-day reproducibility was calculated from the analysis of the same plasma sample that was derivatized each day during 10 consecutive days. The coefficients of variation (CVs) which take into account every step (i.e., reduction, precipitation, centrifugation, derivatization, and injection into the CE-LIF) were 4.9% and 7.8%, respectively, whereas the CVs for injection only were 1.8% and 2.6%.
[FIGURE 1 OMITTED]
To determine the recovery of the method, we added 10 [micro]L of different concentrations of Hcy (2, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 [micro]mol/L) to 90 [micro]L of the same pooled plasma. The average recovery was 100.5% [+ or -] 4.87% (mean [+ or -] SD). The recovery (mean [+ or -] SD) of Hcy added to six plasma samples analyzed in triplicate (Table 1) was 101.1% [+ or -] 6.99% (n = 18).
We used this CE-LIF technique to analyze serum samples from 55 fasting subjects with episodes of deep-vein thrombosis or arterial occlusive disease, and we compared the results with those obtained from an ion-exchange amino acid analyzer (Beckman 6300 analyzer). The mean control values (n = 27) for the amino acid analyzer method (y) were 8.89 [+ or -] 2.85 [micro]mol/L vs 11.73 [+ or -] 4.45 [micro]mol/L for CE-LIF (x). The regression equation was as follows: y = 0.91x + 2.26; n = 55; [S.sub.y|x] =7.49; r = 0.87, which is comparable to previous observations (5, 8,10).
The same calibrator, i.e., homocystine (Sigma), was used in both methods: in the ion-exchange chromatography method, homocystine was diluted in lithium citrate buffer (0.2 mol/L, pH 7.0), and the calibrator curve in aqueous solution was determined using the ninhydrin reaction (specific for amine groups); in the CE-LIF method, homocystine was diluted in carbonate buffer (pH 9.5), and the calibrator curve in plasma solution was determined using IAF, which reacts specifically with thiol groups.
The major advantage of this IAF method is its ability to quantify Hcy, Cys, and glutathione simultaneously in a single analysis. Our derivatized thiols were stable for several months. The method is thiol-specific and the detection limit (0.25 [micro]mol/L Hcy in plasma) is similar to other HPLC or amino acid analyzer procedures (4,5). The IAF method saves time compared with the HPLC method, and we encountered no problems with interference from the reducing agent. When compared with the fluorescein isothiocyanate method, described previously (11), which quantifies Hcy and all amino acids through the reaction with amine groups, the IAF-based procedure allows a more selective detection of thiol-containing amino acids. In addition, in the IAF method, the migration times are shorter, the peak resolution is better, and co-injection of a calibrator is not required. The use of an internal standard [DP or homocysteic acid (11)] also improves the accuracy and the standardization (12) of the method.
The IAF method is simple, low in reagent cost (11), sensible, reproducible, and suitable for routine determination of serum/plasma Hcy concentrations in a clinical laboratory. This method will allow us to investigate and evaluate the potential atherogenic properties of Hcy.
This work was supported by grants from the Conseil Regional Midi-Pyrenees. We thank L. Casey, Washington University, St. Louis, MO for help with manuscript preparation.
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Elizabeth Causse,  * Nathalie Siri,  Helene Bellet,  Sandrine Champagne,  Christophe Bayle,  Pierre Valdiguie,  Robert Salvayre,  and Francoise Couderc  ( Laboratoire de Biochimie/Institut National de la Sante et de la Recherche Medicale, U466, Centre Hospitalier Rangueil, 1 avenue J. Poulhes, 31403 Toulouse Cedex 04, France;  Laboratoire de Biochemie, Centre Hospitalier St Elio, 34295 Montpellier, France;  ZETA Technology, Parc Technologique du Canal, 31520 Ramonville, France;  Universite Paul Sabatier, Institut de Pharmacologie et de Biologie Structurale, 205 Route de Narbonne, 31077 Toulouse, France; author for correspondence: fax 33 5 61 322953)
Table 1. Recovery of the assay. Concentration, [micro]mol/L Mean Patient Sample Added Measured recovery, % 1 9.6 5 14.0 96.0 2 28.5 5 31.3 93.4 3 5.6 10 16.6 106.4 4 68.5 20 84.4 95.4 5 9.6 25 36.2 104.6 6 20.3 40 66.6 110.4
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|Title Annotation:||Technical Briefs|
|Author:||Causse, Elizabeth; Siri, Nathalie; Bellet, Helene; Champagne, Sandrine; Bayle, Christophe; Valdiguie|
|Date:||Mar 1, 1999|
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