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Technical Briefs |
National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341
a author of correspondence: fax 770-488-4609, e-mail cfp8{at}cdc.gov
Although several approaches for measuring plasma total homocysteine by HPLC have been described during the last few years (1)(2)(3)(4), none combines all the desired features for a rapid, user-friendly, and robust assay: (a) a stable, efficient, and nonhazardous reducing agent; (b) incorporation of a suitable internal standard; and (c) rapid, isocratic separation of the thiols of interest, using a mobile phase of mild pH. We have therefore modified the method of Vester and Rasmussen (5) by using tris(2-carboxyethyl)phosphine (TCEP), a newer stable, water-soluble phosphine derivative introduced by Gilfix et al. (6), as the reducing agent, cystamine as the internalnd isocratic separation of the thiols extracted from only 50 µL of pla standard, asma within 6 min.
A mixture of 50 µL of plasma, 25 µL of internal standard, and 25 µL of phosphate-buffered saline (PBS, pH 7.4) was incubated with 10 µL of 100 g/L TCEP (Pierce Chemical Co.) for 30 min at room temperature to reduce and release protein-bound thiols, after which 90 µL of 100 g/L trichloroacetic acid containing 1 mmol/L EDTA was added for deproteinization. After the sample was centrifuged for 10 min at 13000g, 50 µL of the supernatant was added to an autosampler vial containing 10 µL of 1.55 mol/L NaOH; 125 µL of 0.125 mol/L borate buffer containing 4 mmol/L EDTA, pH 9.5; and 50 µL of 1 g/L SBD-F (Wako Chemicals) in the borate buffer. The sample was then incubated for 60 min at 60 °C. HPLC was carried out on a 2690 Alliance solvent delivery system and a 474 scanning fluorescence detector (385 nm excitation, 515 nm emission), both from Waters Technologies Corp. Separation of the SBD-derivatized plasma thiols was performed on a Prodigy ODS2 analytical column, 150 x 3.2-mm, 5 µm (Phenomenex) with an Adsorbosphere C18, 3-cm guard column (Alltech Associates), using a 10-µL injection volume and 0.1 mol/L acetic acid-acetate buffer, pH 5.5, containing 30 mL/L methanol as mobile phase at a flow rate of 0.7 mL/min and a column temperature of 29 °C.
L-Homocystine and L-cysteine calibrators (0–50 µmol/L free thiol in 100-µL assay volume) were prepared in PBS, pH 7.4, and in pooled EDTA plasma. The internal standard was cystamine dihydrochloride, which was added to all samples to achieve a final concentration of 10 µmol/L free thiol (in 100-µL assay volume). All chemicals were obtained from Sigma Chemical Co. Calibration was performed daily in PBS and in plasma (standard addition) and was evaluated as both external and internal calibration (area ratios between the thiol and the internal standard).
Plasma specimens from healthy adult volunteers were obtained from whole blood collected into EDTA-containing tubes (Becton-Dickinson) and cooled on ice water; the plasma was separated by centrifugation within 30 min after venipuncture and stored for a maximum of 3 months at -70 °C before being assayed. Blood specimens were collected by the Emory University Hospital Blood Collection Service under an agreement with the CDC (including an omnibus informed consent and Human Subjects Review protocol).
Under the chromatographic conditions described, the retention times of
all thiols were very stable, with a CV <2% during 6 months. At pH
5.5, homocysteine, cysteine, and the internal standard were clearly
baseline separated from each other and from cysteinylglycine and
glutathione (Fig. 1
A). Increasing the pH of the mobile phase to 6.0 had no
significant effect on the separation or retention times. Lowering the
pH of the mobile phase to 5.0 and 4.5 improved the separation between
cysteinylglycine and glutathione (Fig. 1B
). No interfering peaks were
observed in plasma or serum samples for any of the thiols measured.
Plasma samples with no internal standard added showed no cysteamine
peak.
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Calibration curves for homocysteine were linear up to 200 µmol/L for samples prepared in PBS (r2 = 0.997) or in plasma (r2 = 0.999). The limit of detection for homocysteine was 0.16 µmol/L.
The mean recoveries (± SD) of L-homocystine added to plasma at five different concentrations (3.13–50 µmol/L free thiol), determined on 10 days, were 98.7% ± 2.5% and 96.7% ± 4.7%, calculated with internal and external calibration, respectively. For cysteine, recoveries were 100.6% ± 1.5% and 98.7% ± 3.5%, calculated with internal and external calibration, respectively.
Two plasma specimens containing high total homocysteine (tHcy) concentrations (28.5 and 360 µmol/L) were diluted with PBS 0- to 8-fold. The ratios of the observed/expected values were between 1.0 and 1.1.
The mean intraassay CVs for 20 plasma samples processed in five replicates on 1 day ranged from 1.1% to 1.8% for tHcy and cysteine (tCys). The mean interassay CV of the same 20 plasma samples processed in one replicate on 5 days was 5.6% and 2.4% for tHcy and tCys, respectively. Analyzed over 20 days, the three in-house plasma quality-control (QC) pools showed a variation for tHcy of 6.7% (low pool, 6.5 µmol/L), 5.0% (medium pool, 12.4 µmol/L), and 4.4% (high pool, 29.9 µmol/L) for internal calibration in plasma. The day-to-day variation was higher when internal calibration was performed in PBS or external calibration was performed in plasma. External calibration in PBS produced significantly increased tHcy values for the low and medium QC pools. The slope of the daily calibration curve demonstrated less variation with internal calibration (4.3% vs 8.5% with external calibration).
The correlation between tHcy concentrations for 38 plasma samples covering tHcy concentrations within and greater than the health-related reference range calculated with PBS calibration and with plasma calibration was very good: internal calibration (r2 = 1.0000; slope = 0.9942; intercept = -0.0063), external calibration (r2 = 1.0000; slope = 0.9838; intercept = -1.3309). However, with external calibration the intercept was significantly higher, which especially affected quantification of low homocysteine concentrations.
We performed a direct comparison of the reducing efficiency of TCEP,
the newer reducing agent, and tributyl phosphine (TBP), the older
reductant (Table 1
). Although the relative fluorescence intensities were lower if
TBP was used as the reducing agent (approximately two-thirds of the
TCEP value), this difference was not apparent in the calculated
concentrations of tHcy because of the calibration. For internal
calibration in plasma, tHcy concentrations were indistinguishable
between TCEP and TBP. Internal calibration in PBS or external
calibration in plasma gave the same results if TCEP was used as the
reducing agent. The use of TBP led to measured tHcy concentrations up
to 20% different from the values obtained with TCEP. Finally, external
calibration in PBS led to significantly increased values for TCEP
compared with the other three calculation types. For TBP, we found
signifi-cantly increased values for the low QC pool and significantly
decreased values for the high QC pool. The slopes obtained with
internal calibration were not significantly different for the two
reducing agents: 0.034 (TCEP) and 0.035 (TBP) for calibration in
plasma, and 0.032 (TCEP) and 0.031 (TBP) for calibration in PBS.
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We studied tHcy and tCys plasma concentrations in 70 healthy subjects (27 men and 43 women; mean age, 43.8 ± 10.6 and 40.7 ± 9.0 years, respectively). The mean tHcy and tCys values were 9.1 ± 1.8 and 298 ± 29 µmol/L, respectively, for men, and 7.8 ± 1.7 and 280 ± 32 µmol/L, respectively, for women. For both thiols, men had significantly higher plasma concentrations than women (P = 0.0112 and P = 0.0287 for tHcy and tCys, respectively).
In conclusion, the protocol described is a robust, user-friendly, rapid assay, suitable for clinical and pediatric settings. The use of cystamine as the internal standard significantly improves the precision of this method and overcomes the matrix effect of plasma.
References
The following articles in journals at HighWire Press have cited this article:
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L. B. Bailey, R. L. Duhaney, D. R. Maneval, G. P.A. Kauwell, E. P. Quinlivan, S. R. Davis, A. Cuadras, A. D. Hutson, and J. F. Gregory III. Vitamin B-12 Status Is Inversely Associated with Plasma Homocysteine in Young Women with C677T and/or A1298C Methylenetetrahydrofolate Reductase Polymorphisms J. Nutr., July 1, 2002; 132(7): 1872 - 1878. [Abstract] [Full Text] [PDF]
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G. J. Cuskelly, P. W. Stacpoole, J. Williamson, T. G. Baumgartner, and J. F. Gregory III Deficiencies of folate and vitamin B6 exert distinct effects on homocysteine, serine, and methionine kinetics Am J Physiol Endocrinol Metab, December 1, 2001; 281(6): E1182 - E1190. [Abstract] [Full Text] [PDF]
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M. A. Caudill, J. C. Wang, S. Melnyk, I. P. Pogribny, S. Jernigan, M. D. Collins, J. Santos-Guzman, M. E. Swendseid, E. A. Cogger, and S. J. James Intracellular S-Adenosylhomocysteine Concentrations Predict Global DNA Hypomethylation in Tissues of Methyl-Deficient Cystathionine {beta}-Synthase Heterozygous Mice J. Nutr., November 1, 2001; 131(11): 2811 - 2818. [Abstract] [Full Text] [PDF]
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J. Krijt, M. Vackova, and V. Kozich Measurement of Homocysteine and Other Aminothiols in Plasma: Advantages of Using Tris(2-carboxyethyl)phosphine as Reductant Compared with Tri-n-butylphosphine Clin. Chem., October 1, 2001; 47(10): 1821 - 1828. [Abstract] [Full Text] [PDF]
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G. L. Hortin, P. Sullivan, and G. Csako Relationships among Plasma Homocysteine, Cysteine, and Albumin Concentrations: Potential Utility of Assessing the Cysteine/Homocysteine Ratio Clin. Chem., June 1, 2001; 47(6): 1121 - 1124. [Full Text] [PDF]
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M. Zhang, E. W. Gunter, and C. M. Pfeiffer Evaluation of the Drew Scientific DS30 Homocysteine Assay in Comparison with the Centers for Disease Control and Prevention Reference HPLC Method Clin. Chem., May 1, 2001; 47(5): 966 - 967. [Full Text] [PDF]
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 M. A. Caudill, T. Le, S. A. Moonie, S. T. Esfahani, and E. A Cogger Folate Status in Women of Childbearing Age Residing in Southern California after Folic Acid Fortification J. Am. Coll. Nutr., April 1, 2001; 20(2): 129 - 134. [Abstract] [Full Text] [PDF]
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C. M. Pfeiffer, S. P. Caudill, E. W. Gunter, B. A. Bowman, P. F. Jacques, J. Selhub, C. L. Johnson, D. T. Miller, and E. J. Sampson Analysis of Factors Influencing the Comparison of Homocysteine Values between the Third National Health and Nutrition Examination Survey (NHANES) and NHANES 1999+ J. Nutr., November 1, 2000; 130(11): 2850 - 2854. [Abstract] [Full Text] [PDF]
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C. M. Pfeiffer, D. L. Huff, S. J. Smith, D. T. Miller, and E. W. Gunter Comparison of Plasma Total Homocysteine Measurements in 14 Laboratories: An International Study Clin. Chem., August 1, 1999; 45(8): 1261 - 1268. [Abstract] [Full Text] [PDF]
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