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Stability of blood homocysteine and other thiols: EDTA or acidic citrate?

Homocysteine (Hcy), a thiol-containing amino acid resulting from demethylation of methionine (Met), is relevant to the risk of vascular diseases (1). In plasma, total homocysteine (tHcy) includes the free reduced and oxidized forms as well as the Hcy bound by disulfide bonds in proteins. tHcy is frequently increased in patients with coronary, cerebrovascular, or peripheral arterial diseases; the association is independent of most other risk factors for atherosclerosis (2). The simultaneous measurement of other thiols is of interest because most of them are metabolically related and disturbances of their concentrations can correspond to disorders of metabolism. Hcy may either be catabolized to cysteine (Cys) or remethylated to Met (3). In addition, Cys and y-glutamylcysteine are precursors to glutathione (GSH), and cysteinylglycine (CysGly) is a breakdown product of GSH; this latter plays a major role in defense against oxidative and free radical-mediated cell injury, and its measurement permits the evaluation of oxidative status of cells and tissues (4).

It should be expected that less rigorous blood sampling and treatment conditions are needed than those involved for thiol redox status evaluation (5). For plasma, storage conditions have little influence on tHcy values: tHcy in plasma is stable at -20 [degrees]C for at least 3 months and after nine freeze/thaw cycles (6). In contrast, in whole blood, an increase of tHcy is observed after collection because of ongoing metabolism and time-dependent release from erythrocytes (7). The artificial increase in plasma tHcy occurs at a rate of 1 [micro]mol/L per hour at room temperature (6, 7). In most studies, blood is drawn in tubes containing potassium EDTA, which are put immediately in crushed ice and centrifuged "as soon as possible", and then the plasma is frozen. These conditions prevent increases in tHcy in whole blood after collection, but they are not always practical. An alternative procedure is needed, especially for large epidemiological studies involving different sample collection sites, where processing with crushed ice is difficult. Use of sodium fluoride (with heparin as anticoagulant) has been proposed to inhibit Hcy release from blood cells for 2 h at room temperature (8). More recently, Willems et al. (9) have claimed that blood collection in acidic citrate tubes stabilizes tHcy in whole blood: tHcy did not increase markedly for 6 h at room temperature after collection, but an increase of 1.3 [micro]mol/L was found at baseline in tHcy measured in samples collected into acidic citrate compared with EDTA samples. tHcy has been reported to be stable in blood lysate for 2 days at room temperature; however, tHcy concentrations were lower than in plasma, and a different reference range had to be defined (10). Finally, an inhibitor of S-adenosylhomocysteine hydrolase, i.e., 3-deazaadenosine, has been reported to keep tHcy constant in whole blood samples at room temperature for 24 h (11).

Other plasma thiols have been measured. The usual tHcy assays rely on gas chromatography-mass spectrometry (8), HPLC (5-7, 9, 10, 12-15), or fluorescence polarization immunoassay (FPIA) (16). Gas chromatographymass spectrometry is used preferentially to study Hcy and its metabolites, cystathionine and Met. HPLC permits the concomitant measurement of other plasma thiols, mainly Cys, CysGly, and GSH, whereas FPIA is devoted to Hcy alone. However, all of these methods include a plasma sample treatment with a reducing agent, e.g., sodium borohydride (5, 9,11,13), tri-n-butylphosphine (6,10,12), or 1,4-dithio-D,L-threitol (5, 7,8,15-17) to convert free oxidized and bound thiols into their free reduced form. Stability of other thiols in plasma has been studied with HPLC methods. Blood uptake and treatment sequences with similar anticoagulants and operating precautions as for Hcy are usually involved to stabilize the plasma thiols. Additional reagents can be used: (a) L-serine-borate, a specific inhibitor of [gamma]-glutamyltranspeptidase (EC 2.3.2.2), to prevent cleavage of GSH (13,14,17,18); or (b) bathophenanthroline disulfonate disodium salt, as a chelating agent of iron (14,17), and thiol trapping reagents (5,14,17) to avoid further thiol oxidation, especially when their redox status is studied.

We used an HPLC method for the simultaneous measurement of tHcy and other total plasma thiols, i.e., tCys, tCysGly, and tGSH, to evaluate the use of acidic citrate-treated blood at room temperature instead of EDTA-treated blood at 0 [degrees]C for the assay of all of the cited thiols. Different time intervals between blood collection and centrifugation were considered, ranging from 15 min to 6 h and including the minimum (15 min) and maximum (2 h) delay time periods existing between these two steps in our own laboratory practices. Moreover, we investigated the difference between the two blood-collection media on the tHcy concentration by use of a commercially available FPIA.

Blood was drawn by venipuncture of the antecubital vein from healthy laboratory coworkers (n = 21); informed consent was obtained, and the research protocol was in agreement with the Helsinki Declaration. Blood was collected in tubes containing either 1.8 g/L [K.sub.3]EDTA (Vacutainer[TM] Tubes; Becton Dickinson) or 0.5 mol/L acidic citrate (Biopool Stabilyte[TM]). The EDTA-treated blood was immediately put on crushed ice, and the acidic citrate-treated blood was kept at room temperature (i.e., 24 [degrees]C in the present case). Two independent studies were performed with HPLC and FPIA, respectively. The HPLC study used blood from seven subjects (three men and four women; ages, 28-52 years). Within 15 min, each blood sample was divided into six equal volumes; these aliquots were centrifuged (1000g for 15 min at 4 [degrees]C for EDTA-treated samples and at room temperature for acidic citrate-treated samples) at 15 and 30 min and 1, 2, 4, and 6 h after blood collection. The FPIA study was done with blood from 14 subjects (8 men and 6 women; ages, 27-56 years), using the same methodology at 15 min and 2 h. Plasma samples resulting from the two studies were frozen at -80 [degrees]C until analysis.

The plasma thiols were measured with a slightly modified HPLC technique (11) including reduction of disulfides with tri-n-butylphosphine, precolumn derivatization with 7-fluoro-2,1,3-benzoxadiazole-4-sulfonamide, isocratic reversed-phase chromatography, and spectrofluorometric detection. The stock solution of each thiol calibrator was prepared at a concentration of 1.0 mmol/L in 10.0 mmol/L HCl containing 1 mmol/L EDTA and kept at -80 [degrees]C for a maximum of 2 months. Calibration curves including five points were constructed daily with further dilutions of stock solutions in 9 g/L NaCl containing 4 mmol/L EDTA, at concentrations of 50-300 [micro]mol Cys/L, 10-50 [micro]mol CysGly/L, 2.5-15 [micro]mol Hcy/L, and 1-10 [micro]mol GSH/L. Plasma samples were quickly thawed at 37 [degrees]C. A 200-[micro]L aliquot of plasma or calibrator was transferred into a 1.5-mL polypropylene tube (in crushed ice), to which we added 100 [micro]L of the internal standard solution [thioglycolic acid at a concentration of 300 [micro]mol/L] and 50 [micro]L of tri-n-butylphosphine (50 mL/L) in dimethylformamide. After the sample was mixed for 10 s with a vortex-mixer, a nitrogen stream was introduced into the tube for 10 s before it was capped. The resulting mixture was incubated in the dark at 4 [degrees]C for 30 min, and 200 [micro]L of a trichloroacetic acid solution (100 g/L) was added for protein precipitation. After centrifugation at 1500g for 15 min at 4 [degrees]C, 100 [micro]L of the supernatant was transferred into a 1-mL autosampler vial and mixed with 30 [micro]L of 0.5 mol/L NaOH, 250 [micro]L of 0.2 mol/L borate buffer, pH 9.0, and 50 [micro]L of a 2.3 [micro]mol/L 7-fluoro-2,1,3-benzoxadiazole-4-sulfonamide solution in dimethylformamide. The resulting mixture was incubated at 50 [degrees]C for 20 min under gentle stirring, and then the derivatization reaction was stopped by the addition of 50 [micro]L of 1.0 mol/L HCl and chilling in crushed ice.

A 20-[micro]L aliquot was injected into a guard column (4 x 4 mm i.d.) packed with end-capped LiChrospher RP18 (5 [micro]m bead size; Merck Darmstadt) and a short-length analytical column (70 x 4 mm i.d.) packed with Nucleosil 100 [C.sub.18] (5 [micro]m bead size; Macherey Nagel). The sample was eluted with 50 mL/L acetonitrile-950 mL/L phosphate buffer (0.1 mol/L), pH 2.5, at a column temperature of 35 [degrees]C and a flow rate of 1.0 mL/min. Excitation and emission wavelengths were set at 385 and 515 nm, respectively.

We used the Abbott IMx[R] plasma assay for tHcy, as described by Shipchandler and Moore (16). The thiol concentrations from the samples collected in the tubes containing acidic citrate were corrected by the dilution factor caused by the fluid (0.5 mL) initially present in the tube.

The HPLC method was linear for the concentrations indicated above (r >0.990), and its recoveries were >98%. The within-day imprecision (CV; n = 6) measured with a plasma pool was 2.2% for tCys, 1.7% for tCysGly, 1.9% for tHcy, and 7.2% for tGSH at concentrations of 178.0, 27.8, 7.9, and 3.4 [micro]mol/L, respectively. For the IMx assay of tHcy, the intraday CV (n = 6) was 1.0% at a concentration of 12.5 [micro]mol/L. ANOVA with repeated measurements was used to test the significance of differences between thiol concentrations.

Plasma thiol concentrations measured by HPLC using two different blood collection and storage conditions (EDTA at 0 [degrees]C and acidic citrate at room temperature) for delay periods before centrifugation of 15 min to 6 h are shown in Fig. 1.

Differences of thiol concentrations noted between the two collection media were as follows: whatever the delay before centrifugation, tCys concentrations were higher in EDTA plasma than in acidic citrate plasma (6.1% at baseline and 5.3% at 6 h), whereas tGSH concentration were lower (-10.9% at baseline and -8.3% at 6 h), all tests being statistically significant (P <0.05). tHcy concentrations were higher in EDTA than in acidic citrate plasma (5.2% at baseline and 4.6% at 6 h). A previous report (9) found that tHcy was significantly lower in EDTA than in acidic citrate medium at baseline and at any delay period (-9.3% at baseline and -10% at 6 h) in the same conditions of temperature and storage time as in the present work. No difference in tCysGly concentrations was observed.

Over the time points studied, the time to centrifugation after collection of blood in either EDTA or acidic citrate collection tubes did not significantly affect the concentrations for these thiols except for tCysGly, the concentration of which varied slightly with time (0.5 [micro]mol/L per hour in both collection media). We noticed no significant difference in tCysGly concentrations between EDTA and acidic citrate media at the delay time of 2 h, as reported previously (7); this fact could be also related to the changes of tCys observed between EDTA and acidic citrate media. A hypothetical mechanism could be a leak of GSH from erythrocytes, in which its concentration is much higher than in plasma [3.6 [micro]mol/L in plasma and 0.9 mmol/L in whole blood, respectively (15)], followed by a degradation pathway via reactions catalyzed by [gamma]-glutamyltranspeptidase and dipeptidases (EC 3.4.13.6). The tGSH concentration in plasma is also lower in EDTA than in acidic citrate medium, probably because the same enzymatic reactions are involved. As a matter of fact, a simultaneous decrease of GSH and increase of Cys and CysGly in plasma have been observed as a function of blood storage period between collection and centrifugation (5,13), and a specific [gamma]-glutamyltranspeptidase inhibitor such as L-serine-borate added to the collection medium (14, 15,17, 18) seems to be a good alternative to keep the concentration of these thiols stable as a function of time.

The results of the FPIA assays are shown in Table 1. During the restricted delay period (2 h) corresponding to a more practical approach in our laboratory, a slight but significant increase as a function of time was observed (4.9% between 15 min and 2 h in both media). The difference in tHcy measurement by FPIA between the two collection media was not significant (P >0.05). Such an absence of difference was opposite to data obtained with HPLC in the present study and in a previous study (9). It could be explained by less selectivity of FPIA than HPLC methods.

[FIGURE 1 OMITTED]

In conclusion, tCys, tHcy, and tGSH concentrations measured with HPLC remain stable for 6 h in whole blood, whatever the anticoagulant used: EDTA at 0 [degrees]C or acidic citrate at room temperature. This latter approach is a good alternative when the use of EDTA medium collection is not possible, in particular in epidemiological surveys involving several centers. However, because of the significant differences between the mean concentrations in these two media, reference values need to be established that take into account the anticoagulant used. For CysGly, a different strategy must be defined to keep its concentration stable over the same blood storage period. The slight increase in tHcy as a function of time in both collection media that we observed only when using FPIA cannot be fully explained at the present time. The prime priority is thus to define optimal collection and treatment conditions and to keep them constant in an overall study.

We are indebted to the preclinic staff and laboratory department of the Centre de M6decine Pr6ventive (Vandoeuvre-les-Nancy, France) and to the participating subjects who made this study possible. We particularly wish to thank Dominique Aguillon, Maryvonne Chaussard, Chantal Lafaurie, and Marie-Pierre Recouvreur. We also thank Abbott Laboratories (Abbott Park, IL) for providing FPIA IMx reagents free of charge.

References

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(2.) Malinow MR. Homocyst(e)ine and arterial occlusive diseases. J Intern Med 1994;236:603-17.

(3.) Jacobsen DW. Homocysteine and vitamins in cardiovascular disease. Clin Chem 1998;44:1833-43.

(4.) Stamler JS, Slivka A. Biological chemistry of thiols in the vasculature and in vascular-related disease. Nutr Rev 1996;54:1-30.

(5.) Mansoor MA, Svardal AM, Ueland PM. Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma. Anal Biochem 1992;200:218-29.

(6.) Vester B, Rasmussen K. High performance liquid chromatography method for rapid and accurate determination of homocysteine in plasma and serum. Eur J Clin Chem Clin Biochem 1991;29:549-54.

(7.) Andersson A, Isaksson A, Hultberg B. Homocysteine export from erythrocytes and its implication for plasma sampling. Clin Chem 1992;38:1311-5.

(8.) Moller J, Rasmussen K. Homocysteine in plasma: stabilization of blood samples with fluoride. Clin Chem 1995;41:758-9.

(9.) Willems HPJ, Bos GMJ, Gerrits WBJ, den Heijer M, Vloet S, Blom HJ. Acidic citrate stabilizes blood samples for assay of total homocysteine. Clin Chem 1998;44:342-5.

(10.) Probst R, Brandl R, Blumke M, Neumeier D. Stabilization of homocysteine in whole blood. Clin Chem 1998;44:1567-9.

(11.) Al-Khafaji F, Bowron A, Day AP, Scott J, Stansbie D. Stabilization of blood homocysteine by 3-deazaadenosine. Ann Clin Biochem 1998;35:780-2.

(12.) Jacob N, Guillaume L, Garcon L, Foglietti MJ. Dosage de l'homocysteine plasmatique totale et autres aminothiols par chromatographie liquide couplee a la detection par fluorescence. Ann Biol Clin 1997;55:583-91.

(13.) Fiskemtrand T, Refsum H, Kvalheim G, Ueland PM. Homocysteine and other thiols in plasma and urine: automated determination and sample stability. Clin Chem 1993;39:263-71.

(14.) Jones DP, Carlson JL, Samiec PS. Sternberg P Jr, Mody VC Jr, Reed RL, et al. Glutathione measurement in human plasma. Evaluation of samplecollection, storage and derivatization conditions for analysis of dansyl derivatives by HPLC. Clin Chim Acta 1998;275:175-84.

(15.) Michelet F, Gueguen R, Leroy P, Wellman M, Nicolas A, Siest G. Blood and plasma glutathione measured in healthy subject by HPLC: relation to sex, aging, biological variables, and life habits. Clin Chem 1995;41:1509-17.

(16.) Shipchandler MT, Moore EG. Rapid, fully automated measurement of plasma homocysteine with the Abbott IMx[R] analyzer. Clin Chem 1995;41:991-4.

(17.) Samiec PS, Drews-Botsch C, Flagg EW, Kurtz JC, Sternberg P Jr, Reed RL, et al. Glutathione in human plasma: decline in association with aging, age-related macular degeneration, and diabetes. Free Radic Biol Med 1998;24: 699-704.

(18.) Tate SS, Meister A. Serine-borate complex as a transition-state inhibitor of [gamma]-glutamyl transpeptidase. Proc Natl Acad Sci U S A 1978;75:4806-9.

Jean-Frederic Salazar, [1] Bernard Herbeth, [2] Gerard Siest, [1,2] and Pierre Leroy [1]*

[1] Centre du Medicament, Faculte des Sciences Pharmaceutiques et Biologiques, B.P. 403-54001 Nancy Cedex, France; [2] Centre de Medecine Preventive, UPRES, B.P. 7-54501 Vandoeuvre-les-Nancy Cedex, France; * author for correspondence: fax 33-(0)3-83-32-13-22, e-mail pierre.leroy@pharma.u-nancy.fr
Table 1. Plasma concentrations of tHcy measured by FPIA
as a function of blood anticoagulant and delay times
between collection and centrifugation.

 tHcy, (a) [micro]mol/L

Anticoagulant 15 min 2 h

EDTA(b) 9.2 [+ or -] 3.1 (c) 9.6 [+ or -] 3.3
Acidic citrate 9.4 [+ or -] 2.8 (c) 9.9 [+ or -] 2.8

(a) Mean [+ or -] SD; n = 14.

(b) EDTA at 0 [degrees]C; acidic citrate at room temperature.

(c)Significant medium and time effects (ANOVA for repeated
measurements, statistical significance accepted at P <0.05).
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Title Annotation:Technical Briefs
Author:Salazar, Jean-Frederic; Herbeth, Bernard; Siest, Gerard; Leroy, Pierre
Publication:Clinical Chemistry
Date:Nov 1, 1999
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