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Letters to the Editor |
Dipartimento di Chimica e, Chimica Industriale Università, di Genova and Consiglio, Nazionale delle Ricerche, Genova, Italia
aAddress correspondence to this author at: Dipartimento di Chimica e Chimica Industriale, Consiglio Nazionale delle Ricerche, Via Dodecaneso, 31 I-16146 Genova, Italy. Fax (+39)010 353 6107; e-mail giocev{at}chimica.unige.it.
To the Editor:
The measurement of aminothiols such as cysteine (Cys), cysteinyl-glycine (Cys-Gly), homocysteine (Hcy), and glutathione (GSH), as well as the corresponding disulfides, has gained high interest within the biomedical community because such molecules are important biomarkers for a wide range of diseases. In particular, increased total plasma Hcy is now considered a risk factor for cardiovascular disorders(1) as well as other degenerative conditions. Although various methods for the measurement of aminothiols are available, HPLC coupled with fluorometric detection is one of the most suitable techniques for determination of minute amounts of thiols(2). Most methods require precolumn derivatization, and although different types of fluorogenic reagents for thiols have been proposed, the most commonly used and sensitive reagent is the commercially available ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F)(3).
With this method the completion of the reaction takes a very long time and the conditions required are quite strict, i.e., derivatization must be carried out at 60 °C for a 1-h period in a moderately alkaline medium (pH = 9.5) in the presence of excess reagent. Moreover, fast oxidation or degradation of thiols, as well as of other species participating in the reaction, may take place under these conditions. To circumvent these drawbacks a number of new fluorogenic reagents possessing the benzofurazan structure have been proposed(4), but all require heating and/or long reaction time.
All of the molecules used in aminothiol assays are characterized by the presence of 2 substituents in positions 4 and 7 of the 2,1,3-benzoxadiazole skeleton: the leaving group (halogen) and a 2nd group whose function is to increase both reactivity and solubility in water. In the search for new fluorogenic reagents with benzofurazan structure along with an additional reactivity-enhancing substituent to improve reactivity, we synthesized ammonium 5-bromo-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-BF) and characterized this reagent by means of spectroscopic and analytical data.
The results of a preliminary kinetic study, carried out with Cys and Hcy nucleophiles by means of the ultraviolet-visible detection technique, indicated that SBD-BF reacts about 3 times faster at 25 °C than SBD-F at 60 °C. SBD-BF was therefore more reactive than SBD-F, and it reacted with thiols under milder conditions and during a time period of about 15 min.
This result was supported by time-course studies, carried out with reversed-phase HPLC, on the derivatization reactions of SBD-BF with the aminothiols used in the present work. Moreover, the spectra of the derivatives of Cys with SBD-F and SBD-BF, recorded with a Perkin-Elmer MPF 44 A spectrofluorimeter, were practically superimposable, suggesting that our new reagent SBD-BF is a suitable precolumn derivatization reagent for reversed-phase HPLC fluorometric determination of thiols.
Calibration curves for the derivatives of Cys, Cys-Gly, Hcy, and GSH with SBD-BF, obtained in concentration ranges from 0.125 to 50 µmol/L in borate buffer, exhibited excellent linearity between peak areas and concentrations over the entire range, with r2 values of 0.9992, 0.9993, 0.9989, and 0.9991, respectively. The lower limit of detection (signal-to-noise ratio of 3:1) for the derivative of Hcy was 3 nmol/L, corresponding to 60 fmol (the upper limit was not tested).
Comparative chromatographic runs of standard solution of aminothiols in borate buffer (Fig. 1A
) and human plasma (Fig. 1B
) after derivatization with SBD-BF demonstrate that this new fluorogenic reagent is suitable for thiol determination in biological samples. Briefly, 100 µL of human plasma was mixed with 10 µL of a solution of Tris (2-carboxyethyl)phosphine (75 mmol/L in borate buffer, pH 7.4) and allowed to react at room temperature for 15 min. Then 90 µL of a solution of trichloroacetic acid (100 mL/L in wa-ter with EDTA 1 mmol/L) was then added and the sample was centrifuged for 6 min at 14.5g. An aliquot of the resulting solution (100 µL) was mixed with 380 µL of a solution of SBD-BF (25 mmol/L in 0.125 mol/L borate buffer with EDTA 5 mmol/L, pH 9.5) and 20 µL of NaOH 1 mol/L, incubated at room temperature for 30 min, and then acidified with 50 µL of HCl 1 mol/L. In a final step, 20 µL of the sample was injected into a Agilent 1100 HPLC system equipped with fluorescence and ultraviolet-visible detectors.
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The samples, as well as the starting solutions, were carefully flushed with nitrogen and protected from air until injection onto the column. Because oxygen is indeed a strong florescence quencher, some previously reported unexpected results, i.e., decrease in the fluorescence intensities(5), may be attributable to oxygen exposure.
In conclusion, SBD-BF reacts with thiols under mild conditions, at 25 °C for a time period of approximately 15 min, and seems to be a very promising fluorogenic reagent for aminothiol derivatization.
Acknowledgments
Grant/funding support: Financial support was provided by Ministero dell’Istruzione, dell’Università e della Ricerca (PRIN2005).
Financial disclosures: None declared.
References
ich V. Measurement of homocysteine and other aminothiols in plasma: advantages of using tris(2-carboxyethyl)phosphine as reductant compared with tri-n-butylphosphine. Clin Chem 2001;47:1821-1828.
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