Development of simultaneous determination of thiols, ascorbic acid and their oxidized forms using HPLC with electrochemical detection.
Application to the study of the reaction catalyzed by GSH-DHA oxidoreductase
Nadia KAÏD, Jacques POTUS, Jacques NICOLAS*
RÉSUMÉ Mise au point d’une méthode HPLC avec détection électrochimique permettant le dosage simultané des thiols, de l’acide ascorbique sous leurs formes réduites et oxydées. Application à l’étude de la réaction catalysée par la GSH-DHA oxydoréductase.
Une méthode HPLC rapide et sensible a été mise au point pour le dosage simul- tané de l’acide ascorbique (AA), du glutathion (GSH) et de la cystéine (CSH) en vue de l’appliquer aux pâtes de farine de blé. La détection électrochimique utili- sée permet le dosage des thiols sous leur forme native (sans dérivation). La phase mobile est constituée par une solution de dihydrogénophosphate d’am- monium (100 mM) ajustée à pH 2,8 par l’acide o-phosphorique. La première électrode de mesure dont le potentiel est fixé à 0,6 V permet le dosage de AA alors que la seconde fixée à un potentiel de 1,05 V permet celui des thiols. Les limites de détection sont de 5 et 10 pmoles injectées pour CSH et GSH et de 1 pmole injectée pour AA, respectivement. Les formes oxydées de ces compo- sés sont quantifiées par la même technique HPLC après traitement de l’échan- tillon par le dithiothréitol. Cette méthode a été appliquée à un milieu contenant GSH, CSH et de l’acide déhydroascorbique (DHA) auquel a été ajoutée une pré- paration purifiée de GSH-DHA oxydoréductase. L’analyse du milieu réactionnel après action de l’enzyme a permis de mettre en évidence la formation simulta- née des formes oxydées du glutathion (GSSG), de la cystéine (CSSC), mais aussi du disulfure mixte GSSC.
Mots clés : thiols, acide ascorbique, glutathion, cystéine, GSH-DHA oxydoré- ductase.
SUMMARY
A rapid and sensitive high performance liquid chromatography method has been developed for the simultaneous determination of ascorbic acid (AA), glu- tathione (GSH) and cysteine (CSH) which can be applied to supplemented wheat flour doughs. Electrochemical detection was used and allowed thiol
Chaire de biochimie industrielle et agro-alimentaire, Conservatoire national des arts et métiers, 292 rue Saint-Martin, 75141 Paris cedex 03, France.
* Correspondence [email protected]
detection without chemical derivatization. The mobile phase was composed of 100 mM ammonium dihydrogenphosphate, adjusted to pH 2.8 with o-phos- phoric acid. The first electrode potential was fixed at 0.6 V for AA detection, the second electrode potential was fixed at 1.05 V for thiol detection. The detection limits were 5 and 10 pmoles for CSH and GSH respectively, and 1 pmole for AA. Oxidised forms can be quantified after reduction with dithiothrei- tol. The method has been applied to model system to highlight the mixed disul- fide GSSC formed during the reaction catalysed by GSH-dehydroascorbate oxidoreductase in the presence of its substrates (GSH and dehydroascorbic acid) and CSH.
Key words: thiols, ascorbic acid, glutathione, cysteine, GSH-DHA oxidoreduc- tase.
1 - INTRODUCTION
The redox reactions occuring in wheat flour doughs have long been recogni- zed as playing an important role in bread quality (CHENand SCHOFIELD, 1996).
Cysteine (CSH) and glutathione (GSH) are mainly involved in thiol/disulfide inter- change reactions. In particular, there has been considerable interest in the pos- sible involvement of the tripeptide glutathione (γ-glutamylcysteinylglycine) and CSH in the redox reactions occuring during dough mixing. These compounds are present in the flour and act in the free reduced (GSH, CSH) and oxidised (GSSG, CSSC) forms, as well as in the form of protein-thiol mixed disulfide (PSSG, PSSC) (CHENand SCHOFIELD, 1996).
Chemical bread improvers, such as ascorbic acid (AA), are thought to affect the polymeric structure of the gluten network by redox reactions (GROSCH and WIESER, 1999; KAID, 1999). The biochemistry of these reactions is still poorly understood. AA which is commonly used in breadmaking, is a reducer. It is rapidly oxidised to dehydroascorbic acid (DHA) by AA oxidase in the presence of oxygen during dough mixing (MAIR and GROSCH, 1979). DHA allows gluta- thione oxidation catalyzed by GSH-DHA-oxidoreductase (KAID et al., 1997).
GSH is then unavailable to participate in the thiol/disulfide exchanges with glu- ten proteins (MAIR and GROSCH, 1979), causing an increase in both dough strength and loaf volume. AA oxidase and GSH-DHA oxidoreductase are thus involved in the AA improver effect. Although cysteine is not a substrate for GSH-DHA oxidoreductase, KAIDet al. (1997) found that this thiol was an activa- tor of the reaction catalyzed by this enzyme probably through coupled oxidation reactions. In order to understand and explain the reactions responsible for this phenomenon, we have developed a method allowing the separation and the assay of AA and thiols such as CSH and GSH.
High performance liquid chromatography (HPLC) analysis coupled with elec- trochemical detection (ECD) has been chosen, on the one hand for its sensitivity and selectivity and on the other, because ECD allows simultaneous determina- tion of thiols and AA without chemical derivatization. The field of application of electrochemical detection is not as large as UV or fluorescence detection, however the technique is well adapted to our study, i.e. to highlight the mixed
disulfide GSSC formation in a model system containing GSH-DHA oxidoreduc- tase and its substrates in the presence of CSH.
The method development required a preliminary study for the optimization of the separation and detection conditions. AA, GSH and CSH are the main agents of oxidoreduction reactions when ascorbic acid is added in wheat dough. A number of studies detail the role of AA and its derivatives (KUNINORIand MATSU- MOTO, 1963 and 1964a; TSEN, 1964; CARTERand PACE, 1965; MEREDITH, 1965;
GRANT, 1974; ELKASSABANYet al., 1980, NICOLASet al., 1980), or thiol and disul- fide compounds (KUNINORI and MATSUMOTO, 1964b; MAIR and GROSCH, 1979;
SARWIN et al., 1992; SCHOFIELD and CHEN, 1995; HAHNand GROSCH, 1998).
Concerning thiol determination, two types of techniques are used. The first one is based on direct polarographic assays (TSEN and BUSHUK, 1963; TSEN and HLYNKA, 1963; KUNINORI and MATSUMOTO, 1964a and b; GRAVELAND et al., 1978). The second one is based on spectrophotometric assay after chromogen action (EWART, 1990; CHAN and WASSERMAN, 1993). In contrast to these methods, ECD coupled with HPLC does not require a preliminary thiol derivati- zation. Amongst all of the publications which report AA and thiol determination by ECD, only one has described the simultaneous detection of these com- pounds in biological samples (HONEGGERet al., 1989). These authors developed a technique using a gold electrode which allowed the quantification of the three (AA, GSH and CSH) compounds in less than 20 min. ECD, to the best of our knowledge, has never been used for simultaneous glutathione, cysteine and ascorbic acid quantification in wheat flour doughs.
2 - MATERIAL AND METHODS
2.1 Reagents and materials
Activated charcoal (Norit), m- and o-phosphoric acids were from Prolabo (Paris, France). L-threo AA, CSH, GSH, dithiothreitol (DTT) were obtained from Sigma Chemical Co (St Louis MO, USA). The diethylaminoethyl (DEAE) Sepha- rose CL6B was from Pharmacia (Uppsala, Sweden) and the 5 µm Kromasil C18 column from AIT (Mesnil-le-Roi, France). Purified GSH-DHA oxidoreductase activity was extracted from wheat flour (KAIDet al., 1997).
2.2 Apparatus and conditions
AA, CSH and GSH were separated and quantified by HPLC using a 9012 pump driven by a 9020 workstation (Varian), a Valco C6W injector with a 10 µL sample loop and an electrochemical detector equipped with a dual glassy car- bon working electrode and an Ag/AgCl reference electrode (Eldec 201 model, Chromatofield, France). Separation was achieved using a 5 µm Kromasil C18 column (250 ×4 - mm) with a mobile phase of ammonium dihydrogenphosphate (0.1 M) adjusted to a final pH of 2.8 with o-phosphoric acid. Samples were run isocratically at a flow rate of 0.8 mL–1.min–1. The potential of the first working electrode was fixed at + 0.6 V in oxidation mode (vs Ag/AgCl reference elec- trode) for the quantification of AA. The potential of the second working elec-
trode was fixed in oxidation mode at + 1.05 V for the quantification of thiols like CSH, GSH, γ-glutamylcysteine or cysteinylglycine. For each compound, linearity of the detector response was verified by injecting pure standards in solution at different concentrations.
2.3 Standard AA, GSH and CSH preparation
Standard AA, GSH and CSH solutions (20, 50 and 20 µM in the mobile phase, respectively) were freshly prepared prior to use. An external standard method was used by injecting the standard solution after every five samples in order to take in account electrode passivation.
2.4 Dehydroascorbic acid (DHA) preparation
DHA preparation was developed in our laboratory. The method used was adapted from WALTHERand GROSCH(1987). DHA solution was freshly prepared by oxidation of 2.5 mM AA solution (50 mL) for 15 min with 0.15 g activated charcoal (Norit) under air bubbling. After 15 min, the charcoal was removed by paper filtration (Whatman n° 1). The filtrated DHA solution was stable for 12 h.
The oxidation yield was higher than 90%. The absence of AA in the filtrate was checked both by spectrophotometry at 266 nm and by HPLC.
2.5 DHA and disulfide determinations
DHA was determined as the difference between the total AA after DHA reduction and the AA contents of the original sample. Disulfides (GSSG, CSSC or GSSC) were also determined by the same method. The reduction conditions were adapted from the method described by MORIER-TESSIER et al. (1993). To 0.5 mL of the sample, we added 0.25 mL of 20 mM dithiothreitol (DTT) solution prepared in 0.2 M Tris buffer (pH 8.2) and 0.5 mL of the later buffer. After 30 min of the reaction at 0°C, excess DTT was extracted three times with 2.5 mL ethyl acetate. After a suitable dilution in the mobile phase, the aqueous phase (10 µL) was injected into the HPLC.
2.6 Extraction and purification procedure
The GSH-DHA oxidoreductase was purified according to KAIDet al. (1997).
The enzyme activity was determined by following the AA formation at 266 nm (KAIDet al., 1997). Activity is expressed in nkat (i.e. nmol of AA formed/sec).
2.7 Ion exchange chromatography separation and analysis of thiol and disulfide mixtures
The aim was firstly to separate by ion exchange chromatography the diffe- rent thiols and disulfides contained in the solution, namely reduced (GSH) and oxidised (GSSG) glutathione, cysteine (CSH), cystine (CSSC) and possibly the mixed glutathione-cysteine (GSSC) disulfide. Secondly, the thiol and disulfide contents of each fraction were determined by HPLC. Separation was performed by ion exchange chromatography (DEAE Sepharose CL6B) on a mini-column (Vac-Elut System of Analytichem International, 0.63 cm2section and 2 mL bed volume). The glutathione peptide contains a glutamic acid which brings an
excess of negative charges leading to a better fixation onto the DEAE Sepha- rose gel. Therefore, the thiol compounds which lack this residue, i.e. CSH and CSSC, can be easily separated from those where it is present.
The separation was firstly carried out on 2 mL model solution of buffer A (25 mM sodium acetate at pH 4) containing CSH (5 mM), GSH (2 mM), CSSC (2.5 mM) and GSSG (1 mM) which was loaded onto the mini-column (2 mL bed volume) of DEAE Sepharose CL6B equilibrated with 30 mL of buffer A. CSH and CSSC were eluted by 3 bed volumes of buffer A (Fraction 1). The GSH, GSSG and GSSC were eluted by 2 bed volumes of buffer A and 1 mL of buffer A enri- ched with 0.5 M sodium chloride (Fraction 2) followed by 3 bed volumes of buf- fer A enriched with 0.5 M sodium chloride at pH 4 (Fraction 3). Elution was continued by 3 bed volumes of acetic acid (0.5 M). Thiols (GSH, CSH) and disul- fides (CSSC, GSSG and GSSC) were determined by HPLC in each fraction, before and after reduction by DTT.
Once this method had been validated, it was applied to a mixture (10 mL) containing GSH (1 or 2 mM), DHA (1 mM) and CSH (2 or 1 mM) prepared in a 0.1 M sodium phosphate buffer (pH 6.2). The reaction was started by addition of puri- fied GSH-DHA oxidoreductase (total activity of 2.5 µkat), and followed by spec- trophotometry at 266 nm. After 25 min, the reaction medium was diluted ten fold by the addition of buffer A. 2 mL of the diluted solution was loaded onto the DEAE column and the chromatographic procedure was applied as previously described.
3 - RESULTS AND DISCUSSION
3.1 Optimization of the HPLC analysis
3.1.1 Separation conditions
The separation conditions were built on adequate mobile phase composition.
ECD required the use of a salt solution. Two salt solutions were compared:
ammonium and potassium dihydrogenphosphate (80 mM). The peak areas of CSH, AA and GSH were followed under the two salt conditions (figures 1A, 1B and 1C). For each compound, the peak areas obtained with the ammonium salt were higher than those with the potassium salt. The effect of pH on retention times and peak areas was studied between the values 2.5 and 3 (salt concentra- tion: 80 mM). Only the results obtained with pH 2.5 and 3 are given in figure 2.
For each compound, when pH increased, peak areas increased but retention times decreased leading to an overlapping between the CSH and AA peaks. The best result was obtained with a pH value of 2.8.
The eluant (NH4H2PO4) ionic strength used also affects retention times and peak areas (figure 3). The effect of salt concentration was studied between 50 and 200 mM. High salt concentration (200 mM) led to longer retention times and lower peak areas. The best results were obtained with the NH4H2PO4concen- tration fixed at 100 mM.
Numerous authors (DUPUYand SZABO, 1987; HARVEYet al., 1989; KUNINORI and NISHIYAMA, 1991) added ion-pairing reagents in order to modify the peak
retention time. Thus, n-octyl sodium sulfate (2.5 mM) was tested in order to increase the retention time. However under our chromatographic conditions, its use results in a loss of reproducibility. Methanol can also be used to modify the retention times. However under our conditions, a solution containing m-phos- phoric acid is used for the extraction of thiol compounds and AA from wheat dough. This acid is only partially soluble in methanol which rules out the use of
Figure 1
Effect of mobile phase cations on the peak area of CSH (A), AA (B) and GSH (C)
this alcohol in the mobile phase when the samples analysed contain m-phos- phoric acid. Lastly, we used o-phosphoric acid to acidify the mobile phase at pH 2.8 since, according to HONEGGERet al. (1989), it results in the improvement of the peak shape and column efficiency.
Figure 2
Effect of mobile phase pH on the chromatogram 1: cysteine; 2: homocysteine; 3: ascorbic acid; 4: glutathione.
Figure 3
Effect of mobile phase ionic strengh on the chromatogram 1: cysteine; 2: homocysteine; 3: ascorbic acid; 4: glutathione.
[mobile phase] = 200 mM [mobile phase] = 100 mM pH = 2.5
pH = 3
3.1.2 Detection conditions
Determination of thiols requires a high potential applied to the carbon elec- trode. However, HONEGGERet al. (1989) reported that this leads to a lack of spe- cificity, instability and rapid de-sensitisation of the electrode. Various other electrodes operating at lower potentials have been tested by other authors, such as mercury, gold-mercury amalgam, gold and platinum. The double elec- trode glassy carbon/glassy carbon used in our laboratory was therefore compa- red to a double electrode gold/glassy carbon and a gold-mercury amalgam was first prepared. The potential was set at 0.2 V for the gold-mercury electrode and at 1.05 V for the glassy carbon electrode. A solution of CSH (15 µM) and GSH (12 µM) in the mobile phase was injected five times to compare the responses given by the two electrodes. The results obtained showed clearly that even at a lower potential (0.2 V), the gold-mercury electrode led to higher peak areas than the glassy carbon electrode. The responses were 1.7 and 2.4-fold higher for CSH and GSH respectively. This phenomenon may be due to the fact that thiol oxidation is more effective with mercury (DEMASTER et al., 1984). Reaction (1) occurs more easily than Reaction (2):
2 RSH + Hg → Hg (SR)2+ 2 H++ 2 e– (1) 2 RSH → RSSR + 2 H++ 2 e– (2)
However, as already stated by HONEGGERet al. (1989), we have noticed that in spite of a higher sensitivity with a gold-mercury electrode, the amalgam is delicate to prepare and difficult to maintain, leading to less reproducible results.
Moreover, KRIENet al. (1992) found that thiol pollution of this type of electrode was possible because of the high specificity of reaction (1). We consequently chose the glassy-carbon electrode, which is more stable for our samples, although it requires a higher potential and is less specific than the gold-mercury electrode.
The work potentials of the two glassy carbon electrodes have been determined from voltamograms (figures 4A and B). For this, the same quantity of each com- pound was injected at fixed potentials between 0.2 to 1.2 V. With AA (figure 4A) the detector response increased from 0.2 to 0.5 V and then remained constant at values above 0.5 V. With CSH and GSH, the detector responses increased from 0.75 to 1.1 V (figure 4B). Therefore, the potential of the first electrode was fixed at 0.6 V (in oxidation mode) for the AA detection and the second electrode potential was fixed at 1.05 V (in oxidation mode) for the thiol detection. The 1.05 V value was chosen because higher values of the potential led to an unacceptable base- line noise. Moreover, the potential value is also limited by the risk of electrolysis of either the eluant or other sample compounds (YOSTet al., 1981).
We did not use internal standards because the compounds which could be used (homocysteine or phenolic compounds) may interfere with the oxidoredu- cing compounds present in the analysed samples. Consequently, we used an external standard method. For each redox compound, linearity of the detector response was checked by carrying out a calibration curve. We noticed however that the same mixture composed of CSH (20 µM), AA (20 µM) and GSH (50 µM) injected at different times of the day gave different detector responses, due to passivation of the electrode surface with time. Consequently, a progressive loss of sensitivity occurs. This is unavoidable with this type of detection because electrochemical reactions occuring at the electrode give rise to polymerisation
products. These products come either from the sample or the eluant. Accordin- gly, electrodes are polished weekly with an alumina solution. Furthermore, a standard solution was systematically injected after five samples. In this way, we were able to define a detector response coefficient (ks):
ks= peak area (ps) / injected amount (a)
If ki and kfare the standard response coefficients obtained before and after the five samples sequence, the detector response coefficient kscorresponding to the nthsample can be estimated by the following relation:
ks= ki+ n * (kf-ki)/ 6 (with 1 ≤n ≤5)
and the corresponding concentration (Cs) can be calculated from the sample peak area (ps) by the relation:
Cs= ps/ks Figure 4
Voltamograms (increasing potential) of AA (A), CSH (B) and GSH (B)
3.1.3 Final chromatographic conditions
After these studies, the final chromatographic conditions were the following:
the mobile phase was composed of 100 mM ammonium dihydrogenphosphate, adjusted to pH 2.8 with o-phosphoric acid, the first electrode potential was fixed at 0.6 V in oxidation mode for AA detection, the second one was fixed at 1.05 V in oxidation mode for thiol detection. With these conditions, the retention times of CSH, AA and GSH were 2.8, 4.9 and 7.3 min respectively (figure 5). The detection limits were 1, 5 and 10 pmoles for AA, CSH and GSH respectively. This method can also be applied to isoascorbic acid (D-erythro AA) and homocy- steine for which the retention times were 5.3 and 3.1 min respectively as well as to the 2 dipeptides cys-gly and γ-glu-cys, derived from GSH, for which the reten- tion times were 3.9 and 6.8 min respectively (figure 5).
Figure 5
Chromatogram of CSH, cysteinyl-glycine, L-threo AA, D-erythro AA, γ-glutamyl-cysteine and GSH
1: CSH; 2: cysteinyl-glycine; 3: L-threo AA; 4: D-erythro AA; 5: γ-glutamyl-cysteine; 6: GSH.
3.2 Application: analysis of thiol and disulfide mixtures after separation by ion exchange chromatography
Before applying the HPLC method to a sample which contained a mixture of AA, CSH, GSH and of their oxidized forms (DHA, CSSC, GSSG and GSSC), it was necessary to develop a method for the separation of CSH and CSSC from GSH and GSSG (and possibly GSSC) in order to be able to quantify all the thiols and disufides present in the sample. Thus, ion exchange chromatography on a minicolumn of DEAE Sepharose CL6B was tested using a 2 mL model solution containing CSH (5 mM), CSSC (2.5 mM), GSH (2 mM) and GSSG (1 mM). The
results given in table 1 showed that the chromatography yields were better than 95% irrespective of the thiol or disulfide compound considered. In addition, a good separation of CSH and CSSC from GSH and GSSG was observed. Thus, fraction 1 contained all the CSSC as well as the bulk of CSH, and was devoid of glutathione. Fraction 2 contained a part of the GSH with traces of CSH, and was devoid of disulfide compound. Fraction 3 contained the rest of the GSH, all of the GSSG, and was devoid of CSH and CSSC.
Table 1
HPLC analysis of the thiol and disulfide composition of 2 mL of a solution containing CSH (5 mM), CSSC (2.5 mM), GSH (2 mM) and GSSG (1 mM) before
and after separation by DEAE chromatography at pH 4 Before reduction After reduction
Sample CSH GSH CSH GSH CSSC GSSG
(µmoles) (µmoles) (µmoles) (µmoles) (µmoles) (µmoles)
Before ion Initial amounts
exchange 10 4 20 8 5 2
chromatography
Fraction 1 9.55 0 19.35 0 4.9 0
Fraction 2 0.05 2.04 0.05 2.03 0 0
Fraction 3 0 1.96 0 5.92 0 1.98
Total 9.6 4 19.4 7.95 4.9 1.98
Yield (%) 96 100 97 99 98 99
After validation of the ion exchange chromatography, we applied this method to the study of an enzymatic reaction catalyzed by GSH-DHA oxidore- ductase. The thiol and disulfide composition of a 2 mL solution S1containing DHA (1 mM), GSH (1 mM) and CSH (2 mM) was analyzed after addition of 2.5 µkat of the purified enzyme from wheat flour and a reaction time of 25 min. This duration was chosen because it corresponded to a plateau in the formation of AA (checked by spectrophotometry at 266 nm and by HPLC). The results obtai- ned with solution S1are given in table 2a. Before reduction by DTT, fraction 1 does not contain GSH. After reduction, the GSH released (0.42 µmol) came only from the mixed disulfide GSSC. The CSSC quantity was determined by the dif- ference between the total CSH released after reduction (2.75 – 2.11 = 0.64 µmol) and the CSH contained in GSSC (0.42 µmol) (table 2a). Before DTT reduc- tion, there were traces of CSH in fraction 2. The CSH quantity released after reduction was, in fractions 2 and 3, lower than the quantity of GSH released.
Thus, it was assumed that the CSH released came from the mixed disulfide whereas the GSH released came partly from the mixed disulfide (in the same quantity as that of the CSH released) and partly from GSSG. Both cysteine and glutathione in their reduced and oxidized forms were fully recovered after ion exchange chromatography as shown by the yields which were close to 100% in all cases. It appeared that, in addition to the thiols, the three fractions contained the GSSC. These results demonstrate the ability of GSH-DHA oxidoreductase (with its substrates (DHA and GSH) and in the presence of CSH) to catalyse not only GSH oxidation into GSSG, but also CSH oxidation to form disulfide bonds
between GSH and CSH and between two CSH, even though CSH is not a sub- strate of this enzyme (KAIDet al., 1997).
A similar experiment was carried out with 2 mL of a solution S2containing DHA (1 mM), GSH (2 mM) and CSH (1 mM) i.e. with an initial amount of thiol equivalent to S1 but with the ratio of GSH/CSH 4 times higher. A qualitative similar distribution of the thiols and disulfides species in the 3 fractions was obtained after ion exchange chromatography (table 2b). Again, excellent yields were obtained for all the reduced and oxidized forms of cysteine and gluta- thione. The total amounts of the formed disulfides and residual thiols were simi- lar to those obtained with the solution S1. However, the relative proportions of the different disulfides are largely different. In solution S1, the disulfides are mainly in the GSSC form whereas the main disulfide is GSSG in S2.
Table 2a
HPLC analysis of the thiol and disulfide composition of 2 mL of a solution containing DHA (1mM), CSH (2 mM) and GSH (1 mM) after 25 min of enzymatic reaction,
before and after separation by DEAE chromatography at pH 4 Before reduction After reduction
Sample CSH GSH CSH GSH CSSC GSSG GSSC
(µmoles) (µmoles) (µmoles) (µmoles) (µmoles) (µmoles) (µmoles) Before ion
exchange 2.15 0.48 3.31 2.07 — — —
chromatography
Fraction 1 2.11 0 2.75 0.42 0.11 0 0.42
Fraction 2 0.02 0.23 0.32 0.61 0 0.04 0.30
Fraction 3 0 0.22 0.28 1.02 0 0.26 0.28
Total 2.13 0.45 3.35 2.05 0.11 0.30 1.00
Yield (%) 99 94 101 99 — — —
Table 2b
HPLC analysis of the thiol and disulfide composition of 2 mL of a solution containing DHA (1mM), CSH (1 mM) and GSH (2 mM) after 25 min of enzymatic reaction,
before and after separation by DEAE chromatography at pH 4 Sample Before reduction After reduction
CSH GSH CSH GSH CSSC GSSG GSSC
(µmoles) (µmoles) (µmoles) (µmoles) (µmoles) (µmoles) (µmoles) Before ion
exchange 1.13 1.62 1.65 3.82 — — —
chromatography
Fraction 1 1.14 0 1.40 0.10 0.08 0 0.10
Fraction 2 0.01 0.80 0.09 1.04 0 0.08 0.08
Fraction 3 0 0.75 0.08 2.49 0 0.83 0.08
Total 1.15 1.55 1.57 3.63 0.08 0.91 0.26
Yield (%) 102 96 95 95 — — —
In order to explain these results, we propose an enzymatic mechanism with a transient formation of glutathionyl radicals (GS*):
DHA + 2 GSH → 2 GS* + AA
The GS* is able to react with different compounds:
a) with another GS*, to form GSSG (which is the only reaction occuring in the absence of other thiols):
GS* + GS* → GSSG (1)
b) with a CSH molecule to form a cysteinyl radical (CS*) and GSH:
GS* + CSH → CS* + GSH (2)
c) with a cysteinyl radical to form the mixed disulfide GSSC:
GS* + CS* → GSSC (3)
d) lastly, two cysteinyl radicals can react together to form CSSC:
CS* + CS* → CSSC (4)
The respective concentrations of GSH, GS*, CSH and CS* have an influence upon the rate of reactions 1, 2, 3 and 4. Accordingly, this will modify the relative proportions of the different disulfides formed (GSSG, CSSC and GSSC). Thus, a solution richer in CSH will lead to higher quantities of GSSC. In all cases, CSSC is the disulfide formed in the lowest quantity. KIEFFERet al. (1990) have already proposed a reaction pattern showing the interactions between CSH and GSSG during the GSH-DHA oxidoreductase catalysis. With the help of our results, the reaction pattern has been completed, since in addition to the CSSC formed, GSSC also appeared. Moreover, the thiyl radicals could react with other com- pounds (e.g. oxygen) leading to molecules containing sulfur in a higher oxida- tion state. In particular, sulfonic compounds have been highlighted during glutathione oxidation by hydrogen peroxide, potassium bromate or lipid per- oxides (FINLEYet al., 1981).
4 - CONCLUSION
The development of a simultaneous separation of thiols and AA by HPLC coupled with ECD was useful and convenient to study the evolution of these redox compounds during an enzymatically catalyzed reaction. Working elec- trodes were fixed at 0.6 V for AA determination and 1.05 V for thiol determina- tion. Compared to the HONEGGER et al. (1989) method which simultaneously separates thiols and AA in less than 20 min, our method allows separation of these compounds in less than 10 min. Other thiols like γ-glutamyl-cysteine, cysteinyl-glycine and homocysteine, as well as AA isomers like D-erythro AA can be determined. Moreover, after DTT reduction, their oxidised forms can also be quantified. Applied together with an ion exchange chromatography to the study of the reaction catalyzed by GSH-DHA oxidoreductase in the pre- sence of its substrates and CSH, this method allowed quantification of the
GSSC mixed disulfide formation. Accordingly, this HPLC method will be helpfull in the study of thiol-disulfide interchanges during wheat flour dough mixing.
ACKNOWLEDGMENTS
The skillful assistance of Murielle ASSOUNwas greatly appreciated. This work was supported by a grant from ministère de l’Agriculture (DGAL 94-41).
Receveid 20 September 1999, accepted 15 December 1999.
REFERENCES
CARTER J.E., PACE J., 1965. Some interrela- tionships of ascorbic acid and dehydroascor- bic acid in the presence of flour suspensions and dough. Cereal Chem., 42, 201-208.
CHAN K., WASSERMAN B.P., 1993. Direct colorimetric determination of free thiol group and disulfide bonds in suspensions of solubi- lized and particulate cereal proteins. Cereal Chem., 70, 22-26.
CHEN X., SCHOFIELD J.D., 1996. Effects of dough mixing and oxidising improvers on free reduced and free oxidised glutathione and protein-glutathione mixed disulfides of wheat flour. Z. Lebensm. Unters. Forsch., 203, 255-261.
DEMASTER E.G., SHIROTA F.N., REDFERN B., GOON J.W., NAGASAWA T., 1984. Analy- sis of hepatic reduced glutathione, cysteine and homocysteine by cation-exchange high performance liquid chromatography with electrochemical detection. J. Chrom., 308, 83-91.
DUPUY D., SZABO S., 1987. Measurement of tissue sulfhydryls and disulfides in tissue protein and non-protein fractions by high performance liquid chromatography using electrochemical detection. J. Liquid Chrom., 10, 107-119.
ELKASSABANY M., HOSENEY R.C., SEIB P.A., 1980. Ascorbic acid as an oxidant in wheat flour dough. 1. Conversion to dehy- droascorbic acid. Cereal Chem., 57, 85-87.
EWART J.A.D., 1990. Thiols in flour and baking quality. Food Chem., 38, 41-60.
FINLEY J.W., WHEELER E.L., WITT S.C., 1981. Oxidation of glutathione by hydrogen peroxide and other oxidizing agents. J. Agric.
Food Chem., 29, 404-407.
GRANT D.R., 1974. Studies of the role of ascorbic acid in chemical dough develop- ment. 1. Reaction of ascorbic acid with flour water suspension. Cereal Chem., 51, 684-692.
GRAVELAND A., BOSVELD P., MARSEILLE J.P., 1978. Determination of thiol groups and disulfide bonds in wheat flour and dough. J.
Sci. Food Agric., 29, 53-61.
GROSCH W., WIESER H., 1999. Redox reac- tions in wheat dough as affected by ascorbic acid. J. Cereal Sci., 29, 1-16.
HAHN B., GROSCH W., 1998. Distribution of glutathione in Osborne fractions as affected by addition of ascorbic acid, reduced and oxi- dised glutathione. J. Cereal Sci., 27, 117-125.
HARVEY P.R.C., ILSON R.G., STRASBERG S.M., 1989. The simultaneous determination of oxidized and reduced glutathione in liver tissue by ion pairing reverse phase high per- formance liquid chromatography with a cou- lometric electrochemical detector. Clin.
Chim. Acta, 180, 203-212.
HONEGGER C.G., LANGEMANN H., KREN- GER W., KEMPF A., 1989. Liquid chromato- graphy determination of common
water-soluble antioxidants in biological samples. J. Chrom., 487, 463-468.
KAID N., RAKOTOZAFY L., POTUS J., NICO- LAS J., 1997. Studies on the glutathione- dehydroascorbate oxidoreductase (EC 1.8.5.1) from wheat flour. Cereal Chem., 74, 605-611.
KAID N., 1999. Étude d’oxydoréductases impliquées en technologie boulangère agis- sant sur l’acide ascorbique et les thiols. Thèse de Doctorat, Université de Paris 7, France.
KIEFFER R., KIM J.J., WALTHER C., LAS- KAWY G., GROSCH W., 1990. Influence of glutathione and cysteine on the improver effect of ascorbic acid stereoisomers. J.
Cereal Sci., 11, 143-152.
KRIEN P.M., MARGOU V., KERMICI M., 1992. Electrochemical detection of femto- mole amounts of free reduced and oxidised glutathione. J. Chrom., 176, 255-261.
KUNINORI T., MATSUMOTO H., 1963. L- ascorbic acid oxidising system in dough and dough improvement. Cereal Chem., 40, 647- 657.
KUNINORI T., MATSUMOTO H., 1964a.
Dehydro-L-ascorbic acid reducing system in flour. Cereal Chem., 41, 39-46.
KUNINORI T., MATSUMOTO H., 1964b. Glu- tathione in wheat and wheat flour. Cereal Chem., 41, 252-259.
KUNINORI T., NISHIYAMA J., 1991. Measu- rement of biological thiols and disulfides by high performance liquid chromatography and electrochemical detection of silver mercap- tide formation. Anal. Biochem., 197, 19-24.
MAIR G., GROSCH W., 1979. Changes in glutathione content (reduced and oxidised form) and the effect of ascorbic acid and potassium bromate on glutathione. J. Sci.
Food Agric., 30, 914-920.
MEREDITH P., 1965. The oxidation of ascor- bic acid and its improver effect in bread doughs. J. Sci. Food Agric., 16, 474-480.
MORIER-TESSIER E., MESTDAGH N., BER- NIER J.L., HENICHART J.P., 1993. Reduced and oxidised glutathione ratio in tumor cells:
comparison of two measurements methods using HPLC and electrochemical detection.
J. Liquid. Chrom., 16, 573-596.
NICOLAS J., GUSTAFSSON S., DRAPRON R., 1980. Effects of some parameters on the kinetics of ascorbic acid destruction and dehydroascorbic acid formation in wheat flour doughs. Lebensm. Wiss. Technol., 13, 308-313.
SARWIN R., WALTHER C., LASKAWY G., BUTZ B., GROSCH W., 1992. Determination of free reduced and total glutathione in wheat flour by an isotope dilution assay. Lebensm.
Unters. Forsch, 195, 27-32.
SCHOFIELD J.D., CHEN X., 1995. Analysis of free reduced and free oxidised glutathione in wheat flour. J. Cereal Sci., 21, 127-136.
TSEN C.C., BUSHUK W., 1963. Changes in sulfhydryl and disulfide contents of dough during mixing under various conditions.
Cereal Chem., 40, 399-408.
TSEN C.C., HLYNKA I., 1963. Flour lipids and oxidation of sulfhydryl groups in dough.
Cereal Chem., 40, 145-153.
TSEN C.C., 1964. Ascorbic acid as a flour improver. Baker’s Dig., 38, 44-47.
WALTHER C., GROSCH W., 1987. Substrate specificity of the glutathione dehydrogenase (dehydroascorbate reductase) from wheat flour. J. Cereal Sci., 5, 299-305.
YOST R.W., LETTRE L.S., COULON R.D., 1981. Pratique de la chromatographie liquide.
Tec & Doc Lavoisier, Paris, France.
