Indian Journal of Chemistry Vo l. 42A. April 2003. pp. 739-7j3

Electroanalytical adaptation of the classical Ellman's Assay: Determination of total thiols in incubation media from a biological sample

(excised articular cartilage)

O lga ckrassova, Nathan S Lawrence, Robert B Leet, Jill P G Urbant ~ Ri chard G Compton"

Physica l and Theoreti ca l Chemistry Laboratory, !-lni versity of Ox ford. South Parks Road, Oxford. OX I 3QZ~ U K

tphysiulogy Department. Uni versity of Oxford. South Parks Road. Ox ford . OX I 3PT. U K.

Received 30 Ouober 2002

The total thiols in a medium containing authentic biologica l sample (art icular cartilage) has been determined electrochemicail y. T he detection strategy utili ses the electrochemica l adaptati on of the Ellman's reacti on process via the elec trochemica ll y initiated reac ti on of thi ols w ith diethyl-p-phenylenediamine. T he strategy has been examined over a biolugica ll y signifi cant p H range and the behav iour observed rati onali sed. The detection of thiols w ithin the biolog ica l sample is shown to proceed with micromolar levels of thi ol determined within the sample.

The detecti on of thi ol containing amino acids has recentl y come lO the fore due to their importance in biological processes t. As a consequence they are used as biomarkers for a wide range of diseases; an increased leve l of homocysteine is indicati ve of cardiovascular di seasec, atherosclerosis3

, and is associated w ith common pregnancy complicati ons4

,

where as high concentrati ons of glutathi one can signi fy a pati ent is suffering from AIDS-related dementi a, A lzheimer 's disease or Parkin son's diseases.6 . Therefore the phys iological importance of thiol is well recognised within the medical and analyti cal communi ties.

A large nu mber of comprehensive rev iews on the analytical assays for thi ol detecti on have been publi shed 7., and signify that the majority of the preferred determinati on processes are based on chromatographic separati on before detecti on. However. these protoco ls often require the matrix pre­treatment fo llowed by post-co lumn sample derivati sati on. Therefore there is a press ing need for the development of a protocol which produces fast and sensiti ve responses with minimal sample pre­treatment.

Electroanalytica l techniques are well represented in the literature') and could prov ide a means of producing such a protocol. These have focused on the direct oxidation of thiols (RSH) at bare solid substrates tO

-t6

and the possib ility of suitable redox indicators and

To whom correspondence should be addressed. Direc t Tcl: OJ8()5 275413. r ax 0 1865 2754 10. Emai l : ri chard.compton@chemi stry.ox.ac. uk

electrocatalys ts capable of improving both sensiti vity and selecti vity of the electrode response (for example metal complexes t7, metallo-phthallocyanines I .24 .

b . d ' 25 d ?6 ?7 ) romlne re uctl on an enzy mes-' - . A more recent development in the electroanalytica l

determination of thi ols species has been the electrochemicall y initiated derivati sation of pheny lene-di amine species by thiols (see Scheme 1)2 . The reactio.n procedure has been shown to be generi c in nature and as such an electrochemical adaptat ion of the classical Ellman's tes t29 has been developed (Scheme 2). Thi s involves the reaction of the th iol spec ies and 5,5'-dithiobi s(2-nitrobenzoic acid) (DTNB), producing two equi valents of TBN A and one equi valent of the oxidi sed thiols specie ' (Scheme 2). The newly generated TBNA species can subsequently attack the electrochemically ox idised DEPD species thereby forming the electrochemica lly

9 NH

"'9 NH

¢ "1} ~ I .::::..: ·11 RSI I (T13~A) : I -::::..: -II

~1{' "'11 ',, ·11

N (I (NI (I ( 1 DEI'IJ

Scheme I - T he proposed detection path wJY

Scheme 2- Ellman's reacti on

740 INDIAN J C HEM , SEC A. APRIL 2003

Table I- Producti on of thio ls by bov ine artic ular cartilage

Experiment Incubation Incubatio n Dry weight pH of incubation Concentrat io n of total thiol s conditions period, h of cartil age, medium at end of in incubati on medium at e nd.

g ex periment 11M

Aerobic 20 1.1 3 7.0 0.97

Anoxic 20 1.1 2 6.5 1.65

2 Anoxic 45 1.06 6.3 2.44

Anoxic 45 1.09 7.3 0*

3 Anox ic 45 2.53 5.9 11.0

4 Anoxic 45 .5 1.88 5.9 9.3

5 Anox ic 45 1.90 6.2 1.7

*The bottle cont aining the cartilage was stood in boiling water for 20 minutes The cartilage was then coo led and wa~hed several times in o 15 M NaCI , be fore anox ic incubati on bega n

acti ve TBNA -DEPD adduct which provides the analytical signal 29

. Thi s methodology has been shown (0 work for a wide range of thiol species each producing the same analytical sig nal and therefore allows the determination of the tota l thiol (R-SH) concentrati on in the sample of interest.

In the paper outlined thi s prev iously reported technique has been used to determine the total thiol content in a incubation medium from a biological sample. The results reveal experimental adjustments to those reported prev iously which have improved the sensitivity and allowed the determination of thiol s within the matrix .

Materials and Methods All reagents were of the hi ghes t grade ava ilable and

used without further purificatio n. All solutions and subsequent dilutions were prepared daily using deionised water from an Elgastat (Elga, UK) UHQ grade water system with a resistivity of not less than 18 MQ cm and were refrigerated when not in use. Electrochemical measurements were conducted using an IlAuto lab computer controll ed potentiostat (Eco-Chemie, Utrecht, Netherlands) with a standard three e lec trode configuration and a typical cell vo lume of 5 cm3 mainta ined a t a temperature of 20°C ± 2°C. Glassy carbon (GC, 3 mm diameter, BAS Technicol , UK) served as the working e lec trede with spi ral wound pl at inum wire as the counter e lectrode and a saturated calo mel (Radiometer, Copenhagen) reference electrode. The GC elec trodes were poli shed between experiments with di amo nd pastes of decreasing particle size (6 11m to 111m, Kemet, UK).

Bovine articular cartilage preparatioll The bovine arti cular cartilage was excised on the

day of analys is from the metacarpal-phalangeal joints

of 2-3 feet and washed several times with 0 .15 M NaCI. Thi s was followed by a further three washes wi th a Salts and Glucose medium (see Appendix). The cartilage was then di vided evenly into two samples, with approximately 15 mL of Salts and Glucose added to each sample. The anoxic medi a was bubbled with nitrogen through a can nula for 5 min before the bott le was sealed. These amples were then incubated o n a rotary Hybaid at 37°C from 20 to 45 .5 h (T able I).

After the incubation period the samples were analysed immedi ate ly by means of a standard addit ion ex periments (see below) in o rder to determ ine the concentration of thi o l species present.

General assay protocol for the determination of rotal thiols in the sample

For electrochemical experiments, 2 .5 mL of the biological sample was pl aced into the e lectroche mical

cell. To this solutio n a liquots of DEPD (25 11M) and

DTNB (25 11M) were added . This solut io n was then degassed thoroughly with nitrogen pnor to commencing the e lec trochemi cal measurements. Once the e lectrochemical experiments were being conducted a nitrogen stream was passed across the soluti on to mainta in an inert (oxygen free ) atmosphere.

Linear sweep vo ltammetry (sta rt ing potential +0.5 Y, pre-conditioning time 2 s, end potential - 0.2 Y, scan rate 50 mY S· I) was util ised thro ughout the experiments with a sta ndard add ition protocol bei ng utili sed to determine the concentrat ion of thi o l species. In order to overcome cumu lati ve e lectrode fouling by the spec ies within the samp le the e lectrode was poli shed after each experi ment with the voltammetric response be ing recorded three times

NEKRASSOVA el of.: DETERMINATION OF THIOLS IN INCU BATION MEDIA 74 1

between each addition In order to ensure reproducibility .

Results and Discussion The reaction procedure has been examined in the

incubation solution and over a range of biologically re levant pH .

Total thiols (Ellman 's assay) Initi al studies focused on the examination of the

reaction process in the incubation buffer (see appendix ) at pH 7.4. The cOlTesponding linear sweep vo ltammograms detailing the reduction of DEPD

(25 11M) and DTNB (25 11M) in the incubation buffer

to increasing additions of g lutathione (5 J-lM) are detailed in Fi g. I . In the absence of the g lutathi one a single reductio n peak is observed at +0.075 V (vs. SeE), which can be attributed to the two e lectron reducti on of the ox idi sed DEPD species as detailed in Scheme I. Upon the introduction of the thiol species (g lutathione) to the solution a new reduction wave at -0.025 V is observed. Thi s can be assigned to the reduction of the oxidised TBNA-DEPD adduct as depicted in Scheme 2.

Analys is of thi s new reduction wave by means of a standard additi on plot (inserted in Fig. I) revealed a

linear range fro m 5 to 20 J-lM. In o rde r to show that the limit of detection could be further decreased an

ana logous ex periment was undertaken: I 11M g lutath ione additions were added to the solution. It is seen that on addition of the g lutathione to the sampl e solu tion a new reducti on wave was observed at

-0.0 15 V consistent with that detailed in Scheme I. Analysis o f these sets of data for the I J-lM and SI1M standard additio n plo ts revealed a linear range from I

to 20 11M and a corresponding limit of detectio n of

0.5 111M, which is an enhancement w ith that reported . I 0 <) prevIous y- .

I n order to study the reaction process further the effect of pH o n the ana ly ti cal s ig na l was examined. This is required as the used incubati on medium pH vari es between the samples (Tab le I) and there fore in o rder to minimi se sample pre-treatment (adjusting the pH o f the solutio n) it would be convenient for the reaction process to work over bi o logica lly re levant range o f pH (5-7.5). The vo ltaIl1metric response was examined over a pH range (5 to 7) each solution

conta ining SO 11M DEPD and SO 11M DTNB. The corresponding wave shapes obtained were ana logous to those observed at p H 7.5 (Fig. I); in the absence of any lhi o ls species on ly a s ingle reduction wave was

1.0

<t: 0.5 :::l. 0.25 ....... C 0.00 ~ -0.25 U -0.50

-0.75

O.lO~ -<:

~o 20 ," ~ 0 10 . -'< 1.l "-

0000 50 100 150200

-0.10 -0.3 -0.2 -0. I 0.0 O. I 0.2 0.3 0.4 0.5

POlcntia l / V

Fig . I- Linear sweep vollammograms (scan rate = 50 mVs· l)

detailing the response of 25 ~M concentration of DEPD and DTNB in Salts and Glucose Medium to increasing add itions of 5 ~M glutathione at glassy carbon electrode Insert : A plot of peak current (a t - 0.150V vs SCE) against concentration for a solution contai ning the 25 ~M DEPD and 25 ~lM DTNB and increasing concentrations of glutathione

0.80 < :::l.

::: 0.60 ~ ~ 0.40 .!L

"" :f 0.20

0.00 +---------.------,------, 0.0 10.0 20.0 30.0

Concentrution of Cystei ne / ~M

Fig. 2- The plots of peak height against concentrati on of cysteine for a solution containing the 50 ~M DEPD and 50 ~M DT B in various pH buffers: (_ ) pH 7 phosphate buffer. ( A ) pH 6 phosphate buffer. (. ) p H 5 acetate buffer

observed cOlTesponding to the reduction of the ox id ised DEPD species, and on the addition of a thio l

(cysteine,S to 25 J.lM) to the solution a new reducti on wave was observed at potenti a ls lower than that fo r the reduction of the DEPD species which is attributed to the reduction of the oxidised DEPD-TBNA species.

Analysis of the vo ltammetric waves by means of pl o ts of peak current fo r the reducti on attributed to the reduction of the DEPD-TB A spec ies again t concentration are detailed in Fig. 2. These results reveal that as the pH decreases the sensitivity of the electrochemical Ellman's reaction a lso decreases. Thi s behaviour is confirmed by the analysis of the ana lytical parameters obtained fo r each pH (Table 2). Thi s data revea l that as the pH is dec reased the limit of detection increases consistent with a decrease in the sensitivity. This decrease in sensitivity can be explained by a knowledge of the pK" o f TBNA which is 4.93°. It can be rationali sed that as pH tends towards

742 INDIAN J CI-IEM, SEC A, APRIL 2003

Table 2-A Com pari son of the analyti ca l data obtained for the decrease of 50 11M DEPD and 50 11M DTNB" solution pH cond iti ons

pH studi ed Sens iti vity/A M,l Li ncar range/I1M h LoD/~IMc

7,0 3 ,OI X IO'~ 5-25 2.74

6.0 2,54x I 0'2 5-25 2,99

5,0 0.93x l(y2 5-20 4,2 1

"p H 5 ex perimenl s were carried out in the presence of 100 ~IM DEPD and 100 ~IM DT B hBased on ex peri mental regression data CLoD = limit of Detection based on 3sb

thi s va lue the sulphur moiety of the TBNA species w i II become protonated and as such that the reaction with DEPD will be inhibited.

Analysis of the lIlediulI1 ill which cartilage had been incubated

The assay procedure for the u ed incubation medium has been described prev iously with a standard add ition protoco l being developed in order to determine the quantity of reduced thiol present.

The initial voltammetri c response was recorded in the used incubati on medium before the add ition of the DEPD and DTNB spec ies, to ensure there was no elec troacti ve spec ies in the potential range studi ed. The corresponding linear sweep vo ltammogram (not shown) indeed revealed no appreciable vo ltammetric activ ity in the potential range and therefore allows the total reduced thiol to be determined using the proposed methodology.

After thi s preliminary study the relevant concentrati ons (25 ).lM) of DEPD and DTNB was added to the so lution. The correspond ing reductive linear sweep vo ltammogram for the respon se of a glassy carbon electrode placed in the used incubation mediu lll in the presence of DEPD and DTNB are detai led in Fig. 3. These reveal two reductive waves at +0.135 V and +0.020 V corresponding to the reduction of the oxidi sed DEPD and DEPD-TBNA adducts.

In order to check the reproducibility of the initial voltammetri c signal shown in Fig. 3, several repetitive scans weI e recorded. However, it was observed that upon repetItIve scanning the clari ty of the voltammetri c ~ i g n a l was decreased. This diminishing signal can be tentati ve ly attributed to the fouling of the electrode by species wi th in the biological sample. In order to overcome this problem of electrode fouling the electrode was polished between each voltammetric scan, and the voltammetri c signal was recorded three times between each add iti on in order to

-< 0 41'

~' O 30

-0,10 ~ 0 20 c ~ 010

-0.30 000 00

/ « :::l -0.50

ii -0.70 t: :::l -0.90 u

-I. ID

-1.30 -0.20 -0. 1 () (lOll O,I Il 0.20

POI<:ntial / V

Fig. 3- Linear sweep response of used incubation mediulll con taining 25 ~IM of DEPD and DTNB to increasing additions of thi ol ( 111 M) Insert : The corresponding standard addition plot lIsed to a,scs, the concentrati on of IO tal thi ols

verify the stab ility and reproducibili ty of the electrode response.

Also detailed in Fig. 3 is the effec t of increasing the concentration of glutathi one in the sample solution. Upon the add ition of glutathione (I to 5 ).lM) to the sample the reducti ve wave at +O.O:W V increases in magnitude cons istent with the results detailed abov~ and therefore confi rms th at thi s red ct ion wave is due to the reduction of the DEPD-TBN A species . The corresponding standard addition plot for the sample is inserted in Fig. 3. The results revea l a l inear response to glutathione additions, with the results showi ng that 9.3 ).lM is present in the biological art icular cartil age.

Further analysis of other biologica l arti cu lar cartil age samples were also undertaken in order to exa mine the applicability of the detect ion proceJure for biological procedures. The resulting concentrations of total thiols determined in a range of media, which had undergone vari<;> us preparation conditions are summari sed in Table I , These resu lts show how the concentrati ons of tota l th iol varies between each sample and may be related to the age or history of the animal which is being examined.

It can also be seen from Table I the effect of boiling the cartilage before anoxic incubati on in order to kill the cells. No thiols were detected in the solution from thi s treatment, showing the dependence of thiol production on li ving cell s.

Conclusions The above report illustrates the practical

applicati on of;:: prev iously developed electrochemical detection protocol for the determinat ion of total thiol species produced by biological carti lage. The reaction

EKR ASSOVA el al.: DETERMI NAT IO OFTHIOLS IN INCUBATION MED IA 743

was examined over a range of biologicall y relevant ' pH (5-7.5) and it was found that as the pH was decreased the sensitivity was al 0 found to decrease whil st the limi t of detection increased.

Furthermore the detecti on methodology has been utili sed for incubation medium from a real biolog ical sample with problems caused by surface fouling overcome by the use of repetiti ve poli shing. The fact th at thiol s were detected in the sub-micromolar level in the medium shows the detecti on process works and encourages the adaptation of such protocols into single shot screen printed electrodes which would obv iate the nced for repetitive poli shing in the analyti cal experiment.

Acknowledgements ON expresses her gratitude to the A nalyti ca l

Di vision of the RSC for the award of a studentship. SL thanks the EPRSC and Schlumberger Cambridge

research for financial support .

Append ix COlllpOl/el//.I· of " Sal/.I' al/d GII/cose" M ediI/III Potassium chloridc. Mag nesium sul phate. anhydrous Sodi um chlor ide HEPES Sodi um di hydrogc n orthophosphate. anhydrou s Calcium chlori de. dehydrate Glucose

Ad.iu~ t ed wit h sodi um hydroxide 10 p H 7.4 in air.

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