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1 2 Analyst RSC Publishing 3 4 5 ARTICLE 6 7 8 9 Enhancing permanganate chemiluminescence 10 detection for the determination of glutathione and 11 Cite this: DOI: 10.1039/x0xx00000x 12 glutathione disulfide in biological matrices 13
14 a a a b 15 Zoe M. Smith, Jessica M. Terry, Neil W. Barnett, Laura J. Gray, Dean J. Received 00th January 2012, Wright c and Paul S. Francis a* 16 Accepted 00th January 2012 17 18 DOI: 10.1039/x0xx00000x Acidic potassium permanganate chemiluminescence enables direct post column detection of 19 glutathione, but its application to assess the redox state of a wider range of biological fluids www.rsc.org/ 20 and tissues is limited by its sensitivity. Herein we show that the simple on line addition of an 21 aqueous formaldehyde solution not only enhances the sensitivity of the procedure by two 22 orders of magnitude, but also provides a remarkable improvement in the selectivity of the 23 reagent towards thiols such as glutathione (compared to phenols and amino acids that do not 24 possess a thiol group). This enhanced mode of detection was applied to the determination of 25 glutathione and its corresponding disulfide species in homogenised striatum samples taken 26 from both wild type mice and the R6/1 transgenic mouse model of Huntington's disease, at 27 both 8 and 12 weeks of age. No significant difference was observed between the GSH/GSSG Manuscript 28 ratios of wild type mice and R6/1 mice at either age group, suggesting that the early disease 29 progression had not significantly altered the intracellular redox environment. 30 31
32 Introduction reaction with acidic potassium permanganate and quinine. 15 33 This system actually contains two light producing pathways: (i) 34 The tripeptide glutathione (GSH) is a critical physiological the reduction of permanganate to form an excited state 35 component endogenous to all biological tissues and fluids.1, 2 manganese(II) species, and (ii) the oxidation of the thiols to 36 Existing primarily in its reduced form, an overproduction of generate an excited intermediate capable of transferring energy 37 reactive oxygen species (ROS) initiates the conversion of GSH Accepted to the quinine fluorophore.16 The manganese(II) pathway can 38 to its corresponding disulfide (GSSG).2 4 An alteration in the be considerably enhanced by addition of sodium polyphosphate 39 level of GSH (or the GSH/GSSG ratio) is one of the first rather than quinine, which improves sensitivity by eliminating 40 indications of cellular oxidative stress, a status which has been the background emission resulting from the reaction between 41 implicated in the pathophysiology of conditions such as the sensitiser and oxidant.17, 18 In 2011, McDermott et al. 42 Alzheimer's, Parkinson's and Huntington's disease.2 5 Analytical utilised this approach for the post column chemiluminescence 43 methodologies to determine GSH often involve complex 44 detection of GSH in cultured skeletal muscle cells (C2C12 sample pre treatment steps such as derivatisation of the analyte 11 myotubes) following a rapid isocratic separation. The limit of Analyst 45 for fluorescence detection. 3, 6 8 GSSG is normally measured detection was 5 × 10 7 M, compared to 6.5 × 10 6 M reported by 46 after disulfide bond reduction, in which case it is then treated Li and co workers.11, 15 McDermott and co workers also utilised 47 by the same derivatisation as the native GSH. Subjecting 48 a colloidal manganese(IV) reagent for the direct post column biological samples to these lengthy derivatisation procedures, 49 chemiluminescence detection of thiols and disulfides. The however, can artificially alter the GSH/GSSG ratio through 50 limits of detection of 7 × 10 8 M for GSH and 1 × 10 7 M for auto oxidation. Methods of detection that enable direct 51 GSSG were suitable for their determination in whole blood measurement (without derivatisation), such as electrochemical 9 52 samples, 12 but they are inadequate to measure the relatively low and chemiluminescence, 10 12 are therefore an attractive 53 concentration of GSSG in various other clinically relevant alternative to fluorescence. 6, 7, 13, 14 54 physiological fluids, cells and tissues. Several new approaches for chemiluminescence detection of 55 Herein we investigate a range of enhancers in order to GSH have recently emerged. 10 12, 15 Li et al. reported the 56 improve the sensitivity of acidic potassium permanganate determination of thiols such as cysteine, GSH, N acetylcysteine 57 chemiluminescence reagent towards thiol compounds. We 58 and captopril based on the sensitised chemiluminescence 59 60 This journal is © The Royal Society of Chemistry 2013 J. Name ., 2013, 00 , 1-3 | 1 Analyst Page 2 of 7 ARTICLE Journal Name
1 show that the on line addition of an aqueous formaldehyde point located at the entrance of a 1 m reaction coil. This stream 2 solution can not only improve the sensitivity towards thiol was then combined with the acidic potassium permanganate 3 4 compounds, but also dramatically enhance the selectivity of the reagent just prior to entry into the flow cell. reagent. We apply this enhanced chemiluminescence reagent 5 High Performance Liquid Chromatography 6 system to the determination of GSH and GSSG in mouse 7 striatum. Chromatographic analysis was carried out on an Agilent 8 Technologies 1260 series liquid chromatography system, 9 Experimental equipped with a quaternary pump, solvent degasser system, and 10 autosampler (Agilent Technologies, Forest Hill, Victoria, Chemicals 11 Australia) using an Alltech Alltima C18 column (250 mm × 4.6 12 Deionised water (Continental Water Systems, Victoria, mm i.d., 5 m) equipped with an Alltima C18 guard column, at 13 Australia) and analytical grade reagents were used unless room temperature, with an injection volume of 10 L and a 14 otherwise stated. Chemicals were obtained from the following flow rate of 1 mL min 1. Isocratic elution was performed with 15 sources: N acetylcysteine (Nacys), N cyclohexyl 3 97% solvent A (deionised water adjusted to pH 2.8 with formic 16 aminopropanesulfonic acid (CAPS), L cysteine (cys), L cystine acid) and 3% solvent B (methanol). An analogue to digital 17 (CYSS), N ethylmaleimide (NEM), L glutathione (GSH), interface box (Agilent Technologies) was used to convert the 18 L glutathione disulfide (GSSG), glyoxal, homocysteine (Hcys), signal from the chemiluminescence detector. Before use in the 19 homocystine (HCYSS), L lysine (Lys), 2 mercaptoethanol, L HPLC system, all sample solutions and solvents were filtered 20 methionine (Met), L phenylalanine (Phe), quinine, serotonin through a 0.45 m nylon membrane. For post column 21 hydrochloride (5 HT), sodium polyphosphate, tris(2 chemiluminescence measurements the column eluate (1 mL 1 1 22 carboxyethyl)phosphine hydrochloride (TCEP), L tryptophan min ) and a formaldehyde enhancer (2 M; 1 mL min ) were 23 (Trp) and L tyrosine (Tyr) from Sigma Aldrich (New South merged at a T piece located 10 cm from the entrance of the 24 Wales, Australia); potassium permanganate, and formaldehyde detector. This stream was then combined with the potassium 25 (37%) from Chem Supply (South Australia, Australia); permanganate reagent (2.5 × 10 4 M; 1 mL min 1) just prior to 26 methanol, sulfuric acid, and tris(hydroxymethyl)methylamine entry into the flow cell. A GloCel chemiluminescence detector 27 from Merck (Victoria, Australia); morphine from (Global FIA, WA, USA) containing a dual inlet serpentine Manuscript 28 GlaxoSmithKline (Victoria, Australia), formic acid from flow cell and photomultiplier tube (model 9828SB, ETP, NSW, 29 Hopkin and Williams (Essex, England) and hydrochloric acid Australia) set at a constant voltage of 900 V from a stable 30 (32% w/v) from Ajax Finechem (New South Wales, Australia). power supply (PM20D, ETP) was utilised in this system. The 31 Stock solutions (1 × 10 3 M) of N acetylcysteine, cysteine, reagent and enhancer solutions were propelled by a 12x6 Dual 32 cystine, GSH, GSSG, homocysteine and homocystine were Piston Pump (Scientific Systems Inc., PA, USA) equipped with 33 prepared and diluted as required in 0.01% formic acid (pH 2.8). a pulse damper and self flushing pump heads. 34 Formaldehyde was filtered and diluted to the required 35 Chemiluminescence Spectra concentration with deionised water. The acidic potassium 36 permanganate reagent (2.5 × 10 4 M) was prepared by A Cary Eclipse fluorescence spectrophotometer (Varian, 37 Accepted Mulgrave, Victoria, Australia) equipped with R928 38 dissolving solid potassium permanganate in a solution of photomultiplier tube (Hamamatsu, Japan) was operated in 39 sodium polyphosphate (1% (m/v)) and adjusting to pH 3 with 40 sulfuric acid. bio/chemiluminescence mode to collect chemiluminescence 41 spectra. A two line flow manifold was used to continuously Flow Injection Analysis merge the analyte (5 × 10 5 M; 3.5 mL min 1) and reagent 42 4 1 43 The manifold was constructed from a Gilson Minipuls 3 solutions (2.5 × 10 M; 3.5 mL min ) at a confluence point 44 peristaltic pump (John Morris Scientific, NSW, Australia) with located just prior to the entrance of a coiled PTFE flow cell
45 bridged PVC pump tubing (white/white, 1.02 mm i.d., DKSH, (200 L, 0.8 mm i.d.) that was mounted against the emission Analyst 46 Queensland, Australia), PTFE manifold tubing (0.8 mm i.d., window of the spectrophotometer. A three line continuous flow 47 Cole Parmer Instrument Company, Illinois, USA) and a six manifold was subsequently used to merge the analyte solution 7 1 48 port injection valve (Vici 04W 0192L Valco Instruments, (1 × 10 M; 3.5 mL min ) with the formaldehyde enhancer 49 Texas, USA) equipped with a 70 L sample loop. A custom (2.0 M; 3.5 mL min 1) at a confluence point located at the 50 built flow cell (a tight coil of 0.8 mm i.d. PTFE tubing) was entrance of a short (1 m) reaction coil. This stream was then 51 mounted against an extended range photomultiplier tube combined with the acidic potassium permanganate reagent (2.5 52 (Electron Tubes model 9828SB, ETP, NSW, Australia) and × 10 4 M; 3.5 mL min 1) just prior to entry into the flow cell. 53 encased in a light tight housing. The output signal from the Final spectra were an average of 20 scans (1 s gate time; 1 nm 54 detector was obtained using an e corder 410 data acquisition data interval; 20 nm band pass; PMT, 800 V) and corrected as 55 system (eDAQ, NSW, Australia). Analytes were injected into a previously described.19 56 carrier stream (deionised water) which merged with a stream of 57 either deionised water or formaldehyde (2 M) at a confluence 58 59 60 2 | J. Name ., 2012, 00 , 1-3 This journal is © The Royal Society of Chemistry 2012 Page 3 of 7 Analyst Journal Name ARTICLE
1 Mice than that obtained with only deionised water in the enhancer 2 20 stream. A small improvement in the chemiluminescence signal 3 R6/1 transgenic hemizygote males were originally obtained 4 from the Jackson Laboratory (Bar Harbor, ME, USA) and bred was obtained when using solutions of CAPS, quinine and 5 with CBB6 (CBAÅ~C57/B6) F1 females to establish the R6/1 glyoxal at 1.0 mM (Fig. 1). A much larger increase in signal 6 colony at the Florey Institute of Neuroscience and Mental intensity was observed with a more concentrated solution of 7 Health (FINMH). After weaning, animals were group housed (4 glyoxal (~600 fold enhancement), but this response was lower 8 mice per cage with 2 of each genotype) and maintained on a 12 than that obtained from the corresponding blank (i.e. the 9 h light/dark cycle with access to food and water ad libitum. reaction of permanganate and the enhancer without GSH). The 10 Mice were handled in accordance with the guidelines of the addition of formaldehyde resulted in a significant increase in 11 FINMH Animal Ethics Committee and the National Health and signal intensity (over three orders of magnitude), with a 12 Medical Research Council (NHMRC). relatively small increase in the corresponding blank response. 13 14 Sample Collection 16000 15 Immediately after cervical dislocation, mice brains were 14000 16 dissected on ice and snap frozen in liquid nitrogen, before being 12000 17 stored at ‐80 oC. Tissue was allowed to defrost on ice prior to 10000 18 homogenization. Tissue homogenates were prepared in 0.1% 8000 19 formic acid, using a motorized pestle. Samples were allowed to 6000 20 rest on ice for 10 mins prior to centrifugation at 8000 G for 15 4000 21 mins. Supernatants were removed for analysis. 2000 22 23 Sample Analysis 50 24 For GSH determination the sample was initially diluted 10 fold 40 25 into aqueous formic acid (0.01%). A 50 L aliquot of the 26 30 diluted supernatant was then combined with 450 L of aqueous Chemiluminecence Intensity (mV) 20 27 Manuscript 28 formic acid (5%), immediately prior to analysis using HPLC 10 with acidic potassium permanganate chemiluminescence 29 0 detection. For GSSG determination, a second aliquot of the r S S l e 30 P P ine d de yde A n inine inine hy diluted supernatant (100 L) was combined with a Tris HCl Wate C ui u u ehy 31 CA Q d de ldeh 3 M Q Q al al a mM M M m m rm buffer (0.675 M; 20 L; pH 8.0) and NEM (6.3 × 10 M; 20 mM m or ormaldehydeor 32 .01 0.1 mM1.0 CAPS m 1 0 1.0 M Glyoxal F F F 3 0 .0 0.1 m 1. 1.0 mM Glyoxa L) and mixed for 30 s. 2 Mercaptoethanol (8 × 10 M; 20 L) 0 M Fo .1 M .5 M .0 M .0 33 0 0 1 2 was then added and mixed for a further 30 s. Following 34 Fig 1 Chemiluminescence intensities for GSH (1 × 10 -6 M) upon reaction with 4 35 addition of TCEP (7.8 × 10 M; 20 L) the solution was gently acidic potassium permanganate and various enhancer solutions using flow 36 heated at 50° C for 60 min to allow complete disulfide bond injection analysis.
37 reduction. Finally, aqueous formic acid (5%, 20 L) was Accepted 38 introduced to re acidify the sample prior to filtration and Confirmation of the Emitting Species analysis. 39 The mechanism of enhancement of permanganate 40 chemiluminescence systems by formaldehyde has not yet been 41 Results and Discussion elucidated. 17 It has been postulated that the enhancer is oxidised 42 to form excited singlet oxygen or an unknown intermediate that 43 Preliminary Investigations either emits light or transfers energy to other compounds.24 27 44 In an attempt to provide a more analytically useful signal from For the chemiluminescence reaction of GSH with the acidic 45 Analyst GSH, four compounds previously reported to enhance the potassium permanganate reagent, we observed a broad single 46 emission intensity from the reaction of various organic band with an apparent emission maximum at approximately 47 17, 18, 21 23 compounds with acidic potassium permanganate were 687 nm (Fig. S1a), which is characteristic of the electronically 48 examined: N cyclohexyl 3 aminopropanesulfonic acid (CAPS), 49 excited manganese(II) species formed in the oxidation of quinine, glyoxal and formaldehyde. Using a three line flow organic analytes by permanganate in the presence of sodium 50 6 injection analysis manifold, a GSH solution (1 × 10 M) was polyphosphate (λ = 689 ± 5 nm; 4T →6A transition 28, 29 ). 51 1 max 1 1 injected into a deionised water carrier (3.5 mL min ) which The incorporation of a formaldehyde enhancer (2 M) into the 52 merged with the enhancer solution (3.5 mL min 1) at a 53 system, increased the overall emission intensity but did not alter confluence point located at the entrance of a short reaction coil. the spectral distribution (Fig. S1b). It was therefore concluded 54 4 A continuously flowing stream of the reagent (2.5 × 10 M; 3.5 that the same Mn(II) species was responsible for the emission 55 mL min 1) then merged with the combined analyte enhancer 56 in both cases. solution just prior to entry into the flow cell. Under these 57 conditions, each of the enhancers resulted in emissions greater 58 59 60 This journal is © The Royal Society of Chemistry 2012 J. Name ., 2012, 00 , 1-3 | 3 Analyst Page 4 of 7 ARTICLE Journal Name
1 very intense signals with the acidic potassium permanganate 2 a) (morphine and serotonin) were only enhanced 2 to 3 fold. 3 2600 Therefore, the formaldehyde enhancer not only affords much 4 2500 greater sensitivity, but also provided a remarkable improvement 5 2400 in selectivity towards thiol compounds such as GSH, thus 6 2300 7 reducing potential interferences in complex biological samples. 60 8 High Performance Liquid Chromatography 9 50 10 A mobile phase consisting of 97% deionised water adjusted to 40 11 pH 2.8 with formic acid and 3% methanol enabled the 12 30 separation of four routinely measured thiols in under 15 min with high resolution (Fig. 3). Similar to the observations of the 13 20 14 Chemiluminecence (mV) Intensity preliminary flow injection analysis experiments, the addition of 15 10 2 M formaldehyde to the detection system resulted in an 16 0 increase in the chemiluminescence response (peak area) for et these thiol analytes by approximately two orders of magnitude. 17 he Tyr Trp cys cys SS Gly Lys P M Cys GSH a Y H N CYSS C GSSG 5 HT 18 H orphine M Analyte 1200 16 19 b) Hcys Hcys 20 90000 14 12 21 80000 1000 10 22 8 70000 Cys 6 23 GSH 24 60000 800 4 Nacys 25 2 50000 Cys (a.u.) intensity Chemiluminescence 0 26 600 0 3 6 9 12 15 Time (min) 27 40000 GSH Manuscript 28 30000 400 29 Nacys 30 20000 Chemiluminescence Intensity (mV) Intensity Chemiluminescence 31 10000 (a.u.) intensity Chemiluminescence 200 32 (b) 33 0 (a) y ys ys ys S Tyr Trp SH c S 0 34 Gl L Phe Met Cys G a Hc N CYSS GSSG 5 HT rphine 0 3 6 9 12 15 HCY 35 Mo Analyte Time (min) 36 Fig 2 Chemiluminescence intensities for selected analytes (5 × 10 -6 M) with (a) Fig 3 HPLC separation of a mixture of four biologically important thiols; cysteine Accepted 37 acidic potassium permanganate (2.5 × 10 -4 M in 1% (m/v) sodium polyphosphate, (CYS), homocysteine (HCYSS), glutathione (GSH) and N-acetylcysteine (NACYS) (1 38 pH 3) and (b) the permanganate reagent and formaldehyde, using flow injection × 10 -5 M), using (a) acidic potassium permanganate (2.5 × 10 -4 M) 39 analysis methodology. chemiluminescence detection (20 µl injection volume) and (b) acidic potassium permanganate and formaldehyde (2.0 M) (10 µl injection). The unenhanced 40 separation is also shown inset. 41 Analyte Screen 42 Fifteen analytes (5 × 10 6 M) were screened with the acidic It should be noted that signal increases obtained when using 43 potassium permanganate reagent under reaction conditions a formaldehyde enhancer are typically accompanied by a 44 previously found to afford the greatest chemiluminescence deleterious increase in background emission.17, 18 Peristaltic 45 Analyst response for GSH.11 These responses (Fig. 2a) were then pumps (which are often used to propel reagent and enhancer 46 compared to those obtained when incorporating the solutions into the flow cell for post column chemiluminescence 47 formaldehyde enhancer (Fig. 2b). With the exception of glycine detection) contribute greatly to baseline noise because of the 48 and lysine, the addition of formaldehyde increased the pulsation of solution flow. To minimise this effect, we 49 chemiluminescence intensity for each analyte. Moderate employed two dual piston pumps equipped with pulse dampers 50 to deliver reagent and enhancer streams to the detector. 51 enhancement (59 to 197 fold) was obtained for species containing a disulfide bond (CYSS, HCYSS, GSSG). However, Calibration curves were prepared using eighteen standard 52 9 5 53 this improvement in sensitivity was still insufficient to allow solutions between 1 × 10 M and 1 × 10 M. Each of the thiols 54 direct post column quantification of GSSG in clinical samples. exhibited a highly linear relationship between signal (peak area) The greatest improvements in signal intensity were obtained for and analyte concentration (Table 1). The precision of repeated 55 6 56 the thiol containing species: Cys, Hcys, GSH and N acys, with injections (n=10; 5 × 10 M) was excellent, with relative 57 increases of between 528 and 4417 fold. Conversely, the standard deviations of between 0.49% and 1.46%. Limits of 58 response from two phenolic analytes that are known to produce detection, defined as a signal to noise ratio of 3, were in the 59 60 4 | J. Name ., 2012, 00 , 1-3 This journal is © The Royal Society of Chemistry 2012 Page 5 of 7 Analyst Journal Name ARTICLE
1 range of 1 × 10 8 M and 5 × 10 8 M. Most importantly, the limit determination of GSH and GSSG in mouse striatum. 2 of detection for GSH was 50 fold superior to that of the Progressive degeneration of this brain region is the primary 3 previously published HPLC procedure utilising permanganate neuropathalogic feature of Huntington's disease.30, 31 Several 4 11 5 chemiluminescence detection. studies have reported signs of oxidative stress in the brain tissue 6 of subjects suffering from the late stages of the disease; 7 however, it is not clear if this begins in early stages or if it is Table 1: Analytical figures of merit 31 33 8 secondary to tissue degeneration. Quantification of GSH and GSSG in animal models of Huntington's disease is 9 Retention 10 therefore invaluable in determining the stage at which oxidative stress begins to occur. 11 Time RSD Linear RSD LOD R2 12 (min) (%)* Range ( M) (%)* ( M) We examined homogenised striatum samples taken from 13 both wild type mice and the R6/1 transgenic mouse model of 14 Cys 2.83 0.02 0.996 0.01 – 7.0 0.77 0.01 Huntington's disease, at both 8 weeks of age (when affective 34 15 Hcys 3.47 0.04 0.999 0.03 – 7.0 0.49 0.03 like phenotypes begin to emerge ) and 12 weeks of age (when 16 motor dysfunction occurs 20 ). The acidic supernatant was 17 GSH 5.78 0.17 1.000 0.01 – 7.0 0.72 0.01 initially diluted 10 fold into aqueous formic acid (0.01%). A 18 Nacys 12.89 0.17 0.999 0.05 – 7.0 1.46 0.05 portion of the diluted supernatant was then further diluted 10 fold in 5% formic acid immediately prior to analysis (Fig. 5a). 19 *n = 10 20 For GSSG determination, a second aliquot of dilute supernatant 21 After quantification of the free GSH (Fig. 4a), a second (100 L) was subjected to the thiol blocking and disulfide bond 22 aliquot of sample was used to establish the concentration of reduction procedure as described above. The sample was then 23 GSSG. The free thiol was blocked with the masking agent, re acidified with 20 L of 5% formic acid prior to analysis (Fig. 24 N ethylmaleimide (Fig. 4b) and then the disulfide bond 5b). 25 reduction procedure was performed to liberate GSH, which was 26 quantified as described above (Fig. 4c). As such, the gains in 700 27 Manuscript sensitivity for GSH were also translated to the detection of GSH 28 * GSSG. 600 29 30 31 2000 500 32 GSH 1800 * 33 400 (b) 34 1600 35 300 1400 36 GSH 37 1200 (c) 200 Accepted 38
1000 Chemiluminescenceintensity (a.u.) 39 100 40 800 (a) (b) 41 600 0 42 GSH 0 3 6 9 12 15
43 (a.u.) intensity Chemiluminescence 400 Time (min) 44 200 Fig. 5 Typical acidic potassium permanganate chemiluminescence 45 (a) chromatograms obtained for the analysis of mouse striatum, (a) detection of Analyst 46 0 GSH (total 1/100 dilution) and (b) detection of GSSG as GSH after performing the disulfide bond reduction procedure (total 1/20 dilution). *This peak corresponds 47 0 3 6 9 12 15 Time (min) to the excess 2-mercaptoethanol and does not interfere with the analysis. 48 -5 49 Fig. 4 The analysis of a mixture of GSH and GSSG standards (1 × 10 M) with post-column chemiluminescence detection using acidic potassium Using this method, both GSH and GSSG were detected in 50 permanganate and a formaldehyde enhancer. (a) Detection of GSH, (b) signal all samples tested. Despite the complexity of the sample matrix, 51 after addition of Tris-HCl buffer and NEM (thiol blocking step), (c) detection of the selectivity of the reagent combined with the ability to 52 GSSG as GSH after the addition of 2-mercaptoethanol (to react excess NEM) and TCEP (to break the disulfide bond). *This peak corresponds to the excess perform large dilutions resulted in only a few observable peaks 53 2-mercaptoethanol and does not interfere with the analysis. from other compounds. GSH was present in the range 1.21 3.21 54 mol g 1 whilst GSSG was present in the range 0.16 0.44 mol 55 Determination of GSH/GSSG in Mouse Striatum g 1 (Fig. 6). There was no significant difference in the levels of 56 GSH, GSSG, or the ratio of GSH/GSSG, at either time point 57 To demonstrate the viability of this method for the analysis of (t(8) = 2.89, p = 0.20; see table S1 for further statistical data). 58 biological tissues, the procedure was applied to the 59 60 This journal is © The Royal Society of Chemistry 2012 J. Name ., 2012, 00 , 1-3 | 5 Analyst Page 6 of 7 ARTICLE Journal Name
1 This suggests that the disease progression had not significantly 2 Acknowledgements altered the intracellular redox environment. This preliminary 3 The authors thank Deakin University and the Australian result may indicate that significant changes in antioxidant 4 Research Council (FT100100646) for funding. 5 capacity are not occurring in the prodromal stages of the R6/1 Huntington's disease model. However, it may be that such 6 Notes and references 7 changes in glutathione homeostasis are compartmentalised 8 between different cell types (glia and neurons) and/or aCentre for Chemistry and Biotechnology, Faculty of Science, 9 subcellular structures (mitochondria and cytoplasm). Thus Engineering and Built Environmental, Deakin University, Locked Bag 10 further analysis is needed to confirm if such changes are 20000, Geelong, Victoria 3220, Australia. 11 occurring. These results do, however, demonstrate the bSchool of Medicine, Faculty of Health, Deakin University, Locked Bag 12 analytical utility of this method for the measurement of 20000, Geelong, Victoria 3220, Australia. 13 GSH/GSSG levels in complex biological matrices. cFlorey Institute of Neuroscience and Mental Health, 30 Royale Pde, 14 Parkville, Victoria 3052, Australia. 2.50 1.8 ) 15 ) (a) (b) Electronic Supplementary Information (ESI) available: Corrected 2.25 1.6 chemiluminescence spectra and statistical data for striatum samples. See
16 mol/g mol/g 2.00 1.4 ( 1.75 ( 17 1.2 DOI: 10.1039/b000000x/ 1.50 18 1.0 1. A. Meister and M. E. Anderson, Annu. Rev. Biochem. , 1983, 52 , 711 1.25 0.8 760. 19 1.00 0.6 2. D. Giustarini, I. Dalle Donne, D. Tsikas and R. Rossi, Crit. Rev. Clin. 20 0.75 Lab. Sci. , 2009, 46 , 241 281. 21 0.50 0.4 GSH Concentration GSH Concentration 0.2 3. A. Pastore, G. Federici, E. Bertini and F. Piemonte, Clin. Chim. Acta , 22 0.25 0.00 0.0 2003, 333 , 19 39. WT HD WT HD 23 Genotype Genotype 4. D. M. Townsend, K. D. Tew and H. Tapiero, Biomed.
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25 ( 0.25 ( Telser, Int. J. Biochem. Cell Biol. , 2007, 39 , 44 84. 26 0.20 0.20 6. T. Toyo’oka, J. Chromatogr. B , 2009, 877 , 3318 3330. 27 0.15 7. P. Monostori, G. Wittmann, E. Karg and S. Túri, J. Chromatogr. B , Manuscript 28 0.15 0.10 2009, 877 , 3331 3346. 29 0.10 8. R. E. Hansen and J. R. Winther, Anal. Biochem. , 2009, 394 , 147 158. 30 0.05 0.05 9. J. C. Harfield, C. Batchelor McAuley and R. G. Compton, Analyst , GSSG Concentration GSSG Concentration 31 0.00 0.00 2012, 137 , 2285 2296. WT HD WT HD 10. S. Bai, Q. Chen, C. Lu and J. M. Lin, Anal. Chim. Acta , 2013, 768 , 32 Genotype Genotype 8 (f) 7 (g) 96 101. 33 7 6 11. G. P. McDermott, P. S. Francis, K. J. Holt, K. L. Scott, S. D. Martin, 34 6 5 N. Stupka, N. W. Barnett and X. A. Conlan, Analyst , 2011, 136 , 35 5 2578 2585. 4 36 4 12. G. P. McDermott, J. M. Terry, X. A. Conlan, N. W. Barnett and P. S. 3 37 3 Francis, Anal. Chem. , 2011, 83 , 6034 6039. Accepted 2 13. E. Camera and M. Picardo, J. Chromatogr. B , 2002, 781 , 181 206. 2 38 GSH/GSSG Ratio GSH/GSSG Ratio 14. F. Carlucci and A. Tabucchi, J. Chromatogr. B , 2009, 877 , 3347 1 1 39 3357. 40 0 0 WT HD WT HD 15. Y. Li, A. Zhang, J. Du and J. Lu, Anal. Lett. , 2003, 36 , 871 879. 41 Genotype Genotype 16. J. L. Adcock, P. S. Francis and N. W. Barnett, J. Fluoresc. , 2009, 19 , 42 Fig 6 Influence of Genotype (wild type (WT) or R6/1 (HD)) on intracellular GSH, 867 874. 43 GSSG and the GSH/GSSG ratio in moue striatum taken from (a, c, e) 8 week old 17. J. L. Adcock, P. S. Francis and N. W. Barnett, Anal. Chim. Acta , 44 and (b, d, f) 12 week old animals. Values are reported as means of 5 2007, 601 , 36 67. determinations (± SEM). 18. J. L. Adcock, N. W. Barnett, C. J. Barrow and P. S. Francis, Anal. Analyst 45 Chim. Acta , 2014, 807 , 9 28. 46 19. P. S. Francis, J. L. Adcock and N. W. Barnett, Spectrochim. Acta 47 Conclusions Part A, 2006, 65 , 708 710. 48 20. L. Mangiarini, K. Sathasivam, M. Seller, B. Cozens, A. Harper, C. 49 Applying a formaldehyde enhancer to chemiluminescence Hetherington, M. Lawton, Y. Trottier, H. Lehrach and et al., Cell , 50 reactions with acidic potassium permanganate not only afforded 1996, 87 , 493 506. 21. N. T. Deftereos, A. C. Calokerinos and C. E. Efstathiou, Analyst , 51 significant increases in signal intensities, but also increased selectivity of the reagent towards thiol compounds. Applying 1993, 118 , 627 632. 52 22. J. A. Murillo Pulgarín, P. Fernández López and P. Hoyas Nuño, Anal. this to the previously developed HPLC method for thiol 53 Bioanal. Chem. , 2006, 384 , 423 430. 54 detection, we were able to improve the detection limits for the 23. J. A. Murillo Pulgarín, L. F. García Bermejo and P. Fernández López, 55 important biological thiol GSH by over an order of magnitude Anal. Chim. Acta , 2005, 546 , 60 67. 56 whilst also halving the injection volume. The improved method 24. G. N. Chen, F. X. Huang, X. P. Wu, Z. F. Zhao and J. P. Duan, Anal. was successfully applied to the detection of GSH/GSSG in Bioanal. Chem. , 2003, 376 , 873 878. 57 25. S. Liao, X. Wu and Z. Xie, Anal. Chim. Acta , 2005, 537 , 189 195. 58 mouse striatum. 59 60 6 | J. Name ., 2012, 00 , 1-3 This journal is © The Royal Society of Chemistry 2012 Page 7 of 7 Analyst Journal Name ARTICLE
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19 20 21 22 23 24 25 26
27 Manuscript 28 29 30 31 32 33 34 35 36
37 Accepted 38 39 40 41 42 43 44
45 Analyst 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 This journal is © The Royal Society of Chemistry 2012 J. Name ., 2012, 00 , 1-3 | 7