Clinica Chimica Acta 275 (1998) 175184

Glutathione measurement in human plasma Evaluation of sample collection, storage and derivatization conditions for analysis of dansyl derivatives by HPLC
Dean P. Jones *, Joanne L. Carlson , Paula S. Samiec , Paul Sternberg Jr.b , Vino C. Mody Jr.b , Robyn L. Reed b , Lou Ann S. Brown c
b a Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA Department of Ophthalmology, Emory University School of Medicine, Atlanta, GA 30322, USA c Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA

a,

a ,b

Received 25 August 1997; received in revised form 25 April 1998; accepted 28 April 1998

Abstract Literature values for human plasma GSH vary over 10-fold despite the use of apparently valid analytical procedures for GSH measurement. The purpose of this study was to develop a procedure to minimize error in sample collection, processing and storage that could contribute to such differences. HPLC with uorescence detection of dansyl derivatives was used for quantication. The results show that collection of blood with a buttery needle and syringe reduces overestimation due to limited hemolysis and that use of a preservation solution designed to inhibit autooxidation and enzymatic degradation allows quantitative recovery of both GSH and GSSG. Stability tests showed that non-derivatized samples were stable for at least 2 months at 2 808 while dansyl derivatives were stable in the dark at 048 for 12 months. Results from 59 healthy individuals (2043 years) provided a mean (61 SD) GSH value of 2.0961.14 micromolar. 1998 Elsevier Science B.V. All rights reserved. Keywords: Glutathione; Plasma; HPLC; Fluorescence detection; Antioxidants

*Corresponding author. Present address: Emory University School of Medicine, Department of Biochemistry, Atlanta, GA 30322, USA. Tel.: 1 1 404 727 5970; fax: 1 1 404 727 3231; e-mail: dpjones@emory.edu 0009-8981 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 98 )00089-8

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1. Introduction Plasma GSH in humans has been reported to be altered in several pathophysiologic states, including alcoholic liver disease, human immunodeciency viral infection and ascorbate deciency [1], yet the ndings in different laboratories are difcult to compare in terms of absolute contents of GSH. For instance, the measurement of plasma GSH with monobromobimane has provided values of approximately 36 mM in one laboratory [2] while a variation of this method gave values under 1 mM [3]. An orthophthalaldehyde method gave values of 24 mM [46] while enzymatic recycling methods have produced values from 1 to 11 mM [79]. Because the different analytical methods for GSH have been validated and are calibrated against GSH standards, at least some of this variability is likely to be due to differences in sampling, processing and / or storage. Three types of sample collection and processing errors have been discussed in the literature. Red blood cells contain approximately 500-times higher GSH concentration than plasma so that minor hemolysis (0.1% to 1%) can result in erroneously high plasma values. Alternatively, GSH can be lost by oxidation which occurs with a half-time of about 5 min in plasma at room temperature [1012]. Human plasma also contains g-glutamyltranspeptidase, an enzyme that degrades GSH [13] and can be present at high activities in human plasma in association with liver disease. In the present report, we describe a procedure that we have optimized to collect blood with minimal hemolysis, prevent GSH oxidation and degradation during processing and provide a specic and sensitive measure of GSH and GSSG under clinical conditions. In this procedure, blood is collected with a buttery needle to minimize hemolysis and transferred immediately into a preservation solution. The preservation solution includes heparin to inhibit coagulation, serine ? borate to inhibit degradation of GSH by g-glutamyltranspeptidase [13], bathophenanthroline disulfonate to inhibit GSH oxidation [10] and iodoacetic acid to alkylate GSH [14]. The use of this preservation solution allows for the blood sample to stand at room temperature for up to 30 min without signicant changes in the absolute contents or redox state of the GSH and GSSG. With the greater exibility in timing that this approach allows, a phlebotomist can prepare the plasma after attending to the subject so that a second investigator is not needed to immediately process samples. In the protocol as described, an internal standard (g-glutamylglutamate) is included to facilitate quantitation, and derivatization with dansyl chloride [15] has been optimized to allow quantitation of GSSG as well as GSH. Conditions for HPLC separation on an amine column have been optimized to allow simultaneous measurement of cystine (Cys 2 ) and cysteine (Cys) with GSH and GSSG.

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2. Methods

2.1. Materials
Sodium heparin, bathophenanthroline disulfonate sodium salt (BPDS), iodoacetic acid, dansyl chloride, L-serine, g-glutamylglutamate (g-Glu-Glu), GSH, GSSG, Cys, Cys 2 , g-Glu-Cys, and sodium acetate trihydrate were from Sigma Chemical (St. Louis). The disulde of Cys and GSH, CySH-GSH, was from Toronto Research Chemicals (Toronto). Boric acid, sodium tetraborate, potassium tetraborate, perchloric acid, acetic acid, acetone and chloroform were reagent grade and purchased locally. Methanol was HPLC grade. Distilled, deionized water was used throughout.

2.2. Preservation solution for blood sampling

A solution of 100 mM serine ? borate (pH 8.5) containing (per ml) 0.5 mg sodium heparin, 1 mg BPDS, and 2 mg iodoacetic acid is prepared using stock solutions of 100 mM boric acid (0.62 g / 100 ml) and 100 mM sodium tetraborate (3.81 g / 100 ml). It is made by mixing 8 ml of the boric acid stock, 2 ml of the tetraborate stock, 105 mg L-serine, 5 mg sodium heparin, 10 mg BPDS and 20 mg iodoacetic acid. This solution is used to prepare A tubes, which are used for blood collection. These are clear 1.5-ml graduated microcentrifuge tubes to which 0.5 ml of the preservation solution has been added. These tubes can be stored at 2 808 for six months without adversely affecting the assay.

2.3. Perchloric acid solution

A solution of 10% (w / v) perchloric acid containing 0.2 M boric acid and 10 mM g-Glu-Glu is prepared by adding 6.2 g boric acid and 1.38 mg g-Glu-Glu to approximately 300 ml water, mixing in 71 ml of 70% perchloric acid, and adjusting to 500 ml total volume. This solution is used to prepare B tubes, which are 1.5-ml microcentrifuge tubes to which 0.2 ml of the perchloric acid solution has been added. These tubes can be stored at 2 808 for at least 2 years.

2.4. Derivatization solutions

A KOH / tetrahydroborate solution is used for adjustment of pH to 9.060.2. This solution is prepared by adding 5.6 g KOH to a plastic bottle containing 50 g K 2 B 4 O 7 ? 4H 2 O and 100 ml water. This should be mixed and allowed to stand overnight at room temperature before removing the solution from the remaining precipitate. This solution is stable indenitely at room temperature. The

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iodoacetic acid solution is made by adding 14.8 mg per 2 ml of distilled water. Two milliliters are sufcient for 32 samples. This is made fresh on the day of derivatization. The dansyl chloride solution is prepared on the day of derivatization by dissolving 200 mg dansyl chloride in 10 ml of acetone. This is sufcient for 32 samples.

2.5. HPLC solvents

Solvent A is 80% (v / v) methanol / water. Solvent B is an acetate-buffered (pH 4.6) methanol solution prepared by mixing 640 ml of methanol, 200 ml of acetate stock, 125 ml of glacial acetic acid and 50 ml of water. The acetate stock is prepared by mixing 272 g sodium acetate trihydrate, 122 ml H 2 O and 378 ml glacial acetic acid. Both solvents are ltered with a 0.45-mm pore size lter prior to use.

2.6. Blood sampling procedure

Blood is collected by venipuncture to the antecubital vein with a 23-gauge buttery needle attached to a 5-ml syringe. Approximately 2 ml of blood is drawn into the syringe, the buttery needle is removed (while the syringe remains attached to the needle), and the venipuncture site is covered with gauze. The subject is asked to hold the gauze while the collector removes and disposes of the buttery needle and drips 0.5 ml of blood into an A tube. The tube is gently inverted twice for mixing and placed in a rack at room temperature while the venipuncture site is bandaged. A tubes are then spun in a microcentrifuge for 30 s to remove blood cells. If there is any visual evidence of hemolysis, samples should be discarded. However, in more than 500 blood samples that we have collected by this procedure, we have had no evidence of hemolysis. Aliquots (200 ml) of supernatant from the A tubes are transferred to B tubes, and the tubes are inverted to mix. Collected samples are placed at 2 808 as soon as possible for storage; for multiple collections during a day, samples can be maintained on ice or in a refrigerator. Samples stored at 2 808 are stable for at least 2 months and can be shipped overnight on dry ice without deterioration.

2.7. Derivatization and analysis

B tubes are spun for 2 min in a microcentrifuge to pellet protein. An aliquot (300 ml) of each supernatant is transferred to a fresh microcentrifuge tube. Iodoacetic acid solution (60 ml) is added to each tube. The pH is adjusted to 9.060.2 with the KOH / tetraborate solution (approximately 200 ml). After about 3 min to allow complete precipitation of potassium perchlorate, the pH of at

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least some of the samples should be checked to verify that they are in the correct range. After 20 min, 300 ml of the dansyl chloride solution is added, and the samples are mixed and placed in the dark at room temperature for 16 to 26 h. Chloroform (500 ml) is then added to each tube to extract the unreacted dansyl chloride, and samples are stored in the presence of both the perchlorate precipitate and the chloroform layer at 048 until assay by HPLC. Stability tests show that samples can be stored under these conditions for 12 months with little change in the amounts of GSH and GSSG derivatives (see results).

2.8. HPLC analysis

Samples are centrifuged for 2 min in a microcentrifuge prior to transfer of an aliquot of the upper (aqueous) layer to an autosampler. Typical injection volume is 20 ml. Separation is achieved on a 3-aminopropyl column (5 mm; 4.6 mm 3 25 cm; Custom LC, Houston). Initial solvent conditions are 80% A, 20% B run at 1 ml / min for 10 min. A linear gradient to 20% A, 80% B is run over the period from 10 to 30 min. From 30 to 46 min, the conditions are maintained at 20% A, 80% B and returned to 80% A, 20% B from 46 to 48 min. Equilibration time for the next run is 12 min. These conditions have been established to allow simultaneous measurement of Cys 2 and Cys along with GSH and GSSG; run times can be shortened considerably if resolution of Cys 2 and Cys is not desired. Detection is obtained by uorescence monitoring with bandpass lters, 305 395 nm excitation and 510650 nm emission (Gilson Medical Electronics, Middleton, WI). To facilitate simultaneous measurement of Cys 2 and Cys we routinely use two detectors in series with different sensitivity settings. Fluorometric detectors with monochromators set at 335 nm for excitation and 515 nm emission can also be used with equivalent results. Quantitation is obtained by integration relative to the internal standard.

2.9. Patient recruitment

This study was reviewed and approved by the Investigational Review Board of Emory University and performed in accordance with the ethical standards outlined in the 1975 Declaration of Helsinki, as revised in 1983. Each participant gave their informed consent prior to their inclusion in the study. Participants were recruited from patients of the Retina Service at The Emory Clinic, as well as Emory University employees. Data are presented as mean61 standard deviation. Because inter-individual variation was large, comparisons of repetitive values were normalized prior to statistical analysis. The process used for normalization was to divide each measurement by the mean of all measurements for that individual and multiply

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this ratio times the overall mean for all of the individuals in the respective experiment. Data so treated are indicated as normalized in the text.

3. Results Biologically important compounds separated by the method include Cys 2 , Cys, CySH-GSH, GSH and GSSG (Fig. 1). Studies performed to optimize conditions for derivatization showed that reaction of thiols with iodoacetic acid was complete within 5 min at pH 9.0. The rate of derivatization with dansylchloride was the same for GSH as for the internal standard (complete by 8 h at room temperature); however, disuldes, which have two amino groups, required 16 h for complete derivatization at room temperature. The pH optimum was relatively narrow, with derivatization at 8.6 or lower requiring . 16 h for completion and at pH $ 9.4 not reaching completion apparently because of instability of dansyl chloride. Thus, a borate buffer was included to facilitate pH adjustment to 9.060.2. A representative chromatogram (Fig. 1) shows that GSH and GSSG are readily detectable and quantiable in human plasma by this approach. Direct

Fig. 1. HPLC separation of GSH and related compounds in human plasma following derivatization with iodoacetic acid and dansyl chloride. Ten ml of derivatized human plasma depicting Cys (23.3 min), Cys 2 (25.1 min), g-Glu-Glu (27.7 min), CySH-GSH (32.3 min), GSH (33.7 min) and GSSG (37.7 min). Inset shows a 10-fold amplication of the region containing CySH-GSH, GSH and GSSG derivatives. Co-elution of the peaks with the relevant standards was conrmed with individual additions of known standards to the plasma samples.

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comparison of this uorescent HPLC method to the spectrophotometric assay of Reed et al. [14] was performed for GSH measurement on plasma from seven individuals. Mean values for GSH were 1.6360.29 mM for the dansyl chloride method and 1.5260.11 for the spectrophotometric method. For the uorimetric method, the coefcient of variation was 2.4 within a series and 4.9 between days. In comparing the two methods, the mean difference between individual paired samples was 4.1% (coefcient of variation was 2.3), further showing that the two HPLC methods are comparable for measurement of GSH. The spectrophotometric method does not have sufcient sensitivity to measure GSSG so that direct comparisons could not be made. Studies comparing blood collected into evacuated tubes (Vacutainer; BectonDickinson) and using the syringe method as described showed that GSH was signicantly higher (P , 0.05) in the plasma from samples drawn into evacuated tubes (4.1561.30; n 5 5) than in paired plasma samples from blood drawn into a syringe (1.8960.67; n 5 5). One sample drawn into an evacuated tube showed slight visible evidence of hemolysis, suggesting that the difference was due to limited hemolysis by this approach even though hemolysis was not visibly evident in the other four samples. An alternate explanation for the difference between methods is that there could be a greater loss of GSH in the blood drawn by syringe even though the timing for collection by the two methods was the same. To test for loss of GSH from blood samples drawn by syringe, plasma GSH and GSSG were determined on blood from ve individuals without or with 1 mM GSH or 0.5 mM GSSG added at the time that blood was drawn. The results showed that 10469% of the added GSH was recovered and 98611% of the added GSSG was recovered. Thus, we conclude that the more likely explanation for the difference is that blood drawn into an evacuated tube can have some limited hemolysis. Consequently, unless hemolysis is to be specically measured on each sample, collection of blood by syringe is preferable to evacuated tubes because it reduces the chance of hemolysis. To determine whether GSH recovery was affected by a time delay in transfer of blood from the collection syringe into the microcentrifuge tubes with the serine ? borate, samples were transferred at 0, 30 and 60 s. The results showed that as long as transfer was made to the serine ? borate solution within 1 min, no detectable change in GSH occurred (respectively, 1.5560.08, 1.5260.07 and 1.5160.03; n 5 5; normalized). Longer times were not tested because previous reports show that a 2-min delay results in about 20% loss when plasma is prepared directly [10], and transfer to preservation solution as described can be readily achieved within one min. To determine whether GSH was oxidized due to a delay in processing the blood in the preservation solution, A tubes were centrifuged to obtain plasma at 2, 5, 10, 20 and 30 min and subsequently analyzed. Results (Table 1) showed that the ratio of GSH / GSSG was not

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Table 1 Effect of time in preservation solution on measured GSH, GSSG and GSH / GSSG ratio Time (min) 2 5 10 15 20 30 GSH (mM) 1.5660.07 1.4460.08 1.5260.11 1.6160.02 1.5560.12 1.5560.11 GSSG (mM) 0.2160.02 0.2060.01 0.2060.04 0.2360.03 0.2160.01 0.2160.02 GSH / GSSG Ratio 7.460.9 7.460.7 7.961.4 7.261.0 7.560.7 7.661.0

Samples were collected from ve individuals and analyzed as described in Section 2. Values were normalized to eliminate inter-individual variation. Without normalization, GSH was 1.5360.23 and GSSG was 0.2160.07 for these ve individuals. Values given are mean61 standard deviation.

signicantly different at any time point. Thus, the preservation solution protects against changes in GSH and GSH redox state. Stability studies of non-derivatized samples in perchloric acid solution stored at 2 808 showed that there was no detectable loss of GSH or GSSG with time up to 2 months of storage (GSH: Day 7, 1.4960.06; Day 60, 1.5360.06; GSSG: 0.3060.02; Day 60, 0.2960.02; n 5 3, normalized). Analysis of derivatized samples stored in the dark at 048 showed only a small loss of GSH or GSSG at 12 months (GSH: Initial, 1.8960.14; 12 months, 1.7960.14; GSSG: 0.2060.03; 12 months, 0.1860.03; n 5 20, normalized). Thus, results show that sample stability is sufcient for convenient application of this method to studies which require intermittent sample collection over periods of several days to weeks. Application of this method to measurement of GSH in 59 healthy individuals aged 2043 years showed that values ranged from about 0.6 to 6 mM with a mean of 2.0961.14 mM. The distribution was skewed with a median value of 1.75 mM. There was no signicant age dependence within this range and there was no signicant difference between males (2.0761.29; n 5 26) and females (2.1061.02; n 5 33). Measurements of GSSG provided a mean of 0.08360.090 mM. The mixed disulde of GSH and Cys was 1.6760.75 mM. Cys was 7.6964.68 mM and Cys 2 was 51.3610.9 mM. 4. Discussion Experimental evidence suggests that plasma GSH concentration varies in response to disease and may therefore be a useful clinical measure [1]. Thus, there is a need for a standardized procedure for determination of plasma GSH. Several analytical techniques are available for measurement of GSH at con-

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centrations that are present in human plasma, and these can be calibrated with relevant standards to provide reliable measurement. The present study has focused on aspects of sample collection and processing that could contribute to variations in measurement even with reliable analytical methods for GSH. For this purpose, we have used a modication of the method of Martin and White [15] to detect GSH as the dansyl derivative of S-carboxymethyl-GSH. With the modications as described, this method provides values comparable to the well established method of Reed et al. [14] when used on identical samples. The results indicate that collection via a syringe can reduce the risk of hemolysis which can result in an overestimation of GSH. The results also show that immediate transfer of blood into the preservation solution provides stabilization of GSH, preventing its loss due to oxidation or degradation. The use of this method for detection of GSH allows simultaneous measurement of GSSG. Analysis of the time course of derivatization showed that dansylchloride reacts slowly with GSSG at room temperature and that 16 h was required for complete derivatization. Comparison to values obtained by other methods reveals that the present method results in lower values for GSSG. For instance, Mansoor et al. [2] obtained 1.4860.36 mM with a monobromobimane method in which they calculated GSSG as the difference between GSH and the total GSH following reduction with NaBH 4 . Using a similar approach, Yang et al. [3] obtained values of 1.8 to 2.3 mM. The present data show that these values are probably overestimates because they include CySH-GSH, a compound that is present at about 1.7 mM and will yield GSH upon reduction. Adams et al. [8] and Kretzschmar et al. [9] obtained mean values for GSSG of 0.33 to 0.42 mM with an enzymatic recycling method. Paolisso et al. [16], using the same method, obtained values of approximately 0.8 mM. These values could also be overestimates if the GSSG reductase reduces the mixed disulde of GSH and Cys. In summary, collection of blood with a buttery needle and syringe reduces the chance of an overestimation of GSH due to a minor extent of hemolysis. Use of a preservation solution minimizes loss due to oxidation or degradation during processing. Non-derivatized samples can be stored in 5% perchloric acid / saturated boric acid at 2 808 up to 2 months without changes in GSH, and dansylated samples can be stored in the dark for 12 months at 048. Application of these conditions can be expected to improve the reliability of plasma GSH measurements.

Acknowledgements This research was supported by NIH grant EY07892 and Research to Prevent Blindness Inc..

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References
[1] Jones DP, Brown LAS, Sternberg P. Variability in glutathione-dependent detoxication in vivo and its relevance to detoxication of chemical mixtures. Toxicology 1995;105:26774. [2] Mansoor MA, Svardal AM, Ueland PM. Determination of the in vivo redox status of cyteine, cysteinylglycine, homocysteine, and glutathione in human plasma. Anal Biochem 1992;200:21829. [3] Yang C, Chou S, Liu L, Tsai P, Kuo J. Effect of aging on human plasma glutathione concentrations as determined by high-performance liquid chromatography with uorimetric detection. J Chromatogr B 1995;674:2330. [4] Neuschwander-Tetri BA, Roll FJ. Glutathione measurement by high-performance liquid chromatography separation and uorimetric detection of the glutathione-orthophthalaldehyde adduct. Anal Biochem 1989;179:23641. [5] Michelet F, Gueguen R, Leroy P, et al. Blood and plasma glutathione measure in healthy subjects by HPLC: relation to sex, aging, biological variables, and life habits. Clin Chem 1995;41(10):150917. [6] Paroni R, De Vecchi E, Cighetti G, et al. HPLC with o-phthalaldehyde precolumn derivatization to measure total, oxidized, and protein-bound glutathione in blood, plasma and tissue. Clin Chem 1995;41(3):44854. [7] Mills BJ, Richie Jr. JP, Lang CA. Glutathione disulde variability in normal human blood. Anal Biochem 1990;184:2637. [8] Adams JD, Johannessen JN, Bacon JP. Quantication of glutathione and glutathione disulde in human plasma. Clin Chem 1987;33(9):16758. [9] Kretzschmar M, Muller D, Hubscher J, Main E, Klinger W. Inuence of aging, training and acute physical exercise on plasma glutathione and lipid peroxides in man. Int J Sports Med 1991;12:21822. [10] Lash LH, Jones DP. Distribution of oxidized and reduced forms of glutathione and cysteine in rat plasma. Arch Biochem Biophys 1985;240:58392. [11] Newton GL, Dorian R, Fahey RC. Analysis of biological thiols: derivatization with monobromobimane and separation by reverse-phase high performance liquid chromatography. Anal Biochem 1981;114:3837. [12] Anderson ME, Meister A. Dynamic state of glutathione in blood plasma. J Biol Chem 1980;255:95303. [13] Smith CV, Hansen TN, Martin NE, McMicken HW, Elliott JJ. Oxidant stress responses in premature infants during exposure to hyperoxia. Pediatr Res 1993;34:3605. [14] Reed DJ, Babson JR, Beatty PW, et al. High performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulde, and related thiols and disuldes. Anal Biochem 1980;106:5562. [15] Martin J, White NH. Fluorimetric determination of oxidized and reduced glutathione in cells and tissues by high-performance liquid chromatography following derivatization with dansyl chloride. J Chromatogr 1991;568:21925. [16] Paolisso G, DiMaro G, Pizza G, et al. Plasma GSH / GSSG affects glucose homeostasis in healthy subjects and non-insulin-dependent diabetics. Am J Physiol 1992;263:E43540.