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Measurements of glutathione and other thiols in cells and tissues: A simplified procedure based on the HPLC separation of monobromobimane derivatives of thiols
??Session IV
MEASUREMENTS OF GLUTATHIONE AND OTHER THIOLS IN CELLS AND TISSUES: A SIMPLIFIED PROCEDURE BASED, ON THE HPLC SEPARATION OF MONOBROMOBIMANE DERIVATIVES OF THIOLS ANDREW Gray Laboratory
of the Cancer
Research
Campaign.
I. MINCHINTON, Mount
Vernon
B.Sc.
Hospital.
Northwood.
Middlesex
HA6 ‘RN.
U.K.
Although there is intense interest in the role of thiols in controlling the efficiency of radiosensitizers, and in developing thiols (or pro-drugs liberating thiols) as radioprotectors. there is little information regarding the concentration of specific thiols in cells, tumors and normal tissues. Details are presented of a modified procedure using the thiol binding agent monobromobimane with separation using paired-ion reverse phase high performance liquid chromatography (HPLC). This method has been extended to include measurements of the radiosensitizer misonidazole and its desmethylated metabolite Ro OS-9963 in tissues. Glutathione,
High
performance
liquid chromatography,
Radioprotectors,
INTRODUCTION
Radiosensitizers.
Several high performance liquid chromatography (HPLC) methods exist.‘.J.h.TUsually they involve the derivatization of the thiol function to form a fluorescent chromophore. The derivatization of the amine function present in some thiols has been exploited by some workers.’ the advantage of this approach being the ability to detect disuiphides. In this paper the method of Newton et al’ was selected and modified to meet the criteria mentioned above.
Glutathione constitutes the major proportion of cellular non-protein sulphydryl (NPSH) and hence plays a major role in the involvement of thiols in modifying the response ofcells and tissues to radiation. Buthionine sulphoximine (BSO) is a potent inhibitor of y-glutamyl-cysteine synthetase. its administration to mice or its incorporation in tissue culture media results in rapid depletion of intracellular glutathione. ‘Z To establish the magnitude of the role that glutathione plays in radiobiology it is important to measure accurately the degree of glutathione depletion. after exposure to BSO. in cells in culture and in a variety of normal tissues as well as in tumor tissue. Criteria for an appropriate analytical method include: (i) the ability to differentiate between all thiols of interest (e.g. glutathione (GSH). cystcine and WR-1065). (ii) high sensitivity capable of measurement of thiols down to a few percent of control values in ceils and tissues (e.g. after depletion with BSO). (iii) rapid. simple sample preparation avoiding hydrolysis and autoxidation. (iv) the capability of some degree of automation because of the desirability to process many samples in order to measure fluctuation in thiol concentrations. and (v) the ability to simultaneously measure radiosensitizer concentration.
METHODS
AND
MATERIALS
Monobromobimane (mBBr) was supplied by Calbiochem. Bishops Stortford, England, or synthetized by H. Monney. Brunei University, England. All other reagents were analytical or HPLC grade. WR-2721 was supplied by the NCI. U.S.A. WR-1065 was prepared by acid hydrolysis of WR-272 1. Eyltipmmr
Two 6000A pumps, a 730 data module, a 720 system controller, a 441 UV monitor at 365 nm and a 710B automatic sampler were supplied by Walers.* A Hypersil
Presented at the Conference on Chemical Modifiers of Cancer Treatment. Banff. Canada. 27 Nov.-l Dec. 1983. This work is supported by the Cancer Research Campaign. Ackno#Iedgmenrs-Monobromobimane was synthesized by H. Monney, Brunei University, England. WR-2721 was supplied by NCI, U.S.A. Misonidazole and Ro 05-9963 was supplied by Dr. C. E. Smithen. Roche Products Ltd., Welwyn, England.
Accepted for publication 24 April 1984. * Waters Associates, Hartford, Cheshire, Bennett and Company, Brimpton Common,
U.K.; Hichrom; Berkshire, U.K.;
Whatman. Maidstone, Kent, U.K.; Sigma Chemical Co., Poole, Dorset. U.K.; BDH Ltd., Dagenham, Essex, U.K.; Rathbum Chemicals (Walkerbum) Ltd., Walkerbum, Peebleshire, U.K.; Fisons Scientific Apparatus, Loughborough, U.K. 1503
I504
Radiation Oncology 0 Biology 0 Physics
September 1984. Volume IO. Number 9
ice cold SSA (20 mmol dme3) centrifuged and treated as for cells in suspension. For WR- 1065 analysis tissues were homogenized in ice cold KOH ( 1 mmol dmm3)containing mBBr (5 mmol dm-‘) and treated with SSA ( I mol dm-3) as for attached cells above.
ODS (250 X 4.6 mm i.d.) column and a guard column packed with Co-pell were supplied by Hichrom and Whatman, respectively. The guard column was repacked every 100 analyses. Sample preparation-Cells For the analysis of GSH, cells in suspension were pelleted by centrifugation, washed in phosphate buffered saline (PBS), repelleted and extracted with 5-sulphosalicylic acid (Sigma) (SSA) (20 mmol dmw3). After recentrifugation the pH of the supematant was adjusted to 8.5 and derivatization accomplished with KOH (BDH) (0.2 mol dms3) containing N-ethyl-morpholine (Sigma) (2.0 mol dm-3) and mBBr (20 ~1 of 50 mmol dmm3 to 1 ml extracts). For WR- 1065 analysis, attached cells were rinsed with PBS and extracted with ice cold KOH (1 mmol dm-‘) containing mBBr (5 mmol dme3). The complete lysing of all cells was achieved by repeated withdrawal through a 23 guage hypodermic needle. The pH was then adjusted to 2 with SSA (I mol dm-‘) to precipitate protein and after centrifugation an aliquot was injected on to the HPLC.
Storage Tissue samples extracted in SSA were stored at 4°C until derivatization and HPLC was performed. Tissue samples extracted with KOH/mBBr were treated after 4 to 5 minutes with SSA to precipitate protein. and after centrifugation the supematant was stored at 4°C. Cells were similarly treated and in all cases HPLC was carried out within 24 hours. High performance liquid chromatograph? A binary gradient elution system was used. Solvent A contained: 75% Acetonitrile (Rathbum), 25% water: Solvent B contained 5 mmol dme3 Tetrabutylammonium hydroxide (Fisons) (if GSH, misonidazole and Ro 059963 were being analyzed) or Hexane sulphonic acid (Fisons) (if WR- 1065 was being analyzed) in 50 mmol drnm3 ammonium dihydrogen orthophosphate (BDH). The gradient profile used was: initial conditions: 10% A. 90% B to 10 min; 40% A, 60% B to 12 min; 75% A, 25% B to 14 min; 75% A, 25% B to 16 min; 10% A, 90% B (linear gradients throughout) at a constant flow of 2 ml min-‘.
Tissue For GSH analysis, portions of liver, kidney, muscle and three implantable tumors were excised from mice killed by decapitation and immediately homogenized in
c
i.
L
3
2 time
Fig. I. Stability of GSH-mBBr adduct 0 and WR-1065-mBBr pH 8.5 (derivatizing solution without mBBr).
(
5
.
2L
hours) adduct A. Stability
of GSH
0 and Cysteine ??at
1505
Measurements of glutathione 0 A. I. MINCHINTON
Standurds and peak identification
11
001
Peaks were identified by comparison of retention times with standards of GSH and WR-1065. Thiol authenticity was further confirmed by treatment of duplicate samples with the thiol binding agent 2,2-dithiodipyridine (Sigma), chromatograms of samples thus treated were free of peaks representing thiols. Recalibration with standards was carried out automatically every 10 samples.
AU cl065 /
RESULTS
O-
This method has been used to measure glutathione in V79 379A Chinese hamster fibroblasts attached and cell suspension. GSH can be measured in extracts of as little as 0.25 X lo6 control cells, and depletion studies using BSO followed GSH down to approximately 5% of control levels, e.g., 0.2 fmol/cell, using a proportionally larger number of cells. In tissues, concentrations found in liver, kidney and muscle were similar to concentrations reported elsewhere. Figure 1 illustrates that the stability of the thiol-mBBr adduct at room temperature is good, allowing automatic sample injection. The stability of thiol compounds in the experimental alkaline solutions but without mBBr is shown and indicates the need for immediate derivatization. Figure 2 shows an HPLC chromatogram of GSH, misonidazole and Ro 05-9963. From an acid extract of liver.
I
I
1
0
20 time
lo
(minsl
Fig. 3. HPLC analysis of attached V79 379A cells in culture. 3.5 X lo6 cells were extracted in I ml KOH (1 mmol dm-‘) containing mBBr (5 mmol dmm3) Cells were incubated with WR-1065 (5 mmol dmm3) for 30 minutes. 50 ~1 injection. Details as Figure 2. -
0.0 AU AU
c
E 10 time
20
time
10
(hrs)
2o
(mans)
Fig. 2. HPLC analysis of mouse liver (45 mg) homogenized in 2 mls SSA (20 mmol dme3). 25 ~1 injected volume. Hypersil ODS (250 X 46 mm id) column, monitoring at 365 nm.
Fig. 4. HPLC analysis in 1 ml KOH (1 mmol 45 minutes after i.p. Kg-‘). 50 ~1 injection.
of mouse kidney dme3) containing administration of Details as Figure
(73 mg) homogenized mBBr (5 mmol dm-‘) WR-2721 (1.5 mmol 2.
1506
Radiation Oncology 0 Biology 0 Physics
the glutathione peak represents an on-column injection of approximately 5 nmol. The total analysis time of 20 minutes includes re-equilibration of the column in prep aration for the injection of the next sample. Figure 3 shows WR- 1065 extracted from 3.5 X 1O6cells in a volume of 1 ml. Figure 4 shows WR-1065 extracted from mouse kidneys after i.p. administration of WR-272 1 ( 1.5 mmol kg-‘). The resultant concentration was calculated to be 0.44 mmol kg-‘.
September 1984. Volume 10. Number 9
CONCLUSIONS The results show that the method described is a simple. reliable one for the determination of GSH and WR- 1065 which can be extended to other thiols and, through automatic injection, allows large numbers of samples to be sequentially analyzed. The use of a U.V. detector may compromise the ultimate sensitivity of the assay compared to fluorescence, but it allows the simultaneous determination of radiosensitizers such as misonidazole.
REFERENCES Griffith, O.W.: Mechanism of action, metabolism and toxicity of buthionine sulphoximine and its higher homologs. potent inhibitors of glutathione synthesis. J. Biol. Chem. 257: 13704-13712, 1982. Mesiter, A., Griffith, O.W.. Novogrodsky. A., Tate, S.S.: New aspects of glutathione metabolism and translocation in mammals. In Sulphur in Biology Ciba Foundation SJmposium 72. North Holland, Elsevier, 1980, pp. I35- I6 I. Nakamura, H., Tamuro, Z.: Fluorometric determination of thiols by liquid chromatography with post column derivatization. Anal. Chem. 53: 2 190-2 193, 198 I. Newton, G.L., Dorian, R.. Fahey, R.L.: Analysis of biological thiols: Derivatization with monobromobimane and separation by reverse-phase high performance liquid chromatography. Anal. Biochem. 114: 383-387, 1981.
5. Reed. D.J.. Babson, J.R.. Beatty. P.W..
Brodie. A.E.. Ellis.
High performance liquid chromatography analysis of nanomole levels of glutathione. glutathione disulphide and related thiols and disulphides. .4nal. B&hem. 106: 55-62. 1980. W.W..
Potter. D.W.:
Reeve. J.. Kuhlenkamp. J.. Kaplowitz. N.: Estimation of glutathione in rat liver by reverse-phase high performance liquid chromatography: Separation from cysteine and yglutamyl cysteine. J. Chromatog. 194: 424-428. 1980.
Takahashi. H.. Nara. Y.. Meguro. H.. Tuzimura. Z.: A sensitive fluorometric method for the determination of glutathione and some thiols in blood and mammalian tissues by high performance liquid chromatography. .4Rric. &ol Chem. 43: 1439-1445. 1979.
MEASUREMENTS OF GLUTATHIONE AND OTHER THIOLS IN CELLS AND TISSUES: A SIMPLIFIED PROCEDURE BASED, ON THE HPLC SEPARATION OF MONOBROMOBIMANE DERIVATIVES OF THIOLS ANDREW Gray Laboratory
of the Cancer
Research
Campaign.
I. MINCHINTON, Mount
Vernon
B.Sc.
Hospital.
Northwood.
Middlesex
HA6 ‘RN.
U.K.
Although there is intense interest in the role of thiols in controlling the efficiency of radiosensitizers, and in developing thiols (or pro-drugs liberating thiols) as radioprotectors. there is little information regarding the concentration of specific thiols in cells, tumors and normal tissues. Details are presented of a modified procedure using the thiol binding agent monobromobimane with separation using paired-ion reverse phase high performance liquid chromatography (HPLC). This method has been extended to include measurements of the radiosensitizer misonidazole and its desmethylated metabolite Ro OS-9963 in tissues. Glutathione,
High
performance
liquid chromatography,
Radioprotectors,
INTRODUCTION
Radiosensitizers.
Several high performance liquid chromatography (HPLC) methods exist.‘.J.h.TUsually they involve the derivatization of the thiol function to form a fluorescent chromophore. The derivatization of the amine function present in some thiols has been exploited by some workers.’ the advantage of this approach being the ability to detect disuiphides. In this paper the method of Newton et al’ was selected and modified to meet the criteria mentioned above.
Glutathione constitutes the major proportion of cellular non-protein sulphydryl (NPSH) and hence plays a major role in the involvement of thiols in modifying the response ofcells and tissues to radiation. Buthionine sulphoximine (BSO) is a potent inhibitor of y-glutamyl-cysteine synthetase. its administration to mice or its incorporation in tissue culture media results in rapid depletion of intracellular glutathione. ‘Z To establish the magnitude of the role that glutathione plays in radiobiology it is important to measure accurately the degree of glutathione depletion. after exposure to BSO. in cells in culture and in a variety of normal tissues as well as in tumor tissue. Criteria for an appropriate analytical method include: (i) the ability to differentiate between all thiols of interest (e.g. glutathione (GSH). cystcine and WR-1065). (ii) high sensitivity capable of measurement of thiols down to a few percent of control values in ceils and tissues (e.g. after depletion with BSO). (iii) rapid. simple sample preparation avoiding hydrolysis and autoxidation. (iv) the capability of some degree of automation because of the desirability to process many samples in order to measure fluctuation in thiol concentrations. and (v) the ability to simultaneously measure radiosensitizer concentration.
METHODS
AND
MATERIALS
Monobromobimane (mBBr) was supplied by Calbiochem. Bishops Stortford, England, or synthetized by H. Monney. Brunei University, England. All other reagents were analytical or HPLC grade. WR-2721 was supplied by the NCI. U.S.A. WR-1065 was prepared by acid hydrolysis of WR-272 1. Eyltipmmr
Two 6000A pumps, a 730 data module, a 720 system controller, a 441 UV monitor at 365 nm and a 710B automatic sampler were supplied by Walers.* A Hypersil
Presented at the Conference on Chemical Modifiers of Cancer Treatment. Banff. Canada. 27 Nov.-l Dec. 1983. This work is supported by the Cancer Research Campaign. Ackno#Iedgmenrs-Monobromobimane was synthesized by H. Monney, Brunei University, England. WR-2721 was supplied by NCI, U.S.A. Misonidazole and Ro 05-9963 was supplied by Dr. C. E. Smithen. Roche Products Ltd., Welwyn, England.
Accepted for publication 24 April 1984. * Waters Associates, Hartford, Cheshire, Bennett and Company, Brimpton Common,
U.K.; Hichrom; Berkshire, U.K.;
Whatman. Maidstone, Kent, U.K.; Sigma Chemical Co., Poole, Dorset. U.K.; BDH Ltd., Dagenham, Essex, U.K.; Rathbum Chemicals (Walkerbum) Ltd., Walkerbum, Peebleshire, U.K.; Fisons Scientific Apparatus, Loughborough, U.K. 1503
I504
Radiation Oncology 0 Biology 0 Physics
September 1984. Volume IO. Number 9
ice cold SSA (20 mmol dme3) centrifuged and treated as for cells in suspension. For WR- 1065 analysis tissues were homogenized in ice cold KOH ( 1 mmol dmm3)containing mBBr (5 mmol dm-‘) and treated with SSA ( I mol dm-3) as for attached cells above.
ODS (250 X 4.6 mm i.d.) column and a guard column packed with Co-pell were supplied by Hichrom and Whatman, respectively. The guard column was repacked every 100 analyses. Sample preparation-Cells For the analysis of GSH, cells in suspension were pelleted by centrifugation, washed in phosphate buffered saline (PBS), repelleted and extracted with 5-sulphosalicylic acid (Sigma) (SSA) (20 mmol dmw3). After recentrifugation the pH of the supematant was adjusted to 8.5 and derivatization accomplished with KOH (BDH) (0.2 mol dms3) containing N-ethyl-morpholine (Sigma) (2.0 mol dm-3) and mBBr (20 ~1 of 50 mmol dmm3 to 1 ml extracts). For WR- 1065 analysis, attached cells were rinsed with PBS and extracted with ice cold KOH (1 mmol dm-‘) containing mBBr (5 mmol dme3). The complete lysing of all cells was achieved by repeated withdrawal through a 23 guage hypodermic needle. The pH was then adjusted to 2 with SSA (I mol dm-‘) to precipitate protein and after centrifugation an aliquot was injected on to the HPLC.
Storage Tissue samples extracted in SSA were stored at 4°C until derivatization and HPLC was performed. Tissue samples extracted with KOH/mBBr were treated after 4 to 5 minutes with SSA to precipitate protein. and after centrifugation the supematant was stored at 4°C. Cells were similarly treated and in all cases HPLC was carried out within 24 hours. High performance liquid chromatograph? A binary gradient elution system was used. Solvent A contained: 75% Acetonitrile (Rathbum), 25% water: Solvent B contained 5 mmol dme3 Tetrabutylammonium hydroxide (Fisons) (if GSH, misonidazole and Ro 059963 were being analyzed) or Hexane sulphonic acid (Fisons) (if WR- 1065 was being analyzed) in 50 mmol drnm3 ammonium dihydrogen orthophosphate (BDH). The gradient profile used was: initial conditions: 10% A. 90% B to 10 min; 40% A, 60% B to 12 min; 75% A, 25% B to 14 min; 75% A, 25% B to 16 min; 10% A, 90% B (linear gradients throughout) at a constant flow of 2 ml min-‘.
Tissue For GSH analysis, portions of liver, kidney, muscle and three implantable tumors were excised from mice killed by decapitation and immediately homogenized in
c
i.
L
3
2 time
Fig. I. Stability of GSH-mBBr adduct 0 and WR-1065-mBBr pH 8.5 (derivatizing solution without mBBr).
(
5
.
2L
hours) adduct A. Stability
of GSH
0 and Cysteine ??at
1505
Measurements of glutathione 0 A. I. MINCHINTON
Standurds and peak identification
11
001
Peaks were identified by comparison of retention times with standards of GSH and WR-1065. Thiol authenticity was further confirmed by treatment of duplicate samples with the thiol binding agent 2,2-dithiodipyridine (Sigma), chromatograms of samples thus treated were free of peaks representing thiols. Recalibration with standards was carried out automatically every 10 samples.
AU cl065 /
RESULTS
O-
This method has been used to measure glutathione in V79 379A Chinese hamster fibroblasts attached and cell suspension. GSH can be measured in extracts of as little as 0.25 X lo6 control cells, and depletion studies using BSO followed GSH down to approximately 5% of control levels, e.g., 0.2 fmol/cell, using a proportionally larger number of cells. In tissues, concentrations found in liver, kidney and muscle were similar to concentrations reported elsewhere. Figure 1 illustrates that the stability of the thiol-mBBr adduct at room temperature is good, allowing automatic sample injection. The stability of thiol compounds in the experimental alkaline solutions but without mBBr is shown and indicates the need for immediate derivatization. Figure 2 shows an HPLC chromatogram of GSH, misonidazole and Ro 05-9963. From an acid extract of liver.
I
I
1
0
20 time
lo
(minsl
Fig. 3. HPLC analysis of attached V79 379A cells in culture. 3.5 X lo6 cells were extracted in I ml KOH (1 mmol dm-‘) containing mBBr (5 mmol dmm3) Cells were incubated with WR-1065 (5 mmol dmm3) for 30 minutes. 50 ~1 injection. Details as Figure 2. -
0.0 AU AU
c
E 10 time
20
time
10
(hrs)
2o
(mans)
Fig. 2. HPLC analysis of mouse liver (45 mg) homogenized in 2 mls SSA (20 mmol dme3). 25 ~1 injected volume. Hypersil ODS (250 X 46 mm id) column, monitoring at 365 nm.
Fig. 4. HPLC analysis in 1 ml KOH (1 mmol 45 minutes after i.p. Kg-‘). 50 ~1 injection.
of mouse kidney dme3) containing administration of Details as Figure
(73 mg) homogenized mBBr (5 mmol dm-‘) WR-2721 (1.5 mmol 2.
1506
Radiation Oncology 0 Biology 0 Physics
the glutathione peak represents an on-column injection of approximately 5 nmol. The total analysis time of 20 minutes includes re-equilibration of the column in prep aration for the injection of the next sample. Figure 3 shows WR- 1065 extracted from 3.5 X 1O6cells in a volume of 1 ml. Figure 4 shows WR-1065 extracted from mouse kidneys after i.p. administration of WR-272 1 ( 1.5 mmol kg-‘). The resultant concentration was calculated to be 0.44 mmol kg-‘.
September 1984. Volume 10. Number 9
CONCLUSIONS The results show that the method described is a simple. reliable one for the determination of GSH and WR- 1065 which can be extended to other thiols and, through automatic injection, allows large numbers of samples to be sequentially analyzed. The use of a U.V. detector may compromise the ultimate sensitivity of the assay compared to fluorescence, but it allows the simultaneous determination of radiosensitizers such as misonidazole.
REFERENCES Griffith, O.W.: Mechanism of action, metabolism and toxicity of buthionine sulphoximine and its higher homologs. potent inhibitors of glutathione synthesis. J. Biol. Chem. 257: 13704-13712, 1982. Mesiter, A., Griffith, O.W.. Novogrodsky. A., Tate, S.S.: New aspects of glutathione metabolism and translocation in mammals. In Sulphur in Biology Ciba Foundation SJmposium 72. North Holland, Elsevier, 1980, pp. I35- I6 I. Nakamura, H., Tamuro, Z.: Fluorometric determination of thiols by liquid chromatography with post column derivatization. Anal. Chem. 53: 2 190-2 193, 198 I. Newton, G.L., Dorian, R.. Fahey, R.L.: Analysis of biological thiols: Derivatization with monobromobimane and separation by reverse-phase high performance liquid chromatography. Anal. Biochem. 114: 383-387, 1981.
5. Reed. D.J.. Babson, J.R.. Beatty. P.W..
Brodie. A.E.. Ellis.
High performance liquid chromatography analysis of nanomole levels of glutathione. glutathione disulphide and related thiols and disulphides. .4nal. B&hem. 106: 55-62. 1980. W.W..
Potter. D.W.:
Reeve. J.. Kuhlenkamp. J.. Kaplowitz. N.: Estimation of glutathione in rat liver by reverse-phase high performance liquid chromatography: Separation from cysteine and yglutamyl cysteine. J. Chromatog. 194: 424-428. 1980.
Takahashi. H.. Nara. Y.. Meguro. H.. Tuzimura. Z.: A sensitive fluorometric method for the determination of glutathione and some thiols in blood and mammalian tissues by high performance liquid chromatography. .4Rric. &ol Chem. 43: 1439-1445. 1979.