Some aspects of sulphydryl metabolism in granulocytic leukaemia

Europ. 07. Cancer Vol. 1, pp. 189-194. Pergamon Pre~ 1965. Printed in Great Britain

Some Aspects of Sulphydryl Metabolism in Granulocytic Leukaemia K. R. HARRAP Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, Fulham Road, London, S.W.3.

INTRODUCTION

Calcutt and Connors have shown that the response of certain transplantable tumours in the mouse to treatment with an alkylating agent (to which class of compounds busulphan belongs) may be related to the ratio of proteinfree thiol to protein-bound thiol [6].

THE INTERESTin the metabolism of sulphydryl compounds described in this communication arises from previous studies associated with the response of patients (CGL*) to busulphan therapy [1]. Many patients in the later stages of this disease exhibit the well known blast-cell crisis, which is accompanied by (though not necessarily associated with) an increasing resistance to busulphan therapy. In attempting to understand the biochemistry of this process it is necessary to identify some biochemical property which might be expected to change in accordance with both the progress of the disease and the reaction of the latter to therapy. The task of identifying such a property is eased somewhat by the literature implicating abnormalities of sulphur metabolism in leukaemia [2-4], and by the work of Roberts and Warwick, who have demonstrated that busulphan reacts in vivo in animals with sulphydrylcontaining compounds [5]. Furthermore,

Measurement of the levels of protein-free sulphydryl compounds in the blood cells of patients (CGL) might be expected therefore to reflect the progression of the disease and the response of the patient to treatment with busulphan. Soluble disulphide has also been measured, since a change in the free sulphydryldisulphide ratio found normally in healthy tissues may be a more intrinsic indication of metabolic abnormality than changes in gross sulphydryl concentration [1]. Previous studies [1] have indicated that oxidised glutathione can occur in the blood cells of patients suffering from chronic granulocytic leukaemia, and the results are summarised in Fig. 1 [7]. In the present communication these studies are extended to include a survey of patients suffering from acute granulocytic leukaemia.

*Abbreviations to be used in this paper: CGL - - chronic granulocytic leukaemia; A G L - acute granulocytic leukaemia; G S H - - r e d u c e d glutathione; GSSG-oxidised glutathione; SH-- sulphydryl; SS-- disulphide; NADP--nicotinamide adenine dinucleotide phosphate; N A D P H - reduced nicotinamide adenine dinucleotide phosphate; EDTA - - ethylene-diamine-tetraacetic acid (disodium salt); G 6 P - glucose 6 phosphate; 6PG - - 6 phosphogluconate; R5P - ribulose-5 phosphate,

The oxidation and reduction of glutathione in the erythrocyte is mediated by the system shown in Fig. 2: the present paper also describes an attempt to account for the presence of oxidised glutathione in the erythrocyte in terms of this enzyme system. 189

190

If. R. Harrap SOLUBLE SH/SS LEVELS IN CHRONIC MYELOID LEUKAEMIA Leucocytes

NORMALCONTROLS

~

UNTREATEDCMLG ( ROUPI)

~

BUSULPHANORX-RAY'TREATEDCHL (GROUP"IT)

CML APPROACHINGBLASTCRISIS (GROUPIH)

•@

Eryt.hrQcytes

0% SS

~

0 %. SS

32 % SS

~

0O/oSS

7[% SS

@

.0~ SS

0 °/o SS

@

40 % SS

Fig. 1. Soluble SH/SS levels in chronic myelogenons leukaeraia. The circumfirence of the circles is proportional to the total S H content of the cells (normal cells: erythrocytes O. 18 ~tmoles/lO 9 cells, leucocytes 1" 1 I~mole[lO 9 cells). Patients fall into three groups as shown.

6PG (R5P)

. NADPH

GSSG

H20

for 30 min before removing the stroma by centrifugation at 6 500 g (0°C) for 30 min. The haemolysates were stored for at least 24 hr at --40°C before the enzyme assays were performed.

(BPG)

NADP

GSH

202

Enzyme assays All enzyme assays were carried out in 0.05M triethanolamine-HC1 buffer pH 7.6 [8], and at a temperature of 25°C (except for glutathione peroxidase (GSHP) which was assayed at 0°C). Glutathione reductase (GSSGR) was determined according to Scott et al. [9], and glucose6-phosphate dehydrogenase (G6PD) and 6phosphogluconate dehydrogenase (6PGD) by the method of Shonk and Boxer [8]. Glutathione peroxidase (GSHP) was assayed by a modification of the method of Mills [10] as follows: To 1 ml GSH (4.0× 10-3M in 0 . 0 5 M triethanolamine-HC1 buffer pH 7.6, 0. O06M with respect to EDTA) was added 3.1 ml triethanolamine buffer pH 7.6 and 0.1 mi sodium azide (0.31M), followed by 0.3 ml haemolysate. The solution was equilibrated at 0°C for 5 min, whereafter the enzyme reaction was started by adding 0.5 ml of 4-0 × 10-*M hydrogen peroxide in triethanolamine buffer (0.05M, pH 7.6). One millilitre samples were removed at 0, 3, 6, 9 min intervals and added rapidly to a separate tube containing 2 ml isobutanol and 4 ml 2.5% metaphosphoric acid [11]. These tubes were shaken thoroughly, centrifuged at approximately 400g for 10 min, the supernatant organic layer aspirated off, and residual GSH estimated in 1 ml aliquots of the aqueous layer by the colorimetric method described below.

Fig. 2. Enzyme system responsible for the oxidation and reduction of glutathione within the erythrocyte: Enzymes: glutathione reductase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogennse, glutathione peroxidase.

METHODS

Chemicals were obtained from Hopkin and Williams Ltd. or B.D.H. Ltd. : AnalaR grades were used where available. GSH and GSSG were obtained from Sigma Chemical Company; NADP and NADPH from Boehringer; G6P and 6PG from Wessex Biochemicals Ltd., Bournemouth, England; 5,5'-dithiobis- (2-nitro benzoic acid) from Aldrich Chemical Co. Inc. Venous blood was collected and fractionated as described previously [1]. Erythrocyte haemolysates were prepared by centrifuging whole blood at 350 g for 10 min at 0°C, removing the plasma and buffy coat, and washing the packed red cells twice with icecold physiological saline. The washed cells were then diluted to a measured volume with a further aliquot of saline, counted, removed from suspension by centrifugation at 350 g, and haemolysed by adding ice-cold water until the haemolysate volume was equivalent approximately to that of the blood originally taken. The haemolysate was then allowed to stand at 0°C

Some Aspects of Sulphydryl Metabolism in Granulocytic Leukaemia

191

Table 1. Occurrenceof oxidized thiol in the blood cells of patients : AGL. Cellular thiol content (lamoles SH/109 cells) Erythrocytes Leucocytes

Total WBC

Early granulocytes (%)

Blasts

1,550 6,750 3,800 4,500 280,000 23,700 66,000

8.0 0 6.5 2"0 0 18 1

2.5 0 2"0 0.5 95.5 58 79

Patient

c°g°

P.D. D.H. H.P. M.S. R.G. W.L. (CGL

Total thiol

Oxidized form (%)

0" 15 0.32 0.28 0" 45 0.22 0.21 0.34

0 9 21 16 10 0 18

Total thiol

Oxdized form (%)

0.74 0 0.48 58 0.40 50 None detected 0.03 100 0.06 0 0.9 0

(%)

in "blast

crisis")

Units of enzyme activity GSSGR: ltmoles N A D P H oxidized per minute per 109 ceils. G6PD and 6PGD: ~tmoles NADP reduced per minute per 10~ cells. GSHP: m~tmoles GSH oxidized per minute per 109 cells. GSSG and GSH were estimated as described previously, or by a modification of the colorimetric method of Beutler et al. [12] as follows: to 1 mi of the erythrocyte suspension in isotonic saline was added 9.0 ml ice-cold 3 % sulphosalicylic acid. After 20 min at 0°C the protein precipitate was removed by centrifugation or filtration. Reduced glutathione was determined by adding 1 ml of the filtrate to 4.0 ml of 5,5'-dithiobis-(2-nitrobenzoic acid) - - 0 . 5 × 10-sM in 1.0M phosphate buffer p H 7.0 (10-sM with respect to EDTA). The optical density of the solution was recorded at 412 m ~t and compared with appropriate standards. GSSG was determined by a similar colorimetric procedure following electrolytic reduction [1]. I n order to obtain consistent results it was found necessary to rinse all glassware before use with the buffered reagent, and to measure the

Table 2.

optical density of the solutions within 3 min of colour development. RESULTS

The distribution of soluble thiol (SH) and disulphide (SS) between the erythrocyte and leueoctyte fractions from a series of patients (AGL) is shown in Table 1. In Table 2 are shown the results of SH/SS determinations in the erythrocytes of a further series of patients suffering from granulocytic leukaemia. The analyses in Tables 1 and 2 were carried out on freshly fractionated material. It has already been shown, in a group of 11 healthy controls, that the mean erythrocyte SH content was 0 . 2 0 + 0 . 0 2 pmoles[10 s cells: No GSSG present [1]. Tables 3 and 4 respectively list the results of enzyme assays carried out on the two series of patients in Tables 1 and 2. Haemolysates from the acute leukaemia patients in Table 1 were stored (at --40°C) for a longer period than the haemolysates of the second series of patients in Table 2, but are compared with a set of controls taken at the same time as the patient blood

Occurrenceof oxidized thiol in the erythrocytes of patients: GGL, AGL. Erythrocyte thiol content (~tmoles SH/10 ° cells)

Patient

S.H. F.B. M.P. E.I. H.C. A.C. G.J. J.S.

Diagnosis

AGL AGL AGL CGL CGL CGL CGL CGL

Total thiol

Oxidized form (%)

0.44 0.21 0.33 0.33 0.17 0- 27 0.23 0.28

15 0 0 21 0 0 0 7

Total WBC

Early granulocytes (%)

Blasts (%)

5,700 2,200 3,900 2,100 40,000 14,400 7,600 11,200

0 6 7 4 50 6 5 8" 5

0 5 0 21 10 2 0" 5 0.5

192

K. R. Harrap

Table 3. Enzyme assays on erythrocyte haemolysatesfrom patients: AGL (cf. Table l for SH/SS analysis and blood data) GSSGR G6PD (~tmoles 0xrnoles NADPI-I oxidized NADP reduced per min per 109 per min per 109 cells) cells)

6PGD (Bmoles NADP reduced per min per 109 cells)

GSHP (mgmoles GSH oxidized per min per 109 cells)

Patien~ C.L. P.D.* D.H.* H.P.* M.S.*

R.G. W.L.* Means Standard deviation

0.030 0-170 0.070 0.118 0.087 0.100 0.108 0-096 0.018

0.011 0.029 0.034 0-025 0.015 0.023 0-022 0.023 0.007

0.028 0.044 0.039 0-068 0.032 0.030 0.037 0-039 0.013

1.03 8.20 15.60 17.90 15.50 1.88 8.60 9.81 6-27

0-065 0-089 0.096 0.053 0-071 0.080 0.072 0.067 0.074 0.013

0.015 0.016 0.016 0.016 0-012 0.028 0.033 0.013 0.018 0. 007

0.055 0-030 0.037 0.024 0-019 0.061 0-061 0.034 0.040 0.015

11.10 8.99 8.36 3-55 8.15 24.60 14.10

Contro~ A B C D E F G H Means Standard deviation Compar~on of means ("t" test)

P>0.3

P>0.3

P>0" 9

11.26 2.14

P>0" 9

*Erythrocytes contain GSSG (cf. Table 1).

Table 4.

Enzyme assays on erythrocyte haemolysatesfrom patients: AGL, CGL (cf. Table 2 for SH/SS analysis and blood data) GSSGR G6PD 6PGD (Bmoles (~tmoles (Bmoles NADPH oxidized NADP reduced NADP reduced per rain per 10 9 per min per 109 per min per 109 cells) cells) cells)

GSHP (m~Lmoles GSH oxidized per min per 10 9 cells)

Patients E.I. (CGL)* H.C. (CGL) A.C. (CGL) G.J. (CGL) J.S. (CGL)* S.H. (AGL)* F.B. (AGL) M.P. (AGL) Means Standard deviation

0. 167 0. 125 0.048 0. 108 0. 103 0- 136 0. 122 0- 173 0- 123 0. 023

0.200 0.250 0.066 0. l l 0 0. 160 0.270 0. 130 0. 170 0- 170 0. 064

0. 181 0. 120 0.090 0. 120 0.090 0.024 0. 130 0. 140 0.112 0.021

5.96 14.00 4.35 7.57 8.26 18.30 6.00 12.70 9.64 4.54

J.B. 0. 101 P.W. 0. 099 M.W. 0.112 L.H. 0. 060 K.H. 0-087 Means 0. 091 Standard deviation 0.017 Comparison of means ("t" test) P>0.02

0- 180 0. 160 0. 180 0" 140 0.070 0. 150 0.023

0. 140 0.110 0.110 0.090 0.110 0.110 0.014

17.20 9.58 13-20 11.70 5.08 11.35 4.02

Controls

P>0.5

*Erythrocytes contain GSSG (cf.Table II).

P>0.8

P>0.5

Some Aspects of Sulphyd~yl Metabolism in Granulocytic Leukaemia samples. The assa,ts recorded in Table 4 were performed after only 24 hr storage at --40°C and are compared with controls taken at the same time as the patient blood samples, and stored similarly. Comparison of the control group in Table 3 with that of Table 4 reveals that GSHP and GSSGR are stableunder these storage conditions: G6PD and 6PGD are less stable.

Table 5. Patient ecvthrocyte enzyme activities: expressed as percentage of mean control values. E~ythrocytes containing GSSG P.D. D.H. H.P. M.S. W.L. E.I. J.S. S.H.

GSSGR 230 95 159 116 135 183 113 149

C.L. R.G. H.C. A.C. G.J. F.B. M.P.

GSSGR 41 135 137 53 119 134 190

G6PD 160 189 139 83 122 133 107 180

6PGD 110 98 170 80 93 163 82 22

GSHP 73 140 159 138 76 53 73 161

Ery&rocy~ not con~ing GSSG G6PD 61 128 167 44 73 87 113

6PGD 70 75 109 82 109 118 127

GSHP 9 11 123 38 67 53 112

In Table 5 the enzyme activity figures in patients' erythrocytes are expressed as a percentage of the corresponding mean control values, and are tabulated in two groups according to whether or not GSSG was found in the erythrocytes.

DISCUSSION From an inspection of Tables 1 and 2 it is apparent that patients suffering from AGL do not fall into such clearly defined categories, in terms of the distribution of disulphide between the cell fractions, as has been found with CGL [1]. Also there would seem to be a tendency for the total erytihrocyte thiol content to be raised in those erythrocytes containing GSSG.

193

The activities of the enzymes in the two groups of patients listed in Tables 3 and 4 do not differ significantly from those of the corresponding control groups. In Table 5 the relative enzyme activities of erythrocyte preparations from cells containing GSSG are compared with those from cells conraining only GSH. Only in one patient (SH), whose erythrocytes contained GSSG, did the relative activities of any of the reduced enzyme, studied fall below the control values: in all other cases the enzyme activities were comparable with, or greater than, the mean control values. On the other hand, with patients whose erythrocytes contained only GSH, there were several cases where the activities of the erythrocyte enzymes fell below the mean control values (CL, RG, AC, GJ, EB). It would seem therefore that the appearance of GSSG in the erythrocytes of those patients reported here cannot be attributed to impairment of the apo-enzymes under study: the enzyme assay methods used here for GSSGR, G6PD, 6PGD have, of course, only measured apo-enzyme levels (excess cofactor has been added during assay), and it is feasible that impairment of holoenzyme function in vivo could exist as a result of cofactor deficiency. This aspect of the problem is under study.

Acknowledgements--The author is grateful to Prof. F. Bergel, F.R.S. and Prof. A. Haddow, F.R.S. for their continued interest and encouragement in this work. He also wishes to thank Dr. Sidney Farber (Childrens Cancer Research Foundation, Boston) for his kindness in providing a grant and laboratory facilities while some of the work described herein was carried out, and Dr. W. C. Maloney (Tufts Haematology Laboratory, Boston City Hospital) for referring patient material for study. Thanks are also due to the consultant staff of the Royal Marsden Hospital for referring patient material for study, and to Mr. R. Burch for technical assistance. The work has been supported by grants to the Chester Beatty Research Institute (Institute of Cancer Research: Royal Cancer Hospital) from the Medical Research Council and the British Empire Cancer Campaign for Research, and by the Public Health Service Research Grant No. CA03188-08 from the National Cancer Institute, U.S. Public Health Service.

RESUME

L'auteur a gtudig la rgpartition de composgs solubles sulfhyd~yl et disulfure entre les fractions leucocytaires et grythrocytaires, chez sept malades atteints de leu~gmie mygloblastique aigu~.

194

If. R. Harrap On a essayg d'expliquer la prgsence de glutathion oxydd dans les drythrocytes de malades (leucgmie mydloblastique aigug, leucdmie myglo¢de chronique) en fonction de l'intdgritg du systkme enzymatique responsable de l'oxydation et de la rdduction du glutathion. En conclusion, la fonction des apo-enzymes en cause n' est pas diminude. SUMMARY The distribution of soluble sulphydryl and disulphide compounds between the leucocyte and erythrocyte fractions of a group of patients (AGL) has been investigated. An attempt has been made to account for the presence of oxidised glutathione in the erythrocytes of patients (AGL, GGL ) in terms of the integrity of the enzyme system responsible for oxidation and reduction of glutathione. It is concluded that thefunction of the apoenzymes involved was not impaired. ZUSAMMENFASSUNG Die Verteilung yon liislichen Sulfhydryl- und Disulfidverbindungen zwischen den roten und den weissen Blutk6rperchen wurde bei einer Gruppe von Patienten mit akuter Granulozytenleukiimie (AGL) untersucht. Es wurde versucht die Anwesenheit von oxydiertem Glutathion in den Erythrozyten ovn Patienten mit akuter und chronischer Granulozytenleukiimie durch die Unversehrtheit des Enzymsystems, welchesfiir die Oxydation und fiir die Reduktion des Glutathions verantwortlich ist, zu erklgren. Aus den Ergebnissen wurde der Schluss gezogen, dass die Funktion der in Frage kommenden Apoenzyme nicht beeintrSchtigt ist. REFERENCES 1. K. R. HARRAP and D. E. M. SPEED, Some biochemical aspects of leukaemias: the appearance of a soluble disulphide in the blood in chroniie granulocytie leukaemia. Brit. 07. Cancer 18, 809 (1964). 2. A. N. CONTOPOULAS,and H. H. ANDERSON,Sulphydryl content of blood cells in dyscrasias. 07. Lab. Clin. Med. 36, 929 (1950). 3. J. WHrrE, G. B. Mm~R and W. E. HOSTON,Effect of amino acids on the induction of leukaemia in mice. 07. Nat. Cancer Inst. 4, 409 (1943). 4. A. S. WEXSBEROERand B. L. L~VINE, Incorporation of radioactive L-cystine by normal and leukaemic leukocytes in vivo. Blood 9, 1082 (1954). 5. J. J. ROBERTS and G. P. WARWICK, The mode of action of alkylating agents. II. Studies on the metabolism of Myleran. The reaction of Myleran with some naturally occurring thiols in vitro. Biochem. Pharmacol. 6 205 (1961). J . J . ROBERTSand G. P. WARWICK, The mode of action of alkylating agents. II1. The formation of 3-hydroxytetrahydrothiophene-l:l-dioxide from 1:4 dimethanesulphonyloxybutane (Myleran), S-[3-L-alanyltetrahydrothiophenium mesylate, tetrahydrothiophene, and tetrahydrothiophene-1 :l-dioxide in the rat, rabbit and mouse. Biochem. Pharmacol. 6, 217 (1961). 6. G. CALCtrrT and T. A. CONNORS,Tumour sulphydryl levels and sensitivity to the nitrogen mustard Merophan. Biochem. Pharmacol. 12, 838 (1963). 7. F. BEROEL and K. R. HARRAP, Future possibilities for the development of treatments of leukaemia. In: Conf. on Obstacles to the Control of Acute Leukaemia, Warrenton, Virginia, U.S.A. CancerRes. (1965) In press. 8. C. E. S~ONK and G. E. BOXER, Enzyme patterns in human tissues. I. Methods for the determination of glycolytic enzymes. CancerRes. 24, 709 (1964). 9. E. M. SCOTT, I. W. DUNCANand V. EKSTRAND,Purification and properties of glutathione reductase of human erythrocytes. 07. Biol. Chem. 238, 3928 (I 963). 10. G. C. MInLs, The purification and properties of glutathione peroxidase of erythrocytes. 07. Biol. Chem. 234, 502 (1959). 11. G. COHENand P. HOCttSTEIN,Glutathione peroxidase: the primary agent for the elimination of hydrogen peroxide in erythroeytes. Biochemistry 2, 1420 (1963). 12. E. BEUTLER,O. DURONand B. M. KELLY,Improved method for the determination of blood glutathione. 07. Lab. Clin. Med. 61, 882 (1963).