101

Clinica Chimica Acta, 58 (1975) 101--108 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

CCA 6776

A NEW COLORIMETRIC METHOD FOR THE DETERMINATION OF NADH/NADPH DEPENDENT GLUTATHIONE REDUCTASE IN ERYTHROCYTES AND IN PLASMA

LEE KUM-TATT*, IT-KOON TAN and AI-MEE SEET

Clinical Biochemistry Laboratories, Government Department of Pathology, Outram Road, Singapore 3 (Singapore)

(Received August 6, 1974)

Summary

A simple and rapid colorimetric method for the assay of e ry throcyte and plasma glutathione reductase (GR) activity is described. The method is based on the colorimetric measurement of reduced glutathione (GSH) [1] produced when the enzyme is incubated with oxidised glutathione (GSSG) in the pre- sence of either reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH). Results of investiga- tions on the effects of substrate and coenzyme concentrations, pH, EDTA, sodium/potassium chloride, and time, on enzyme activity are presented. Erythrocyte and plasma NADH-GR and NADPH-GR activities in 100 healthy blood donors, and 85 cord blood samples and plasma NADH-GR and NADPH- GR levels in patients with various disease condit ions are given.

Introduction

In the presence of reduced nicotinamide adenine dinucleotide (NADH) or its phosphate (NADPH) the flavin enzyme glutathione reductase (EC 1.6.4.2) (GR) catalyses the reduction of oxidised glutathione (GSSG) to reduced gluta- thione (GSH).

GR GSSG + NADH/NADPH + H" ~ 2GSH + NAD'/NADP*

This reaction is important to the structural integrity of the red cell as it ensures the maintenance of sufficient quantities o f GSH for the protect ion of the cell

* Presen t add re s s : S i n g a p o r e I n s t i t u t e o f S t a n d a r d s a n d I n d u s t r i a l R e s e a r c h , 1 7 9 R ive r Va l l ey R o a d , S i n g a p o r e 6, R e p u b l i c o f Singapore.

102

against damage caused by oxidising agents [2--4 ]. It is therefore not surprising that absence or deficiency of GR which adversely affects the regeneration of GSH from GSSG would lead to reduced stability of the erythrocyte , particu- larly when the affected erythrocytes are exposed to oxidising agents. Various forms of haemolytic states have been at t r ibuted to the deficiency of erythro. cyte GR. Clinical manifestations range from drug or food-induced haemolysis [5,6] to chronic non-spherocytic haemolytic anaemia [7 ,8] .

Our interest in the investigation of e ry throcyte GSH and enzymes invol- ved in the maintenance of e ry throcyte stability led us to the search for simple and rapid methods suitable for their quantitative determination [9 ,10] . An earlier report described the development of a colorimetric method for the measurement of GSH using palladous (ed) chloride and chlorpromazine (CPZ) hydrochloride as reagents [1] . The present paper describes the application of this method for the assay of NADH and NADPH-dependent GR activity in erythrocytes and in plasma. The amount of GSH generated over a fixed period of incubation of the enzyme with GSSG and the appropriate reduced coen- zyme was measured by its quantitative displacement of CPZ from a preformed colored complex of Pd--CPZ resulting in a proport ionate decrease in absor- bance of the solution. The proposed method has the advantage over procedures based on the direct spectrophotometr ic measurement of the rate of dis- appearance of the reduced coenzyme at 340 nm [11,12] in that it is not limited by the quanti ty of the reduced coenzyme nor the concentration of haemolysate which could be measured accurately at 340 nm.

Using the proposed method, normal values for e ry throcyte and plasma NADH/NADPH dependent GR activities were obtained for 85 cord blood samples and blood collected from 100 healthy blood donors. Plasma enzyme levels were also determined on patients with various disease conditions.

Materials

NADH, NADPH and GSSG in the form of a disodium salt were purchased from Sigma Chemical Co.

CPZ • HC1 was a gift from May and Baker Co. Ltd, Dagenham, England. All other reagents used were of analytical reagent grade. Solutions were pre- pared in glass distilled water.

1. Phosphate buffer, 0.2 M, pH 6.48, 10 -4 M EDTA. 2. Phosphate buffer, 0.067 M, pH 7.38, 2 mM EDTA, 0.5 M KC1. 3. NADH solution in water, 5 mM. 4. NADPH solution in water, 2.2 mM. 5. GSSG solution, 22.8 mM. 6. Trichloroacetic acid (TCA), 4.5% (w/v). 7. Palladous chloride solution containing 60 pg/ml. A stock solution con-

taining 1 mg Pd per ml was prepared by dissolving 1.6664 g palladous chloride in approximately 20 ml 0.1N hydrochloric acid, standing at room temperature overnight and making up to a litre with water. Standardisation of this solution was made by the gravimetric dimethylglyoxime method [13] . Appropriate dilution of this solution was made to give a working solution containing 60 gg Pd per ml.

103

8. Orthophosphoric acid, 85% (w/v). 9. Chlorpromazine hydrochloride aqueous solution, 1% (w/v). 10. Ascorbic acid. 11. Pd--CPZ colour reagent for the determination of GSH. This was pre-

pared by mixing reagents (7), (8) and (9) in the proportion of 1 : 7 : 1 and allowing the solution to stand for 5 min before use. A few mg of ascorbic acid was added to prevent the oxidation of CPZ to its free radical [14] .

Methods

Preparation of haemolysate Fresh whole blood was collected in EDTA (1.5 mg/ml blood). The cells

were packed by centrifuging the blood at 1500 × g in an M.S.E. Minor centrifuge for 15 min. The plasma and buffy layer were removed and a 1 : 40 haemolysate solution of the packed cells was prepared in cold distilled water. The haemoly- sate solution was left to stand for 10 min with occasional mixing before assaying for GR activity.

Assay procedure for NADH-GR To two tubes marked 'Blank' and 'Test ' were added the

solutions: following

Blank Test

Phosphate buffer, 0.2M, pH 6.48, 10 -4 M EDTA 0.80 ml NADH, 5 mM 1 : 40 haemolysate/plasma/serum 0.20 ml

Preincubated for approximately 5 min at 37 ° GSSG, 22.8 mM

Incubated exactly 10 rain at 37 ° TCA, 4.5%

0.50 ml 0.25 ml 0.20 ml

0.05 ml

1.00 ml 1.00 ml

The mixtures were well stirred and centrifuged to remove the protein. One ml of the supernatant from each tube was added to 4.5 ml of the Pd--CPZ reagent. After thorough mixing, the solutions were left to stand for 30 rain at room temperature (27 ° to 30 °), preferably in a dark place, before absorbance readings were taken at 565 nm.

Individual blanks were required for each haemolysate sample, to correc for erythrocyte GSH content. As plasma and serum contain virtually no GSH only one common reagent blank was required for an entire batch of serum ol plasma enzyme determinations.

Assay procedure for NADPH-GR To two tubes marked 'Blank' and 'Test ' were added the following solu-

tions:

104

Blank Test

Phosphate buffer, 0.067M, pH 7.38, 2 mM EDTA, 0.5M KC1 0.8 ml NADPH, 2.2 mM 1 : 40 haemolysate/plasma/serum 0.2 ml

Preincubated for approximately 5 r.fin at 37 ° GSSG, 22.8 mM

Incubated exact ly 10 rain at 37 ° TCA, 4.5% 1.0 ml

0.5 ml 0.2 ml 0.2 ml

0.1 ml

1.0 ml

The mixtures were well stirred and centrifuged. One ml of the supernatant from each tube was then assayed for GSH con ten t using the method described earlier.

As in the case of NADH-GR assay procedure, individual blanks were required for haemolysates but no t for plasma or serum GR determinations.

Enzyme unit One unit of enzyme activity is defined as that producing 1 #mol of GSH

per min per litre e ry throcyte /p lasma/serum at 37 °. One pmol of GSH in a total volume of 5.5 ml gave an absorbance dif-

ference (AA) of 3.374.

AA Ery throcy te GR activity - 3.374

, 0 0 0 - - - - X ~ X ~ X X units

= AA X 11,850 units

AA Plasma/serum GR activity - 3.374

1000 2 - - - X ~ X ~X units

= AA X 296 units

Results and Discussion

Effect o f GSSG concentration Effect of GSSG concentra t ion on GR activity is shown in Fig. 1.

Maximum NADH-GR activity was obtained for GSSG concent ra t ion ranging from 0.5 to 2 mM and maximum NADPH-GR activity was observed with GSSG concentrat ions of from 1.5 mM to 6 mM. For routine purposes, NADH and NADPH concentra t ions of 1.14 mM and 2.28 mM were used for the assay of NADH-GR and NADPH-GR, respectively.

Effect o f reduced coenzyme concentration The influence of NADH and NADPH concentra t ions on GR activity is

shown in Fig. 2. NADH and NADPH concentra t ions of from 1.2 mM to 2.0 mM and from 0.44 mM to 1.60 mM, respectively, in the reaction mixture were required for opt imum NADH-GR and NADPH-GR activity, i

1 0 5

0 7

E o.~ o ~. OA / / NADH- GR

o.~ ~n

0.2

0.1

A A ' I I i 0 0:2 0.4 O.6 O:8 1.0 1.2 2 4 &

GSSG concentrotion (raM)

Fig. 1. E f f e c t o f G S S G c o n c e n t r a t i o n o n N A D H - G R a n d N A D P H - G R act ivi t ies . R e a c t i o n c o n d i t i o n s w e r e as d e s c r i b e d i n the t e x t e x c e p t that G S S G c o n c e n t r a t / o n s w e r e v a r i e d f r o m 0 t o 6 • 1 0 -3 M.

Effect of pH The pH opt imum for NADH-GR activity was found to be at 6.4. A broad

pH optimum ranging from 6.9 to 7.4 was observed for NADPH-GR activity (Fig. 3).

Effect of EDTA EDTA had a marked activation effect on GR activity. Addit ion of EDTA

in increasing quanti ty resulted in a corresponding rise in enzyme activity which reached a maximum when the EDTA used was from l f f 5 to 10 -3 M for the NADH<lependent reaction and from 8 . 0 . 1 0 - 4 to 1 2 . 5 . 1 0 - 3 M for the NADPH~tependent reaction. Therefore for routine assay of NADH-GR and NADPH-GR, EDTA concentrations of 5 • 10 -5 M and 10 -3 M, respectively, were used.

The activation effect of EDTA on GR activity may be at tr ibuted to its ability to sequester traces of heavy metal ion inhibitors such as Ni 2÷, Cu 2., and Zn 2÷. The effectiveness of the chelating agent in preventing inhibition by these ions has been fully investigated by Mize and Langdon [12] .

The kinetics of the erythrocyte and plasma GR action on GSSG was studied on 10 different blood specimens. For each blood specimen, the rate of

.~ 0.8 ~ . . . . . NADPH *

o o.~ I " f °

0.2

0 IL - , , ~ i , i i i 0'.2 c;.4 0.6 0.8 ,12 1.4 1.6

Concen t ra t i on ( r aM)

Fig. 2. E f f e c t o f N A D H / N A D P H c o n c e n t r a t i o n s o n G R a c t i v i t i e s . R e a c t i o n c o n d i t i o n s w e r e as descr ibed in the t e x t e x c e p t t h a t N A D H / N A D P H c o n c e n t r a t / o n s w e r e v a r i e d f r o m 0 t o 1 .6 • 1 0 -3 M.

106

0.8

c "~ 0 6

0

o.

0.2

0

5K~

~ A ~ A D P H GR / NADH - GR

~ o.# 6'.~ / o - z% 8'.0 pH

Fig. 3. E f f e c t o f p H o n G R act iv i t i e s . R e a c t i o n c o n d i t i o n s w e r e as d e s c r i b e d in t h e t e x t . Citrate-- phosphate b u f f e r s w e r e u s e d f o r t h e p H r a n g e 4 . 7 t o 5 .9 a n d p h o s p h a t e b u f f e r s f o r t h e pH range 5.96 to

7.9.

GSSG reduct ion was found to be linear with respect to t ime for only the initial 4 to 5 min when EDTA was no t included in the reaction mixture, the forma- tion of GSH after 10 min proceeded at a greatly reduced rate. The reduct ion of GSSG remained linear for at least 30 min in the presence of EDTA.

The lack of linearity in GSH generation when EDTA was omi t ted from the reaction mixture could be caused by trace metals catalysing the oxidation of GSH. Removal of the interfering metal ions by chelation could account for the relatively long period of zero order reaction kinetics observed when GR measurement was conducted in the presence of the chelating agent.

Effect of salts NADH-GR activity was maximally activated by 0.2 M sodium or potas-

sium chloride. NADH-GR activity was, however, strongly inhibited by both salts. About 100% inhibition of NADH-GR activity was observed at 0.2 M salt concentrat ion.

Effect of time The reduction of GSSG by NADH-GR and NADPH-GR of red blood cells

and plasma was found to be linear for a minimum period of 30 min under the given assay conditions.

Reproducibility of assay The reproducibil i ty of the proposed procedure was studied by performing

15 replicate determinat ions on the same plasma and haemolysate. Coefficients of variation for e ry th rocy te and plasma GR determinat ions were found to be within 5%.

Stability of NADH/NADPH-dependent GR activity No loss in NADH/NADPH dependent GR activity was observed when

whole blood, plasma or serum was stored at 0 to 5 ° for a week.

107

TABLE I

N A D H ' G R A N D N A D P H - G R L E V E L S IN C O R D B L O O D A N D IN H E A L T H Y B L O O D D O N O R S

Unit of e n z y m e ac t iv i ty is d e f i n e d as t h a t p r o d u c i n g 1 ~umol o f GSH per rain p e r l i t re e r y t h r o c y t e / p l a s m a /

serum at 37 ° .

Ery t h r o c y te P lasma

N A D H - G R N A D P H - G R N A D H - G R N A D P H - G R

Cord b l o o d No. of samples 85 85 85 85 Mean 5460 5750 72 72 Standard dev ia t ion 1270 1390 15 12 Range 2 9 2 0 - - 8 0 0 0 2 9 7 0 - - 8 5 3 0 4 2 - - 1 0 2 48- - 96

Heal thy b lood d o n o r s No. of samples 100 100 100 100 Mean 4 5 3 0 4 5 3 0 76 88 Standard dev ia t ion 750 750 12 13 Range 3 0 3 0 - - 6 0 3 0 3030-- -6030 5 2 - - 1 0 0 6 2 - - 1 1 4

GR levels in erythrocytes and plasma Table I shows GR levels in blood from 100 healthy adult blood donors

and in 85 cord blood samples. On the average, erythrocyte enzyme activities of cord blood were found to be higher than those of blood donors. There was, however, no significant difference in plasma NADH-GR and NADPH-GR levels in the two groups studied.

Table II shows the relationship between NADH-GR and NADPH-GR activ- ities in 100 healthy blood donors. Good correlation was observed between the two enzyme activities both in the erythrocytes and in the plasma. The ratio for NADPH/NADH GR activities was approximately 1 for erythrocyte GR and from 0.9 to 1.2 for plasma GR. These results do not compare with the value of 6 to 7 : 1 obtained by Icen [15] and about 8 to 10 : 1 as reported by Waller [16]. The relatively high ratio obtained by these two groups of workers may be explained by the subopt imum and limiting NADH concentrations used in their assays resulting in lower NADH-GR activities. Although the present obser-

TABLE II

C O R R E L A T I O N OF N A D H AND N A D P H - G R A C T I V I T I E S IN N O R M A L AND D I S E A S E S T A T E S

N A D H - G R N A D P H - G R Corre la t ion coef f i c ien t

Mean S.D. Mean S.D. (r)

Normal adu l t e r y t h r o c y t e s 100 4 5 3 0 Nornlal adu l t p l a sma 100 76 Liver cance r (p lasma) 87 224 Live r cirrhosis (p lasma) 56 100 Infective hepa t i t i s (p lasma) 34 148 Active chron ic hepa t i t i s (p lasma) 21 96 Nasophanyngeal c a n c e r (p lasma) 30 94 Megaloblastic anaem i a 8 108

750 4 5 3 0 750 0 .828 12 88 13 0 .799

108 206 78 0 .843 28 98 30 0 .702 55 149 60 0 .814 36 103 40 0 .8 7 8 26 101 27 0 .887 28 110 40 0 .8 0 0

108

vations do no t conclusively prove that one single enzyme is responsible for the two activities, the relatively constant ratio between the NADPH and NADH. linked activities suggests that these are given by one enzyme which is non- specific for NADPH or NADH. Depending on the availability of pyridine nucleotide and upon the ionic and pH changes, the enzyme functions as NADPH-GR or NADH-GR.

To provide further evidence for this point, plasma NADH-GR and NADPH-GR levels were determined for different disease states. Should there be two enzymes catalysing the reduction of GSSG, one would expect variation in the ratio of NADPH/NADH GR activities in different diseases. Results presen. ted in Table II show that there was good correlation for plasma NADH--GR and NADPH-GR in the various disease condit ions studied. Raised plasma GR values were obtained for liver cancer and infective hepatitis cases. Enzyme activities were normal or slightly raised in cases of liver cirrhosis, active chronic hepatitis, nasopharyngeal carcinoma and megaloblastic anaemia. Plasma GR values were observed to be normal in cases of pulmonary tuberculosis and nephritis and in patients with ischaemic heart condition.

It is significant to note that Scot t et al [11] and Icen [15] were unable to separate the two enzyme activities despite numerous fractionations. Blume, Riidiger and LShr [17] found that when ery throcyte GR was subjected to high voltage electrophoresis two isoenzyme bands were obtained, both of which gave NADH and NADPH activity. Our findings add further evidence to the hypothesis that the reduction of GSSG is catalysed by one enzyme which is non-specific for NADH and NADPH.

References

1 ICT. Lee and I.K. Tan, CI/n. Chim. Acta, 53 (1974) 153 2 D.W. Allen and J.H. Jandl, J. Clin. Invest., 40 (1961) 464 3 H.S. Jacob and J.H. Jandl, J. Clin. Invest., 41 (1962) 779 4 H.S. Jacob and J.H. Jandl, J. Clin. Invest., 41 (1962) 1514 5 P.E. Carson, G.J. Brewer and C.E. lckes, J. Lab. Clin, Med., 58 (1961) 804 6 H.D. Waller. W. Kaufman. W. Gerok and M. Eggstein. Klin. Wochenschr.. 42 (1964) 613 7 G.W. Lohr and H.D. Waller, Meal. Klin. (Munich) 36 (1962) 1521 8 H.D. Waller, Klin. Wochenschr.. 45 (1967) 827 9 A.M. Sect and K.T. Lee, Mikrochlm. Acta , in t h e press

10 A.M. Seet and H.C. Teoh, Clin. Chim. Acts, s u b m i t t e d 11 E.M. Scott , I.W. Duncan and V. Ekstrand, J. Biol. Chem., 238 (1963) 3928 12 C.E. Mize and R.G, Langdon0 J. Biol. Chem., 237 (1962) 1589 13 A.I. Vogel, Quanti tat ive Inorganic Analysis, Longman's , London, 1961. p. 511 14 K.T. Lee, Anal. Chim. Acta, 26 (1962) 285 15 A. Icen, Scand. J. Clin. Invest., 20 Suppl. (1967) 96 16 H.D. Waller in E. Beutler (Ed.), H e r e d i t a r y Di sorders o f Erythrocyte Metabolism, Grune and Stratton,

N e w York and London, 1968, p. 185 17 K.G. Blume0 H.W. R~idiger and G.W. I~h r , Biochim. Biophys. Acta, 151 (1968) 686