Chem.-Biol. Interactions, 23 (1978) 281--291

281

© Elsevier/North-Holland Scientific Publishers Ltd.

STUDIES ON THE REDUCTION OF N I T R O B L U E T E T R A Z O L I U M C H L O R I D E MEDIATED T H R O U G H THE ACTION O F NADH AND PHENAZINE METHOSULPHATE V. PONTI, M.U. DIANZANI, K. C H E E S E M A N *

and T.F. S L A T E R *

Institute of General Pathology, Corso .Raffaelto 30, Torino 10125 (Italy) and *Biochemistry Department, Brunel University, Uxbridge, Middx. (United Kingdom)

(Received February 4th, 1978) (Accepted June 3rd, 1978)

SUMMARY

The general features of the reduction of nitroblue tetrazolium chloride (NBT) by NADH and phenazine methosulphate (PMS) have been studied under aerobic and anaerobic conditions. Under aerobic conditions the reduction appears to be mediated through the intermediate formation of the superoxide anion radical O ( ' ; this reaction is strongly inhibited by superoxide dismutase and by a number of O(" scavengers such as propyl gallate, (+)-catechin, manganous ions, reduced glutathione and benzoquinone. Cupric ipns inhibited the overall reaction by reoxidising reduced PMS. Under anaerobic conditions, superoxide dismutase had only a small inhibitory action and, with the exception of cupric ions, the other substances mentioned above were ineffective as inhibitors. The data presented show that the use of NBT to detect the presence o f OC" is fraught with difficulties due to an equally rapid reduction of NBT b y NADH and PMS under anaerobic conditions.

INTRODUCTION It has been known for approx. 20 years that the reduced nicotinamide adenine dinucleotides (NADH, NADPH) do not directly reduce tetrazolium salts at an appreciable rate (see, e.g., Slater, 1959). Phenazine methosulphate (PMS) can, however, couple NAD(P)H with tetrazolium salts such that significant reduction rates are observed with the formation of highly coloured formazans. This non-enzymic coupling has been made use of in methods developed for the estimation of NAD(P)H in tissue extracts (e.g. see refs. 2, 5, 9, 11). PMS has been widely used to couple dehydrogenases to various acceptors; for example, the coupling of succinate oxidation via succinate dehydrogenase to ferricyanide reduction [ 7 ] . This facility of PMS Abbreviations: DMSO, dimethyl sulphoxide; GSH, glutathione; NBT, nitroblue tetrazolium chloride; PMS, phenazine methosulphate.

282 to couple dehydrogenases to various acceptors has been much used also in histochemical procedures (for refs. see ref. 6). In quantitative cytochemistry, PMS has been used to estimate the total available reducing flux from NAD(P)H in cytoplasm [1,3]. The interactions of NAD(P)H, PMS and tetrazolium salts are therefore of some general interest, and y e t a number of features of the overall interaction remain unclear. It was shown by Nishikimi et al [4] that reduction of PMS by NADH under aerobic conditions followed by reduction of oxygen to yield the superoxide anion radical; this was capable of reducing nitroblue tetrazolium chloride (NBT): NADH + t F + PMS -~NAD÷ + PMSH2 PMSH2 + 202 ~202-" + 2H + + PMS NBT 2 + 2C1- + 4 0 ( " + 4 I F > diformazan + 402 + 2HC1

(i) (ii) (iii)

The overall reaction should be inhibited therefore not only by oxidants of NADH and PMSH2, but by scavengers of 02-. We have studied the characteristics of this reaction and have also compared the effectiveness of various free radical scavengers on the system. METHODS

The standard conditions for the reaction were as follows: Tris--HC1 buffer, pH 8.0, final concentration 0.016 M; NADH (1.13 mM) in Tris buffer (pH 8.0, 0.1 M) to give a final concentration of NADH of 73 t~M; PMS, in aqueous solution, final concentration 5.2 pM; NBT in aqueous solution, final concentration 80 #M; the total vol. used in assay was 3.1 ml, and the reaction was studied at ambient temperature which was between 15--20°C. NADH solutions were prepared freshly and kept in ice; phenazine methosulphate and nitroblue tetrazolium were kept cold in vessels shielded from the light. Anaerobic reactions were studied using an anaerobic 'Thunberg-type' spectrophotometer cell with PMS in the side-arm. Before adding the solutions to the anaerobic cell, the solutions were thoroughly purged with pure nitrogen in an anaerobic box containing N2 : H2 (90 : 10) and platinum catalyst to remove residual oxygen. The reaction was started by mixing PMS with the other components and the increase in absorption at 560 nm was measured using either an SP-1800 (Pye-Unicam) or a Beckmann DB recording spectrophotometer; the initial rate of reduction of NBT was derived from the recorder traces so obtained. All materials used were of the highest purity available; the metal salts were of AnalaR quality. NBT was recrystallised from water before use and was obtained from Sigma Chemical Co. Ltd. The sources of other chemicals used were as follows ( + ) - c a t e c h i n (Zyma, S.A., Nyon, Switzerland); diphenylfuran (Eastman Kodak, Rochester, N.Y.) ~-carotene {Sigma); dimethyl sulphoxide {Merck); superoxide dismutase ('Ontosein', Fisons Ltd.,

283 Loughborough, U.K.). Inosine, reduced glutathione, and NADH were obtained from Boehringer (U.K.) Ltd., mannitol, 3-aminotriazole, imidazole, propyl gallate, and PMS were obtained from Sigma (U.K.) Ltd. Vitamin E succinate polyethylene glycol 1000 was obtained from Distillation Products Industries, Rochester, N.Y. Some rapid reactions described in the Results section were performed using a Nortech model SF-3A stopped-flow spectrometer coupled to an Advance Electronics 2000 storage oscilloscope. The effective mixing time of the solutions used in this technique was 3 ms. RESULTS

Dependence on PMS Fig. 1 shows that the initial rate of reaction increases with PMS concentration over the range 0--10 ~M; when the reaction is assessed after 4 min in terms of absolute extinction change then the increase with low PMS concentrations is more pronounced. Dependence on N B T Fig. 2 gives data for the effects of varying NBT concentration on the initial rate of reaction, and on the reaction as assessed at 4 min after starting. It can be seen that a half maximal rate of reaction occurred with a concentration of NBT of 80 aM. Dependence on N A D H The dependence of the reaction on the NADH concentration is shown in Fig. 3. It can be seen that a half maximal rate of reaction was obtained with NADH present at 450 uM. 0e-

Q

Q O.6-

*S-

c

0.4-

O2-

la

~o NBT ~M)

Fig. 1. E f f e c t o f varying t h e p h e n a z i n e m e t h o s u l p h a t e c o n c e n t r a t i o n o n N B T r e d u c t i o n u n d e r aerobic c o n d i t i o n s . S t a n d a r d c o n d i t i o n s as d e s c r i b e d in t h e M e t h o d s S e c t i o n were used. (--o--) initial rate o f r e d u c t i o n c a l c u l a t e d f r o m t a n g e n t t o r e d u c t i o n curve over the first m i n ; (--~--) r e d u c t i o n assessed by c h a n g e in e x t i n c t i o n at 5 6 0 n m over first 4 rain o f reaction. Fig. 2. E f f e c t o f varying t h e N B T c o n c e n t r a t i o n o n t h e N A D H - P M S - N B T r e a c t i o n u n d e r aerobic c o n d i t i o n s . S t a n d a r d c o n d i t i o n s as d e s c r i b e d in the Methods Section were used. The d a t a s h o w n were c a l c u l a t e d f r o m t h e initial r a t e of a p p e a r a n c e o f blue c o l o u r ( f o r m a z a n ) as m e a s u r e d at 560 nm.

284

E

Solvent

ol5

I

I 15

I 0

TI~ 20

4D

NADH(mM)

Fig. 3. E f f e c t o f varying the N A D H c o n c e n t r a t i o n o n the initial rate o f r e d u c t i o n o f N B T as measured by the increased e x t i n c t i o n at 5 6 0 rim. S t a n d a r d c o n d i t i o n s as described in the M e t h o d s S e c t i o n were used. Fig. 4. E f f e c t s o f the solvents ethanol (o), a c e t o n e (A) and d i m e t h y l s u l p h o x i d e ( o ) o n the standard reaction c o n d i t i o n s for the N A D H - P M S - N B T reaction under aerobic c o n d i t i o n s . The results o b t a i n e d are s h o w n as percentage deviations from the c o n t r o l reaction d o n e in the absence o f solvent and measured by the initial rate o f reaction.

Effects of solven ts Some of the free radical scavengers used in this study, e.g./3-carotene are poorly soluble in aqueous solution and relatively high concentrations of appropriate organic solvents must be used in these studies. Three such solvents have been used (ethanol, acetone and dimethyl sulphoxide) and the results obtained concerning their effects on NBT reduction are shown in Fig. 4. It can be seen that DMSO and acetone inhibit the reaction by approximately 50% at final concentrations of 4 M; ethanol is less inhibitory. The variable effects seen with lower concentrations of the solvents may be due to solubility effects on the formazan product; when ethanol was studied for its effects on NBT reduction using NBT solutions after various times of storage, '40 ( ~

i Eth~ol iMI

'~

?0

S~0c0n d s '0

50

~

Second s

Fig. 5. Effect of ethanol on the N A D H - P M S - N B T reaction under standard conditions but with the N B T solution being stored in the dark at room temperature for 7 days (o), 14 days (A) compared to fresh N B T solution (e). Fig. 6. Fig. 6a s h o w s the decrease in e x t i n c t i o n at 3 4 0 n m resulting from the rapid m i x i n g o f N A D H ( 1 0 0 uM) in tris buffer pH 8.0 with PMS ( 1 0 0 ~M) at r o o m t e m p e r a t u r e under aerobic c o n d i t i o n s . Fig. 6 b gives c o r r e s p o n d i n g data for the anaerobic r e a c t i o n ; measurem e n t m a d e at 3 8 5 nm.

285 the results shown in Fig. 5 were obtained. The changing response seen with 'old' solutions of NBT is probably due to ethanol solubilising photochemically produced formazan.

Interaction of N A D H and PMS As part of the study of the overall reaction, the rate of reaction of NADH with PMS was studied by following the decrease in absorption at 340 nm. The reaction was also done under anaerobic conditions and the results so obtained are shown in Fig 6. It can be seen that NADH reduces PMS relatively rapidly at room temperature; the tlh for the reaction was 8.7 s under aerobic conditions and 8 s anaerobically.

Reaction o f reduced PMS with 02 PMS was reduced by an equivalent a m o u n t of NADH under anaerobic conditions and the reduced PMS was then rapidly mixed with oxygen containing buffer (mixing time 3 ms). The appearance of oxidised PMS was followed at 400 nm using a stopped flow technique. The results are given in Fig. 7 from where it can be seen that tv, for the reaction P M S r e d +202

"

20~" + PMSox

is approximately 7 s.

Effects o f metal ions Since the overall reaction leading to NBT reduction involves redox reactions it was thought of interest to test the effects of various metal salts on the system. The following were tested: ferrous sulphate, zinc chloride,

©

2~T

,0

20

a0 S~¢o.ds

40

5.

~

0,

;

~'0

,~

,&o

LoU [ e o . ~ . t , a , b o . . m l

Fig. 7. Reaction of reduced PMS with oxygen using stopped-flow spectrophotometry. PMS (100 uM) was reduced with NADH (100 uM) in Tris buffer, pH 8.0 under strict anaerobic conditions. The resultant solution was then rapidly mixed with air saturated Tris buffer pH 8.0 (oxygen concentration approx. 250 ~M) and the absorption change at 400 nm recorded. Fig. 8. Effects of copper (*) and cobalt ions (o) on the NADH-PMS-NBT reaction under aerobic conditions. Standard conditions of reaction were used (see Methods Section). The results obtained are expressed as percentage inhibition produced by the metal ion on the initial rates of reaction relative to the control situation in the absence of added metal ion.

286

Log Icon~.1~Jtlo., .ml

Fig. 9. Effects of manganese (.) and molybdenum (o) ions on the NADH-PMS-NBT reaction under aerobic conditions ; other details as in the legend for Fig. 8. Fig. 10. Effects of ~-carotene (-) and diphenyl furan (o) on the NADH-PMS-NBT reaction under aerobic conditions. Both materials were added in DMSO solution and the results are shown as percentage changes in the initial rates of reaction relative to the rate produced in the presence of DMSO. lead acetate, m o l y b d e n u m p e n t a c h l o r i d e , m a n g a n o u s chloride, c o b a l t o u s chloride, cupric sulphate. No significant e f f e c t was p r o d u c e d b y lead, ferrous or zinc salts over t h e c o n c e n t r a t i o n range t e s t e d ( 0 . 1 - - 1 0 0 taM). T h e results f o r C o 2+ and C u 2÷ (Fig. 8) Mn =+ and Mo s÷ (Fig. 9) s h o w some p o w e r f u l inhibitions o n the overall r e a c t i o n measured. C o p p e r ions p r o d u c e d a 50% i n h i b i t i o n at 2 taM; c o b a l t o u s ions at a p p r o x i m a t e l y 35 taM. Molybd e n u m ions gave a 50% inhibition at 100 taM whereas m a n g a n o u s ions were the m o s t e f f e c t i v e o f the m e t a l ions tested, p r o d u c i n g a 50% inhibition at a p p r o x i m a t e l y 0.1 taM final c o n c e n t r a t i o n . It is e v i d e n t f r o m these results t h a t v e r y low c o n c e n t r a t i o n s o f Cu 2+ or Mn 2÷ are effective inhibitors o f NBT r e d u c t i o n u n d e r aerobic c o n d i t i o n s . U n d e r s t a n d a r d c o n d i t i o n s used, E D T A (5, 10 or 50 taM) h a d n o significant e f f e c t on the r e a c t i o n .

Effects of free radical scavengers T h e effects o f / J - c a r o t e n e and o f d i p h e n y l furan, b o t h in 4.5 M d i m e t h y l s u l p h o x i d e (DMSO) are s h o w n in Fig. 10. T h e solvent itself inhibits the r e a c t i o n a p p r o x i m a t e l y 50%; d i p h e n y l f u r a n in DMSO h a d no significant e f f e c t c o m p a r e d t o DMSO itself a l t h o u g h at high c o n c e n t r a t i o n ( 5 0 - - 1 0 0 pM) t h e r e was an i n d i c a t i o n o f a small increase in the rate o f reaction. ~-carotene in DMSO increased the i n h i b i t i o n seen with DMSO alone as the c o n c e n t r a t i o n of/3-carotene was increased o v e r 25 taM. T h e results o b t a i n e d with t h r e e well k n o w n inhibitors o f lipid p e r o x i d a t i o n ( P r o m e t h a z i n e , p r o p y l gallate, and ( + ) - c a t e c h i n ) are s h o w n in Fig. 11. P r o p y l gallate p r o d u c e d a 50% i n h i b i t i o n o f N B T r e d u c t i o n at a final c o n c e n t r a t i o n o f a p p r o x i m a t e l y 5 taM; ( + ) - - c a t e c h i n was similarly effective at a b o u t 80 taM; P r o m e t h a z i n e was n o t i n h i b i t o r y and, indeed, p r o d u c e d a small increase in r e a c t i o n r a t e w h e n p r e s e n t in c o n c e n t r a t i o n s greater t h a n 100 taM. A similar increase in N B T r e a c t i o n rate was f o u n d with vitamin E

287

Fig. 11. Effects of the free radical scavengers propyl gallate (o), promethazine (o) and (+)--catechin (a) on the NADH-PMS-NBT reaction under aerobic conditions. The results are shown as percentage changes in the initial rates of reaction relative to the control situation in the absence of added free radical scavenger.

polyethylene glycol succinate except at very high concentration where the stimulation seen with lower concentrations 'crossed-over' to an inhibition. A similar cross-over effect with vitamin E has been reported in relation to the microsomal stimulation of lipid peroxidation [9]. Strong inhibitory effects on NBT reduction were found with reduced glutathione (GSH), benzoquinone and hydroquinone. These results are summarised in Table I. Weak effects, or no significant effects on NBT reduction were observed with the following substances over a range of tested concentrations (the maximum concentration used is shown in parenthesis): indomethacin (100 ~M), mannitol (0.6 M), iso-propanol (3 M), aniline (5 mM) imidazole (4.9 mM), 3-amino-l,2,4-triazole (5 mM), p-chloromercuribenzoate (90 pM), inosine (240 uM), sodium salicylate (200 pM), oxidised glutathione (500 uM).

TABLE I EF F EC TS OF REDUCED GLUTATHIONE (GSH), BENZOQUINONE AND HYDROQUINONE ON THE NADH/PMS/NBT REACTION UNDER AEROBIC CONDITIONS For experimental details see the text. Agent

Final concentration (~M)

% Inhibition

GSH

27 272

30 100

Benzoquinone

1.2 5.9 59

12 31 100

Hydroquinone

1.5 146

55 100

288

10 Superoxldedism~la~ L ~

10 (ml umlsl]

Fig. 12. E f f e c t s o f s u p e r o x i d e d i s m u t a s e o n t h e initial rate of t h e N A D H - P M S - N B T r e a c t i o n u n d e r a e r o b i c c o n d i t i o n s . T h e results are s h o w n as p e r c e n t a g e c h a n g e s relative t o the c o n t r o l s i t u a t i o n .

Superoxide dismutase The effects of superoxide dismutase on the rate of reduction of NBT under aerobic conditions are shown in Fig 12. It can be seen that a 50% inhibition of the reaction was obtained with approximately 1 int. unit superoxide dismutase. Maximum inhibition of about 90% was obtained with 30 int. units.

Anaerobic reduction o f N B T It was observed that NBT is reduced anaerobically by NADH and PMS to the blue formazan at about the same rate as aerobically. The ratio obtained for the anaerobic rate to the aerobic rate (mean of 4 experiments) was 135%. Anaerobic reduction was not greatly affected by superoxide dismutase added in excess (Table II) indicating that a different mechanism for reduction operates anaerobically compared to aerobically. The substances found to strongly inhibit aerobic reduction of NBT (propyl gallate, catechin, imidazole, cupric ions, and manganous ions) were tested for their effects on the anaerobic reduction of NBT. Only cupric ions had any significant effect suggesting that the other substances were effective T A B L E II R E D U C T I O N O F N B T B Y N A D H A N D PMS U N D E R A E R O B I C O R A N A E R O B I C CONDITIONS, WITH OR WITHOUT THE ADDITION OF SUPEROXIDE DISMUTASE ( 1 0 0 INT. U N I T S ) . F o r o t h e r details see the text. Reaction conditions

Aerobic Aerobic Anaerobic Anaerobic

Superoxide dismutase

+ +

Initial rate of reaction (E56Jmin) 0.42 0.00 0.56 0.41

289 aerobically through an interaction with O~'. The inhibitory action of copper ions appears due to a rapid reoxidation of reduced PMS.

Reaction of N A D H (+_PMS) with strong inhibitors To eliminate the possibility that the strong inhibitors of NBT reduction were acting by chemical oxidation of NADH directly, or were interfering with the reduction of PMS b y NADH, the effects of those inhibitors on these two processes were studied. None caused any significant change in the extinction at 340 nm of NADH when tested in concentrations up to 100 uM. With the exceptions of acetone and dimethyl sulphoxide, none affected the decrease in E340 consequent upon mixing NADH with PMS; acetone and DMSO both inhibited the rate of decrease in E340 by approximately 50% at final concentrations of 4 M.

DISCUSSION As a first approximation, a probable sequence of reactions in the aerobic reduction of NBT is as shown below: NADH + I-I+ + PMS PMSH2 + 202 402-" + NBT + 4H ÷ 2PMSH2 + MBT

kl k2 k3 k4

~ ~ , ,

NAD+ + PMSH2 PMS + 2H ÷ + 202-" diformazan + 402 + 2HC1 diformazan + 2PMS

I II III IV

The strong inhibition produced by superoxide dismutase (Fig. 10) is consistent with the superoxide anion radical being an essential intermediate in the aerobic NBT reduction. In this respect, our data are consistent with those of Nishikimi et al (1972). Other strong inhibitors of the aerobic reduction of NBT were propyl gallate, (+)-catechin, reduced glutathione, benzoquinone, hydroquinone, cupric ions and manganous ions. Of these substances, cupric ions interfered also with the anaerobic reaction (see Results) and probably was effective by reoxidising reduced PMS. The other substances did n o t directly oxidise NADH, or affect the NADH/PMS reaction, or affect the anaerobic reduction of NBT. It seems probable therefore that t h e y are acting as effective scavengers of O2-'. It is interesting to note that neither mannitol nor iso-propanol, which are often used as OH" scavengers due to the high rate constants for their reaction with OH" (iso-propanol, 2.1 × 10 9 M-I/s -I, [12] mannitol, 2 × 10 9 M-I/s -I, R.L. Willson and A.F. Searle, pers. comm.) had any significant effect on aerobic N B T reduction. Moreover, Promethazine and vitamin E which are very effective in scavenging electrophilic radicals such as CCI~ or OH" [8] had no inhibitory action on aerobic N B T reduction. This indicates a degree of selectivity in free radical trapping that is not always appreciated. Promethazine, for example, effectively inhibits carbon tetrachloride stimulated lipid peroxidation in rat liver microsomes at less than 0.1 ~ M [9] : it has no

290 inhibitory effect on the aerobic reduction of NBT at 100 uM. Its reactivity with 02" compared to CC13" must therefore be different by several orders of magnitude. Propyl gallate, on the other hand, is an equally good scavenger of O2-" (Fig. 10) and CC13". The anaerobic results were not expected. The data reported show that NBT is reduced anaerobically at about the same rate as aerobically. Moreover, the anaerobic reaction is not greatly inhibited by superoxide dismutase. These results suggest that under anaerobic conditions the following reaction occurs readily: PMSH2 + NBT

k4

= Formazan + PMS

and t h a t oxygen strongly interferes with this reaction under aerobic conditions. The anaerobic results reported here show that NBT reduction by itself cannot be used as a demonstration of 02-" involvement from an NADH/PMS/ 02 interaction. Clearly, NBT reduction can occur under anaerobic conditions where O2-" is n o t an essential intermediate. This suggests that caution has to be exercised, for example, in interpreting the mechanism of NBT reduction patterns in tissue sections or cell suspensions where the conditions of the reactions may lead to heterogeneous oxygen tensions. A similar conclusion has been reached by Woolf and Gregory [13] in a study on NBT reduction by human renal tissue. ACKNOWLEDGEMENT

We are grateful to the Cancer Research Campaign for financial assistance to one of us (K.C.). The help provided by Mr. Detlef Bahnemann in the stopped flow experiments is warmly acknowledged. REFERENCES 1 F.P. Altman, A comparison of dehydrogenase activities in tissue homogenates and tissue sections, Biochem. J., 114 (1969) 13. 2 C. Bernofsky and M. Swan, An improved cycling assay for nicotinamide adenine dinucleotide, Anal. Biochem., 53 (1973) 452. 3 J. Chayen, L. Bitenksky, R.G. Butcher and F.P. Altman, Cellular biochemical assessment of steroid activity, Adv. Steroid Biochem. Pharmacol., 4 (1974) 1. 4 M. Nishikimi, N.A. Rao and K. Yagi, The occurrence of superoxide anion in the reaction of reduced phenozine methosulphate and molecular oxygen, Biochem. Biophys, Res. Commun., 46 (1972) 849. 5 J.S. Nisselbaum and S. Green, A simple ultramicro method for determination of pyridine nucleotides in tissues, Anal. Biochem., 27 (1969) 212. 6 A.G.E. Pearse, in Histochemistry: Theoretical and Applied 3rd Edn. Vol. 2., Churchill Livingstone, London 1972. 7 T.P. Singer and E.B. Kearney, Chemistry, metabolism and scope of action of the pyridine nucleotide coenzymes, Adv. Enzymol., 15 (1954) 79. 8 T.F. Slater, in Free Radical Mechanisms in Tissue Injury, Pion Ltd., London, 1972. 9 T.F. Slater and B.C. Sawyer., The stimulatory effects of Carbon tetrachloride on peroxidative reactions in rat liver fractions in vitro, Biochem. J., 123 (1971 ) 823.

291 10

T.F. Slater, B. Sawyer and U.D. Straiili, An assay procedure for nicotinamideadenine dinucleotides in rat liver and other tissues, Arch. Int. Physiol. Biochem., 72 (1964) 427. 11 P.J.C. Smith, Assay of pyridine nucleotides, coenzymes and associated dehydrogenases with phenozine methosulphate, Nature, 190 ( 1961 ) 84. 12 R.L. Willson, C.L. Greenstock, G.E. Adams, R. Wageman and L.M. Dorfman., The standardisation of hydroxyl radical rate data from radiation chemistry, Int. J. Radiat. Phys. Chem., 3 (1971) 211. 13 J.H. Woolf and E.M. Gregory., Superoxide anion independent reduction of nitroblue tetrazolium by human renal tissue, Clin. Biochem., 9 (1976) 241.

Studies on the reduction of nitroblue tetrazolium chloride mediated through the action of NADH and phenazine methosulphate.

Chem.-Biol. Interactions, 23 (1978) 281--291 281 © Elsevier/North-Holland Scientific Publishers Ltd. STUDIES ON THE REDUCTION OF N I T R O B L U E...
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