INFECTION AND IMMUNrrY, Sept. 1974, p. 528-534Copyright 0 1974 American Society for Microbiology

Vol. 10, No. 3Printed in U.S.A.

Reduced Nicotinamide Adenine Dinucleotide and ReducedNicotinamide Adenine Dinucleotide Phosphate Diaphorase

Activity in Human Polymorphonuclear LeukocytesLAWRENCE R. DECHATELET, LINDA C. McPHAIL, DEBRA MULLIKIN, AND CHARLES E. McCALL

Departments of Biochemistry and Medicine, The Bowman Gray School of Medicine, Winston-Salem,North Carolina 27103

Received for publication 17 January 1974

Total reduced nicotinamide adenine dinucleotide (NADH) and reducednicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activitieswere examined in human neutrophils. Approximately two-thirds of each enzymeactivity was located in the granule fraction with the remainder in the soluble.The activities in a 27,000 x g supernatant from a sonic extract of humanpolymorphonuclear leukocytes were characterized. Both NADH and NADPHdiaphorase were insensitive to cyanide and azide and showed greater activity atacid pH. Km values for nitroblue tetrazolium were not markedly different (33 OMwith NADH and 12 AM with NADPH), but there was a 40-fold difference in Kmfor the reduced pyridine nucleotides (10 ,M with NADH and 400 ,uM forNADPH). Since the intracellular concentration of both nucleotides is estimatedto be about 50 MM, it is much more likely, from a kinetic argument, that therespiratory burst of phagocytosis is intiated by the oxidation of NADH ratherthan of NADPH.

The term "diaphorase," as used here, is adescriptive one which is applied to any enzymethat can transfer electrons from reduced pyri-dine nucleotides to an artificial (i.e., nonphysio-logical) acceptor molecule. Thus, it is evidentthat a "reduced nicotinamide adenine dinucleo-tide (NADH) diaphorase activity" does notdescribe a single unique enzyme activity; infact, there may be a number of different en-zymes within a single cell (including pyridinenucleotide oxidases) which exhibit diaphoraseactivity. Furthermore, since the activity is de-fined in terms of an artificial acceptor molecule,the true function of the enzyme(s) within thecell is not known.Baehner and Nathan (8) devised a test re-

volving around the ability of a cell to reducenitroblue tetrazolium (NBT) dye as a measureof the metabolic competency of the cell. Normalcells reduce a certain amount of the dye underresting conditions, and the degree of reductionincreases markedly during phagocytosis. Meta-bolically deficient cells, as in chronic granuloma-tous disease (CGD) or leukocyte glucose-6-phos-phate dehydrogenase deficiency, fail to reducethe dye under either resting or phagocytizingconditions (7, 11), and this phenomenon hasbeen used successfully as a screening test forCGD. The inability of the cells to reduce NBTdye does not appear to be due to a defect in

either ingestion or degranulation (6). Further-more, Baehner and Nathan have reported thatthe diaphorase activity of broken cell prepara-tions from patients with CGD is essentiallynormal, when NADH or reduced nicotinamideadenine dinucleotide phosphate (NADPH) isemployed as a cofactor (21). Similar resultshave been observed in severe glucose-6-phos-phate dehydrogenase deficiency (11). Thus, agenetic defect resulting in a totally inactivediaphorase seems relatively unlikely. The dem-onstration that normal diaphorase activity waspresent in the defective cells was made underoptimal conditions of enzyme activity. It seemsmost likely that a subtle change in the enzymemight have occurred, resulting in an inability ofthe enzyme to express its activity under condi-tions of substrate concentration, etc., whichexist within the cell.

This study describes some of the properties ofthe normal diaphorase enzymes found in humanpolymorphonuclear leukocytes (PMNL). This isnecessary so that future work may compare theproperties of enzymes from normal and defi-cient neutrophils.

MATERIALS AND METHODSPreparation of leukocyte extracts. Leukocytes

were isolated from the blood of apparently healthyvolunteer subjects by a method described previously

528

on February 21, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

DIAPHORASE ACTIVITY IN PMNL

(12). Contaminating erythrocytes were removed byhypotonic lysis, and the leukocyte pellet was washedtwo times with Dulbecco phosphate-buffered saline,containing both magnesium and calcium ions, and ad-justed to a final concentration of 5 x 107 phagocytesper ml by the addition of phosphate-buffered saline.Phagocytes are defined here as segmented neutro-phils, band neutrophils, eosinophils, and monocytes.Lymphocytes accounted for less than 10% of theisolated cell suspension. Lymphocytes were separatedfrom normal human blood in 95% purity by centrifu-gation in a Ficoll-Hypaque gradient (10). The cellsuspensions were disrupted by sonic treatment for 45s in three 15-s intervals with cooling in between. ABranson sonifier with a power output of 20 W was

used for disruption. Cellular debris was removed bycentrifugation at 27,000 x g for 15 min; the clearsupernatant solution was assayed immediately be-cause of loss of activity during storage. Attempts torelease latent activity with 0.1% Triton X-100 were

unsuccessful due to enzyme inhibition by the deter-gent.

Determination of diaphorase activities. Leuko-cyte diaphorase activity was determined by a modifi-cation of the assay of Nishikimi et al. (22) forsuperoxide dismutase activity. The following mate-rials were incubated in a total volume of 3.0 ml in a

glass cuvette: 50 umol of sodium phosphate (pH 7.0),0.15 Mmol of NBT dye, and 0.23 ,mol of NADH or 5.401Imol of NADPH. Reaction was initiated by theaddition of leukocyte sonic extract (usually 0.10 mlcontaining 0.2 to 0.5 mg of protein), and the increasein absorbancy at 560 nm was followed for at least 5min on a recording spectrophotometer. Initial rates ofNBT reduction were determined from the slopes ofthe lines.

Lysozyme was assayed with a recording spectro-photometer by following the decrease in absorbancyat 450 nm due to lysis of a suspension of MicrococcusIysodeikticus in 0.10 M phosphate buffer, pH 6.2 (19).Beta-glucuronidase activity was assayed by measur-

ing the liberation of free phenolphthalein from phe-nolphthalein glucuronide at pH 5.0 (27). Proteinconcentrations of the leukocytic extracts were deter-mined by the biuret procedure of Gornall et al. (13),using bovine serum albumin as a standard.

RESULTS

The specific activities of both NADH andNADPH diaphorase in sonic extracts of humanleukocytes are shown in Table 1. The activity ofsonic extracts from PMNL with NADH as sub-strate was generally slightly greater than thatobtained with NADPH as substrate, althoughthere was considerable variation from sample tosample. In two experiments, the diaphoraseactivities of sonic extracts from lymphocyteswere of the same order of magnitude as thosefrom the PMNL. Since the purity of the PMNLsuspensions was always 90% or greater, thecontribution of lymphocytic enzyme to the totalactivity measured was less than 10%.

Although the data in Table 1 were derivedfrom whole sonic extracts, the specific activi-ties of both diaphorase enzymes were compar-able in the whole sonic extract or in a 27,000 xg supernatant of the whole sonic extract (datanot shown). Attempts to release additional en-zyme into the soluble fraction by the use ofdetergents (including Triton X-100, sodiumdodecyl sulfate, and deoxycholate) were unsuc-cessful due to inhibition of the enzyme by thecompounds in question. Similarly, sonic treat-ment in alkaline isotonic KCl (20) did not re-lease any more activity than did sonic treatmentin phosphate-buffered saline. Because of tur-bidity problems encountered when the wholesonic extract was employed, subsequent assayswere performed on the 27,000 x g superna-tant of the sonic extract.To localize the diaphorase activities within

the cell, a modified procedure was employed.Because a relatively large number of cells wasrequired, a patient with polycythemia vera wasused as the donor; however, we have not ob-served any reproducible differences (eitherqualitative or quantitative) between diaphoraseactivities of normal or polycythemia vera cellsto date. Cells were isolated from 150 ml of bloodwithout being subjected to hypotonic lysis. Thepacked cells (0.60 ml) were suspended in 0.34 Msucrose to a final concentration of 1.5 x 108 cellsper ml. The cells were disrupted by homogeni-zation in a Potter-Elvejhem-type homogenizeruntil approximately 90% disruption wasachieved, as estimated by phase microscopy.Intact cells and cellular debris were removed bycentrifugation at 400 x g for 10 min. Thesupernatant was centrifuged at 12,000 x g tosediment the lysosomes. The lysosomal pelletwas suspended in 8.0 ml of 0.34 M sucrose andsubjected to five cycles of freezing and thawingin liquid nitrogen. The 12,000 x g supernatantwas centrifuged at 100,000 x g for 30 min, andenzyme assays were performed on the resus-pended 12,000 x g pellet (lysosomal fraction)and 100,000 x g supernatant (soluble fraction).

TABLE 1. Specific activity of human leukocytediaphorases

Sonic No. A OD per min per mg'extracta assayed NADH NADPH

PMNL 7 0.0183 + 0.004 0.0142 + 0.006Lymphocyte 2 0.0154 0.0134

a Values for PMNL represent the mean standarddeviation.

b Determined in triplicate at three different proteinconcentrations and averaged.

VOL. 10, 1974 529

on February 21, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

DECHATELET ET AL.

Both diaphorase activities were found pri-marily in the granule fraction, although therewas significant activity in the soluble fraction as

well (Table 2). Approximately two-thirds ofeach diaphorase activity was found to be associ-ated with the lysosomal fraction under theconditions employed.Because of the possibility that some of the

soluble diaphorase activity might be due toerythrocyte contamination, the experiment wasrepeated with the following modifications. Theisolated cells were subjected to hypotonic lysisto remove erythrocyte contamination prior tohomogenization, and the lysosomal pellet waswashed twice with phosphate-buffered salineprior to freeze-thawing to minimize the adsorp-tion of soluble diaphorase activity to the lyso-somes. The results exactly paralleled thoseillustrated in Table 2. The specific activity oflysozyme in the lysosomal fraction was 40 timesthat in the soluble fraction, whereas the specificactivity of beta-glucuronidase was eight timeshigher in the lysosomal fraction. The diapho-rase activities were observed in both the lysoso-mal and soluble fractions; the ratio of thespecific activities of both NADH and NADPHdiaphorase was approximately 3:1 (data notshown). These procedures do not differentiatebetween an enzyme that is localized in thegranule membrane rather than in the contentsof the granule. It is possible that the diaphoraseactivity, or a part of the activity, is actuallymembrane bound.The effect of reduced pyridine nucleotide

concentration on diaphorase activity is illus-trated in Fig. 1. Km values estimated from thegraph (as the substrate concentration requiredfor half-maximal velocity) are approximately0.40 mM for NADPH and 0.01 mM for NADH.Although there was a 40-fold difference in Kmvalue, the maximal velocities were roughlyequal (0.016 A optical density (OD) per min permg for NADPH and 0.010 A OD per min per mgfor NADH). Because of the very high affinity ofthe enzyme for NADH, it was difficult todetermine initial velocities at nonsaturatingconcentrations of this nucleotide. At concentra-

tions below about 20 uM, the absolute amountof nucleotide became limiting in the first min-utes of reaction. The assay was complicatedfurther by a relative lack of sensitivity due tothe small extinction coefficient of reducedNBT. This resulted in a lack of precision whensmall amounts of reduced NBT were measured.For these reasons, the data could not be ex-pressed as the usual double reciprocal plot. Itshould also be evident that the experimentalKm for NADH represented a maximal value; theactual Km may be considerably lower than .01mM, but this could not be demonstrated dueto limitations in the assay procedure.The effect of NBT concentration on the

various diaphorase activities is illustrated in

018r

016

014

012

010

008

006

, 004

002\S

r~~~~~~~

A

I0 20 30 40 50 60 70 80

NADPH (mM)

012

0I0/

008-

006 rI-

004,

002

20 4C

B

t0 80 100 120 140 160

NADH (SuM)

FIG. 1. Effect of reduced pyridine nucleotide con-

centration on initial rate of diaphorase reaction.Concentration of NBT was 50 ,.M in all cases. A,effect of NADPH concentration; B, effect of NADHconcentration.

TABLE 2. Localization of leukocyte diaphorase activitya

A OD per min per mg of protein j-Glucuronidase(AMmol of

Fraction NADH NADPH phenolphthalein

diaphorase diaphorase Lysozyme per h per mgof protein)

Lysosomal (12,000 x g pellet) 0.0136 0.0181 0.2009 0.091Soluble (100,000 x g supernatant) 0.0067 0.0060 0.0048 0.010

a Each value represents the mean of triplicate determinations run at different levels of protein concentration.

530 INFECT. IMMUNITY

on February 21, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

DIAPHORASE ACTIVITY IN PMNL

Fig. 2. Optimal concentrations of pyridine nu-cleotides were employed in this experiment (i.e,78 MM NADH and 1.80 mM NADPH). Incontrast to the preceding experiment, thesedata lend themselves quite nicely to the Line-weaver-Burke plot. The Km for NBT withNADH as substrate was estimated to be ap-proximately 33 gM, whereas the Km withNADPH was somewhat less, approximately 12MM. The maximal velocities obtained in thisexperiment are quite similar, 0.026 A OD permin per mg for the NADPH diaphorase and0.021 A OD per min per mg for the NADHenzyme.Because of these results, the standard condi-

tions chosen for assay of diaphorase activityutilized 50,MM NBT, 78 MM NADH, and 1.80mM NADPH, as outlined above.

FIG. 2. Double reciprocal plot of initial velocityversus NBT concentration. Velocity (V) is in A ODper min per mg; NBT concentration is in uM. Sym-bols: 0, 78 uM with NADH as substrate; 0, 1.80mM with NADPH as substrate.

03

02/A

C .05

ci 04

03 B

.02-

.0I

10 20Protein (mg)

FIG. 3. Effect of protein concentration on initialvelocity of diaphorase reaction. A, NADPH concen-tration of 1.8 mM; B, NADH concentration of 78 uM.The concentration of NBT was 50 MM in all cases.

The validity of this procedure for assay of thediaphorase activities is demonstrated in Fig. 3.Using these conditions, the change in absorp-tion per minute was proportional to the proteinconcentration up to at least 2.0 mg of leukocyteprotein. Similarly, at relatively low concentra-tions of protein (0.50 mg), the reaction waslinear with respect to incubation time for atleast 10 min (data not shown).The pH profile of the two activities is illus-

trated in Fig. 4. Both the NADH- and theNADPH-dependent reactions showed increasedactivity at acidic pH, although the change wasconsiderably greater and more abrupt in thecase of the NADH-dependent diaphorase.Both enzyme activities were completely in-

sensitive to either cyanide or azide as illustratedin Table 3. The small degree of apparentstimulation observed in some cases was notconsidered significant.

DISCUSSIONThe importance of diaphorase activities in

the human PMNL is deduced from the factthat physiologically incompetent cells (e.g.,from patients with CGD or leukocyte glu-cose-6-phosphate dehydrogenase deficiency) donot exhibit diaphorase activity during phagocy-tosis (7, 11, 14), and this is associated with animpaired ability to destroy certain types ofbacterial pathogens (11, 17). Other metabolicchanges, including increases in oxygen con-sumption, hexose monophosphate shunt ac-tivity, and hydrogen peroxide production, arelikewise lacking during phagocytosis by thesecells, although ingestion appears to be normal

02 F

01

04

03

02

.01

. A

B

45 55 65 75 8.5 9 5

pH

FIG. 4. Effect of pH on initial velocity of diapho-rase reaction. A, NADPH concentration of 1.8 mM; B,NADH concentration of 78 AM.

VOL. 10, 1974 531

INOT

03 r

on February 21, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

DECHATELET ET AL.

TABLE 3. Effect of cyanide and azide on leukocytediaphorase activity

A OD per min per mgaConditions

NADH NADPH

Control .......0.......0.0231 0.0231+ CN- (0.33 mM) ...... 0.0231 0.0254+ CN- (1.65 mM) ...... 0.0233 0.0267+ Azide (0.33 mM) ..... 0.0280 0.0225+ Azide (1.65 mM) ..... 0.0278 0.0231

a Each value representsdeterminations.

the mean of duplicate

(11, 14, 17). The precise basis for these meta-bolic changes is unknown, although most inves-tigators feel that activation of an oxidase is themost likely explanation for the initiating event.Baehner and Karnovsky (5) have postulatedthat the initiating enzyme is NADH oxidase,based on their finding that cells from patientswith CGD have low levels of this enzyme (about50% of control) when compared with cells ob-tained from normal infected patients. Otherinvestigators have disputed this finding (16),and some propose NADPH oxidase to be thecritical enzyme (15). Indeed, a recent abstract(D. C. Hohn and R. I. Lehrer, Clin. Res.22:394A, 1974) has described a deficiency of thisenzyme in CGD cells.From a quantitative viewpoint, the activity of

either diaphorase can readily explain the respi-ratory burst. It is possible to completely reduceNBT dye by the use of alkaline ascorbate. Whenthis was done, we observed that 0.005 ,umol ofreduced NBT/ml gave an absorbancy change of0.100 U. If the typical diaphorase activity isapproximately 0.015 A OD per min per mg(Table 1), this corresponds to 2.3 nmol of re-duced NBT per min per mg or 138 nmol per hper mg. Baehner et al. (3) have calculated thatthe change in oxygen consumption of humancells under similar conditions amounts to 63nmol per h per mg, whereas the activity ofNADH oxidase is 39 nmol per h per mg, and thechange in glucose oxidation is still less at 20nmol per h per mg. The diaphorase activitieswithin the phagocytic vacuole might be ex-

pected to be significantly greater due to thelowered pH, in keeping with the data in Fig. 4.Thus, the activity of either NADH or NADPHdiaphorase is more than sufficient to explainthe respiratory burst.The reduction of NBT is highly nonspecific

and can be accomplished in a number of waysthat would not require a specific diaphorase. Inparticular, superoxide anion (02), which can

be generated by PMNL (2), will itself cause thereduction of NBT (9). This does not seem to bea problem in the present system, since reduc-tion of the dye is absolutely dependent upon theaddition of reduced pyridine nucleotides. Fur-thermore, the addition of purified superoxidedismutase (Truett Labs, Dallas, Tex.) gaveminimal inhibition of the reduction of NBT(less than 20% inhibition) under the assayconditions employed (data not shown). Simi-larly, the granule diaphorase activity cannot beascribed to myeloperoxidase, since purified my-eloperoxidase (kindly supplied by JuliusSchultz) failed to reduce NBT in either thepresence or absence of CN-.

It seems reasonable that at least a portion ofthe diaphorase activity of human PMNL is dueto NADH and NADPH oxidase activity (13).We undertook to investigate the diaphoraseactivity for several reasons. First, the oxidaseenzymes are low in activity and are difficult tomeasure. Furthermore, it is extremely difficultto adequately control the reaction since the onlyreactants are oxygen and reduced pyridine nu-cleotide; other cellular reactions could be re-sponsible for the changes in oxygen consump-tion or oxidation of reduced nucleotide by whichthe oxidase activities are typically measured.Finally, since a defect in NBT reduction is welldocumented in phagocytizing CGD cells, thisreaction might provide the best opportunity toprobe the metabolic basis of this particulardisease. Although the reduction of NBT byCGD cells is lacking during phagocytosis, thecells apparently have a normal complement ofactive enzyme when measured in a cell-freesystem (21). Thus, a more subtle defect than asimple enzyme deficiency is implicated. Such adefect might involve a deficiency in a particularisoenzyme which might not be observed whenthe total activity is determined, or it might bethe result of a change in the properties of anenzyme so that it is unable to function atconditions which are present within the cell.The present investigation reports some of the

properties of the diaphorase activities of normalcells. Both NADH and NADPH diaphorasewere found in both the granule and solublefractions of the cell (Table 2). The solubleactivity could not be ascribed to leakage due tolysosomal damage, since a far smaller percent-age of lysosomal marker enzymes (beta-glucuronidase and lysozyme) was seen in thesoluble fraction. The dual location suggests thatthere were at least two diaphorases, not distin-guishable by the catalytic assay, which werelocated differently within the cell. It seems

532 INFECT. IMMUNITY

on February 21, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

DIAPHORASE ACTIVITY IN PMNL

likely that the granule diaphorase activity is oneof importance during phagocytosis, since Na-than et al. have reported that the reduction ofNBT dye occurs only within the phagocyticvacuole (21). The diaphorase activity would bedelivered to the vacuole during the process ofdegranulation.Some of the properties of the various diapho-

rases are of interest. Both NADH and NADPHdiaphorase were totally insensitive to both cya-nide and azide; this is consistent with a role foreither in initiating the respiratory burst, whichis itself cyanide insensitive.Both enzyme activities were more active at

acid pH, although the NADH diaphoraseshowed the more dramatic change. This mightserve as an initiating mechanism, since theintravacuolar pH is known to decrease duringphagocytosis (25).Perhaps the most interesting data is obtained

in the kinetic studies. Although both NADHand NADPH diaphorase had about the sameaffinity for NBT (Fig. 2), there was a markeddifference in the affinity of the enzymes for theirrespective pyridine nucleotide substrates. TheKm value for NADH was about 10,uM, whereasthe corresponding value for NADPH was about400 MM. It is revealing to compare these valueswith the intracellular concentrations of thesecompounds. Baehner (4) has reported that theconcentration of both NADH and NADPH is 2.4nmol per 108 PMNL. Assuming that the cell is70% water and that 1.0 ml of packed cells equals1.5 x 109 PMNL (L. DeChatelet, unpublisheddata), then the intracellular concentrations ofboth nucleotides may be calculated to be about51 uM. Although this concentration is suffi-cient to saturate the NADH diaphorase, it is farbelow the optimal concentration for theNADPH-requiring enzyme. Although not con-clusive, these data argue in favor of an NADHoxidase as initiator of the respiratory burstwhich accompanies phagocytosis. Factors whichmight militate against this conclusion includeactivation of a specific diaphorase by phagocy-tosis, or the presence of different isozymes of adiaphorase with greatly different Km values.Patriarca et al. (24) have reported that the Kmfor NADPH oxidase in guinea pig PMNL de-creases by a factor of 10 during phagocytosis. Ifconfirmed, this would make the values for thetwo enzymes considerably closer, although theNADH-requiring enzyme would still have alower Km by a factor of four. A recent abstract(D. C. Hohn and R. I. Lehrer, Clin. Res.22:394A, 1974) suggests that a similar activa-tion NADPH oxidase might occur in phago-

cytizing human PMNL. It will be important todetermine whether the Km values for the diaph-orase enzymes change with phagocytosis.

Also, Holmes (16) has demonstrated thatthere are four isozymes of NADPH diaphoraseand three of NADH diaphorase in the humanPMNL. It is quite possible that only a specificisozyme is involved in the respiratory burst.Since the present investigation was concernedonly with total diaphorase activity, we cannotexclude the possibility that a specific isozyme ofNADPH has a much lower Km than the averagevalue for the total NADPH diaphorase activity.The linearity observed with increasing pro-

tein concentration (Fig. 3) demonstrates thatthe conditions employed gave a valid estimatefor enzyme activity. This assay system shouldprove useful in determining whether cells thatdemonstrate a high histochemical reduction ofNBT (as in severe infection [23] or polycythe-mia vera [1]) do so because of an increasedquantity of diaphorase, or because of someother change in the metabolism of the cell.Future experiments include an examina-

tion of the various isozymes of the diaphoraseactivities in both normal and CGD cells. Altera-tions in enzyme activity upon phagocytosis willalso be examined.

ACKNOWLEDGMENTS

This research was supported by a grant from the ForsythCancer Service and Public Health Service grants Al-10732,from the National Institute of Allergy and Infectious Disease,and CA-12197, from the National Cancer Institute.

LITERATURE CITED

1. Ashburn, P., M. R. Cooper, C. E. McCall, and L. R.DeChatelet. 1973. Nitroblue tetrazolium reduction:false positive and false negative results. Blood41:921-925.

2. Babior, B. M., R. S. Kipnes, and J. T. Curnutte. 1973.Biological defense mechanisms. The production byleukocytes of superoxide, a potential bactericidalagent. J. Clin. Invest. 52:741-744.

3. Baehner, R. L., N. Gilman, and M. L. Karnovsky. 1970.Respiration and glucose oxidation in human andguinea pig leukocytes: comparative studies. J. Clin.Invest. 49:692-700.

4. Baehner, R. L., R. B. Johnston, Jr., and D. G. Nathan.1972. Comparative study of the metabolic and bacteri-cidal characteristics of severely glucose-6-phosphatedehydrogenase-deficient polymorphonuclear leuko-cytes and leukocytes from children with chronic granu-lomatous disease. J. Reticuloendothel. Soc. 12:150-169.

5. Baehner, R. L., and M. L. Karnovsky. 1968. Deficiency ofreduced nicotinamide adenine dinucleotide oxidase inchronic granulomatous disease. Science 162:1277-1279.

6. Baehner, R. L., M. T. Karnovsky, and M. L. Karnovsky.1969. Degranulation of leukocytes in chronic granulo-matous disease. J. Clin. Invest. 48:187-192.

7. Baehner, R. L., and D. G. Nathan. 1967. Leukocyteoxidase: defective activity in chronic granulomatous

VOL. 10, 1974 533

on February 21, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

534 DECHATELET ET AL.

disease. Science 155:835-836.8. Baehner, R. L., and D. G. Nathan. 1968. Quantitative

nitroblue tetrazolium test and chronic granulomatousdisease. N. Engl. J. Med. 278:971-976.

9. Beauchamp, C., and I. Fridovich. 1971. Superoxidedismutase: improved assays and an assay applicable toacrylamide gels. Anal. Biochem. 44:276-287.

10. Boyum, A. 1967. Isolation of mononuclear cells andgranulocytes from human blood: isolation of mononu-clear cells by one centrifugation, and of granulocytes bycombining centrifugation and sedimentation at 1 g.Scand. J. Clin. Invest. 21(Suppl. 97):77-89.

11. Cooper, M. R., L. R. DeChatelet, C. E. McCall, M. F.LaVia, C. L. Spurr, and R. L. Baehner. 1972. Completedeficiency of leukocyte glucose-6-phosphate dehydro-genase with defective bactericidal activity. J. Clin.Invest. 51:769-778.

12. DeChatelet, L. R., and M. R. Cooper. 1970. A modifiedprocedure for the determination of leukocyte alkalinephosphatase. Biochem. Med. 4:61-68.

13. Gornall, A. G., C. J. Bardawill, and M. M. David. 1949.Determination of serum proteins by means of the biuretreaction. J. Biol. Chem. 177:751-766.

14. Gray, G. R., G. Stamatoyannopoulos, S. C. Naiman, M.R. Kliman, S. J. Klebanoff, T. Austin, A. Yoshida, andG. C. F. Robinson. 1973. Neutrophil dysfunction,chronic granulomatous disease, and nonspherocytichaemolytic anemia caused by complete deficiency ofglucose-6-phosphate dehydrogenase. Lancet2:530-534.

15. Holmes, B., and R. A. Good. 1972. Laboratory models ofchronic granulomatous disease. J. Reticuloendothel.Soc. 12:216-237.

16. Holmes, B., and R. A. Good. 1972. Metabolic andfunctional abnormalities of human neutrophils, p.51-66. In R. D. Williams and H. H. Fudenberg (ed.),Phagocytic mechanisms in health and disease. Inter-continental Medical Book Corp., New York.

17. Holmes, B., A. R. Page, and R. A. Good. 1967. Studies ofthe metabolic activity of leukocytes from patients witha genetic abnormality of phagocytic function. J. Clin.Invest. 46:1422-1432.

INFECT. IMMUNITY

18. Iyer, G. Y. N., M. F. Islam, and J. H. Quastel. 1961.Biochemical aspects of phagocytosis. Nature (London)192:535-536.

19. Jolles, P. 1962. Lysozymes from rabbit spleen and dogspleen, p. 137-140. In S. P. Colowick and N. 0. Kaplan(ed.), Methods in enzymology, vol. 5. Academic PressInc., New York.

20. Karnovsky, M. L., S. Simmons, M. J. Karnovsky, J.Noseworthy, and E. A. Glass. 1972. Comparativestudies on the metabolic basis of bactericidal activityin leukocytes. p. 67-82. In R. D. Williams and H. H.Fudenberg (ed.), Phagocytic mechanisms in health anddisease. Intercontinental Medical Book Corp., NewYork.

21. Nathan, D. G., R. L. Baehner, and D. K. Weaver. 1969.Failure of nitro blue tetrazolium reduction in thephagocytic vacuoles of leukocytes in chronic granulo-matous disease. J. Clin. Invest. 48:1895-1904.

22. Nishikimi, M., N. A. Rao, and K. Yagi. 1972. Theoccurrence of superoxide anion in the reaction ofreduced phenazine methosulfate and molecular oxy-gen. Biochem. Biophys. Res. Commun. 46:849-854.

23. Park, B. H., S. M. Fikrig, and E. M. Smithwick. 1968.Infection and nitrobluetetrazolium reduction by neu-trophils. Lancet 2:532-534.

24. Patriarca, P., R. Cramer, S. Moncalvo, F. Rossi, and D.Romeo. 1971. Enzymatic basis of metabolic stimula-tion in leukocytes during phagocytosis: the role ofactivated NADPH oxidase. Arch. Biochem. Biophys.145:255-262.

25. Rous, P. 1925. The relative reaction within living mam-malian tissues. II. On the mobilization of acid materialwithin cells, and the reaction as influenced by the cellstate. J. Exp. Med. 41:399-411.

26. Sbarra, A. J., and M. L. Karnovsky. 1959. The biochemi-cal basis of phagocytosis. I. Metabolic changes duringthe ingestion of particles by polymorphonuclear leuko-cytes. J. Biol. Chem. 234:1355-1362.

27. Talalay, P., W. H. Fishman, and C. Huggins. 1946.Chromogenic substrates. II. Phenolphthalein glucu-ronic acid as substrate for the assay of glucuronidaseactivity. J. Biol. Chem. 166:757-772.

on February 21, 2020 by guest

http://iai.asm.org/

Dow

nloaded from