THE JOURNAL OF INFECTIOUS DISEASES. VOL. 138, NO.5. NOVEMBER 1978 © 1978 by The University of Chicago. 0022·1899/78/3805·0022$00.75

Comparison of Myeloperoxidase Activity in Leukocytes from Normal Subjects and Patients with Chronic Granulomatous Disease From the Departments of Infectious Diseases, Immunology, and Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina

P. Samuel Pegram, Jr., Lawrence R. DeChatelet, and Charles E. McCall

The phagocytic activity of human polymorphonuclear leukocytes (PMNLs) has been linked to a number of intracellular metabolic events (the "respiratory burst") ultimately related to bacterial killing [I, 2]. The nature of the oxygenconsuming enzyme responsible for initiating the respiratory burst is controversial, and its identity is not firmly established, but prevailing data favor a reduced pyridine nucleotide oxidase [3]. However, myeloperoxidase (MPO) continues to be mentioned as a possible candidate principally because of the large quantity of this substance within leukocyte azurophilic granules and its ability to catalyze the oxidation of a variety of substrates, including pyridine nucleotides [4, 5]. Neutrophils from patients with chronic granulomatous disease (CGD) are incapable of initiating the respiratory burst despite normal phagocytic activity [6-8]. The present study was undertaken to compare more definitively MPO activity of leukocytes from normal subjects and patients Received for publication April 7, 1978, and in revised form June 16, 1978. This research was supported by U.S. Public Health Service grants no. Al-10732 and AI-09169 from the National Institute of Allergy and Infectious Diseases, no. CA-12197 from the National Cancer Institute, and no. HL-16769 from the National Heart and LungInstitute. Please address requests for reprints to Dr. Lawrence R. DeChatelet, Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, WinstonSalem, North Carolina 27103.

with CGD; similar activity in the two populations would argue against MPO being the enzyme responsible for the oxidative response to phagocytosis. Materials and Methods Population studied. Data from nine patients meeting the diagnostic criteria for CGD [7, 8] form the basis of this report. Patients studied included both those with the classic X-linked recessive and those with the autosomal recessive forms of the disease; details of the patient population have been published [9]. Blood specimens from the patient population and from normal controls were collected from August 1975 to August 1976, processed (granule fraction isolation), and then frozen until the assays were performed. Preparation of the granule fraction. Isolated PMNLs were designated as resting cells or as phagocytizing cells according to a technique and criteria published earlier [9]. The isolation of granule fractions from PMNLs has been described [10]. The granule fraction thus obtained represents a resuspended pellet obtained by centrifugation at 27,000 g, with heterogenous composition and activity reflecting predominantly intracellular granule constituents. The protein content of each granule fraction was determined in triplicate by the method of Lowry et al. [11]; preparations generally contained 1.0-1.5 mg of proteirr/ml.


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A study was undertaken to compare myeloperoxidase (MPO) activity in leukocytes from normal subjects and those with chronic granulomatous disease (CGD) and to eliminate further consideration of MPO as the oxidase responsible for the post-phagocytic respiratory burst and subsequent oxidative microbicidal sequence lacking in leukocytes from patients with CGD. With use of granule fractions isolated from a number of samples, MPO activity in several MPO-mediated biochemical systems (peroxidase assays, protein iodination, and amino acid decarboxylation) was measured. No difference was demonstrated between granule fraction preparations from normal subjects and those with CGD. These data show that MPO activity is normal in CGD leukocytes and are inconsistent with a role for MPO in initiating the postphagocyticrespiratory burst.


Pegram, DeChatelet, and McCall

mosan particles. Granule fraction preparations at a 1:5 dilution (0.1 ml) were added to flasks containing Cav-free Dulbecco's phosphatebuffered saline (PBS) (0.35 ml), 0.2 mM H 202 (0.05 ml), zymosan (-1.67 X 108 particles), and 1?5I (0.1 ml to yield approximately 200,000 cpm). After incubation for 1 hr at 37 C with constant shaking, 5% TCA was added, the resultant precipitate was washed four times with TCA, and radioactivity was counted in a gamma well scintillation counter. Blanks were routinely included in which the zymosan particles were omitted; the average blank value was 1,461 cpm. Results from triplicate samples were averaged. Results are expressed as cpm of 1251 incorporated/ 0.02 mg of protein. Results Because few samples of resting granule fractions from subjects with CGD were available for assay, it was first necessary to compare activities in normal resting and phagocytizing granule fractions to ensure that there was no alteration in activity induced by phagocytosis. Table 1 compares the mean MPO activity along with SE values of normal resting vs. phagocytizing granule fractions utilizing as many as 15 samples per assay. In the spectrophotometric peroxidase assay using as hydrogen donors two different dye substrates, 0dianisidine and o-tolidine, MPO activity was not significantly different in normal resting and phagocytizing preparations. Activity towards o-to1idine was slightly greater than that towards o-dianisidine. In addition, quantitative leukocyte iodination as well as decarboxylation of t-alanine-P4C was similar in normal resting and phagocytizing granule fractions. Table 2 compares normal phagocytizing vs.

Table 1. Myeloperoxidase activity of normal resting vs.. normal phagocytizing granule fractions. Peroxidase assay (~OD/min per mg of protein) * o-dianisidine (460 nm) Parameter Mean SE

No. of observations

o-tolidine (400nm)

Resting Phagocytizing Resting Phagocytizing 2.33 0.30 11

*ll.OD = change in optical density.

2.13 0.27 15

2.76 0.31 11

2,49 0.27 15

Iodination (cpm/0.02 mg of protein) Resting

Phagocy tizing

36,968 3,738 11

35,369 2,707 15

Decarboxylation (cpm/0.02 mg of protein) Resting Phagocytizing 4,300 673 8

5,065 512 15

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Peroxidase assay. Peroxidase activity was assessed spectrophotometrically using as hydrogen donors the dye substrates o-dianisidine and 0tolidine [12-15]. Assays were performed utilizing 2.9 ml of 0.01 M N-[2-acetamido]-2-iminodiacetic acid (ADA) buffer, pH 6.0, containing 0.22 mM H 202 and 25 JLl of 1% dye in ethanol. The reaction was initiated with the enzyme-containing granule fraction (0.02, 0.04, and 0.06 ml), and the change in absorbance was measured continuously with a Beckman DU spectrophotometer (Beckman Instruments, Fullerton, Calif.) coupled to a Gilford recorder (Gilford Instrument Laboratories, Oberlin, Ohio) at 460 nm for o-dianisidine and 440 nm for o-tolidine. Activities were expressed as change in optical density (!10D)/ min per mg of protein and were dependent upon the presence of sonicate, H 202, and reduced substrate. Decarboxylation assay. t-alanine-l-t-C was utilized to measure MPO-mediated decarboxylation by modification of a previously described method [16]. A volume of 2.5 ml of 0.02 M KSP04 buffer (pH 5.5) in 0.22 mM H 202 was added to each flask containing 0.3 ml of 1 M KCl, 0.1 ml of 0.05 M L-alanine, and 0.05 ml of t-alanine-l-t-C (5 JLCi/ml). Granule fraction preparations at a dilution of 1:5 (0.1 ml) were added to initiate the reaction at 37 C with constant shaking. After 60 min, the reaction was terminated with 1.0 ml of trichloroacetic acid (TCA), released 14C0 2 was collected in 0.5 ml of hyamine hydroxide, and radioactivity was counted in a liquid scintillation counter. All assays were performed in triplicate; activities were expressed as cpm of 14C0 2/O.02 mg of protein. Iodination assay. Protein iodination was determined by a modification of the technique described by Pincus and Klebanoff [17] using zy-


Myeioperoxidase in CGD Leukocytes

Table 2. Myeloperoxidase activity of granule fractions from normal subjects vs. those with chronic granulomatous disease (CGD) during phagocytization. Peroxidase assay (AOD{min per mg of protein)" o-dianisidine




2.13 0.27 12


No. of observations '~OD =

CGD 2.80 0.40 15

Iodination Decarboxylation (cpm{0.02 mg of protein) (cpm{0.02 mg of protein)








2.49 0.27 12

2.79 0.29 15

36,369 3,707 12

37,079 3,720 15

5,065 512 10

4,899 895 15

change in optical density.

sults are incompatible with a role for MPO in the initiation of the respiratory burst during phagocytosis.

References Discussion I. Klebanoff, S. J. Antimicrobial mechanisms in neutro-

MPO was first proposed as a candidate for initiating the respiratory burst in 1964 [4]. However, evidence has been accumulating to suggest that MPO is not the responsible oxidative enzyme. Patients with a congenital absence of MPO reportedly do not suffer from recurrent bacterial infections and exhibit a normal or even elevated respiratory burst [18-20]. Although several studies have reported normal MPO activity in cells of patients with CGD [3, 21], these studies have generally utilized few patients and a single spectrophotometric reaction for measurement of the enzyme. MPO is capable of oxidizing a variety of artificial substrates with varying efficiency [12, 15] as well as of iodinating various proteins [17] and of decarboxylating amino acids [16]. These different activities of MPO have not previously been investigated in patients with CGD. Table 1 verifies previous reports [3, 18] that MPO in normal cells is not activated by phagocytosis and extends those studies by measuring several substrates in spectrophotometric assays and by determining MPO-mediated iodination and decarboxylation reactions. Table 2 shows normal activity with regard to these parameters in cells from patients with CGD as compared with normal cells. The present investigation demonstrates that MPO from a number of patients with CGD is equally efficient in all of these activities compared with MPO from control cells. These re-






7. 8.




philic polymorphonuclear leukocytes. Semin. Hematol. 12:117-142, 1975. Allen, R. C., Stjernholm, R. L., Steele, R. H. Evidence for the generation of an electronic excitation staters) in human polymorphonuclear leukocytes and its participation in bactericidal activity. Biochern. Biophys. Res. Commun. 47:67~84, 1972. Cheson, B. D., Curnutte, J. T., Babior, B. M. The oxidative killing mechanisms of the neutrophil. In R. S. Schwartz [ed.], Progress in clinical immunology. Vol. 3. Grune and Stratton, New York, 1977, p. 1-65. Roberts, J., Quastel, J. H. Oxidation of reduced triphosphopyridine nucleotide by guinea pig polymorphonuclear leucocytes. Nature 202:85-86, 1964. Patriarca, P., Dri, D., Rossi, F. The NAD (P)H oxidase activity of PMNL revisited [abstract no. 24]. J. Reticuloendothel. Soc. 18 (Suppl.):12a, 1975. Baehner, R. L., Nathan, D. G. Leukocyte oxidase: defective activity in chronic granulomatous disease. Science 155:835-836,1967. Schmalzer, E. M., Miller, D. R. Chronic granulomatous disease. Prog. Med. Genet. 1:145-184, 1976. Johnston, R. B., Jr., Newman, S. L. Chronic granulomatous disease. Pediatr. Clin. North Am. 24:365376,1977. McPhail, L. C., DeChatelet, L. R., Shirley, P. S., Wilfert, C., Johnston, R. B., Jr., McCall, C. E. Deficiency of NADPH oxidase activity in chronic granulomatous disease. J. Pediatr. 90:213-217,1977. DeChatelet, L. R., McPhail, L. C., Mullikin, D., McCall, C. E. An isotopic assay for NADPH oxidase activity and some characteristics of the enzyme from human polymorphonuclear leukocytes. J. Clin. Invest. 55:714-721, 1975. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. Protein measurement with the folin phenol reagent. J. BioI. Chern. 193:265-275, 1951.

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CGD phagocytizing granule fractions. No statistically significant differences were noted in the peroxidase assays, the protein iodination, or the amino acid decarboxylation.


18. Klebanoff, S. J., Hamon, C. B. Role of myeloperoxidase-mediated antimicrobial systems in intact leukocytes. J. Reticuloendothel. Soc. 12:170-196, 1972. 19. Klebanoff, S. J., Pincus, S. H. Hydrogen peroxide utilization in myeloperoxidase-deficient leukocytes: a possible microbicidal control mechanism. J. Clin. Invest. 50:2226-2229,1971. 20. Patriarca, P., Cramer, R., Tedesco, F., Kakinuma, K. Studies on the mechanism of metabolic stimulation in polymorphonuclear leucocytes during phagocytosis. II. Presence of the NADPH 2 oxidizing activity in a myeloperoxidase-deficient subject. Biochim. Biophys. Acta 385:387-393, 1975. 21. Baehner, R. L., Karnovsky, M. J., Karnovsky, M. L. Degranulation of leukocytes in chronic granulomatous disease. J. Clin. Invest. 48:187-192,1969.

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12. Anonymous. Peroxidase. In Worthington enzyme manual. Worthington Biochemical Corp., Freehold, N.J., 1972,p. 43-45. 13. Purr, A. Zur bestimmung pflanzlicher peroxydasen. Biochemische Zeitschrift 321:1-18, 1950. 14. Luck, H. Peroxidase. In H. Bergmeyer [ed.]. Methods of enzymatic analysis. Academic Press, New York, 1965,p. 895-897. 15. Maehly, A. C., Chance, B. The assay of catalases and peroxidases. Part I. General assay methods. Methods Biochem. Anal. 1:357-408, 1954. 16. Strauss, R. R., Paul, B. B., Jacobs, A. A., Sbarra, A. J. The role of the phagocyte in host-parasite interactions. XXII. H 2 0 2·dependent decarboxylation and deamination by myeloperoxidase and its relationship to antimicrobial activity. J. Reticuloendothel. Soc.7:754-761,1970. 17. Pincus, S. H., Klebanoff, S. J. Quantitative leukocyte iodination. N. Engl. J. Med. 284:744-750,1971.

Pegram, DeChatelet, and McCall

Comparison of myeloperoxidase activity in leukocytes from normal subjects and patients with chronic granulomatous disease.

THE JOURNAL OF INFECTIOUS DISEASES. VOL. 138, NO.5. NOVEMBER 1978 © 1978 by The University of Chicago. 0022·1899/78/3805·0022$00.75 Comparison of Mye...
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