Effects of Promethazine-Hydrochloride on Human Polymorphonuclear Leukocytes - Infection ...
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INFCTON AND IMMUNITY, Mar. 1973, p. 403-408 Vol. 7, No. 3
Copyright © 1973 American Society for Microbiology 1'rinted in U.S.A.
Effects of Promethazine-Hydrochloride on Human
Polymorphonuclear Leukocytes
LAWRENCE R. DECHATELET, DIANE QUALLIOTINE-MANN, RONALD CALDWELL,
CHARLES E. McCALL, AND JOHN P. GUSI)ON
Departments of Biochemistry, Medicine, and Obstetrics and Gynecology, Bowman Gray School of Medicine,
Wake Forest University, Winston-Salem, North Carolina 27103
Received for publication 6 November 1972
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Promethazine hydrochloride at a concentration of 0.033 mg/ml has pronounced
effects on leukocyte metabolism and function. The drug inhibits the phagocytosis-
induced increases in 02 consumption and hexose monophosphate shunt activity.
Associated with these effects is an inhibition of the iodination of zymosan particles
and an inhibition of bacterial killing by the cell. At least two mechanisms appear
to be involved. Many of the effects can be explained by an inhibition of phago-
cytosis, but promethazine also inhibits the decarboxylation of amino acids and
iodide fixation in a cell-free system, indicating a specific effect on metabolism.
These results may partially account for the action of the drug in ameliorating the
effects of erythroblastosis.
Bierme-Alie-Enjalbert (Doctoral thesis, Cen- under a stream of nitrogen, and the isotope was
tre Regional de Transfusion Sanguine et d'Hema- dissolved in deionized water to give an activity of
tologie, Toulouse, France, 1967) first suggested 5.0 uCi/ml. Carrier-free Na'15I was obtained at a
an amelioration of the effects of erythroblastosis concentration of 5 mCi in 0.10 ml. A stock solution
in babies whose mothers had been treated with of 50 ICi/0.10 ml was prepared in 10 ml of sterile
deionized water. Since the half-life of 1251- is 54
promethazine-hydrochloride during pregnancy. days, the activity was adjusted weekly. All bio-
She ascribed the effectiveness of the drug to its chemicals were obtained from the Sigma Chemical
diuretic properties. Gusdon et al. (4) then demon- Co., St. Louis, Mo.; all other chemicals were of
strated that administrationi of l)romethazine to reagent grade quality. Hanks balanced salt solu-
experimental animals resulted in inhibition of tion (HBSS) and latex particles (0.8-,um diameter)
both the primary and secondary immune re- were obtained from Difco Laboratories, Detroit,
sponse and delayed hypersensitivity. However, Mich. The latex particles were washed three times
depression of circulating anti-Rh antibody titers in cold deionized water by repeatedsuspension and
has been noted in only some of the patients centrifugation (6,040 X g for 15 min). The washed
treated with the drug (J. P. Gusdon, unpub- pellet was suspended in isotonic saline, such that a
1 to 100 dilution would have an absorbance of 0.42
lished data). The present investigation was under- at 540 nm. The final concentration of latex was
taken to study the effects of the drug on a specific approximately 3 X 109 particles/ml. Plasma gel,
cell associated with the inflammatory response in used in sedimenting the red blood cells, was ob-
an effort to more clearly define the mechanism of tained from the HTI Corp., Buffalo, N.Y. Zymo-
action of the drug. san particles were obtained from Nutritional
Biochemicals Corp., Cleveland, Ohio. They were
MATERIALS AND METHODS suspended in HBSS to a concentration of approxi-
All isotopes were obtained from the New Eng- mately 6 X 109 particles per ml. Serum was derived
land Nuclear Corp., Boston, Mass. D-Glucose-1 -'4C from a pool of human AB type serum kept frozen
and D-glucose-6-14C (specific activities 54.2 mCi/ in small samples at -70 C. Promethazine-hydro-
mmol and 36.5 mCi/mmol, respectively) were dis- chloride (Phenergan) was supplied as the pure
solved in deionized water to give an activity of powder by the Wyeth Laboratories, Philadelphia,
2.0 JMCi/ml. D-Glucose-1-14C-6-phosphate was ob- Pa. Purified human myeloperoxidase was the
tained as the disodium salt and diluted with de- generous gift of Jutlius Schultz.
ionized water to anl activity of 2.0 ;sCi/ml; unla- Isolation of leukocytes. Leukocytes were
beled glucose-6-phosphate was added to give a isolated from the blood of apparently healthy
specific activity of 0.60 mCi/mmol. L-Alanine-1 - volunteer subjects by a method previously de-
14C (specific activity 13.2 mCi/mmol) was obtained scribed (2). For experiments requiring intact cells,
in 0.5 ml of 0.1 N HCI. The HCI was evaporated the cells were counted by conventional means and
403404404DECHATELET ET AL. INFECT. IMMUNITY
the concentration was adjusted to 5 X 106 cells/ml described (10). The ingestion of latex particles
by the addition of HBSS. Differential counts were was estimated by determining the phagocytic
done in a counting chamber by classifying cells as index. The phagocytes (1 X 107) were incubated in
phagocytes (mature and band form neutrophils, 10% serum in HBSS with latex particles at a ratio
eosinophils, and monocytes) and lymphocytes. of 100 particles per phagocyte for 15 min. Five
Lymphocytes accounted for less than 10%/ of the hundred cells were then examined by phase mi-
isolated cell suspension. Cell viability was deter- croscopy, and the number of particles in each cell
mined by staining with 1% trypan blue dye. was estimated. The index was calculated on the
For experiments in which broken cell prepara- basis of 0 to 5 for each individual phagocyte. Thus,
tions were used, the cells were suspended in phos- the phagocytic score could range from 0 to 2,500.
phate-buffered saline and disrupted by sonic lodination reaction. The ability of the intact
treatment for 1 min using a Branson sonifier with a cell to iodinate ingested particles was measured by
power output of 20 W. Examination of the soni- a modification of the method of Pincus and
cally treated material by phase-contrast micros- Klebanoff (9) as previously described (10). lodina-
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copy indicated total cell disruption. tion of zymosan particles in a cell-free system was
Measurement of glucose utilization. Glu- determined by the procedure of Klebanoff (6).
cose utilization via the hexose monophosphate Each incubation tube contained in a total volume
shunt (HMS) was estimated by a modification of a of 1.0 ml: 200 pmol of sodium acetate buffer, pH
previously described method (5), employing glu- 5.0; 0.2 pmol of H202; 0.2 pmol of Nal"'I containing
cose differentially labeled in the C-1 or C-6 posi- 0.50 uCi of radioactivity; 2 X 108 zymosan parti-
tion. Each flask contained 0.20 ml of serum, 0.10 cles; 10 pg of purified human myeloperoxidase
ml of KCN (0.01 M), and 0.10 ml of either glucose- (200-o-dianisidine units); and varying amounts of
1-14C or glucose-6-4C. Promethazine-hydrochlo- promethazine-hydrochloride. Controls in which
ride was added to experimental flasks dis- either the zymosan or the myeloperoxidase were
solved in HBSS; an equal volume of HBSS was omitted were run. The reaction was stopped after
added to control flasks. Phagocytosis was initiated 1 h at 37 C by the addition of 1.0 ml of cold 5%
by the addition of 1.0 ml of a suspension of heat- trichloroacetic acid. The resulting precipitates
killed Escherichia coli (1.0 X 1011 bacteria/ml). were washed and counted as described by
This concentration was demonstrated experi- Klebanoff (6).
mentally to give maximal stimulation of the HMS. Aldehyde reaction. The decarboxylation of
The volume of all flasks was brought to a final total L-alanine-1 -14C was taken as a measure of aldehyde
of 3.0 ml (after the addition of cells) with HBSS. formation according to the procedure of Strauss
Reaction was initiated by the addition of 1.0 ml et al. (12).
of cell suspension (containing 5 X 106 cells) and Enzyme assays. Glucose-6-phosphate dehy-
allowed to proceed for 1 h at 37 C. 14CO2 was drogenase and 6-phosphogluconate dehydrogenase
collected in 0.5 ml of hyamine hydroxide and were measured by observing the initial rate of
counted in a liquid scintillation spectrometer as formation of nicotinamide adenine dinucleotide
previously described (5). The oxidation of glucose- phosphate (reduced) at 340 nm in a Beckman DU
6-phosphate-1-14C in a cell-free system was meas- spectrophotometer with a Gilford recorder (7).
ured as previously described (12). Myeloperoxidase activity was determinded by the
Leukocyte oxygen consumption. Oxygen o-dianisidine dye procedure (13).
consumption was measured by use of a Clark
oxygen electrode with an automatic recorder RESULTS
according to a modification of a previously re- The effect of promethazine on the HMS ac-
ported method (11). Each chamber contained in a tivity of phagocytizing leukocytes is demon-
final volume of 3.0 ml:0.50 ml of serum, 0.10 ml strated in Fig. 1. It is apparent that a concentra-
of KCN (0.01 M), 1.0 ml of cell suspension (5 X 106 tion of promethazine as low as 0.033 mg/ml (0.1
cells per ml), and HBSS to give total volume of
3.0 ml). Promethazine-hydrochloride dissolved in mM) completely abolishes the respiratory burst.
0.10 ml of HBSS was added where indicated. This effect is not due to an inhibition of the
Phagocytosis was initiated in appropriate flasks by HMS enzymes glucose-6-phosphate dehydroge-
the addition of 1.0 ml of heat-killed E. coli. The nase or 6-phosphogluconate dehydrogenase as
oxygen consumption was calculated as micro- demonstrated by the data in Table 1. In this
moles of oxygen consumed per hour per 5 X 106 experiment, the oxidation of glucose-6-phosphate-
cells. 1-14C by sonic extracts of polymorphonuclear
Bactericidal assay. The clearance of bacteria leukocytes in the presence of exogenous nicotina-
by the polymorphonuclear leukocytes was deter- mide adenine dinucleotide phosphate was deter-
mined by the plate dilution technique of Maal0e mined as previously described (11). The fact that
(8) as previously described. The drug was added promethazine exerts no effect on this system
to appropriate flasks in a minimal volume (0.10
ml) of HBSS. indicates that it does not act as an inhibitor of
Phagocytosis assay. Phagocytosis of micro- the HMS enzymes, as has been demonstrated for
organisms was quantitated by measuring leuko- colchicine (3). This was confirmed by spectro-
cyte uptake of radiolabeled E. coli as previously photometric experiments in which a concentra-VOL. 7, 1973 PROMETHAZINE AND PMN LEUKOCYTES 405
plained by a decrease in cell viability. Under the
40 4 conditions of the experiments, greater than 95%
of the cells were viable after 1 h in the presence
3.0 I -
of 0.033 mg/ml promethazine, and approximately
70% were viable after 1 h in the presence of
2.0 I - 0.17 mg of the drug per ml. Cell viability in the
presence of promethazine, however, was markedly
1.0 influenced by the presence of serum. In the
absence of serum, only 50% viability was ob-
I served after 1 h with 0.033 mg of promethazine
0.017 0.033 per ml, and 0 to 5% viability was seen at the
Promethozine (mg/ml ) higher (0.17 mg/mo) concentration. Apparently,
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FIG. 1. Effect of promethazine concentration on serum exerts a protective effect against prome-
glucose oxidation of phagocytizing polymorphonu- thazine-induced cell destruction. For this reason,
clear leukocytes. Values represent the oxidation of all assays employing whole cells were routinely
glucose-1-"4C corrected for oxidation of glucose-6- run in the presence of serum.
"4C. Each value represents the mean of three de- Figure 2 illustrates the effect of the drug on the
terminations.
TABLE 1. Effect of promethazine on glucose-6- TABLE 2. Effect of promethazinea on
phosphate- -"4C oxidation in sonic extracts oxygen consumption of human
of polymorphonuclear leukocytes polymorphonuclear leukocytes
14C02 from glucose-6- O2 uptakeb
Description phosphate-I-14C Description
(counts/min) Expt 1 Expt 2
Control 95 Promethazine alone 1.6 1.5
(90-98) Resting cells 9.0 5.0
+ NADPb 2,714 Resting cells (+ promethazine) 3.6 2.0
(2,474-2,928) Phagocytizing cells 24.3 13.1
+ NADP + promethazine 2,694 Phagocytizing cells (+ pro- 1.8 4.4
(0-033 mg/ml) (2,644-2,769) methazine)
+ NADP + promethazine 2,702
(0.167 mg/mil) (2,655-2,764) a The concentration of promethazine in experi-
a Each value represents the average of three ment 1 was 0.167 mg/ml; in experiment 2 it was
0.033 mg/ml. The calculated values are based on
determinations; numbers in parentheses represent the change in percent saturation from 10 to 15
the range. min and assume that 1.0 ml of solution holds 5.0
b NADP, Nicotinamide adenine dinucleotide
,uliters of 0 when saturated.
phosphate. bResults are expressed as microliters of 02 Up-
take per hour per 5 X 106 cells.
tion of promethazine of 0.17 mg/ml had no effect
on the activity of leukocyte glucose-6-phosphate
dehydrogenase or 6-phosphogluconate dehydro-
x1Id Ko7 6/
p
Kd
'p
genase (data not shown).
Table 2 illustrates the effect of promethazine- 4ic-
X S albus
\E.cIi If
hydrochloride on the oxygen consumption of id' \ V' K \
human polymorphonuclear leukocytes. A con- Ti 0
centration of promethaziine of 0.033 mg/ml in- 04 0
hibits the oxygen consumption of resting cells by 00
more than 50% and lowers the oxygen consump- 0 60 120
Time (min)
tion of phagocytizing cells to below the normal
resting level. The differenices seeni between the FIG. 2. Effect of promnethazine on the clearance
two experiments for either resting or phagocy- of three strains of bacteria. Solid line, bacteria
tizing cells are due in large part to biological alone; dashed line, bacteria plus cells; dotted line,
bacteria plus cells plus 0.083 mg of promethazine
variatiois; the experiments were runi on differenit per ml; dashed and dotted line, bacteria plus cells
davs with different cell donors. plus promethazine (0.17 mg/ml). Each value repre-
The effects of promethazine cannot be ex- sents the mean of four determinations.406 DECHATELET ET AL. INFECT. IMMUNITY
clearance of three strains of bacteria. Prometha- TABLE 4. Effect of promethazine on inge8tion
zine (0.033 mg/ml) inhibits the ability of the cell of polystyrene particles
to kill Staphylococcus albus, E. coli, and Psudo-
monas aeruginosa. A higher concentration of drug Description indexy
(0.17 mg/ml) causes a greater inhibition of killing
in all cases.
}3ecause the ability of the cell to destroy bac- Control 2,140
teria has been correlated with iodination of the + Promethazinie (0.33 mg/ml) 233
ingested particles, we studied the effects of + Promethazine (0.067 mg/ml) 217
promethazine on the fixation of 125I to zymosan a Cells (500) were scored on an arbitrary scale
particles. The data (Table 3) demonstrate a 70% from 0 (no ingestion) to 5 (completely filled).
inhibition of iodination at a concentration of Hence, the phagocytic index could range from 0
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0.033 mg of promethazine per ml. Iodination is to 2,500.
completely abolished at the higher concentration
of the drug. 241-
Since all the parameters studied to this point
utilized whole cells, they were dependent upon
the ability of particles to enter the cell as well as 20 /.-
the subsequent intracellular events. Hence, it was
considered important to determine the effects of 16
promethazine on particle ingestion per se. The
data in Table 4 and Fig. 3 indicate that prometha- ftI
zine can exert a pronounced effect on the ingestion 01 12
of either latex particles or radiolabeled E. coli.
Another reaction which has been implicated in
bactericidal activity is the myeloperoxidase- 8hI
.%
catalyzed formation of aldehydes from amino
acids. Figure 4 shows that this reaction, too, is 4 L~~~~
markedly suppressed by the drug. This is of
particular interest because this reaction is per-
formed in a cell-free system and is therefore inde- 5
10 15 20
pendent of any effects on phagocytosis. Time
Since we had previously demonstrated that FIG. 3. Effect of promethazine on the uptake of
promethazine could inhibit iodination of zymosan radiolabeled Escherichia coli by polymorphonuclear
by intact leukocytes (Table 3), it now became of
leukocytes. Dotted line, control values; solid line,
promethazine added (0.17 mg/ml). Each value
interest to determine whether the drug would represents the mean of three determination&s.
affect iodination in a cell-free system. The results
in Table 5 clearly indicate that promethazine will
inhibit iodination independently of an effect on zine was an inhibitor of myeloperoxidase. To test
particle uptake. The iodination reaction, how- this hypothesis, the enzyme was assayed by the
ever, is not nearly as sensitive to the drug as is o-dianisidine procedure in the presence and
the formation of aldehydes from amino acids. absence of the drug. The data in Table 6 indicate
Both iodination and aldehyde formation are that a concentration of promethazine as high as
dependent upon the enzyme myeloperoxidase. 0.167 mg/ml has absolutely no effect on the
Accordingly, it was hypothesized that prometha- activity of myeloperoxidase, indicating that the
TABLE 3. Effect of promethazine on iodination of zymosan particles
Counts/min4
Description Net counts/minb
-Zymosan + Zymosan
Control 2,116 41,470 39,354
+ Promethazine (0.033 mg/ml) 1,032 13,991 12,959
+ Promethazine (0.167 mg/mi) 463 563 100
a Each value represents the average of three determinations. The assay employed intact cells.
b Net counts/min represents 1211 specifically associated with particle ingestion.VOL. 7, 1973 PROMETHAZINE AND PMN LEUKOCYTES 407
the respiratory burst following phagocytosis.
40' Associated with the inhibition of oxygen con-
sumption and HMS activity is a concomitant
~30 inhibition of the clearance of bacteria by the cell.
Most of these effects could be explained by an
.k 20 inhibition of phagocytosis; however, the fact that
promethazine also inhibits 02 consumption and
10 HMS activity of resting cells indicates that inhi-
bition of particle ingestion is not the sole mecha-
nism of action of the drug. This observation is
0.017 0.033 substantiated by the experiments in which
Promethazine (mg/ml) promethazine was shown to inhibit both aldehyde
formation and iodination in a cell-free system.
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FIG. 4. Effect of promethazine concentration The mechanism of this inhibition is not clear,
on the decarboxylation of L-alanine-_4C by a sonic but the drug cannot be interfering with H202
extract of polymorphonuclear leukocytes. Each
point represents the mean of three determinations. production since this compound is added to the
reaction mixture. Likewise, the drug does not
TA\BLE 5. Iodination of zymosan in a inhibit myeloperoxidase as shown by its lack of
cell-free system effect on the o-dianisidine assay. Perhaps pro-
methazine exerts its effect by interfering with
5I incor- some reactive intermediate such as hypochlorite
Description poration
(countsn or chloramines in the case of the aldehyde reaction
(counts/ (14) or an oxidized species of iodine (6) in the
min)a
iodination eaction. These two reactions are not
inhibited to the same extent by a given concen-
Complete system 92,969 tration of promethazine; this may be a reflection
- Zymosan 5,629 of the relative reactivity of the intermediate
- Myeloperoxidase 6,441
+ Promethazine (0.05 mg/ml) 67,538 involved in the respective reactions. The differ-
+ Promethazine (0.10 mg/ml) 56,999 ence in susceptibility of the two reactions to
+ Promethazine (0.15 mg/ml) 45,123 promethazine suggests that this compound
+ Promethazine (0.20 mg/ml) 31,927 might be useful in assessing the relative contribu-
tions of the reactionis to the bactericidal activity
a Each value represenits the meanl of three de- of the intact cell.
terminations. Promethazine was first reported to be effective
in ameliorating the effects of erythroblastosis in
TABLE 6. Effect of promethazine on babies (S. Bierne-Alie-Enjalbert, Doctoral thesis,
myeloperoxidase activity in vitro Cenltre Regional de Transfusion Sanguine et
d'Hematologie, Toulouse, France, 1967). Clinical
Expt Conditions Myeloper- experiments conducted at this institution (J. P.
Gusdon, manuscript in prepraration) have gen-
erally substantiated this observation. In her
1 Control + promethazine 0.275 thesis, Bierme-Alie-Enjalbert had empirically
(0.33 mg/ml) 0.293
attributed the clinical effectiveness of the drug to
2 Control + promethazine 0.263 its diuretic action (Doctoral thesis, Centre
(0.167 mg/mi) 0.278 Regional de Transfusion Sanguine et d'Hema-
tologie, Toulouse, France, 1967). Gusdon et al.
3 Control + promethazine 0.296 (4) demonstrated a possible mechanism of action
(0.167 mg/ml) 0.286 by showing that the drug is an inhibitor of both
a Results expressed as micromoles of product
the primarv and secondary immune responses
formed per minute per milligram. and of delayed hypersensitivity in rats and guinea
pigs at a dosage of 30 mg/kg. In four of six
drug is nlot simply an inhibitor of the eiizyme's patients treated with this drug, and followed with
activity. serial indirect coombs antibody titers, a signifi-
cant depression of the titer was noted. No
DISCUSSION elevatioji of the antibody titer was found in the
The preseint results demonstrate that pro- other two cases (J. P. Gusdon, manuscript in
methazine-hydrochloride is a potent inhibitor of l)rel)aration). These observations led to the pres-408S481),CHATELET ET AL. INFECT. IMMUNITY
ent investigation of the effects of the drug on the 5. Holmes, B., P. G., Quie, D. B. Windhorst, and
polymorphonuclear leukocytes. R. A. Good. 1966. Fatal granulomatous disease
The administration of promethazine-hydro- of childhood-an inborn abnormality of phago-
cytic function. Lancet 1:1225-1228.
chloride to the mother results in a transfer of the 6. Klebanoff, S. J. 1967. Iodination of bacteria: a
drug to the fetus (1). The amelioration of erythro- bactericidal mechanism. J. Exp. Med. 126:
blastosis by promethazine might be due to the 1063-1078.
inhibition of the metabolism of the fetal reticulo- 7. Kornberg, A., and B. L. Horecker. 1955. Glucose-
6-phosphate dehydrogenase, p. 323-326. In
endothelial cells responsible for red cell lysis. S. P. Colowick and N. 0. Kaplan (ed.), Methods
Studies of the effects of promethazine on other in enzymology, vol. 1. Academic Press Inc.,
cells of the reticuloendothelial system are in New York.
8. Maal0e, 0. 1946. On the relation of alexin and
progress. opsonin. Ejnae Munksgaard, Copenhagen, Den-
ACKNOWLEDGMENTS mark.
9. Pincus, S. H., and S. J. Klebanoff. 1971. Quanti-
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The excellent technical assistance of Pamela Shirley
is gratefully acknowledged. tative leukocyte iodination. N. Engl. J. Med.
This research was supported by a grant from the 284:744-750.
Forsyth Cancer Service; by Public Health Service 10. Qualliotine, D., L. R. DeChatelet, C. E. McCall,
grants AI-10732 from the National Institute of Al- and M. R. Cooper. 1972. Effect of catechol-
lergy and Infectious Disease and CA-12197 from the amines on the bactericidal activity of poly-
National Cancer Institute; and by grant RF-72019 morphonuclear leukocytes. Infect. Immunity
from the Rockefeller Foundation. 6:211-217.
11. Qualliotine, D., L. R. DeChatelet, C. E. McCall,
LITERATURE CITED and M. R. Cooper. 1972. Stimulation of oxida-
tive metabolism in polymorphonuclear leuko-
1. Corby, D. G., and I. Schulman. 1971. The effects cytes by catecholamines. J. Reticuloendothel.
of antenatal drug administration on aggregation Soc. 11:263-276.
of platelets of newborn infants. J. Pediat. 79: 12. Strauss, R. R., B. B. Paul, A. A. Jacobs, and A. J.
307-313. Sbarra. 1970. Role of the phagocyte in host-
2. DeChatelet, L. R., and M. R. Cooper. 1970. A parasite interactions. XXII. H202-dependent
modified procedure for the determination of decarboxylation and deamination by myelo-
leukocyte alkaline phosphatase. Biochem. Med. peroxidase and its relationship to antimicrobial
4:61-68. activity. J. Reticuloendothel. Soc. 7:754-761.
3. DeChatelet, L. R., M. R. Cooper, and C. E. 13. Worthington Biochemical Corporation. 1972.
McCall. 1971. Dissociation by colchicine of Worthington enzyme manual: peroxidase. Worth-
the hexose monophosphate shunt activation ington Biochemical Corp., Freehold, N.J.
from the bactericidal activity of the leukocyte. 14. Zgl czynski, J. M., T. Stelmaszynska, J. Doman-
Infect. Immunity 3:66-72. ski, and W. Ostrowski. 1971. Chloramines as
4. Gusdon, J. P., V. L. Moore, Q. N. Myrvik, and intermediates of oxidation reaction of amino
P. A. Holyfield. 1972. Promethazine-HCI as an
immunosuppressant. J. Immunol. 108:1340- acids by myeloperoxidase. Biochim. Biophys.
1344. Acta 235:419-424.You can also read
