polymorphonuclear leukocytes ( pmns ) play an important role in host defense against microbial infections .
the microbicidal mechanism consists of phagocytosis of pathogens , production and subsequent release of reactive oxygen species ( ros ) and bactericidal proteins to phagosomes .
the process involves activation of nadph oxidase ( nox ) , which catalyzes the reduction of oxygen molecular to o2 at the expense of nadph in phagocytes [ 13 ] .
noxs are a group of plasma membrane - associated multicomponent enzymes consisting of at least two components bound to plasma membrane ( gp91 and p22 that together form the flavocytochrome b558 ) , and three cytosolic components ( p47 , p67 , and p40 ) , and a small gtpase rac . in resting pmns ,
noxs are dormant and their components separately exist in the membrane and in the cytosol . once pmns are primed by appropriate stimuli , noxs are activated to produce o2 by association of these cytosolic components with the plasma or phagosome membrane components [ 58 ] .
besides the bactericidal action as a beneficial effect , the uncontrolled production of ros by phagocytic granulocytes may lead to crippling disorders such as shock and inflammation .
phorbol 12-myristate 13-acetate ( pma , fig .  1 ) , a potent activator of protein kinase c ( pkc ) , induces the phosphorylation of p47 and subsequent o2 production by the assembled nox components .
more in detail , phosphorylation of proteins plays an essential role in the regulation of nox activity , and the activation of pkc with pma promotes the phosphorylation of serine and threonine residues of the p47 and its translocation to the plasma membrane [ 1113 ] .
the respiratory burst is accompanied by tyrosine phosphorylation in cytosolic and membrane proteins , and it is also well known that tyrosine phosphorylation of proteins occurs during priming of pmns . as shown in fig . 
1 , a side chain group of pma is myristic acid ( mya ) , a 14-carbon , straight chain saturated fatty acid .
mya is mainly contained in vegetable oils such as coconut oil and palm oil , and likely affects lipid metabolism such as hypercholesterolemia [ 1618 ] , i.e. mya causes high ldl cholesterol and apob levels and low hdl to ldl ratios in healthy men and women , a positive regression between the plasma cholesterol level and the dietary level of mya was observed in hamsters , and incorporation of trimyristin in the oil resulted in marked rise in serum cholesterol of rats . in term of oxidative metabolism of pmns , it was reported that mya has an ability to stimulate the generation of o2 from human neutrophils .
it was reported that mya induces the hydrolysis of phosphatidylcholine leading to generation of arachidonic acid ( aa ) .
aa when added with membrane protein separated from bovine pmns can activate nox of bovine pmns .
in addition , it was reported that apocynin ( a specific inhibitor of nadph oxidase ) suppressed this stimuli ( including mya ) -dependent ros generation of undifferentiated hl-60 cells .
thus , mya may prime pmns through two routes , one is nox activated by aa from phosphatidylcholine and the other is direct effect on nox . thus , to assess the effect of mya ,
when absorbed into the circulation , on ros generation , we need to estimate the potency of aa to generate ros in addition to the potency of mya itself .
these findings tempted us to compare the stimulative effect of mya to release ros with that of pma , and to examine the stimulative effect of aa as an indirect effect of mya . in this study , we examined stimuli - dependent ros release from human pmns by applying electron paramagnetic resonance ( epr)-spin - trapping method .
reagents were purchased from the following sources : dextran , pma , mya and diethylenetriamine - n , n , n',n'',n''-pentaacetic acid ( dtpa ) from wako pure chemical industries ( osaka , japan ) , aa from icn biomedicals , inc .
( costa mesa , ca ) , superoxide dismutase ( sod from bovine erythrocytes ) and 4-hydroxyl-2,2,6,6-tetrametylpiperidin-1-oxyl ( tempol ) from sigma - aldrich ( st .
lous , mo ) , ficoll - paque plus from amersham biosciences ( buckinghamshire , uk ) and 5,5-dimethyl-1-pyrroline - n - oxide ( dmpo ) from labotec inc .
pmns were prepared from the blood by density gradient centrifugation using a ficoll - paque plus ( d  =  1.077 ) following dextran sedimentation as previously described .
after the pellet of pmns were subjected to rapid hypotonic lysis of erythrocyte , the cells were suspended in hank s balanced salt solution ( hbss ) to be 1.0    10  cell / ml .
pma and mya were dissolved in dimethyl sulfoxide ( dmso ) to be 0.80 to 4.0  mm .
dtpa was dissolved in 0.1  m phosphate buffer solution ( pb , ph  7.4 ) to be 20  mm , sod was to be 5000  u / ml in pb , and dmpo was to be 2.25  m in hbss .
one hundred and twenty l of cell suspension was mixed with 16  l of dmpo and 16  l of dtpa solution .
after addition of 8  l of pma , mya or aa , the resultant mixture was transferred to a quartz flat epr cell within 40  s. epr spectra of dmpo spin adducts were recorded at given times .
the measurement conditions for epr ( jes - fa100 , jeol , tokyo , japan ) were as follows : scan rate , 10  mt / min ; scan field , 335.5    5  mt ; modulation width , 0.1  mt ; modulation frequency , 100  khz ; time constant , 0.03  s ; microwave power , 8  mw ; and microwave frequency , 9.5  ghz .
peak areas of epr spectra of dmpo spin adducts , which are compensated by the peak area of mn , were compared with those of the given concentrations of tempol standard .
statistical significances between the two mean values of each time point were assessed by student s t test or welch t test following analysis of variance .
reagents were purchased from the following sources : dextran , pma , mya and diethylenetriamine - n , n , n',n'',n''-pentaacetic acid ( dtpa ) from wako pure chemical industries ( osaka , japan ) , aa from icn biomedicals , inc .
( costa mesa , ca ) , superoxide dismutase ( sod from bovine erythrocytes ) and 4-hydroxyl-2,2,6,6-tetrametylpiperidin-1-oxyl ( tempol ) from sigma - aldrich ( st .
lous , mo ) , ficoll - paque plus from amersham biosciences ( buckinghamshire , uk ) and 5,5-dimethyl-1-pyrroline - n - oxide ( dmpo ) from labotec inc .
pmns were prepared from the blood by density gradient centrifugation using a ficoll - paque plus ( d  =  1.077 ) following dextran sedimentation as previously described .
after the pellet of pmns were subjected to rapid hypotonic lysis of erythrocyte , the cells were suspended in hank s balanced salt solution ( hbss ) to be 1.0    10  cell / ml .
pma and mya were dissolved in dimethyl sulfoxide ( dmso ) to be 0.80 to 4.0  mm .
dtpa was dissolved in 0.1  m phosphate buffer solution ( pb , ph  7.4 ) to be 20  mm , sod was to be 5000  u / ml in pb , and dmpo was to be 2.25  m in hbss .
one hundred and twenty l of cell suspension was mixed with 16  l of dmpo and 16  l of dtpa solution .
after addition of 8  l of pma , mya or aa , the resultant mixture was transferred to a quartz flat epr cell within 40  s. epr spectra of dmpo spin adducts were recorded at given times .
the measurement conditions for epr ( jes - fa100 , jeol , tokyo , japan ) were as follows : scan rate , 10  mt / min ; scan field , 335.5    5  mt ; modulation width , 0.1  mt ; modulation frequency , 100  khz ; time constant , 0.03  s ; microwave power , 8  mw ; and microwave frequency , 9.5  ghz .
peak areas of epr spectra of dmpo spin adducts , which are compensated by the peak area of mn , were compared with those of the given concentrations of tempol standard .
statistical significances between the two mean values of each time point were assessed by student s t test or welch t test following analysis of variance .
fig .  2 shows the typical epr spectra of dmpo spin adducts of oxygen radicals generated from human pmns stimulated by pma or mya . as shown in fig .  2a , multiple - lines of epr spectra of human pmns primed by pma were detected .
the hyperfine coupling constants ( hfcc ) of a  =  1.48 and a  =  1.48  mt ( closed circle ) at the four - line spectrum were typical dmpo spin adducts of oh ( dmpo - oh ) [ 22 , 23 ] , and those of a  =  1.425 , a  =  1.145 and a  =  0.124 ( open circle ) were dmpo spin aducts of o2 ( dmpo - ooh ) [ 21 , 22 ] .
2b shows the representative epr spectra obtained from human pmns primed by mya . as is the case with fig .  2a , spin adducts of dmpo - oh and
dmpo - ooh were observed , suggesting that mya can stimulate human pmns to release o2 and oh . to confirm that the dmpo - ooh was derived from o2 , the pmns were treated with sod , a potent o2 scavenger , prior to pma and mya stimulation .
2c and d show characteristic epr spectra of dmpo - oh obtained from pmns primed with pma and mya following sod treatment , respectively .
since the twelve - line spectrum with the hfcc of a  =  1.425 , a  =  1.145 and a  =  0.124 ( open circle ) was completely disappeared in either of fig . 
2c and d , these spectra was derived from spin adduct of dmpo and o2 .
3a shows time course changes in dmpo - ooh production after the addition of pma , and of mya to be a concentration of 81  m .
apparently mya stimulated pmns to produce o2 more strongly than did pma when the same concentrations were added .
3b shows time course changes in dmpo - oh production after the addition of pma , and mya to be a concentration of 81  m , indicating that oh production was time - dependently increased by either pma or mya .
similar to the case of o2 , mya stimulated pmns to produce oh more strongly than did pma when the same concentrations were added .
these results clearly showed that mya has an ability to prime human pmns to produce oxygen radicals such as o2 more potently than pma .
it was reported that mya induces chemiluminescence of human promyelocytic leukemia cells with a luminol analogue l-012 .
however , qualitative analysis of ros has not been conducted yet because chemiluminescence is induced by various ros . on the other hands ,
the epr spin - trapping method could detect o2 and oh simultaneously as dmpo - spin adducts as shown in our study .
4 shows the typical epr spectra of dmpo - ooh and dmpo - oh by human pmns with 81  m mya , 150  m aa , and 1.5  mm aa . the epr spectra were observed at 8  min after additions of mya or aa .
these findings show that aa can also prime human pmns to relerase o2 and oh , even though the potency is weaker than that of mya . as for the mechanism
by which aa primes pmns , it was reported that aa activates nox and proton channels of dormant neutrophils .
5 . unlike c5a and n - formyl - l - methionyl - l - leucyl - l - phenylalanine ,
both of which can stimulate pmns to produce o2 by inducing calcium ( ca ) influx , mya - functions , like pma - functions , bypass ca influx since it has been reported that mya induces o2 production from human neutrophils in the absence of ca and magnesium [ 19 , 26 ] .
although the potency of aa to stimulate the release of ros is much weaker than that of mya , the possibility of involvement of aa to the effect of mya when absorbed into the circulation should be considered .
our study shows that mya is a more potent activator of pmns than pma , and this fact calls attention that too much intake of mya can lead to crippling effect through uncontrolled production of ros .
we found that mya is a stronger inducer of o2 and oh generation by pmns than pma .
mya is mainly contained in vegetable oils and likely affects lipid metabolism [ 1618 ] .
too much intake of mya - rich vegetable oil may work as an inducer of pmns activation , which may lead to undesirable conditions of the body . in the future study
 myristic acid ( mya ) , which is a saturated fatty acid ( c14:0 ) and a side chain of phorbol 12-myristate 13-acetate ( pma ) , was examined if mya stimulates human polymorphonuclear leukocytes ( pmns ) to release oxygen radicals comparable to pma by applying electron paramagnetic resonance ( epr)-spin - trapping method . when mya was added to isolated human pmns , spin adducts of 5,5-dimethyl-1-pyrroline - n - oxide ( dmpo)-oh and dmpo - ooh 
 were time - dependently observed . 
 the amounts of these spin adducts were larger than those of pmns stimulated by pma . 
 these results clearly show that mya is more potent agent to prime human pmns than pma , in a point of view of not only o2 but also oh production . 
 this fact calls attention that too much intake of mya that is known to be contained vegetable oils can lead to crippling effect through uncontrolled production of reactive oxygen species . 
Introduction
Materials and Methods
Reagents
Isolation of human PMNs
EPR-spin trapping determination of oxygen free radicals generated from human PMNs
Statistical analysis
Results and Discussion
Conclusions