prostaglandins ( pgs ) are critical mediators of a variety of physiological and pathophysiological processes . the initial step in the biosynthesis of these bioactive lipids is accomplished by cyclooxygenase ( cox ) , which catalyzes the conversion of arachidonic acid ( aa ) to pgg2 and h2 . cox-1 is ubiquitous enzyme and it appears to perform many homeostatic functions [ 24 ] . cox-2 is generally thought to be an enzyme synthesized in response to inflammatory stimuli [ 24 ] . there are lots of published data concerning the differential roles of cox-1 and -2 on the physiological and pathophysiological functions , but it is unclear about differences in the endogenous regulation of these two isoforms in the body . up to date , it has been widely accepted that linoleic acid ( la ) is a competitive inhibitor of cox-1 activity [ 57 ] . have found that la is also an inhibitor of cox-2 , and the inhibitory potency on the cox-2 is more than that on the cox-1 . on the other hand , many investigators [ 9 , 10 ] including us   have shown that la is transformed into hydroperoxides by cox reactions . have shown that both cox-1 and -2 prepared from cos-1 cells metabolized la , and that 13s - hydroperoxy-9z,11e - octadecadienoic acid ( 13-hpode ) was the main product of both reactions . there is little information concerning a difference of potency of 13-hpode on the cox-1 and -2 activities . therefore , we investigated the effects of la without or with preincubation using cox-1 or -2 on the pg formation from aa by the respective enzyme . cox-1 ( isolated from ram seminal vesicles ) and cox-2 ( isolated from sheep placenta ) were purchased from cayman ( ann arbor , mi ) . 13-hpode and 13s - hydroxy-9z,11e - octadecadienoic acid ( 13-hode ) were purchased from cascade biochem . ten units of cox-1 or -2 were incubated with 100  m aa in 0.5  ml of 0.1  m tris / hcl buffer ( ph  8.0 ) containing hematin ( 0.1  m ) and phenol ( 2  mm ) as cofactors for 2  min at 37c . 13-hpode , 13-hode or la was added in the incubation mixture at t  =  0 . in table  1 , la was also preincubated with cox-1 or -2 for 10  min prior to the incubation with aa . after incubation , the reaction mixture was extracted with 6 vol . of n - hexane the aqueous phase was then acidified ( approximately ph  3 ) with 2  n hcl and extracted with 6 vol . of ethyl acetate . pge2 , d2 and f2 in the extracted lipid were simultaneously determined by an hplc using adam for derivatization , as described in our previous studies [ 13 , 14 ] . peak heights were measured for the quantification of adam derivatives of pgs relative to the standard derivatives prepared from authentic pgs . cox-1 ( isolated from ram seminal vesicles ) and cox-2 ( isolated from sheep placenta ) were purchased from cayman ( ann arbor , mi ) . 13-hpode and 13s - hydroxy-9z,11e - octadecadienoic acid ( 13-hode ) were purchased from cascade biochem . ten units of cox-1 or -2 were incubated with 100  m aa in 0.5  ml of 0.1  m tris / hcl buffer ( ph  8.0 ) containing hematin ( 0.1  m ) and phenol ( 2  mm ) as cofactors for 2  min at 37c . 13-hpode , 13-hode or la was added in the incubation mixture at t  =  0 . in table  1 , la was also preincubated with cox-1 or -2 for 10  min prior to the incubation with aa . after incubation , the reaction mixture was extracted with 6 vol . of n - hexane the aqueous phase was then acidified ( approximately ph  3 ) with 2  n hcl and extracted with 6 vol . of ethyl acetate . pge2 , d2 and f2 in the extracted lipid were simultaneously determined by an hplc using adam for derivatization , as described in our previous studies [ 13 , 14 ] . peak heights were measured for the quantification of adam derivatives of pgs relative to the standard derivatives prepared from authentic pgs . in the present study , when either cox-1 or -2 was incubated with aa , pge2 was mainly formed in both reaction mixtures ( 7779% ) . lesser amounts of pgd2 and f2 were also made by these two enzymes ( pgd2 , 1820% ; pgf2 , 3% ) . it has been reported that indomethacin inhibits both cox-1 and -2 activities , but ns-398 selectively inhibits cox-2 activity . our previous study   utilizing indomethacin and ns-398 has demonstrated that pge2 formations by the present assay conditions exactly reflect the activities of cox-1 and -2 . therefore , we evaluated the cox-1 and -2 activities by measuring the amounts of pge2 formed from aa . table  1 illustrated the effects of la without or with preincubation using cox-1 or -2 on pg formation from aa catalyzed by the respective enzyme . co - addition of 5  m la in the reaction mixture did not show any significant change in pg formation catalyzed by both enzymes . this inability of 5  m la is explained by the previous finding by ringbom et al . , which showed that a competitive inhibition by la on the cox-1 and -2 activities became clear at much higher concentrations ( ic50 : 170  m for cox-1 ; 94  m for cox-2 ) . in contrast , 5  m la preincubated for 10  min with cox-1 or -2 strongly inhibited the pg formation catalyzed by the respective enzyme , and the inhibition on the cox-2 ( 95% inhibition ) was somewhat stronger than that on the cox-1 ( 74% inhibition ) . it has been shown that la is transformed into 13-hpode by cox-1 and -2 , and that 13-hpode is further decomposed to form 13-hode by enzymatic and non - enzymatic pathways [ 912 ] . to investigate the inhibitory effect of preincubated la on cox-1 or -2 activity , the effects of 13-hpode and 13-hode on the enzyme activities were examined ( table  2 ) . 13-hpode inhibited pg formation catalyzed by both cox-1 and -2 , and the concentrations required to inhibit the enzyme activities by 50% ( ic50 ) were 3.1  m for cox-1 and 1.5  m for cox-2 . so , the inhibitory effect of 13-hpode on the pg formation catalyzed by cox-2 was about two times stronger than that by cox-1 . on the other hand , 13-hode did not affect pg formation catalyzed by both cox-1 and -2 ( ic50 , over 10  m ) . we also confirmed that la was rapidly converted into 13-hode by cox-2 ; 13-hode ( 1.2  nmol ) was detected by hplc - uv apparatus , when 100  m la was incubated with cox-2 ( 10 units ) for 1  min ( n  =  2 ) . therefore , these findings indicate that 13-hpode formed from la during preincubation with cox-1 or -2 can be a potent inhibitor of pg formation from aa by the respective enzyme . it is unclear about the mechanism by which 13-hpode has an inhibitory effect on the cox-1 and -2 activities , and the effect on cox-2 activity is stronger than that on cox-1 activity . but , very low concentrations of hydroperoxides are required to activate cox , and in the absence , there is very little enzyme activity . on the other hand , higher concentrations of these substances can inhibit cox [ 18 , 19 ] . since a discovery of cox-1 and -2 isoforms in living cells , chen et al .   and liu et al . have reported that exogenously added peroxides decrease or abolish pgg2-derived self - activation of cox-1 and -2 , with a higher efficacy on the cox-2 . therefore , it seems likely that the inhibitory effect on cox-1 and -2 activities is ascribed to lipid hydroperoxide - derived inactivation mechanism , and cox-2 is more sensitive to lipid hydroperoxides than cox-1 . thus , it is thought that pgs produced by cox-1 being constitutively expressed and acting for homeostatic control are maintained at extremely low levels . have shown that a competitive inhibition by la on the cox-1 and -2 activities become clear at much higher concentrations ( ic50 : 170  m for cox-1 ; 94  m for cox-2 ) in - vitro . however , we can consider that basal appearance of free la in vivo is continuous upon physiological stimuli ; namely free la in physiological conditions is rather high compared to free aa because of a difference of total amounts of la and aa in vivo [ 22 , 23 ] . therefore , it appears that 13-hpode preformed by constitutively expressed cox-1 might be an important homeostatic suppressor of pg formation by this enzyme . further , its stronger inhibition on the activity of cox-2 by 13-hpode observed in the present study led us to speculate its protective role on excess production of pgs by cox-2 in early pathophysiological states .
linoleic acid ( la ) preincubated with cyclooxygenase ( cox)-1 or -2 inhibited prostaglandin ( pg ) formation from arachidonic acid ( aa ) catalyzed by the respective enzyme , but la without the preincubation did not . 
 13s - hydroperoxy-9z,11e - octadecadienoic acid ( 13-hpode ) a hydroperoxy adduct of la inhibited pg formation catalyzed by cox-1 or -2 . 
 13s - hydroxy-9z,11e - octadecadienoic acid had no effect on both cox-1 and -2 activities . 
 these results suggest that 13-hpode which is preformed from la by cox reaction under normal physiological conditions can be a basal suppressor of pg formation from aa .