Patent Application: US-22570002-A

Abstract:
the disclosure describes a method for detecting conditions indicative of sepsis . in one embodiment of the invention , an increase in the level of 3 - chlorotyrosine or 3 - bromotyrosine from the normal level in a sample of body fluid or tissue is indicative of early sepsis or infection . in another embodiment of the invention , the level of 3 - chlorotyrosine or 3 - bromotyrosine is measured or monitored to determine the response to therapeutic treatment of the infective condition in which a reduction in the level that existed prior to the treatment is an early sign or indication that the treatment is working in vivo . in a preferred embodiment , the method of the invention is illustrated in a clinically relevant mouse model of sepsis .

Description:
while the specification concludes with claims particularly pointing out and distinctly claiming the subject matter regarded as forming the invention , it is believed that the invention will be better understood from the following preferred embodiments of the invention taken in conjunction with the accompanying drawings . [ 0020 ] fig1 is a graphical showing that myeloperoxidase deficiency impairs survival in a cecal ligation and puncture ( clp ) model of sepsis in which percent of survival is plotted against time in days . mortality was monitored in myeloperoxidase - deficient ( mpo −/− ) mice and littermate wild - type ( wt ) controls . [ 0021 ] fig2 shows ec - nci gc / ms analysis of the ethyl heptafluorobutyrate , mtbstfa derivatives of 3 - bromotyrosine and 3 - chlorotyrosine in peritoneal inflammatory fluid of a wild - type mouse subjected to clp . note the simultaneous monitoring of [ 0025 ] fig3 shows isotope dilution gc / ms quantification of a , 3 - chlorotyrosine ( 3 - cl - tyr ) and b , 3 - bromotyrosine ( 3 - br - tyr ) in the peritoneal inflammatory fluid of sham - operated and clp - subjected mice . oxidation products were monitored in myeloperoxidase - deficient ( mpo −/− ) mice , and in wild - type ( wt ) mice in the 129 / svj and c57bl / 6j background . [ 0026 ] fig4 shows reverse - phase hplc analysis of n - acetyl - l - tyrosine ( n - ac - tyrosine ) exposed to the myeloperoxidase - h 2 o 2 — cl − — br — system . reactions proceeded for 60 min at 37 ° c . in chelex - treated buffer a ( 100 mm nacl , 50 mm sodium phosphate , 100 μm dtpa , ph 4 . 5 ) supplemented with 3 nm myeloperoxidase , 1 mm n - ac - tyrosine , 10 μm nabr , and 50 μm h 2 o 2 . the reactions were initiated with h 2 o 2 and terminated with 0 . 1 mm methionine . [ 0027 ] fig5 is a graphical showing of reaction requirements for the generation of n - acetyl - l - 3 - bromotyrosine by phagocyte peroxidases and hypohalous acids at neutral ph . reactions were carried out in buffer a supplemented with 10 μm br − a , shows effect of ph on the generation of n - acetyl - bromotyrosine by myeloperoxidase . b - e , shows effect of taurine ( 200 μm ) on the generation of n - acetyl - bromotyrosine by , respectively , hocl , hobr , myeloperoxidase or eosinophil peroxidase at ph 7 . amino acids were quantified by reverse - phase hplc . results are representative of those found in three independent experiments . mpo , myeloperoxidase ; epo , eosinophil peroxidase ; ac , acetyl . [ 0031 ] fig6 is a graphical showing of reaction requirements for the generation of n - acetyl - l - 3 - chlorotyrosine and n - acetyl - l - 3 - bromotyrosine by phagocyte peroxidases and hypohalous acids under acidic conditions . effect of [ br − ] on the generation of n - acetyl - chlorotyrosine and n - acetyl - bromotyrosine by : effect of taurine on the generation of n - acetyl chlorotyrosine and n - acetyl - bromotyrosine by : reactions for a and b were carried out as described in the description of fig4 . reactions for c and d were performed as described in the description of fig4 except the ph was 5 . 9 . amino acids were quantified by reverse - phase hplc . results are representative of those found in 3 independent experiments . mpo , myelo - peroxidase ; epo , eosinophil peroxidase ; ac , acetyl . in order to illustrate the invention in greater detail , the following specific laboratory examples were carried out . although specific examples are thus illustrated herein , it will be appreciated that the invention is not limited to these specific , illustrative examples or the details therein . materials . all materials were purchased from sigma - aldrich ( st . louis , mo .) or fisher ( pittsburgh , pa .) unless otherwise indicated . isotope - labeled amino acids were from cambridge isotope laboratories ( andover , mass .). rabbit polyclonal antibody was raised against a peptide present in mouse myeloperoxidase ( ntlpklnltswket ) [ seq id no : 1 ] ( ref . 14 ). animals . the animal studies committee of washington university school of medicine approved all animal studies . myeloperoxidase - deficient mice were generated in a 129 / svj background ; they were backcrossed at least 4 generations into the c57bl / 6j background prior to use ( ref . 14 ). c57b1 / 6j and sv129 / j mice were from jackson laboratories ( bar harbor , mass .). mice were maintained under pathogen - free conditions on a 12 h light - dark schedule and allowed ad libitum access to rodent diet 5001 ( harlan - teklad , madison , wis .). general procedures . myeloperoxidase was purified from hl60 cells ( ref . 19 ). enzyme was apparently pure as assessed by its absorption spectrum ( a 430 / a 280 ratio & gt ; 0 . 8 ), sds - page , and peroxidase activity gel electrophoresis ( ref . 20 ). porcine eosinophil peroxidase ( a 415 / a 280 & gt ; 0 . 9 ) was from exoxemis ( little rock , ark .). chloride - free naocl and hobr were prepared by conventional procedures as described previously ( refs . 21 , 22 ). peritoneal white blood cells were elicited with intraperitoneal ( i . p .) injection of 4 % thioglycollate and harvested 24 h after injection ( ref . 23 ). sds - page was performed using 10 % bis - tris nupage gels ( invitrogen ; carlsbad , calif .). the separated proteins were transferred onto a nitrocellulose membrane , which was blocked overnight at 4 ° c . with 5 % milk and 0 . 05 % tween - 20 in phosphate - buffered saline ( pbs ; 10 mm phosphate , 138 mm nacl , 2 . 7 mm kcl , ph 7 . 4 ). the blot was then incubated ( 1 h ) with rabbit anti - mouse myeloperoxidase antibody ( 1 : 1 , 000 ) followed by goat anti - rabbit immunoglobulin antibody ( 1 : 10 , 000 ; santa cruz biotechnology ; santa cruz , calif .) conjugated with horseradish peroxidase . west pico chemiluminescent substrate ( pierce ; rockford , ill .) was added , and the membrane was exposed to autoradiography film and developed . reaction conditions . reactions were carried out for 60 min at 37 ° c . in buffer a ( 100 mm nacl , 50 mm sodium phosphate , 100 μm diethylenetriaminepentaacetic acid ( dtpa )) supplemented with 1 mm n - acetyl - l - tyrosine and either 50 μm hypohalous acid or 3 nm peroxidase and 50 μm h 2 o 2 . buffer a was passed over a chelex resin column prior to use to remove redox active transition metal ions . reactions were initiated by the addition of oxidant and terminated with 0 . 1 mm methionine . reverse - phase hplc analyses of halogenated amino acids . analyses were performed using a flow rate of 1 ml / min and an ultrasphere ods reverse - phase column ( 4 . 6 mm × 25 cm ; 5 μm ; beckman instruments ; berkeley , calif .) coupled to a waters 484 tunable absorbance detector with monitoring at 275 nm . solvent a was 0 . 1 % trifluoroacetic acid ( tfa , ph 2 . 5 ), and solvent b was 0 . 1 % tfa in methanol ( ph 2 . 5 ). the gradient profile was : isocratic elution at 66 : 34 ( v / v ) a : b over 2 . 1 min ; linear increase of b from 34 % to 40 % over 30 min ; isocratic elution at 60 : 40 a : b for 3 min ; increase of b from 40 % to 100 % over 2 min ; and isocratic elution at 100 % b for 3 min . products were quantified using integrated peak areas by comparison to standard curves of authentic n - ac - chlorotyrosine and n - ac - bromotyrosine . sepsis model . cecal ligation and puncture of age - matched ( 8 - 16 weeks ) and sex - matched mice was performed by conventional procedure as previously described ( ref . 16 ). the peritoneum was lavaged 24 h after surgery with 5 ml of pbs or with pbs supplemented with 50 μm butylated hydroxytoluene , 100 μm dtpa , 1 mm sodium azide , and 10 mm aminotriazole . lavage fluid was stored at − 80 ° c . until analysis . control experiments demonstrated that inclusion of antioxidants had no effect on the amount of products detected . for survival studies , animals were given i . p . injections of metrinodizole ( 35 mg / kg ) and ceftriazone ( 50 mg / kg ) approximately 1 h after surgery . antibiotic injections were repeated once 24 h after surgery . mice were allowed ad libitum access to water and food . cell analysis . cells were pelleted onto slides by centrifugation and stained with a modified wright &# 39 ; s stain ( dimscio & amp ; associates ; manchester , mo .). cell counts were performed by the department of comparative medicine ( washington university school of medicine ). peroxidase activity assay . peritoneal lavage fluid was centrifuged at 4 ° c . for 10 min at 16 , 000 × g . cetyltrimethylammonium bromide buffer ( 0 . 3 %; 500 μl ) containing 25 % glycerol and 35 mm β - alanine ( ph 4 . 5 ) was added to the pellet , and the mixture was sonicated for 10 min . after a second 10 min centrifugation at 16 , 000 × g , soluble proteins in the supernatant were subjected to native page and peroxidase activity staining ( ref . 20 ). gc / ms analysis . isotope - labeled internal standards ( 1 pmol l - 3 - chloro [ 13 c 6 ] tyrosine , 500 fmol l - 3 - bromo [ 13 c 6 ] tyrosine , 10 nmol l -[ 13 c 9 , 15 n ] tyrosine ) were added to 500 μl of peritoneal fluid and prepared for gc / ms analysis by conventional procedure as previously described ( refs . 24 , 25 ). briefly , amino acids were isolated using solid - phase extraction with a c18 column followed by a supelco chrom p column ( refs . 24 , 25 ). samples were dried under anhydrous n 2 and stored at − 20 ° c . until analysis . following derivatization with ethyl heptafluorobutyrate and n - methyl - n -( t - butyldimethylsilyl )- trifluoroacetamide + 1 % trimethylchlorosilane ( mtbstfa ), the samples were dried under anhydrous n 2 and resuspended in 50 μl of undecane / mtbstfa ( 3 : 1 v / v ). a 1 μl sample was analyzed by selected ion monitoring using a hewlett - packard ( hp ) 6890 gas chromatograph coupled to a hp 5973 mass detector in the electron capture negative chemical ionization ( ec - nci ) mode . the injector , transfer line , and source temperatures were initially set at 183 ° c , 300 ° c ., and 250 ° c ., respectively . the injector and transfer line temperatures were set to increase incrementally with the temperature gradient ( 180 ° c . to 300 ° c . at 40 ° c ./ min .). 3 - chlorotyrosine and 3 - bromotyrosine were quantified using the respective ions at m / z 489 ([ m - halide - t - butyl - dimethylsilyl ] − ) and their isotopically ring - labeled [ 13 c 6 ]- internal standards ( m / z 495 ). potential artifact formation during sample work - up was monitored as the appearance of ions at m / z 499 derived from l -[ 13 c 9 , 15 n ] tyrosine ( refs . 24 , 25 ). liquid chromatography - electrospray ionization - ms . n - acetyl amino acids were separated by reverse - phase hplc and analyzed with a finnigan lcq ( ref . 26 ). full mass scanning ( m / z 100 - 350 ) and tandem ms analyses were performed in the negative ionization mode . bacterial killing . the bactericidal activity of myeloperoxidase was quantified by conventional procedures as described previously ( ref . 27 ). briefly , klebsiella pneumoniae were cultured in luria - bertani broth to mid - log growth phase , washed with pbs , and used at 10 9 bacteria / ml . bacteria were incubated with 5 nm enzyme , 5 nm h 2 o 2 , and 100 mm nacl in pbs ( ph 5 . 5 ) for 15 min at 37 ° c . serial dilutions were immediately spread on agar plates and the colony forming units determined after overnight incubation at 37 ° c . statistical analysis . the survival study was evaluated using a fisher &# 39 ; s exact p test . all other data were analyzed with a student &# 39 ; s t test . significance was accepted at p & lt ; 0 . 05 . myeloperoxidase deficiency increases mortality in the cecal ligation and puncture ( clp ) model of sepsis . to evaluate the potential role of the myeloperoxidase system in host defense , myeloperoxidase - deficient mice and a clinically relevant and widely utilized model of intra - abdominal infection and sepsis were used . in the cecal ligation and puncture ( clp ) model , the blind - ended cecum is ligated and punctured ( ref . 16 ), releasing intestinal microflora into the abdominal cavity . previous studies have demonstrated that 35 - 45 % of wild - type mice survive this procedure for longer than 7 days ( refs . 16 , 18 ). when myeloperoxidase - deficient mice were subjected to clp , none of the animals were alive five ( 5 ) days after surgery ( fig1 ). in contrast , 63 % of the wild - type littermate control mice were alive after five ( 5 ) days , and 38 % were alive after 1 week ( fig1 ; p = 0 . 02 ). neutrophils predominated in the cell populations elicited from both the wild - type mice ( n = 7 ; 77 ± 3 % neutrophils , 23 ± 3 % macrophages , 0 . 3 ± 0 . 2 % eosinophils ) and the myeloperoxidase - deficient mice ( n = 11 ; 77 ± 4 % neutrophils , 23 ± 3 % macrophages , 0 . 9 ± 0 . 5 % eosinophils ). red blood cells , cellular debris and microorganisms were also apparent in the peritoneal fluid after the mice were subjected to clp . importantly , the cellular response to sepsis of the myeloperoxidase - deficient animals was comparable to that of the wild - type animals . levels of free 3 - chlorotyrosine and 3 - bromotyrosine rise markedly during sepsis . to determine whether products of the myeloperoxidase system accumulate during sepsis , attention was directed to 3 - chlorotyrosine , a product of cytotoxic hocl ( ref . 28 ). to explore the physiological significance of bromination by myeloperoxidase , levels of 3 - bromotyrosine were also quantified . to recover free 3 - chlorotyrosine and 3 - bromotyrosine in the cellular and extracellular components of the lavage fluid , wild - type mice were subjected to clp . after 24 hours , inflammatory fluid was harvested from the peritoneum by lavage . the lavage fluid , which contained white blood cells , bacteria and inflammatory exudate , was frozen . to recover free amino acids , thawed fluid was centrifuged to pellet cellular debris and the supernatant was harvested . amino acids were derivatized with ethyl heptafluorobutyrate and mtbstfa and analyzed by gc / ms in the ec - nci mode . to prevent the generation of halogenated tyrosines during sample preparation ( ref . 24 ), a highly sensitive (& lt ; 100 amol ) and specific gc / ms method that avoids acidic conditions was used . ( refs . 24 , 25 ). the procedure detected compounds that exhibited major ions and retention times identical to those of authentic 3 - chlorotyrosine and 3 - bromotyrosine ( fig2 a ). selected ion monitoring showed that the ions derived from the amino acids co - eluted with those derived from 13 c - labeled internal standards ( fig2 a , b ). the identity of each compound was confirmed by comparison with authentic standards , using mtbstfa derivatives of each oxidized amino acid . to monitor any artifactual generation of halogenated amino acids , l -[ 13 c 9 , 15 n ] tyrosine was included in the samples and observation was made for l - 3 - chloro [ 13 c 9 , 15 n ] tyrosine or l - 3 - bromo [ 13 c 9 , 15 n ] tyrosine ( fig2 c ). artifact formation was negligible when either biological material or pure tyrosine was analyzed with this method . peritoneal fluid from the sham - operated animals contained only low levels of 3 - chlorotyrosine and 3 - bromotyrosine ( fig3 a , b ). fluid from the clp animals contained 16 - fold higher levels of 3 - chlorotyrosine ( p = 0 . 03 ) and 6 - fold higher levels of 3 - bromotyrosine ( p = 0 . 03 ). these observations indicate that generation of halogenating intermediates increases dramatically during acute inflammation induced by sepsis . myeloperoxidase generates 3 - chlorotyrosine and 3 - bromotyrosine during sepsis . to determine whether a pathway involving myeloperoxidase is responsible for the elevated levels of halogenated l - tyrosines seen in inflammatory exudates , levels of the amino acids in septic myeloperoxidase - deficient mice were quantified . whereas lavage fluid levels of free 3 - chlorotyrosine and 3 - bromotyrosine rose when the wild - type mice became septic , 3 - chlorotyrosine levels barely increased after the myeloperoxidase - deficient animals were subjected to clp ( p = 0 . 0004 ). the genetically altered mice also produced 59 % less 3 - bromotyrosine after clp than the wild - type mice ( p = 0 . 009 ). the myeloperoxidase - deficient animals used for these experiments had been backcrossed at least 4 generations into the c57b1 / 6j background . to ensure that variability in the animals &# 39 ; genetic background had no bearing on the results , clp was performed on two groups of wild - type mice that represented the two backgrounds of the myeloperoxidase - deficient mice . the c57bi / 6j ( n = 11 ) and sv129 / j ( n = 9 ) wild - type animals displayed similar patterns of tyrosine chlorination and bromination . myeloperoxidase was undetectable when peritoneal cells ( predominantly macrophages ) isolated from the sham - operated animals were immunoblotted . peritoneal cells from wild - type mice subjected to clp had markedly higher levels of immunoreactive myeloperoxidase , as would be expected for an acute neutrophilic inflammatory response . in contrast , peritoneal cells isolated from the septic myeloperoxidase - deficient mice contained no immunoreactive protein . these results indicate that the number of myeloperoxidase - containing cells increases markedly in the normal mouse peritoneum during sepsis and that myeloperoxidase - deficient mice lack immunoreactive enzyme . the decreased ability of myeloperoxidase - deficient animals to generate 3 - bromotyrosine suggested that myeloperoxidase produces brominating intermediates in vivo . alternatively , the genetic manipulation might have deleted eosinophil peroxidase as well as myeloperoxidase . in humans , the gene for this brominating enzyme lies near the myeloperoxidase gene , on chromosome 17 ( ref . 29 ). to exclude the possibility of a double deletion , peroxidase activity was measured in inflammatory cells isolated from wild - type and myeloperoxidase - deficient mice were subjected to clp . the wild - type cells generated a single major band of material with peroxidase activity that co - migrated with human myeloperoxidase . this material was undetectable in cells isolated from myeloperoxidase - deficient clp - treated mice . thus , the peroxidase in the inflammatory cells that appears in the peritoneum after clp is myeloperoxidase rather than eosinophil peroxidase . in contrast , extracts of white blood cells isolated from the peritoneal cavity of wild - type mice injected with thioglycollate produced two bands of peroxidase activity on non - denaturating polyacrylamide gel electrophoresis . the rapidly and slowly migrating bands co - migrated with human eosinophil peroxidase and human myeloperoxidase , respectively . cell extracts from the myeloperoxidase - deficient mice produced only the rapidly migrating band that corresponded to eosinophil peroxidase . these results suggest that both the wild - type mice and the genetically altered mice were able to make eosinophil peroxidase . collectively , these observations strongly suggest that myeloperoxidase was the enzyme that generated 3 - chorotyrosine and 3 - bromotyrosine in septic wild - type mice subjected to clp . physiological concentrations of br − modulate the bactericidal activity of myeloperoxidase in vitro . having shown that mice without myeloperoxidase are more vulnerable to sepsis than wild - type mice and also generate much lower levels of halogenated tyrosines , attention was focused directly at the effects of halides and myeloperoxidase on a bacterial pathogen , klebsiella pneumoniae . the complete myeloperoxidase - h 2 o 2 — cl − system , as described above , killed 47 % of the bacteria in the incubation mixture . adding a low concentration ( 1 μm ) of br markedly increased bacteria killing ( 72 % dead ); higher concentrations ( 10 μm ) of br − were completely inhibitory . therefore , physiologically plausible variations in [ br − ] markedly affect the ability of myeloperoxidase to kill k . pneumoniae in vitro . myeloperoxidase brominates tyrosine in vitro . exploration was made for possible mechanisms of this cytotoxic effect of bromide by determining whether the transhalogen pathway that brominates nucleosides and nucleobases ( ref . 12 ) can also halogenate tyrosine , which was brominated in the clp experiments . for these experiments , n - acetyl - l - tyrosine was used rather than l - tyrosine itself to avoid chloramine formation and to prevent the conversion of l - tyrosine to p - hydroxyphenylacetaldehyde ( ref . 22 ). after exposure of n - acetyl - l - tyrosine to enzyme , h 2 o 2 , and plasma concentrations of halide ( 100 mm cl − and 10 μm br − ), reverse - phase hplc detected early and late eluting products that respectively co - migrated with authentic n - acetyl - l - 3 - chlorotyrosine and n - acetyl - l - 3 - bromotyrosine ( fig4 ). negative - ion electrospray ionization tandem mass spectrometry confirmed the identities of the halogenated amino acids . both chlorination and bromination of n - acetyl - l - tyrosine were optimal under acidic conditions , but significant levels of the halogenated amino acids were also generated at neutral ph ( fig5 a ). under acidic ( ph 5 . 9 ) and neutral conditions , bromination by myeloperoxidase required both enzyme and h 2 o 2 ; it was inhibited by catalase ( a peroxide scavenger ), sodium azide ( a heme poison ), and taurine ( a scavenger of hypohalous acids ). myeloperoxidase brominates n - acetyl - l - tyrosine at physiologically plausible concentrations of halide ion . it was next determined whether myeloperoxidase prefers to use bromide or chloride when it oxidizes tyrosine under physiological conditions . in the presence of 100 mm cl − alone , n - acetyl - l - 3 - chlorotyrosine was the principal product when either reagent hocl or the myeloperoxidase - h 2 o 2 system oxidized n - acetyl - l - tyrosine under acidic ( fig6 a , b ) or neutral conditions . adding 1m levels of br − ( in the presence of 100 mm cl − ) to either oxidation system generated n - acetyl - l - 3 - bromotyrosine and caused a corresponding decrease in n - acetyl - l - 3 - chlorotyrosine production ( fig5 a , fig6 a , b ). the relative yields of n - acetyl - l - 3 - bromotyrosine and n - acetyl - l - 3 - chlorotyrosine depended on the ph and [ br − ] in the reaction mixture ( fig5 a ; fig6 a , b ). under mildly acidic conditions ( ph 5 . 9 ), taurine — a scavenger of hocl — inhibited the formation of n - acetyl - l - 3 - bromotyrosine by the hocl - cl − — br − system ( fig6 c ). in striking contrast , it stimulated n - acetyl - l - 3 - bromotyrosine formation by hypobromous acid ( hobr ), suggesting that bromamines are potent brominating agents . taurine also inhibited l - tyrosine bromination by the myeloperoxidase - h 2 o 2 — cl − — br − system ( fig6 d ). however , it failed to affect production of n - acetyl - l - 3 - bromotyrosine by the eosinophil peroxidase - h 2 o 2 — cl − — br − system ( fig6 d ). these observations strongly imply that myeloperoxidase first oxidizes cl − to hocl and that hocl then reacts with br to generate reactive brominating intermediates . under neutral conditions , taurine completely inhibited the formation of n - acetyl - l - 3 - bromotyrosine by the hocl — cl − — br − system but only partially inhibited the myeloperoxidase - h 2 o 2 — cl − — br − system ( fig5 c , d ). in contrast , it stimulated n - acetyl - l - 3 - bromotyrosine formation by hobr and had little affect on the eosinophil peroxidase - h 2 o 2 — cl − — br − system ( fig5 c , d ). these results suggest that myeloperoxidase oxidizes br − by two different pathways . in the pathway that operates under mildly acidic and neutral conditions , the enzyme initially generates hocl , which then oxidizes br − . in the pathway that operates only at neutral ph , myeloperoxidase directly oxidizes br . although the inventor is not bound by theory , it is believed that the foregoing results can be explained as follows : more than 30 years ago , klebanoff proposed that halogenating intermediates generated by myeloperoxidase are of major importance in killing bacteria ( ref . 30 ). however , the enzyme &# 39 ; s role in host defense against invading pathogens has remained unclear . the examples herein revealed that myeloperoxidase - deficient mice were more likely than wild - type mice to die after clp , a clinically relevant model of sepsis that releases bacteria into the peritoneum . this observation supports the understanding that myeloperoxidase generates bacterial cytotoxins in vivo . moreover , the enzyme was able to use halides to kill k . pneumoniae in vitro . it has also been discovered that myeloperoxidase - deficient mice are more likely than wild - type mice to die after k . pneumoniae is injected into the peritoneum . thus , myeloperoxidase appears critical to antibacterial defense mechanisms in mice . further evidence that myeloperoxidase contributes to antibacterial defense in vivo came from the observation that levels of 3 - chorotyrosine , one of the enzyme &# 39 ; s characteristic products , rise markedly in peritoneal lavage fluid after wild - type mice are subjected to clp . levels of 3 - bromotyrosine were also increased . septic mice that were deficient in myeloperoxidase failed to generate 3 - chlorotyrosine at levels above those observed in sham - operated animals , indicating that myeloperoxidase is the major source of chlorinating oxidants in this sepsis model . 3 - bromotyrosine levels were also markedly lower in the septic myeloperoxidase - deficient animals , though they were slightly higher than in non - septic wild - type mice , presumably because of eosinophil peroxidase activity . these observations indicate that myeloperoxidase generates chlorinating and brominating oxidants , whose production was previously ascribed solely to eosinophil peroxidase ( refs . 11 , 31 , 32 ). thus , both myeloperoxidase - dependent chlorination and myeloperoxidase - dependent bromination may represent physiologically relevant pathways for bacterial killing . hypothiocyanite and other oxidants derived from thiocyante may also play a role because this pseudohalide is present at high concentrations in extracellular fluids and is readily oxidized by peroxidases ( ref . 33 ). the halogenated intermediates produced by myeloperoxidase include hocl ( refs . 3 , 4 ), a potent cytotoxic oxidant that converts tyrosine to 3 - chlorotyrosine . the observations indicate that myeloperoxidase will also brominate tyrosine in vitro at plasma concentrations of halide ions . remarkably , the bromination pathway operates when cl − concentrations are 1 , 000 - fold to 10 , 000 - fold higher than br − concentrations . therefore , it is believed to be physiologically relevant . n - acetyl - bromotyrosine production by myeloperoxidase occurred at neutral ph but was optimal under acidic conditions . in vivo , therefore , myeloperoxidase might halogenate tyrosine extracellularly at neutral ph and also in the phagolysosome ( or hypoxic inflamed tissue ) under acidic conditions . at acidic ph , taurine almost completely inhibited bromination , suggesting that hocl is an intermediate in the pathway . in contrast , taurine only partly inhibited n - acetyl - l - bromotyrosine production by myeloperoxidase at neutral ph , suggesting the existence of a bromination pathway not involving hocl . one possibility for this second pathway is that myeloperoxidase might directly oxidize br − to hobr , as does eosinophil peroxidase ( refs . 11 , 20 ). at neutral ph and 10 μm br − , myeloperoxidase produced similar concentrations of n - acetyl - l - bromotyrosine and n - acetyl - l - chlorotyrosine , suggesting that the enzyme could produce halogenating intermediates in the extracellular environment . in contrast , wu et al . found that neutrophils generated protein - bound 3 - chlorotyrosine much more effectively than 3 - bromotyrosine ( ref . 34 ). one possible explanation for the discrepancy is that wu et al . studied halogenation of proteins , whereas the present experiments focused on halogenation of free amino acids . these observations indicate that mice lacking functional myeloperoxidase are more likely to die from polymicrobial sepsis , that in vivo levels of free 3 - chorotyrosine and 3 - bromotyrosine rise during sepsis , and that production of these halogenated amino acids is markedly reduced when myeloperoxidase is absent . thus , myeloperoxidase can produce a variety of chlorinating and brominating intermediates that appear capable of defending mice against clp - induced sepsis . in the enzyme &# 39 ; s absence , end products of these intermediates fail to accumulate in peritoneal fluid , and mice become more vulnerable to infection . various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention . it is intended that all such other examples be included within the scope of the appended claims . thus , the presence and level of the 3 - chlorotyrosine and 3 - bromotyrosine in the sample of body fluid or tissue also can be determined by procedures other than gc / ms as specifically illustrated herein . for example , the level of these markers can be determined by conventional immunoprecipitation procedures in an immunoassay with polyclonal or monoclonal antibodies to the marker . one - and two - site radioimmunoassay and enzyme immunoassays , e . g ., enzyme - linked immunosorbent assay ( elisa ) procedures as described by engvall and perlmann , j . immunol ., vol . 109 , pp . 129 - 135 ( 1972 ), can be used for determination of the level of the marker . monoclonal antibodies for use in immunoassay procedures can be prepared by conventional hybridoma methodology as described by kohler and milstein , nature , vol . 256 , pp . 495 - 497 ( 1975 ), and eur . j . immunol ., vol . 6 , pp . 511 - 519 ( 1976 ), and goding , “ monoclonal antibodies : principles and practice ”, academic press inc ., new york ., 1983 . 3 . harrison , j . e . & amp ; 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