m1dg ( 3-(2-deoxy--d - erythro - pentofuranosyl)-pyrimido[1,2-]purine-10(3h)-one ) is one of many endogenous exocyclic lesions derived 
 from the reaction of dna with bifunctional electrophiles generated 
 by lipid , protein , or dna peroxidation .
being an endogenous constituent 
 of human and rodent genomes , m1dg is mutagenic in 
 bacterial and mammalian cells and leads to mispairing 
 when replicated in vitro by multiple different dna polymerases .
we have previously developed 
 a monoclonal antibody against m1dg and incorporated the antibody 
 into analytical schemes using immunoaffinity purification of m1dg followed by mass spectrometric detection .
these studies indicated that adult humans excrete m1dg 
 at a rate of 12 fmol / kgd in urine .
subsequent investigations into the metabolism and elimination 
 of m1dg revealed that administration of m1dg 
 to rodents at doses from 8 mg / kg to 6 pg / kg leads to its rapid disappearance 
 from plasma due to oxidation and formation of the single , stable metabolite , 
 6-oxo - m1dg ( 3-(2-deoxy--d - erythro - pentofuranosyl)-pyrimido[1,2-]purine-6,10(3h,5h)-dione ) . in both humans and rats ,
xanthine 
 oxidase appears to be responsible for this oxidation , while aldehyde 
 oxidase also plays a role in m1dg metabolism in humans .
because 6-oxo - m1dg is the sole metabolite of m1dg in rats , it appears to be an ideal surrogate for m1dg as an in vivo biomarker for oxidative stress .
all studies 
 of m1dg metabolism in vivo have been conducted 
 with exogenously administered deoxynucleoside . 

thus , it is not known if 6-oxo - m1dg is actually present 
 in unadulterated intact animals . in order to address this question
, 
 we developed a highly specific monoclonal antibody ( mab ) against 6-oxo - m1dg and covalently linked the antibody to sepharose beads . 

the resultant antibody - sepharose matrix ( the gel ) was used for the 
 immunoaffinity purification of 6-oxo - m1dg from urine and 
 feces followed by lc - ms / ms quantification against the stable isotope - labeled 
 internal standard , [ n5]-6-oxo - m1dg . utilizing this method , we report for the first time that 6-oxo - m1dg is endogenously produced in rodents and is excreted in 
 urine and feces .
na2co3 , 
 nahco3 , nacl , water , acetonitrile , 
 mariculture keyhole limpet hemocyanin ( mcklh ) , hcl , acetic acid , and 
 methanol were obtained from thermo fisher scientific ( waltham , ma , 
 usa ) .
abts ( diammonium salt ) , thimerosal , kh2po4 , na2hpo4 , kcl , 30% h2o2 , tween 20 , bovine serum albumin ( bsa ) , dimethylformamide ( dmf ) , 
 dimethylsulfoxide ( dmso ) , k2co3 , sodium periodate , 
 sodium methoxide , nabh4 , hypoxanthine , xanthosine , xanthine , 
 ethyl cis-3-iodoacrylate , ethyl cis-3-bromoacrylate aminopterin , ethanolamine , cyanogen bromide - activated 
 sepharose beads , diethylene glycol , 2-deoxyguanosine , sodium 
 hydroxide , formic acid , sodium azide , glycine , and citric acid were 
 obtained from sigma - aldrich ( st . louis , mo , usa ) .
horseradish peroxidase - conjugated 
 goat antimouse igg ( h + l ) [ 0.8 mg / ml ] and igg ( fc ) were purchased 
 from jackson immunoresearch ( west grove , pa , usa ) .
phosphate buffered 
 saline ( pbs ) was purchased from invitrogen ( carlsbad , ca , usa ) .
the synthesis of 
 6-oxo - m1guo was based on a previously described synthetic 
 scheme with some modifications .
guanosine 
 was dissolved in a minimum amount of a dmf / dmso mixture ( 1:1 , v / v ) . 

k2co3 ( 1.5 equiv ) was added to the solution 
 and the reaction mixture was held at 6065 c for 18 h. 
 ethyl cis-3-iodoacrylate ( 0.25 equiv per h for 5 
 h ) was added to the reaction mixture .
the residue was dissolved into a 
 minimum amount of methanol , and sodium methoxide ( 1.5 equiv ) was added 
 ( dropwise in a 0.5 m solution ) .
6-oxo - m1guo was purified 
 on biotage sp1 flash chromatography system ( biotage , uppsala , sweden ) 
 using the following gradient ; 1% to 5% acetonitrile in 30 column volumes 
 followed by a 5% to 15% gradient in 10 column volumes .
separation 
 was achieved on a 12 + m c18 column , and the purified compound showed 
 a single sharp peak by reverse - phase hplc .
h nmr ( 600 
 mhz , dmso - d6 ) :  8.65 ( d , 1h , h8 , j = 7.8 hz ) , 8.29 ( s , 1h , h2 ) , 6.24 ( d , 1h , h7 , j = 8.4 hz ) , 5.82 ( d , 1h , h1 ) , 5.48 ( d , 1h , h2oh ) , 
 5.20 ( d , 1h , h3oh ) , 5.03 ( d , 1h , h5oh ) , 
 4.49 ( d , 1h , h2 ) , 4.15 ( d , 1h , h3 ) , 3.91 ( d , 1h , h4 ) , 
 3.633.55 ( m , 2h , h5 ) . the h nmr may be 
 seen in figure a of the supporting information .
6-oxo - m1dg and its stable isotope - labeled analogue , 
 [ n5]-6-oxo - m1dg , were synthesized 
 as previously described .
briefly , anhydrous 
 dg ( 1.6 g , 6 mmol ) and anhydrous k2co3 ( 0.93 
 g , 6.75 mmol ) were dissolved in anhydrous dmf ( 18 ml ) .
one milliliter 
 of a 0.55 m solution of ethyl cis-3-bromoacrylate 
 in anhydrous dmf was added to the reaction every 15 min for 4 h. the 
 reaction was stirred for an additional 2 h then cooled and filtered . 

the filtrate was evaporated under high vacuum and the residue was 
 dissolved in water ( 4 ml ) and 5% acetic acid ( 4 ml ) .
the product was 
 purified by reversed - phase ( c18 ) medium pressure liquid chromatography 
 with a biotage sp1 apparatus ( biotage ) using a prepacked 25  
 75 mm biotage flash 25 + s cartridge ( kp - c18-hs ) and a flash 25 c18 
 samplet for sample loading .
a pure final product , 6-oxo - m1dg ( 0.92 g , 50.6% ) , was obtained .
[ n5]-6-oxo - m1dg was synthesized by substituting [ n5]-dg for dg ( cambridge isotope laboratories , andover , ma , usa ) and 
 employing the scheme described above .
the h nmr of 6-oxo - m1dg and its stably labeled analogue matched the spectrum previously 
 reported for this compound .
mass spectrometric 
 analysis of 6-oxo - m1dg showed an [ m + h ] peak 
 at m / z 320 , while analysis of [ n5]-6-oxo - m1dg showed an [ m + h ] peak at m / z 325 .
the m / z = 325 corresponds to the 6-oxo - m1dg value plus 5 additional mass units from the 5 n atoms incorporated into the [ n5]-dg starting 
 material .
a m / z 320 peak was not 
 observed for [ n5]-6-oxo - m1dg , indicating 
 that no unlabeled 6-oxo - m1dg was present in the internal 
 standard .
6-oxo - m1guo ( 12 mg ) was dissolved in 750 l of 100 
 mm aqueous sodium periodate .
protein ( 20 mg of bsa or mcklh ) was reconstituted 
 with 700 l of pbs preadjusted to a ph of 9.5 with 5% k2co3 . the 6-oxo - m1guo solution and the 
 protein
after 1 h , 45 l of a 1 
 m diethylene glycol solution was added to the mixture ( to quench excess 
 oxidizing agent ) followed by 700 l of 0.45 m nabh4 ( aqueous ) .
after another 12 h , the ph of the mixture was adjusted 
 to 7.0 with 1.0 m formic acid .
the mixture was kept at this 
 ph for 1 h. then , the ph was increased to 8.5 by the careful addition 
 of 1 m aqueous ammonium hydroxide solution .
this mixture was dialyzed 
 against pbs buffer twice for 24 h. the sample was lyophilized and 
 stored at 4 c .
scheme 1 shows the conjugation 
 reaction of 6-oxo - m1guo with a lysine residue of the carrier 
 protein .
figure b of the supporting information depicts the scheme for preparation of the conjugated protein .
four balb / cj 
 mice and four a / j mice ( the jackson laboratory , bar harbor , me , usa ) 
 were injected subcutaneously with 50 g of 6-oxo - m1guo - klh and freund s complete adjuvant ( primary boost ) .
four 
 wk after the initial immunization , the mice were boosted subcutaneously 
 ( first boost ) with the same dose of conjugate , but with the substitution 
 of incomplete adjuvant ( which was also used in subsequent boosts ) . 

two wk after the first boost , the mice were tail bled and antibody 
 titers were assessed by direct and competitive elisa as described 
 below .
a second boost was administered 4 wk subsequent to the first , 
 and after 2 wk , sera were again extracted and subjected to elisa evaluation . 

a third boost was administered 25 wk after boost two , and the sera 
 were collected and screened approximately 2 wk later . on the basis 
 of the cumulative elisa data , a single balb / cj mouse ( balb / cj r ) showing 
 the most selective and concentrated anti-6-oxo - m1dg titer 
 was chosen for a fourth and final boost given intraperitoneally and 
 lacking adjuvant to prepare it for splenocyte extraction .
four days 
 after the final boost , the mouse was sacrificed by cervical dislocation 
 and the spleen harvested .
splenocytes were isolated and subjected 
 to polyethylene glycol - mediated fusion with both sp/20 and ns1 murine 
 myeloma cells ( obtained from the university of virginia and the university 
 of nebraska medical univeristy school of dentistry , respectively ) 
 and allowed to recover for 24 h in liquid culture .
the products of 
 the fusion were evenly distributed into 24 96-well plates , with 12 
 plates corresponding to fusions of sp/20 cells and splenocytes and 
 12 plates corresponding to fusions of ns1 cells and splenocytes .
cells 
 were grown in iscove s modified dulbecco s medium with 
 20% fetal bovine serum ( invitrogen ) .
hybridomas were selected by growing 
 in the presence of aminopterin ( 5  10 m , 
 sigma ) and ht supplement ( 1:100 dilution , invitrogen ) for 14 days 
 with media and aminopterin replenishment every 3 days .
supernatant 
 was removed and screened for antigen - specific antibodies 
 by elisa , with 6-oxo - m1dguo - bsa conjugate used as the antigen 
 ( described below ) .
positive hybridoma supernatants were rescreened 
 in the presence of xanthine and 6-oxo - m1dg .
xanthine was 
 employed as a competitor to identify antibodies not specific to 6-oxo - m1dg .
clones exhibiting anti-6-oxo - m1dg activity 
 and specificity in addition to positive growth and productivity were 
 selected and plated on 24-well plates . of these
, five clones were 
 chosen based on activity , specificity , growth , and productivity as 
 determined by direct and competitive elisa analysis ( described below ) 
 and then subsequently subjected to two rounds of subcloning .
the clones 
 were subsequently cryopreserved . under vanderbilt institutional care and use committee 
 protocol # m-07 - 109 ,
the vanderbilt antibody and protein resource core 
 is permitted to use freund s complete adjuvant in the manner 
 described above .
the following solutions were prepared 
 as follows and their use is described below : ( a ) carbonate - bicarbonate 
 coating buffer ( ph 9.6 ) was prepared from na2co3 ( 1.59
g / l ) , and thimerosal ( 0.10 g / l ) ; 
 ( b ) pbs - tween ( ph 7.4 ) was prepared from nacl ( 8.00
g / l ) , tween 20 ( 1.00 ml / l ) , and thimerosal ( 0.10 g
/ l ) ; ( c ) 
 bsa ( 5.0 g ) mixed in pbs - tween ( 500 ml ) ; ( d ) 1 nm abts solution in 
 70 mm citrate - phosphate buffer ( ph 4.2 ) was prepared from citric acid 
 ( 5.64
elisa quality microtiter plates 
 ( thermo fisher scientific ; 96-well immulon 2hb flat bottom microtiter 
 plates or 384-well 4hb flat bottom plates ) were coated with 50 , 75 , 
 or 100 l of a 10 g / ml solution of 6-oxo - m1guo - bsa antigen in carbonate - bicarbonate coating buffer ( a ) and incubated 
 at 4 c overnight .
plates were washed three times with 100 l 
 of pbs - tween ( b ) using a bio - tek elx405 automatic microplate washer 
 ( winooski , vt , usa ) , then incubated with 100 l of pbs - tween 
 ( b ) for 30 min at 37 c .
aliquots of murine serum dilutions , hybridoma supernatants , 
 or purified antibody ( depending on the stage of the antibody development 
 process ) were then incubated in the coated wells with or without the 
 addition of varying concentrations of 6-oxo - m1dg and structural 
 analogues ( figure 1 ) in a final volume of 100 
 l pbs for 60 min at 37 c . for wells that included
6-oxo - m1dg or structural analogues , the serum dilution used was 1:5000 
 in pbs - tween ( b ) , and the compounds depicted in figure 1 were preincubated in the dilute murine serum for 45 min prior 
 to the serum addition step .
this allowed the nonspecific antibodies 
 to bind the structural analogues , theoretically leaving behind only 
 6-oxo - m1dg - specific antibodies .
initial analysis revealed 
 that xanthine was recognized nearly as well as 6-oxo - m1dg , which led to the use of this structural analogue in further competition 
 studies .
6-oxo - m1dg , m1dg , and structural analogues 
 that were used as competitive antigens during elisa analysis of murine 
 sera , hybridomal supernatant , and purified antibodies .
the plates were then washed three times with pbs - tween 
 ( b ) .
aliquots 
 of diluted horseradish peroxidase - conjugated goat antimouse igg fc 
 region - specific or igg ( h + l ) secondary antibody ( diluted 1:5000 
 in pbs - tween / bsa ) were added , and the plates were incubated at 37 
 c for 60 min . after 60 min , the plates were washed three times 
 and 100 l of a freshly prepared abts solution ( 1.8 l 
 of h2o2 per 1 ml of abts ) was immediately added 
 to each well . to determine peroxidase activity ,
the absorbance at 
 414 nm was measured after 15 and 30 min for each well using a bio - tek 
 powerwave ht 340 plate reader with gen5 software .
selected 
 clones were scaled up , inoculated into one - liter bioreactors ( wilson 
 wolf manufacturing , new brighton , mn , usa ) and grown for 34 
 wk .
purification of mab from the bioreactor supernatants was achieved 
 by affinity chromatography on protein - g sepharose ( ge healthcare , 
 piscataway , nj , usa ) followed by a final desalting step into pbs . 
 purified mab were isotyped and subsequently quantified by sds - page 
 electrophoresis followed by infrared coomassie staining .
purified anti-6-oxo - m1dg mab was loaded onto pd-10 columns ( ge healthcare ) equilibrated 
 with 25 ml coupling buffer ( 0.1 m nahco3 , 0.5 m nacl , ph 
 8.0 in water ) .
cyanogen bromide - activated sepharose beads 
 ( 3.75 g ) were prepared for conjugation by rapid swelling in 1 mm hcl 
 followed by filtering , rinsing with 400 ml of 1 mm hcl and then 10 
 ml of coupling buffer .
the sepharose beads were transferred to the 
 antibody solution and gently rotated at 4 c for about 20 h. 
 the mixture was then filtered and washed with 500 ml of coupling buffer , 
 and the residue was divided into four parts .
each quarter was placed 
 in 14 ml of ethanolamine solution ( 1.0 m , ph 8.0 ) and gently rotated 
 overnight at 4 c .
the sepharose beads were then filtered and 
 washed with a low ph acetate buffer ( 0.1 m acetic acid , 0.1 m naoh , 
 and 0.5 m nacl ) followed by a 0.1 m tris hcl buffer ( 0.5 m nacl , ph 
 8.0 ) .
each wash used 75 ml of buffer , and the wash cycle was repeated 
 two more times .
the resulting residue was suspended in 20 ml of 0.1 
 m tris buffer with 0.5 m nacl and 0.2% nan3 ( ph 8.0 ) , and 
 the final product ( the gel ) was stored at 4 c .
the 
 gel was used to recover 6-oxo - m1dg and [ n5]- 6-oxo - m1dg from various fluids by the following 
 general procedure .
an aliquot of gel was prepared for use by rinsing 
 with the following solutions : 50 ml of 0.1 m glycine ( ph 2.7 ) , 20 
 ml h20 , 50 ml methanol , and 50 ml pbs .
this cleaning procedure was necessary 
 to remove any 6-oxo - m1dg that had bound to the mab during 
 preparation .
the ph of the fluid to be analyzed was adjusted 
 to 7.08.0 with acetic acid or ammonium hydroxide .
an aliquot 
 of the washed and reconditioned gel was added to the sample , and the 
 suspension was mixed gently for 14 h. the sample / gel suspension 
 was filtered through a precleaned empty polypropylene spe cartridge 
 fitted with a polypropylene frit ( sigma - aldrich ) .
the filtered gel 
 was washed with 10 ml of pbs , and the analytes were eluted with 2 
 ml of methanol .
the eluant was dried under n2 gas , capped , 
 and stored at 20 c .
the sample was reconstituted in 
 9:1 water / methanol ( v / v ) immediately prior to lc - ms / ms analysis .
urine 
 and feces were collected at 24 h intervals and stored at 20 
 c following collection .
urine samples were analyzed by thawing , 
 adding 200500 fmol of [ n5]-6-oxo - m1dg , and centrifuging the samples at 4000 g for 25 min at 4 c .
an aliquot of supernatant was diluted 1015-fold 
 with pbs , and the ph of the final solution was adjusted to 7.5  
 0.5 with either acetic acid or sodium hydroxide .
the gel ( prepared 
 as described above ) was introduced after ph adjustment , and the suspension 
 was rotated gently end - over - end for 14 h. the suspension was 
 filtered and the analyte eluted from the gel as described above .
the weighed sample was transferred 
 to a clean vessel with 40 ml of pbs , and 200500 fmol of [ n5]-6-oxo - m1dg were added .
dispersion 
 of the sample was accomplished by rotating end - over - end for 23 
 h followed by sonication for 35 min .
the suspension was then 
 filtered through a mesh screen , with the collected solid material 
 rinsed with 10 ml pbs .
the supernatant was transferred 
 to a clean vessel , diluted roughly 5 times with pbs , and the antibody 
 gel ( prepared as described above ) was added .
the suspension was rotated 
 gently end - over - end for 14 h , filtered , and the analyte eluted 
 from the gel as described above .
final lc - ms / ms analysis of gel - purified 
 samples was accomplished on a thermo surveyor autosampler and ms pump 
 in - line with a thermo quantum triple quadrupole mass spectrometer 
 ( thermo fisher scientific ) .
the mass spectrometer was equipped with 
 an electrospray source and operated in positive ion mode .
6-oxo - m1dg and its internal standard , [ n5]-6-oxo - m1dg , were detected via selected reaction monitoring ( srm ) with 
 the following transitions , respectively ; m / z 320  204 and 325  209 . in both cases
, 
 the transition corresponds to cleavage of the glycosidic bond and 
 loss of the deoxyribose moiety , with the positive charge remaining 
 on the base .
the analytes were chromatographed on the following 
 reverse - phase gradient system ; 2% b to 15% b in 5 min , 15% b to 80% 
 b in 1.5 min , followed by a 1 min hold at 80% b. component a was water 
 and component b was methanol / acetonitrile ( 3:1 ) , and each component 
 contained 0.1% acetic acid ( v / v ) .
the column used was a phenomenex c18 ( phenomenex , torrence , ca , usa ; 
 10.0  0.2 cm , 3 m ) held at 3540 c .
the 
 column was equilibrated at the initial conditions for 3 min prior 
 to each injection .
6-oxo - m1dg was 
 quantitated by stable isotope dilution against [ n5]-6-oxo - m1dg .
lc - ms / ms data was acquired and processed 
 by xcalibur software ( thermo fisher scientific ) .
the overall scheme of the antibody generation and isolation process 
 is depicted in figure 2 .
eight mice ( four balb / cj 
 and four a / j ) were injected with 6-oxo - m1 g conjugated to 
 klh as described in the materials and methods section . at 6 , 10 , and 35
wk after the initial innoculation , the 
 mice were tail bled , and direct and competitive elisa analyses of 
 the sera were performed ( figure 2 , step 1 ) . 

all three bleeds from all 8 mice showed robust responses against the 
 6-oxo - m1g - bsa antigen , indicating that the immune systems 
 of the mice were producing significant amounts of antibodies against 
 the 6-oxo - m1 g portion of the innoculant .
competitive 
 elisa screenings were executed to assess antibody specificity , with 
 6-oxo - m1dg and a series of structural analogues ( figure 1 ) serving as competing antigens .
eight mice were inoculated with adduct / protein conjugate , followed 
 by tail bleeding and testing for the presence of 6-oxo - m1dg antibodies using direct elisa analysis ( step 1 ) .
splenocytes from 
 a single mouse , balb / cj r , were fused with myeloma cells , plated on 
 twenty - four 96-well plates , and screened using direct and competitive 
 elisa analyses ( step 2 ) . from this screen ,
five parental cell lines were chosen for subcloning 
 and further screening ( step 4 ) .
on the basis of these final screenings , 
 the cell line 6c9ba4c12 was chosen for scale - up and antibody purification 
 ( step 5 ) .
figure 3 depicts a competitive 
 elisa analysis 
 of the 35-week serum from mouse balb / cj r. this analysis employed 
 6-oxo - m1dg as the competing antigen and a robust concentration 
 dependent decrease in optical density is observed , indicating the 
 presence of anti-6-oxo - m1dg antibodies in the sera from 
 this mouse .
the results of similar analyses of the sera of all 8 inoculated 
 mice can be seen in supporting information figure c. supporting information figure 
 d displays the results from competitive elisa analyses of the 35-week 
 serum from all 8 inoculated mice where m1dg is employed 
 as the competing antigen . increasing amounts of m1dg do 
 not result in a decrease in optical density , indicating that m1dg does not bind to the anti-6-oxo - m1dg antibodie(s ) 
 present in the sera .
6-oxo - m1dg antibodies are present in the serum 
 of mouse 
 balb / cj r. the serum of mouse balb / cj r was subjected to competitive 
 elisa analysis with 6-oxo - m1dg used as the competitor . 

the displayed results represent the average of spectroscopic readings 
 at 15 and 30 min postaddition of abts substrate . on the basis of the elisa analysis of murine sera , 

a single mouse 
 ( balb / cj r ) was chosen for hybridoma production .
splenocytes from 
 balb / cj r were fused with both sp/20 and ns1 myeloma cells ( figure 2 , step 2 ) , and 1670 hybridomas were formed from 
 2304 fusions wells .
elisa analysis revealed that of the 1670 hybridomas , 
 180 showed production of anti-6-oxo - m1dg antibodies .
these 
 were grown in 24-well plates ( figure 2 , step 
 3 ) , and the supernatants were subjected to further elisa screenings . 

on the basis of the screening data , five hybridomas were selected 
 for subcloning and further screenings ( figure 2 , step 4 ) .
figure 4 shows a representative 
 elisa analysis 
 of the supernatants from two subclones ( b and e2 ) of parental hybridoma 
 cell line 6c9 in which xanthine and 6-oxo - m1dg are employed 
 as competing antigens .
the presence of unbound 6-oxo - m1dg decreases the optical density of both b and e2 supernatants , indicating 
 the presence of anti-6-oxo - m1dg antibodies . for subclone 

e2 , xanthine has a similar effect , demonstrating that e2 s 
 mab are not specific to 6-oxo - m1dg . however , xanthine has 
 no effect on the optical density of subclone b supernatant , suggesting 
 that mab from subclone b are specific to 6-oxo - m1dg .
on 
 the basis of these data , subclone b was subjected to further subcloning 
 but e2 was not . supporting information figure 

e illustrates that cell line 6c9ba4 , a subclone of hybridoma 6c9b , 
 retained activity against 6-oxo - m1dg but did not react 
 to xanthine .
the specificity of antibodies produced by two hybridoma cell lines 
 ( b and e2 ) subcloned from the parental hybridoma cell line 6c9 were 
 assessed using competitive elisa analysis .
the displayed results 
 represent the average of spectroscopic readings of subclones e2 and 
 b at 15 and 30 min postaddition of abts substrate . on the basis of a review of the cumulative elisa 
 data ,
a final 
 daughter cell line was selected from each parental hybridoma line , 
 and these daughter cell lines were cryopreserved .
the elisa data indicated 
 that antibodies from the cell line 6c9ba4c12 displayed the most promising 
 expression and specificity .
thus , this cell line was subjected to 
 large - scale expression and antibody purification .
figure 5 shows 
 the elisa responses of the 
 mab purified from 6c9ba4c12 in the presence of 6-oxo - m1dg and structural analogues .
only 6-oxo - m1dg attenuated 
 the optical density , indicating a specificity of the purified mab 
 for 6-oxo - m1dg over close structural analogues .
purified antibodies from the hybridomal subclone ( 6c9ba4c12 ) 
 were screened for specificity for 6-oxo - m1dg in the presence 
 of structural analogues using competitive elisa analyses . the purified anti-6-oxo - m1dg mab was covalently linked 
 to sepharose beads as described above .
the purification and analysis 
 protocol outlined in materials and methods was used to quantify aliquots of 10 mm potassium phosphate ( ph 8.0 ) 
 spiked with varying amounts of 6-oxo - m1dg ( 0.25 , 2.5 , and 
 25 pmol ) and 1.0 pmol of the internal standard , [ n5]-6-oxo - m1dg ( n = 3 for each concentration 
 of 6-oxo - m1dg ) .
as shown in table 1 , the experimentally determined amount of 6-oxo - m1dg was 
 within 12% of the known amount at each level .
the average percent 
 recovery of 6-oxo - m1dg ranged from 94% to 74% , while the 
 internal standard was recovered at an average rate of 79% .
thus , the 
 gel was able to bind 6-oxo - m1dg from an aqueous solution 
 and released the analyte in the presence of methanol .
the amount detected , % accuracy , 
 and % recovery values are shown as the mean  s.d .
the limit of detection ( lod ) of the described lc - ms / ms 
 system was 
 established at approximately 10 fmol 6-oxo - m1dg on - column 
 ( supporting information figure f , top ) 
 by analyzing a series of increasingly dilute 6-oxo - m1dg 
 solutions . given this lod and the percent recovery observed from urine 
 and fecal samples ( approximately 30% ) , the limit of quantification 
 of the assay was estimated to be 50 fmol per sample . additionally , 
 the lc - ms / ms system provided a linear response to 6-oxo - m1dg solutions over a 5000-fold concentration range and a single [ n5]-6-oxo - m1dg concentration ( supporting information f , bottom ) .
these 
 results indicate that the gel provided a sufficient level 
 of recovery of the analyte and its internal standard as well as sufficient 
 capacity for the expected levels of 6-oxo - m1dg present 
 in rat urine and feces .
they also demonstrate that the lc - ms / ms analysis 
 of the purified sample provided sensitive and accurate quantification . urine and feces collected from male sprague 
dawley 
 rats over 3 days were analyzed as described in the materials and methods section .
6-oxo - m1dg was detected 
 in urine from only one subject , animal 3 , at a rate of 188 fmol / kgd . 
 however
, 6-oxo - m1dg was eliminated in feces in all the 
 animals at a rate of 3501893 fmol / kgd .
thus , it appears 
 fecal elimination is the main route by which that 6-oxo - m1dg is excreted from the body .
values are given as the mean 
 amount observed in fmol / kgd  s.d .
( n = 35 for feces and 2 for urine ) . while the amounts of 6-oxo - m1dg are low , 
 the assay provided 
 a sufficient signal - to - noise ratio to permit accurate quantification . 

the upper trace ( m / z 320  204 ) shows the analyte , while the bottom 
 trace ( m / z 325  209 ) represents 
 the [ n5]-6-oxo - m1dg internal standard . 

the inset of figure 6 is 
 the internal standard alone after recovery from pbs .
there is no peak 
 in the 6-oxo - m1dg trace of the inset , which is illustrative 
 of the fact that there was no isotopic impurity in the internal standard 
 that could contribute to the 6-oxo - m1dg signal .
a representative 
 lc - ms / ms chromatogram of 6-oxo - m1dg and the internal standard 
 [ n5]-6-oxo - m1dg isolated from rat 
 feces is displayed .
m1dg is an endogenous dna adduct 
 detectable in the genomic 
 dna of humans and rodents .
its presence is associated 
 with peroxidative damage to cellular constituents , particularly polyunsaturated 
 fatty acids , and dna .
although many studies have shown that the lipid 
 peroxidation product , malondialdehyde , can react in vitro with deoxyguanosine 
 to form m1dg , recent studies 
 in e. coli , with defined polyunsaturated fatty acid 
 content in membrane phospholipids , indicate that this is not a major 
 source of m1dg . rather , the 
 dna peroxidation products , base propenals , appear to account for nearly 
 all of the m1dg generated when bacteria are exposed to 
 peroxynitrite ( onoo ) .
thus , although m1dg appears to be a reliable marker of 
 oxidative damage to cells , it primarily reflects oxidative damage 
 to dna .
m1dg is repaired by nucleotide excision repair and is excreted in urine at rather low levels 
 in humans ( 12  3.8 fmol / kgd ) .
a major contributor to the low levels of m1dg is its 
 oxidative metabolism to 6-oxo - m1dg .
this suggests 
 6-oxo - m1dg may be superior to m1dg as a noninvasive 
 marker of oxidative damage . in the present manuscript
, we describe 
 the generation of a monoclonal antibody that exhibits sensitive and 
 specific binding to 6-oxo - m1dg and its use in a quantitative 
 assay suitable for analysis of urine and feces . in the assay
, 
 the sepharose - bound mab is used to isolate 6-oxo - m1dg from 
 urine and feces .
the purified sample is subjected 
 to lc - ms / ms analysis where 6-oxo - m1dg is quantified via 
 stable isotope dilution against the synthetic internal standard , [ n5]-6-oxo - m1dg , which is introduced 
 to the sample prior to antibody purification .
the antibody gel 
 shows excellent recovery of 6-oxo - m1dg from buffer solutions . 

the analyte is recovered at 74% or greater 
 from 10 mm potassium phosphate over a 100-fold range of 6-oxo - m1dg , 0.25 pmol to 25.0 pmol .
the internal standard was recovered 
 at 79% ( 1.0 pmol ) from these same solutions , and spiked buffer solutions 
 were quantified accurately to 12% .
these data demonstrate that 
 100 l of the antibody gel is able to bind at least 26 pmol 
 of 6-oxo - m1dg from solution , and lc - ms / ms detection of 
 the purified sample is quantitative .
the recovery of [ n5]-6-oxo - m1dg from saline solutions was greater 
 than 78% but its recovery from 
 urine and feces was only 29% .
this suggests a matrix effect 
 where other nucleosides , deoxynucleosides , and/or endogenous congeners 
 compete with 6-oxo - m1dg for antibody binding sites .
although 
 there was minimal cross - reactivity of the antibody with individual 
 members of a panel of exocyclic adducts and purine oxidation products 
 ( figures 1 and 5 ) , it 
 is possible that high levels of low - avidity nucleosides in urine and 
 feces compete with the low levels of endogenous 6-oxo - m1dg for binding sites on the mab .
6-oxo - m1dg was 
 detected in the feces of each of three 
 different sprague 
dawley rats but was detected in the urine 
 of only one of the rats ( table 2 ) .
the fecal 
 levels of 6-oxo - m1dg ( 3501893 fmol / kgday ) 
 were significantly higher than the urinary levels ( 188 fmol / kgday ) . 

this is consistent with previous findings that demonstrate m1dg is excreted mainly in urine , whereas 6-oxo - m1dg is 
 excreted mainly in feces . because 6-oxo - m1dg is present endogenously in rats , 
 it suggests that its metabolic precursor , m1dg , is also 
 produced endogenously in rats and metabolized to 6-oxo - m1dg .
this idea is consistent with the finding that m1dg 
 is present in rodent and human genomic dna and 
 with studies from this laboratory demonstrating that 6-oxo - m1dg is the sole metabolite of m1dg .
it may be argued that endogenous 6-oxo - m1dg is generated 
 in the nucleoside pool rather than arising from direct damage to dna
. 
 however , this seems unlikely if one considers that base propenals 
 are the principal precursors to m1dg .
base propenal formation is triggered by abstraction of 
 the hydrogen from the c-4 position of the deoxyribosyl unit 
 of double - stranded dna .
the base propenal produced then reacts with 
 a deoxyguanosine residue in duplex dna to form m1dg . in 
 comparison ,
the yields of base propenals are much lower when deoxynucleosides 
 are oxidized instead of duplex dna .
furthermore , m1dg formation 
 as a reaction of base propenals and free deoxyguanosine is a bimolecular 
 process .
if base propenals are formed in duplex dna , they are generated 
 in close proximity to deoxyguanosine residues in the dna duplex , which 
 should maximize the probability of their reaction with a deoxyguanosine 
 residue .
one or both of these compounds 
 may have been ingested , 
 then excreted or metabolized .
it was not possible to control for this 
 possibility under the reported experimental regime , so future studies 
 with animals fed nucleic acid - free diets will be required to test 
 this hypothesis .
the ability to measure excreted 6-oxo - m1dg has the potential 
 for clinical relevance in assessing the levels of oxidative dna damage 
 in humans .
since the basal levels of both deoxynucleoside adducts 
 are low , they should provide high sensitivity to increases triggered 
 by oxidative damage .
given that the analytical method described herein 
 is applicable to feces and urine , the possibility exists for preclinical 
 or clinical studies conducted in a noninvasive fashion .
 oxidative stress triggers dna and lipid peroxidation , 
 leading to 
 the formation of electrophiles that react with dna to form adducts . 
 
 a product of this pathway , ( 3-(2-deoxy--d - erythro - pentofuranosyl)-pyrimido[1,2-]purine-10(3h)-one ) , or m1dg , is mutagenic in bacterial and 
 mammalian cells and is repaired by the nucleotide excision repair 
 pathway . in vivo , m1dg is oxidized to a primary metabolite , 
 ( 3-(2-deoxy--d - erythro - pentofuranosyl)-pyrimido[1,2-]purine-6,10(3h,5h)-dione , or 6-oxo - m1dg , 
 which is excreted in urine , bile , and feces . 
 we have developed a specific 
 monoclonal antibody against 6-oxo - m1dg and have incorporated 
 this antibody into a procedure for the immunoaffinity isolation of 
 6-oxo - m1dg from biological matrices . 
 the purified analyte 
 is quantified by lc - ms / ms using a stable isotope - labeled analogue 
 ( [ 15n5]-6-oxo - m1dg ) as an internal 
 standard . 
 healthy male sprague  dawley rats excreted 6-oxo - m1dg at a rate of 3501893 fmol / kgd in feces . 
 this 
 is the first report of the presence of the major metabolite of m1dg in rodents without exogenous introduction of m1dg . 
Introduction
Materials and Methods
Results
Discussion