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Quantitative determination of pterins in biological fluids by high-performance liquid chromatography
During OUT continuing study of p&dine metabolism, the need arose for a more rapid and quantitativedeterminationof pterinsia biological fluids. By adopting and mocl@ing previously developed techniques, we have obtained a rapid and sensitivemethod that allows the simultaneousdetesmiaationof eight differentpf,erin in human urineand blood. When examined over a lo-day period, the levelsof pterins excretedby a normal individualaveragedthe followingvaluesexpressedin picomoles per mg of creatinine: biopteti, 9104; neopterin, 6018; xanthopterin,6561; p&n, 1136; isoxanthoptcrin,636; pteri&-carboxylate, 483; 2nd 6-hydroxymethylptesin, 3 15. Moreover, 6-hydroxymethylpterinand pterinA-ca&oxaldehyde were detected for the kst time in the blood of normal individua.Is_
The rapid and quantitativemeasurementof pterinsin urine, blood and other biological fluids has assumed a major importancebecause of reports describingthe occurrenceof ekv2ted kvels of certainpferidinesin tissueculturemedia of mzlignant cellsr, in the urine9 and in the blood of cancer p&en&, as well as in the urines of mice carryingthe Ehrlichascitestumor’. In order to establishf&e si@ace of such observations,and to finther the stiy of p&e* metabolism in nomizd as well as pathoXogiczdconditions, 8 simple, rapid and sensitivemethod of analysisis required.We have recentlyshown that separation of all the major pterinsknown to occur in human urinescan be accomplished by high-performanceliquid chromatography(HPLC) on a strong cationexchange coiumns~This method has also been shownto be highly sensitive,since,when coupkA to &x0rescace detection, it permitsthe detectionof peeks in the picomolar range.
The sensitiv& of the measurements, however, is strictly dependent on the state of
oxidation of the pterins,s&e t&y are kno~rnto yield their greatestffuorescenoein the fMy oxidizd state-We have thus examineda varietyofm&ods for the oxidation offe&ccd pterinspreLse&t in urine!%S&e a pu.ri&ationstep is need&b&ore aQalysis by HPLC, we have modiGed some existing m&hods so that eight di&rezEtpterins could be readilypur&d from urinesand subsequentXy analyzed by HPLC5. I3 this communication, we also report a0 HPLC system for the excfusive de&m&a&n of pterin&carboxyEc acid. Fiiy, the method so developed was )appEedto the quautitativedeterminationof unconjugated pterins excreted in the urine of a be&by subjectand to the study of the diurnalvariationin the excretionof thesecompounds-
Colietron of
specimens
Urines wereco&cted in brown-glassbottles or darkenedcontainersto protect pterinsfrom photo-oxidation and then frozen at -20”. When aliquots were needed for analysis, a specimenwas melted in a water-bathat 4045” with vigorous mixing and a 10 m3 aiiquot removed, Creatininelevels were determinedby the method of J~e(as~~inrrf_6)onaLiquots~had~~i~~ppHIwi~66N HCL We found that warming of the u&e to about Zoo and acitication to pH 1 were aecestary to obtain reproducibleresults. Bloods were co!lected in hepasinizd n&es and the plasmawas separatedfrom the c&Isby ~~tiugation in an International desk-top c&G& celitriige_ These specimenswere aIso stored at -20° uatil used. All of the foliowing operationswere carriedout in dim lighttXxi&ztim of redizcefipteriks have been reportedso occur in urine Biopterin,neopterin’**and xanthopterinp ais their dibydro- Oi tetrahydrdetivatives_ Tiie same has also beCn EpXtd fOr biopterin present in bzood3s*_Since our detection metbud strictly depends OQ the to adopt an oxidation fIuorcscence&ciency of the compound, it became nv method thai would convert the non-flu0 resent hydrogznatedforms of pterins into the highly ftuorescent,f&y oxidized forms, Three merent procedureswere contideied and the resultscompared, (i) It has been report&O &at dibydro- and tetrabydrobiopterincould be easily convertedto their oxiditi form by incubationof an acidic sofutioo (0.05 N HCl) of the compounds for 20 min in a boiling water-batbTen differenturineswere thus incubatedas describedabove after partialpurification by a two-step ion-exchangeproceduredescribedfater.(ii) The same set of urineswere also subjectedto an oxidation proceduseadopted from a recentlypublishedmet&d*. In short, oxidation was accomplishedby addition to the acidified(pH t) urine of a soiution of 0.5% ITI % KI in 0.1 N HCI to a final concentration of 0.05% I&Al % ILIor until excess I2 pessisted in solution. We found that biologicai fiuids diEer vtidelyfrom one anotker in the content of compounds cap&e of reducingI, to I-. Thus, the amount of Ir#I soiution needed to obtain an excess of Iz differedfrom sample to sample_F urfhermore,it has been repon&@ that incubationof the sample in the presenceof I, for 15 min would be sticient to oxidize fully ail the reduced p&ins. We found this to be correctin the majority of the cases, but occasionallywe encounteredsome urires that requireda ionget incubationtime in the presenceof EL-
1s 30 45 60
9% 70.56 10.759 9352
3741 3%3 3593
3855 10~10 12&s 10,552
134 247 288 280
'NeopterinieveIsrepresent~sumoibo~~~erythroand~e
583 1?09 1438 1219
7568 a463 28,517 24,436
t-tkreoisomers.
An exampleis reportedin Table I wherefour ahquotsof the same urineweresubjected to IZ oxidation for differentlengths of time. Clearly, in this case, an incubationfor 45 min was necessaryto maximizethe yield ofpterins that could be detected.We thus adopted a 45 min incubation time as the standardoperatingprocedure.In order to ensure that excess IZ was present in solution for the entire incubation period, the biologicai fluid being oxidized was tested periodicallywith starch which imparts an intense blue color to solutions containing the triiodideion. At the end of the incubation time, the excessIZwas removedby additionof a few drops of 1% ascorbicacid until the starch test was negative. (iii) Finally, a third method was attempted for oxidizing reducedpterinspresentin urines_This consistedof addition of H&D, to a finat concen’ation of 1% and incubation at room temperaturefor 15 min. In this case, too, the oxidation was stoppedby additionof a small amount of an appropriate ascorbic acid solution. When three differentaliquots of the same set of urines were oxidized in the three differentways and the resuhs obtained were compared, we found that the I2 and the H,O, proceduregave consistentlybetter results than the incubation in acidic medium in a boiling water-bath.The I, and the H& method appeared to give consistent and comparable resuhs. However, we adopted the I2 method becauseit had alreadybeen extensivelyused in other laboratoriesand proven to be very effectivein the oxidationof tetrahydro-,7,S-d.ihydro-and quinoid dihydrobiopteri#. Purijicuhm of sampks bejiore analysis by HPLC
In order to analyze biological materiak by HPLC, a preliminarypurification step is often necessa@*, because these samples contain salts, compounds and highmolecular-weightmokcuks that would otherwiseinterferewith the separationas web as with the detection of the compounds of interest_Furthermore,if the purifkation step were omitted, ffie microparticulatecolumn used for the separation in HPLC wou!d irreversiblydegeneratewith a few chromatographicrtms, thus making repetitive analysis of biological samplesprohibitivelyexpensive.FOFthesereasons,all the biological materialsanalyzedby HPLC werepm-i&d as follows. A certainvohime of. the biological fluid (for urines, the voIume containing 2 mg of creatinine)was first acid&d to pH 1 with 6 N HCI. Then 100 ~1 of a 30 FM solution of internalstandard werequantitativefyadded to the sample. The use of an internalstandardis necessary
accomf for any pQssibk day&day variati6min the ptlrikatim pnx&m& ~~asintbe~~onvo~~d~the;perfo~~of~d~t.fn~~gan intend standard, av&abilility;-stability, sohzbility, &%ech~-and~nm+ti&mee to
as
with the-oamponadsof-intenz& were tzo+xsiM Wecbse-6,~~y~ &N3ME') as it is cumavaiIabIc bigUyfbzo rescent,m~&&m~to
dete&, very stableto rkg oxidationand, mxe importantly,it chromatographedon a strongcation exchangecohunnwitha retentiontime Iongerthan any other&~~rescezkt cuxnpomd presentin urines. The sampIe to be ana&& was then subjected to an oxidation step w&h I, as clescriw above and GMuy p&ai by a Emdikation of 8 mctbod previomly dcscriibed~2, The sample is appliedto a 6 x 20 mm Dower SO-X$ c&mm (X3*, 10%2OQmesh), followed by 15 ml of double water. This wash is culkzctedmd ana&mi for iso~tboptiti which is not retainedon the cation exchmge c&mm because of its low p& u_ The Dower 50 column is &en stripped with6mlof1N~OHwbichisdirectlyappliedtoaSxSrnmDowexl-XS coluxm (OH-, 2oo400 mesh). ‘Es column is washed with 6 ml of a suhzticmof ~OHad~topH9and~wi~0_5mLof~~r_BotfEof~ese~ahe c-ikard&. Fii, the DUWZX1 c&lml is e!uted sumively with 2 ml of I N acetic tid-7 % metbanoi-5 % acetonitrilefollowedby 2 ml of 0.5 N HCC7 % methanoL5 % zkc&omitcfe_ The fonon+Qg pa are ehrted in tzteacetic a.ca fraction: xz+n&q~ zt-eg&ru-n~-hw-n~p~ biopterin, pterin, pteriu~boxakkhyde (FM-CHO~, 64ydroxymefhylpWn (R-6-CH2QH) and the internal standard, 6,7DMP. The HCI skate co&&is @.erk&kqlic acid (Pt-6-COOH) which binds tcrthe anion exchange c&mm zather strongly because of the io&abEe carb~xylic group_Tbe 15 ml of water-washcontainingisoxanthopti axe alkalinizedwith concentsated M&OH to pH 11, and tbm the entire &k&ion is applied to another 5 x 8 mm DOWZXl-X8 column (OH-, I?BO+IOmesh);thiscohzmniswasbedinthe same way as descxii above with 6 ml NH+OH pH 9 and 0.5 ml water, then elut& with 3 ml of 0.5 N NC%-7% mtioLS% acetotitsile. Methanol and acetonitrile were added to the deting bufferto ovenxme hydrophobicintezactionsof tbe pterin ring with the Dowa backbone. The purikatioz~ scheme is !mmmmzd in Fig- lThe xecoveryof eachpterinfrom urinepurikd by tbis procedm~~ is shown in Table IL Wii the exceptionof PM-CKO, all the other paerinswere recoveredin y-i&s that rmgcd between ?7% and lCIO%_Ozdy 35-46)x of PtGCHO was recoveredwhen added to urine and szxbjectedto the purifkation procedure.Fkarly laO% recovery was obtained instead when Pt-6CHO was subjectedto the pur%icationscheme in the absence of urine. Thus Pta0 reams with some compontnt(s) in the urine and is not I-emqxmtiawy. Blood and other biofogkal sxz&xi& were fks~ depropein;zta by ztdditi~~~of impropanol to-a final conoentrationof 70% and imx&atiu~ at 4O fOr st ka.S 2 h; dmproteins were mnovetd by cmtrif&atio~ and isqropaml by extiztion with six volumes of toIuena The aqueous extra.&was Grsta,tzim to pH 1 and then subjectedto I, oxidation as desc&ed previoaslyfor urines; &tally, tk solution was purikd by the same Cm-step ioxx-exchan~ m&mL At&&
of pieri& pressrt in bia~fl& by APLG The~ticaeidelnate~~the~~Icol~was~byHPL6:oraa IWiz3ill_oscx(whatman, csfkoq NJ., USA.) c&l2mn as dlzscrii
pEviousW-
367
15 ~1 K@ rash altalinized to pH 11 with KK@K
a) 2) Elute with 3 01 o.!jN ha71 CK,OK-5: CK3CK
2 ml 1N Kzc7: CK3oK52 CIyN
b) 2 ~1 O.SN KCl7: CH3OK5: cK3CK
0 Q I==_-_-_< __._ -.-.-.-_-
:_:__I_: ____ .-.-_ .._-_-_-_-_-.-_-_-_-__
XAKTMPTEKIK KEOPTEfXK BIOPTERIK Pt-CCKO Pt-6-CH$K PTEKIU 6.7~LW
ISOIAHTfX!PTERlH
Frg_ 1_ -tion
scheme ior
.-_-_ --_-_ _-_-_ .-_-_
--.-_ _-_-_ _-_-_ ::::: _-_-_ --_-_ :_:_
Pt-6-COOH
pm&s_
Becarrse of its exceedinglyhigh retenticntime, the intend standardwas elutedfrom the strong cationexchange column by increasingcolumn temperatureto 55” and flow-rate to 2.5 rrd/minafter alI the compounds of interest had been eMed at a flow of 1 ml/tin and at room temper&me. The HCl elate containing isoxanthopterinwas chromatographed on two Partis SCX columnsjoined in tandem,and also equippedtiti a 5-cm guardc&mm. chromatography was pedosrued with ffie same solvent system used for analysis of the acetic acid eluate (1 m.M NH&PQ pH 2%7% methanoL5% acetonitde) at
Bml/min and 4”_ This low temperatureis necessaryin order to separate the isoxanthoptesiupeak from other eariy eluting unknown peaks which would otherwise c0mi5te with isoxanthopterinat room tem~ture. Finally, for analysisof FWGCOQH, the HCI eluatecontainingthis compound is Gst Iyophiked to remove the HCI as the uAumn used to anaIyze this fraction is sensitiveto the acid. The residueso obtained is dissolvedin ! ml of the same buffer used for HPLC Chromato5phic analysis is performedin this case with a E%tisil iO-!U.X column (Whatman) equipped with 5-011prec~Iumn containing the same sacking as the main column. Isocratic ehition is perfok-medwith 20 mM K&PO4 Cadjusted to pH 33 withH,IQ)-5 % n-propanolat I.5 m&Go and room temperatureDetecticmwas performedin everycasewitha Farrand-A4fiuorometer(Farrand Optical, ValhaUa,N-Y., U.S.A.) equippedwith an excitationfiltercenteredat 360 nm and a narrow band emissionfiftepat 450 MI_ The aperture of the fluorometerwas set at a VaIne of 5 and the range varied between the vahxs of 0.3 and 10 dependingon the doncentrationof t&e samplesinjected. Quamifafion ofpierins
Quantitativedeterminationof pterinspresentin urines was accomplishedby means of linear calibrationcuryes wnstsucted by adding dXerent amounts of each pterin zlong with the same amount of internal standard tO 0.5ml urine aliquots. These aiiquotsweresubjectedto the purificationschemedkscrii above(Fig. 1) and t0 HPLC The peak area ratios of each pteri~ peak to the 6,7-DMP peak were plotted agai.nssthe correspondingamount of standardpresesltin the aliquot injected(100 rr)C~r~~~~trationvzlaes of xanthoptcrh~,~c0pterin, biopterin, ptcrin, PM-CEZ~ and PMXHJDH present in urines were obW.ned through this type of standard curveAn example is showy onIy for neoptehin(Fig_2)_ PtDH and isoxa~thoptcxk ecr~lcentrations instead were determinedfrom tieat standa& curves 0braiued by
369
Fig. 2. Scaudard curve for oeopcerin. Enereasingamounts of authentic n-erytlzr~aeopterin we.? added to five differesztu&e "s (0.5 ml) together w
Standard pterinswere obtained as describedin ref. 5; 6,TDMP was purchased from Regis (Morton Grove, Ill., U.S.A.). Salts used for HPLC buffers were of the highestgrade available, and *&eorganic solventsused as componentsof thesebuffers we%-of the HPLC grade (Burdick & 3ackson Labs., Muskegon, Mich., U.S.A.). RESUL’IS
During our initial study on the excretionof unconjugated pterins in human urines, we observed that if urines were not oxidized, and no precautionswere taken to keep pterins in the reduced state, increasingamounts of bioptcrin, neopterm and xanthopterinwould be detectedas a function of time when the acetic acid fraction of a puritkd urine was analyzed by HPLC. This indicated to us that the non-Buorescent reduced forms of those pterins were being spontaneousIy oxiciked while
StaQdiQg at room temperatuteill acetic acid thus becoming fluoresceQt and detectabt at 450 sun This conkned previouslypublisbcd data on t&e OcarrrenGeof redWed pterinsin nrines‘=*_We also found that isx&atiarXfor 20 min oft&e aceticacid f%acti~n axm&ing reducedptcrins in a boiling water-bathwovld immf&iateIyimxease the level of detectablepterins_The levels obtained t&isway, however, were always lower tba~~those obtained when the urine bad beep oxidized either by the Z, or ffie H& method before purification-Fig_ 3 shows a typical cbromatograpbk profile obtained whenthe aceticacid fracticmof a purifiedurinefrom a normal individualwas analyzd by HPLC. Peak assignmentswere made strictly by retention times which remain relativeIycons*%ntuntil the column starts to deteriorate*when this bappe&we found that by simply decreaskg by a few degreesthe temperatureat which the cbromato$apbic run is performed,we could restorethe origkal reteatio~ times. This is possible because migration of pterinson a Part&l IO-XX column is very sensitive to temperatur@. It is evident from fig. 3 &at pterins occur in lmman mines at difErent coiiceatrations,as it was neuzsszy to change the sensitivityof the fluorometer over a ten-fold range to obtain a good signal/~oise ratio for some peaks. A peak having the same mobiity as authenticPt-6-CH,OH was c0nsistent.l~seen in all tk urinesexamined.This cumpoundwas presentin very minuteSLELLOKKSS when wmparedwithmore abundantpterins*suchas neoptexinand biopterk (seeTable I or m).
EZPLCOFPlERINS
371
KNBrOLaGrcA4LFLw3Ds
m
TABLE
Days Avt?opiet7iP Bioptain Pt-hCHzOH 1
5592
s 4 5 6 7 8 9 IO
6370 5963 4848 6370 6630 5852 8444 5222 4888
6758 11,448 7414 9424 8241 9655 11,138 9828 9690 7448
Ptenh
Xanftiopienh Isoxanthopterin PM-COOH
264
%9
6110
ND”
408
461 2.92 177 306 373 293 458 239 269
1156 a55 994 1312 1139 1290 1461 1225 956
9073 9258 5830 6234 602.5 5376 6357 57% 5555
719 831 ND 744 837 575 ZE
520 372 349 563 596 562 612 510 341
425
- Neopterin levels sepxtxes~t the sum of both the a-erytkro and the rhueo konms. -- ND = not determined
only idedfied by its retentiontime on HPLC, to OIKknowledge,this is the first report describingthe occurrenceof Pt-GCH,OH in human urine. Its presence in very small amounts has preventedits positive identificationby other means. The pterinspurifiedfrom urinesand presentin the acetic acid frxtion appearto be stable indefinitelywhen kept at -20” and in the dark, with the singleexceptionof xanthopterinwhich slowiy decreas~ with time over a period of severaldays. The separationof peaks presentin the HCI fraction containing P&6-COOH and the idenacation of this pterin‘ are shown in Fig. 4. As can be seen from the cbromatogcapbicprome, this pterin is also presentin small amounts in the urine of this normal individual.fig. 5 depictsthe chromatogrzphicprofileof the HCI faction containing isoxznthopterin.Many other peaks were present in this and the other two fractions,but no 2ttempts were made to identifythem_ Peaks attributedto p&ins were identifiedeitherdirectlyby co&ration with authentic standardsor indirectlyby adsorption on charcoal with concomitant disappearanceof tie pe&s attributedto pterins, a criterionpreviouslyused by other investigatorsto verig the identityof certainpeak$*. In order to gather data on the urinary levels of pterinsin normal individuals and obtain clues on factors controlling &eir exaretiat, ten consecutive urines were cokcted daily from a young healthy adult and analyzed for theircontent of pterins. Table III shows the levelsof pterinspresentin these ten urines.The mean output for each pterin as well as the maximum and minimum levels excretedare shown graphicaEy in Fig. 6. It is clear that urinaryexcretionleveis of pterinsvary less than twofold duringthe USday period.‘li”li”li”li”li’ reasonfor this variationis not known and requires flier inves@tion- However, the values reported in Table III for the individual under study are typical for people in the same age group as shown elsewhere”. XII addition to studyingthe daily variationin the urinaryexcretionlevels of pterins,we also followed excretion during a 24-h period by examining timed urines coJ.lected Though
Fip. A Elution proiik of the HCl fraction containing PtAC0OH from a u&e of a Wthy indi~_~~ulumtafurinemntainirrg2mgofa~~flml)nasoxidEsedandpurifiedasdcs&bed in the kgeud of Fig. 3_ The HCl frastion obtahed after &tio~ with acetic acid was in lml ofTDmMKH~?O. pH3.3-5% rycJ@iEzd to rcams~tlxcHCluldtkr&ducrcdisolvcd
every6 h_The res&s of this study are shown in Fig. 7. With the possibleexceptionof xzntthcpterin,au the Other pterins ex2mined were excreted in amslant amounts during the 24-h period, Xanthoptet;ll levels instead appeared to decreasebetweeQ &e morning and the night specima; this change, however, is not as great ss the day-today variation(Table III). These resultsvalidateour practiceof cukcting urine specimensat any time of the day, sinceptmins’ excretionappearsto remainrelativeiy consant during a 24-h period. Finally, the metbad desiibed in this paper can afso be applied to quantitate the Pevelsof pterim presentin blood. Fig_ 8 shows the typical pattern obti& for the &sma &action of the blood of a normal individualafter 1, oxidation and pwifkation as describedin Methods. From the pro& &own in Fig. 8, it is appme~t that p=ks having the same chmzatogmphic m~brlty as Pa-0 and Pt-6-CK@H are prcsmt in the biuod of this normal in&vi&a& in additionto neopteiir~,bioptezin,
373
La
81
e
i
Fii. 6. Mean urinary excretion IeveIs of pterins in a h&y individual over 2 IO-day period. The of urine containing 2 mg of CreatIninewas proczssed in each case as descsibed in Table III. The bars indicate tke avenge daiiy excretion level while the brackets indicate the Iowcst and +&e highest level excreted during the period of study. Neopterin fevek represent the sum of both the D e@iro and tEwL-rhreo isomers. volume
&-I
Pt-6-cof% Pt*-c&OH
excretion kveIs of pcerius in a healthy IndividuaL Timed Fii7.Dium?lv&ationintbell&aXy urincs~co~cvery6has~o~~.‘EhevoIumeofurineequivalentto2mgofcreatininewas process& for each specimm Analysis was performed by HPLC as described in the md of Figs. 3-5. Neoptcrin levels repzsent the sun of both r.be.~+~~~&o and the r-rfzreo isomexs.
xanthopterinand p&sin_ Moreover, the relativelevel of Ft-6CH20H appear to be mu& greaterin the blood than in the urine. Although it seems unlikeiythat other compounds could copurify and corn&rate01).HPLC with Pt-6-CHO or FM-C&OH and aIso-fluoresceat 450 nm, we f=I that a more positiveititi=tioa is neededfor these two new p&ins fomrd in the blood of normal individuals.Pt-6-CCOI-Z and smalI amounts of isoxanthopterinwere also fouad in the plasma of the individual under study (resultsoat sbawn). DISCUSSION The method described in this publication is a rapid, highly sensitive and relatively inexpensive technique to analyze and quantitate alK the major ptezins present in hunran urine, blood and other biological &ids. Although otkr rapid or reWrsed-phase methods uti.l&g gas chromatography-mass sp&&&&y’%= HJ?LP have previousIybeen describedfor the quantitaticmof pterins, none, however, addressesthe question of the simukmeous detcrm&tion of eight d3Semt pterinsfrom a singlespeciwn, Most of the fzxktingllX&ho&concxm~V~witb the q?XWitation of onxy one Of two pterins_OExrf3Qcri~ in the dete&oEl ztzkd measaremexitof pterinsin urines of cana patientshas sfcown8s that quantitaticm of ali the kImwT.l ptelins can be of great diagnostk im@cR%uNe’~. It is our hope that because of its simplicityand rep~oducibiiity,this method will find wide application in the study of foIateand p&in m&ab&sm in the &M& & well as the patboi6gical
ACKNOWLEDGEMENDS This work was suppated by the Wius and Dorothy Fried Research Foundation and by the Celh&arand Molecukr Biology Training Grant No. GM 07185-W
1 B. Sea., P. S. BacIchn~d,Jr., P. B. Bekclcy.A. K. C&o, E. C. HaIpem, R M. E&&ternand R A. sni&,cmcca a_. 38 (197Q 237% 2 RM.Ifalpan,B.C_~~B.Stca.A_~p.K,ConLLin.B.CIark,~~,L_SperPing J. A. HXpcm, D. Hardy and R A. Smith. Pax. Nat. A& Sci U.S.. 74 (1977) 587. 3 N. K&otis and I. Ziq#a, Cheer ,%ochcm.wbphy., 2 (1977) 79. 4 EL Wachter. K. E rasxuays and A. Ehsen, Ckcer Lctt.. 6 (1979) 61. 5 B. Stea, R. M. H&em and R A. Smith, I. Cfwasztogr.. 168 (1979) 385. 6RJ.Henry,K.C.CaMonandJ.W.Winkkman, ChiazI Chenris~ry:Pticipks and Tedyriques, Hasper an! Row, Efagcrstown, Md., 1974,pp. tiff--552. 7 T. Fukushma snd T. Shiota, 1. BzX CYrent.,247 (1972) 4549. S T_ Fw K. Kobayashi L Efo and T. ‘&iota, A&. Biocfwz.. 89 (197.3)71. 9 P. SchImr, B. hi. Watson, R G. K. Cotton and D. M. Dar&s, Ch. Chin. Acft& 92 (1979) 187. IO E. M. Gbl znd A. D. Shaman, Prep. Biod?em., 7 (1977) 155. 11 A. Bye and M. E. Brown, I. Chrunazfugr.Scf., IS (1977) 365. 12 R RiitbIer and M. Karobafh, Clin. Chin. Acra. 69 (1?76) 457. 13 R. C Blakky, Ike i3kxheremimy of Folic Acidand Re~tedPhmZnes, North Holhnd, Amsterdam, 1969. pp. 61-615. 14 B. Stca, R M. liS&cm, B. C HaIpcm and R. A. Smith, in preparation. 15 T. Fukushima and J. C. Nixon, in R L. KisIiuIc and G. M. Brown (Editors). Chemistry and Bhbgy of Pteridks, Ekcvier/North Holland, New Ymk, 1979.pp. 31-36.
The rapid and quantitativemeasurementof pterinsin urine, blood and other biological fluids has assumed a major importancebecause of reports describingthe occurrenceof ekv2ted kvels of certainpferidinesin tissueculturemedia of mzlignant cellsr, in the urine9 and in the blood of cancer p&en&, as well as in the urines of mice carryingthe Ehrlichascitestumor’. In order to establishf&e si@ace of such observations,and to finther the stiy of p&e* metabolism in nomizd as well as pathoXogiczdconditions, 8 simple, rapid and sensitivemethod of analysisis required.We have recentlyshown that separation of all the major pterinsknown to occur in human urinescan be accomplished by high-performanceliquid chromatography(HPLC) on a strong cationexchange coiumns~This method has also been shownto be highly sensitive,since,when coupkA to &x0rescace detection, it permitsthe detectionof peeks in the picomolar range.
The sensitiv& of the measurements, however, is strictly dependent on the state of
oxidation of the pterins,s&e t&y are kno~rnto yield their greatestffuorescenoein the fMy oxidizd state-We have thus examineda varietyofm&ods for the oxidation offe&ccd pterinspreLse&t in urine!%S&e a pu.ri&ationstep is need&b&ore aQalysis by HPLC, we have modiGed some existing m&hods so that eight di&rezEtpterins could be readilypur&d from urinesand subsequentXy analyzed by HPLC5. I3 this communication, we also report a0 HPLC system for the excfusive de&m&a&n of pterin&carboxyEc acid. Fiiy, the method so developed was )appEedto the quautitativedeterminationof unconjugated pterins excreted in the urine of a be&by subjectand to the study of the diurnalvariationin the excretionof thesecompounds-
Colietron of
specimens
Urines wereco&cted in brown-glassbottles or darkenedcontainersto protect pterinsfrom photo-oxidation and then frozen at -20”. When aliquots were needed for analysis, a specimenwas melted in a water-bathat 4045” with vigorous mixing and a 10 m3 aiiquot removed, Creatininelevels were determinedby the method of J~e(as~~inrrf_6)onaLiquots~had~~i~~ppHIwi~66N HCL We found that warming of the u&e to about Zoo and acitication to pH 1 were aecestary to obtain reproducibleresults. Bloods were co!lected in hepasinizd n&es and the plasmawas separatedfrom the c&Isby ~~tiugation in an International desk-top c&G& celitriige_ These specimenswere aIso stored at -20° uatil used. All of the foliowing operationswere carriedout in dim lighttXxi&ztim of redizcefipteriks have been reportedso occur in urine Biopterin,neopterin’**and xanthopterinp ais their dibydro- Oi tetrahydrdetivatives_ Tiie same has also beCn EpXtd fOr biopterin present in bzood3s*_Since our detection metbud strictly depends OQ the to adopt an oxidation fIuorcscence&ciency of the compound, it became nv method thai would convert the non-flu0 resent hydrogznatedforms of pterins into the highly ftuorescent,f&y oxidized forms, Three merent procedureswere contideied and the resultscompared, (i) It has been report&O &at dibydro- and tetrabydrobiopterincould be easily convertedto their oxiditi form by incubationof an acidic sofutioo (0.05 N HCl) of the compounds for 20 min in a boiling water-batbTen differenturineswere thus incubatedas describedabove after partialpurification by a two-step ion-exchangeproceduredescribedfater.(ii) The same set of urineswere also subjectedto an oxidation proceduseadopted from a recentlypublishedmet&d*. In short, oxidation was accomplishedby addition to the acidified(pH t) urine of a soiution of 0.5% ITI % KI in 0.1 N HCI to a final concentration of 0.05% I&Al % ILIor until excess I2 pessisted in solution. We found that biologicai fiuids diEer vtidelyfrom one anotker in the content of compounds cap&e of reducingI, to I-. Thus, the amount of Ir#I soiution needed to obtain an excess of Iz differedfrom sample to sample_F urfhermore,it has been repon&@ that incubationof the sample in the presenceof I, for 15 min would be sticient to oxidize fully ail the reduced p&ins. We found this to be correctin the majority of the cases, but occasionallywe encounteredsome urires that requireda ionget incubationtime in the presenceof EL-
1s 30 45 60
9% 70.56 10.759 9352
3741 3%3 3593
3855 10~10 12&s 10,552
134 247 288 280
'NeopterinieveIsrepresent~sumoibo~~~erythroand~e
583 1?09 1438 1219
7568 a463 28,517 24,436
t-tkreoisomers.
An exampleis reportedin Table I wherefour ahquotsof the same urineweresubjected to IZ oxidation for differentlengths of time. Clearly, in this case, an incubationfor 45 min was necessaryto maximizethe yield ofpterins that could be detected.We thus adopted a 45 min incubation time as the standardoperatingprocedure.In order to ensure that excess IZ was present in solution for the entire incubation period, the biologicai fluid being oxidized was tested periodicallywith starch which imparts an intense blue color to solutions containing the triiodideion. At the end of the incubation time, the excessIZwas removedby additionof a few drops of 1% ascorbicacid until the starch test was negative. (iii) Finally, a third method was attempted for oxidizing reducedpterinspresentin urines_This consistedof addition of H&D, to a finat concen’ation of 1% and incubation at room temperaturefor 15 min. In this case, too, the oxidation was stoppedby additionof a small amount of an appropriate ascorbic acid solution. When three differentaliquots of the same set of urines were oxidized in the three differentways and the resuhs obtained were compared, we found that the I2 and the H,O, proceduregave consistentlybetter results than the incubation in acidic medium in a boiling water-bath.The I, and the H& method appeared to give consistent and comparable resuhs. However, we adopted the I2 method becauseit had alreadybeen extensivelyused in other laboratoriesand proven to be very effectivein the oxidationof tetrahydro-,7,S-d.ihydro-and quinoid dihydrobiopteri#. Purijicuhm of sampks bejiore analysis by HPLC
In order to analyze biological materiak by HPLC, a preliminarypurification step is often necessa@*, because these samples contain salts, compounds and highmolecular-weightmokcuks that would otherwiseinterferewith the separationas web as with the detection of the compounds of interest_Furthermore,if the purifkation step were omitted, ffie microparticulatecolumn used for the separation in HPLC wou!d irreversiblydegeneratewith a few chromatographicrtms, thus making repetitive analysis of biological samplesprohibitivelyexpensive.FOFthesereasons,all the biological materialsanalyzedby HPLC werepm-i&d as follows. A certainvohime of. the biological fluid (for urines, the voIume containing 2 mg of creatinine)was first acid&d to pH 1 with 6 N HCI. Then 100 ~1 of a 30 FM solution of internalstandard werequantitativefyadded to the sample. The use of an internalstandardis necessary
accomf for any pQssibk day&day variati6min the ptlrikatim pnx&m& ~~asintbe~~onvo~~d~the;perfo~~of~d~t.fn~~gan intend standard, av&abilility;-stability, sohzbility, &%ech~-and~nm+ti&mee to
as
with the-oamponadsof-intenz& were tzo+xsiM Wecbse-6,~~y~ &N3ME') as it is cumavaiIabIc bigUyfbzo rescent,m~&&m~to
dete&, very stableto rkg oxidationand, mxe importantly,it chromatographedon a strongcation exchangecohunnwitha retentiontime Iongerthan any other&~~rescezkt cuxnpomd presentin urines. The sampIe to be ana&& was then subjected to an oxidation step w&h I, as clescriw above and GMuy p&ai by a Emdikation of 8 mctbod previomly dcscriibed~2, The sample is appliedto a 6 x 20 mm Dower SO-X$ c&mm (X3*, 10%2OQmesh), followed by 15 ml of double water. This wash is culkzctedmd ana&mi for iso~tboptiti which is not retainedon the cation exchmge c&mm because of its low p& u_ The Dower 50 column is &en stripped with6mlof1N~OHwbichisdirectlyappliedtoaSxSrnmDowexl-XS coluxm (OH-, 2oo400 mesh). ‘Es column is washed with 6 ml of a suhzticmof ~OHad~topH9and~wi~0_5mLof~~r_BotfEof~ese~ahe c-ikard&. Fii, the DUWZX1 c&lml is e!uted sumively with 2 ml of I N acetic tid-7 % metbanoi-5 % acetonitrilefollowedby 2 ml of 0.5 N HCC7 % methanoL5 % zkc&omitcfe_ The fonon+Qg pa are ehrted in tzteacetic a.ca fraction: xz+n&q~ zt-eg&ru-n~-hw-n~p~ biopterin, pterin, pteriu~boxakkhyde (FM-CHO~, 64ydroxymefhylpWn (R-6-CH2QH) and the internal standard, 6,7DMP. The HCI skate co&&is @.erk&kqlic acid (Pt-6-COOH) which binds tcrthe anion exchange c&mm zather strongly because of the io&abEe carb~xylic group_Tbe 15 ml of water-washcontainingisoxanthopti axe alkalinizedwith concentsated M&OH to pH 11, and tbm the entire &k&ion is applied to another 5 x 8 mm DOWZXl-X8 column (OH-, I?BO+IOmesh);thiscohzmniswasbedinthe same way as descxii above with 6 ml NH+OH pH 9 and 0.5 ml water, then elut& with 3 ml of 0.5 N NC%-7% mtioLS% acetotitsile. Methanol and acetonitrile were added to the deting bufferto ovenxme hydrophobicintezactionsof tbe pterin ring with the Dowa backbone. The purikatioz~ scheme is !mmmmzd in Fig- lThe xecoveryof eachpterinfrom urinepurikd by tbis procedm~~ is shown in Table IL Wii the exceptionof PM-CKO, all the other paerinswere recoveredin y-i&s that rmgcd between ?7% and lCIO%_Ozdy 35-46)x of PtGCHO was recoveredwhen added to urine and szxbjectedto the purifkation procedure.Fkarly laO% recovery was obtained instead when Pt-6CHO was subjectedto the pur%icationscheme in the absence of urine. Thus Pta0 reams with some compontnt(s) in the urine and is not I-emqxmtiawy. Blood and other biofogkal sxz&xi& were fks~ depropein;zta by ztdditi~~~of impropanol to-a final conoentrationof 70% and imx&atiu~ at 4O fOr st ka.S 2 h; dmproteins were mnovetd by cmtrif&atio~ and isqropaml by extiztion with six volumes of toIuena The aqueous extra.&was Grsta,tzim to pH 1 and then subjectedto I, oxidation as desc&ed previoaslyfor urines; &tally, tk solution was purikd by the same Cm-step ioxx-exchan~ m&mL At&&
of pieri& pressrt in bia~fl& by APLG The~ticaeidelnate~~the~~Icol~was~byHPL6:oraa IWiz3ill_oscx(whatman, csfkoq NJ., USA.) c&l2mn as dlzscrii
pEviousW-
367
15 ~1 K@ rash altalinized to pH 11 with KK@K
a) 2) Elute with 3 01 o.!jN ha71 CK,OK-5: CK3CK
2 ml 1N Kzc7: CK3oK52 CIyN
b) 2 ~1 O.SN KCl7: CH3OK5: cK3CK
0 Q I==_-_-_< __._ -.-.-.-_-
:_:__I_: ____ .-.-_ .._-_-_-_-_-.-_-_-_-__
XAKTMPTEKIK KEOPTEfXK BIOPTERIK Pt-CCKO Pt-6-CH$K PTEKIU 6.7~LW
ISOIAHTfX!PTERlH
Frg_ 1_ -tion
scheme ior
.-_-_ --_-_ _-_-_ .-_-_
--.-_ _-_-_ _-_-_ ::::: _-_-_ --_-_ :_:_
Pt-6-COOH
pm&s_
Becarrse of its exceedinglyhigh retenticntime, the intend standardwas elutedfrom the strong cationexchange column by increasingcolumn temperatureto 55” and flow-rate to 2.5 rrd/minafter alI the compounds of interest had been eMed at a flow of 1 ml/tin and at room temper&me. The HCl elate containing isoxanthopterinwas chromatographed on two Partis SCX columnsjoined in tandem,and also equippedtiti a 5-cm guardc&mm. chromatography was pedosrued with ffie same solvent system used for analysis of the acetic acid eluate (1 m.M NH&PQ pH 2%7% methanoL5% acetonitde) at
Bml/min and 4”_ This low temperatureis necessaryin order to separate the isoxanthoptesiupeak from other eariy eluting unknown peaks which would otherwise c0mi5te with isoxanthopterinat room tem~ture. Finally, for analysisof FWGCOQH, the HCI eluatecontainingthis compound is Gst Iyophiked to remove the HCI as the uAumn used to anaIyze this fraction is sensitiveto the acid. The residueso obtained is dissolvedin ! ml of the same buffer used for HPLC Chromato5phic analysis is performedin this case with a E%tisil iO-!U.X column (Whatman) equipped with 5-011prec~Iumn containing the same sacking as the main column. Isocratic ehition is perfok-medwith 20 mM K&PO4 Cadjusted to pH 33 withH,IQ)-5 % n-propanolat I.5 m&Go and room temperatureDetecticmwas performedin everycasewitha Farrand-A4fiuorometer(Farrand Optical, ValhaUa,N-Y., U.S.A.) equippedwith an excitationfiltercenteredat 360 nm and a narrow band emissionfiftepat 450 MI_ The aperture of the fluorometerwas set at a VaIne of 5 and the range varied between the vahxs of 0.3 and 10 dependingon the doncentrationof t&e samplesinjected. Quamifafion ofpierins
Quantitativedeterminationof pterinspresentin urines was accomplishedby means of linear calibrationcuryes wnstsucted by adding dXerent amounts of each pterin zlong with the same amount of internal standard tO 0.5ml urine aliquots. These aiiquotsweresubjectedto the purificationschemedkscrii above(Fig. 1) and t0 HPLC The peak area ratios of each pteri~ peak to the 6,7-DMP peak were plotted agai.nssthe correspondingamount of standardpresesltin the aliquot injected(100 rr)C~r~~~~trationvzlaes of xanthoptcrh~,~c0pterin, biopterin, ptcrin, PM-CEZ~ and PMXHJDH present in urines were obW.ned through this type of standard curveAn example is showy onIy for neoptehin(Fig_2)_ PtDH and isoxa~thoptcxk ecr~lcentrations instead were determinedfrom tieat standa& curves 0braiued by
369
Fig. 2. Scaudard curve for oeopcerin. Enereasingamounts of authentic n-erytlzr~aeopterin we.? added to five differesztu&e "s (0.5 ml) together w
Standard pterinswere obtained as describedin ref. 5; 6,TDMP was purchased from Regis (Morton Grove, Ill., U.S.A.). Salts used for HPLC buffers were of the highestgrade available, and *&eorganic solventsused as componentsof thesebuffers we%-of the HPLC grade (Burdick & 3ackson Labs., Muskegon, Mich., U.S.A.). RESUL’IS
During our initial study on the excretionof unconjugated pterins in human urines, we observed that if urines were not oxidized, and no precautionswere taken to keep pterins in the reduced state, increasingamounts of bioptcrin, neopterm and xanthopterinwould be detectedas a function of time when the acetic acid fraction of a puritkd urine was analyzed by HPLC. This indicated to us that the non-Buorescent reduced forms of those pterins were being spontaneousIy oxiciked while
StaQdiQg at room temperatuteill acetic acid thus becoming fluoresceQt and detectabt at 450 sun This conkned previouslypublisbcd data on t&e OcarrrenGeof redWed pterinsin nrines‘=*_We also found that isx&atiarXfor 20 min oft&e aceticacid f%acti~n axm&ing reducedptcrins in a boiling water-bathwovld immf&iateIyimxease the level of detectablepterins_The levels obtained t&isway, however, were always lower tba~~those obtained when the urine bad beep oxidized either by the Z, or ffie H& method before purification-Fig_ 3 shows a typical cbromatograpbk profile obtained whenthe aceticacid fracticmof a purifiedurinefrom a normal individualwas analyzd by HPLC. Peak assignmentswere made strictly by retention times which remain relativeIycons*%ntuntil the column starts to deteriorate*when this bappe&we found that by simply decreaskg by a few degreesthe temperatureat which the cbromato$apbic run is performed,we could restorethe origkal reteatio~ times. This is possible because migration of pterinson a Part&l IO-XX column is very sensitive to temperatur@. It is evident from fig. 3 &at pterins occur in lmman mines at difErent coiiceatrations,as it was neuzsszy to change the sensitivityof the fluorometer over a ten-fold range to obtain a good signal/~oise ratio for some peaks. A peak having the same mobiity as authenticPt-6-CH,OH was c0nsistent.l~seen in all tk urinesexamined.This cumpoundwas presentin very minuteSLELLOKKSS when wmparedwithmore abundantpterins*suchas neoptexinand biopterk (seeTable I or m).
EZPLCOFPlERINS
371
KNBrOLaGrcA4LFLw3Ds
m
TABLE
Days Avt?opiet7iP Bioptain Pt-hCHzOH 1
5592
s 4 5 6 7 8 9 IO
6370 5963 4848 6370 6630 5852 8444 5222 4888
6758 11,448 7414 9424 8241 9655 11,138 9828 9690 7448
Ptenh
Xanftiopienh Isoxanthopterin PM-COOH
264
%9
6110
ND”
408
461 2.92 177 306 373 293 458 239 269
1156 a55 994 1312 1139 1290 1461 1225 956
9073 9258 5830 6234 602.5 5376 6357 57% 5555
719 831 ND 744 837 575 ZE
520 372 349 563 596 562 612 510 341
425
- Neopterin levels sepxtxes~t the sum of both the a-erytkro and the rhueo konms. -- ND = not determined
only idedfied by its retentiontime on HPLC, to OIKknowledge,this is the first report describingthe occurrenceof Pt-GCH,OH in human urine. Its presence in very small amounts has preventedits positive identificationby other means. The pterinspurifiedfrom urinesand presentin the acetic acid frxtion appearto be stable indefinitelywhen kept at -20” and in the dark, with the singleexceptionof xanthopterinwhich slowiy decreas~ with time over a period of severaldays. The separationof peaks presentin the HCI fraction containing P&6-COOH and the idenacation of this pterin‘ are shown in Fig. 4. As can be seen from the cbromatogcapbicprome, this pterin is also presentin small amounts in the urine of this normal individual.fig. 5 depictsthe chromatogrzphicprofileof the HCI faction containing isoxznthopterin.Many other peaks were present in this and the other two fractions,but no 2ttempts were made to identifythem_ Peaks attributedto p&ins were identifiedeitherdirectlyby co&ration with authentic standardsor indirectlyby adsorption on charcoal with concomitant disappearanceof tie pe&s attributedto pterins, a criterionpreviouslyused by other investigatorsto verig the identityof certainpeak$*. In order to gather data on the urinary levels of pterinsin normal individuals and obtain clues on factors controlling &eir exaretiat, ten consecutive urines were cokcted daily from a young healthy adult and analyzed for theircontent of pterins. Table III shows the levelsof pterinspresentin these ten urines.The mean output for each pterin as well as the maximum and minimum levels excretedare shown graphicaEy in Fig. 6. It is clear that urinaryexcretionleveis of pterinsvary less than twofold duringthe USday period.‘li”li”li”li”li’ reasonfor this variationis not known and requires flier inves@tion- However, the values reported in Table III for the individual under study are typical for people in the same age group as shown elsewhere”. XII addition to studyingthe daily variationin the urinaryexcretionlevels of pterins,we also followed excretion during a 24-h period by examining timed urines coJ.lected Though
Fip. A Elution proiik of the HCl fraction containing PtAC0OH from a u&e of a Wthy indi~_~~ulumtafurinemntainirrg2mgofa~~flml)nasoxidEsedandpurifiedasdcs&bed in the kgeud of Fig. 3_ The HCl frastion obtahed after &tio~ with acetic acid was in lml ofTDmMKH~?O. pH3.3-5% rycJ@iEzd to rcams~tlxcHCluldtkr&ducrcdisolvcd
every6 h_The res&s of this study are shown in Fig. 7. With the possibleexceptionof xzntthcpterin,au the Other pterins ex2mined were excreted in amslant amounts during the 24-h period, Xanthoptet;ll levels instead appeared to decreasebetweeQ &e morning and the night specima; this change, however, is not as great ss the day-today variation(Table III). These resultsvalidateour practiceof cukcting urine specimensat any time of the day, sinceptmins’ excretionappearsto remainrelativeiy consant during a 24-h period. Finally, the metbad desiibed in this paper can afso be applied to quantitate the Pevelsof pterim presentin blood. Fig_ 8 shows the typical pattern obti& for the &sma &action of the blood of a normal individualafter 1, oxidation and pwifkation as describedin Methods. From the pro& &own in Fig. 8, it is appme~t that p=ks having the same chmzatogmphic m~brlty as Pa-0 and Pt-6-CK@H are prcsmt in the biuod of this normal in&vi&a& in additionto neopteiir~,bioptezin,
373
La
81
e
i
Fii. 6. Mean urinary excretion IeveIs of pterins in a h&y individual over 2 IO-day period. The of urine containing 2 mg of CreatIninewas proczssed in each case as descsibed in Table III. The bars indicate tke avenge daiiy excretion level while the brackets indicate the Iowcst and +&e highest level excreted during the period of study. Neopterin fevek represent the sum of both the D e@iro and tEwL-rhreo isomers. volume
&-I
Pt-6-cof% Pt*-c&OH
excretion kveIs of pcerius in a healthy IndividuaL Timed Fii7.Dium?lv&ationintbell&aXy urincs~co~cvery6has~o~~.‘EhevoIumeofurineequivalentto2mgofcreatininewas process& for each specimm Analysis was performed by HPLC as described in the md of Figs. 3-5. Neoptcrin levels repzsent the sun of both r.be.~+~~~&o and the r-rfzreo isomexs.
xanthopterinand p&sin_ Moreover, the relativelevel of Ft-6CH20H appear to be mu& greaterin the blood than in the urine. Although it seems unlikeiythat other compounds could copurify and corn&rate01).HPLC with Pt-6-CHO or FM-C&OH and aIso-fluoresceat 450 nm, we f=I that a more positiveititi=tioa is neededfor these two new p&ins fomrd in the blood of normal individuals.Pt-6-CCOI-Z and smalI amounts of isoxanthopterinwere also fouad in the plasma of the individual under study (resultsoat sbawn). DISCUSSION The method described in this publication is a rapid, highly sensitive and relatively inexpensive technique to analyze and quantitate alK the major ptezins present in hunran urine, blood and other biological &ids. Although otkr rapid or reWrsed-phase methods uti.l&g gas chromatography-mass sp&&&&y’%= HJ?LP have previousIybeen describedfor the quantitaticmof pterins, none, however, addressesthe question of the simukmeous detcrm&tion of eight d3Semt pterinsfrom a singlespeciwn, Most of the fzxktingllX&ho&concxm~V~witb the q?XWitation of onxy one Of two pterins_OExrf3Qcri~ in the dete&oEl ztzkd measaremexitof pterinsin urines of cana patientshas sfcown8s that quantitaticm of ali the kImwT.l ptelins can be of great diagnostk im@cR%uNe’~. It is our hope that because of its simplicityand rep~oducibiiity,this method will find wide application in the study of foIateand p&in m&ab&sm in the &M& & well as the patboi6gical
ACKNOWLEDGEMENDS This work was suppated by the Wius and Dorothy Fried Research Foundation and by the Celh&arand Molecukr Biology Training Grant No. GM 07185-W
1 B. Sea., P. S. BacIchn~d,Jr., P. B. Bekclcy.A. K. C&o, E. C. HaIpem, R M. E&&ternand R A. sni&,cmcca a_. 38 (197Q 237% 2 RM.Ifalpan,B.C_~~B.Stca.A_~p.K,ConLLin.B.CIark,~~,L_SperPing J. A. HXpcm, D. Hardy and R A. Smith. Pax. Nat. A& Sci U.S.. 74 (1977) 587. 3 N. K&otis and I. Ziq#a, Cheer ,%ochcm.wbphy., 2 (1977) 79. 4 EL Wachter. K. E rasxuays and A. Ehsen, Ckcer Lctt.. 6 (1979) 61. 5 B. Stea, R. M. H&em and R A. Smith, I. Cfwasztogr.. 168 (1979) 385. 6RJ.Henry,K.C.CaMonandJ.W.Winkkman, ChiazI Chenris~ry:Pticipks and Tedyriques, Hasper an! Row, Efagcrstown, Md., 1974,pp. tiff--552. 7 T. Fukushma snd T. Shiota, 1. BzX CYrent.,247 (1972) 4549. S T_ Fw K. Kobayashi L Efo and T. ‘&iota, A&. Biocfwz.. 89 (197.3)71. 9 P. SchImr, B. hi. Watson, R G. K. Cotton and D. M. Dar&s, Ch. Chin. Acft& 92 (1979) 187. IO E. M. Gbl znd A. D. Shaman, Prep. Biod?em., 7 (1977) 155. 11 A. Bye and M. E. Brown, I. Chrunazfugr.Scf., IS (1977) 365. 12 R RiitbIer and M. Karobafh, Clin. Chin. Acra. 69 (1?76) 457. 13 R. C Blakky, Ike i3kxheremimy of Folic Acidand Re~tedPhmZnes, North Holhnd, Amsterdam, 1969. pp. 61-615. 14 B. Stca, R M. liS&cm, B. C HaIpcm and R. A. Smith, in preparation. 15 T. Fukushima and J. C. Nixon, in R L. KisIiuIc and G. M. Brown (Editors). Chemistry and Bhbgy of Pteridks, Ekcvier/North Holland, New Ymk, 1979.pp. 31-36.