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Determination of C14-lactate adapted to liquid scintillation counting
The investigation reported here :UWC from the need for :t simple micromethod for the determination of ratlioactivity in the l-carbon :md in the 2,3-carbon moieties of lactate in whole blood adapted to liquid scint.illation counting. The method described is relatively simple, reproducible, and accurate, which makes it’ especially valuable in the away of low levels of radioactivity in this metabolitc cncountercd in esperiments conducted in the large animal or humans. W-Lactate to which a carrier amount of unlnlwled lactate has bceu added is quantitatively osidized to ncet~aldehyde and carbon dioxide by ceric su1fat.e (1). The 1~roduct.s of this react,ion are carried over in a CO,-free and carbonyl-free air stream and trapped as t,he acetaldehydesemicarbazone and the Hynmine-carbonate. respect,ively. Concentration of t,he semicarbazone is IIKW~NY~ by FY atworption, and the amount. of CO, t,rapped is determined by difference titrnt,ion. Radioact,ivit8y of the products is detcnnined dirrctly by j11wnb of liquitl wintillation rounting. mmr~ras
.\sI)
~~~:nw~)s
All reagents were prep:trcbd in di~tillwl. tlcionizwl n-atcr from c*lwlnic:\l~ meet~ing ACS standards. Set&m-bnzide, 0.4 X irr (I 0.1 3‘ .Ircfccfc B~ufier cct pfl @S. X weighed out. amount of senric:trbazitle h~tlrochloride n-as dissolved in a small amount of water. The pH TW:: adjusted to pH 4.25 with 1OS NaOT-I and the solution mndc up to volume wit’h 0.1 iA’ acctatc buffer, pH 4.25. El~a~nin~. 0.04 111i/c Xelhonol. This was prepared fresh wxkly 1q diluting Hyamine 10X (I J1 1T;vm~~ine in met,hanol M supplied 1);~Pnckawl) with methanol. ’ TIIIS investigation IV:,.. ~~I~I~Kx~~~ IIF H~~‘scYII.~~~~ C+r:m(s HE-083ii and in 11art. 1)~ H-3130 of rhc S:~tion:ll Inditutc; 01 TTIYII~II. Rcc,l]Gc,nt of n Il. P. I’. H. S. l’o~t~lo~~iord Vc~llo\wllil~ ( l-l%H I~~-?~.IX!bW) front I 1~ X:LI ion:rl HIYI~~ 1n.t it IItr,. UK1
I ‘( vi,, ,Su/jtrtc, 0.2 ;\.. (‘cric wii:ltcl IV;ISclisrolvc~, l in 1 .\- I-1 $0, wit,h warming and stirring, filtered. and tit,ratecl against f(xrrou:: 0.1 .\’ mlmonium sulfate using o-~~lienant~ltrolctt~-ferrous Sulf:ltc colll]‘lm. 0.025 1-u in water, as an indicator. The ferr0u.c :~mmoniuttt sulf:ttc WI,. 111:uh~iwll for titration by dissolving 1.96 gm ferrous: anttt1oniu111 sulfate.tiH,O in 100 ml water. The titer of ceric sulfate was :~djlrstc~cl :trcor(lingly witlt 1 ,V H,SO, to a ceric ion concentration of 0.2 X. Radioisotope Xohtions. Carrier lactate solutions were pr~~p:~r~.~~lt’r0t11 lithium lactate (Nutritional Biocllclnical~). I -t,-C1l-L:rc~t:ttc~ ~1.. ol)tained from Volk Radiochcttticals. 2-~-C"-L:xt:~t,e vv?i:w p*~~~~rc~l II~ incubat.ion of 2-C”-sodium pyruvate (New Engl:ttttl NuclcnFt \vitlt L-la&e acid deltydrogcrtase and SAD (Sigma) at 25,‘C in a plto>l)ltatc buffcl at pH 7.5 as described by Koeppe et al. (2). The reaction was stopped wit’lt perchloric acid and the mixture was centrifuged. The perchlorate ion WI:: removed by neutralization to pH 5 with 10N KOH in an ice bath with 30 min standing. The supernat,ant was retnoved by centrifugation and filt,ration and placed on a Dowex l-58 colunm for purification. Lactate was eluted with 0.15M SaCl as described by the mcthofl of Piatnek-Leunissen and Leunissen (3). ll’aphthalene-Dioaane Xcintillator. This liquid scintillator describc~l by Bray (4) consists of the following: naphthalenc 60 gm; PPO, 2.5 diphenyloxazolc, 4 gm; POPOP, 1,4-tli-Z-(5-pltenyloxazolyl) benzene). 0.2 gm; methanol 100 ml; ethylene glycol 20 ml; made up to n volume oi’ 1000 ml with p-dioxanc. P,YWP,~UW. To an aliquot of protc~in-frcbc filtrate, of which the lncr:ltt~ content ltnd been determined previously by the metltod of Barker and Summerson (5), carrier lactate is added to bring the total lactate in a for I~clllc,~:~l of working range of 40-50 Pmolee. The filtrate t,'? trentct1 glucose and pyruvate by ion-exchange chromatography (3). Tltc ~tntl)le is then added to a 3-neck pear-shaped 100 ml flask for the tlegracl:ltion of tltcl l;tct:iic. The dcgr:ul:ltiott tr:liti i.. (lc~l)ic~tc~llin E’ig. I. l)iffcBrc%t cli.+tillation setups were used, and it was found t,hat the ljrcsettt nrrangem~ttt gave t,lte least rise to condensation of water vapor* int,o the sctnicarllazide trap. After introduction of t,ltc sample, the fla,sk is attacltcd by iL,< centet neck to a water-cooled Liebig condenser, 20 cm long with an id. of 1 CI~I. =\ Gl:i+C’ol ltcating tti:~titlc~ c*ottrtc~c*tc~llto :i vario*t:it tran~fornter :tncl supported by an adjustable ring is placed under the flask. -Mjustability of the support allows a bett,er regulation of the flask temperature. The glass :lc>rat,or is inserted into one of t,ltc necks, while the third neck ’ TIN, glassware ~;I:I~sw.:II.c Coml’nnp,
necdrd
for
Woo~lh~~r,v,
this N.
study J.
~3s
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from
Jo1111
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Iil)\.(by
.5;lwr.r:11
serves to hold M I O/30 ground-joint thermometer. with 75 mm immersion. The condcnwr via a connecting tube fitted with lo/30 joint thermometer leads to a series of traps. The thermometer :~llows constant monitoring of the vapor, SO that the temperature may be maintained just above t,he boiling point of ucetnldehyde C’l”C I. The t,rnin of t,rnps ron%ts of the following consecutively nrrnngecl: 1 tube tube 1 tulle tion. 1 tube
of 2.5 ml 0.4111 seiiiic:~riJazick in acet,atc buffer (this receiving is kept immersed in a11 ice bath). of :~pprosiniatc~ly 5 111101’ :Ilwolutc methanol for ~~nlwr ~aturaof rj ml 0.04 M Hyamine
in llwtlrnnol.
1 tube of 5 ml concentrated H,SO, as a vapor trq whic~h then is (VUnect,cd via a drying tube of Drierite to a Fisher flowmetcr. -411 ground-glass joints were of 19, 22 size unlcas rpccificd otherwise. The length of the receiving tubes used was 15 ~111,and the delivery t#ube was fitted to almost reach the bott.om. These receiving tube:: Inay be either simple test tubes with side arm fit’tccl with n rubbrr rt’oppcr through which a glass delivery tube is passed or test tubes with groun(lglass joints and fitted gas-dispersion delivery tube?. With the fl:~k and train as~eml~l~~l. the vapor thermomctel~ above the condenstlr is removed, nncl a prenerntion period is begun during which the heating mant’le is turned on. When t.he working tcml)ernture of :i;i60” is reached, the vapor thermomctcr is reinserted into the condcnscar and the air flow quickly adjusted to 300 ml/min. The flask thcrmomrtt~r is removed and 4 ml of 0.2 A’ ceric .+ulfatc is injcc’tcacl into the flask by means of a plastic syringe providrtl with a plastic cnnnula. The flxsk thermometer is quickly reinserted nncl the flow readjusted. The vapor by temperat,ure is kept above 21”. the boiling point of acctnltlchyde. means of regulating the dcgrcc of water cooling. A reaction time of 20 min results in a recovery of 95-lOOF, if the init,ial snmplc~ volume in the degradation flask was 10 ml. At complct’ion of the clegr:&~tion the flask thermometer is rrtnovcc-l and the traps arc clisconncc+ctl. The contents of the scmicarbazide trap containing the 2,3-c:~rbon moic$ :I- :tn :tcc,taldehydc-~:crnicarl~~lzone are transferred into a glass-st,opperc(l T.VXW~ without any furthrr clilut.ion. A 0.5 ml aliquot is maclct 1111to 1UO iill with watcbr anal tllc> optic:tl density is recorded at 224 ml.{. in :I spectrophotometcr against ;i ‘.rcagent blank” similarly Radioactivity of t11c sclnicarbazonc is “oxidized.” determined in a Packard Tri-Carl) scintillation spcct,romcter by diluting 1 ml of the semicarhnziclc tr:ip in I5 ml of Bray’s scintiliat.or solution. Counting was done wit,11 ;I backgrounfl of 30 cpm and an efficiency of 48% (2 ml of the trap coul~l be counte(d at an efficicnry of 34% \. The contents of the Hyamine-lllethanol trap containing t~hc carl~osyl moiety as Hyamine-carbonate are quantitatively t~ransfcrrcd into a 10 ml volumetric flask with methanol and made up to volume. Cart sboul~l bc taken to rinse both reccivcr and cleliverp t~ube in RS short :t time as possible to prcsvcnt contamination by room-air CO,. The amount of C’f j1 t,rappe(l is determined by diffrrence titration of 2 ml aliquot:: of ~a~nplc a.nd clegradat,ion blank wit11 0.01 :Y HC’l in methanol in 10 ml Erlt~nmeyer flasks containing 1 1111 of 0.8’/: I&Cl, with phcnolpl~thnlcin t.nclpoint. Radioactivity is dctcrmined on a 3 ml aliquot in 15 ml Bray’s solution with an rfficiencp of 409 and :L background of 30 cpltr. ‘I%~
uw of Brag’s solution for the counting of the Hyamine-carbonate the count*ing of a larger aliquot with less quenching than when was the scintillator solvent. Efficiency of the diffcrcnt counting Rntlionctive counts were WIS cletennined with CL’-toluenv. with :i 1-2s error (6).
allowed toluene system obtainelI
13‘~. 2. Schematic repreac,ntntion of the CO,-cnrbonyl scrub train: (&i) reducing valve, (B) wash bottle containing glass wool filter, (S) check valve (Dynalab. catalog #1268), (C) 500 ml 20% KOH, (D) 150 ml 3% semicarbazide dissolv4 in water and made to volume with concc~ntrntcd J&SO,, (E) cartridge containing intlirat,ing Drieritc, 6-20 mesh, (F) Ascnrite, 6-20 mesh, (G) Tygon tubing, length 80 cm, i.d. 0.6 cm, also containing Ascarite. (H) T-tube for adjustment. of flow, (I) screw clamp, (J) Drierite. All gas-wvnshing hottlcs were provided with a doul)lr a(‘t of dog-ears for securing the two parts with lr~~ary duty springs.
assembly the towers of the scrub train require a 24 hr aeration period to remove all of the CO, present in the large drutl space. This initial scrubbing should be done with Xmlritf tubw disconnected to Ilrcbvent early saturation of the ,4scnrit,e. 1ViCh this scrub t’rnin no semicnrbazone was formed during :t blank degradation and CO, ~nlues wrc 3 to .i /moles. It, is imperntjive that a degradation blnnk IX> obtained on each day that the train is used. Rcttlotwl of Zn.terfeGg Sub.st~~ctcs.During :I cwic snlfatc osicl:ition both pyruvate and glucose will be degraded. In fact, ceric sulfate osiclation was originally used for the assay of pyruvate by Fronmgeot and Ikwuelle (7)) since it is completely oxidized to acetic a&d and car*bon
dioxide. Using uniformly labeled glucose-C14 it was found hew that, during a 20 min degradation at 55-60”, 0.5% of thr glucose is 31~ oxidized to CO,. While the absolute value is small, this could lead to considerable interference in radiochemical assay?: whw the glucone is present in a relatively high specific activity. The glucose can bc rcmovcd I 1) by copper-Iinic treat,ment or (2 ) by ion-exchange chron~ntogrnphy. It is recommended that carrier lactate be added to the sample before trcntmcnt by either proccdurc. -4 Gnglc copper-lime treatnlent removed 88p of the glucose. A ScccJllti copperlime treatment w-n+ con~iclered to t)t’ impractical. Since our interests centered around the int,erlnbeling of glucose, lactate, ant1 pyruvnt.e in the intact. animal, an ion-escliangc prowlurc was dcsignc(l to allow -ep:trntion. complete recovery, and meawrement of the specific activity of csch of the abore substratw in the smile .~~nall blootl sanlple (3). \I’hile t.he 25 ml fraction eluted from the column which contain.~ the lact#atc may be degraded as such, this l:trgw ~olunw clock lend to :I, slightly lower recovery a:: will he discus& later. Othrr
sut)Strtttrs
in
thtb
fJlOOll
tllfit
Illigllt
t,hcOWtically
iIlt(‘l’l’CI’t’
:il‘f
acetoacetak and acet’onc,. I’nclcr normal conditions, howcvcr. the conwntratioti of tII(w’ +uI)>t:~iiw~ in thcx I~loocl is wry low. In any c\.ent the prcucration at 60°C in the nlcrliunl of strong acid here ~houl~l he sufficient. to decwbosylatc the acctoacetate and cwporate the acetow (b.1). 55”) wit.hout contamination of the trap:: on slllwqnent lnct:ltc> degradat,ion. lIR.‘;ULTS Stnndatd &rzje of dcctnldeh!Jlle-SenLicarbazolze. A standard curvy’ war constructed using a serie? of Rolutions prepared by diluting acctnldehyde (Eastman Kodak AR) with water. =Iliquots containing O-70.8 ;moles were mixed with 2.5 ml of the eemicarhazide reagent. This reaction mixturcl was then diluted 1:200 with water for measurcrncnt~of the optical density of t,hc acetaldehycle-remicarbazone at 224 m/l.. All prq)urations, swept the final dilution, wcrc carried out in a cold room at 4°C. The mean data obtained from 4 determinations of each of 3 chosen c*onccntrations arc presented in Tablr 1. Since the c,onc~~ntr:at,ion-al)sor~)tioll relationship was linear over the range O-53.1 ~,.moles,a kttistirally dctcrmined standard curve was con&twcted and all futrlro concrntrationc calculated using this curve. Fo,nmtion and Stnbility of the ilcetnlclehu~le-Se~~~i~~~bu~oi~.e. The* formation and stabilit’y of the semicarbazone derivative are dependent on pH, temperature, and molar ratio of the two reart’ants. Conant and Ikwtlett (8) found that the optimum pH for t,he reactSion wa+ npprosi-
‘I The acetaldehyde w:w 1 :‘LW with water, 31~1 1he * I
0.4 .lf at pH 4.2.5, diluted misrtl with 2.5 ml of semicw$:uitlt, :~IJsorption re:~l in a al)~ot,roI)hotolll~,l(~~ at 224 mp. at 224 mp with 1.00 cm light lxtt h. tlrtwmirl:~tiolk: wit tr thr rtxndartl tlvvi:ttioll. for Illc I)cst slr:iigtrl. liw which fits I hrse p)itrt,s ii !I = I).OlIT
n~atclp i..?. In the prcscnt study the tienlicarl)nziclc rcugcnt was, thercfort, lxelwxl in an acctutc buffer of pH 4.25. Tmniwion of thcb trapping vc~scl in an ice Imth further enhances the reaction by lnaintnining the tenqwrature of the tral)ping aenlicarbazide wag& well bclo~\- tllc boiling l)oint of :~cctaldeh)-clt (21 G). In preliminary stwlies it was founcl that a 4: 1 inolar ratio of the two resctant~ ycmirarbazide: wetal~lchyde rebultctl in :a relatively unstable rlcrix-ati\-? :I> refkctcfl in continually tlccrcwing opt.ical tlcnGtiw. -in increase in the concentration of the scn~icarbazidc reagent to effect, a final nlolnr ratio of 20-25 senlicarbwzi~lc : 1 ncct:~ldehyde procluced a derivative observed to be stable for 20 hr. It nppcarccl that pH was also a factor. I3urbridgc et trl. (91, wing :I pho~phntc~ I)l~ff(~ :lt IIH 7 :incl tlrcb sanle high niolar ratio, reportctl a stability of only 2 hr. The ~~illicarbazonc~ cl(~rirati\-e is relativc,ly 5tnhlc in tlw Bray’s solution enqAoyec1 for rxlioactiw counting. Thcrc is no :~ppreciahle dccrcaw in act,ivity in the first 24 hr. Thcwaftcr. t,lle rnclio;act,ivit~ gradually decrcas(+ presiunnbly due to color c~~~cnching. X J& yellow\ color n-a: observed to forni with t’his prolongecl standing even at, the 101~ teinlwrxtiire of the frcczcr conil~:~rtnirnt of thcb I’acliard liclllill wintillation wuntcr. Rcrol’f ‘.,I/ trd Kclinbilit!/ of Spscific ;IctG,it!/. Tllt~ l)(‘r ccant r(~(71~c~l~~of known aiil0unt.i of lactic acid degradccl in tlic sptcnl clc~cril)(~cl here i.i generally dependrnt on the volunlc of the ~;:unplc introducc(l into thtl tlcgraclation fln>k ant1 the duration of tlic rlcgrxdation-distillation procc.. durc. Tlict inost practical conditions producing optinluln reco~-,~ric~ \verc fount1 to IIC 10 in1 eainplc 1 size and 20 nGn clk:tillation. I’ndcr these conditions with 50 poles lactic acid the recovery of t,hc :~cet:~ldehyclp fraction is 9%100% ant1 the H\ramine-cnl,l)orIntc. 100yO. The slight discrcpnc*y brtween nrct:1l~lcl~prlc and (‘0, rccovcrica is rnos:t likc*jy ~1~1~~
416
LEUNISSIW
AND
PIATNEK-LElJKlsSKN
to a further oxidation of acetaldehyde to acetic acid in the degradation flask (10). Acetate-l-C’“, if formed during degradation of 2-Cl,‘-lactate, is not carried over. After degradation of 50 ,.molcs of acetic acid-l-C’“, 0.32 and 0.700j0 of the radioactivity mere detcctcd in the scmicarhazide a,nd the Hya.mine traps, respectively. When the volume of the snmplc in the degradation flask was 25 ml, the per cent recovery was reduced to 90% evrn though the distillation time was increased to 30 or 40 min. Thus, for our purposes, the 25 ml lactate fraction eluted from the ion-exchange column was evaporated under reduced pressure to 1-2 ml and transferred quantitatively to the degradation flask. Table 2 presents data drmonstratin, u the reliability and accuracy of the dcterminnt,ion of specific artivit,y for a S;cric>s of clegrndations of TAml,I~: ” D~~ERMITATIOS
OF
Stvwrsrc
Amrrvu
W-LACTATIC
my
D~:oa.\r~.~~ro~
06
*P~SI).ZRI)
SOLUTIONS
Degradation flasks were prepwctl coni :tining 0.5 rul of 0.1 .I[ lithium ktate (50 pmoles) and 1 ml of 10 W H&O,. T(k #I--#. ’ 5 wew adtletl I ml cd L’-CLi-l:~ctate and I ml of I-CF4-lactate; to #6, 1 ml of 1-C’” lactate only; lo 17. I ml of ?-C!lq lactate only, and the fin:J r(Jume T~\VUS adjusted t,o 10 ml wN1 deionized w:~ter. The degradations were carried solution was out. as described in the text. The mdionctivity of the 2-c”‘- lact:tte stantl:ud 10,883 cpm/ml; that. of the I-CWlactnte, with irlfitlitesimally small 30,OO 3 cpm/ml amomlts of chemical lactate. The t.heoretic:tl specific activities were, therefore, 218 cpm/pmole for the 2-C14-lactnte and 601 cpm/pmole for l-c’L1-l:wi ut t’. (‘0,
:\cetnldehvdz x0
1 2 :; 4 5 6 7 Mean *RD. y0 deviation
p111OlC4 (P-W 1
<‘,lW
~~~-..
-
10,3ss 10,387 0,737 10,289 10,434 4 lO,.i%l
47.S6 4s. 06 4i.u 47 .!I6 47.71 4S.S 4s “7
rpm /pM 215 “16 304 215 210 neg. “17 214 5 3
C(,ll, 30,451 27,050 :30,2‘30 30,369 30 ( 485 :;o ,058 II
prllolw
(jl‘ll)
50.45 44.35 49.75 .50. 10 50 75 50.60 40 so
cpm/fiM 604 610 609 606 601 504 neg. 604 15 ::
standard C14-lactate solutions. Radioactivity was measured with a counting error of 1%. Under the conditions described above the mean value obtained for the acetaldehydc-semicarbazone was 214.3 f 5.4 (S.D.1 cpm/pmole as compared to the theoretical value of 217.7 cpm/ Pmole. The mean value obtained for the Hyamine-carbonate was 604 k 15.3 IS.D.) as compared t,o a theoretical value of 600.5 cpm/pmole.
Specificity of the Trnps. Cross-contamination of either oi the traps did not occur, as illust,rat8ed in Table 2. When only I-Cl”-lactate was present (degradation .#6), no radioactivity ins found in the acetaldehyde trap. Likewise, when only 2-Cl&-lactate was present, (degradat,ion #7), no mdionctirity was found in the cnrl~osyl trap. DI,SCUSSION
The principle employe~l in t,hc methotl clcs:cribed is an osidative degradation of the lactic acid molecule to acetaldehydc and CO,. Scvera1 reagents are available for this purpose. Potassium pcrnianganate was used by Fricdemann. Cotonio, and Shaffcr (II), but., as stated by these authors, the removal of glucose is mandatory bincc it is also extensively oxidized. For general use the ceric sulfatcl oxidation of Gordon and Quastel (1) woultl stem to I)e prefernblc since only 0.4% of the glucose is degraded under the conditions of th(l re:tction (20 min at 60°C). However, for experiment’al conditions in which the glucose may be present in very high ppccific activity the gluco>c ~l~oulcl also be removed prior to degradation of t’he lactate. The conditions of the ceric sulfate reaction as to air flow, clurat,ion, temperature, and molarity of ccric sulfat,e hare been thoroughly invcstigated by Elsden and Gibson (12). It would seem from t’heir data that, at 53°C with a eerie sulfat,e concentration of 0.1 M, the formation of acetaldehyde requires less than a few minutes. Prolonged reaction times result in a further oxidation to acetic acid as sugge&d previously by I,ong (10). Eleden and Gibson, Ulerefore, increased the flow rate in their steam-distillation system to 500-600 ml,‘min in orcler to rcmovc the acetaldehyde from the degradation flask immediately on formation. Recoveries of 100% were obtainecl using a t’otal reaction tiltic of 45 min. For our purposes a flow rate of 250-300 ml/min was found to he more desirable (a) to prevent working at, too high air ~)rw~urw, (b) to improve efficiency of the CO,-carbonyl scrub train, which is ncccssa~y for accurate CO, measurement, and (c) to increase the efficicncg of acetaladehyde and CO, trapping in the dist,illation train. Tht lower recoveries of acetaldehyde (95%‘) as compared to CO, (100% 1 may thus be due to lose t’hrough acetic acid formation at the slower rate of the acctaldehyde. In our hands the dropwise addition of ceric sulfate prescribed by Elsden and Gibson did not appear to be required. The simpler, single addition technique of Long (10) was therefore adopted. The introduction of t’rapping agents for the products of lactic acid degradation which would allow their isolation and raclionrt’ivc assay was first presented by Rrin and Olson (13). Using the permanganat!e osida-
tion technique of Friedemann et CCL (12)) t,hesc authors trapped the acetaldehyde as its dimedon derivative and the CO, as sodium curl)onatcl. Both products were then prepared for plan&t count,ing. Reeovcries in these trapping agents as inclicat,ed by these nuthor~ mere in the order of 40%. Using this method initially, we were able to incareast the yield of the acetaldehyde fraction to 70% by employing a higher molarity of dimedon dissolved in methanol. However, precipitation of the tliniedon derivative required prolonged treatment, overnight standing, and recrystallizations to obtain a pure product of con&ant specific activity and characterist’ic melting point. These rather time-consuming features led US to examine other t’rapping reagents that woul(l allow titrimetric or spectrophotometric assays which would also be more suitable to liquid .qcintillation counting. For the trapping of the acetaldehyde fraction we elected to use semicarbazide hyclrochloritle, a well-known reagent for earbonyl compounds. Two features make it a desirable trapping agent: (1) the product can be assayed directly by spectrophotometric measurement and (21 since the ol~timmn condition for ncetaldehyde-semicarbazone formation was found to be in an acetate buffer of apl)roximatc pH 4.5 IS), the l-carbon moiety of the lactic acid degradation, the CO,, is not simultaneously trapped. Conditions for t,he t,rapping of acetaldehyde by this reagent in such a distillation train were made optimal by keeping the vapor temperature in the condenser slightly above the boiling point of acetaldehyde and by immersing the trapping vessel in an ice bath. Recovery of acct:~ldc~hpck with t,his reagent under these condition+ w:I‘: ~~t~l~~‘o~lr~c~ii)ly 95%. The (‘0, evolved in the degradation is trapped directly in Hyamine after the mncrornethod of Fredrickson and Ono (141, who employed a similar technique in the determination of the specificsactivity of C”(L of expired air. According to Passman, Radin, and Cooper (15), a minimal amount. of 1.1 nrolcs of Hyamine will efficiently trap 1 mole of CO,. At the flow rates used in our degradation system, a molar ratio of Hyamine to CO, of 5: 1 provided t,he most efficient trapping. ~~%ile only 3OClrof the CO.. t.rap was gtnernllv us;fd for the r:~cliochemi~al assay. IxrgtJr :unounts could be cmployc*cl by first reducing the methanol concentration by evaporation. It was found, however, that ext8rcmc c:\re had to he cixercised to prevent splashover and loss. The range of the method is 15 to 60 pmolea. It is advisable not to n-ork below 15 ~moles since then a slight variation in thr CO, blank may cause a larger per cent error. In extending the range beyond 60 +oles it should be noted that, while the amount, of semicarhazide used is sufficient
to trap 100 pmoles of acetaldehyde, accuracy may be sacrificed in obtaining a sufficient dilution for the spectrophotometric assay. The latt,er, as previously noted, was found to be linear from 0 to 53 ,pmoles acetaldehyde when dilut~ed 1: 200. In working with biological filtrates, it has been our procedure to degrade an equivalent8 of approximately 2.5 to 3 ml of whole blood and 40 ,moles of carrier lactate. With a normal range oi arterial blood lactate of 1 to 2 ~~moles,~ml,the total amount degraded varied from 42 to 46 ~molcs. If the blood level of la&ate is expected to be higlrer (during csercisc or systemic hypoxia’) , t INLcl~~gr:rtlat.ionc:m 1~ carried out with less carrier lactate. The degr:tdxtion method clrsrribc~l is relatively eiml~le and easy to perform. With home experienrc: (i to 8 degradations could be handled daily, including the time required for chemical :rn(l rn~liochcmic~nlanalpw of the final product.~. SLinIKmlI. X method is preicntcd which :tllo~ the dctrl,lliin:ttic,li of radioAivity in the l-carbon anal the 2,3-carbon moieties of I:u%ic acid adapted to liquid scintillation counting. 2. T11c lactic acid is osidizcd by ccric sulfate to :rcctnltl~hy~lr: and CO,, which are carried over into :I s;(+c.- of t 1x1’~ by nrc:rn+ of :r CO,:mcl cnrbonyl-frer air stream. 3. The acetnlclehydc is trappe(l :LS its FClllic:Ir~):Izol1(~:un(l :tF>ayed sl~ect~~opl~otometri~allp.The CC), is tr:rl)pctl as its II?-:llllinc-~:lrl-)oll:~t~, and measured by difference titration. 1~:~~liochemicnl:rs;eny~ arc performctl directly using Bray’s solution :ts the counting system. 4. ,4 descript.ion is gircn of the clistillation-tlegl,:~(l:~tioti setup 311~1 of the CO1-~arhonpl rcrub train u~cl. Optimal condition:: a-; to :tir flow. t8rrul)c~r:rturc. all11 tluration of tlrc reaction arc given. 5. Tbc methotl (*:rn lx u~e~l iii tllcl r:rrr~;c~of 15 to 60 pl\lolrs :tncI i., reproducible and nccur:rtc. .\CI~~o~~‘r,7SDC;I~~T~ Tht :wsislnncr.
:IUI 1111rs nr~
incivl)trll
IO Mrr.
S. Rogers
and
Mr.
F. Gil
[or
(~q)c’r(
If,,.llnic.:lJ
7. FROMAGEOT, 8. CONAZIT, J. 9. BURBRIDGE,
C., AND DESNUELLE, P., &o&en. 2. 279, 174 (19%). B., AND BARTLETT, P. D., J. Am. Chem. Sot. 54, 2881 t 1932~ T. N., HINE, C. H., AND Scmx, A. F., .I. Lc~ij. (‘lin. .\f~l. .?5, ~3
(1950). 10. LONQ, C., Biochem. J. 40, 27 (1946). 11. FRIEDEK~SS. T. E., Cnmsro, Ibl., AND SH.WEXK, P. A., J. Sol. (Ihrn~. 73, 335 (1927). 12. ELSDEN, S. R., AND GIBSON, Q. H., Biochem. J. 58, 154 (1954). 13. BRIN, M., AND OLSON, R. E., 1. Biol. Chem. 199, 475 (1952). 14. FREDRICKSON, D. S., AND ONO, K., J. Lab. Clin. Med. 51, 147 (1958). 15. PA’SMANN, J. M., RADIN, N. S., AND COOPER, J. A. D., Anal. Chern. 28, 484 (1956).
.\sI)
~~~:nw~)s
All reagents were prep:trcbd in di~tillwl. tlcionizwl n-atcr from c*lwlnic:\l~ meet~ing ACS standards. Set&m-bnzide, 0.4 X irr (I 0.1 3‘ .Ircfccfc B~ufier cct pfl @S. X weighed out. amount of senric:trbazitle h~tlrochloride n-as dissolved in a small amount of water. The pH TW:: adjusted to pH 4.25 with 1OS NaOT-I and the solution mndc up to volume wit’h 0.1 iA’ acctatc buffer, pH 4.25. El~a~nin~. 0.04 111i/c Xelhonol. This was prepared fresh wxkly 1q diluting Hyamine 10X (I J1 1T;vm~~ine in met,hanol M supplied 1);~Pnckawl) with methanol. ’ TIIIS investigation IV:,.. ~~I~I~Kx~~~ IIF H~~‘scYII.~~~~ C+r:m(s HE-083ii and in 11art. 1)~ H-3130 of rhc S:~tion:ll Inditutc; 01 TTIYII~II. Rcc,l]Gc,nt of n Il. P. I’. H. S. l’o~t~lo~~iord Vc~llo\wllil~ ( l-l%H I~~-?~.IX!bW) front I 1~ X:LI ion:rl HIYI~~ 1n.t it IItr,. UK1
I ‘( vi,, ,Su/jtrtc, 0.2 ;\.. (‘cric wii:ltcl IV;ISclisrolvc~, l in 1 .\- I-1 $0, wit,h warming and stirring, filtered. and tit,ratecl against f(xrrou:: 0.1 .\’ mlmonium sulfate using o-~~lienant~ltrolctt~-ferrous Sulf:ltc colll]‘lm. 0.025 1-u in water, as an indicator. The ferr0u.c :~mmoniuttt sulf:ttc WI,. 111:uh~iwll for titration by dissolving 1.96 gm ferrous: anttt1oniu111 sulfate.tiH,O in 100 ml water. The titer of ceric sulfate was :~djlrstc~cl :trcor(lingly witlt 1 ,V H,SO, to a ceric ion concentration of 0.2 X. Radioisotope Xohtions. Carrier lactate solutions were pr~~p:~r~.~~lt’r0t11 lithium lactate (Nutritional Biocllclnical~). I -t,-C1l-L:rc~t:ttc~ ~1.. ol)tained from Volk Radiochcttticals. 2-~-C"-L:xt:~t,e vv?i:w p*~~~~rc~l II~ incubat.ion of 2-C”-sodium pyruvate (New Engl:ttttl NuclcnFt \vitlt L-la&e acid deltydrogcrtase and SAD (Sigma) at 25,‘C in a plto>l)ltatc buffcl at pH 7.5 as described by Koeppe et al. (2). The reaction was stopped wit’lt perchloric acid and the mixture was centrifuged. The perchlorate ion WI:: removed by neutralization to pH 5 with 10N KOH in an ice bath with 30 min standing. The supernat,ant was retnoved by centrifugation and filt,ration and placed on a Dowex l-58 colunm for purification. Lactate was eluted with 0.15M SaCl as described by the mcthofl of Piatnek-Leunissen and Leunissen (3). ll’aphthalene-Dioaane Xcintillator. This liquid scintillator describc~l by Bray (4) consists of the following: naphthalenc 60 gm; PPO, 2.5 diphenyloxazolc, 4 gm; POPOP, 1,4-tli-Z-(5-pltenyloxazolyl) benzene). 0.2 gm; methanol 100 ml; ethylene glycol 20 ml; made up to n volume oi’ 1000 ml with p-dioxanc. P,YWP,~UW. To an aliquot of protc~in-frcbc filtrate, of which the lncr:ltt~ content ltnd been determined previously by the metltod of Barker and Summerson (5), carrier lactate is added to bring the total lactate in a for I~clllc,~:~l of working range of 40-50 Pmolee. The filtrate t,'? trentct1 glucose and pyruvate by ion-exchange chromatography (3). Tltc ~tntl)le is then added to a 3-neck pear-shaped 100 ml flask for the tlegracl:ltion of tltcl l;tct:iic. The dcgr:ul:ltiott tr:liti i.. (lc~l)ic~tc~llin E’ig. I. l)iffcBrc%t cli.+tillation setups were used, and it was found t,hat the ljrcsettt nrrangem~ttt gave t,lte least rise to condensation of water vapor* int,o the sctnicarllazide trap. After introduction of t,ltc sample, the fla,sk is attacltcd by iL,< centet neck to a water-cooled Liebig condenser, 20 cm long with an id. of 1 CI~I. =\ Gl:i+C’ol ltcating tti:~titlc~ c*ottrtc~c*tc~llto :i vario*t:it tran~fornter :tncl supported by an adjustable ring is placed under the flask. -Mjustability of the support allows a bett,er regulation of the flask temperature. The glass :lc>rat,or is inserted into one of t,ltc necks, while the third neck ’ TIN, glassware ~;I:I~sw.:II.c Coml’nnp,
necdrd
for
Woo~lh~~r,v,
this N.
study J.
~3s
obtnined
from
Jo1111
$.
Iil)\.(by
.5;lwr.r:11
serves to hold M I O/30 ground-joint thermometer. with 75 mm immersion. The condcnwr via a connecting tube fitted with lo/30 joint thermometer leads to a series of traps. The thermometer :~llows constant monitoring of the vapor, SO that the temperature may be maintained just above t,he boiling point of ucetnldehyde C’l”C I. The t,rnin of t,rnps ron%ts of the following consecutively nrrnngecl: 1 tube tube 1 tulle tion. 1 tube
of 2.5 ml 0.4111 seiiiic:~riJazick in acet,atc buffer (this receiving is kept immersed in a11 ice bath). of :~pprosiniatc~ly 5 111101’ :Ilwolutc methanol for ~~nlwr ~aturaof rj ml 0.04 M Hyamine
in llwtlrnnol.
1 tube of 5 ml concentrated H,SO, as a vapor trq whic~h then is (VUnect,cd via a drying tube of Drierite to a Fisher flowmetcr. -411 ground-glass joints were of 19, 22 size unlcas rpccificd otherwise. The length of the receiving tubes used was 15 ~111,and the delivery t#ube was fitted to almost reach the bott.om. These receiving tube:: Inay be either simple test tubes with side arm fit’tccl with n rubbrr rt’oppcr through which a glass delivery tube is passed or test tubes with groun(lglass joints and fitted gas-dispersion delivery tube?. With the fl:~k and train as~eml~l~~l. the vapor thermomctel~ above the condenstlr is removed, nncl a prenerntion period is begun during which the heating mant’le is turned on. When t.he working tcml)ernture of :i;i60” is reached, the vapor thermomctcr is reinserted into the condcnscar and the air flow quickly adjusted to 300 ml/min. The flask thcrmomrtt~r is removed and 4 ml of 0.2 A’ ceric .+ulfatc is injcc’tcacl into the flask by means of a plastic syringe providrtl with a plastic cnnnula. The flxsk thermometer is quickly reinserted nncl the flow readjusted. The vapor by temperat,ure is kept above 21”. the boiling point of acctnltlchyde. means of regulating the dcgrcc of water cooling. A reaction time of 20 min results in a recovery of 95-lOOF, if the init,ial snmplc~ volume in the degradation flask was 10 ml. At complct’ion of the clegr:&~tion the flask thermometer is rrtnovcc-l and the traps arc clisconncc+ctl. The contents of the scmicarbazide trap containing the 2,3-c:~rbon moic$ :I- :tn :tcc,taldehydc-~:crnicarl~~lzone are transferred into a glass-st,opperc(l T.VXW~ without any furthrr clilut.ion. A 0.5 ml aliquot is maclct 1111to 1UO iill with watcbr anal tllc> optic:tl density is recorded at 224 ml.{. in :I spectrophotometcr against ;i ‘.rcagent blank” similarly Radioactivity of t11c sclnicarbazonc is “oxidized.” determined in a Packard Tri-Carl) scintillation spcct,romcter by diluting 1 ml of the semicarhnziclc tr:ip in I5 ml of Bray’s scintiliat.or solution. Counting was done wit,11 ;I backgrounfl of 30 cpm and an efficiency of 48% (2 ml of the trap coul~l be counte(d at an efficicnry of 34% \. The contents of the Hyamine-lllethanol trap containing t~hc carl~osyl moiety as Hyamine-carbonate are quantitatively t~ransfcrrcd into a 10 ml volumetric flask with methanol and made up to volume. Cart sboul~l bc taken to rinse both reccivcr and cleliverp t~ube in RS short :t time as possible to prcsvcnt contamination by room-air CO,. The amount of C’f j1 t,rappe(l is determined by diffrrence titration of 2 ml aliquot:: of ~a~nplc a.nd clegradat,ion blank wit11 0.01 :Y HC’l in methanol in 10 ml Erlt~nmeyer flasks containing 1 1111 of 0.8’/: I&Cl, with phcnolpl~thnlcin t.nclpoint. Radioactivity is dctcrmined on a 3 ml aliquot in 15 ml Bray’s solution with an rfficiencp of 409 and :L background of 30 cpltr. ‘I%~
uw of Brag’s solution for the counting of the Hyamine-carbonate the count*ing of a larger aliquot with less quenching than when was the scintillator solvent. Efficiency of the diffcrcnt counting Rntlionctive counts were WIS cletennined with CL’-toluenv. with :i 1-2s error (6).
allowed toluene system obtainelI
13‘~. 2. Schematic repreac,ntntion of the CO,-cnrbonyl scrub train: (&i) reducing valve, (B) wash bottle containing glass wool filter, (S) check valve (Dynalab. catalog #1268), (C) 500 ml 20% KOH, (D) 150 ml 3% semicarbazide dissolv4 in water and made to volume with concc~ntrntcd J&SO,, (E) cartridge containing intlirat,ing Drieritc, 6-20 mesh, (F) Ascnrite, 6-20 mesh, (G) Tygon tubing, length 80 cm, i.d. 0.6 cm, also containing Ascarite. (H) T-tube for adjustment. of flow, (I) screw clamp, (J) Drierite. All gas-wvnshing hottlcs were provided with a doul)lr a(‘t of dog-ears for securing the two parts with lr~~ary duty springs.
assembly the towers of the scrub train require a 24 hr aeration period to remove all of the CO, present in the large drutl space. This initial scrubbing should be done with Xmlritf tubw disconnected to Ilrcbvent early saturation of the ,4scnrit,e. 1ViCh this scrub t’rnin no semicnrbazone was formed during :t blank degradation and CO, ~nlues wrc 3 to .i /moles. It, is imperntjive that a degradation blnnk IX> obtained on each day that the train is used. Rcttlotwl of Zn.terfeGg Sub.st~~ctcs.During :I cwic snlfatc osicl:ition both pyruvate and glucose will be degraded. In fact, ceric sulfate osiclation was originally used for the assay of pyruvate by Fronmgeot and Ikwuelle (7)) since it is completely oxidized to acetic a&d and car*bon
dioxide. Using uniformly labeled glucose-C14 it was found hew that, during a 20 min degradation at 55-60”, 0.5% of thr glucose is 31~ oxidized to CO,. While the absolute value is small, this could lead to considerable interference in radiochemical assay?: whw the glucone is present in a relatively high specific activity. The glucose can bc rcmovcd I 1) by copper-Iinic treat,ment or (2 ) by ion-exchange chron~ntogrnphy. It is recommended that carrier lactate be added to the sample before trcntmcnt by either proccdurc. -4 Gnglc copper-lime treatnlent removed 88p of the glucose. A ScccJllti copperlime treatment w-n+ con~iclered to t)t’ impractical. Since our interests centered around the int,erlnbeling of glucose, lactate, ant1 pyruvnt.e in the intact. animal, an ion-escliangc prowlurc was dcsignc(l to allow -ep:trntion. complete recovery, and meawrement of the specific activity of csch of the abore substratw in the smile .~~nall blootl sanlple (3). \I’hile t.he 25 ml fraction eluted from the column which contain.~ the lact#atc may be degraded as such, this l:trgw ~olunw clock lend to :I, slightly lower recovery a:: will he discus& later. Othrr
sut)Strtttrs
in
thtb
fJlOOll
tllfit
Illigllt
t,hcOWtically
iIlt(‘l’l’CI’t’
:il‘f
acetoacetak and acet’onc,. I’nclcr normal conditions, howcvcr. the conwntratioti of tII(w’ +uI)>t:~iiw~ in thcx I~loocl is wry low. In any c\.ent the prcucration at 60°C in the nlcrliunl of strong acid here ~houl~l he sufficient. to decwbosylatc the acctoacetate and cwporate the acetow (b.1). 55”) wit.hout contamination of the trap:: on slllwqnent lnct:ltc> degradat,ion. lIR.‘;ULTS Stnndatd &rzje of dcctnldeh!Jlle-SenLicarbazolze. A standard curvy’ war constructed using a serie? of Rolutions prepared by diluting acctnldehyde (Eastman Kodak AR) with water. =Iliquots containing O-70.8 ;moles were mixed with 2.5 ml of the eemicarhazide reagent. This reaction mixturcl was then diluted 1:200 with water for measurcrncnt~of the optical density of t,hc acetaldehycle-remicarbazone at 224 m/l.. All prq)urations, swept the final dilution, wcrc carried out in a cold room at 4°C. The mean data obtained from 4 determinations of each of 3 chosen c*onccntrations arc presented in Tablr 1. Since the c,onc~~ntr:at,ion-al)sor~)tioll relationship was linear over the range O-53.1 ~,.moles,a kttistirally dctcrmined standard curve was con&twcted and all futrlro concrntrationc calculated using this curve. Fo,nmtion and Stnbility of the ilcetnlclehu~le-Se~~~i~~~bu~oi~.e. The* formation and stabilit’y of the semicarbazone derivative are dependent on pH, temperature, and molar ratio of the two reart’ants. Conant and Ikwtlett (8) found that the optimum pH for t,he reactSion wa+ npprosi-
‘I The acetaldehyde w:w 1 :‘LW with water, 31~1 1he * I
0.4 .lf at pH 4.2.5, diluted misrtl with 2.5 ml of semicw$:uitlt, :~IJsorption re:~l in a al)~ot,roI)hotolll~,l(~~ at 224 mp. at 224 mp with 1.00 cm light lxtt h. tlrtwmirl:~tiolk: wit tr thr rtxndartl tlvvi:ttioll. for Illc I)cst slr:iigtrl. liw which fits I hrse p)itrt,s ii !I = I).OlIT
n~atclp i..?. In the prcscnt study the tienlicarl)nziclc rcugcnt was, thercfort, lxelwxl in an acctutc buffer of pH 4.25. Tmniwion of thcb trapping vc~scl in an ice Imth further enhances the reaction by lnaintnining the tenqwrature of the tral)ping aenlicarbazide wag& well bclo~\- tllc boiling l)oint of :~cctaldeh)-clt (21 G). In preliminary stwlies it was founcl that a 4: 1 inolar ratio of the two resctant~ ycmirarbazide: wetal~lchyde rebultctl in :a relatively unstable rlcrix-ati\-? :I> refkctcfl in continually tlccrcwing opt.ical tlcnGtiw. -in increase in the concentration of the scn~icarbazidc reagent to effect, a final nlolnr ratio of 20-25 senlicarbwzi~lc : 1 ncct:~ldehyde procluced a derivative observed to be stable for 20 hr. It nppcarccl that pH was also a factor. I3urbridgc et trl. (91, wing :I pho~phntc~ I)l~ff(~ :lt IIH 7 :incl tlrcb sanle high niolar ratio, reportctl a stability of only 2 hr. The ~~illicarbazonc~ cl(~rirati\-e is relativc,ly 5tnhlc in tlw Bray’s solution enqAoyec1 for rxlioactiw counting. Thcrc is no :~ppreciahle dccrcaw in act,ivity in the first 24 hr. Thcwaftcr. t,lle rnclio;act,ivit~ gradually decrcas(+ presiunnbly due to color c~~~cnching. X J& yellow\ color n-a: observed to forni with t’his prolongecl standing even at, the 101~ teinlwrxtiire of the frcczcr conil~:~rtnirnt of thcb I’acliard liclllill wintillation wuntcr. Rcrol’f ‘.,I/ trd Kclinbilit!/ of Spscific ;IctG,it!/. Tllt~ l)(‘r ccant r(~(71~c~l~~of known aiil0unt.i of lactic acid degradccl in tlic sptcnl clc~cril)(~cl here i.i generally dependrnt on the volunlc of the ~;:unplc introducc(l into thtl tlcgraclation fln>k ant1 the duration of tlic rlcgrxdation-distillation procc.. durc. Tlict inost practical conditions producing optinluln reco~-,~ric~ \verc fount1 to IIC 10 in1 eainplc 1 size and 20 nGn clk:tillation. I’ndcr these conditions with 50 poles lactic acid the recovery of t,hc :~cet:~ldehyclp fraction is 9%100% ant1 the H\ramine-cnl,l)orIntc. 100yO. The slight discrcpnc*y brtween nrct:1l~lcl~prlc and (‘0, rccovcrica is rnos:t likc*jy ~1~1~~
416
LEUNISSIW
AND
PIATNEK-LElJKlsSKN
to a further oxidation of acetaldehyde to acetic acid in the degradation flask (10). Acetate-l-C’“, if formed during degradation of 2-Cl,‘-lactate, is not carried over. After degradation of 50 ,.molcs of acetic acid-l-C’“, 0.32 and 0.700j0 of the radioactivity mere detcctcd in the scmicarhazide a,nd the Hya.mine traps, respectively. When the volume of the snmplc in the degradation flask was 25 ml, the per cent recovery was reduced to 90% evrn though the distillation time was increased to 30 or 40 min. Thus, for our purposes, the 25 ml lactate fraction eluted from the ion-exchange column was evaporated under reduced pressure to 1-2 ml and transferred quantitatively to the degradation flask. Table 2 presents data drmonstratin, u the reliability and accuracy of the dcterminnt,ion of specific artivit,y for a S;cric>s of clegrndations of TAml,I~: ” D~~ERMITATIOS
OF
Stvwrsrc
Amrrvu
W-LACTATIC
my
D~:oa.\r~.~~ro~
06
*P~SI).ZRI)
SOLUTIONS
Degradation flasks were prepwctl coni :tining 0.5 rul of 0.1 .I[ lithium ktate (50 pmoles) and 1 ml of 10 W H&O,. T(k #I--#. ’ 5 wew adtletl I ml cd L’-CLi-l:~ctate and I ml of I-CF4-lactate; to #6, 1 ml of 1-C’” lactate only; lo 17. I ml of ?-C!lq lactate only, and the fin:J r(Jume T~\VUS adjusted t,o 10 ml wN1 deionized w:~ter. The degradations were carried solution was out. as described in the text. The mdionctivity of the 2-c”‘- lact:tte stantl:ud 10,883 cpm/ml; that. of the I-CWlactnte, with irlfitlitesimally small 30,OO 3 cpm/ml amomlts of chemical lactate. The t.heoretic:tl specific activities were, therefore, 218 cpm/pmole for the 2-C14-lactnte and 601 cpm/pmole for l-c’L1-l:wi ut t’. (‘0,
:\cetnldehvdz x0
1 2 :; 4 5 6 7 Mean *RD. y0 deviation
p111OlC4 (P-W 1
<‘,lW
~~~-..
-
10,3ss 10,387 0,737 10,289 10,434 4 lO,.i%l
47.S6 4s. 06 4i.u 47 .!I6 47.71 4S.S 4s “7
rpm /pM 215 “16 304 215 210 neg. “17 214 5 3
C(,ll, 30,451 27,050 :30,2‘30 30,369 30 ( 485 :;o ,058 II
prllolw
(jl‘ll)
50.45 44.35 49.75 .50. 10 50 75 50.60 40 so
cpm/fiM 604 610 609 606 601 504 neg. 604 15 ::
standard C14-lactate solutions. Radioactivity was measured with a counting error of 1%. Under the conditions described above the mean value obtained for the acetaldehydc-semicarbazone was 214.3 f 5.4 (S.D.1 cpm/pmole as compared to the theoretical value of 217.7 cpm/ Pmole. The mean value obtained for the Hyamine-carbonate was 604 k 15.3 IS.D.) as compared t,o a theoretical value of 600.5 cpm/pmole.
Specificity of the Trnps. Cross-contamination of either oi the traps did not occur, as illust,rat8ed in Table 2. When only I-Cl”-lactate was present (degradation .#6), no radioactivity ins found in the acetaldehyde trap. Likewise, when only 2-Cl&-lactate was present, (degradat,ion #7), no mdionctirity was found in the cnrl~osyl trap. DI,SCUSSION
The principle employe~l in t,hc methotl clcs:cribed is an osidative degradation of the lactic acid molecule to acetaldehydc and CO,. Scvera1 reagents are available for this purpose. Potassium pcrnianganate was used by Fricdemann. Cotonio, and Shaffcr (II), but., as stated by these authors, the removal of glucose is mandatory bincc it is also extensively oxidized. For general use the ceric sulfatcl oxidation of Gordon and Quastel (1) woultl stem to I)e prefernblc since only 0.4% of the glucose is degraded under the conditions of th(l re:tction (20 min at 60°C). However, for experiment’al conditions in which the glucose may be present in very high ppccific activity the gluco>c ~l~oulcl also be removed prior to degradation of t’he lactate. The conditions of the ceric sulfate reaction as to air flow, clurat,ion, temperature, and molarity of ccric sulfat,e hare been thoroughly invcstigated by Elsden and Gibson (12). It would seem from t’heir data that, at 53°C with a eerie sulfat,e concentration of 0.1 M, the formation of acetaldehyde requires less than a few minutes. Prolonged reaction times result in a further oxidation to acetic acid as sugge&d previously by I,ong (10). Eleden and Gibson, Ulerefore, increased the flow rate in their steam-distillation system to 500-600 ml,‘min in orcler to rcmovc the acetaldehyde from the degradation flask immediately on formation. Recoveries of 100% were obtainecl using a t’otal reaction tiltic of 45 min. For our purposes a flow rate of 250-300 ml/min was found to he more desirable (a) to prevent working at, too high air ~)rw~urw, (b) to improve efficiency of the CO,-carbonyl scrub train, which is ncccssa~y for accurate CO, measurement, and (c) to increase the efficicncg of acetaladehyde and CO, trapping in the dist,illation train. Tht lower recoveries of acetaldehyde (95%‘) as compared to CO, (100% 1 may thus be due to lose t’hrough acetic acid formation at the slower rate of the acctaldehyde. In our hands the dropwise addition of ceric sulfate prescribed by Elsden and Gibson did not appear to be required. The simpler, single addition technique of Long (10) was therefore adopted. The introduction of t’rapping agents for the products of lactic acid degradation which would allow their isolation and raclionrt’ivc assay was first presented by Rrin and Olson (13). Using the permanganat!e osida-
tion technique of Friedemann et CCL (12)) t,hesc authors trapped the acetaldehyde as its dimedon derivative and the CO, as sodium curl)onatcl. Both products were then prepared for plan&t count,ing. Reeovcries in these trapping agents as inclicat,ed by these nuthor~ mere in the order of 40%. Using this method initially, we were able to incareast the yield of the acetaldehyde fraction to 70% by employing a higher molarity of dimedon dissolved in methanol. However, precipitation of the tliniedon derivative required prolonged treatment, overnight standing, and recrystallizations to obtain a pure product of con&ant specific activity and characterist’ic melting point. These rather time-consuming features led US to examine other t’rapping reagents that woul(l allow titrimetric or spectrophotometric assays which would also be more suitable to liquid .qcintillation counting. For the trapping of the acetaldehyde fraction we elected to use semicarbazide hyclrochloritle, a well-known reagent for earbonyl compounds. Two features make it a desirable trapping agent: (1) the product can be assayed directly by spectrophotometric measurement and (21 since the ol~timmn condition for ncetaldehyde-semicarbazone formation was found to be in an acetate buffer of apl)roximatc pH 4.5 IS), the l-carbon moiety of the lactic acid degradation, the CO,, is not simultaneously trapped. Conditions for t,he t,rapping of acetaldehyde by this reagent in such a distillation train were made optimal by keeping the vapor temperature in the condenser slightly above the boiling point of acetaldehyde and by immersing the trapping vessel in an ice bath. Recovery of acct:~ldc~hpck with t,his reagent under these condition+ w:I‘: ~~t~l~~‘o~lr~c~ii)ly 95%. The (‘0, evolved in the degradation is trapped directly in Hyamine after the mncrornethod of Fredrickson and Ono (141, who employed a similar technique in the determination of the specificsactivity of C”(L of expired air. According to Passman, Radin, and Cooper (15), a minimal amount. of 1.1 nrolcs of Hyamine will efficiently trap 1 mole of CO,. At the flow rates used in our degradation system, a molar ratio of Hyamine to CO, of 5: 1 provided t,he most efficient trapping. ~~%ile only 3OClrof the CO.. t.rap was gtnernllv us;fd for the r:~cliochemi~al assay. IxrgtJr :unounts could be cmployc*cl by first reducing the methanol concentration by evaporation. It was found, however, that ext8rcmc c:\re had to he cixercised to prevent splashover and loss. The range of the method is 15 to 60 pmolea. It is advisable not to n-ork below 15 ~moles since then a slight variation in thr CO, blank may cause a larger per cent error. In extending the range beyond 60 +oles it should be noted that, while the amount, of semicarhazide used is sufficient
to trap 100 pmoles of acetaldehyde, accuracy may be sacrificed in obtaining a sufficient dilution for the spectrophotometric assay. The latt,er, as previously noted, was found to be linear from 0 to 53 ,pmoles acetaldehyde when dilut~ed 1: 200. In working with biological filtrates, it has been our procedure to degrade an equivalent8 of approximately 2.5 to 3 ml of whole blood and 40 ,moles of carrier lactate. With a normal range oi arterial blood lactate of 1 to 2 ~~moles,~ml,the total amount degraded varied from 42 to 46 ~molcs. If the blood level of la&ate is expected to be higlrer (during csercisc or systemic hypoxia’) , t INLcl~~gr:rtlat.ionc:m 1~ carried out with less carrier lactate. The degr:tdxtion method clrsrribc~l is relatively eiml~le and easy to perform. With home experienrc: (i to 8 degradations could be handled daily, including the time required for chemical :rn(l rn~liochcmic~nlanalpw of the final product.~. SLinIKmlI. X method is preicntcd which :tllo~ the dctrl,lliin:ttic,li of radioAivity in the l-carbon anal the 2,3-carbon moieties of I:u%ic acid adapted to liquid scintillation counting. 2. T11c lactic acid is osidizcd by ccric sulfate to :rcctnltl~hy~lr: and CO,, which are carried over into :I s;(+c.- of t 1x1’~ by nrc:rn+ of :r CO,:mcl cnrbonyl-frer air stream. 3. The acetnlclehydc is trappe(l :LS its FClllic:Ir~):Izol1(~:un(l :tF>ayed sl~ect~~opl~otometri~allp.The CC), is tr:rl)pctl as its II?-:llllinc-~:lrl-)oll:~t~, and measured by difference titration. 1~:~~liochemicnl:rs;eny~ arc performctl directly using Bray’s solution :ts the counting system. 4. ,4 descript.ion is gircn of the clistillation-tlegl,:~(l:~tioti setup 311~1 of the CO1-~arhonpl rcrub train u~cl. Optimal condition:: a-; to :tir flow. t8rrul)c~r:rturc. all11 tluration of tlrc reaction arc given. 5. Tbc methotl (*:rn lx u~e~l iii tllcl r:rrr~;c~of 15 to 60 pl\lolrs :tncI i., reproducible and nccur:rtc. .\CI~~o~~‘r,7SDC;I~~T~ Tht :wsislnncr.
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7. FROMAGEOT, 8. CONAZIT, J. 9. BURBRIDGE,
C., AND DESNUELLE, P., &o&en. 2. 279, 174 (19%). B., AND BARTLETT, P. D., J. Am. Chem. Sot. 54, 2881 t 1932~ T. N., HINE, C. H., AND Scmx, A. F., .I. Lc~ij. (‘lin. .\f~l. .?5, ~3
(1950). 10. LONQ, C., Biochem. J. 40, 27 (1946). 11. FRIEDEK~SS. T. E., Cnmsro, Ibl., AND SH.WEXK, P. A., J. Sol. (Ihrn~. 73, 335 (1927). 12. ELSDEN, S. R., AND GIBSON, Q. H., Biochem. J. 58, 154 (1954). 13. BRIN, M., AND OLSON, R. E., 1. Biol. Chem. 199, 475 (1952). 14. FREDRICKSON, D. S., AND ONO, K., J. Lab. Clin. Med. 51, 147 (1958). 15. PA’SMANN, J. M., RADIN, N. S., AND COOPER, J. A. D., Anal. Chern. 28, 484 (1956).