Simultaneous extraction and fluorometric measurement of brain serotonin, catecholamines, 5-hydroxy-indoleacetic acid and homovanillic acid

.~NALTTI(‘AL

55,

BIOCHEMIHTHY

306312

SHORT

Simultaneous

Extraction

of Brain

Serotonin,

indoleacetic

(19i3)

COMMUNICATIONS

and

Fluorometric

Catecholamines, Acid

and

Measurement 5Hydroxy-

Homovanillic

Acid

Assay procedures currently availnble cl0 not, perniit~ the simultaneous estimation of 5-11~-vclrosyinclole:tcetic acid (5-HIAA), homo~anillic acid (HVA), the catecholsmines and serotonin in the ssn1e tissue snmple. Thw, :i wparatc l1omogeiiizat~ion nn(l cxtraciion ljrocedure is required to ntcasurc clolxm1ine :ud I-ET-A (1) or scrotonin and 5-HIAA (2,3). The conccnttxtions of HVA and 5-HIAA may be measured in the same tissue ~~:ttii~~lc,l)ut tl:cx extract must be dividecl in half for ael)arnte extraction ant1 annI\-sis of (Y~(~~Iacid (4,.5). This paler clcscribce n procedure for the &ultnncou:: cstrtwtion nnrl fluoromctric mcwtu-cmet1t of 5-HIAA. HVA, +ctrotonin, tlopaminc, and norcl,ittel)ltrirte. The a.w:ny is moclifictl from lwc~viously l~ublishetl tnctltotl~ i B-IO) Methods. Rats, weighing 100-I 50 g, were dec:tpitntetl, and their brains ww clriic*kly rcmovcrl. 13r:~iit :: n-erc clisscctcd nrcortling to guiclelines of (;lowinzki nu(l Tvcxtwt1 (I I 1. Spinal fluid was w-ithrlran-n from rhesus tnottliey.~ through 3 cmnul:~ pcrmnnentl~ implnntcd itit’ the lateral wntriclc. E.rt~~trrfiori

of

IZYA,

6-IIIAA,

scrota~lin,

no~~epincphrinc.

crud

tlopn-

M~‘TWfn~m tisslse. Tissue samples were homogenized by use of a Polytrort hotnogcnizcr (Et-ittktuan Inst. Co.) in colt1 nciclificd (IO IIIRI HCl) l)ut:tnol to girt a final tissue concentration of 2.5-75 m&ml. After the samples had been centrifuged for 20 min at, 6000!/, n 4.0-ml aliquot of the dtpcrnatant fluid n-as added to a FiO-ml glass-st’oppered conical centrifuge tube containing 10 ml of w~1shcti ,l-hcptane and 1.5 ml of 0.01 x HCl. A mixture of internal standards (0.5-2.0 nmoles of each statlclard in 10 11.1of 0.01 iv HCl) n-as ad~lcd t,o several samples at this stel). After having been shnkcn for 5 min to extract the amines, the tuber were centrifttf;ed for 10 min at IOOOg. A lo-ml aliquot, of the organic phase, containing the acids, n’:W thtn a&led to a 15-ml glassCopyright AI1 rights

306 @ 1973 by iZcademic Press. Inc. of reproduction in any form rcsrrrcd.

~toplwred

cent,rifuge tube containing 1.5 ml of 10 mM Tris-HCl and the .5-HIAA and HVA were extracted into the Tris-He1 buffer by being shaken for 5 min. Biter centrifugation of the tulwe for 5 min at IOOOg, the organic phase wad :wl~irnte(l and discarded, ;lt1(1 the 5-HIAA :~lltl HVA in the aquc~ous1~1~:~ Tverc estimated as ticxcribeil I~OJY. If the acpeous phase apl~~w~l cloudy after asl~iration, t.hv tubes \serc cent,rifugctl again until the x:m~l)lcs nppcnrecl clear. L4 1.O-ml :iliquot of the :wid pli:ise, cont:Gniii,0‘ thv amines:, was nddcd to tllc’ 15-in1 glass-stoppcrcd conicnl wutrifuge t,ube containing 200 mg of :tluininn (prepared as dewribed by Anton and Pnyrc. 12 I, ad 1.5 ml of 0.5 31 Tris-HCl bufier (pH 8.3‘) contnining 0.13 M ET)TA and 13 mM sodilnn ill(‘t,3l)iHlllfit,C’. Tlw tubw n-crc drdwn (5 mill) to :~(lsorh the c.ntccliulnliliii~~, then ccntrifugctl for 2 min at lOOOq,nncl n Z-ml portion of t,lw supernat:wit, fluid was tr:msferrcd to a 50-ml gl:ws-stopped conical centrifuge tube containing 6.0 ml of wbutanol i salt- nn(l watersaturatecl) , 1.5 g of KaCll sncl 2.0 ml of 0.35 11 borate Idffer IpH 10). This mixture w:t~ shaken for 5 min, thaw reutrifugetl, ant1 nn diquot (5.0 ml 1 was trnnafcrwtl to another SO-ml tld~c contniniug 10 ml of n-heptn~tc and 0.4 ml of 0.1 s HCi. After 5 more minutes of siding, follow(1 by cvntrifugition, 0.2 ml of the acid ~~llnsewas remowcl for the :~ssay of scrotonin~ a8 tle~cribctl btlolv. The rcmainrlcr of the supcrnatnnt liquid over t,he alumina was aspirated and cliscnrtlcd, and tlic ndsorhant was washed ly being shaken for 5 min with 2 ml of distilled water. The wash vater lvas removed by aspiration, and. the alumina was then shaken for 5 min with 1.0 ml of 0.2 pi acetic acid to elute the cntecholnminee. A portion (0.5 ml) of the supernat,ant liquid was transferred to a separate t’ubc for the assay of catccholnminta ar described by (%nng ( 6). To extract HBA and 5-HI-4&4 from cercbrospinwl fluid, nn nliquot (0.1.5-0.3 ml) of spinal fluit was shskcn togcthtr vith 1.5 ml of 0.1 N WC1 ant1 10 ml of n-hel)t’:tne and acidified (10 mu HCli butanol (2: 1 rnixt’urci. hftcr ccntrifugation, the HV,4 and 5-HItZA in 9.0 ml of the organic phase wrt estrnctcd into the Tris buffer, as described above. Fluomaefric anal,ysis. Fluorescence was rend on an Aminco-Bowman SI’ertrophotofluoromcter (American Instrument, Co.‘1. The tvavelcngths reported below iescit3tioii/ciilission) arc uncorrected dues. The 5-HIL4L4 was estimnted in the Tris buffer 1)~ measuring n:itivp fluoresCelIW of the inclole ring at 295/350 nm (101. Fluorescence was stnblp in Tris huffc~r for at’ le:tst 4 hr. After the s:lmples had been red for 5-HIAQI, 1 ml of the Tris buffer was transferred to another t,ube for the cstimntion of HYA by tl~c proccduw of Anden et al. (9). Tltc pH of the medium wna first incrrased by the nddition of 0.1 ml of 7 p; ammonium l)uffcr

conical

(,$H

7.0;),

308

SHORT

COMMUNICATIONS

hydroxide. Potassium ferricyanide (0.01 ml) was t,hen added to oxidize the HVA. After 4 min, the oxidation was stopped by the addition of 0.02 ml of cysteine, and fluorescence was read within 1 hr at 310/420 nm. Tissue blanks were preparetl by addin g the potassium ferricyanide and cpsteine in reverse order to several tistiue samples prepared from the unused portion of the Tris-buffer extract. A fluorc~ccnt derivative of scrotonin was produced by heating a 0.2-1~~1 aliquot of the final acid extract for 10 min with 0.4 ml of a-pht~halaldehydc reagent (50 mg/lOO ml of 10 N HCl, prepared 4-8 hr before use) (8). hfter being cooled in tap water, the samples were extracted briefly (Yortex; 5 set) with 0.6 ml of chloroform to reduce the blank fluorescence (13‘1. Fluorescence was read at 350/470 nm. Results u~l I)iscussion. With the preticnt assay, it is possible to obt’ain rapid simultaneolls estimates of changes in the met,abolism of two transmitter systems in the same animal, a feature particularly useful in studies involving long periods of treatment’ or training, where maximum information is desirable from a limited nlunber of animals. The procedure seems to be specific for all five compounds in cerebral t.issuc. The following compounds did not. interfere when 5 nmoles of each was cnrricxtl through the ext,rnction procedure: tyrosinc, 4-hydroxy-3-mcthosy-phenylglpcol, normetanephrinr. dihydroxymandelic acid, metancphrine, tyraminc, vanillyl-mandelic acid, dihydrosyphenylacctic acid, octopamine, dihydroxyp1icnylgl;vcol, t~ryptopl~nn, j-l~ydroxyt~r~l~topl~nn, and tymmine. ~il~yclrospl~hen~lalanine (DOPA) will interfere in the assay of dopamine. but the concentration of DOPA in brain is normally very low 114). Likewise, melatSonin, n-acetylserotonin, and 5-hydroxytryptophol would all interfcrc n-it,!] S-HIAA, and 6-mcthox~tr~ptazrniIlc mit.h serotonin, bllt these coml~o~mtl:: are eit,hcr not present or are not formed to an ap1)recixhlc extent n-ithin the brain (15,16). When serotonin was measured in tht initial ncaid cxtrnct without the borat,c buffer ITash, using the l)rocedure of Maickel et crl. (8)) the tissue conrcntration of serotonin was con&tently higher il.5-20%) than when borate buffer was used, +uggestinp that washing with borate buffer may, indeed, be necessary to remove substances t’hat would othcm-isc interfere in the assay of serotonin by the a-phtl~alnltlel~yde method. The fluoresrcnce charncteristice of serotonin, dopaminc, norcpinephrine. TWA nnrl h-I-ILL4 extracted from rat brain were shown to bc idcntiral with tho:e of authentir st.andards of the amines ant1 aci(ls. The sensitivity was assessed I+ carrying 1 ntnole of each standard through the extraction procedure and comparing the fluorescence read-

ing of tile stnnclnrtl wit,11 tllnt for the tissue or reagent bld~ The sensitivity, esl)ressed as the number of nanomolcs of amine Or Xid that must be Jmwnt, in the original tissue ~xmple to theoret.ically yield a fluorcs~enre rentling twice that for the blank, WC+ for serotonin, 0.17; $I{IXX, 0.12; norcl)inel)hrillc, 0.14; tlolxw~ine, 0.72; anal IIT-A, 0.45. In actual practice, dEckid, t,isslie is estrnc~ted to produce :I fluoreticence reading nt lend tlircc times the blanli value. The fluorwcence rcxlings of tissue \,lanks for norepinephrine and dopof :LY ~uuch a8 500 mg of tissue, were amine, prel)ared from estrart:: ue:lrl?- cq11:11to the rcngcnt hlsuks. Ti~suc Idnnks for HVA gave ~IUOPCSonce rw(ling-: ecpnl t.0 the reagent Idanka when 100 mg of brain tk.bue or 0.3 ml of spinal nliid \r-as usc~l. X:iking the :tmount of tissue to 2.50 mg clo~~bled the fluorescence rwcling for HJ’A ldank. P. tisslIe ldnnk for serotonin! prepared witllout llvating the kw~~ple, gn\:e a reading lvss than that, for the reagent Irl:mk. The c~oncentr:~tions of acrotonin and S-HIAA were cnlculatcd hy 11se of the redng:: f’or the reagent I)lanks. \\:c have modifwcl the ositlation lw.wcdurc of Anclen et al. (9) fur H\Th to give x lligllcr find concentration of the aci(l: whi~~li reslilts in :L 7572 increaSe in wndtivitp from t.hat of the original mctlio~l. 130th acilds map tw mensllrrl1

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Frlrtlwmwe, by inc:wlring HYA :~icl 5-HLdA in the tiain(: nliqlot of sl)inal fluid or in the same l)iccc of tissue, the sensiti\-it;\- is t.hcoreticall>clouhlccl. ns wmpnrecl with other proccrlurc~ in nhich the sample mud lx, hnlvc~l for tlie estimation of both acids i4,5). Tlic conditions clex%l~~l for dtl\-cloI)ing acrutonin fluorcwence with ~-l~ht,hnl:~ldeli~dc gave :I m:kmllm rat.io of r:tmple/hlnnk, and use of the c*hloroform wash (13) m:~rkctlly reduced tlw flII0rwwnw of the rctlgent hifink without affcctirq rccovcry. Fluorcwencc rcndinp for stantlartls carried through the extraction Iwoccclllrc n-cre linear for all five c~~ml~ouncls from 0.5 nmoles ul) to 10 1111101~~~. Linear fluorwwiicc rcailings lwre also ohtninetl for the acids nnrl amino~ estrnctccl frolu 50 to 500 mg of tiwle. Thp average rrcovery of st:111(1wls wa:: eerotonin. 42FS ; 5-HIAL1. 71% ; norel’inephrinc. 67% ; rlnpnminc, G3Th rind HT.\, .X5$. Tll(~ (I:Lt:L in T:lhlc 1 illustr:Lk tile applicntion 0f the mp[li0~1 t0 l)rnin ti~~~lv. Homuvnnillic acid could he cletcctd only in t,lle c0rpus striatum of tllo rnt brain. This region also contained the higllevt concentration of clop:lmil1c~. The wxlwntrnt8ioiw of ilorcpincphrine nnrl wrotonin n-ere highest in the h~l~otlldam~ls nnrl medulln-pens and lowest in t,he ccre11~~11111n :~nd ca0rtes. Similar results have lwcn obtained 11~ different :~n;~lytic~nl metllo~ls i 1,4,7-lO,17-19’I. The finding 0f a high concentration pi’ 5-HIA4.i in the lnedulla-loons, l~ppotldnmus :,,nd st,riatllm, and 0f a

310

SIlORT

COMMUNICATIONS

312

LSIIORT

COAlM\IIINICATIONS

18. IWRSEN. L. L. (1967) Tlrc Uptake and Storage of Soradrenaline in Symlut,hetic Kerves. Cambridge CnivclGty Press, Sew York. 19. EWPAMER. V. (1966) in Handbuch der Eqxrimentc~llcn Pharmaltologie (Eichler, A. and Fnrnh. A.. rds.), Vol. 19. 11, 132. Sljringer-Verlng, Berlin. 20. AA-TON, A. H., AND SAYRE, D. F. (1971) d. Phn, MICO/. Exp. Ther. 179, 207. 21. ALPERS. H. 8.. .AP*‘D HIMVJCH. H. E. (1972) J. I’ilo,~v/~rc~,/. Esp. Ticev. 180, 531. 22. cr-luolv, c+.. AN11 Cinms, ii. R. (1970) Hi,it. J. I’ii~i r~rrco/. 39, 653. 23. MOIR. A. T. 13.. ASHCROFT. G. TV., CR.AWNJRD. T. I!. 13.. KWLESTOS. I).. .\ND GULP BERG, H. C. (1970) Biai,~ 93, 357. DEAN JOAN

R. S.

HAUBRICH DEKZER