isotope derivative method for quantitative determination ... ·...

ISOTOPE DERIVATIVE METHODFOR

QUANTITATIVE DETERMINATIONOF

HISTAl^NE

rnH

BOTOPE DERIVATIVE METHOD

FOR

QUAHTTTATIVE DETEttMINATION OF HISTAMWE

Abstimct of

A TliMi*

Presented in Partial Fulfillment of the Requirements

for the Degree Master of Science

by

Richard Raphael i^ntwhisae, Ch. E.

The Ohio State University

1IS3

Approved hy:

Adviser

Library

U. S. I-Taval Postgraduate School

Monterey, California

ISOTOPE DERTVATIVJB MJCTHODFOR

QUANTITATIVE DETEEMINATION OF HISTAMINE

RICHARD RAPHAEL ENTWHISTLE

CH. E. , UNIVERSITY OF CINCINNATI, 1935

Dvpartmeat of Phyilcs(Approved by WiUiaiaQ G. Myers)

A review of varioas methods for separating histamine from inter*

fering substances and the quantitative determination of histamine by

chemical methods is presented. The Isotope derivative method of

analysis is then described and its adoption as a method for the quantl*

tativc determination of histamine is disctissed. Detailed methods,

together with flow diagrams, are presented for the preparation of

plpsyl*chloride, radioactive pipsyl-chloride, and the pipsyl*histamine

derivative. By the method described herein, pipsyl-chlorlde tagged

with S-thirtyfive was prepared at the five thousandths mole level

from radioactive sulfknilic acid. The pipsyl-chloride contained forty*

four percent of the activity of ^e sulfaniUc acid. Two equivalents of

pipsyl*chloride appeared to combine with one equivalent of histamias

to form a derivative which was difficult to dissolve. Of the many sol*

vents tested, only chloroform, toluene and acetone dissolved the

pip8yl*histamine derivative. The solubility was approximately one part

of the pipsyl*histamine derivative to one"thousand parts of solvent.

\'\m

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The isotope derivative method using the cmrrler techaic and chloro*

form as the solvent was applied to a sample containing seventy-five

hundredths of a microgram of histamine. The histamine values as

determined by this method were high by a factor of two-hoiulred to

four*handred percent, indicating the presence of radioactive con*

tmminattts. Activity measurements showed that self-abaorptloa of

the weak beta•particle emitted by S^-thirty-five renders the use of the

radioactive sulfur isotope undesirable as a tag la this method of

analysis unless a correction factor, a ftuction of the surface density*

is affiled. Histamine was separated from histidine and arglnine by

paper chromatography using isopentanol saturated with two normal

ammonium hydroxide as a developer; the R^ Sectors for histamine*

arglnine and histidine were forty-one hundredths, sero and zero,

respectively.

ISOTOPE DEarVATIVE METHOD

FOR

QUANTITATIVE DETERMIRATION OF HISTAMINE

A Thesis

Presented in Partial Falfillment of the Hequlrements

for the Degree Master of Science

by

Biehard Haphael £titwhistle« Ch. £.

The Ohio State University

19$2

Approved by:

Adviser

TABLE OF CONTENTS

Pftg«

t. Introduction 1

U, Isotope l>erlTatlTe Method 3

m. Choice of taotopicalljr THi^ged Reagent 5

Hf- Preperetton of Pipejl'^ehioride f

V, Preparetion of Pipeyl-histamine Derivative .... 16

VI. Sei^ration of Histamine froxrj Basic AminoAeida by Paper Chromatography tl

VU. Experimental Procedure 24

vm. DaU iT

XX. X>iacaesion of Data 31

X, Cencluaiona 39

XX. Bibliography 35

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I. INTaODUCTION :

Histamine and histainine<>like substances have been named as the

probable causative agents In the response of the body to anaphylactic

shock, serum sickness, allergy, injury to cold (1), exposure to ionizing

radiation <2), and other Intrinsic ejects. Perhaps much of the doubt as

to whether histamine is the causative agent results from the fact that

there is no quick simple accurate method for detecting histamine In low

concentrations . Numerous estimates have been made of the amounts of

histamine in animal tissue. Blood, however, has received the most

tiMirough study because of the ease of obtaining samples and the fact that

successive samples can k>e taken in the course of an observation. Since

the histamine content of blood is rather low and deviations from the nor-

mal are small, an extremely sensitive method of detection must be em-

ployed. In order better to understand the order of sensitivity demanded,

the concentration of histamine in micrograms per ml. of rabbit blood,

which has the highest concentration, and of a normal man are cited as

about 1.25 and 0.05. respectively (3). While the naain research effort

relative to histamine determination has been devoted to blood with the

major emphasis on the separation of histamine from interfering sub-

stances and improving the sensitivity at the sub-micrograai level, the

technics developed are not restricted to blood analysis alone.

A search of the literature reveals that purely chemical methods

for the detection and quantitative determination of histamine have been

flaAaot oi einaofx* ,(I) tkXOd ot ^fuitai nXZ^- t jiUois Mi:t;i«^^ ,ji9orf«

Ml t4m6b •ill to Amtoi t^l#i»^ .«!••&• oianlTicd -xsflic i»ii> a£> r.oiijsibjrx

vol al MdSAiaM ytfi»itt»l^ vA hmftaig slsvtxiftads At^mle jItAuso c-v. ?' s'^dHl

to alawini itHi lo alMua a»*d •v«<i •»i«oii"^ -

': r;ao'>

lAdt t^jA 9dS fa«ji ••Iqoum solnlAlcio let »•«» »dl lo •ftti«9Mi t^biu* li^myioii^

-ntCHi sdl mor) aooiÂJvsb boa wol isiitfln si b«odU lo ten^ .tain a^

-ai» ad tOMB iMll9«lab lo bofU»A avitlaoat \r > rrî-<f?^ An ,Ur.> . .^ Imr

tbdHMMtib x'^viliij!9« lo taino 5di lM(£ta'Itli^flll ta t'*tim& *i»biro sil .boôlq

«bo«I<f tlcfafjn )o .lAf i»q wnAitgoâlax ol aaloiiilti^ H aoiiAoinâood aitt

Ml I^l9 axa oAca I«anoB m \o nam «aolia*tlnaaaot> laailî! aiit auui ilalihr

Hilla ifa<»MMi alMi a«ti altiCW .«) xiovJtaa^Mi .eo .0 baa dS . i hMMbi

•A JIhr koaM Qi kolofab a»#d Mrf aolJuMtaiiotafe MtoMtald ol arllaia^

-dM yiHaHalal aia^ aaliwafM to m^êim^% %A% m» tlvMiqaM taian

•dl «l«Tal ai«i$o^dlia»difa adl Ya ytlvDlmsaa adi sshronqml ka« taaatf*

•fradtooi toalmaifo xiô-tucf ladl atao'rai anuHsna^)! %Ai to ddHAoa A

oaaa avad aimaraia zo wortaBiimaiap avUrjfTaaay jbeus ncuLraeiso am sax

-I-

devUied. Each of these methods employed a prelimliiary pttrificatlon

or separatioii of the histamine followed by a spectrophotometric deter*

minatioft. The following methods of preliminary purification have been

employed:

a. Precipitation and extraction procedures <4. S» 6).

b. Adsorption on synthetic resins, cotton acid succinate (S,7.8K

amberlite <9)« and by paper chromatography (10).

JDa the final determination of histamine, color reactions involving dla*

Botisation (Pauly reaction) with p*Diasobettsene sulfonate (4), p«Bromoa*

niline (5« 10), or 4»}4itroanUine i^,B) have been employed, or optical

dinsities have been measured after Uie reaction of histamine with 2,4-

Dinitroflsorobeaaene (II). The earlier procedures lacked sufficient sen*

sitivity for the detection of histamine concentrations normally found in

•mall volumes of blood.

The most widely used and accepted procedures involve some initial

purification procedure of the histaniiie fbllowed by its biological assay.

The bioassay method has, ia general, been more seositive than colori-

Bietry in the quantitative eetlmatlon of histamine but it has also been

found to be less accurate (IS). The initial chemical procedures have

been directed toward liberating histamine from siitetaaees and structures

with which it Is bound, separating it from other similar substances which

iSMild interfere with its accurate biological determination or accurate

chemical determination while sin^ltaueously maintainiag a sufficiently

:brjvolr:aif.

.(9«£,i^) ««-iMfc&r7caq acU-J^^rrJx** baa nc:i."..;k!i,'>»*i'=l .«

,<S,T,8) •txnloaua M3fi aottoo ^ftntssi 9ilS!r?tmr« nc r70t:h7i;n3b4 .<f

.<0i> V -•^- .>o - .- xiouK

laltlid •oiott •viovflj ••i«ii»30iq i}«lq993« bixA bi^av i^i»blw taooi silT

•lnoioo ii»ui iiyiilf*«»ft •Hen m»m( «Li%Mi»A *^ •*<< bmidmsa. xikiii T

•tm( a*i»fe»oa*iq i«oJ;m»i4^ Ifiitl/irjt 1 3Ki 01 band!

^tfUNr 9»nmBi94um i«Jiaii« ^»dio men) ll 9al>inAq4a .ianiiocf &

coii8taitt and high yield of the histamine originally present to allow a

satisfactory quantitative estimation. The final biological assay employ*

lag guinea pig gat« uterus or bronchi must be sufficiently sensitive to

detect the minute quantities of histamine normally present (13).

n. ISQTOPIC DERIVATIVE METHOD :

1. General : It is api»irent that the presently accepted and employed

methods all depend on an initial chemical separation of histamine from

interfering substances before the final quantitative determination is

made. This procedure has its analogue in gravimetric anadysis whereby

a desired compound is quantitatively precipitated from solution. In em*

ploying the latter procedure it is realized that a small anAOunt of the

compound sought, limited by the solubility product, is lost to the solu*

ttoa. When analysing for macro-amounts of material, the amount lost

to the solution usually contributes a negligible error in the final result.

l&wever, when dealing with micro-quantities, i.e. , at the microgram

and sub-microgram level, the amount of material not recovered by the

extraction may be large enough to render worthless the final result of

the analysis. Since the isotope derivative method, using the carrier

technlc, would eliminate this source of error, it was chosen fbr explo-

ration as a possible method for the quantitative detection of histamine.

The isotope derivative method lends itself admirably to the present situa-

tion since it has been demonstrated to have an extremely high sensitivity,

being operable below the microgram level (14).

-8-

A lAol^. 'mXk &

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mii lo MUWOUI UftOia «i iA'^'^: Lif ^ilat^'i :i: , .y\p .:':- .o-i'T -.'i-ai sell |tiil<t4k^l

-Ilia* mH «« l«fi< tl JaaHmq, tItfMMlM m(» ytf ^llrjii . Iwiiinw

.llsaai IjoA 341 oi lorxs slidlittfSMi a Vftlacfiiiro:) yJLiwam isaliBiM Mft «t

&di yd bd'YSVf^sftn ton IdT&fjft£B to UnmnA sif} . fsrsf ^«Tftirxalat<x^ra boa

-mkTU loaBftiq dxi.* oi \id«*iiisl># titail tiaaal ft Q|lt»w v<§kUt9tt»b B jtHwrnk mlf

2. Outline of Method : In brief, this method coosistc of the follow-

ing step! (14):

a. Treating the unknown mixture with a reagent containing a

radioactive isotope under such conditions that the component to be esti-

mated is quantitatively converted to the derivative of radioactive reagent.

b. Adding a large excess « accurately weighed, of the unlabelled

derivative to the unknown mixture.

G. Separating and purifying the desired derivative to a constant

molal isotope concentration. <Note: This doesn't imply that the deriva-

tive must be recovered quantitatively. Howevar, that fraction which is

recovered must be isotopically pure. 1. e. , counts per mole per second

for successive fractions must be constant).

d. Preparing an Isotopic derivative, using the same isotopically

labelled reagent employed in step "a' . of a known amount of the compo*

ant sought and purifying to constant molal Isotopic concentration.

s. ]>etermining the quantity of the desired component present

in the unkitown mixture by comparison of the constant specific molal

aetivities according to the following formula:

W5 P<m •« W)K

where

«

w • amount of Isotopic derivative which was present in

the unknown mixture.

W 9 amount of unlabelled derivative (carrier) added in

excess.

•mrtnt 9iti tm^ X^. 'i»rb vMT tsMK) «rtH»d f>tfo»oal IsfcMai

te«M« 1^ •foot iMi atHMis «<tifq ic^XaiUjtiMrt 94 fanati h»i«v>0Mr«

t«i<g»aumwiMif •%• «"«*' ^« at tnnEiiiMii ^»t»a*«^-« it^MimkA

.^-

U * constant molal isotope conceatratioa resulting Iroxa

tuoJknown mixture.

K « constant xnolal Isotope concentration insulting fronc

reaction involving the known amount of the component•ought

.

When relatively large anaouata of carrier are added, the formula re-

duces to the form;

w WEK

Brief and simple though the procedure may be. It Is emphasised

that in order for the final result to be meaningful (14):

a. The reaction between the isotopic reagent and the compound

sought must be complete.

b. The carrier which is isolated from the reaction mixture

UMt be rigorously purified from radioactive costamliiaats.

e. The measurement of the quantities U and K must be precise.

HL CHOICE OF ISQTOPICALLY TAOQED REAGENT ;

Acid chlorides were decided upon as the reagents which would be

Viattd to reset with histamine. It was assoxaed a priori that histamine

would react with acid chk>rides la the same manner as the amino acids

(14) since it contained a primary and secondary reactive amlno-group.

2» 6-diiodo8ulfaniUc acid, provided it could be converted to 2, 6*

diiodobenzene sulfonyl chloride* was suggested since it could be pre*

pared at the 0. 001 mole level in high yield (85%), an important factor in

preparing radioactive compounds. The diiodobenzene sulfonyl chloride

•5-

aIa. -ts »t«

..n•

Ml tWMB X fem U —mHwy iHii to Umm^immm ^ '^

mufEM ttiil »• itiMfwi •«#• tiil «l tt«MiHiUh» hIoa dsiw t9«n MiNMr

tagged with 1-131 would prove an excellent reagent because it could be

prepared with an extremely high apeclfic activity due to the two <2)

iodine atoms, representing 59% by weight, which are an Integral part of

the molecule. The high specific activity would permit a greater sensi-

tivity in detecting components with which it reacts. Efforts to replace

the amiao-group of the diiodosulfanilic acid by hydrosfen.prlor to con-

iwrsion to an acid chloride, resulted in failure.

Itie methods employed to replace the amino-g^roup with h3rdro^en

was first to diasotiste the diiodosulfanilic acid and then treat with excess

l^pophosphorous acid (15) or 95% ethanol <16). The resuUlA;^ products

showed evidence of iodine decomposition upon reerystallizatios from

aqueous or alcohol solution at temperatures as low as 50*' C. The re*

covered product had a melting point range 131 - 180^ C, indicating a

mixture of components.

It was finally decided to wie pipsyl-ehloride (p-iodobenzene sul*

fDByl chloride) as the reagent to react with histamine since the literature

indicated that this compound could be prepared and isolated in pure form.

IV. PREPARATION OF PIPSYL-CHI.X)RroE (P-IQDOBENZENE SUL-FOisYL CHiX>KID£ ):

1. Non-radioactive; Reactions of the type required to prepare com-

pounds similar to pipsyl-ehloride were found in the literature (17, 18).

iiowever, since there were no specific procedures for the preparation of

pipsyl-ehloride, the steps are outlined herein: (Reference is flow sheet

No. 1 for diagram of process.

)

-6-

ittfw i«©ij jb*:.^ !--.e^ -• ..:V '. -'- i}u*rftfj>« oi ^«ilt msw

•ai«9 siAqsT^ ot b*i|yp»i ftq^t •dl )o mq41ftg»>i ^iiu4 i

.{8 "i 9-r»w 3blTrolrf:>-ix»Oi <iIb

-8-

a. Dissolve sulfanilic acid in 8% NaOH« add Nal^O and run

slowly with stirring into a mixture of 20% U^SO. and ice.

CgH4(NH2)SO H + NaOH^-CgH^(NHJSOjONa -^ H O

2CgH^<NH2)S020Na -4- 2 NaKOj 4- 3H SO^-h* ZNa^SO^ H- 4H2O

-KC6H^<N2)S03H)2SO^

b. Permit diazotized compound to stand I hour in order to

settle oat.

c. Decant ott supernatant liquid in order to eliminate excess

NaNOj used in dlazotization. (Note 1)

d. Add a concentrated solution of KI to diazotized salt and permit

Sandmeyer reaction to take place at room temperature. Complete reac-

tion by placing in a boiling water bath.

(CgH4<N)2SOgH)^SO^ -^ aKI—-ZCgH^CDSOjH -f ZNj * K^SO^

•. Permit reaction mixture to cool, make alkaline with NaOH,

and salt out of sodium salt of p-iodobenzene sulfonate with NaCl.

CgH^(I>SOjH -f NaOH -*- CgH^(I>30 ONa -f HjO

f. Filter through Buchner ftinnel and dry at 140 ^^ C for 3 hours.

g. Mix finely powdered sodium p-iodobenzene sulfonate with

PCI. and POCU in ratio of 1 mole of salt to 0. 8 moles of POCl, and 0.

3

9 3 «}

moles of P€l.. Heflux mixture at 170 - 180® C for 18 hours. Cool re-ft

action mixture for 5 minutes at the end of each 4 hour period and shake

until mixture becomes pasty. The shaking brings the unreacted com-

ponents together and thereby increases the yield. (Note 2)

-7-

X

<kf iftfcnro si nwBif 1 luuifs of CmujC4|3F09 ts'SStto^Atb Jffsnv^ .cf

.itoo *tttM

tlJV«HI*Mt llM b9«l«Mwli^ <i» n? >'^ tfOii . . ^^sdaes a M^a ^

.lilMf idiftw jollied • mk yU-^^^^ t> .—

.

«iIOMI 4Hw —Wrilii a^mb .Iom ot rutfxtM ottlttiMn Itanv^ --

»

S .0 tea ^J[30<| to ••IMB 9 .0 •! «Im Io •Imb I )o oktvi mk JLOO% tea ^lOV

•Jtehi tea teliHNi immd k iC9m Io te* 9di $m wm$maim t i«l »*utJxlai aolHia

-?•

3CgH^a)S OjONa - PCl^—^ 3CgH (I)SO CI -I- 2NaCl + NaP03

2C^HJDSO ONa -*- POCl —^ 2CrH <I)SO-Cl -4- NaCl * NaPO,042 304* *

h. Cool reaction mixture and extract with benzene, grinding the

•olid material with the benzene to facilitate the extraction.

t. Waah the benzene fraction 3 times with ice water to remove

dissolved unreacted phosphorous halldes and Inorganic salts.

J. Dry benzene fraction over anhydrous Na SO..

k. Evaporate to dryness on a water bath and dissolve in a slight

excess of ethyl ether.

1. Add activated carbon and warm for a few minutes to remove

color.

m. Filter through Buchner funnel to remove carbon.

n. Reduce volume of ether to about one-half by evaporation and

place in ice bath. Crystals of pipsyl-chloride now appear.

6. Filter off plpsyl-chloride and wash crystals with petroleum

ether. Pipsyl-chloride is insoluble in petroleum ether.

p. Add petroleum ether wash to mother liquor and again reduce

volume by about one-half. Cool and pipsyl-chloride crystals separate

out. Wash crystals with petroleum ether. This procedure was repeated

until a pink oily liquid Instead of pipsyl-chloride crystals separated out

on cooling. The pink oily liquid was discarded.

Mote 1: This reduces the final yield of p*iodobenzene sulfonic acid

.8-

of lEsSaBln wiA A Tdi nr^. •.,f

•t»io9«« afjîo^^^ 9ktutUia*ixmil^ bam iooD .U«h««« ImkUi ^d •AMl«r

l^»laa«»i tmr »mliti>w| UdT .VMtto t—fiwilo^ fiilw ttUtsti^ daaW .too

.l>ol»vAMik «Mr blfloU Ûo iAiq sdT t«rUo«» •bk9» ;Mactip» »sefta9dgBfzi«^ ItF Milt <*aa ^^ c»;îi^»oi vldT : I »H«C

but it eliminated the necessity of adding urea to remove excess

NaNOj before addition of KI.

Note 2: This ratio of phosphorous halldes to p-lodobenzense sulfonic

acid gives the best yield of the sulfonyl chloride.

Each batch of crystals was kept separate and their melting point

determined. The melting point for each batch ranged between 84^-85° C

(literature 86-87**C) (19). To identify the crystals further as plpsyl-

chlorlde, they were reacted with glycine and alanine. Melting points of

the glycine derivative and alanine derivative were found to be 204 -205^ C

and 194 -195^C« respectively. The literature reports the melting point

of glycine and alanine derivatives of pipsyl-chloride to be 20S^C and

194. 5**C, respectively (14). Thus It can be condbded that the product

was pipsyl-chloride. The yield of plpsyl'chlorlde based upon the original

amount of sulfanlllc acid used was 59%.

2. Radioactive;

^' Preparation : Sulfanlllc acid tagged with S-35 was used as the

starting product because this reagent was made available in the labora-

tory. The procedure for converting sulfanlllc acid to p'-iodobenzens sulfonyl*

chloride at the 0.0005 mole level is given in detail below. A' cold run

was conducted in parallel with the hot run to act as a control at each

stage of the process. The sulfanlllc acid (0. 09 grams) tagged with 7. 75

mllllcuries of S-35 was received as the sodium salt dissolved in 25 ml. of

water. ^(Reference is flow sheet No. 2 for diagram of process.

)

* Radioactive sulfanilic acid was prepared by David Imhof , a student In

the Arts and Science College, Ohio State University.-9-

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f~/oi/]/ 5heGt No 1 • Fr&fDanofion ofA/on-

Radio o(zt'i\/e Pibs\/ 1- chloride

P*unifiecj

P'psyl-'^hlonide

To \ivasten1 1

^ ^'iyJ.

to-

M

-I

to Mil

tun

ai H

(1) Place sodium eulfanilate soluticm in 40 mi. ceatrifug*

tube

(2) Evmporate volume to 3 ml. * frequently washing down

the sides of the tube with hot distilled water.

<3) Add 0.0375 g. NatiO<| (very slight excess over stoichio*

metric amount) dissolved in 0. 25 ml. distilled water.

(4) Add the mixture of the sodium suUSnilate and NaNO^2

dropwise to a 40 ml. centrifuge tube containing 0. 5 ml.

20% HjSO^ maiaUiacd at 0-So C. The sulfuric acid was

stirred vigorously during the addition of the mixture and

stirring was continued for 30 minutes to insure complete-

ness of reaction. The tube containing the sodium sulfanl-

late was rinsed careftilly, using a total of 3 ml. of dls«»

tilled water.

(5) Permit dlasotlced salt to stand for 1 hour at 10® C.

(6) Add 0. 0935 g. KI (excess over stoichimetrlc requirement)

and permit to react 1 hour at room temperature. Com*

plete reaction by placing in boiling water bath for 15

minutes

.

(7) Evaporate reaction mixture to 2 ml. and add C.P. NaCl

to salt out p-iadobenzene sulfonic acid.

(t) Centrifuge mixture for 5 minutes to throw down p-Iodo-

benzene sulfonic acid, plpet off mother liquor and wash

-11-

•«lii«toN <!•¥• «M9X* MbU« ti»v) ||C>il«M ^ dTfO .0 bkA (e|

s

iatHag mbmUma Mil nalsiAlwM iidiilit tdCT .Moi^MHi^ lo mma

-a^b to lâ C tn tmicf m «idao ^Wfi/l»-tjra tissaht 9£W At^

.i»t«ir teOit

{n»m9tia^9rg QkiHuaidoioSm isvo M9»Jt9> Dl .^ it00.0 libA <0)

êaoO ,9%tiiMfâm mtn f* ivoil 1 idan €# ll«n»^ tea

0«H ."7 fate teo .Ifli t ^r vifflsdifli Mlio«n •sarof^mm (T)

.faiOil llMllfMI If—llO>»i q iJM ItMl 4tf

««tel*^ ffvob «r«i^ at mImUis lol •nuDvlfli •îAIyIksD <0)

precipitate with 1 ml. cold saturated brine.

(9)Centriliige tube containing precipitate for 5 minotes and

pipet off supernatant liquid. Add supernatant to mother

liquor and set aside p-Iodobenzene for drying.

(10) Warm mixture of supernatant from previous step and

mother liquor, and add 0.04 g. p-Iodobenzene sulfonic

acid Ctton>radioactiTe) as a saturated solution in warm

water. Mix and cool to 5^ C. p-Iodobenzene sulfonic

aeid precipitates out.

<ll) Centrifuge mixture for 5 minutes; pipet off supernatant;

wash precipitate with 1 ml. cold saturated brine; again

centrifuge; pipet off supernatant and combine It with

mother liquor; and set aside p<»Iodobenxene sulfonic acid

lor drying.

(IS) Repeat steps 10 and 11 once more. Tht final wash water

was still slightly radioactive.

(IS) Dry the 3 precipitates at IJO® C. for 3 hours.

(14) Add 1 ml. POClg saturated with PClg to each of the 3

tubes and pool contents of the 3 tubes into the one contain-

ing the first precipitated p«Iodobenzene sulfonic acid.

Carefully wash the tubes with a total of 1 ml. of the

phosphorous halide mixture to insure complete transfer.

-12-

kmm niiiw i tA •iBtNi9»*t9 iMiHtfafiu •Art wâAttmaOW

ainîni ASSCfflBCfoftfil^ci .- Il^ !),.:' bf.^ f-^-N- . -'tu,'. if :%:-.(£

m^ . ilAa • maitf'filaaoitei-fK-

-i*l«v 4mmm itrnH »dT .^n^mt 90am ti jemm ^1 tÛi is«iQ»A (tl)

-flitiaos •«• M» Qtal ••4fff C Alii )• >fi twill fe«<F Imw wa4^

Mom auii|i#» 3iX9xa9qipbnL«4 osmyiiigg^ loxa Aiîiiii

-tl-

(15) Heat mixture in an oil bath at 100 - 110^ C. for 5 hours.

(16) Cool reaction mixture and transfer it to a separatery fun-

nel containing 50 ml. benezene and 25 ml. ice water. Use

a small volume of benzene to make the transfer.

<1T) Add 0»2 g. pure non-isotopic pipsyl-chloride to the mix*

ture of benscne and water.

<18) Wash the bensene layer 3 times, using IS • 20 ml. ice

water for each wash. The final wash water was only

slightly radloactiTS.

(18) Add approximately 20 g. anhydrous Na.SO to benzene

fraction and allow to stand 12 hours to remove water.

(20) Filter off bensene fraction and wash Na SO 10 times.

using a total of 100 ml. benzene. Na^SO. still remained^ 2 4

quite radioactive.

(21) Evaporate benzene fraction to one-half original volume.

(22) Again wash Na^SO . 5 times, using a toUl of 50 ml. hot2 4

benzene, and add to bensene fraction in step 21. ^"^9^^

Showed a marked decrease in radioactivity.

(23) Return benzene fraction to water bath at 60 - 65^ C. and

evaporate to dryness.

(24) Wash down sides of beaker with 10 ml. ethyl ether; add

approximately 0. 3 g. activated carbon; filter; wash car-

bon with 5 ml. ethyl ether; and evaporate to dryness.

Colorless crystals now appear.-IS*

-siii T>«l«%Mpiw c •$ 11 lEHlMHnt bam wvittMim maAtmrna^ ht<

•«U .if«^«w ^ as 1)132 :Mi^«at»d .ig& 4M iBM«lM<.>.'

ttiio SAW •Mtpw îi#w Xsftll •fft .ff»iiiir d9m t«l T«*4w

Vi»»«l«p v^QttîM 49mtm»tl AmWl ^ ijifis « sMlWr

.XflB M to XaivI « suj«u ««MBil 4 A^f^*^ ^"""^ KliÂ (ft^

. CMîiH . it if»f9t Hi «Qft3«rt1 mnmuimI ftf Mm fe«« ,<

lKx« .D «lf - 0^ is fUttd -«»i«w ol inm«l •m»mm4 mmMli (ei)

bbm :'VMiH i cfito .Ins 01 ritlw ttNtoMr le i*i^l» £fw«lb if—W (M)

*<M» d«Mr |t*Stft iMMlUft tMSnUMA .Bt.U

wo '^-.'.oD

Sol sod

su/foniiofe

i-a<3qed 5 35

Svci/t> or -

ate f-o 3

No AJOj^ Mi>^

hour- at

KI f<t ecji-f ton

1

/-o Z ml

1

/VoC/

^CjJ/- Gutl/v , t r. '\/aC.I

5ot ô/ /b

lodo hen

Sot Sol. fy-

I oda bensu/f cicid

7~o l/ÔSte

L

CenfnfuK.

ffr .-' rriii

•So//.

rnother\ liquor-

\AIOr -—

Coo/ to

5°C

Centrifuge

for ^ m in-

ute^

rn other iicfuor

Vyarm

CZool {-<

s°c

"?'

'Oent-/-/ fuoe

fan ^ rrtiri-

afes.

< e.n.

su,/-

Vâsit) i/vitn

cold so/.

tiiîne

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/ZO°C /"re-

.3 tloor^

t f'l.

Cold e>at.

t>i~ine

C OlfJ &Qti:>r~iine

Or^ ai

3 hour- &

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t>t~m e

Cold e,Qf.

IAlQs.h i^v/t-h

co/c/ so/-

br- irie

uf ^ r ncj tonr

Oiy of

/Xo'C for-

•^ hours

/V7 /^ Kv/MPOCI^ ona

f^<r-i^

t-fGOt o/

/ /o° G /"o r^

~> tiaur^__

POCI^

and

.l/Cl/t n /•-\

7~o *vQ s/ê

l^vciAer- ^ -

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/V?, //h

/^<C)CI^ ondPcifi

J

andPc/s

PGCl^

Cind

PaisT

PQCI^ end

/" O o rr7 /e fn^

Tr-on s /e»^

t-<^r-j funntî

tê. /^^frM /Oyer-

l/Va^h (r~^ -

f:>c-ot ^<^t-al

0/5 t-irrjc^)

C>r-y over"

O Dtiydrou S

/i/o. SQ, >VÔ

iA/a^h until

*~\ free rromrod act /xîty

(^ vo/b o r- -

ncn- rod act

pure p /yfc -

chlcr.Ue

&en^.ane

and

iâtêr

\/siatêr

(4o /?y C//~ou s

A/a SC^

S^mêne

/4-~/~lo\f\j 3heet NoZ : Pre-purofian of Rodioacfiue iîp^yl- chlonide

/l^' /s sc/v'e

in e fh er

Aad czurocn

\/\ ar rr

P il tf^r

/-tirauQh

Gtcjch ne

\

t-a Cir yneî^

LI'ôdio ac tive

lîp ^y I -chlor

ide (zrys>fcils>

C or be r> y-c

Hty\/oe.h

<^î i-h

1

A

xoa .1

,"%'"

him'f

Hhm carbon residue from the final step of the recovery of ^tut

pipsyl-chlorlde remained extremely radioactive despite repeated wash^

tugs with bensene and ether (these washing were not added to the final

product). This high activity Is understandable from the fact that 0. 087 g

.

of product was retained by the carbon. The final yield of pipsyl-chloridc

was 0.301 g.

b. Assay : The radioactive pipsyl*chloride was assayed by weigh-

lag oat a small sample, dissolving it in benzene, taking allquots, eva«

porating to dryness on an aluminum planehet« and counting by Model

SC 1 Autoscaler« Tracerlab, using flow chamber with gas mixture 99%

belium and 0. 95% isobatane.

Mg. Sampls Net Count Seeondf!! Counts per mg.

Used per see.xlO

0.019 4067 It. 4 1.240.009S 4042 31.6 1.340.0019 3871 133.5 1.53

Ave 1.37

The amount of radioactivity incorporated into the recovered pip*

syl«<:hloride« assuming a counting efficiency of 33% due to geometry, is:

Amount of radioactivity

incorporated Into « 1.37x10^ x 3.01x10^pipsyl-chloride 3.8x10"^

- 12. 5 X 10® counts per sec.

number of mUUcurles « 12.5 x 10^ • 3.4

represented by samplt 3.7 x IC^

.15.

•jfaov bfttAscfn slhmab ^-^t^^âihrn-i -/fAmstht* bo«ljuii»^ •JbHolad*!^^^

i«ra dfif ot b*fabs loft ettiw wâitltmm •«mnU| 'XMfi* iuM ffoiowf dlhr «^

• S XOC.O fkam

#•« »i«lmlM Ml lillw i»dat«il9 woQ jftlM ,d«l i^. .v.r .^»i«aMi|0A I Di

.MMiMl^Bi î% M Imm Hi«i»d

^fi.i ».ri rao* ^lo.o^r.I 8.1C ffMi^ --- n

foil. J^ » tf^coial

Yield in terms of

initial radio- • 3.4x10-activity Incorporated 7,75

into final product « 44%

V. PREPARATION OF PIPSYL DERIVATIVES;

1. Preparation of Pipsyl -Histamine Derivative : The pipsyl-

histamine derivative was prepared as follows:

a. Dissolve histamine dihydrochloride in water, and add enough

NaHCOj to neutralize the HCl associated with the molecule and that

which would be formed during the reaction.

b. Place solution in a boiling water bath and add« with stirring,

excess plpsyl-chloride.

c. Stir vigorously for 30 minutes while maintaining the tempera-

ture near the boiling point.

d. To recover precipitate formed during step above.

(1) Decant off supernatant.

(2) Wash precipitate with ethyl ether to removeunreacted pipsyl-chloride and p-iodophenylsulfonic acid arising from Iqrdrolysis of excesspipsyl-chloride.

(3) Wash precipitate with water.

(4) Dry precipitate at 120^C for 2 hours.

AUquots of the supernatant liquid recovered in step d (1) were

acidified with dilute and concentrated HCl and H2S04# an<* cooled to 0°C.

Only the slightest turbidity resulted. Extraction with ether, returning

to a small volumf of NH40H, acidification and cooling still resulted in

-16-

T

t?r

->Rq^

i-..iies 3uiitT;Cia.iii

'iiC /7trt

«Ji w «&SA j:^iu; aj£c; 'isjjsw yuiiC43 £ £;i :

'

only a slight turbidity.

The roAction between histamine and pipsyl-chloride is wholly

unlike that of the amino acids and pipayl-chloride; the amino acid de*

rivatives are soluble in a basic mediazs whereas the histamine derivative

is insoluble In both acid and basic media. A precipitate was obtained

regardless of whether histaroine or pipsyl-chlorlde was used in ex-

cess. Applying the excess plp8yl*chloride in steps did not alter the

reaction.

No attempt was made rigorously to identify the precipitate. How-

•vsr, it is believed to be a ternary salt resulting from the coupling of

two equivalents of plpsyl^chlorlde with one equivalent of histamine. It

was initially discovered that aromatic sulfonic acids act as precipitants

for basic amino acids; later it was shown that sulfonic acids as a class

will precipitate all amino acids » with the less basic amino acids usually

requiring a more complex sulfonic acid (20). It is realised that hista*

mine is not an amino acid but It is relatively basic and is structurally

similar to histidine* a basic amino acid. Attempts to prepare a pre*

cipitate of histamine resulted in failure using benzene sulfonic acid

and 2, 6-diiodobenzene sulfonic acid according to the established met-

hod of preparing pipsyl-chloride derivatives and that described by

Doherty, et al (21). Another reason for believing that two equivalents

of histamine react with one equivalent of plpsyl-chlorlde are the

stoichiometric relationships involved which are summarised below.

-17-

b—liKfi tt«v »l«ti 3d bnm bi:/« d^nd ci sldbdcani al

Mil YslU fon bU^ w^$m td »fciiniii i\WH0% iiwKixt

9ffiaEst«lrf lo >( JV9 tiio dtlw ffttr oviTi

wmM9 • •« 8blo« 9liiQ^0« lerft oworf* 9Rr fl toIsI t'^bfoA ofilaic slft^ *»•

Xffa— ailMi wtUmM 3/»ad «a«l »isj diiw «aUa« ooJom i-'--: :^ '^qi. -> ..i Uivr

XiX«'Uit9ini« ai bam al««' ^r.'si o r.fafr./^ rui too ai mI«

•Mm b«ifmllil«tli9 aift of Î&iossa Mta rlQcUiia •ii9Sc»dabolli»*t «S bos

^ bMliioveb l«(l tes wrrltwliob nbiioUio^l jiitijm«-iq lo bod

•dl 9ta sbmofil^-lx^^cr lo }«9lsvif;;po mio Hitw tom»t •n loi lo

.wQ£»d bftsliAfCMBMi eiAi liî^ttf h«..vIoval a^lifaf^ < 'fr^ â

-fl*

Actual wt. *

ppt. recovered Teat for

theoretical wt. Melting Histamine in

Amouiit In ppt. resulting Point Saper-Excess from reaction ®C natant**

Excess histamlAe 59%Excess pipsyl-chloride 45%

*Based on reaction: MI

H-C = C - CH, - CHj - NI I

' *I

H-N H H\//c

I

H -H

ClI

o=s» o

CiI

o« s= oI

0.880.875

169*170169

Pes.Neg.

H

H - C * C - CH2 - CH2 - N

-N-N\ //

C— H

o» s=o

o • s

** A drop of supernatant was placed on No. 2 Whatman filter paper« dried

for 1 hour at 110**C, sprayed with a solution of nlnhydrln (0. 1% dissolved

in n-butanol)^ and dried at 80^C for 5 minntes. A purple color resulted.

2. Determination of Solvent for Derivative :

a. General: It was necessary to find a suitable solvent for the

pipsyl-histamine derivative since the extract of the analysis mixture

must be added to the carrier before proceeding with the analysis. The

search for a solvent was a difficult task for it ftgures critically In the

separation of the desired derivative from contaminaBts. The criteria

to be met by the solvent are:

-18-

.Jwl

80^ an G

H

VI

BI

O • <5 • o

I

1

H

M

O re -O

u

W /

r-l

9qaq i<>iiri £;* - Ao bsojBiq »«w iai^fir

fi* ,"•>». ^ fflKs, "i* rjIT'

rf.-.:?

BAW

»-rt .'•' -fTrtJ^V^-. fc,.. > -,.

' >i ',,

ID Dissolve pipsyl-histamine derivative readily.

(2) Immiscible with water or ethyl ether since the pipsyl

amino acid derivatives, which would asually be present

ia an analysis, are sohible in these media.

(3) Insoluble to p-io(ik>pheityl sulfonic acid resulting from

hydrolysis of excess pipsyl-^chlorlde used in the reaction.

(4) Not react with pipsyl-chloride to form a contaminating

product. (This eliminates the alcohols).

(5) Permit ready recovery of the pipsyl-histamine cmrrier

after separation has been performed.

While criteria (2) through <5) are not absolutely essential, they

greatly simplify and facilitate the recovery of the pipsyl*hlstamiae de-

rivative in pure form.

b. Solvents: Precise quantitative measurements of the solubility

of the pipsyl'histamine derivative in the various solvents were not made;

only qualitative answers were sought. The solubilities, stated qualita*

tively, are as follows:

(1) Insoluble in hot and cold:water; dilate and concentrated

NH OH. NaOH, HCl, H SO : bensene; ethyl ether; petro-

leum ether; carbon tetrachloride; butyl acetate; n-butyl

ether; and ethyl butyl acetate.

(2) Slightly soluble in cold: acetone, chloroform and toluene.

(3) Moderately soluble in hot: acetone, chloroform and toluene.

Sxm^q •di •»«*» isjtt» Ifiiifi — • f « •^^w (il^»«liaial U)

.bMKiM^vq nsaa sad aoijjruK^s 'imnm

Xi^ili «{*NmMn» ^•lfllot4« >«ii nil {9} ffi»«^ JlHo •llrfW

b»intMo««» taA •liiUlb r%mimm:kim9 tea lotf ol »Mal»>iil CD

No reaUy good eolvent, of those tested, was found. Acetone was

the best of the three moderately good solvents, howercr, the fact that it

ii Sdiscible with water makes it undesirable. Chloroform and toluene

were equally good solvents, dissolving 1 g. of plpsyl-histamine deri-

vative per 1000 g. solvent at 20^ C. Both chloroform and toluene meet

the criteria established in all but one respect, i.s. , thsy both dissolve

p^lodopheayl sulfonic acid to aa appreciable extent. This may not nsces-

mrily niake them unsuitable as solvents. However, when radioactive

pipsyl*chloride was hydrolysed in NaHCO solutton, acidified, extracted

with chloroform, and counted; the aqueous fractton was slightly less radio-

active than the chloroform fractton, indicating a slight preferei^e of the

sulfonic acid for the aqueous layer.

3- Preparattoa of Pipsyl-Derivatives of Dl-amino Acids ; Since

the available quantity of histamine was rather limited mad the amount of

histldine relatively abundant, it was considered expedient to gain experi-

ence preparing th^ pipsyl-chloride derivative using histidine rather than

histamine (assuming that because of their structural similarity the two

compounds would react alike to pipsyl-chloride). The results of this

diversion from the main problem proved to be interestiag . Pipsyl de-

rivatives of histidine were prepared according to the procedure used by

Xsston, Udezifriend and Caanan <14) using either an excess of amino acid

or pipsyl-chloride. It was always possible to obtain a precipitate upon

acidification of the reaction mixture of histidine and pipsyl-chloride but

-20-

9aaU.

•«*»'**«^ ^* - -^ - >i«i i»|jiw Atim mm»Um •!

•H»i>*ain lo .» :Hm|m -*" -

#••01 MMMii«)# i-|i;>« . . vol'XMt «VilCT

«Viott^i i^lHo »il»

•••aea Joa v,:*. a«ix» •ici«i99^(|(|« tic ©r 'iMiq^feotoq

aryUaaotftBi ar. a* 4* did*;; . v*"'**

•<Mq

•^h£1 a«e*f vftr iHa S«W acilO«^) EUJl^Jrf^,^ v... . J.' 43i>/JK.KrU -iiiw

lA^ •Vtt9«

»9ttia ;tribdj>4j|Bfa;

qx» hmr»blmtma mmm

su .; i^y 4i7iL«?7. Si:i .iC C«(|1n| '-^ -'«»! billow

-as-

the precipitate could not be recovered. The precipitate would disaiêar

if attempts were made to recover It ijumediately» either fc^ filtration or

centrifttgatloa at -S^ C* or reduce iteelf to a asiall tan sticky mass if

permitted to stand several hours at 0^ C Acidification with various

acids; dilute and concentrated HCl, H^SO^, and HC HÔ ; produced

similar results. Precipitates wonld not appear unless the pH was

reduced to a ran.'ê 5.2- 5. •; acidification beyond this range did not

assist in the recovery. The pipsyl*chloride derivative of arginine

behaved in a similar manner It Is to be noted that both histidine and

arglaine are di*>amino acids. Aceordinc^ly, it appears as if pipsyl-

ehloride can be used successfully only with meno-amino acids.

VI. SEPARATION OF HISTAMINE FROM BASIC AMINO ACIDS BYPAPER CHROMATOGiÂ PHY ;

^' Proce<faire : Histamine was separated from a solution of hlsta*

mine, histidine and arginine on a paper chromatogram. The H* fsctor

for histamine was found to be 0. 41; the R| fsctor for both histidine aad

arginine. approximately zero. The experimental procedure used to

effect the separation is described below:

a. XSmt a strip of Eaton-Dikeman filter paper « 1. 5 cm. wide

1^ 40-45 em. long, for the chromatogram and a 500 ml. glass-stoppered

graduate for the chamber.

b. Place a drop of solution containing about 5 micrograsos of

amino acid or histamine. 6 cm. from one end of the paper; designate

this position as the aero-point. "Weight the end of the paper on which

-21-

•««»l#f»^ «j<t

*nm •%»<

. ^ atiuuuim A mora t>9$m%mvm mtm iMAammn !»v*t .^

ivoltii feMtH»iM» tti iMatst*|!i« 6^:

1 rtsciv flMus;^. i^^ ^j - .' ;i-'

bdl^4|qol«»llM|^ .1^ 90i a jtMM «UnB«iMMl4» Mil 1«l «B«oi

I© aMnaoiîm e tuotfc âUlnco nc li»i<« )« ô^ « m«|s

f.;T'.la<>?"T :i-i'i/*r **f^t \0 J •varfw? ^îw'

rlw B« T«f«4 Ailf lo tei •iH Idâv

.'iiîujusin 'w Dzoti ^'"^'''^n

i£ £L«tli»gq fnUH

*IC*

solution has been placed with a small lead weight.

e. Place paper, weighted end dowB« into chamber so that the

lead weight Juct tonchec bottom . The bottom of the chamber contains

25 ml. of isopentanol saturated with 2M ammonium hydroxide. Secure

other end of paper with glass stopper « being careful to keep paper vertical

and away from side of graduate.

d. Develop for 10 hours at 83^ C.

«. Remove paper from chamber and mark solvent front.

f. Dry paper in oven at 110^ C.

g. Spray paper with ninhydrin (0. 1% solution in n-butanol).

h. Air dry paper.

i. Heat paper at 80^C for 5 minutes to produce color.

J. Determine R. factor by comparing the distance the center

of each spot has moved from the zero-point to the distance the solvent

front has moved from the sero*point.

When a mixture of components are to be separated, a drop of

solution containing about S micrograms of each of the components is

added to ths p^psr strip and the procedure carried out as described

above.

S. Determination of H Factors: The chromatographic separa*

tlon produced the following results:

-22-

C' .9bix0^^J fStficoixinA lit riiiw bs^st&liiia !c<^ X.it fit

^ImrbBty. Jr> iihfft inc'i'J vawfl fccfJI

Off t« tiaori 01 icA

. taO*7t infiyrlov. ^^&fn fine Ti*d^?ii^«fT mt^'A f»iqj|<4 .^'r.

. 'J ^Uli SB lt9VO ai n»<|4H|

.talocf*<n»a Mil bviI Iwtom Md ItHrtl

-SS-

Added to

Column

Distance of

Distance of Center of coloredSolvent front froro epota from aero*

zero -point point

(cm,) (cm.) Factor

Color of Spots

Developedwith

Ninhydrln

HlttamineHistamineHistidine

Arginine

17.716.5

17.417.7

8.06.7

0.450.41

purplepurple

purplepurple

Mixture of <>

histamine

»

histidine andarginine

17.9 7.5 0.41purplepurple

^ Two purple spots ai^peared. The spot with Hm m Q wasdue to histidine and arginine; the spot with R «

was due to histamine.0.41

-M*

«q ^ if Ai

aj«ar joci* «rr .t > : tv ; -:^ji '.,;-•

1- VJ ..? .vl-.

-«t-

Vn. EXPERIMENTAL PROCEDURE FOR ANALYSIS OF SAMPLE CON -

TAINING HISTAMINE ; (Reference is Flow Sheet No. 3 for dia-

gram of procedure.)

1. Pipet known amount of histamine solution (less than 0.$ ml.)

where ' U is to be determined or unknown solution (less than 0.6 ml.)

where "fC* is to be determined into a Folin sugar tube.

2. Add 0.015 g. NaHCOs.

3. Pipet I ml. of ethfr solution of radioactive pipsjl*chloride con-

taining 0.010 g. , tagged with about 0.02 millicurie S~33 into a Folin tube.

4. Place Folin tube in 50^C water bath to evaporate ether. Pipsyl-

chloride sinks to bottom. Add distilled water to make volume to

0. 6 ml.

.

5. Raise temperature of water bath to boiling and agitate Folin

tube to emulsify pipsyl-chloride. A hand electric sander was used for

agitation. Agitate for 5 minutes.

6. Permit tube to remain in water bath for 10 minutes and then

cool.

7. Add 0. 2 ml. N HCl and agitate.

8. Add 2ml. chloroform and agitate.

9. Transfer aqueous layer to small test tube, add 2 ml. chloro*'

form, agitate and transfer aqueous layer to a small test tube. Repeat

same process once more.

10. Transfer chloroform fractions from the 3 tubes to a separatory

funnel containing a known weight, approximately 0.1 g. . of non-radio-*

-24-

.ITf

^ ' r I-

UiU.

.aiOJlOQ CJ S.SXUA

Am 9.9

.^fmtli^ bmM ion n ,lmt.e hfc# .T

active pipsjl-hlstamiae derivative diaaohrcd Ia chloroform. Wash tubatf

with 1 ml. chloroform and transfer to a aeparatory funnel.

11. Wash chloroform lajer 3 times wlthNHCl; agitate for 5 min-

utes and permit to settle for 13 minutes before separation. Use 20 ml.

of acid for each wash.

12. Determine activity of 0. & ml. aliquot of acid wash to find ef«

fsetivcness of washing process. *

13. Wash chloroform layer with 0.2 N NH OH; agiUte for 5 min-

tttes and permit to settle for 15 minutes before separation. Use 20 ml.

of base for each wash. Continue to wash until constant specific activity

ts reached.

14. Determine activity of 0. 5 ml. aliquot of each base wash to

find effectiveness of washing process.*

1$. Determine specific activity of chloroform layer after each

NH OH wash by evaporating 5 ml. aliquot to dryness in aluminum plan-

ehet at 5S^ C. followed by weighing and counting. *

16. After constant specific activity has been reached, evaporate

remainder of chloroform fraction to dryness in aluminum planchet at

55<>C« weigh* count and determine "U" or "K".*

•All counting was performed in flow chamber* gas mixture of

99% helium and 0.93% isobutane, using Model SCI Auto-

scaler, Tracerlab.

-25-

>. /A.

r'W d3A9 left t

^* ' «iioi»« ••liiflifiB ex i«| ftfjdot oi ôftMi fen

^ ^^âw 4*^wr el 9MU4UV. afcaw xssâ* id »•«« ta

«t if«mr sasd d-.im ^« ,©.^41 .!« « .0 U> x»-»vij;xG ^^.n-wiu<, .*x

ifo^ -59fta -.i^.ât miolotaiWa to t*î^,, 3iii3»<|« ^fîtttq .«i

*• T" €»alxTft'j<i#»Jb boB tm«.»^ ..ir.^sw ,riOdi

"oiuk IDS is»bQltf gajitf .•cuiftMfosi v ôs aii.=i3;^ ^<-':'

Htf-

SompJGdoni-am-

1

-

/-f&at Of

30' C /-o

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NaHCO., - minuf-es> at

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in e+her

Heat at/oo"*C /-on

C3dd i-f lo min

(Z>Oal /o

noom i-emp

r\i Hci /^qi i-aê

;e<5-

CHdÔ..^N ^ t vupcjnat-e

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s^

u

c

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u

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dHCi^ A^/ f-ateCHCI,

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funncti

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in <ZHC.I^

Sx^lutl-on

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funnelVêigh/oyer .

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y U V V

CHCZI^ Acfi i-ofe

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Oef-ennninâ CLf l\y i ty

aeneous /oyer

y V 1 r

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^<=-t-tle /on

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^€T^t/G /on

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C otcuJa /•«

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V•'

A c id /ay<^ r- ^s/beat- /VC/

êt-t/e ti/^'ice

<C/Vc/ /oyeraddit y Qaitofe,

s>e.t-fle <jnti/

cons-tont

S/foe-c/Z^/c act-

toyen IS

<=ii-tQined

r^MôHOet-ennnine

actit/i /-y ofal, -

acid Qi'icfuof \Cjl'KfUai-

1 1

De.f-G-r-mineOct-/ 'yi^y ofeoch \A/as,h

O^/erm'ineOc-//'i///y or

(S.a<it) v\icif>h

<ZHc.lO^t-ennîneSyfoe-o Oct.

c^f each i^Q-E>h

ah -

F/oiAi 5iheet No.5 ; f-'^refi?arot ion of His t -\

/cjyer-

r

and Analy

'^yl Oeni\/o/'\/e AtUÔtt toy-

5/S ProcGdure <srr fo UV0 5 te

J., r»

-tel

.Utt

im$

to n

via. DATA

1. Preparation of Reference Standarcl to Determine V t

a. Quantity of histamine used: 2. 5/g.

b. ^naatity of carrier added: 0. 104 g.

c. Activity per 0. 5 ml. aliquot of acid wash: (Background *

1. 03 ct. per sec. . Counted 4096 fross counts.

)

Wajih Net Counts per Second

1 9102 55.53 8.0

d. Activity per 0. 5 ml. aliquot of NB^OH wash: (Backgrounds

1.03 ct. per sec. . Counted 4090 gross counts.

)

Wash Net Counts per S^econd

1 103

f 1.83 1.73

e. Activity per approximately 5 ml. aliquot of chloroform layer

after each NH.OH wash: (Background • 1. 03 ct. per sec. , Counted 4096

counts.)

Net Counts Net Counts K CountsAliquot Taken Mg. Of Net Counts Per Second Per Second Per Sec.After NH4OH Aliquot Per Second Per Mg. of Per //g. Per MoleV'ash Counted Aliquot Histamine rlO^^

1 8.5 35.2 4.15 172.8 1.92

t 7.5 22.3 2.98 124.0 1.38

s 7.7 22.3 2.90 120.8 1.34

# 15.4 fil.l 3.98 164.8 1.83

Ave. 1.62

^This sample represents result of addition of another 5 ml. chloroformsolution to sample 13

-27-

f . Activity of remiklxider of chloroform fraction which was

evaporated to dryneee: (Background • 1.15. Counted 4006 groaa counts).

Nat CoimtJi

Mg. of Mat Coitata Par Saeond Net Counts perSaznpla Sanapla Par Saeond Par Mg. Second par /^g.

lumbar Countad .....^............^Of Saxapie Hlstamina

1*aaasaaa

93.333.2

62*697.290.5

1.S62.932.72

78.3121.6108.8

* Sampla II had most of evaporated material on vertical wall of plan-

ehet; almost nothing in bottom.

*4i Sampla #2 Is sample #1 which has had the material removed from its

wUlfm wmd spread as uniformly as possible across bottom by dissolving

in acetone and evaporating to dryness at room temperature. Sample

was finally heated at 80^ C fbr 15 minutes to complete drying opera-

tion.

Sample 13 is sampla 13 which has been treated again with acetone

dried in an attempt to produce a more uniform distribution of eva*

porated material.

-28-

I iJBViSârTJZ I âmsjKsU. . » SflHBSxL 2:

e.8r sî.i 8.CC1 e.ee î

•m$i% lo Ujnr ImolM*^ no >.n kÛioqa^ to l«««i bail II if^wrt ^

•li flBO^ kwtomit lal-^îMm 91O fa«f Mil 4«i4ir II •l<|u«i al tl •lufmnti ^^

ôivlosalb xd onlHitf MindA slcttaaoq a« xiâitdUAif ai3 '^0910:; baft sdbia

^fofHiilt .flnffl«TMrin»l macn im aasmnh ot îiltsiooavfi boa snotsDJs oi

Kifi dikw mUm bft|4#:tt atii mmA dolitv tl •IqniM at €1 W^pwril ^^^

-jrvb . .. —,itMdtii%kb onoUoi} »ioqx « sidxibdiq ot }îs»I1jb Juk isi buJhib tea

-6ii-

2. Determination of Hlitamtne Content of Unknown Sample ;

Histamine alone waa included in tie sample and was taJcen from the same

solution of histamine used to prepare reference standard.

a. Qaaatity of histamine in sample: Unknown initially to me

hut later reported to be 0.7S /*%, .

b. Quantity of carrier added: 0. 1013 g.

e. Activity per 0. 5 ml. aliquot of acid wash .(Background «1U& et. "^v sec. . Counted 4096 gross counts.)

Wttili Met Counts Per Second

1 Its2 10

3 o.a

d. Activity ^v 0. 5 ml. aliquot of NH4OH wash; (Backgrowad*

1. 14 ct. |Mr sac. . Coantsd 512 gross counts. \

Wash Wet Counta i^'er Second

I

2 0.3SS 0.4«

•. Activity per approximately S ml. aliquot of chloroform

layer after each NH4OH wash. (Background" 1.14 ct. per sec. . Counted

40N gross counts . )

Aliquot Mg. Of Net Counts Net CounU Net Counts U CoaaU 'V in*

Tafcsa AUq^t Per Sceond Per Second Per Second Per Second /x-g.

After Counted "Pmr Mg. Per QikioiB Per Mole Hista-

NH4OH ofAlliuot Sample xlO^ xaln«

Wash

1 i.e 27.3 3.42 »47 3.64 2.37

t 7.4 20.4 2.7f no 3.10 1.91

% t.3 30. e 3.M 374 4.15 2.56

% •.4 28 5 3 40-29-

345 3.a2 2.36

ftl7t

^4^/ ^tto Afc|«ra8 teiipUAto

#a.c rM( s*.c £ tS o.i iMl >r •it •T.f T s

*?€ •a.s 4< «A . . w iM

-«f-

o^.t •J t

* These calculated quantities are based on an average K* 1.S2 xlO .

f . Activity of remainder of chloroform fraction which was

evaporated to dryness. <Backgrottnds I. IS. Counted 4096 gross counts.)

Mg. of

Sample Sample Net Cduats Net Counts Per Second PerNuaaber Counted Per Second M^. of Sample

•i 41.0 93. S 2.28•^ 40.5 99.4 2.21

>»««3 40.4 89.9 2.22

^Sample #1 had a concentration of evaporated material on vertical v«ill

of planchet* however, some material was spread uniformly across

bottom.

*^Sample 12 is sample #1 which has bad the material removed from its

sides and spread as uniformly as possible across bottom by dissolving

in acetone and evaporating to dryness at room temperature. Sample

was finally heated at 60^C. for 15 minutes to complete drying operation.

***Sample 13 is sample #2 which had been treated again with acetone and

dried in an attempt to produce a more uniform distribution of evaporated

material.

-19-

^AtarrfT* A

.'uxL .

.

lis, itj ii^tMim

f^ t . a

^»Sf bmmamSi rvH «*w«-''5 t*^.^i

<l .«;«i

^ .«3

f .RJi

MA

-.Of-

IX. DISCUSSION OF DATA ;

The hUtamlne values, ranging from 1 . 91-2. 59/ g. , obtained by

the isotope derivative method for the imkaown sample are between

200-400% in excess of the correct valne, 0. 7S/.g. . Even though the

error is great, the result is better than should have been expected

because the actual total activity of the histamine reference standard after

the final NH^OH wash is higher than the theoretical toUl activity that should

have been present even if the initially formed pipsyl-histamine deri*

vative was carried through the entire operation without loss aad self-

absorption was absent. The basis fbr the above conclusion is as

follows: the activity of the pipsyl-chloride added was approximately

7 counts per second per/ig. and, since 5. 4Syag. of pipsyl-chloridc

should combine with l/g. histamine, the maximum total theoretical

activity should have been approximately 95 counts per second <5.45 x 7

X 2.5); the actual total count was of the order of 300 counts per second.

This indicates that some contaminant is being carried along in the

chloroform fraction and is not removed by the acid and ammonium

hydroxide wash. The contaminant may be pipsyl-chloride itself because

it is not too easily hydrolyxed aad is readily dissolved by the same

solvents which dissolve the pipsyl-chloride derivative.

It is quite evideat from the data that self-absorption of the soft

bsta-particle by the sample makes all activity readings uncertain.

One is uncertain as to whether a constant molal activity is a £alse one

dae to a change in counting geometry or a true one indicating the

-31-

tsftfi {yiJdt«Ata ssos'sslsn eataiJttalct &jtt to ^^l^ftas fiitot lA^t^j.^ E^rf*

'UsfS b«s maud nfoiisiw aektanm^n^ »ijia» •lit 4%#e: }o ««w sylisr

.btmoi— %mq aiavod M€ lo 'i*fei« 94^ U» mam Uuro9 Ua€4 la»t9« tiii ;(e .t x

iKgjNifnif ha< blaa »A >ftii t<ia al isos asi: ionolrf?

»jua«99ii listVI fitiafiaui3*ixa!iil au \msL iaifmim wraqj fttf''^

.iil«riM«r siiMlMMii x;ttviia« Oa suUob •it^aMNi mtu %a i^Mnaq^asaa

amUi m ml xtfTlf9« latooi >miWJi> • ^taMmdm of u olitt^vDfte Hi mO

absence of contaminants; whether the observed netlvlty i^ directly pro^

portional to the quaiitity of material present or U less by the amount

being seif-absorbed. The data indicate that even when the amount of

solid material in the planchet was increased from 7.7 mg. to 15.4 mg.

,

the counting rate per mg. increased from 2. 19 to 3.96« a S05b change,

due to a change in the counting geometry. The data also show that when

S). 8 mg. of materiai are re -distributed more uniformly oY9r the count*

ing surface* the counting rate per mg. Increased from 1. 8a to 2.S2« a

&4% increase. The magnitude of the error introduced by self-absorption

luM been illustrated by a study of the measured activity of S^Z5 in bari-

um sulfate of constant specific activity (22):

Mg. per sq.

cm. Ba$04 Activity

a. ft 9005.0 1§W7.5 asM

1&.SX aftoo

IS.O 2800ao.o 300040.0 3100

The data above reveal that even with a surface density as low as 5 mg.

per sq. cm. the activity ceases to be linear. In the experiment per-

formed, the surface density of the evaporated pipsyl-hlstamioe deriva-

tive, based upon the total area of the planchet, was always less than 5

mg. per sq. cm. . However, the evaporated material could not be spread

uniformly over the bottom, the sample showing a tendency to concen-

trate in smaU clumpH during the evaporation process.-32-

ti to ki»«»iq If :t'j.'sm lo

t»itM« ty 9\wt. tf, ,*i? . . ci t! .J aioaj caiaaiaiii .;gi,cK i«*q 9ttti T^£$tmoo »«il

J ol iJt-? \ii j* b«*i!fi'>j««m »<lt 1o Yfci't* « vr* bstmr -Mid nmd

:(&£) x^Xv^i^** alUMMH lM«i«ioo 1« •toUvt m»

00319tiUooes

OOiii 0. .

.t|« 9 «8 «•! UM x^i»n9if :n • xiliw n«rr» Ssdt U'^v^-j STuds Jilsfe %dT

Mil i«Hiifr aqfli iMa ! •linU

X. CONCLUSIONS;

1. Plpsyl*chloride, tagged with radioactive S-3S« caa be success*

fully prepared from radioactive snlfanillc acid at the 0.0005 mole level.

Forty-four percent of the Initial activity of the i^ulfaailic acid was con-

tained la the final product. There is reaisonable evidence to believe that

one equ valeat of histamine and two equivalents of pipsyl-chloride react

to form a pipsyl-histamlne derivative which is Insoluble In water, acid

mad bane but is sparingly soluble in chloroform, acetone, and toluene.

Labelled with a suitable radioactive Isotope and employed with a

flilAUriMts solYS&t, the plpsyl-htstamlne derivative holds promise for use

as a tool to determine histamine quantitatively by the isotope derivative

method employing the carrier technic.

2. Chloroform is not a suitable solvent for extraction of the reaction

mixture because it dissolves radioactive contamlnanttR as well a;^ th«

|4psyl-histamin« derivative*

3. In employing the isotope derivative method for quantitative

aaalysia using the added carrier technic, it is highly desirable to be able

to recover the component sought from the solvent by a method other

than evaporation. The separation will provide a possible means of

freeing the desired component from contaminants.

4. When the isotope derivative method with carrier technic is em-

ployed in quantitative determinations, S-35 is not a suitable tag, due

to self^absorption of the soft beta-emission, unless the ixiethod is mod-

ified according to a technic patterned after that of Henriques et al (23)

./

?c «1

ii boa

J 'v.- I, iîJii ;ii:

113 D?njbin

'^lm» t '.jt^y-i '_

:»i|*?

i.îfj tfv.isviisu »^^-..Tci. Siiii ^*ijci<ixss«t ai .8

-31

-^ «

liio dthv foorftêa

;-i CUp^-^.iiîl lo IJK4> Y«ft/

I^«Mmi sdi j|ale0 Bi«xi«0«

1 >•» oi

l^

l|ni»»il

3i*ti»b b*xoi<|

{«) fJoe 9^ \n ot

sdd*t « oi fifkihtooom hm'Ak

-ee -

U> give reliable resolta. In H«nriques' metlKxi the sulfur is converted

to the sulfiiie , precipitated «« the benxidine sulfate, dried^ weighed

and cottBted. In order to arrive at the true activity of the aas^ple

connted« a correction factor baaed upon the sample aiirfbce density Is

ai^Ued to the observed activity.

5. S»35 cottld be used to advantage as a tag in the Isotope derivative

method without carrier being added in which the derivative soaght is

recovered by paper chromatography. It was discovered that when a

miictare of histamine, histidlne and arginine In solution were placed on

a paper chromatogram and developed with Isopentanol saturated with

3!f ammoalnm hydroxide for a period of 10 hours, the R| factor for

histamine was 0. 41 and eero for histidlne and arginine. Since the dis*

tance separating histamine from the basic amino acids was relatively

great, it is possible that this method could be used to separate the

plpsyl'^derivatives of these compounds ftH>m each other even though the

compounds are made more similar by the addition of the plpsyl-groi^.

6. Pi^yl-»chlorlde appears to be specific for mono«amiBO acids,

the di-amino derivativea being non-recoverable by the methods used here.

7. S-35 is an ideal radioactive Isotope to woxic with due to its rela*

tlvely long half-life, 87. 3 days, and its weak beta*dmission which Is

shielded out by the laboratory glassware.

-34-

fe«W««>:, II. 4i/iu.s, «^ fe„^^ .^^.^^^ ^ .•il*^^ .W»tt^ ,Hrls Ot

b«l^i»w ,b^Hfe •tiMiw faffclMMii* *^4 n jp * •>. *i,»

-^ ..V > •^. ,

'^' tvui ^i-^ -^ ,^

,i/»icr a

-(£•<;•

XI. BIBLIOGRAPHY :

1. Alexander, H. L. . Synopsis of Allergy. The C, V. MosbyCo.,

St. Louis, 1847.

2. Behrens, C. F. (editor). Atomic MmAlciae. Thomas Nelson

and Soah<, 1949.

3. Rose, B. . Role of Bistamiae In Anaphylaxis and Allergy. Am.

J. Med. 3. 545 - 559 (1947).

4. Hanke* M. T. and Koessler K. IC». A Method for the Quantita-

tive Coloriinetric Estimation of Histamine in Protein and Pro-

tein Containing Matter. J. Biol. Chem. 43, 543 - 556 (1920).

5. Baraud J., Genevois L., Mandielon G., and Ringenbach G.

.

Dosage de V Histamine dans le Sang au Cours de Diverges In-

toxications. Compt. rend. acad. Sci. 222, 760 - 781 (1946).

6. Rosenthal, S« M. and Tabor. H.. An Improved Colorimetrlc

MethcKi for the Estimation of Histamine. J. Pharmacol. Exp.

Therap. 92. 425 - 431 (1948).

7. Mclntire F. C, Roth L, W., and Shaw J. L. . Purification

of Histamine for Bloassay. J. Biol. Chem. 170, 537 - 544

(1947).

8. Mclntire F. C, Sproull M.. and White F. B. . A New Chemical

Method for the Detection of Histamine in Biological Materials.

Fed. Proc. 8. 102 (1949).

9. Lubsches, R. . Chemical Determination of Histamine in Humaa

*35-

im jK

vdst 'i > ?=ft-:a:

'% v .-- ft

.

,??V /--,tfT>

u K nft; V <r ?»:.T.i, *i t5I '^^^ *

-«lJUfl£>

^•«.'A«j.^ I-

ifrfi3fff

.(

if» ' »'. -tt^j^v

u»i» 9r.<ij\m^f

.(U*^ bns'

:>vvo~qail r OS .A'

vS «d* 'loTi b

,<^^vij ;&» - cs» ,^'$ .qjrt^Al

.ns&ii ; icl i

Blood. J. Biol. Chem. J^, 731 - 738 (1950).

10. Urbach, K. F. and Giscafre, L..Identlficmtion of Histamine in

Blood by Paper Chromatography. Proc. Soc. Exp. Biol, and

Med. 68. 430 - 431 (1940).

U. Mclntire F. C, Roth L. W., and Shaw J. L. . The in vitro

Release of Histamine from the Blood Cells of Sensitised Rab-

bits. Am. J. Physiol.159, 332 - 336 (1949).

12. Code, C. F. . Histamine in Blood. Physiol. Rev. 32, 47 - 66

(1952).

IS. Gaddam, J. H. . Histamine. Brit. Med. J. I, 667 -B73 (1948).

14. Keston A. S., Udenfriend S. , and Canaan R. K, . A Method

for Determination of Organic Acids in Form of Isotopic Deriv-

atives. J. Amer. Chem. Soc. U, 249 - 257 (1949).

15. Adams R., Adkins H., Biatt A. U. , Cope A. C, Uc Grew

F. C, N>^emanC.. and Snyder H. R. (editors). Organic He*

actions Vol. 2. John Wiley and Sons, Inc., New York, 1944.

16. Shober, W. B. . Oi the Decomposition of Diaso Compounds.

Am. Chem* J, lb, 379 -391(1693).

17. Weygand, C. . Organic Preparations*. Interscieace Publii:»h-

ingCo. , Inc., New York« 194S.

la. Gilman, H, and filatt A. H. . Organic Synthesis. Vol. 1. Sec-

ond Edition. John Wiley and Sons, Inc., New Toric, 1941.

19. Lens, W.. Ber. 10, 1135-1137(1377).

-36-

(C,

r-'

Ai

( «a;

J ..* .^I

-Cjjj''. J. i'- Hi' , j.'

' •"/".A sliifi^'sO Ite r-fil*«i-jIff«">«-»l*i«T t^^

20. Steio. W. H. and Moore, S. . The Use of Specific Precipi-

tants in tiae Amino Acid Analysis of Proteins. Ann.N. Y.

Acad. Sc. 47j 95 - U9 (1947).

21. Dotierty D. G., Stein W. H., and Bergman M. . Aromatic

Salf(»aic Acids as Reagents for Amino Acids. J. Biol. Chem.

135, 4«7 - 493 (1940).

32. Graf W. i^., Comar C. JL., and Whitney I, B,. Relative Sen-

sitivities of Windowless and End- Window Counters. Nucle-

onics 9 No. 4, 22 - 2/ (1951).

2S. Henriques F. C. Jr., Kistiakows&y G. B., MargnettiC,

and Schneider W. G. . Analytical Procedure for Measure-

ment of Long-JUived Hadioactive Sulfur, S-35, with a Laur-

itsen Electroscope and Comparison of Electroscope with

Special Geiger Counter. lad. ii^ng. Cinem., Anal. Ed. Id^

No. 6, 349 > 363 (1946).

-37-

fjt ,*".

-lie? •JJAl?>f ..'^.

.

,.8 .f

15 Ti? - f d aoiico

..^ ...^fcOfl"^"' T* «»<ffi-»;».-f '»'»?'

((<i)rv*^ '•-*

Ui

Thesis17229

E55 Entwhistleisotope derivative^

n,ethod for quantitative

determination of hista-

mine.

Thesis 17229

E55 EntwhistleIsotope derivative

method for quantitative

determination of hista-

mine.

aiNDERi

isotope derivative method for quantitative determination ... ·...

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