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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
AiJ»T'-j*>r •:? ".Tf-r A
•:'mm'nrTn :-r^i-r>r;
Li; "'V A/'
.T<4jf»f --;>»f»vf jtii>< :;ni ».*;/? rC ^„.fi Jf C-** /rf»j' idbedl^m ftiiv
^ f^ 1 to «'
.^^ -^odiaixi •vilii'^^'' -
•llnsop »jii Tcd !>odJ
stvUsJiiifSML
^V^m
o .'J ** >^ «1
^sl
t»i.. ^Al ,'« , J •.'i«a« ^1 L.^
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to AoAJwu
f:y ':i»iiv.;r^-^-fv :., 'j»X!a L^
t..' J
I,. I . ! . ^«
ft^i^r/
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
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) aooi^AJvsb boa wol isiitfln si b«odU lo ten^ .tain a^
-ai» ad tOMB iMll9«lab lo bofU»A avitlaoat \r > rr^i-<f?^ An ,Ur.> . .^ Imr
tbdHMMtib x'^viliij!9« lo taino 5di lM(£ta'Itli^flll ta t'*tim& *i»biro sil .bo^olq
«bo«I<f tlcfafjn )o .lAf i»q wnAitgo^alax ol aaloiiilti^ H aoiiAoin^aood aitt
Ml I^l9 axa oAca I«anoB m \o nam «aolia*tlnaaaot> laail^i! 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^^eim^% %A% m» tlvMiqaM taian
•dl «l«Tal ai«i$o^dlia»difa adl Ya ytlvDlmsaa adi sshronqml ka« taaatf*
•fradtooi toalmaifo xi^o-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 &
.UX) ImM-^
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«« afj^io^^^ 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 ^Uo iAiq sdT t«rUo«» •bk9» ;Mactip» »sefta9dgBfzi«^ ItF Milt <*aa ^^ c»;^ii^»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-
%Sm.'.\: if
oiMiMuh
•.
!: af.'riT .(^1) rl'/
''- '^'•' HufmM
i ,:»ijcu r: m^ i
f^f b-jvIoB^.^]:. tfaB ttiifli
S> olu // on of 5oli oui
/VaCI
^a^h uwith l/Vos=h
i/water /o
v\/oe>te
Gvopor^ate/yo NO^ y- sod-
/um stuifoniJate
\wirn
Ala CI
.r* ice \^ate:r1
to one -ha/f
r r
'
y
0>lQZ.(DtlZ.^
Z07. H^^O^
f-'ilfraie f ,11(7/-
Ihnoacph
e>uchncr
Or-y ovenon hyc/nou e> to
i/vQste
Cool in
ice both
[
r^ail o P • t.,/ h-in
f
.-=>up&rno ton-h OG-cont
fry al
14 o° C for."5 hr^
6^ vcfponofe to
dryness m\/^/ot-e.r both
^f^e trol-
<sunr)
<etnfcr
f- i/t-er
^hrow^hS> uahne r}}
<alazo - so//'
r r
t>
^ondmfyerf?eacf/on
f^cflUK al
for lb hri>
So/c/A/on
of PCJ^
,n POCi^
Oi's,s>ol\JC in
^f-hyl ether
e/-hyl
<sther
/Vo^h *A/ith
pietno/eamerther
ti vciporci1~e l-o
one -halfvolume
lA/OS>>
\
J '
\
Heal af lod'c
<Zy CIO 1 /o
Oecioloriz_e Carh on
Purified
pi/=}Syl-
chlonde
Cool mice hath
\ 1
'' ^
/o
Benzenein solubles
E >^ fr-oct
yvifh
ernz.ene
P><cnz.ene.
Filter
throuahBuchner
Carbonto
i/\/a ste
P'/ll 'ir
throuyhG>uchner
u 0)
1
be nz.ene ex.troc. tfether• solution
it
U
l/]/as>h lA/ifh
yfee"tr oteanoether- Gther
'
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-
(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 l^a 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)
^eaoO ,9%tiiMf^am 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 •^iAIyIksD <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 w^aAttmaOW
ain^ini 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 t^Ui is«iQ»A (tl)
-flitiaos •«• M» Qtal ••4fff C Alii )• >fi twill fe«<F Imw wa4^
Mom auii|i#» 3iX9xa9qipbnL«4 osmyiiigg^ loxa Ai^iiiii
-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 ^ii#w Xsftll •fft .ff»iiiir d9m t«l T«*4w
Vi»»«l«p v^Qtt^iM 49mtm»tl AmWl ^ ijifis « sMlWr
.XflB M to XaivI « suj«u ««MBil 4 A^f^*^ ^"""^ Kli^A (ft^
. CM^iiH . 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 ^o/ /b
lodo hen
Sot Sol. fy-
I oda bensu/f cicid
7~o l/^OSte
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,/-
V^asit) i/vitn
cold so/.
tii^ine
.-o//Oi-y at
/ZO°C /"re-
.3 tloor^
t f'l.
Cold e>at.
t>i~ine
C OlfJ &Qti:>r~iine
Or^ ai
3 hour- &
Ci<-fJ
Cold Sett.
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/^e
l^vciAer- ^ -
,^
/o ver-
/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 funnt^i
t^e. /^^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, >V^O
iA/a^h until
*~\ free rromrod act /x^ity
(^ vo/b o r- -
ncn- rod act
pure p /yfc -
chlcr.Ue
&en^.ane
and
i^at^er
\/siat^er
(4o /?y C//~ou s
A/a SC^
S^m^ene
/4-~/~lo\f\j 3heet NoZ : Pre-purofian of Rodioacfiue i^ip^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 yne^i^
LI'^odio ac tive
l^ip ^y I -chlor
ide (zrys>fcils>
C or be r> y-c
Hty\/oe.h
<^^i i-h
1
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^^^aihrn-i -/fAmstht* bo«ljuii»^ •JbHolad*!^^^
i«ra dfif ot b*fabs loft ettiw w^aitltmm •«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 ^i% 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-
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 ^I&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 li^i^ttf h«..vIoval a^lifaf^ < 'fr^ ^a
-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 disai^ear
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 H^O ; produced
similar results. Precipitates wonld not appear unless the pH was
reduced to a ran.'^e 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 CHROMATOGi^A 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© aMnaoi^im e tuotfc ^aUlnco nc li»i<« )« ^o^ « m«|s
f.;T'.la<>?"T :i-i'i/*r **f^t \0 J •varfw? ^^iw'
rlw B« T«f«4 Ailf lo tei •iH Id^av
.'ii^iujusin '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*
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-
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 ^oftMi fen
^ ^^^aw 4*^wr el 9MU4UV. afcaw xss^a* id »•«« ta
«t if«mr sasd d-.im ^« ,©.^41 .!« « .0 U> x»-»vij;xG ^^.n-wiu<, .*x
ifo^ -59fta -.i^.^at miolotaiWa to t*^i^,, 3iii3»<|« ^f^itttq .«i
*• T" €»alxTft'j<i#»Jb boB tm«.»^ ..ir.^sw ,riOdi
"oiuk IDS is»bQltf gajitf .•cuiftMfosi v ^os aii.=i3;^ ^<-':'
Htf-
SompJGdoni-am-
1
-
/-f&at Of
30' C /-o
'
NaHCO., - minuf-es> at
i'
^od o ci-
ts - chlomde
in e+her
Heat at/oo"*C /-on
C3dd i-f lo min
(Z>Oal /o
noom i-emp
r\i Hci /^qi i-a^e
;e<5-
CHd^O..^N ^ t vupcjnat-e
/ic^irar^
c
/M t-H^I ^H^UHu
u
s^
u
c
>^
(ft
c
-C
^^
u
fo CJnyne^s, in
f
U
u.^
'1 \
'
dHCi^ A^/ f-ateCHCI,
5 e/bora-f-ory
funncti
CjOrrisn
in <ZHC.I^
Sx^lutl-on
5epQnQ i-ory
funnelV^eigh/oyer .
"
y U V V
CHCZI^ Acfi i-ofe
Agif-ate
S minutes ~3 no/nc/fe^
Oef-ennnin^a CLf l\y i ty
aeneous /oyer
y V 1 r
TIcj i/va^fe
^<=-t-tle /on
/3 nninc^te^
^€T^t/G /on
15 m/nut-es
C otcuJa /•«
"C " on " « "
V•'
A c id /ay<^ r- ^s/beat- /VC/
^et-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^M^oHOet-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-enn^ineSyfoe-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 AtU^Ott toy-
5/S ProcGdure <srr fo UV0 5 te
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-
JUV
»?«
||'|>t>.v:i'~: .!«?
X
t
I C.Hjs->. a<3
e
.i^i
.1 1© «!
€X>htt
v.? t
.«vA
€% sJqmiM of aolti/ioa
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 iJBViS^arTJZ I ^amsjKsU. . » SflHBSxL 2:
e.8r s^i.i 8.CC1 e.ee ^i
•m$i% lo Ujnr ImolM*^ no >.n k^Uioqa^ to l«««i bail II if^wrt ^
•li flBO^ kwtomit lal-^^iMm 91O fa«f Mil 4«i4ir II •l<|u«i al tl •lufmnti ^^
^oivlosalb xd onlHitf MindA slcttaaoq a« xi^aitdUAif ai3 '^0910:; baft sdbia
^fofHiilt .flnffl«TMrin»l macn im aasmnh ot ^iiltsiooavfi 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 }^is»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, i^iJii ;ii:
113 D?njbin
'^lm» t '.jt^y-i '_
:»i|*?
i.^ifj tfv.isviisu »^^-..Tci. Siiii ^*ijci<ixss«t ai .8
-31
-^ «
liio dthv foorft^ea
;-i CUp^-^.ii^il 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
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