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'. ■ . :. >/: '■: submitted■ The Degree of faootor; of- Philosophy' ;'Jhe faculty of Veterinary Modlpihe.■ - : - , o f ' . . y ' . ,; ■ '; V : : ' the University p f Gl asgow' . D u g a I 'd '.P ra n o is Ma o d p p g a l l ^ ^ B, « V*M»S R e C m V ^ S , , v'Dephrtmbnt p f 'Aetèrlmrÿ.i'Physlology»' / ' ■ UniversityvOf Glasgow. -April (, ,1972ProQuest Number: 10647768All rights reservedINFORMATION TO ALL USERS The qua lity of this reproduction is d e p e n d e n t upon the qua lity of the copy subm itted.In the unlikely e ve n t that the au tho r did not send a co m p le te m anuscrip t and there are missing pages, these will be no ted . Also, if m ateria l had to be rem oved,a no te will ind ica te the de le tion .uesLProQuest 10647768Published by ProQuest LLO (2017). C opyrigh t of the Dissertation is held by the Author.All rights reserved.This work is protected aga inst unauthorized copying under Title 17, United States C o deM icroform Edition © ProQuest LLO.ProQuest LLO.789 East Eisenhower Parkway P.Q. Box 1346 Ann Arbor, Ml 4 81 06 - 1346. Thé authp3C wishes to :expzess h is appreciation for thé assistah d e received during the course o f th is wpadc*.' He' is' 'ospepiaiiy indpbted "td ’i>rofessor''W* M uliigan, upder # 6 so ; sup eryi sion the in y estig a tiq n s w ere.carried o u tÿ for the expert guidance and/support & so rea d ily and ■ably.;given.;- -ÿ/ 'I , \ÿ"vt; Thàhks oKO' e x p r e s s e d ; , t b - ; M i D u n l o p -for sk il le d tech n ica l a ss ista n ce end to Dr* ï i Belman for h is advice and assistance* :thanks -are aiso-e%presséd. to Dr# J.D*/Dargi'e, ■Dr>v.-, PmH#. Holmes and Mr J*M* Mcheah for valued d iscu ssion andF in a lly , the author wishes to express b is .grateful: thanks to h is w ife , Sli^abeth:MacdOugall for preparation o f the text#V, ': General Introduction ^ ■ ' • ' \ "■ g ^ i g n ^ " Général M aterials and Mothods'The;Preparation Of jBpvino Fast IgG (IgG,) immunoglobulin .'M siiôâ-’M' . ';ïho ,Met'ab:)llsm o f Bovine FastThe M #abolism o f Bovine IgM The E ffic ien cy o f Absorption . o f d p lo stra i Fast IgG (IgG,^ )Plasma P rotein hoss. via the'" ; >%• k " ■ .èastro in tdstihal. Tracts o f , t h e ./ ' :, ' Neonatal Calf Section VIXI General D iscussionÈ âS â 16 : : 42103142165191194..G B fsffiA L h m m u i s amo;■ ’ ' E x p é r im e n ta i A n im a là r ' ' v-^ ' '' 17.iwtaboi 1:#;'Gagoà' - 7. . 18',; Gi: Isotope, Techniques Y y % , ^ ' ''■20,.B# : <: /■". ■' ' -31V:;H#ï: Ifiïïïiunochêïbioai Tochntlquoa . '32: • T#/; # a n ç .Blue' ( T ; ■ P lm m "%lme : . , : 40•;,'-è.-'rëtatiotlàèl-,Motbodo - 41t h e ; > h b p a r à t iü h . ' " o p DovTNÉ f m 1# / # % ) ^ ■; ' ;' ,■ 7 . , ,Introduction ••.'-.■■ '''■”' 43TW:oriaiS::and''MethodC' -- . ' ' J,'' 83iu\- Starting Matoriaio' 533-4 y MolWcuiar/Biove ^.8 # .. lon*^BKchanso-3hromato9?0phy ^ . .. 0 0éiàouésion ''V •'■"/■; .■ -.'v. '-:''' ^ 04' : ' ' ' ' V ^ ' ' 0 5THE teïAlSOUSM OE iÉOVIWB :.FÂ8Ï ïgG ,(lgG. ) 'IHB.sMEtABOLISM- pF BOVINÈE a r nïntroduetibn" • - ,' ' ^ v ' % ' 6tMaterials and Methods 80ip su its :: ' ■ ' : / l . /: : " ;; ; " ' ' v ,-;-34''A«3 Group '^1 IgG. Metabolism ■ ' ' '■ = -84,'B* ''‘''Oroup; 2 Ri^eat Detéminatipns 86C* Group 3 ^ Albumin Metabolism ■; ) < , ';'.-\87D* Thé Effect pf Diarrhoea à , 88'Discussion'" - ' "'3:: 90% ^ J : / . V.§«#ary:, ' ,1 0 2.. ïntïoduetlPn- ■■;■-. \ "■ , % ., - , :i4(M/M aterials and Methods - : •' ' / , , 110: A v / / î n W à l ^ T # § t u # v V ' ?, ÿ \ i i g'B,. :'First- I.gM' ^epeqK #u()y :/' / ,115. v ,-, ; .G*' /Second IgMTWpeàt Study • /,.. ;■ ' ■;,.■• 119P/: Third Repeat Study , 122V 3 / / / . ^ .3 ' ^ . ^ / / .' - 'Eb Additional Hxamiriàtlon of ;îg l^. Preparationi): ( i l l ) in the Rabbit .':r 127<î>éy ■ ,: 1 /e/,.■ A:. - y / : ' \ j y & y y \ r : . E a r nf% - ^ r : / : : ' ' : { y : : : : ' y ■- ' , ^ v y : : -/ y . , F y / F i m # B x p e à m M t - ' '■ - ■/, : ' : : , / , „ y '. ; :j ■',■>■ ' y ' 1 \ , , -y y - ■ . • ■ - ■ . : '.. ■ - , . . ■ ■ \ -y.Gc/'-Ari 3s|im ate. ofr'the Plasma Hall^hlfa of'--': //v .,; " ■ - - '/ ■• ., ■ .,y:, ■ .:BovW:l'9M.y:'!#'/AadlAl,#ë#i - fv ‘ ''129 . . - y- , y - ■ - , 1 / y / y y . y y T y / y /y . ' ' .' r' f: I ":hP 3vP fs c u s s ib n#1:' y /yyf' , -, : ' y . " ' y " ‘y.^Àÿ /s§ g a ||a lfc S■|'y yTHE , EFEICiENCY OF ABSORPTION OF # W sW L: f:y ./ y :y/- y - ' . -.■' ‘y- . 'FAST IgG (igG^)'..,/ ■• y -y ^ _y;.ry 'y yy I' - Y . ” . y- VL ;y-' ■'■ ■ ' y/■y'-.. '3- y'.' ' - 3in S r o d u c tW y ,; , /? ;" v " '; / / ' : : ■ : -"y f p ê k.M aterials "'/v/- 3 ... 3 t , .;: 14Ty y r CBiscuâsion'.-y- " ,yry ■•,. : y . / / y 156. . ' , r- . V ■ -, y. y Vf - . - ;/ p ™ n a r y y y y - ;/y --'y ,,, , - y - , : - , T .. " r : - ' %:"y r:yy- .. ' ':-Tyy,.y y / y 'vy, - ■ '■ ' ■ ’ * - ' ■■' '^ y y - , r " y ' \ y : , :' , C. ^ . . . . , . _ - ^y - . ; '/y:. ' / ' '' ^1y:;r' ' ' . \ / ' ' ^ " y : y . , , - - .. ,' .-ÿ. / - y.; , 1 / ' : y %- , v-y, -v ■ ■ : y / - ' / i y Î î y ' , : y y ’- ' : - - : y ^ y y y y ■ y , y y :îi- ■• '?i.y % . / y 3 % / : :% -À/ -l#4aT % peaF anc@ ,: . y > .i51y . ,- / y , , 'v ; y y - ; -y-'B.' «IpotopicV E ffic ien cy , / y V - y / ' ’ ; y ' ',#2:.' - L .'/ ': .., -t ' ■' : ■ . - : '-y ■ - - • ;y «■ , --%. Radial Biffusien ResuMe 153' ' y . ' y, . . y" :y y , '-y - y . . y \ ; y y - - ' y . " ' : , y " y / / . / yyy- y - ' y , - : / " y / ' " ' .P* '3 lrst'':# rèe.ypays' ^ / iy /iëp :/; y;' , 'y 3 - -yy/'-y y " - > ,. : " y / / ' y' : ' ' g y # # # # w e y / y . ■ ,y.:...;-yy- , ' y y / y ,:C y 155 '. . / y . ' y .y,,:y{y j : / y"y / y : ^ ^, l; -: / - y y : ' C - : ' c : , y ' . . ,y ' 'Section VII:y., VIAY THE GASTWINTESTINAI./; y;-'y - ' ; v TRACT ,,:0F THE , NEONATAL CALF ■ ,y./^166:y :y :# te r ia i ,^ \e n d Methods/ y- / ' ■ / / / ; /■■; . ' , y, , - y ■./;'^,721y ' y‘ ■'■'y - ' . / y - y v '■- ' / : % - % / - l a i ' yy. y. -y' . ■ y . '; V A# FàéGal axcfetip n o f r'^'^i^pvp in Diarrhoelc\ : yy\y y^y/'and Control. Gaivës, ' v y yl73: y y B, Paocal Recovery of T^ PVP (40,Qp0 Gv.Mbl *#* ),Y -; ': :- y , : ' y ÿ / y : ^ % " P # g 'à 6 0 ; p o 6 Y a v . : # : :a f^ '^ ^ C rC l, . :» y : : , :yy.'-'-' y : ' ; y y .r y ' : \ ; # ^ " ' 9 # % ^ y^.y.;^' ; yy-. / / ' : ' -y y : 'y ' 'y yy G* Repeat Determination o f the Faecal Recovery" " 0*y PlG'smb-jLbs$yin'':Noh^ ^ 'Measured"':/yy.', y,":,.:^ ' " : y W y - \ / / / t /, - y . '■ D iscussion' ■ ■/.■•>,"'. I r ; 182-yy: y7:: y ':y ;y \;8 ^ b ry ." 3 - '^ y^ y ''^ - y" / 190- ■ i: •The development p f our knowledge about the plasma proteihsy has. been very dependentf On the techniques available at any one time.. V Part o f th l s development h a à been a growing awareness of the importance and conplexity ,bf antibodies# Landstoiner, mrlclng from 1906 to 1943 showed that the phenomena of immunity are ' essen tia lly chemical, antibodies being adapted or s lig h tly modified serum globulins# He established the concept of immune specificity*;thé f ir s t techniques applied to the investigation of plasma proteins depended on their solubi 1 ity andyrelated properties*T iseliu s (1937) introduced electrophoreMs vihich along with the doyelopmont o f the uitracentrifuge by Svedberg, has proved invaluable# Ooh^ y.^ .; f ractionatlony #ith ethanol^ aty;iow teiinperatures'provided: ■ ■a method for the production of large quantities o f individual plasma proteins .for YfUrther,■studies# ' 7‘y-■In h is review* %)rmall (1948) en^^hasised that in the future, more modern weapons Wuld be used. immUnochemistry,in "particular,:: the use Of iso topic tracers# Prior to th is * i t had been possible to study plasma protein; metabolism using balance studios and infusions (Jaxnum* 1965)# Thus, Welchv Adams and Wakofiold (1937) observed a high faecal nitrogen output in a casé of''Severe'UlCGrative:;opli tie*-’. .Suchr.'studies ■ '..were : however very limited# IsotopiCally labelled proteins proved to be a . major advance#-/Barlÿylsqtopic'.stWiëG' w re 'con%>l.icated. by problems.''., associated,,x-yitiv the purity of plasma ptotein fractions Ond wlth - ' ■ 'the^riskj&f/dohatur&tion'both before a^nd,' afterylabolling*’'■ :'This was emphasised by Kekwick (1966) vlp pointed but how ia b ile some ■' pur i f lech^plasmo''protein werë^ * - thé .'avoidance: of - damage ^ .during■./The ''development ■■of'-the ,iëohniqwé'iof,ïmmuno'èlect3?qphoresis"''' by Grabar and IVill iams (1953) demonstrated hovy hbierbgohous ' ■ many' of' the .plasma -protein .‘fractions-: were,iri, particular garma globulin, as previously definéd by eToctrpphbrèsis*/- ■ 7 '". The 'iiiproved'yseparatl'op. techniques - of # lé q u la r - .Giqvey chromatography and ion’^ exchange chromatography* developed in the 1950’ s# have over come jnany of the problems '.associated..; td,th'y/ earlier tochnlquee- Finailyi the deveiopmbnt of an in vitro ' method of isotoplc labelling (McFarlane*- 1959) ;Miich- did .not ■//'.- a lter thé metabolism Of thé protein under investigation made possib le/the ;V0ry,/extGnsive'ystudy^ b.f plasma protpip metabolism, Which..'has'■ "subséqtientiy >tafebn ' placé# ■ This' ihvos.tigatioh ■ i s àn ■ application of these inproyedboporationan^ labeliing;/ technique^ to a.study o f ; the metabolism of plasma ,proteins in . ,the::young c a lf , 7 / ,, V . .■ , ^ : /' -. The p'lasma /proteins' o f ' the;ne%bprn .cal'f. cah' bo, divided ': into, those/present' in. the 'foOtus ."and thofefo.re at birth* and %those’ absorbed' frdm'the -traot 'following' th e .ingistloh -. of, colostrum*/ ,. th is i s 'not/a'/rigid*'subdivision; ho-the-.':/...foetus may produce antibodies U nder/c#talh:bohditionë#/.'/%t. may/:-' : b(ï further or sim ilarly complicated In the weeks.foliowln^/bl#h%;; by tho developing capacity of the c a l f to;, syn thoslzo .'its/p# . / - ; antibodies* ■ Both these- p o s s ib il it ie s M il bo dlsCusoèd# ,/Bvm /' ; ;;\# 'the main change:in thé cerum protein%\6f/tho.'/nt^ Àÿtaî^ ^^ is:'- "/'the acqu isition 'of'îàatêrnàl antibodies frdM/the ooloatrum.*''^ /thé / lack o f such protë.ctlôhlhas boon linked xvlth the high mortality ,. among no#borh\..c.qlVeW^ ^^ ^ Bictlon;DisoUWlOn:/} ^ majb%'.#use#. ;; /; -, ,/" \^ ^rdah-(l93S)';.hotod a '40^:te ÿ |a l l ty r xdiicli^yia^aiiy-., . "; //,■ disappeared a$ p###% -;:W ith#e',(# 8 3 ) reported' the-résu lts / - from ,.# /h er# .; th # p # o u t B rl UJ n #v- bn Individuel &rms' he •■ found that tW rote might' exceed Po;1' ai#bugh:th#''ovorall; ://mortality' ./ih ls xms: among heifer oalvéa;* üp,’ïd-'6 -::'■ „■■' fflohthë’-çld^/i'n .d{&y^,herdsf repbrtW 26^ los^ës / . „ ■ •; qf-paivec #ém%- comiqn{##éri^ • Otto son (19%) In .a . ;postmortem survey concluded that 48^ o f the calves examined, had . ' - ; ''-died from CollbacillosiSé- .Oay y AndWson* Fisher ehd McEwan (190S)' ' ' ' ^ '" I : - : 'reported a I?*#; m o rta lity ;# o n g m a#et b%jtli ca lves. In the rawest of Scotland, ; / ' . • /;' - ^ :'-r -■ ' ■ ' More recently Sellers* Smith and Wdod'{1968) ' found-a 2X%" . ’•";, ' .' ‘ r-i " _ . . " R S ' -S ' t ' ' ' ' ■' . f ,, ; >'• V 'm ortttity In l i x e k p # life* /w ith an,;additlphai 4$ in ' ;f ^the second WePk# dp'.le. Fhente ( 197Ô) In asfarm survey carried but.'" ;.':r \ ; /' \ " A, y. / ;in the ;%at/pf. .Bwtland#' ehbmd that/the-highest incidence of dpath/ '- . ' ' ^ / ' ; . / % ; - y ^frpm,:Whn##i dl#r#oea.;bbc0#d" ■- / . : y "-''''' . . '! < / %'/ '/"'' / -\. A -. / i /ta îvéâ* ■ Other reVlewe-,, A lch- associa te a-high - p roportion ofneonatal c a lf mp#aliky 'ûf / '- , ' " .. L ovell (195S)» P e # a le (1908)1 W k a ^ M s ) * MoEmn (1968)m : a m y i - - / / / / Y y , ? : : .y,.’ .The " in v es tig 'a tiq n ;# iith#re#rç\.concentrate on those p i#m e proteins- acquired'by the oail^'dbring/the--first few hours o f/ ' r : ' r . ' / ' = - ' v , - ^life# 'ginc#. %lrichy(;IB92)';'demonstrated ^ #0...transfer of •;;’'C p lo e t# l antibod ies I-n#ic#.@ ' # e \'s# d ÿ of th is acquired i t m u n l t Yhas been t ie d up with the in v estig a tio n s o f antlW diW aS a .-; - ; v : : v - ; : ' '- ■/- Landstainèr and Vap de 'gcheer (1986) demonstrated the \ /production o f antlbbdies qf/yarylhg sp b a lflc ity # ; %6at/end %e^breën (1938) using th e 'n lt# c # & îfp 9 è found'^'WtgrPùP of'■‘. / r / / " ' - / ; ' ; " / ‘ -'■ ' : ' - ■ ■' r ' - , %' ' ' 'aM ibpdiee with vary d W e r W sbdiM c o e ff ic ie n ts*\ ' ' I ' " ' Y ' . ' : . ' v ' ';6 ife ■and' lS#78 respectively# lisbtius and Rabat (1939) -çpnfirpiéd" th e ’speclbs -difference found by Kabat and/Pedersen# ' l%hn» beutsCh and Wbtt^ ^ reported the existence o f an/.additional group o f antlbod^es , sediXQonting between 80 and 13s’ , J(l9ô3) oxmipW mhtlbodle# qf tho'/p##- éedimeùtâtlqri' / -value Tend found à' whblb;zonge_of; biéctiopW rëilo mobilities^'and:"'; ' ' / : isoelectric points# , Franklin and Kunkel-..:(I9B7) shqfed tliet / ‘^ : ,.' /. 3;the'19s fraction a constant constituent of .norAl f 'gioW lin/ i ./,:,.;,ani/#Wigenl tb;ihb/meipr' 7a' cor^one#!; 'B a ÿ n # & ' y y ; ; ; ; '(1959) demonstrated thb marked heterooenoity of dlpthbrla antitoxin*,..,:p # d u W ;ih Wracs#:^ ^ ' ,3 3 / 3 ' / / / - _3ÿ <" '3 3 .3 '• , Y ■’ .;%e,?#dvent of # c h i# fo v W aepaWtibh; toohniqubé.-l# '' t # m ;// ' ;/ /pohçidétahle increase/in/our^knowîWâë -df/thla heterbgénbitÿt ' ""3> y B éh # and Horbett (19591" separated' 19#. # d '7$/co*%)0no#a%y i o # .;. "'3■■■y'/ôxéhühge chromatography and Idund■iiîlèféinbàs I r i /ê ië e tlç p b ^ o tic ' y./-’ ' /' o d # é h t and /##ÿqdy/d istW W t^ the' M'y^y/y ,y /çq0qnbhtax#ré/:;#W ld#i^ "ahtioenibdlly'"xbXitodl - ^8|pd#i-/Pjr#erg ^' Moiley and/Mli’i '('#5#:)\'and Par tor,.(i960) àrrlW d .a$%miîW° ' : /■ /33• . çonclusibna# 3Talmhga'(19'59) on3tha'o ther hand eiïÿhàeised-' t h e -Im a 3 // ;/ ' / ' ^ l y i e l & l i t ÿ : ' b f ' - a r t i h é d i e â * ^ o f ' & o l ÿ L # : # r a i % y # 3 / ,/'■'■'■ A . ■ Elaen and Siskind (1964) continued thé rauch oariior vmk. -of Landatoinof# Tho^r findings wore slmiior to thhsq/bf/ibhr (1964)- 33 . ' /i'h ''his' invostigaLlon ofythe/mdïtlcomponent' ^ ##om # 3 Kobat ' -' ■ 3';ÿ..^(l#6-a nd 't)- a'0"''(|967) ë^phapîbëd'-#1^ 'h # # o g d n d ity ? ^vM'ôh/- 3'3- / Kunk#. (190?)' # # d '.dtàggêrin9fy#%^^ largo;-ë^ay a f , 3 '//b-yyy-dlap0ès3Ohd;subclacaOùÿ forming a heterogeneity aho# and ''Beyond ' -.3'/; : ' th a t -d i# o tlÿ ./ih # lÿ é$ '/ m tïhody '%)oaIfi# t y # . 1# estimated: #â t 3' ' an individual dan iorÉsahoU^'^é^ïÔ diflerëp/aM lfoodios:# 3 ; / \3.;36 \, Bÿ IrimunoelGGtrophbrosis Herëmans (1959) and ( X9ôô) found a populatloh;of s in !lar but eibctrophoretically heterogeneousp ro te in s /in ;th e and ÿ.- regions a l l called T globulins ,■ ... ' ' " ' 3., ■ 3 / . , y-' 3' ' ■ / . ' ;■for iihich he suggested the term immunoglobulin. Schultze, (1959) put .forward a slMllOr/concept/ and Mayer. (1961) found .that although one'3pf these*Im m unoglobulin; (three had so, far been discoveredI,spread oyer, a considerable range bn electrophoresis, antlgenically Ib was qhe, prgtslh. Franklin (1902), Kunkel (1^)62) and Fahey and Mcbayghlin (190%)'a l l / investigated and commented. on the elec.tfophoretic heterogexxelty of the immunoglobulins,Fahey and McLaughlin demonstratecl class sp ecific and common * ' antigonlc; déterminants# .showing that thdyh could be defined oh /. immunochemical grounds. Working with myeloma pro te lh s , M%nnlk and KUnkel (1969), disCovored ,the présence of two antigbnlcally 3/, 3' d istinct, groupe,o f i n man,;3 ,Grabar (1963) tried to tidy up thé;confused nomenclature but th is .was fin a lly achibved by the World ^balth Organisation >3(1964),, The claèsés were named using the. abbreviation I.g or the symbol Y thus, IgG or r^, or or y^ e tc , Fudenberg(1965) stated'that''each \of/these, groups:-contains, ari indeterminate .■ number of: proteins # ic h share; certain structural# physieo/chemical and antigenic .properties' and at the same time certain sp ecific - : individual properties# Rabat. ( 1966%) anciBurnet; ( 1969).yy,'/ y ■ " considered-'that l i had &oxi' 'thë%dc/vëlbpmë;#mfr^immùaqol#tr6' .^, / Z y-A / :. phoroelë "ühich had so increased ouf insight into these heterogenous/:,/ '' ' "■'//. y / ' Subséquent research Into the boolc/structure of'\the:;li#UhOT':/,,;/ 3 globulins was greatly fa c ilita ted by the a v a ila b ility of myeloma'■ and Bence-^dones proteins, Kunkol, KiHander and Monoik (1966)' 3' found that a ll the evidence Was' ih//|ayqur' of the c lo te 't;ql0'i6hship3"'3/y. -:333: A'- . ' V between'these proteins and. antibodies#)Furthermore;,' yçbïd'ra'g : 3:/v;,3i >.'3, #hd rhoumutdld aioti Vi tv hhd beon dombhstf Wed in' some #ld0nstrOni)r 3 'f"/-/:Rr':/:-, ■/ ' màçrbglbWllhs* Kë%at;( 1907) V Fahey ( 1968)/"■ .:/ ' ''and-..Rowe-(1968’j/ure biAtyatfeW/df;thb workors'.iAâio have investigatedr / '::..x 3 . 3 .. : : ' - \ 'V 3 ' d n i'discussed'-the hétclo.9@n01ty o'i}'^antibodies'from/every .angle, ■ < / . 3 a3%: 3lhoXWng\the%t^ 3'3RR_|:3 v, A ■ - A ’'''/A'/qimilar complexity hhs b dOiWonétrèted ^ in,boyihQ serum3 ;3 /Vy#3" ; thé'-'inltlai' 'differehtlatloh 'Betweéh3thè3tm compOhOhtB ' 3^;=%]#1 3 3 / ; 3 : 3 . : : 3 3 ' . / 3 x - : - 3 ; 3 . ‘ 3 3 . . . Y " :: Bépend0d\lârgoiÿA on;éleetrophoresls, .With ItsV lim ita tio n s ,-’- , - '//C iK ’'3 3 " \'T-hus Tmith ''( l948) showed th a t the co lo s tra l InmUne globulins wore3 . /3 ç iosely related#t6' the YR-Qh&Ÿ/séfu ^ g iob U iin s') %bëeiqWhtly, - -I % / '35?33- , ; 3;;’/ . Sxalth .and |Wlm:/(l948) fouhd ho change ih.^^&bl'Tlly'::-after 'absoiptldh - 33' 33/3-■;■ , . : ^ o f oolostral itmuno qloW lin/by th e ,ca lf *3>%bss and DeuLsch' 3" ' 3'''/3 ,;33:3%' 3 '3‘' (:1948)* Using a modified3cbid ethanol3fracAilôhàtlon p ro ceed u re :3 ■3--'/3'-'.3' 3 - 3 ' ' " 3 3 ^ : , i k # & 3 3 3 / ^ ^ W 3 . , ; - 3 ' " 3 ’ ' # > :: 3 ',^ ,,AA#reparé ,^"tif# closely: related,Y jlo b u lin rractrons from bovine serum, -y :V a:other...Wrk'erS' Used : ttiel'Y : o#i- hW^OflatUrë, ; Thus P o i# h ( I9u2) ^ - .y ; / ' yR re fe rs to y, and,;Yg/9XbWlin$*/Pl$roe3''t^ Yi 3 Tn- y ' ’ ^ ^ '. ' 3 / 3 A / \ ,,globul:Lns*j:Tarson3'(\l95g ,to_#^\and Yy\9X#%iD$ and P orter and Press (1957) to 'p a r t o f faovipe x globulin olosely re la ted ;to - the (iRglobuUn’fy The main; conclueloh Trom a l l . t h i s work#'''/; ,„;R'- - ^ . 33 ,r qohoorning the pres^ncéRqf two olopply roletecl Y g lobulin T ractions' ;; Awith* d iffe ring . eiqctrophd#tiO;Lmohili ';ha''s- subsequently, been'. - .3 3' "3:, ; # i 3 '\# s taM tia% d ÿ .. . y / y ; , , . , :: . A "A.y., / I; 3' %3G#riol/ii90i)-'% ho#d ''that# jGOApghonts. v a r ie d /a s"tq/iheR. :/; conditions un îér uhlch they^-'wélë;’ elytdd In ibOTexCMnge Chromatography, ,;'.pie%eR(i!9ô| ) %qgnfirmùd '.tho.;ido%ity(Of c o lq s tr a l ,i&iuhë#globulinR= .R-" with T or component of serum. Mur|)hy ^ Aalund# Osoboid and ' 'A Gqrr^ ^.(.19#),, anci%urphy, .Oseboid;:;and ,A#l^ - " ’slow and fa s t 7s components/in bovine/laCtebl'3seoretio.hs* th e ' - R / /'rélative/am oUntsA'b.fyjwhich'/Varied.depending/on•'the•character of '' “■ À/3"' ' A - 'Y .-va'” ■ ' y ■ -., y, . . y*y ' , . - .r' . y ■>■■ - -the secretidn'3"i.fe,/ Yçolost## /showed; a.nR#mostR,co^iéte ’ ,/.absencet'df :.y;the. slow ;,bo#b#ht3 /AThby:^ ^^ thiç._ a l l ''th e more R... '■ TemarkablW* 3in3fchoAlight;.of..thè-s im ila r itie s ‘between, the two' ■ ‘/-y‘-prb,^ins>.;vélcliAdiîfërèd3only';§s3régorda/hot o id c trie 'ch a rg e , - / ^ ; ;'"a.mihd^3.s'0Mo.h% difference* a miner e lu tion // ' -./position v aria tiqn R#en;pRf ragmen be were-chroma to g raph# ' and , ' /" ' ■'the asso6iatidh''bf;/,Gqmpl'##'#- w/ïth> t h ç f a s t , frac tion « '-y ...The antigenic, d ifference between them was associated w itli // : / " V : / 3 9 - - y : : A :y /;A À / ' -Y-'' / th e / tp # ' formation o b ta in e d /# ImmmpblectrophqrqsxSA- —: / / / - A a i U n d - 131X964)-referfd io # # /Fëst and 810^ 10 of tk%3;:/ -3''. 'guinea\pi.g -.WheW ^ thoRtwo- fraotfotiR can' K# defined on th e ir Mbpaolty ' ''':3r ' , . y ► ,' ' Î V” -, . ' :>■■■■ - » ; '' : --< - \ ;tormedi'atd - particular;,Mbipgical/Rfunc cldn# ( see :BènaG'èrraf * ■ ^ ■ -3-3;;', .ÔVary # ' Block ; and- Franklin * 1963* 3ant( Nüaseh^vféig /and „ iohacetraf;, ■, - - - ;/'31967)# Mansa (1965) refers to3#q/fa%t 'component'as " ' . 3 _' Ry ^ ' /3 3 ' . ' / ; ^ - 3 .R .'" / ' . Pierce'arid F#nstein..| 1905) carried 'out/further.'Studios . , - % .,:a3. into the relationship .betWe# cnlostral/and serum iirinunqglobulins# ' ' /' 3 ' Subsequently Pierce ( 1966) designated/' the slow and-" fa s t Tractions- 3//'80 euh"species/IgG. -.Gbhdh ahd3Mllsteih;XlW ih'-"'' Rr././■'their'roviow 'of; tbè iMunoglô'buHhs " sta te th a t it/dsRihd/Fo- - , \pbrtiona/of therespective'heavy' qhains. 'A i oh' aoboUht. for/the -3 ' /y.. s lig h t 'a n tIgëni^/dlfférenGe,.between Y0f'-'andX^« '.Additional ' ■ "'R"';' A a; ■" ' - - ; ■ . 4: - 3, 4 % - 3 - ' ■>"'"evidence' ofTKe-xaiffaronces'dnd.:,similarities 'between-them/was- -r '/y'- . /p rb d ü c è d ^ 'b y ’^ M i i ^ t e i h ànd ' ’' F 0 i h s t è i h - ' / / / - / - \ ; ' / / / / /_/ ; ;; / Aalund (196# ) shdwed ^thaf^the bpyihe'|gGs ' dross 1 reacted with .- human/10# 3He found :thê same, héjKOso SqhtentRand ' p l a s m a ;"/-'for /colbstral ''ahd''S##'.Fast-Tg^^ :/Klçkhdfén, Hammer and.SchqelR/ „ ^' (l968)/;fbuhd, Some/ evidenceRbf/ slight ;aiterA&tlon during ; absorption , : ■ „.of c o l d s t f a ' i ' : ^ / w h K ^ k / ' t h e y - d i f f f r o m ’serum Xj/yahd/XR#: :%;/. - 3 3/ '/ ;:3.3'"Butler/■(.19091 emphaiiSod' thé;iWt#qgëhêo.us/na 'both a.r -’rR / //, 00vihe 1 gës'R'-comp'ar'èd uo qti(er-’spëoièsR ,No' # % e s ts ' 'that' thé' ' .3 3/a■Rtwd subclasses may''be''an.^oy# simpli'#ioâ%'on#-'-/" • R%. - 3' \ /'■■" "■ 3 '3^ ,T %'#ï ce Car ri ere " ' ( 809 foqnd /ré#tiVely high, cbn i^ement fixing y/; À/. call:■'Sera/containing l i t t l e or ho olqW'tgOR/ Büllivan# Prendergast* -.yy'" nntihosR B11 verçtûih; and Toma s i /( 1969) found, tha t fast-.igG . ^ , R" # #'3/,ivàé/;alao the prihcipal WvlW iWunOglobuiln in.. saliw -and /.^ " R : 3, - ' ' - y yTearsvin addition to GoiqqtrWn.. 'Canaan. (1970) snowed that . . / / /■ thè,,metahqiiam/bf ■ 19% e i Al à# to, that;toi other mammalian- -/a/ ; ■ -I^8* #qro6s f àst IgG-appeared to deviate from the typ ioai '/"'■;y : :\/ypa..tterh# ' ^O.the#/,,# oke^uR/nr.indley Morgan" (1967) * ^Klaus ahd - . ' "JonesX1908), and/0LOpo-and G ittof-,(1969) have studied bovineIgG* without /rèférehce to the two sUhclaaees # : % .' /R J y ' ; / / ' ^ y / / R R \ . . RyR . ' , .. ^Johnson ahd Rierce (.195%) identified a small proportion ' \/r.o f iKaQrôgiohuiin3(^ ^ in opiostrql Wioy* Kiaraer (1963)',, ■•,■, :' /’ // .detected m$Xima.l''E.Goli- agglutinin' ac tlv ity /ln the pm,region ■ i; • - '/'/#'- ^and purified; thia; Tract ion 7/ -Murphy et 91 (1964à) noted the ' ' presence of X r IM immnpglqoUlin in bovine laçteàl secretions, ..'/;/% The same group "of # r k # s * ;:(MurphypAalitn'd and Ùsehold* 1964b) demonstrated Shd investigated^ the. héterogeneity of thiS;|)r6tein : ■- j.j# with which they -àÈsociàted c # ÿ l# e h t .fixing;.activity* /They/ / ;y' /" ■ also investigated its* jbhy## y*. chemical properties* (Muiphyy;- ' ' -, Osehold # d A'alund#/I905)" idontifyingAit on the basis that-it / "■■y‘- y prqdyold an arc on imunoelectrophoresis identical to th a t'o f ■'/,■■ r- human % .M* ■' JenneosR, Andaison /and -#ùgh (1965) and Gough*;: lennesq / ; '#3- Drucalla agglutinins associatedR"\_withUieyIgM%^ . / / ' v ÿ / . R / ' / ; : :R-RR/ '''Rr-r' /RTlqrcf ,(1966) and"-'(|907)ASUppprlod/the'9aHier‘eVidenca ^ R 'rr/'R .' , ; . Fresénçér^ 1 9 #- mqo%oglôbul|%ïn' bpvînb'=sWqR' ' Brindley ■ ’;:RRR3R' Morgan' (19671 ïndicatW 'tho importance of TgM'antibpdiis to ' r ./:/-3" ■RBrUüeiiàR Hammer, 'KlcMwfen'bnd-Wemm^g- ^ In th e lf studies'"R/■-■/'‘',3 ' of primary and aocondar'y'-responses to:R#tigen$' in # a tt ie *;3R/ :-R y 3 d i f f orontlatéd bétwëën 19$ Y glbbulln /and thé compohent* R ' ""3/;"/"r ; 'r'" They pqR$afpd3YM' and''l^A''#ti%b:b|e Klaus and Jones "( 1968) :RR:"/ '■■■"- / foimdRrâiëed, I ^ R |% I s in oxperlmehtali , -Rv"3 S. 33 / "'. inf#otions/|h^Gâlvès., The same vxjrkers"/(klaua,-Bfnhbt^ and Jon##3//R.:3"3:3 1969) investigated the absorption of ïgM3and TgG31h..'the -new-*-, 3 ./R, :3 bbfhRpalf #,33'Üsi0 th e .same- t'#hhiqiW'':R# ahd/G hfH tie (1969) -^l,,R 3' , ' ,. quan tita ted % immunoglobuli ns, in'hdul'-t plUsma a colostrum ; -r' ra''"' .3 3and^$ubséOuuhtTy:|n3néonatai calyeô/(Férihal©.ÿ'Chal'etléR McEwan*. -■/ryR/ 'R^ ^^ _ RHeher and,BoIï#h*. 1978)3 ’-Elco. and'Garriere: (1969) ,id en tif ied ' ' R R'3a-/'-'3^ ’;rR'&w 1 éyel9 :6f IgM inRthè sérum #nd colostrum ofRcpws; wlth-,non«3.R 3 / 3a-/:r; 3 /'b iih iO ai Johnrs Dibëa&eiR/AcooWingRto "Butler '('I969.)'3the antigëhiG' ,■ 3" 33 #/. ■ 3’ ' dfetihetlva; com )^onLntRof \Bovine'TgM'"appp'Ors to reside.; in: the Fc ■- 3 ' : rfragment which ihar#''n6Rantî#nlc, deteimihants v#th/bbviné IgÙ,^mp 1 ement/3fixàtlOQ ' sh o'# . 'thatR!#vlne_^tRc ,, t" ' 3cross react with a n t i human- 'K#'T.'-Ohain$« ' ' -T-R333 / ' - 3 - 3/ '%;3 It was origihëllyA^thought'thàt.oaïyes'wëre-W#^without Y globulin and that the foetus was unable to produce, antibodies* Hansen and P h illip s (1947) and P ierce (1955)- detected very low le v e ls o f y globulin in p re^ co lostfa l serum" R '" ’ ' ' , / ' 'R r /R lÿR R / / r R y R Ro f now - born ca lv es# Fennostad and Borg - Petersen (1957) found evidence that the bovine foetus ' can produce antibod ies against Lqptoqpira sax^oebiha* Subsequently the same workers (Fennestad and Borg -* P etersen , 1962) experiment e l l y in fected foetu ses andsubsequently detected antibodies in them* Schmid and Buschmann- ‘/ ' " .A " \ A ' : 3 , A y Xk R / ' c i R J y / v ’. ' Y ; R f /(1962) were unable to d etect blood group antibodies in the 311 . fo e ta l sera vMch they examined* As. a r e su lt o f the development o f sp ecia l h igh ly sp e c if ic typing systems for gen etic y g lobulin factors* Martensson and Fudenberg (1965) were able to demonstrate that the human foetu s may be able to. syn th esise small amounts o f IgG* KnioEeff, Runner and Gaeta (1967) found s ig n if ic a n t amounts o f Y globulin in about 40/ ^ o f fo e ta l sera examined* There i s thus : considerable evidence that the Toétus can produce antibod ies thus explaining the low le v e ls o f immunoglobulin detected in fo e ta l and prsR colostral c a lf sera by many vjorkors*J : . Other workers have in vestiga ted the b acteric id a l, a c t iv ity A q l pre-nata l and post natal c a lf sera* Turk (1959) suggested ’! ■■tnât 'th is ^ a ctiv ity was ■'■■due YtOAThd? in tera ctio n o f properdin and Cptep'lcmonT*. ,Mucchel ( Î90O) however, considered that....thOre" 'Wa;$-' in s u ff ic ie n t evidence for t h is , vM le Osawa. and Muschel (I960)R:a;/://:R:lt :a; //:::& < . ' - 3 ^ ; / T R A \ Y ; ' m : - . / 3 ' / ’ '''■ R R : 3 3 : y , : R/"R- A V 3: cMolüded thët/iA'tWA-#%6ënôe ô:fRm3#body$'prnperdW\mlgh^ ' / .//i-#/■-■ . -, , :;A, . .-, . ’ ■ A-A \ . : ... ■ V ' '. ':-% "'.'■ ■ ■. ,■ /■ ' ''.A - . '-V '/ A." . th e /.|b ë# d ricW 8 l'i.itre #/-% sligb i'degrW # -W nW .e (I9 6 5 )./'W # tlg u 'W W# ' A\R ArA.../-':'/: ' R ;R ' y R/V' :R'A\§R- , 3 the/prë#o|oGt#%30eruË^^^^ (KSRPf.)'* .#e,G O A #W W d'itto'% R /R •y u AJ,;A. sim il'ët 6 dWbordln'QW,.di8tlh^^ IgUI'Cnk ' H©"demnstr6tW'''\.3A -- - VR\R"A :_: V - T " 3 \ " " '''3 : '^ "i t s ’' h aoteH b ld àL ^ op ù rtieë* TW ë. reobh%|y#'ColHno# GarmlI# "R- : ' \ ' R' ' R. 3 - : ^ / ,.3 '3 3 / . 3 ' 3 " 3 : - / - ' / 3 / . 3 Rand' Jolh (|970}-:#nfi#W ,TW 'W A''Uv3cW ^^ - \;RA3-;3 - 3 3 - 3' V ;■ ‘ ;/j- - '"A ' . -A' A ' / , A ‘à; ,A . ' vR ' ' / %ô f -foetâ'i'end pr#W M '#'W l- Wrn*3'-it/MlL/lW #0foi^o' f.R y . /.A, ■' R/ARR' \ ' ::3R.^ y /srlién e01matlhg ;çWn#sA@s8pol#t#,:vâ^ ^ coloë&M% /'v A@ntil!3fJële,!3,',;t« ÿpLoïfhirte- ths J.svfeï''pf-' iriy'pro“G0 lO0iyëïA'';itaiïün$tjîokvîii|i,- ' A i■'A ' RJ'A ;; A' - ' \. A,. Ay . ,. ;«,, , ■.■, .- % - - ' ' ' ■*' A. vÀ -' -A. ' ' A AA”-' , :3 . /# . A ^ ^ R R A ' . ^ R R , R . . A ' \ \ A / ' - ^ - 3 - R . R.-m ie-ôf tW e^'Wôtral imuW'GloWllns@Apr0WWy\knt :qumtltmtlV0iy A - %- ".:; ' : R 3 : 33/ \ - R " 1333" ' -r . R / . . ' ^ R: V ; - R y . R ' 3 - ;; -^ A ' ' "; - 3_3. . y 3 3 / R- 3 3'R //' 'RRARL RR'. .' À : :/ ' '% ' ' ÂR^ ’ f "-À "-R 'nëow tôi .an lm le Im gèm ràl -âW' ' ^TW/cdW respoW-/# antlgmln' le ,# . 3 R' A'AR-AA'ÀayoilaWq''%m a '# # o # " 0 f $0iirO0e*AAi#qrr:and Êolÿpÿtëbh (1954)' '3■A’ ' ' ■■'' R. ' ' ' •■/ ' . ' .--A'-- / a. -" -. aa ; A '■ 'using ùWMe-. $8 ' :3A,/RR/u w r ë^nth eM'jtoÿl', (lëùôÿ/Àund- tMtR-fiVo^ut 0T âaiYoe ■ --R3 33h' rRR- 3- ' ' 3 \ / ' - ^' R RRA/RR3- / A \ - # ' R.R'#4\'n0t '###MRtoB#üGGM8dr$8i^^^^ - .'^^RR3. / / : / '3-, ' ' : ' / .^R\ A ^ ..R/ ' '^R .-" . '/ R^ ' /3 .^3' g≫|^R'ôtràinà{’ '■■ . ' a ;' ' ' , , à / ‘ a .. ARa R " . , A ■ R ‘.„_. /aa ' /■" -' ' • " ;■ "' A: " .28 ' # 3 # ë^ÿq'ppet ihlTW 'O ut o f oalWs.: '* : A ' ' ' ' . t ' ,'î ' ■, . 5 t ’r t, ^ '^r . - A: , j . ^In thrqe I^ypogqœaglobullmcmiG oalye^^y'antlWdleS' wëro 'présent-#béen TWe:.8rom.;.( 1986j - .W l# 6 ■ vlabiiÿ .Biadeipept le p lm W v iru s'd e tm tW - # ' t lb o # e s nt '21iW ÿ8 àM W # l l d r # p W ith ëhawW14‘ that .infants' given Salpon0lla"#0gen$Aat-Mrth d e v e l o p e d ' h i g B /, 3 ;3 ■ / t i t r# 3 u tT :to ; , i4 days* 'iABmi'th3aW;'in'grWà :(l#5j.AGoncludëd th a t -aR ' ■ iWunoldgioal"/qompet#nqe .dG^fitC a t d iffé re n t fgeaR/d^ëndiog:; # ; ^ ; ^ o q / t h e . ' G f i i a ÿ a e t ë r o f the . - o n t i g # # ’ '%%e# R 0 ; # Ÿ i o ü é l ÿ / ë ' " RA / # _ % . . : "R/" s p é ë i é s , . v a r i a t i o n # © w e l l / ' ' ' ' . ’ ■ , , / r ; r R R R r " R - a R . . . ' R R - ''R ' /Thé in h ib ito ry ''éffoot oh the productloh. qf/'antibodles Bÿ: R;; V passively acquired matexnni antibodies hqs/ been démonâtratéd R -33,R ■R; and qqmmentqd/upoh’by R: number of m rkôré# ;B rofc '(■ 1958)3showed'. ■/,;: R'r that .calvbs. bOrn/ tO Bindo4:)est Immune éé#:#idRnqt.TéBporid;-tôRÀ “a Rr . /' ^ Vacclnatloh.. under■ 3;:Whths;*,''A#bhfi#^ -;(19ô4:):/nbtédÂ -\ . tho strong Inhibition/o.f synthesis-/InRyddhg- r# b its y ^dMeyR/-/' RR - '^ahdRFitch- (^1964) found that pasétyçammbnisatibh’ «nhibitèd the -" :.:R'-'; primary : response in Rats , ifhl $ ; may/ bb _ a rélâtèd phenoWonoh»; '"R. /Thÿ'inhibitqry ..efféôt of/Cblo0t#l//antiW was dlscussêd by\ aRR ■,PÏ‘:&ce ( 196:1:) and #mlth -%nd ïh^wsv X-19'6$)# ■ Another approach /has'been tq /investigate Y globulin le v e l# In children the lowest '■ R'Y glqbulJn leve ls #e'found at # 3 # /.8 /# 8 # $ / (Ulstrom* :Tmith/.' ' r-- ;^- a '/ , 'and'Hcimllck R'i956;* qnd--lPut#h.»'Ry#Di43'"-The inlM ' %.■ çübséqiient risé: w erejéléa#iy demqhst%ated' by G ltlin and3 , r' r ..JanewayRLl95?)' ^and; cbnf.if#©#; '#obbq/ %iW6:)3' Wid#S'R"'Ocmdie.g r-a / A# -:À^ sk and;épo- METABOLISM . CASËS . : ~ - V' O i v . , - , , : .4 M'■:/''V'%"’ " 2 ' ; ’ Two types o f cage ■wefe/hëéid^//%//'■ //.' ' ' ' ' . '' Cages were required which would o ff a c tiv e ly bonfine the' . , , . , - ' , ' ' % " ^' ca lv e s , allow easy access and " 'ensurethat-àli'''UHno a.nd jaecéC'could he co llected * In, an tic ip ation o f the p articu lar problems!. ;;•Ï4-.- 4:-4 - • .o f c o lle c t in g faeces from diarrhoeic ca lv es , i t was decided to have cages b u ilt to s p e c if ic a t io n / (Construction was under#// taken by C*F. Howdon Ltd,* Glasgow),The f in a l design (see Fig* X) con sisted o f an JrinOr /: and outer chamber. The dimensions o f the inner chamber were such that the c a lf was discouraged from turning round. I t had three w alls o f 3Æ inch v er tica l rods at 6 inch in te r v a ls , a, door, at the front covered by ÿ* wire mesh and a ÿ* metal grid as base. The outer chamber had three s id es o f so lid metal sheeting*The top of. the ç%gé\was p a rtly enclosed by t # lïlhgéd Wli^ e mesh : 7tf/wide/, running the fu ll 1 oAgthvof the cage* / Each b h a d a ■ ’ ; /■■'/pair of moveable collecting trays# All excreted material produced % /'' / \ 'by: the calf:-i^s;-^eeîlcctcd'lh;e. b #k ct placed under ^bhr'trayv '//'v/' ' '■' ■ ,. //‘The Liay nnd'-'cage/could bewiehëd’'/çnd brushed'# w to/Achieve - . /% /##;:"r/v . • / ,;;/a Cûïïp]cte'''Coliectton^: /4^ :/' ' / W///-, " "/... 4// %/ //"/##/ . : , . . .>2.. ,^ / y : / , . : / ' ' / : / y / : /'.:, ; # #:////. ./': These ^hagéS'were :$;.s§'d''ih/./e^ mçre/%ah/twè oegés /.//; /////"' ' /' '/'^ ■/ 'were'required'W ;iiàeh i t 1fes/:heceeèa3cy té. separate4.hHne''.and . //' / /" - :/S//::/:^^ / / ' , ■:. ;/ The..&amew6rk^^^ made/fWm/l-*/'/.geivanl#^ . jelhéd-/.:/. '■ , , , , , , / - ' tpgetWr, Gascglgnes'yltd.'; \ ' / : . / / 5 / ' { 4//■•■''//' ./;'.■ //.""' ' It'/oonsisted/df >a'^'maih/outë^/fî^mé;:iàlch fomed; ^the'/support;■. // //'■■-''■'///' ' _ é.iov üï%/:ç'al fj.gr id Ihher ;cage//\It/aiac)' ,' 4/-prd'vided support for^m',.sldpihg-''grbdvad ^sheot, positioned/ / i"'" ' ; / ' ' ' ùnder/the/.:ihh cage//for the collection of urihO /^'/ The/yihhér //; ' /_ : / :Cag#'0OUld be adjusted for width and length as required* The 4//^" ■•,'■/:. ,;-gèTÿahioéd metal g r i d ? " o recLan mesh 1". by 2" was/ / / ' c - : / ma#by'W *M .aoid&Sphp,:/Gi^/ /? /? /'-'These cages wOre/UsQd in two different ways a- ' -. 'V r ' , / ;a« :|g a substitute'for the solid galvanised cages? by/ /, ://://■/' enclosing the cago in 500 guage polythene' .shedking''(Transatlantic'//'/^If'fil "I t =m i : : V: / /., . ■."" ■ ..■, . Ï ■ I I. ■ - . . ,' !. ' .s., ■ ■ ■ ■ /. , '.' ■ J ,) ■.,■ ■ ■- '“. ■ • ■ " , -. ‘.' _ '% " ..:r, J. .. -P la§#q$ - I s ie / o f ; :to # e / s h ë # îÿ f / / /y f ç - .:%ifC:;-f elisninivm ond tho outer frame in ewch s vay that the only .. ^ , ' - , ■ . , v ’:.,: :t; ;4j; . . access to th.o Calf >vas from the front of the cage* - Additional' ■ . access was provided on one side by making a flap in idie polythene.I??'• ■'"■m./ialhfprced hole was thon m&lo'of the lowest point in the?-// /#//%. ''polythene 0090'% whoxo tho polythono wan attaohecl'to the olminim . -/"'///- shoot# All excrotod motoxlal ûmlê thon bo col looted*■ ■ , . ' b# by using tho fnqcol' cbllnotion method dovolopod by do la'/I/*:': ■ :■:■• ^ ’ ^uonto (19Y0)* Tho urine was allowed to mn down thb nlumWmÆ - ^ . - _ , ;in#' $00008 wore oollootod %-long■.,:. ... ■ ,. . ' '/ " % ■ ■■/ ■ ' . ^ ./ /.; ■ .'•.:. , V,.pdiy#%eno bogs attached to tho roar end of the oéif/by, means bf,m ^ ' " ’ ' ................. -i * - 4f^ -:.4 ; , "furthérldotails see de lo FueMt#'?l|)70),':'Ï 4 ; /:â f, ......44 . . f 4 ‘; i vi? ;l.\; , i« r^-i 4 -# 4 ;3^."'l'ÿ'4 ' 4 . iôd iW 'ém cC h icrid e method o f M # Q r îë W ld ^ # ) as 4 '^ ;^,'.:, '}-% 44:%g; ^ . .. / : / . / ' '" 1#.:. (4 ,V'%dëooXlbW. by'ib#gi#%(4%9) vas used throoihbbii' , - .(1)iho stock _ solution ms itpdo # according to tho methodII . I'"- k% ~ ..■ . 11. . 4-' , V "' '"' " - > - ■ ■ '■ :4 ' . ' .4V,." 4 i-s , , " •' ■' , , ' ■" =/4-4|- 4 S , 4 , , Cof Vogel i l9U)* Por labelling, a 1 ,ln 350 dilution in- ./ . ' , / / M l # #n :& eW ily l##p arW *'-l . . 4 / : f i l l - -f4 -1 .4 . i I 'VIt.? -A/I. A -.. ' ÿ \ , -y ,, / ' . -4 ( / ' .?','4 I . r 'I I , , - 4 ■ „.,?•■/ .À %f, 4',.4% A 4':, ■ 4 .4 : / : . ' 4:4: : ^ ■ ', - 44. ■:,4A4•a:- ? ' - I , 4 . :4.., . ' XOCtoî. oi' n / j Oiycino in M /4 sa line vos prcpf-U’cd. ■' / . !. ‘ -V À , ' ' ' . ' . ' ' . 4 4-' -Buffer A woo (hado by adding NaOH td 40ml df / t W ' - "/.'giÿdlA'è iè lu t io n /tW m&TBaB#- $u#br..B '1sim ilarly io 4&1' @f Itbe ’ . _,-i/î. î' 4.4,.,;4 #"'■ ' ' ■ ' ■ \'‘: ' . -■'•■■ .■'-■■y;:' '4. . y * 4 4 4. : . ■ ' V , >■■. . . •.. , ' • y y y ' . 'i/.;- ,yy., ‘i-4' 'A'-âf'ôoMlôn of th'o p sïte î?# $ büffèiMIvâ-th;;’. ' . ' | . y./y|?4: ''0$ p r ê t# # /* . /' ’’ 4."i '4 4 - -4 :A:U .y ) ‘î i to i- lo M 'iy 'A - / . ■ ■■■■'. 4 '4 . 4 , , ; ;- I'-''■■'/'* ■ ' ' ' ■' - y I ■ 4 ■’ ■< -yl-'y, -, . 4 ; ' '44 s_ ■'■ .■ ■ ■ ' - , ' , y .,' / ' . ---" y-‘ . j y- ;..SM*’®!’' À iès'stosi^ in 'O 'M 'én ro'dueing agent'''#«e. tlJloswïpM te frèè),' M e r .W'$GhLllM#'''AM'Wl%!m@ was 'lw .*w sed 'Wm . . y l ? "’.' : ' X y- 4 ' l,'.. ■ : .........., l'y,; 4-i, . ' - y ;" 4^-/ 1 4 / . ; ^ i / i / ? , iI ' -?l' ' -■ . ;4' 4 .■ . , 4 " ' y 44 , 'y ., -f '4 ;.,($) a W B n a ci# 4 ‘ ; ‘ "'1.4: '., ' . . ' ' y 4, 4 " r ,, 4 ■' ■ ' ■ 4 4 . y - , - y , 4 , ■ y ■;;idotop# i n # ^ p W # t e W W , ^:lÿdW #/Âd0hIW W G ( #ah-%hat thè\hW bbr':of/. _ - .4' - %/ - / ' / y / . / /.y ' '% . '/J''# e n .b u ff# r W W i# # $ f e r A4(,,tW aW e yd lm oia# Bu#&" B) * /#.-îW - solution"wâB: ihd» 44.I?: ' ■4?'"'X Af€o% -^';%Wrôu^h/#K lA o l le d w #'"/ "o W fu liy p d u W -W to 'd ëmc aonW hlhg^moi;#.'. x. 'b o # n e éibùmln: to''\roduae--tW' # d 6 1 $ ic 'a e tiV # y 0 $ly l.' 444.4.■f' %to less #$n The prdtelri'was then. ?_ X- >- .-'• ...; dieiysed ip;25x 30 litre s W Isotdnio saline,/fpr-48 ■ ■ / - ■/;■ w.;^ /XS 4 , hôurs?-wlth''oneichéhgéh$ ca lln e*:' ' , /-?'x ; '. . . : ''/ y.-y.,Before Ih jeotlon dr/sterag^ ^^ ^^ ^ the percentage - ' ' -, > // /: /£ ;y V; (Bound activ ity ) was. determined: in 0 * Imi of-/the .label 1 ed " ’ " ' ' i ‘■ ■ /■,?._ p ro te in , \ .lt^>-waSypredlpitatèd/wlth'' 4,9m l 'p f / i# ' T rich loracetic ■açidxànd after'/standing' for # minutes at 4-%:%$. centrifuged*-.,;■ The :prboipatë' /suspended /in ' 5mi' d f dilute/MaOH-: In- a _ - ////- /? :£ !cbunting ;tubç'''àfter''the Bù|>ernat§ht/had been trpnsfered to.-"' ' ' ' another -tubèl -Both tubes ■ # re /th en ■ counted ( soe.-iection ' ./-: y. y. II :.a''7) 'ànd th ë ’porci^niage/bôünd/'h#iv£ calculated,#y coùnting/çàmlés of th e 'saïlné used for dialyèlë, # // - /'iX^^/'/v/'estimate 4 wàé'-al-sp' obtained ' of/the Jiabelling efficiency* , / /P '' .'Weighed syfinges' Were filled-, with the label!ed>preparation ^ XX\/x'“’on# 'syringe for each calf # and/dne.fdf a radi#activé, standard, " .. / / / £ The prdtMn ; was/then .injected Via'O'-Jugular ééLhètèr (Portland / .////X , plastics l t d , , ^By the#. Kent) -pfevi# sly introduced 'under local . ' / .- : :;/. ,XX.^ahaèsfhesia* After injection# the/catheter was flushed:With 7 / / / / / / '#sa lin e -' The ék%)ty syrihge was la te r rowoighed, X '/./ ' ;X’-V;'• , .. When i t was Intended- to carry out repeat Plasma’. Volume . ;determ inations, th e catheter was seoufed to the:heck o f theT . ' ' / .f . »'./ L . ' ,•• r, •: ^ • • •-42 3. -ça lf with 2" elàstûp lèst t one pièce forming Sypanel round the top of the catheter and:a second one holding, thé panel In position? by going right; round"'the ' cA lf/ls neck) , Tho^catheter was closed , with a nylon s tile tto * (1mm diameter) m ich could be subsequently withdrawn, ' During the period'-that;.th é-catheter was le f t in ■■-position? antib iotic cover was provided. The calves were given, daily Intramusculaf injectiona o f 600,000 unit$/3GK9 of Proaaihe, . Pen! ci 11 en G ( * My 1 ip eh ' * 01 exp » afeénfordj/é'nd'i^ ^ ofBtreptomycin/bihydrostraptonycin ( 'Dimycin# # Giaxo* Greenford)2:I, Method o f p repara tion , a^.aboye (Section I I C 1 ) * For- ' fu rth e r d é ta ils of, adm inistration sée/Section^V.,, ' ■/ . '3: ? l n j j # o M m ^ . f ^ f \ a , Hadioi odi na ted 'poly vihyipyrf o lido no (P, ¥ *P, ) was suppl î ed . by th e Eadio chemical .Qentre, Amor sham? with an average xmolecular tvelght o f 40,000^ , vb>; Chromic Ch 1 o r i d e ( 7 Or GÏ ^ ) as supplied-by Amer sham. *: Both of these were in jec ted i n , thé same way ©s the labelledplasma p ro te in s4 i . '/ x .4 #a, Pxy.P* with an average molecular weight: of 40,000 as above. b> PoVeP* with an average molecular weight of 160*000# from Amer sham. /-.x2451c . GrGlg# as above.^These la b e lled compounds were ca re fu lly syringed in to the back o f the mouth. tSee Section VII) .5. § » J j5 S ■ , ,a- B m â : ■ X-Blood samples Were taken from the opposite jugular vein using a 5 or 10; rnl vacutainer, (Bectdn# Dickinson & Go. # Rutherford, Hew Jersey# U.S.Ao)5 heparinised for plasma. Four timed blood samples ( for plasma ) 5 to 15 minutes p ost in je c tio n and additional samples were taken over a 14 day period? at regular in te r v a ls ..Serum samples were taken at the beginning, the middle and the end o f the experiment*b, ,The m ajority o f ca lves were kept in metabolism cages (Section II B ) for the c o lle c t io n o f urine and fn eces. /( i ) c o lle c te d t ogether,Using e ith er type o f metabolism cage* the to ta l excreted m aterial for.each 24 hour period was co lle c te d in a bucket* and where necessary the cage and c o lle c t in g tray and polythene sheet were brushed and washed down* V&ere calves were contaminOted with faeces# th is was scraped o f f and added along with the washings e tc .* to the co llec tio n *The c o lle c t io n was l e f t to stand t o ,a s s i s t in the breakdown o f the faecal m aterial* X25; Twenty four hours la te r i t was homogenised by s t ir r in g / with a metal rod and rapid ly pouring from one bucket to another u n til a l l the sediment was broken up àhd p a rtly suspended*F in ally j. iM le the c o ilo c t io n was being vigorously s t ir r e d , a 5 0 0 ml wide necked polythene b o tt le was f i l l e d and retained for / radio a'c'tiye' déterm inations ? , y-( l i ) ,/ '/>' i l 'The urine wa s co 11 edted in a buck e t , the Vb lume detem ii ned using a measuring cy lind er and recordedX £ \The weight o f faeces was determined by d lffereh ce* Small / q u a n titie s were hoinqgonised (-Ato-^mlxerV? | Crawley, Sussex)à known weight o f . water added i f necessary*/ la rg er q u a n titie s V o f f lu id faeces wore transferred to a bucket and a represen tative X'sample obtaihed'as.?abpve> ; 7 6# /Ffenaration o f Samples :îo t::X ^m M m / } .A ll c a # l 0 8 and standards were made up to the f in a l volume (5 or 15 m l, depending on the counter used, and the counting b p ttles/tu b q s a v a ila b le ) with 0*plH HaOH* "imi o f plasma was p ip etted Tnto. a counting b o t t le and made up to the required volume* V ■/■■ ^b ,:'^ T /b f urine was p ip etted in to a counting b o tt le ( and made :i;up jto /1 5 m l/ff req u ired /)* £ / X x / /■ . ;x26c . Eiomoaeniqed Excreted Material (MiXéd urine and faeces oir ju s t faeces) ; -^I t was prepared as follow s*^Approximately 5 or 15 ml o f homogenate was wlthdravm from the polythene b o tt le or the homogentSer. (Section IX C 5fo ( i ) and (11) ) containing th e represen tative sample, using an adapted disposabieisyringe* I t ivas transfered to balanced counting b o t t le s , the f in a l weight to be counted being determined by difference* I n i t ia l l y , four ,^ 1 samples were counted? but subsequently th is was changed to one 15mi sample* d* Radioactive StandardsA 1 in 200 or 1 in 250 d ilu tio n o f the rad ioactive sanple was preparedj and standards mqde up for counting as for plasma (see above)* . . . / / v 'Y* Radioactive Measurements. ^ (counting o f the samples)These were carried out in one o f two w ell ^ type s in t i l la t io n counters, ; . ,a* A manuaiiyx)peratod counter..(VHKOO* Tole^Garo, Glasgow) using M l counting tubes*b* An automatic By stem (/N uclear Chicago^ G*0* Searlo & Co*,High WycoW)#, % cks) using 5mi tubes or 15ml counting b o ttle s*%ore two iso to p lc M bels .were present in the samples, i t was necessary to count the samples a t tm s e t t in g s , as there was ,/ y . ...y: : -' ' / ..'applying the d ilu tio n p r in c ip le , i/ x7 /./"7^ ( i i ) P l a à m j ^ J P i s a o P é a r a n G e ,A i l / subsequent plasma sëmples were expressed as a percentage o f tho value, and p lo tted on semi^logarithmic 'graph paper, for the Wiqle o f the/expeM mental peridd#( i i i ) ■ App_q^pnt?_Rl'aWL-fr#;-# -7' (%-) S ter lin g (1951)The i n i t i a l part o f the plasma disappearance curve i s steep# la rg e ly representing d is tr ib u tio n , but a fte r t h e , f i r s t 2 to 3 days, the d ec lin e becomes linear*; The slope ca lcu lated ' by lin ea r regression g ives the* apparent plasma h a l f - l i f e (% ) expressed in hours or days # 7 1 t i e an approximate index o f "catabolism* .. :/ - .x , ■ , ' /I ( iv ) P i& W W W '.; ( s X r iln g y 195%)'-The in tercep t obtained by extrapolating the lin ea r part o f the plasma curve to the ordinate i s taken as in d ica tin g the fraction o f the to ta l which would have been present in the plasma at zero tim e, i f equilibrium between the compartments had been .Instantaneous* The fraction ou tsid e the plasma would then represent the extràvascular cOnpartment* We, can thus ; ,derive a ra tio o f the r e la t iv e s iz e s o f the two compartments , i • e* Extravascuiar ( E*V.) ; / Intravascular ( I *V .) . b. Total Excreted A ctiv ity -( i ) .Retained7A ctiv ity (Qr)?'tha excreted activity?.dn .0 doily basis V0 s'oalcuiatW and used to- derive -the -oumulatiVe. excreted ■■■■activity*; This was/ subtracted■' from ■'the'-’ injected ^dose to 'giye-.the a c tiv ity 'retained in, the calf* Theretained .ac:tlvlty expreacod ' as -a percentage of/the'Injected activity?'was then |?lottbd I# on'a -logarithmic ■:coale* ■-"^ ':-■■.■ ,. ( l i ) ' .. '■'■ ’ïhla tvas obtained. Wm' tb o’'fôlopÇ:Of the' retained: ■aotiylty (Qr)--.,*'curvè*' by^ linear regreeaion*'■ X/Ç0anpbeIl,/Cïüthbertcoh, Matthews, and McFarlano# 1956)X. The daily.-excreted'- a c tiv ity 'was; cxpreaoed7aa/'a7fraction'Of the to ta l intravaecdlar'-aotlvity.in the middle ,of-tho-' \ 'col'leotion period*;'The-mean yaluo- fbr the experimental period; ■mà then,.ca I Gti I at cd?,, leaving;'out/tho f ir s t 4B hours? when the plasm, ■activity 'i s .changing;,rapidly# ’ ' r ; ; no. carreotion^.was;made for any change'in"'■pla;m'ç ..vçlme -during tho/e#èrimmt*....- U v ) ■T0.tal/mdy..qata!%liQ..'.'Raie.-.;■ -The daily'eX créted/activity wa# 'aiao,.expr.eG#d 'Ca■'.a .fraction /of'thê/rotaihed aç (Qr)' in tho, middle of;:thO.:;co'll#tion''P^ mean .^va.lve: for,, the. experimental-poriodsWao then; oaloulated?",;.' "■■..'■'', 7;/^ '277.-always some overlap o f the rad ioactive spectra. Thus with i t was p o ss ib le to c o u n t a n d then /'^^I.X / . ' wi'th^ sOme / 4^ I^ overl ap ^ . (Simiiarly with ) * The ;' 77degree7o f overlap in individual samples v/as subsequently calcu lated,from the count ra te 0 f the I .or -Cr standard at i t s own' . ' -£x"X.';.7:X£;^/X4. , x ' '- ' ^/x'i25''.'^7'7 ' . 7se tt in g r e la t iv e to th a t at the I se tt in g and then subtracted.The part o f thej rad ioactive spectrum used for counting" ■ ■ i#e*. the^peak? for'7the- isotopds used w as;as/fo llow s(r ,:; ,v" " 1 .4 'I'l' :.V 4 ' ' V'Oi36 Me?' -I . : .■4..,; . .. -'I ; X / t / , - 4 : . - 4 : / ' ' \I 4 ' ' ' , ' 4 Oj.32 Mev / ' .., Standards and samples v/oré counted tw ice , the mean countrate being corrected for background. The samples were then corrected $or fàd ioactlve\d eoày Of; the isotope,# by ca lcu la tin g a decay factor ;from the change in the standard counts. A ll 7k y counts were expressed, per u n it tim e, and per un it volume or■7-.,. ,.-7 " e l # , K : 7 ; ■7 , . 4 ; : .. , ,:4 , / ■; ,8 , C alculations a. Plasma A ctiv ity ' ( iV '-Plasma teiume■7'; ; 7 X/' ■ Thé\re#uit;S.;Of'vthe four p p st- in j oction plasma - samples .(expressed as the lo g .„ ) were used to /c a lc u la te the **t '* value ;I: by 7extrap olation . The. plasma vulume vfâs then ca lcu lated by' 4/iK,» 3 0(y ) ExW vasculay A ctlv ttv (Qè)Thi é was obtained by, svbtraoting the plasma a c t iv ity (Cp): for the middle o f the c o lle c t io h period from the retained a c t iv ity (Qr) te ,7g ive7the'éxtravdscù lat/ Qe*/. /;..{v i) D lstrifoutloh (Campbell e t a l , 1956)'Where -the extravascuiar a c t iv ity Qe i s maximal? the sp o o if ic ,,a c t iv ity o f the la b e lle d p ro te in . in both eonpartments i s eqpal i$ê*. therei'la.- no 'net" tran sfer o f la b e lled m olecules in e ith er d irection# At t h i s ,"equilibrium" time (m) the ra tio o f the aXtravàsGÛlar/ihtravascUlat a c t iv i t ie s is ' equal to the7 \ // '■ ;; I ' v . / t I . . : .ra tio o f the s iz e s o f the two c o m p a r tm e n ti.o # at m * d , paqcpl: A c tiv ity Alone Ivihefe/d ëto M n o d ï( I ) ■ ■Cumulative faeco l output .Was ealculatod as a ■ , .porpehtage o f .th e ln jo ctec i'-d o sef/■ -( i i ) £piPg3i„.Sl#PftPg•'Paédai çiéaraüoo! o f plaéma (ml/day) wa? determinedby d iv id ing th e to ta l faeca l a c t iv ity in each 24 hour period o f c o lle c t io n p f faeces by the a c t iv ity par ml o f plasma at the beginning o f the c o lle c t io n period# : ■. 3 1 ^HABMTÛLOGYlo Packed d e ll Volume \ ' ■The packed b e ll volume (P*0*V*) percentage Was determined by thé ml oro-haiematocri t method * Heparin ised ca p illa ry , tubes containing the blood aanples v/ere sealed at one end? by heat or. with Cristaseal (Hav/ksley & Sons#/London) and centrifuged for 6 minutes in a mlcro-haematocrit centrifuge 7 (HaWksley & Sons# London), The pereontage P. G* V* Was determined from tho scale on a Hswksley Micrphaematocrit Reader#The to ta l serum p rotein concentration was estim ated by the b iu te t method o f Weischelbaum (1946).'3 , Albwain^lobul i n; i :A fe . ,a# V The serum p rotein fra ctio n a tio n s Wore carried out by eleetrophoréëis* C ellu lose acetate s tr ip s (Oxoid Ltd*, London) Were ca re fu lly spakW In barbi to he buffer (pH 8 .6 ) , l ig h t ly b lo tted and then la id across the supports o f the e lectrop h oresis tank/;(Shandon S c ie n t if ic C o,, London)» The samples (O.ODSml , o f serum) Gppiipd to str ip s; about four centim etres from the cathode end using a micro-pipCttè# A constant vo ltage o f 150 v o lts was thoh applied for one hour, Using a Vokam power pack/ ( Shandon S c ie n t if ic CO,, London)•The s tr ip s were then removed, dried in a hot a ir ovenand developed with 0*2 5^ Ponceau S. (G,T. feurr Ltd# ? London) in a 73^ iaqueous tr ic h lo r a c e tic acid? fo r f iv e minutes# . The s tr ip s were then scanned using a #irompscan recording d en sim eter (Joyce Loebl and Go* ? Gateshead), the r e su lts being expressed .as a ra tio o f albumin;to globulin* , /b#I The e lectrop h oretic method (above).was subsequently replaced .by the d ire c t speCtrophotometric determination o f Albumin (Rodltey? T965)^ ^ ' X- £ .x: 7;^ X /v 'B#7' IMMUNOCHEMICAL//TECHNIQUES 1# Preparation o f: AntiS^raA ntiseraw ere produced in rabbits* Hew Zealand White,Dutch or Rahere Ginger, weighing 2 to 4 kilograms*' For immunisation, a 50/50 mixture o f 2/& p rotein so lu tion and complete Freunds Adjuvant (Di fco Laboratori o s , D é tro it , Michigan) was prepared and em u lsified , manually7br using a. homogoniser (M*S.E*:, Grawley,;Sussex)* The rabb its were then innoculated with TO - 140 mg o f em ulsified p ro te in , given intram uscularly, in to one or both hind legs# A repeat in jec tio n was given 21 - 28 days a fte r the primary in ocu lation and the rabbit b led 10 days a fte r th is# Booster in je c tio n s w ere given a t 2 months in terva ls# The rab b its were bled from the marginal ear vliî-n and artery , using Toluene to increase the flow o f blood where necessary* By th is means, 20 to 40 ml o f blood was co lle c te d33:in universal b o t t le s , allowed to c lo t overnight a t room temperature, and the serum removed* A ll an tisera were stored at -20^C t i l l required#2 . : Adsorption o f AntiseraFor Radial D iffu sion (B eçtion II B 5) a;pure an ti serum i s required# To prepare t h is , rab b its were inoculated with a pure p rotein fra c tio n { prepared by ge l chromatography and / or ion-exchange chrom atography/Section i l l / ) . When the re su ltin g antiserum was te s te d by Immunoelectrophoresis (S ection XI H 3) antisera ra ised against immunoglobulins were found to cross react with other immunoglobulins* I t was therefore necessary to adsorb the anti serum i # e* to remove the "cross reacting" an tib od ies, thus rendering the antiserum sp ec ific#Adsorption was carried out as follovjs M a small amount o f a su ita b le p rotein or p rotein m ixture, (vjhlch w u ld p r e c ip ita te with the cross reacting a n tib o d ies , but hot with ''specific '*antibodies) was added to the antiserum and l e f t to stand at room temperature for one hour#. The mixture was then stored at 5^C overnight to ensure complete p recip ita tion # F in a lly i t was centrifuged at 5^0 for 30 minutes and thé supernatant retained* This process was repeated t i l l there was no further p rec ip ita tio n # The s p e c if ic ity o f the antiserum was then te s ted by immunoelectrophoresis and double d iffu s io n ^ - .34 - : '3* - K. .a# .I he..-.Aat .i $ff,™For routine examinations the antiëérum used was e ith er anti-bovine-serum or an ti-b ov in e-g lob u lin (with some a n tl- aXbumin a o t iv ity ) . The an tisera were stored at -20^0, in 1ml a liq u ots and thawed as required#Two i i t r e s .o f barbitone b uffer were made up as fo llow s 520#62gm sodium barbitone3/684gm d ieth y l barb ituric acid .8*2 gm sodium a ce ta te/ 200 mg . m erthiolateand the volume made up with deion ised water*To propare the agar for Immunoelectrophoresis, the follow ingmixture was. c a r e fu lly b o iled t i l l /a l l the agar had d isso lv ed : .250 ml foarbitone buffer (a s above)700 ml deion ised water ‘10 gm agar (D ifco Labotatoiries, Detroit?; Michigan)100 mg ,mer th io l ate , .The hot agar was poured in to universal b o tt le s and when cool? "ostored at 4 C* Impregnation agar was made by b o ilin g 1 # o f agar in 500 ml o f water p lus 100 mg o f m erthiolate and 1 mi o f glycine* The mixture was cooled and:500 ml o f water added#/ '/£ :£ ' -- , f ^35I t was si6red/atv4 G ?;7tlii;rëéuired# , Thé'• alcohol cleaned élidé© ■were prepared by bo iling in /th e imprégnation,.'agar and-.than'"dryingà.t";ropm(tempe#ture#i'''' - . ; ■'''r' ■' /■■;'>;'7'^G#■■( droycloh?-/3R^^®y/) '^-équipm,eht v;as Used#-7‘The / : -Ke lid e s were placed in the ",holders 'previously fitted - in to the - le v e ll in g tablé* :■ The table., was adjusted sc that the s l id e s ' W0 ré /co n |ïlé te ly ;'level • ,'i0..-,mi;o.f:-thé liq u id 'a g a r , m s poured/ onto éaôh se t of three slides.# - ' A fter coèilng...a .central - trough - 7/%/ (. 6*5 mm-% l lm ) and tm-% elis X i.l œ . Id dlameter ^ î-and "9 nm'/apart 'were ou t in . each..sl.ide* ■■"■^ Thev;wells ware removed by , suGtion£and-,.theh f i l l e d with antigen using/a' m icrolltre.syrlnge*/':,'T he/slide holders.- were then placed''in-. theYëledtrophoresld-âank? ' the, c ir c u it completed w ith/batm an Ghrdmatogpphy paper ,( Ho 3mm) wicks,-, soaked in the barhitone buffer and the current -applied' - ' 7^ , for 1 hour a t '250■'volts?;, using a /Vbkam p o#r/p ack (Bhandon S c ie n t if ic London).; ,. ; ■,. ... / ■ ' - .:d .,v S fS tiÉ 2 fl,....K ' / ■ ' ; / . : 7 ' ' ■# e n ë ièc trd j^ o reç is was c6% lete? the central troughs4 were ..removed with,''hn;7ag©r/knife; àj#--filXed-'wlth/ântiaorum«'' The ' . ' slides,w ere.,then incubated/overnlghS'at-JT O*. In #ome cases? 'i t 'Was/,'neces$#ry to 'allow the 7slidés7té-vdiffU se for up to 48 hours* Thd s l id e s 'were examined a t ; th is stdge and the r e s u lts ■36recorded* In some oases were aieo photographed*oé; A W m " '£ - ' ...'; %The s lid e s wore washed in sa lin e for one hour and thenovernight (fo r 15 hours). They were f in a l ly washed in water for at ledSt an hour# The washed s lid e s were then dried in an incuhator at 87 C# The dried s l id e s were stained with saturated. AmidoBiack for 5 minutes and then washed in an A cetic Acid/Methanol; w ash till the/unbound dye was removed# The s lid e s were f in a l ly dried ’and stored* .•4» Pbuble^-âdffuslon : y : / 'a . ' y ' y i y - 1';The anti sera usedw ero the same as those used in Section II E 3 ( see above"),* ■ ;/y % '■ .-v15 ml o f liq u id agar ( as in Section XI E 2) was poured ..in to a F e tr i£ d ish and any surface;bubbles removed* The desired pattern o f w ellé Was then cut using one o f two cu tters (Shandon, S c ie n t if ic Co*, London) to provide a ! central w ell for the antiserum and s ix peripheral w ails , for the samples#/ £ The w ells ware. carofu llY ;.fiiied using f in e Pasteur p ip e tte s , and the p la te incubated for 24 to 72 hours, at 37^C, to allow the preG ipitin lin e s to deyeiope*0 " J7Tho p le to s v/ero. washed in sa lin e for 48 hours? with three changes? and in water for a further \;48 : h o u r s T h e gel ; m s ' then e ith er removed from the P e tr ie dlshi.and dried on a g la ss slide? , or dried in eitu? 7 The dried gel was then .stained with saturated Amido Black for 15 minutes? unbound dye then being removed in an A cetic Aoid/^iethanol wash* Tho s l id e or p la to v;aa;iTnally dried .’and photographed or stored*SingleThe method used was b a s ic a lly th at o f Mancini ? Carbonara and Heromons (l9&T)*. , ■ 7 :: .a*. ,Before the estim ations could be carried out? i t was necessary to determine a su ita b le d ilu tio n o f the sp e c if ic anti Serum and o f the samples ? where necessary# To th is end? ^ t r ia l' runs were •aarriod..outj using a divided P o tr i.'•■/.dish*Four samples lik e ly : to represent 7the:'7range o f concentrations to : be encountered in the sanples to be subsequently te s te d were allowed to d iffu se in agoic containing the undiluted antiserum and three d ilu tio n s o f it* .I f at an apparently optimum antiserum concentration? as judged by the s izo and c la r ity o f t|ie p r e c ip itin ring? one or more sam^Ues wore too concentrated? 11*0*' gave, large rings? vdth a r isk o f overlap? these wore a lso . su itab ly d ilu ted * / , ^ . .7„. .- ' The,:89ar,;.wa5,prepared 'Bectioh XI E 3b, except thatthe concentration o f : agar wàs increased frorn to 3 ^ i . e . 30 gm ' o f agar were Used# I t was stored in 15 ml a liq u ots at 4 C, t i l l required* Prior to pouring the p la te s , a b o tt le o f agar was melted and then placed in a water bath at 60^C. The antiserum was d ilu ted ( as above) Up to a final; volume o f 7 ml and placed in a water bath a t'55 ;.C. . •:■'//£'.. - 7 /A g la ss plate: wUs prepared, by b o ilih g i t in impregnation , :.a'gar':'(Section .TI/E 3h ) and allowing i t to dry# A second p la te was coated M th s il ic o n e j e l l y and the Perspex mould placed on top* , The top and; in s id e surfaces o f the mould were a lso coated with thé je lly * F in a lly the inpregnated p la te was placed on . top. and the tvb p la te s tvith the mould between? clamped together* %/ . When/thé ' tenp the w te r baths were constant ,? ml o f the agar: was added, with a warmed p ip e tte , to the a n ti- . serum# The m ix tu re ,/a fter gentle shaking w^s then p ipetted ; ca refu lly ;ihtq t h e / A f t e r y l 5 minutes,; v^en th e /g e l had s o t , the mould and the s ii ic o n ise d p ia te were ca refu lly removed* 12 or 16 w ells (2 mm in diameter) were then cut inagar/plugs removed; by su ctio n * /;//■C^ J & f f u s i ^ / 4 ■ ■ . ■ ; //'■'■/. . . 4,//;/£''y''7 - .\ p#4pl;bf each samp 1 e ; was applied tvith a micro 1 i t r e syringe39(G riffin â George? Best K ilbride) along tvith a standard. and three or more d ilu tio n s o f the standard* Wien the samples had d iffu sed in to "the gel? one o f two p o ssib le proc edures was carried out ; - y: ' ., '( i ) The p la te was placed in a shallow tray and covered v4th 2 cm o f liq u id p araffin in Wiich a few cr y sta ls o f thymol had been d isso lv ed ; The p la te was then incubated at 37^0 t i l l d iffu sio n was con^lete* D iffusion/w as Judged to be con^lete when th e .la r g e s t p r e c ip itin rings did not change in diameter betv/éen two readings?. 24 hours apart# . The diameter o f the rings was measured using a magnified sca le (Telemaster? Verebes & Go*? London) ? tv/o readings being made at r igh t angles? and the mean value taken.as the diameter*( l i ) the p la te was p lated in a moist a ir t ig h t container and l e f t a t room temperature t i l l d iffu sio n was complete*d* M A m m ■ -The washing prod edure was carried out as for double d iffu sio n (XÎ E 4d)v To prevent the agar from cracking v i^en the s l id e s were drying? a wet f i l t e r paper was placed on top o f the s lid e s and a hole pierced, with a noedlo over each w ell (Yamamoto?Hashimoto and Yokoyama, 196B)* The.plate was dried at.37^C and the dry f i l t e r paper carefu lly removed* The p la te was then stained*, washed and dried*. In some cases? the diameter o f th e . rings was again measured*- 40.e . C alculation o f R esults y;-'. ' .A ll the r e su lts were expressed as the square o f the ring : . sr.: K: ' K ' V : ' . ' ■ . / • , ' K " ; - - . - •diameter ( d ) . : For the standards? d w a s p lo tted againstconcentration (m g/m l ) , determined by the b u iret method(Weischelbaun, 1946)* The concentrations o f th é other samples werethen read from th is ; graph# Any sanple above the range coveredby the standards was d ilu ted and then the quantitation repeated*F* . EVANS BLUE (T aS24) PLASMA VOLUME' The proceedure/followed was sim ilar to that used for the la b e lled protein* Two: syringes were f i l l e d and weighed? 2 ml (o f a 1$ so lu tion In sa lin e ) for the c a lf and 0*5 ml for the standard* A 10 ml pro in jec tio n heparinised blood sample was taken* The c a lf was then in jected v ia the jugular catheter* Four 5 ml heparinised blood samples were taken from the opposite jugular v e in , 5 to 15 minutes post in jection? using vacutainers* .The samples w ere centrifuged at 3?0p0r*p.m* (M.S*E.? Crawley, Sussex) and then d ilu ted to ,2 or 4 ml with sa lin e , for reading*The standard was made up to 500, m l.w ith .sa lin e and 1 ml o f i t added.to T ml, pf thé pre in jec tio n plasma* This waé d ilu ted to 4 ml m th s a lin e , i f ,required*During the s tu d ie s , tv/o methods were used to read the,■samples t - ■ / /.» the prepared 2 ml samples and standard were read in an.41 -E.E.L, Colorimeter (Baird and Tatlock, Glasgow) using a 607 f i l t e r , ■ . ■Subsequently, the colorim eter method was replaced by the use o f a speotrophotometet', the SP 600 (Pye Unicam, Cambridge) « The prepared samples, th is time d ilu ted to 4 ml were read at a wavelength o f 620m^ i#the plasma volume v/as calcu lated by applying the d ilu tio n 'p r in c ip le , a fter the concentration o f Evans Blue at *'t^" had been determined by extrapolation ( the plasma concentration being expressed as the log^p)*Gt STATISTICAL METHODSThe s t a t i s t i c a l methods used were those described by Shedecor (1956) and Bishop (1966), The exponential phase o f the ra d io a c tiv ity curves was calcu lated by lin ear regression , analysis* correla tion c o e ff ic ie n ts o f fe were .accepted.D eviations ; from th e mean were expressed as the Standard Deviation (S .D .) and the Standard.Error (S*E.) o f the mean. The "t" values were taken to be s ig n if ic a n t at p < 0.05*- 4 2SECTION ll ïTHE PREPARATION OF BOVINE FAST laG (loG- V IMMUNOGLOBULIN■.... ■> ■ : ■ - 43 -INTAODUCriONThe in v estig a tio n o f the d istr ib u tio n o f Bovine Fast Ig6 in the neonatal c a lf required the preparation o f a pure protein fra ctio n , undenatured, in q uan tities su itab le for la b e llin g with the iso to p e s ,o f iod in e , i . e . up to 500 mg* Pierce (1966),Schultze and Heremans (1966) and Weir (1967) give the follow ing methods for the separation and p u r ifica tio n o f plasma proteins*S alfinq o u t. vAich may Be combined with other methods Electrophoresis - in part superc ;eded by ion-^exchange chromatography U ltracentrifugation - d iffer en tia tin g between proteins with d ifferen t sedimentation values*I so la tio h o f SDeciflo_ Antibody - from complexes o f antigen and antibody M61ecular Sieve Ghromato draohv ïon^exchange Chrdmatoaraphv D ifferen t combinations o f two or more methods may also be used. , ' ■ ■■:Thèse methods were then Considered in the l ig h t o f the three requirements already stated above* S alting out was excluded because o f the r isk o f dénaturation* E lectrophoresis and u ltracen trifu gation are lim ited by-the amount o f sample thatprphiema'Mth tegard,\t6 obtaining ' ■..a # f # c lé # iy \p ù r X as'dooo; salting" out ) . ■;, ,The,-Iso la tio n ■of '^ .ee lfic ; antibody,,carries ;thô risk ,'o f a ltera tion ; \ ,,o,f,th0 -moleçulôs and '^;#'s p é c if ie ,antibody was.not'..reqyired*':XTba'' objoct w s\'tq -ob ta ih 'an.- IgG "ff action containing ".'a # b l e rangé o f -specif ic' antibodies * Theobjéétions. raisëd to. those ■ methods. o f '■.. ' - ;.y ; ? - - . / ' :-separation-, a r e 'exh'auëtlvo but; they ,are- s u ff ic ie n t to { ' V-/ ?.,.'■y j u s t i f y eoneehtfatl^ .on" 'the' tm T^naining 'methods t h a t ,of' molecular". s,ioÿe. '-chromatography and lo-n-oxchahgO ehfomatography.. .:• ; i^üÀaiecular-'„siove effects ‘ in gels wefe-'.obseryed by,.Synge ' -- ' and-^Tiseiius (1950)/;,In agar t u t ;b à#e and':Ruthven''(-1956)»:.. using' starch," were'^the 'f ir s t to dm dhstrate th at the'.technique o f im>Xëëüiârysièye- 'chfamatpgraphy- can be,/applied,.to ' bio lo g ica l:' coapounds*. .The''' breakthrough. In - .th is i eld- came # t h / the development bf \ a ;new'’;type of'?gol.prepared-by cfosS*llnking doxtren to -form';, / a macromd.leQUlàr!;het\vork'of-great s t a b i l i t y (porath’and F l o d i i i , . : / i # 9 ) * -''They-coihéd th e name%* - gel'-'flltratioh\!^;/to' d idtlhguish ■/'/'■■ I t IronipthQr ,form'S';of chromatography» 'Many'other, .workers . however prefer-the, term molecular: sieve phromato gr aphy (Anderson,, / and Stoddart^^/1966) ■/ , . ' ' ’ ' / 'y /.# e . gel'.ihtroduced by 'Porath'et/.al:.(l959) had a high ' : ; / c-■’ '' degréë^^^of/pîosa linkage-and thus le n t i t s e l f to-- the -desalting ■' ...;pf :-proteins':'(Porethi/' 1 9 # » -"Flodin ;1'96I# Granath'/and Flodinp 1961». ''-''Porath'-lWs-' ond-.-Andrevm- ^ .;'Poratlvet,--al {1959)-..considered ' ,- , ■ . / - ,that m olecules were separated according; to th e ir size», but a y à r ieÿ o f other factors-w ere subsequentiy shown to in fluence separations# Aromatic and h eterocyclic compounds were found to be adsorbed (G elotie» I960) but other e f fe c t s could be prevented by the use o f su ita b le buffers* These factors were f urther in v e s t i gated by Gl axer and Wellner (1962) » Marsden ( 1 # 5 ) , Andrews (1966) and Stevensen (1968)» 'AS a re su lt o f the pioneer mrk o f Porath and Flodin» a Whole range o f doxtran g e ls with vafying degrees o f cross- linkage were made cpmmercially a y a ila b lé under the trade hame o f Sephadex. They are designated G-10» G-25» G-50» G"75, G-100, 0-150 and G-20C) 1b order o f decreasing cfoss-lih k age and increasing water régain and fraction ation range#porath (1962) reported the a b il i ty o f Sephadex G-200 to d iv id e serum in to three main fraction s (peaks) on a molecular weight basis# M m ilar r e su lts were obtained by Flodln and K illander (1962)» G elo tte , Flodln and K iliander (1962)»K illander and F lg d ih ( 1962)» Fahey and McLaughlin (1963)» K iliander and %gman ( 1963) » lAen separ and 195human blood group antibodies» K il la h d e r (1963)^Hogman» K iliander and Johansson (1964)» K iliander (1964)» Rpthstein (1965) and .Boskes and Thompson (1963) vdio used the G-200 separation to d iffe r e n tia te between normal and pathological sera*//- 46 ' -, The experimental d e ta ils vary frôm/v^orker to worker but are u sually based on the pioneer w rk o f Porath and Flodln (1959) » Porath (1960)» Blod|n (1961)» f lo d in and K iliander (1962) and Porath and Flodln (1962)* In la t e r reviews»;,Andrews (1964)» . Selby and Maitland (1965)» Andrews (1966 j and B ailey (1967), the technique was further developéd*Since la th e and: Buthven (1956) used starch columns to determine the molecular: weights o f : co lu tes , other workers, have used agar (Andrewsf 1 9 6 2 ) 1 polyacryl^^ (H jerten, 1962) -. and Sephadex (Whitaker, ,1963»/iAdkètt^4?64V Andrews'1964 ,and ' ' Andrms 1 9 6 5 and 1966) for th ië puippse^ This p articu lar ^ -sp p lica tib h , developed oh ah': empirical b a i l s , has been ■; hai^ ered by disagieëmént and lic k : o'f ;:Undirstanding'- vd.th regard to the fundamental méchaniemB %iqh bring about the; molecular, , s ie v e e ffec t* ^two main th eo ries hove evolved, both having th e ir adherents* Lathe and-Euthven (1956) f i r s t suggested that; the - extent o f penetration o f 'the stationary phase depends prim arily ;bn thé molecular weight o f thé splUtes concerned. This idea , vas developed fu ff her by Porath and Flodln ( 1962) vho cepncluded that, Sephadek àctèd as an aImost porfé ç t molecular s ie v e , the : optimum # h g e .of m oleculaf séparation for;a partiC ulaf gel; being determined by the' extent -té f^ich' I t sw ells in the so lv en t.. - 47 ';.,.,vThls i s determined by thé amount o f crpss'^linkagei Porath ■;'(■ ':l:;:'.'r;\/(I963)'''éiabéfaté theory':Was put forward '/'-:-by:,Poisoh\'('196l);/ &0m his- work'wiih;.granuiar ' 'f'-’ ■; : :;-K:;a#rv:.that'''ihe':^^ ^ i S "léfgêly' dependent; On: / / : " d ifferences' in -'d lffh sio h -cp effio ien t , Expérimental. évidence-hae;beèn!püt 'fqfw afd-ihsuppprt o f "both' concepts* Exclusion ' :'i:;'from'':thç\matrix; wasy'f by Squire ( 1964), Laurent and ;; ■ ; ; '■ Kiliandef (1964) and AndreWÈ/f 1966) * ;$teefé/and Ackers ( 1962) ■ ■. euppbrted the; cohoept of restricted dlfftision in: ègar'gel columns■; ; éhd' êUbsequéntlÿ Ackers (1964)7 ^ ^results' suggesting '/ : --'V -’ 'that both- theories should ■ be considered» depending. oh the - ■ 7 '" i; degree VO f:;*croSs' linkage'' o:f : thé-.^ë^hadex# ;/ In'reviWs'-of "thesubject Anderson and Btoddart (19,66) ' and Ogston :( 1966) .: ' . ^ cone1uded that practiça1 app11catien had inevitably outstripped7-: ' theOrétlbalv treatment/-and;, th a t”it ' was-a'çüfoject/Of vgreat '. ■-"Vvi. : d i f f i c u l t * //' - ;/7;. / J:./;Apà'rt''from..%ep.h^dex,':other'i.media/haye been'/used%#'^ ve*g# %/ ■, ■r.polyacryiamidC 9el;7and/poiyvinyL-;aiQohol ■ f Laurent 7and % <-V : . Kiilander, 1964) »/ glass (Pederson 1962 and Haller 1965) andagaf, and starth X as above )* ~ ^ ■7 ; "./ 7/r": %e: spp 11 cation; o f /ion-ekchahge chromatography to th e■:, sep4;ratlon;;.of7proteins,:.was;:iimit0 need for à very/ - .-large:..süffacéiéreà;:and:t the protein molecule: / / ,( fo te r so n ##|Sqber.»::i959': apd ^ Huisman I960, P e s te r s . 196Q4Band T isç liu s 1967). . Porter (19M ), referring to the use o f Ainberllto IRG-50 re s in in p a rticu la r , coneiuded that ,tho method was'Confined to proteins most r e s is ta n t to dénaturation* Bowman (1955) and Bowman and Westland (1956) used Dowex 2 ,An a ltern a tiv e technique using Calcium Phosphate as adsorbent was reported by Svvihgle and-T isellus (1951) and developed by T ise liu o »■ Hjerten and Levin (195Ô); and Hj or ten.,(1959)* Ion-exchange :ma made generally applicable to the separation of, p^rbeino by the introduction o f su bstitu ted c e llu lo s e s , anionic (DEAB) and ca tio n ic (CM) exchangers '( Sober, and Peterson» 195^ ) thus - reducing the r isk o f changes. in the configuration o f the protein m olecule, (Peterson, end Sober» 1956). Thoir app lication to plasma p rotein s was describod by Sober»,.Gutter, Wycoff and Peterson (1956) and.Sober» Wycoff and Peter son (1957 ) . .The i n i t i a l observations v/éro included in a comprehensive rbvieiv o f - lon ’^ exchonge chromatography by .Mooro and S te in (1956). Subsequently Sober and Peterson (1957) % and (1958) and Fahey»'McCoy and Goulian (1958) developed the technique-further# The I n i t ia l work was reviewed'and consolidated ;by Peterson and, Sober 11960a)..T h e'th eoretica l cots'' were ' also:'considered by Pet or son andE)Obef ( Sober. and Peterson» 1950 ; Pbtorsop and Sober » 1960a /and Petereon and Sober» ;1962)» .as..well as by Footers (i960)* '7- v , / . , ; - . : ' . 7" , r ' ■ ' . ■ * 4 9 -The foroés cau&ing the binding o f the plasma proteins; to the c e llu lo se particX.es bearing the opposite charge arq .? electrosta.tiO j although othér forces are also believed , to p lay a part* The bonds can be broken or disconnected by a small change in the:pH or an increase in the io n ic strength .The former reduces the nhmher o f charges on the protein molecule -''While''th e^ la ttér v4Ti7 decrease the e ffec tiv e n e ss o f the.7ex istin g . , ’ bonds by promoting t h e ir 'dissociatâdn* Thus Sober and :; Peterson (1958) found that» as a rule» proteins v4th high iso te io ctr ic points ,were eluted earlieri A number of factors,“ pH d^endént d ifferenG es, the presence o f conplexes, sp e c if ic - ; ion*eleG trbstatic a f f I h i t y - for the adsorbent and the s iz e o f the protein molecule^ may tend to a lt e r ,th is situation*; ■ . The tm typ es : o f g.radlent described by Aim, Wi Î 1 lams _ - and T is e l iu s , (.19%)' were-- both : applied by $pber and "Peterson ,.. ' (1#B ) » , Gradient e lu tion provided ithe reQUirèd range o f e lu tin g power :%vlthout sharp changes in the cpèpositioh o f thb eluant.; Stepwise elution on the Cther hand gave a poorer resolution and ^7, made interpretation hazardous» with thé appearance of extra ,, p;èak's. Xt requires prelimihary separations to determine the7 ;'.best c o n d i t i o n s ^ : / / ■\7^': Gradient: e lu tio n was inprovèà by the introduction o fthe 7varigrad (P eterson 'and.Sober 1959 and 1960b) which enabled60small changea to be made/in sp ec ific -p o r tio n s o f tho gradient# .The. equipment; was 4RT>.: o^ved''by Peterson'"and Rowland (1961) and used by-Peterson and Chiazzù (1962) to stan'clardizo-.-the. . . , technique# ...' ^ , ' ' . ' % . /Many vprkors.have used DEAh c e llu lo se for t h e .separation of-serum»: plasma:. ancVpiasaB;protoih;'frac-tiohs* '.Humphrey and porter ( 1957 ) obtained 4, y globulin components * Sober , and -.Peterson .( 1968) ' recovered gcg o f the to ta l y g lobulin in the broakthmugh peak# - Those find ings'ivw o'confim od .by-other - m rkors (Fehoy» ,McCby'and Goulian*:1950,-Fahey and Horbett 1959» Pragor » ■. %oer» ' Hi 11 » # iT lams' and .Ooernor » ' 1959, 'and Fahoy. ' I960) * Sober e t s i (1958) and P èété fs .(--Ï96b)- eonsidorèd'th at the : method vas most su ita b le for' largo .poalo separation under mild ' conditions* Btradss» Kemp»:'Vannier Wd Ooodman (1 9 # ), incroasod , th e ir ylold'-by carrying, o u t . in i t i a l p rd olp ita tion with Sodium Bulpliato# Other tNorkors . scaled i t do\m for ;dlagnostlc;pulpqaea>; ' (Fahey o f a l 1958 aW Progor ot a l 1959)$ Cooke, Tombst-\Weston»,Soutèr-and,)4Q0ld9arr(l957)''Obtainec1-’ - conplox peaks Wien separating serum#Pure V globulin was obtained by 21 f f ; and Lospalluto (1959)» Loyy and. iîober (i960 ):and-.by Stahviprtiv (1960),» vàp 'used a:: "' / : .batch prooeeduro# Work by Fahey et al (1958)» (1959) and (I960).and Fahoy (1960) on the separation o f 78 and 198 -51 -;Erttibodlé$ ,was confirmed, by Goodmanj dé St Gyr,. Clave and Grabar (I960)» Goodman, Eabèy and.Malmgfan (i960) and Albert and Johnston Ç196I)* ,Carroll » 1961 applied the technique to thé Reparation:of , colostral proteins using stepwise/elution, foliowing thé methbd:of Vagüchl»; Taraasuk ahd,Hy for separatingmilk^prptelns,; ^ V: 'V-- '■ : : - . ' '''-Gelotte,: Flodln and,Kiliander referred to the introduction / o f DEAË Sephadex A-5Ô ‘which becàme commercially available in 196i * I t has about 8 times the capacity q f DÈAÉ eellulose# . ;With the recognition; o f the co%lex nature of the immunoglobulins, Vaerman » Heremans and .Vaerman (1963) » found that i t was only pos sib le to obtain 50 ^ o f the seruifn IgG in a pure ; form , in the breakthrough peak. #odm # ( 1964) while reviowing ■ the method, showed how .in it ia l Ammonium Sulphate precipitation could increase; . , ;X -the y ield substantially# > % ' : '■ 7 The tw methods o f sepa3ration,fmpiecUlaf, sieve chromatography and ion;exchange chfomatography have both been applied to the séparât ion o f bovine serum7 and colostral v\hey proteins; by a number of Workers* ., 7/ ■/ '■/ - 'Ga:;:rpll ; ( 1961) uaed/bEAB cellu lose and st^ w ise elution .. as performed by Vaguchi et al (1961) to examine; çô lostfa l v^ey ' and # e y prepared from drying o f f i secretions .and m astitis milk*V/ :■ / ' "5-: / : Z : ' ■ .Thé f i r s t four peaks a l l contained immune globulins#Murphy et a l (1964a) Used two d ifferen t gradient systems to compare the Gamma globulin compohents o f bovine serum with those o f bovine colostrum and dry secretion# Using DEAE Sephadex A-50, th e ir f i r s t péajc consisted o f IgG ^ and the second, eluted as the gradient predominated was m ostly I The same workers (Murphy, e t a l , 1964b) to prépare bovine IgM» used a Sephadex 0%00 column ( 7 x 70 cm ) separating bovine éerum into three major groups on a molecular weight b a s is , as in man. Subsequently they (Murphy e t a l , 1965) applied both techniques in an in v estig a tio n o f the physical heterogeneity o f Bovine y - Globulins^ They showed that th e ir ion-exchange chroma to grams o f bovine sérum Were analogous to the chromatograms obtained with hùman serum, co n sistin g o f ,3 main peaks* : The f i r s t (breakthrough) peak c o n sis ts o f unbound protein in the void volume (IgG^ )*^ ^ The second peak, larger than in man, contains é fa ste r migrating IgG (IgG,) and transferrin* The th ird peak contains albumin and other plasma proteins*\ ....m u m m m ,,,..AT • ■ ; ■ . ‘Blood was obtained from a three day o ld Ayrshire b u ll c a l f , supplied by Dr# I# Selmon, Department o f Veterinary Medicine,- U niversity o f Olas^w# The c a lf had received colostrum and at 40 hours p ost partum I t s serum gave a Zinc Sulphate reading (MoEv/an, 1968) o f 36 units# The blood, in 100 ml beakers, was allo^ved to c lo t at room tenperature# The c lo ts wore ^ringed’ and the serum removed the follow ing day# I t was centrifuged at 3000r#p#m* and the supernatant s p l i t up I n to '20 ml a liq u ots in universal b o t t le s . The to ta l protein coneeritration and A/G ra tio -wore determined, and the a liq u ots stored in the deep freeze at -*20^0 u n til required*:R esults o f p rotein concentration determinations*T.P. 7 .4 gm/lOOmlA/b ra tio . ;/0#38Tills wa s prepared, from fresh/ bovi no co.lo strum ' supp 11 ed by Dr#l#8eiman# 50 ml batches o f colostrum in large centrifuge .tubes'were.-raised to .37^0, In; a water■ bath and 0 .7 ml o f commercial rennet added (J#Jaap & O s., Glasgov;). When the54casein had se t and the whey was/beginning to separate, i t was centrifuged at S000r*p#m. enabling the whey to be separated from the c lo t and frCm the layer p f fa t vMeh formed on top o f the ïfthey* The whey thus obtained; was stored in 20 ml aliq u ots at I t had a Total Protein o f 9*6 gm/lOOmland an A/G ra tio o f 0.21*B. y4o,lecUlpn Sieve, ..QirornatogrppThe medium used was Sephadex G-200, supplied by Pharmacia (G*B.) L td ., 75 Uxbridge Hoad, London W.5. The sta rtin g m aterial i . e . the sairple, was neonatal c a lf serum (a s above). A O.IM T ris 7* hGI -, 0.5M Nad buffer » pH 8 .0 was used for sw elling the Sq^hadex and as éluânti ^ Sodium Azide (0.02%) was added to prevent b acteria l growth (W illiams . and Chase, 1968). 25 gm o f Sephadex (ah excess , ca lcu latedfrom the v/ator regain value o f 30 to 40 ml/gm) was slow ly added to 1 l i t r e o f buffer with continuous stirr in g# . After 24 hours at room temperature, the buffer on top o f the p artly swollen gel was removed, along with any f in e p a r t ic le s . More buffer was added and mixed with the g e l . The process was repeated at 48 and 72 hours. A fter the f in a l mixing, the gel was ready for packing the column.A ll column work was carried ou t;a t a te«perature o f 8 - 10 0 . This temperature was maintained by the use o f awater jacket and / or a 'cold* room.- Cùïum PreDaratioh " ^. , ; ; / The glasSkcolWn» 100 cm by 3 cm diameter was f i t t e d with .tW rubber buhgë, p ierced by perte/cohnèctors (Portland P la s t ie s Ltd#» Byihe, Kent)*' At, th e bottom qf the column a pad o f g la ss wool had; beentrapped on top o f ih e hung, thus providing a mesh which v^uld re ta in the Sephadex but allow- free passage o f büffèr in to tubing attached to the conhector ( w i t h : l i t t le or no dead sp,aoe> ,i*ev space which m uld allow Wixing o f the p2t)texn fractions» as: they came o f f the colunin)^ r / A l i t t l e pre-sw ollen Sephadex was then poured gently into the cblumh) care being taken to avoid a ir bubbles# I f there ■ . was no :evidence Of leakage o f . the Bcphadex, the colùrah was ; _ completely f i l l e d and the le v e l o f the, o u t- le t tubing adjiisted so ih à t thé head.of pressure on the column did not exceed 10 cm*, Over a period o f 6 to T hours the column was p e r io d ic a lly topped up a fte r removal o f the layer o f buffer vjhich formed at . the top o f the column, due to gel packing down. When the le v e l o f the ge l in the columh had ceased to fally:the column was connected to à 1 l i t r e reservoir containing more o f the eluant b u ffer , at constant pressufo* The le v e l of thé reservo ir and thé o u tle t Were again adjusted and the^column l e f t to flow under gravity overnights I f the le v e l of the bed had; fa lle n overnight, more Sephadex was éddèd to bring the'' .height o f the Sephadex bed to about 90 cm; F in a lly , before applying the sample, the top o f the bed was s ta b ilis e d by the. application o f a p iece o f f i l t e r paper, cut to f i t the column; .2 . Column Opération ' ' 'A 4mi sample o f the c a lf serum was carefu lly layered on to the top o f the column, a fter removal o f the buffer present there. When the sample had passed in to the g e l , a layer, o f buffer was s im ila r ly layered on top. In both, cases, great care was taken to ensure that thé top o f the bed. was not d isturbed.I f th is did occur, a small g la ss rod was used to thoroughly mix the sanple With the Sephadex at ,the top o f ,the column and \Mhen i t se tt le d down again buffer was layered on top , as before*. The column was reconnected to the reservoir and the o u tle t connected to the Fraction G ollector (L.K.B#, Croydon, Surrey), : The e fflu en t from the column was then co llec ted in 4ml fraction s oyer the next 46 "^ 72 hours. The to ta l duration o f the separation depended on the flow rate vhich was;found to vary from column to column.: While th e o r e t ic a lly , once a column i s se t up, i t can be used time a fter tim e, in p ractice i t was found th a t a fter being used 2 ,or 3 times» for no apparent reason the flow rate gradually became so slow» that th e column had to be emptied and re-packed, O ccasionally columns accidently ran dry, a lso n ecess ita tin g repacking*F l o u r s 2SEPHADEX G-200 CHROMATOGRAM OF PfEONATAL C A LF SERUM (4ml) (BED VOLUME 3 x 90cm and FLOW RATE 15m l/H O U R )0 .5 0605030 40 70NUMBER O F TUBES* / 5 7 * v;the 4 ml fraction s were removed in batches and th e ir protein content examined at SBOmp in .an S.P* 500 spectrophotometer (Pyè Unlearn, Cambridge)* they were stored at 5 C u n til t h e ‘separation was complete, A graph o f absorbance against e fflu en t volume was p lo tted . The separation was continued t i l l no more protein could be d etected . The chromatogram (Fig 2) obtained showed the ch a ra cter istic 3 peakis (see Introduction)*The fraction s containing the top o f the middle peak were then se lec ted by estim ating the amount o f overlap, seen in Fig 2 as the shaded area, e ith er side o f the middle peak. These fraction s were then excluded and the remaining fraction s bulked* 3» G oncentrationof FractionsThe fraction s were concentrated by making use o f the osmotic p roperties o f Sephadex 0-25 (F lodln , O elotte and Porath, 1960, and Deutsh, Levere and Levine, 1968), the Sephadex (1 gm per 5 ml to be concentrated ) was placed in a Buchner funnel, on top o f a m oist f i l t e r paper* The bulked fraction s from the column were poured onto the Sephadex v h ile s t ir r in g , u n til a l l the d ilu te protein has been added* The protein was then allowed to drip through in to a flask with a sid e arm*A fter f iv e m inutes, or vhen the Sephadex has become semi‘•so lid , negative pressure (using a f i l t e r pumji) was applied to remove: the r e s t .o f the concentrated p rote in . To minimise the production o f bubbles the pressure was ca refu lly con tro lled . The process .was repeated u n til the volume had been reduced to about 10 ml*The concentrated fra ctio n was then examined by Smmunoeiectrophoresis and Double D iffu sio n , prior to storage at '^ 20*^ 0 (see Section xr'H 3 ’ and.4 ) , ' ' T ■ -4*For subsequent separations the p o sit io n o f the middle peak! was ascertained from the p o sitio n o f the tjrouÿüs on e ith er sid e o f i t , and the equivalent fraction s kef>t for concentration.,A further forty one G-2Ô0 separations were carried out in the : manner in d icated #)%The.resUltS; o f the ImmUnoelectrophorotic examination -were"OS f o l lo w s ) ' ;'19' samples .gavo'-^ono l in e corresponding to Fast.-lgG 15 sanpies gave no lin e ( i .e * negative)' ^ - 6. s.amp’lo s 'showed Fast'Tg.G contaminated with otherp lam a p rotein s -' 2 samples showed contaminating p rotein s and w F ast’-igQ ' : :The 15 negative s a b le s were then re*examined by Double D iffusion* Ton o f them gave one lin e i .e * a s in g le protein ■ ' fraction p resen t, ■mc>;st lik ely ' to be Fast IgG* The other f iv e showed, in addition to th is l in e , evidence o f contaminating proteins* the samples that were^^negative on ImmunoeXoctro--Examination o f the grouped IgG ^ Fractions (a fte r concentration) by iramunoelectrophoresis and Double D iffusionGroup Iimuno electrophoresis Double D iffusion% A . Pure Trace impurityPure Trace impurityG B light impurity Trace iap urityD S lig h t irrpurity Trace, inpurityH : Pure Trace impurity■'■■■ F ■ i: Pure Trace inç>urity' G : - S lig h t impurity ContaminatedH : Pure Not tested^ , S lig h t impurity Not tested .59phoresis were probably too d ilu te*A ll the samples were then grouped together (se e Table 1 ) and concentrated to a volume o f about 10 ml* The resu ltan t concentrates were agsin examined by im iunpelectrophoresi s »-|he r e su lts were disappointing and rather variab le . Of the bulked "pure" fra ctio n s Group 0 now showed d e f in ite signs of . other protein fraction s being present and Groups D and G showed a s lig h t impurity to be presen t. One of the impure groups was now showing up as a pure fraction* Vflien Double D iffu sion was carried out on the same sangles there was the suggestion o f a trace impurity in a i l .o f them, except G, which was very d e f in ite ly contaminated* The concentration o f the main coritaminatihg protein appeared to be poised at a le v e l v4iere i t s presence might or might not be detected by Immunoelectrophoresis, whereas i t was always detected.by. Double D iffusion* Thus, although re su lts from individual separations had suggested that i t might be p o ss ib le to obtain a pure fraction; using .G-200 alpne, th is was c lea r ly seen to be im possib le, . ■The presence, in thé middle G-200 peak, along with the Fast IgG, o f the contaminating protein indicated that i t had a sim ilar molecular weight (assuming sim ilar ly shaped m olecules, and no "adsorption" o f e ith er molecule by Sephadex)60 -Provided there was some d ifforenco Ih molecular w eiglit, i t might be p o ssib le to gradually separate the tvx) protoins by rechromatography, u n til a s u ff ic ie n t ly pure preparation was obtained* /'part-.from the- time and labour involved in = such a proç.eduroÿ i t might a lso su ffer from an Increased r isk o f dénaturation and a lo s s o f protoin each time i t was concentrated* I t was therefore decided to try and separate the p rotein s on an io n ic b a s is , using ionrexChange chromatography* ; ;Ç». .iRRrJüfihagfla. „The medium used was Sephadex DBAB A*50 supplied by Pharmacia Ltd * $ 75 Uxbridge Read, London W,5*The bulked IgG fra c tio n s , obtained by molecular s iev e chromatography ..were grouped: 'E, F. and H in'Croup i -C %.B and C in Croup 2■ G and I in Croup 3 V, ’The three groups were then concentrated dom to about 20 ml j^ th Sephadex C#25, to be used as ■■starting m aterial fo r ’ the ion-exchange s#ar'ation ,- -A 0*OIM PhOf^hato; buffer,'pH 8*0 'was Used to sw ell the DBAS S^hadex and m th e in i t ia l eluant* Subsequently an approxlmately Tlnoar gradient was applied , using equal volumes- '6 1of thé: 0*01M buffer and a Phosphate buffer, pH B»0 'i*o* 'a molarity gradient, a t constant pH# . The l i t r e reservoirs, containing 500 ml of the respective buffers were, conneoted by a siphon so that a drop in the level of the iovj m la r ity buffer^ connected d irectly to the column, caused the difference in volume, to bo made up by the high molarity buffer* $lx gns of ‘the Bçp'hadex was èXdwlÿ added to 500 ml ■ of the stertiïitg buffer (0#0lM) with contimous stirring#TW buffer was then changed 3 .times in 24 hours, to ensure donpiôté; equilibration# The'method er^loyed for owolXxng the gol vas basically the same as that used for the G-200 ■ Sephadex* '• After thè-fin a l mixing.'.the gel was ready for ■packing tho'-column* ' ' ‘.1*.The glass column, 40 cm by 3 cm diameter was assembled, as for G-200* Tlio' column was packed in a similar allowing a head of pressure up to 30 cms* It complete In . 2,/to .3, hours, 'to. g i v e a f in a l'v e l# e o f 30 cm-by 3 cm diameter* For Other ''details,, see BectionTt-B 1, above* '2* '. iïhe-sample waé applied fmd after the buffer had been layered on top ,'the column v/aa conWotdd to the 0*01M phosphate reservoir, and to the fraction co llector. ThoFigure 3Immunoelectrophoretic Comparison of Bovine Fast and Slow IgG FractionsFast IgGB.S. Slow IgGFast IgG Slow IgGB.S.A. B. GA. B. G.A. B.G.B.S . - Bovine SerumA. B.G. - Anti-bovine-globulin (with some anti-albumin activity)■ - 6 2 - . , : v - „ ,, ,b u ffe r ,in th is reservoir was stirred continuously. The 5ml fra ctio n s were te sted for p ro te in , with ;5/& T*C*A,, ,as they were co llec ted and a s l ig h t break through peak detected .V)lheh th is peak had been e lu ted , the m olarity gradient was applied , to e lu te the bound prbtein* Prior to t h i s , considerable shrinkage o f the bed volume occurred, due to the increase in m olarity resu ltin g ih it ia X ly in a reduced flow rate* When the bed volume s ta b ilis e d the flow rate increased, and the second.peak was e lu ted , i t s presence being detected with 5%TeC.A* A ll the fraction s were then read in the SP 500 at 280 1% and a chromatogram p lo tted . The third peak was not c o lle c te d , the separation having been stopped lAhile the remaining protein was s t i l l bound to the Sephadex.6 n c e n tr ^ t io m ^ o f .6 ^ t io #The small break through peak was concentrated with G-25*The second, much larger peak was divided in to two p arts and each part concentrated* The concentrated fra c tio n s ,5 * 10 ml. were then checked by Double D iffusion and Immuno-, , . , ■ , v ■ : ' ■ ■■ . ■ ■ ■ ■ , - . .e lectrop h oresis . Both peaks were found to be IgG fra c tio n s, the f i r s t designated Slow IgG (IgGL) and the second Fast IgG (ig G ,) , sëé Figure 3# Groups 1 , 2 and 3 'were a l l separated and yielded the follow ing concentrations o f Fast IgG; r Group Î •:* , 20 ml o f 2 * 5/tiFiqure 43 .023 &DEAE SEPHADEX A -50 CHROMATOGRAM OF COLOSTRAL WHEY (20m l) USING A 0 .0 1 -0 . 5 M PHOSPHATE BUFFER GRADIENT (pH 8 .0 )(BED VOLUME 3 x 30cm AND FLOW RATE 40m l h r. GRADUALLY CHANGING TO3 X 23 AND 80ml hr)F ra c tio n s ftivUif; a P re c ip ita te w ith T .C . A.G rad ien t applied .F ra c tio n s pooled, co n cen tra ted and and exiim U iedby Im m u n o e le c tro p h o res is (along w ith 1 ).G rad ien t d iscon tinued .2.01 . 0NUMBER OF TUBES63 -Group 2. 10 ml o f 1*6%Group 3.- *«.*«k«4 «»:*••«« \- 10 ml o f 1 *8%4* ': Repeat Separations -Although th is method yielded Fast IgG su ita b le for la b e llin g with the iso top es o f iod inej i t suffered from the major disadvantage that the whole process took up to three months to m mplete# The lim itin g factor was the 0^200 separation* ta k in g ,2 t^o 3 days for the separation o f only 4 ml o f serum# To overcome th is d if f ic u lty ) two changes were made. The order o f chromatographic processes was reversed i . e . the DEAE separation was carried out f i r s t and the startin g m aterial, was changed from neonatal, c a lf serum to co lo stra l whey Four DEAE separations,were carried out as described* using 20 ml o f co lo stra l v^ey instead o f the p artly p u rifiedIgG fraction (Fig 4 ) / Hie large second peaks were c o lle c te d ,concentrated separately ahd examined by, immunoelectrophoresis. The 10 ml concentrated fraction s were then separated on Sephadex (^2Q0 as before. This tim e, the f i r s t and la rg est peak was concentrated and 03 Nansen (1970) assumed that i t would be d i f f ic u l t to avoid a s l ig h t admixture o f the two IgG subclasses when separating serum containing IgG^. He therefore prepared IgGj ,^ from serum vÿiçh did not contain IgO^, obtained from a cow with lymphatic leucaemia and chronic purulent broncho pneumonia ( i . e . the breakthrough , peak was absent when the serum was chromatographed on DEAE Sephadex A^bO). However* using the method suggested above, i t should be p o ss ib le to obtain a s u f f lc ie h t ly pure Fast IgG "(igG^) preparation, using adUlt bovine serum as the startin g m ateria l, instead o f the c a lf serum and co lo stra l whey used , in ..thèseastudies*;"" . %.V /: \ ■ ’ /; ; The application o f molecular sieve chromatography to, the preparation o f Bovine past IgG (IgG .)$ fa ile d to y ie ld a s u ff ic ie n t iy pure fraction* When the IgO ^ onrichéd fraction s thus obtained were subjected to ion**exchange chromatography, the m ajority of, the contaminants v/ere removed. The method o f preparation .was in^)roved by the use o f co lo stra l , vdiey as the Ctarting m aterial and by carrying out the ion*"Gxchange separation f ir s t# A method had thus been found that w ill provide Fast IgG, su ita b le for la b e llin g with the iso top es o f iodine*- 6 6SECTION IV THE METABOLISM OF BOVINE FAST .IgG.. ' iNTm puciim ! -The ex isten ce o£ an extravascuXar. *■ circulation** o f plasma proteins has been knovm for a long time* Levtf ^ (1927) showed that injured c a p i l la r ie s . became highly permeable to 'p rotein s *Krpgh (1930), vM le recognising the perm eability o f l iv e r and in te s t in a l oap illar ies ,.con sid ered - thé other ca p illa ry b ed s■ to be inpermeable to co llo id s* , F ield and Drinker (1931) and, Drinker and F ield (1933) suggested j on the, b asis o f lymphatic s tu d ie s , that c a p illa r ie s u n iversa lly -leak protein* Landis (1934) considered that the presence o f blood protein in the lymph makes i t U nlikely that the ca p illa ry endothelium i s t o ta l ly inpermeable to plasma proteins* He suggested that h is finding that 95% o f the plasma proteins are reta ined , could be most read ily explained in torms o f the présence o f a few large pores, perm itting the leak o f albumin and g lob u lin s, in addition to re g io n a l.v a r ia tio n ,/ Krogh (1980) found that c o llo id s fo ile d to return d ir e c t ly to the' c ircu la tio n , but in stead returned via the lynfphatiGS# Subsequent work .‘concentrated on an in v estig a tio n o f these "pores**, using d ext ran s o f known molecular Weight a n d is o to p ic a lly la b elled plasma p ro te in s .The re su lts é f .Wassermah and'Mayèrson (1951) usingalbumin leave no doubt that- in healthy animals, the c a p illa r ie sare p a r t ia lly permeable to albumin, some more thah others*Bollman (1953) and Pappenheimer(1953) wefe o f a s im ilar .6Bopinion* Manery (1954) # i l e accepting that perm oability had been anply demonstrated, suggested that workers varied :in their, views with regard to the quantitative sig n ifica n ce o f th is "leak". -Grotte (1956), G rotte, Juhlin and Sandberg (I960) and Mayerson, Wolfram, Sh irley and Wasserman (1960) produced evidence for tvx) Sets o f d iscre te pores, operating completely independently* However, they disagreed as to the upper lim it o f the small s e t . Mayerson et ai (i960) found évidence o f thé passage o f m olecules up to a mol. wt. o f =250,000 in contrast to pores o f ;35 to 45 A (G rotte, 1956) which, allow passage o f m olecules with a 30,000 to 40,000 molecular weight.Subsequently Renkin (1954) and Renkin and Gariick (1969) found 35 to 4 0 A pores ( i . e . a steep f a l l for m olecules with a molecular weight o f between 10,000 and 40 ,000). Above 100,000 (TO^A) Renkin et a l , 1969, found l i t t l e d eclin e which could be accounted for on the b asis o f increasing molecular w eight. In the d iscu ssion o f th e ir paper, a mol. wt. o f 80,000w as given as the upper lim it o f the small pores. There i s thus some doubt about the s iz e o f the small pores *With regard to the large pore system i t i s generally accepted that m olecules with a molecular weight o f 412,000 to 500,000 o f larger :Wiil pass through (Mayerson e t a l , I960, Grotte 1960 and Renkin and Gariick 1969).‘ ; : : ■ : • _ - 6 9 - , , ■ ■ . - ,Opinion varies as to vvhother th is system i s rspfesented. by pores v^ich raight bo d etectab le by electron microscopy - (Mayerson e t a l , 196d and Goltran, 1967),o r ,in fa c t takes the fom o f vesicu lar transport» as dmio net rat éd by Jennings,Marchesi and Florey ( 1962 ) » Abramson (196?) » Bruns and Pol ado(1968) "and Shea» Karnovsky and :'Bos'set',( 1969)* The process» "cytopem'phslQ** » has been considered in great'depth by Jennings ■ and Florey ( 196?)# ' 'Vv: Likewise »V,lt h as■ been suggested that the. ■sma.l 1 .pores 'ore in functional terns# mi crop 1 no cy to t i c v e s ic le s and the inter-' coU u lar . juhctions (Ç oltran, 196?) as : demonstrated using horse' radish peroxidase (40,000 mol* v4i*)* Reeve and Chen ( 1970) >.ere o f ; the, op in ion 'th at .vesicular transport i s th e main" pathway ■ by Wilch the p lam a p ro te in s enter the in t e r s t i t ia l flu id# .The mechanism i s thus independent of water in flow and i s able /to maintain the largo mass o f in t e r s t i t ia l protein in the skin: ;and muscle» so that only slow changes occur in in t e r s t i t ia l ■C.Ü.P. Thus expérimentation i s slow ly g iv ing a more accurate . p ictu re o f the ro le o f the perm eability o f the ca p illa r ie s*■ Valuable infprm otionhas;:also:been'obtedned.from the '■ 'in v estig a tio n o f bulk: movement o f plasma p rotein s and th e ir ./ , . - ' Thus , G ltlln and Janemy (1953) ■ quantitation in. t i s s u .showed that viiion. the plasma m s depleted there mO a rapid moymB'nt ( s h if t ) ' 'o f p rotein back in to it» from th e \e x t r o vascular7 0p oo l. Rothschild». Bauman,.Valow and Beroen (1954) found, that the skin and muscle con ta in .h a lf o f the to ta l extravascular albumin whereas the l iv e r contains lo s s than 1%. Four to f iv e days were-needed for coir^lété eq u ilib ration o f a l l the exchangeable extravascular albumin in the skin and muscle* Friedman (1957) broadly confirmed the conclusions o f Benson,Kim and Bollmpn (1955) that eq u ilib ration o f albumin i s rapid in ; the. l iy o r arid-the in te s t in e s , .intermediate in muscle and slow in the kidney, spleen and lung* .Friedman found that skin eq u ilib ration yms incomplete at 12 hours * Taylor, Kinmonth, R oliihson , Rotblat .and Francis (1957) using iso to p ic a lly la b elled plasma p ro te in s 'in jec te d subcuta neously, found no evidence o f d irect : return to,, the ca p illa r ie s# They, concluded that the lyR phàtics,yjere the most Important mechanism for the transport o f protein from th e -t is su e f lu id s ,; back to the blood. Dewey, (1959), e s se n tia lly found no d iffereh ce between the d istr ib u tio n o f albumin and y globulin in the t is su e s (although both p rotein s varied considerably). Using homologous and heterologous p rotein s in the rat he found that 90% of the extravascular plasma proteins are present in the muscle, fa t and sk in . The large lymphatics represent 2 to 3 % at the most.Our knowledge o f : thé nature and d istr ib u tio n o f the- 71extravascular p rotein s i s lim ited and mostly ind irect# A few workers have su ccessfu lly obtained and examined samples o f i n t e r s t i t i a l . f lu id from d ifferen t t is s u e s (Maurer, 1938,G it lin and Janem y, 1954, Dewey, 1959, Fricke, 1961 and Creese,D*Silva and Shaw, 1962)# The presence o f a l l major plasma protein fraction s has been demonatrated, although not in the same proportions# Drinker and Yoffey (1941) considered that the only controversy was with regard to the amount o f protein in the in t e r s t i t ia l flu id*Shultze and Heremans ( 1966) eïïphaslsed the bound nature o f th e in t e r s t i t ia l flu id* I t co n sis ts o f tm p a r ts , a semiso lid gel phase in \M ch the water i s firm ly bound and a f lu id sol phase contained in in clu sion vacuoles embedded in the matrix# A major con stituent o f th is matrix i s l ik e ly to be acid mucopolysaccharides, the properties o f viiich have been in vestiga ted in v itro by Duddeke (1960), Laurent (1966) and(1969) and H ellsu ig (1969)* Laurent ( and Reeve and Chen,197Ô) has suggested that as well as acting as ion^exchangersthere w ill be an exclusion e f fe c t very sim ilar to thatoperating In molecular sieve chromatography* Thus highermolecular w eight,substances lA ich w ill be largely excluded,w ill pass through the in t e r s t i t ia l f lu id more rapidly thanlower molecular weight so lu tes vMeh as a r e su lt o f d iffu sio nw ill be present in the in t e r s t i t ia l f lu id at a higher concentration*■■ .The q uan titative signlficanoG o f th is exclusion in v iv o , ,' hos -ÿèt to be detexmihed# ■ the in te r o t ia i f lu id appears ..to . move Bs i f restrained by in v is ib le :channels running aiongsid© conneotivo t is s u e f ib r es (Schuitro and Heremans, 1966) p o ssib ly as a consequenco.of tho exclusion e ffec t*The in v estig a tio n o f plasma protein metabolism using is o to p ic à ily io b e iled plasma proteins and in p articu lar the use o f the iso top es o f iodinebihas led to the development o f L a number o f mathematical modoXs a l l o f which assume extras vascu lar,,d istr ib u tion * >'Hash ■model;,makes certa in -assumptions - about one or both subdivisions o f the A c la body d istrib ution »■ # i c h may or -^may -.nbt‘:be ' true' under - p articu lar erimenta 1 '' conditions* Most models are an over**.slmi>lificetion but may give, reasonablo r e s u lt s , all' errors involved being l o s s than = the erro rs ,involved in , the quantitation i t s e l f * These'models have been d iscussed .by Freeman and Matthews (1958),. Jarnum^C1963), Anderson (1966),:;Donato (1966). an as the S ter lin g d istribution* V ntil sooh time as i t i s p o ss ib le to allpVLfor a l l the additional variab les (e*g* by conputèr sim ulation) i t : >viH hot be p o ss ib le to $ub*"dlvlde the extra*- 'Vaseuiar 'compartment*/. I^wever), in relation: to the measurement , - o f the e ff lc ien o y o f absorption o f the, co lo stra l immunoglobulins, i t i s not necessary to ;tak e account o f any slow ly exchanging ■, \ parts o f th is ooïipartmeht ( see Section VI ^ Disbusslbn )*The Campben method thus becomes aoceptable and the S terlin g method* was also used, as i t does not require the. c o lle c tio n . of, urine and fa eces , and i s thus much m ore,easily applied*I f the re su lts obtained by both methods show a c lo se agreement, i t may be p o ss ib le , in future^ stu d ie s , to ,use the S terlin g .; method alone* ^. ^ An iGssential requirement for a study o f th is kind i s as u ff ic ie n t quantity, o f pure' IgG;^ , ( see Section I I I , for the , method o f preparation ); for la b e llin g with thé iso top es o f lodihh$,'',''ThG Concept o f p u rity with regard to p ^ te in s has : developed a s .new techniques have been applied* , Thus jihen76P ir ie (1940) examined the problemo. o f purity in re la tio n to large m olecules, a fter d iscussing the.various c r ite r ia in current use, he concluded that purity Was a concept vhich has no meaning except with reference to the methods used and assun^tions made* These methods were further examined by Shedlovsky (1943) and Chow (1944) * Edsall (1947) v/as c r it ic a l o f those workers # o considered the method o f preparation as s u ff ic ie n t evidence o f purity*Colvin, Smith and Cook (1954) suggested that a preparation may be considered pure for a given purpose and should be regarded as a population o f more or le s s c lo se ly re la ted molecules* Further support for a r e la t iv e concept o f purity came from Steinberg and Mihalyi (1957) who also warned against a r t ifa c ts re su ltin g in fa lsa conclusions. Vaughan and Steinberg (1959) recognised that the best that could be hoped for was , the accumulation o f evidence o f the, absence o f in ^ u r itie s vMoh increases the p rob ab ility that a protein i s in fact pure* Thus ava ilab le methods could,only esta b lish homogeneity w ith fesp ect to certa in physical properties*The development o f immunochemical methods, in p articu lar Double D iffusion and Immunoelectrophoresis has provided very se n s it iv e methods for the detection o f contaminants in protein preparations^. (Kabat and Meyer, 1961, to p t ie , 1961, Freeman, 1970" 77 "and Grant and B utt, 1970)* Anderson (1966) used several methods, bases on d ifferen t p hysical properties to ensure o high probality that h is IgO preparation was pure* Once I t i s estab lish ed by several c r ite r ia that a preparation i s s u f f ic ie n t ly pure, the other question v^hich must be asked i s whether or not i t i s denaturedk (Freeman, 1970)* Part o f the answer I s provided by the t e s t s for purity i*e* that the preparation i s uniform.This subsequently arose as a p articu lar problem in re la tio n to Bovine XgM (se e Section V).For the purpose o f these stu d ie s , E lectrophoresis, ïrnmuno" electro p h o resis , Double D iffusion and a fte r la b e ll in g , Rechromatography, were chosen for determining the s u ita b il ity o f the IgG ^ preparations for the metabolic studies# Only a trace im purity, detected by Immunoelectrophoresis and Double D iffu sion ( see-Section IXX B and C ) was accepted, the error involved being le s s than the errors Involved in the metabolic ^study*In sp ite o f the overirthelmlng evidence for the extravascular. c ircu la tio n o f plasma proteins reviewed above, i t has s t i l l been p o ssib le for some workers to ignore i t or forget about it* Thus F ierce and Smith (1967) tr ied to quantitate absorption in the neonatal p ig le t using plasma lev e ls* Klaus Bonnet and Jones (1969) using radial d iffu sio n claimed to ; quantitate the absorption o f bovine co lo stra l IgG and IgM,although they com pletely om iitted the extravascular d istr ib u tio n o f these immunoglobulins*% w- -Balfour and Oomlino, (■ 1962) ayoided. the :probIm in .th o ir study J o f factors Gccolorating absorption o f colostrum by coiXocting , thoracic duet lyïJph oyor. a 3CK) minute period# During th is . . tim o, they recovered 12 to 25 %, o f th o ir la b e lled globulin* th e Calves wotd anaostbotised throughout the exporiment* ,-. Other workers ;#1X q ■'recognising ..Ihe ' ,iri^)brtaVice ■ o i taking tho s i zo - o f '-the ' ' ex trava scular pool''! nto ' ac count » were forced . to ostim ate i t s -size» on the b a sis o f figures! ava ilab le at tho tim e# . 'Thps. McEwan' tX96B) - .used: th e ' îbctravacoular/întrovascuiar (E#V#/I#V« ) r a t io -o f ■0#7/i (Nansen, and NleXsm^ 1966;-and N ielsen -and- Nansen# 1967) lo r 81oW:'l'gG in :aduXt'.;-. ' "ca ttle* ' ■ Kruse '(X969)-;on the other hand, - applied data \ irom / ' mrk with Human IgG. ' -' Solman. ,(1969) con fin ed , h im self to ' conparisens of'plasma " le v e ls of-immunoglobuiihs, thus-avoiding'bringing-the extra^' vascular sizo' in to .h is , calouiatlbns#-, McBmn (1968)-; and 8elrnah . ' (1969) ■aaaumed-that:;dlistributlon-between'intra m û extravascular,- compartments w uld' bo complete-by' 48 hours after'.thb in gestion o f colostrum# .--a' . -. Therefore, before quantitating the. ..bfficienoy o f ■' absorption of.-'the co lo str a l immùhoglobulin in tho.neonatal' c a lf i t , was necessary to ' know more- about th e ir , metabolism and in particu lar, the d istr ib u tio n .botv^en in tra and - extras" 79vascular con^artments, thé rate o f equ llibratipu betvjeen the compartments and the rate o f degradation o f the immuno globulins^ ( Anderson 1964, Macdougall arid M ulligan, 1969). Of the immunoglobulins present in bovine colostrum, Fast IgG (IgG^) i s present in the h ighest Goncentration* I t s metabolism was therefore studied f ir s t* 'Figure 6Examination of Fast IgG, prior to Isotop ic Labelling1, ElectrophoresisB. S. Fast IgG2. Double Diffusion Fast IgGA. E.G.3. Immunoelectrophoresis Fast IgGSlow IgGA. B. G.B.S.B.S. - Bovine SerumA. B. G. - Anti-bovine-globulin (with some anti-albumin activity)80MATmÀLsA. ■,; :Calves 1, 4 , 5 , 6 and 7 were supplied by. Dr* ï* Selman,: Department o f Veterinài'y Medlçxne,. University; o l Glasgow*. ^They were a i l 48 hours old* Calves A to H were market ca lv es , purchased through a dealer* They were, up to 'one v/eek. old*Calves 8 to 14 were purchased from Mr, H* Guthie, Moss Side Farm, Kilmarnock* they were 2 to; 5 days o ld .B* Examination Of the IgG ^ preparation - Big* 6 * /( see Section III for the method o f preparation )( see Section II D 3aThe preparations gave a s in g le broad band on electrop h osesis , in the gy region*2* Double D iffusionOne main p r e c ip itin lin e developed, which showed complete; id e n tity with one o f the lin e s in thé reference serum*( see Section II E 3 )Immunoelectrophoresis o f the f in a l IgG preparations was carried out using Ahti-Bovine Serum and Anti-Bovlhe Globulin*They were run opposite whole adult bovine serum and a p u rified Slow IgG ( I ^ g ) preparation, for comparison* Each préparation showed the ch a ra cter is tic arc o f Fast IgG (see Fig* 3)*8 1 "Some o f the preparations ( see Section i l l C 4 ) showed: s lig h t traces o f a contaminant by Iir»mùnoelectrophore,sis and, / or Double D iffusion# This was p o ssib ly Bovine XgA, The degree o f contamination was:considered to be low enough to be disregarded for the metabolic study# s4* A cti.yitoThe method used was that o f Kueppers and Bearn (1966) as described by Shim, Kang, Kim, Cho and Lee (1969) for the examination o f human co lo str a l XgA. p u rified preparations o f , 2% IgGj ,^ from co lo stra l whey and serum were compared against whey and serum respectively* While both v^ey and serum showed marked an ti’^ tryptic a c t iv i t y , neither 0 f the IgG^preparations showed any#Ç. Bovine Albumin r ‘A 2^ so lu tion o f cornier c l a 1 bovine albumin (GSohn Fr V - Armour, Eastbourne, Sussex) was made up for lab ellin g# I t ’ was examined by Electrophoresis and a fter la b e ll in g , by molecular s iev e chromatography on Sephadex Q -2dO {see F ig . 12).B*’ W W llm ■( see Section IT C la':)"' A.,\ .As ; the experiments were spread over, a period o f 10 months, IgG preparations were la b elled as required# LessFigure 7CHROMATOGRAM OF CALF SERUM CONTAINING LABELLED IgG^ . ON SEPHADEX G200O ABSORBANCE O RADIOACTIVITYHIg 0.25I<60 70NUMBER OF TUBES2010- .8 2than lo5 atoms o f iodino, ware, -incorporated per molecule, o f S im ilarly , le s s than 1 .0 atoms X/mdlecule o f aibuniin were -incorporated ( see Dargle, 1969, for the method o f coloulatlon)* i l^ore, than 95% o f the ra d io a ctiv ity in the, f in a l IgG, and albumin preparations ^ was protein «bound#. 'G.' Hechromatoqrap^v. of. tobolXod IgG - , ,d#l ml o f the .iafoelied-preparatlon was added to 4 ml o f c a lf serum and applied to a Sephadex G -200colim n ( s e e Section III B f o r mol ècular s i eye. chroma to gràphy# The ra d io a c tiv ity .was deteimined .ln the 4 ml fraction s and p lo tted to show it s .r e la t io n s h ip to the protein concentration /( See F ig . 7 .)* The ra d io a c tiv ity v^ as always, found to foe asGociatod vvith the middle peak o f the chxomatpgram# VF y im m M m ,The experimental animals were .divided in to 3 groups#Group 1 consisted o f 12 ca lv es , fpr the in i t ia l m etabolic s tu d ie s# ; . -. Group 2 consisted o f 4 ca lv es , for repeat jioterm inations o f d istr ib u tio n and catabolism o f IgG.# An additional c a lf from. Group 1 was. a lso included* ' 'Group 3 consisted o f 7 calves for the study o f Albumin m etabolism ,. to provide a..comparison for the IgG ^ resu lts*The calves in ,Groups 1 and. 2 each redoived 2 to 5 mo- 8 3o f or la b e lled IgG, * The ca lves in Group 3 each131 / ' / -; rece ive approximately 1 .5 me. o f I - la b e lle d bovine : albumin. tVhen in j octed , a l l ca lves were at 1 ea st 7^ : hours ■ bid* '■; G# Metabolic •These were carried out for 14 days, during vibich time : the ca lves v/ere kept in m etabolic cages#: H* ' Furtheïr/,Expe]plm■ See Section II C 1 to 7 #' .ï; :See Section II C 8j . Pi (prho e i c Ca 1 ve s ..Galves were considered to be di arrhoeic\ vjhen they ; produced faeces corresponding to the. v i t , and / or . i t t c la s s if ic a t io n o f de la Fuente, 1970»8.turnoverQrtooQ e.mO eS Or a d i o a c t i v i t y 0 Î V o of i n j e c t e d d o s eDKTRIRUTION _ (EV/_L^^Q e . m / O p / n - ' V i lOQ-Intercept = H/t Intercept1, C am pbell —2, S terling —lO3 5 0ISO 200 250t i m e AFTER I N J E C T I O N (H r)too5 0■V.iïsMâ-kThe Distribution (bÿ Campbell and Sterling methods)W * ft tt'awf -^---------- - . —------:;r innS-'CiTves " Gïôûp'l"" . ....Calf No.» wrvi « M»e^ wA wrw a -iTW * eE.V./l.V. ; SterlingRatio - ^»MMAi»# #,wi#*jtoUftJMfcawrw #%*#<1 *'*1, » I»#*#*#* #Apparent Canpbeli Hquli ibrium Ratio : Time (hours)Total Body Catabolic,Raté,; ;FractionalCatabolicRate%9) 1 .2 /1 . 1 .2A ■ 0.026 0.063 ,4 1.1/1 :" '-.i.iA '-\/ 60 0.023 : 0.0515 ■ 1 .4 /1 ; , 1 .4/ 1- '., 60 0.029 ' 0.0676 • 1 .2 /1 , ‘V 1% ':; - 46 0.040 0.0827 l . s / l ,, ■ 1 .2 /1 : 43 0i056 0.130' ; l . t / l 1.% .^ 36- 0.044 0.092:F . 1'4/i : 1 .2 /1 ■ . 36 0.055 0.099G , 1 .2 /1 " ’ 1.3/1 : / '( '"36 / 0.052 , 0.100H " 1.8A' : 1 .2 /1 J - 0.042 0.08312 1 .9 /1 \ ' 1,3/1 60 0.050 0.09913 1 . 6 /1 :- 1 .3 /1 (:() ' 0.041 0.08414 1 . 7 /1 ' ■ ■' 1 .2 /1 , % ' 86 0.055 0.112Mean 1 .5 /1 ’ 1 .3 /1 ' 45 0.043 0.089StDk* 0 .25/ - 0 .11/ . ■ 16 . 0.012 0.022S t 0.07/ " 0 :03/ 4 .6 0.003 , 0.006 ,84 *A# Crotip 1 Metabeilsm ^: . (Tables 2 , 3 end 4). The d istr ib u tio n was eaiculated by two methods (se e F ig, B)é The equilibrium time method o f Caaipbéli e t al (1956) gave a mean Extravascular/lntravescular (B .V */l * V*) ra tio o f 1*3 & 0 , l l / i * The extrapolation method o f S ter lin g (1951) gave a sim ilar d istr ib u tio n o f 1*4 0*25/l (S*V*/l,V*) #There was a s ig n if ic a n t p o s it iv e correlation between the tv«> methods at the 5^ le v e l (Fig* 9)#, / ' . ' . rThe apparent equilibrium time was 45 * /16 hours*8 ,9 & 2 » 2 g, o f the plasma pool was catabolised per day» and 4*3 it. 1*2-II o f the Total Body p oo l. The apparent plasma and to ta l body wore 16,1 & 2*7 and _ 16*4> 5 ,7 (days)respectively* Very s ig n if ica n t p o s it iv e corre la tion s were found between the Csmphell, d istr ib u tio n and.à l l four o f the above measures o f catabolism (see Hg^ lO)* Sim ilar corre la tion s were found between the F.C*H, ,aud the Total Body C,R* . (Fig* 11) and between the Plasma and Total Body T*|’ s ( r,=' 0*874» p -: 14^» ranging from *12 upTable:ia.:. / ; ■Half h 'Uvgs: and-additiQnal data -for Calves In GroùiP l.h ü :!oV.-Plasma Vblumé ?" (ml/kg)k 4>mnm W" 'Ml—Apparent = Plasma #Total ‘ îtodÿ T-&/ ( # y s )'S Change ih %■ Change In ^P.V« (l4days) Body Weight (14days)l / 17,8 ■ ' ■ 26.0 ... ■.» ■■' ■•" \ \ , 5 .> ' ■' >.4 ^ / 20 ,0 30.0 ■»*': >■ :. ■, 28 _' 76 " ' 17.0 : 24 ,6' ' ^ 2 3 /'' ,. 60 'l;' 16*7.1 ^ / ■* ' . r “i l ' ; ■; ■ ; " ' ■ :)7 49 % 11*6;.' ' 12.3 \ *12.' ' -66 15.5 ■ ** ' •' . 18 , .: ■ . . ,. 63 r. /^ ;i7»o 12 .# '.;■ :79 10,5 - 13.2 „5 ...: 10 , % -'' ,H " . 60 15»1- "■ .17 .9 . . ’ ,1 4 '; ;■ .;■ .> '12 ?-> 45. 17,6 1 6 . ? ; . ' 2 “7 ^ : . ■:- ;X3'j, '-""16,7'' ■ ■ "7.' \ .- ? '•.14:/ , . ' 56 :;18.r, 13.6# m ^ 1M 4|l| M> #ip W,«* W'#% #» ^, -13■H M «4 m >W •* M *# #p WP , • *H. -8 . : ' >îGn ; . . 63,,/' '>•18.4’." " .,■- .-1.3 ■ ; ; ' 5' > .D.* . 11.6 ;> ‘üi?:;-?' . . . ,8 . . ;. 3*4;"' '1 y o .8 - ' , 1.7 '.'Died Day 12m L j âGolf lea nm* ÿ p*c,v■,Total - ’ ProteinA/GRatioTotal Protein 'a/ g. ' Ratio '.Total A/G.P rotein Ratio35 .' o,5s>: ' 6 .3 0.624 29 ..4»! ; . 0 .47 3,7 0 ,80 4*0 .0,65.5 /; ' 30 '' : j .. 0.42, 3 .8 ■ 0 .56 ' 4 .2 . 0 .606 . \ '■ 31 / . ■. 5*1 ' ■" 0,52 . : 4 .9 0 .54 - ; 5 . 1 . . / 0*59T, ■ '~ :44-t " 0.46 6^3 V■ 0.38: "5:* 5/ 0*432$ ; ■ 5*7 ; 0.44 ., ■ 5 .6 ' 0 ,57 e .ô ' 0 ,48F.., ' 33 : 8*6'' 0 ,65 3.1 0.70Gv:,; , 28 : 5*7 : , 0*42 ' 5 .5 ‘ o;53 6*6 ■ -: 0 ,4ôa . -;,: '3? ' ' : -5*0 0,43 ; ’4.7.' 0 .56 ../■ 6*0 0,60ia . '48 , /' : -ÿ\5' , 0.57 ..: 5 ,6 -0 , 5 6 ".' ' ' 5 ,7 0*69X3\, ■' 36 4*2 0,80 4 .6 ; ■ 0.74 4*8 ; 0.6814 . 0 .68 y .4 ,7 0 ,77 ; 4,9 0*66Mean" -• 36 ■-; 4,9 0*53 4*8 0*63. 5,1 : 0*597.7 ■ 0 ,70 ' ; 0.12 ' 6*96 ^ 0*18 . 0.57 0.08" 2 ,2 : 0,23 ■ 0.03 , ;'6é29 0 .04 0.17 0,03Figure 9THE CORRELATION BETWEEN THE CAM PBELL AND STERLING DISTRIBUTIONS (BOTH EXPRESSED AS THE RELATIVE SIZE OF THE EXTRA VASCULAR COMPARTMENT) OF IgG, IN 12 CALVES (GROUP 1)asA§HPCQ;1.1.0 .= 0 .556 = 0.05DISTRIBUTION (STERLING)Figure..,, i.0CORRELATIONS BETWEEN THE CAMPBELL DISTRIBUTION (EXPRESSED AS THE RELATIVE SIZE OF THE EXTRA VASCULAR COMPARTMENT) AND THE FOUR MEASURES OF CATABOLISMIH8mi30 0.745< 0.012010 _ j —1.51.0r = 0.837p = < 0. 001U 0.1000.0501.0 1.5Ir = 0.875p = <0.0012015101,0 1.50.050ÜQ§ 0.025 aÈHr = 0. 813p = < 0. 0011.0 1.5DISTRIBUTION (CAMPBELL)Figure 11THE CORRELATION BETWEEN THE FRACTIONAL CATABOLIC RATE AND THE TOTAL BODY CATABOLIC RATE OF IgG^ . IN 12 CALVES (GROUP 1)0.1500.0500.050 0.100TOTAL BODY CATABOLIC RATE85t o '428g# Wo ê içr ïifico n t corréla tion wao found between the Campbell d is tr ib u tio n and the Plasma Illume ( ml/Kg ) ,' The ca lves had a mean V/ o f 26 i B* The plasma.'protein , changes are shown in Table 4* .The three ca lves ( I , 4 and 5 ) which did not develop: diarrhoea, had an V# ra tio o f 1%Q/1$ (Gsmpbell andS te r lin g ) , an ^ipparent equilibrium time o f 60 hours, an o f d.O^rof the in travascu lar pool/24 .hours, a Total Body. Catabolic Ha to o f 2*9ï'/24 hours, ah apparent plasma T|- o f :XB days and ,a Total Bodÿ;!Ti|' o f ST days { a i l .exprressed, as-the. mean v a lu e), -The follow ing additional information was a lso obtained from Calves H to H, with regard, to the appearance o f radio** a c t iv ity in th e ,u r in e* , Calves H and F f i r s t passed urine vdthin 2 to 3 hours a fte r in je c tio n ( three tim es in the f i r s t 11 hours). Calf G passed urine only once between 8 and 11 ‘ hours'p'C^t in jection * Calf H -'first'passed urine \d th in 2 hours* (tw ice in 11 hours). A ll the urine s a # Ios co llec te d contained s ig n if ic a n t le v e ls o f ra d io a c tiv ity , when counted* There was thus (0 nsiderabl© v a ria tio n , in both the in terv a l between in je c t io n and the f i r s t urination and also In the frequency ■ o f urination*'Repeat Determinations o f the D istribution and Catabolism . o f Labelled IgG, In Four Calves - ,Calf No. ,Age(weeks)E ,V ./l,V .S ter lin gRatioE .V ./l.V .CambellHatioto ta l BodyCatabolicRateFractionalCatabolicRate8(1) 1 , 1 .4 / r 1 . 0 /1 0,071 0*0149(1)$ : 1 l u A 1 .1 /1 0,045 0.08510(1) I . ' 1 *3 /1 1 .4 /1 0,051 0.12311(1) ■ 1 1 .9 /1 1 . 7 /1 '/ 0e042 0,1208(2) 4 1 . 5 /19(2)9 4 1 . 2 /1 1 . 1 /1 0*020 0,04510(2) 4 0 ;8 9 /l 0 .87 /1 0.049 0,08611(2) 4 1 . 0 4 /1 1 . 0 /1 0.043 0,08413(2) 12 0 .92 /1 0 . 9 8 /1 0,044 0,0979(3)9 15 0 .96 /1 0 .64 /1 0,047 0,089- 86A s ig n if ic a n t negative correlation ( . r - *6*66j p «< 0*05 ) was found between the age o f the calves and the Campbell d istr ib u tio n l*é* as the age o f the calves increased , the r e la t iv e s iz e o f the eKtravascular compartment decreased* The n egative .corre la tion with the S ter lin g d istr ib u tio n was not s ig n ifica n t* Although the. d ifferen ce between the ca lves at 1 and 4 weeks was not s ig n if ica n t for Campbell or S ter lin g d is tr ib u tio n s , ca lves 10 and 11 had d istr ib u tio n s below the range o f 1*1 - 1*5/1 (Campbell) and 1*1 1 i9 / l(S ter lin g ) found in Oropp 1, A highly s ig n if ica n t p o s it iv e correla tion ( r = 0*875, p = 0*001 ) was found bétWeen the F,C.R* and the to ta l body catabolic rate* , No s ig n if ica n t correla tion was found between the F.G*R* and the age o f the ca lv es , p o ssib ly r e fle c t in g the in fluence o f diarrhoea in the i n i t i a l ’ study, at .1 week o f age* However, idien the F.C.R* s at 1 week were-replaced by those obtained for the non- diarrhoeic calves ( 1 , 4 and 5 in Group 1, see Table 2 ) , a s ig n if ic a n t p o s i t iv e ,correlation was found between the age o f the calves and the Fractional Catabolic Rate ( p « 0*05 )•1CHROMATOGRAM OF CALF SERUM CONTAINING ‘^ ^I-LABELLED BOVINE ALBUMINON SEPHADEX G200O ABSORBANCE O RADIOACTIVITY1<70NUMBER OF TUBEST a b le 6The D istrib u tion and Gatabû11am o f Labelled Albumin in 7 Calves « Group 3CalfNo*E .V ./l.V .S ter lin gRatioE .V ./l.V .CampbellRatioApparent Equilibrium Time (hours)Total Body CatabolicRate .FractionalCatabolicRateA 1 .6 /x . 1 .5 /1 ~ T ~ 0.024 ; 0.084B 1 .9 / i 1 .9 /1 36 0.016 0.061G 1 .9 /1 1 .8 /1 36 0.021 0.090D 1 .9 /1 2 .0 /1 36 0.0.30 , 0*12012 . 2 ,4 /1 2 ,2 /1 72 0.029 0*11613 1 .7 /1 1 .7 /1 84 0.026 0.07714 2 .0 /1 1 .8 /1 48 0.041 0.109Mean 1 .9 /1 1 .9 /1 50 0.027 0.094S.D.6 0 .2 5 / 0 .2 2 / 20 0.008 0.022S.E.& 0 .0 9 / 0 .0 8 / 8' 0.003 0.008■ ^ 87 - \ ■ ■C. Group 8 -Albumin CatabolismThe same parameters, were deteiïnined .( as for Group 1 ) .The Ganipbell. and S ter lin g methods, both gave an H*V./l* V* ra tio o f i* 9 /U A h igh ly s ig n if ica n t p o s it iv e corréla tion Was thus found between them ( r - 0*928, p - G.OOl )*The apparent equilibrium time was 50 ( 20 ) hours*9 ,4 ( & 2*2 ) % o f the p$asma pool was patabolised per day and 2*7 ( & 0 .8 ) % o$ the to ta l body pool* ■ The.apparent plasma and to ta l body h a lf - l iv e s were 19*2 ( ^ 4*6 ) and V 23*8 ( 3*9 ) days respectively.*the plasma volumes at the sta r t o f th e experiment were 60 Efc 6 ml/kg* A h igh ly s ig n if ica n t hegotivo correlation was found between, plasma volume and the E,V*/l*V* ( Carrpbell ) ra tio { p = <0*001 ) i*e* the larger/th e r e la t iv e s iz e o f the extravascular compartment, the lower the plasma volume ( ml/Kg ) .see,F ig 13* P o sit iv e correla tions found were between,the to ta l body h a l f - l i f e and the to ta l body catabolic rate f ( r F 0 ,663 , p = < 0 * 0 5 ), The correlation between the to ta l body h a lf~ i i f e and the fraction al catabolic raté Vv?as hot s ig n if ic a n t at the 55^ level:*: S ign ifican t correla tion s v/ero not found between the H*V,/I*V* ra tio and the four measures o f catabolism* The p o s it iv e correlation between the 0 P*V*' change and the ^ body weight ^change was sig n ifica n t*: ( r = 0 ,918 , p =<0.01),Figure 13THE CORRELATION BETWEEN THE ALBUMIN DISTRIBUTION (CAM PBELL) EXPRESSED AS THE RELATIVE SIZE OF THE EXTRA VASCULAR COMPARTMENT AND THE PLASMA VOLUME (m l/K ^)§HIp = < 0. 001r = -0 .9 6 92.01 .51.00 .5 50 100PLASMA VOLUME (m l/K g)150. Hali^y U v e s and additional, xiatè.Caif Plasma No* Volume (ml/Kg)Apparent Total Plasma T|r Body %% P*V, Oiange $ Body Weight In 14 days , changeIn 14 days*W lrteerfrinA tv»r>»«ï.V iÈ PT î,^**riiiJiepw .âxr.*ili PABÇ , D 121314Mean69576456506261606.1SfEUa 2 .318.525,2,24,815.418.712.7 18.919;24 /61.72?i827.621,421.324.726.4 17,232514-71323 «8 3.9 1.51357201610«7*•012.5• r .......................Plasma Protein Results (om/lOOml) and Maan P.G.V.for.. Group, 3 CalvesC alf No #Mean P.Ç.V, :TotalProteinA/S R atio \- ■ Total: Protein7Ratio 'EascJà ,.Total; A/G Protein. RatioA 34 0.56 \ 3 .4 0.56 3 .3 0.68B',' : '39 " .3 ,8 . 0*66 '3.9 ■ 0 .75 .. 3 .6 . 0 .83c : 35 6 .6 0*31 6 .0 0 .40 5 .8 0.440 '■ 41 ' , 5*0 0.42 '.\'5*1 0.46 5.3, '■ 0 .4 712 , ',48' -' ■ 5*5 . 0 .57 ■ 5*6 , 0 .56 5.7 0 .6913 .3 6 /.' - ,4*2 . ' 0 .80 4*6 0.74 4 .8 0 .6814 5 b 4*6 0.68 / 7 4 .7 0 .77 4 .9 0.66Mean . ■ 4 b ' ,- / w ' ' 0 .57 4#8 ; 0.61 4 .7 7 0 .63SfD.a 6 .3 1*0 0 .16 0.91 0 .15 0.98 , Q.IO; 2 .4 ' 0 .4 0*06 ^ 0.34 0.06 ' 0 .37 0.00A" 8 8 ,p. The E ffect o fA number o f ivorkers ( see Introduction, Section VII) have demonstrated the hypercatabolism o f plasma p rotein s associated with d iseases o f -the alimentary tr a c t , p a rticu la r ly those causing diarrhoea*; ^ -1 . M iM linIn Group III there did not appear to be any re la tio n s ship between the sev er ity and duration o f the, diarrhoea and the catabolism o f albumin* As none o f the calves remained non-diarrhoeic during the experimental period i t i s not p o ssib le to come to any d e f in ite conclusions as to the in fluence o f diarrhoea on the catabolism o f albumin in the neonatal ca lf*Hov/ever i t d s in terestin g to note that the mean, fraction al catab olic r a te , and plasma h a lf l iv e s o f the seven ca lves vare c lo se to the control values N ielsen (1966) obtained in adult ca ttle* ^2* IgG^A, conparisen .was made betwoon the three ca lves 1, 4 and 5 Miioh did not develop;: diarrhoea and the remaining calves in Group 1 , along with four ca lves from Group 2 ( at one week )* The ’*t’* t e s t showed s ig n if ica n t d ifferen ces between the two groups, for the fo llbm ng parameters;Figure 14COMPARISON o f PLASMA and EXCRETED ACTIVITY in g CONTROL CALF (4) and a DIARRHO EIC CALF (7) (see Tobies Zand 3 for ^he ol-her measures o f coMbolism)1005 0l.x.T'6(days)-20 CALF 7 ; X— X— X'OoRADIOÆTIVITY as % o f INJECTED DOSE1010a URINE + FAECAL 6 ACTIVITY os % o f , INJECTED ACTIVITY86CALF 74CALF 422 6 0O6 0 3 4 0100 140 180 22020TIME AFTER INJECTION (H O U R S )- 8 9Fractional Catabolic Rate p <0#0ITotal Body Catabolic Rate p - < 0.001Total Body H alf-L ife p = <0*001S ig n ifica n t d ifferen ces were hot found for the d istr ib u tio n ( by both methods ) and the apparent plasma h a l f - l i f e ( the le a s t sa tis fa c to ry measure o f catabolism ).Figure 14 shows a comparison between a control and a diarrhoeic ca lf*■- 90 -, ^■ ' D I S C U S S I O N ^ ;o th er’workers have confined th e ir in v estig a tio n o f the metabolism o f albumin and IgG in the bovine* to. stu d ies in more mature animals* Thus N ielsen (,1966a) determined the d istr ib u tio n of. Albumin in 6 f iv e year old Jersey, cows#Including one repeat determ ination, he obtained a mean d istr ib u tio n o f 1*22 ^ 0#24/l (H«V*/l*VO by an equilibrium time method# Thèse animals had a plasma h a lf l i f e o f 2D#4 * 3 .8 days and a fraction a l catab olic rate o f 7#9% * 1#1# Holliday* Mulligan and Dalton (1968) obtained sim ilar values for th e ir 7 control animals i . e . a plasm a-half l i f e , o f 24*6 2*6 days,and a fraction a l ca tab o lic rate o f 6*7% ^ 0.8,# Holmes (1970) found à lower B*V«/l.V- ra tio than H ell sen* o f 0 .82 * 0 #07/1 (H .V ./I .V .) in 5 yearlings* using the s te r lin g method# The plasma h a lf l iv e s and fraction a l catabolic ra tes o f these anlAals were 21*5 ^ 0*2 days and 6*9# & 0*5 respectively*The d istr ib u tio n (E .V ./l.V # ) o f I . 9 / I for albumin ( see Table 6) in the neonatal ca lf* thus represents a proportionately larger extravaecular p oo l, the,plasma h a lf l iv e s and fraction a l catab olic ra tes are however o f a sim ilar order#The only other Information about the metabolism o f Fast IgG in healthy c a t t le has been obtained by Hansen (1970)In on# cow ( # \ ye#$ old; )■ used;.to':compar0;;Sldw'and Fast igQ# ■■■-■Ha fotmd-/# idçâtiaai; dietfIWtloh- ef '0*61/1 far-Slow ■ :■ :'/.cW 0#62/l-■ for ■ Past 'TgG*:, W%différé#' piasma;halfflives ;ènd ■ . ;, _ :catabolic, rates- offl8#5/7#9 ,dayo'-aM. #W#/I4.*l#, :,(■- Slov/ï%ot / . keep actively) $ - in the $#e study Ih:1 4 /Oatti0 { Average ■:„■ ' ,, . ago -8 years- Irhetfound a^distflbytldn-bf ,0*dS/l' for 'Slow I # / a Piamm of 17*6 dayà a.od-a l%aotiooal', 0atebolic,ratO'' , ; ;o f - t h o s e rosuitc wtré'almilâr # ' thoaa,'from m earlier Study’-ming the Matthews method of yàlch gave - , .cUctrlbutlph of"0*69/l\ln 6 .ahlmalç* "'\. ■ \ , " •;’Holmec (1971 ) uMng fact, 1 0 ' in sheep 'obtained - a .vory similar ., . . :dictrihution of 1*13/1 to -that prevloucly'found'for''-Slow'-IgO, of itX /l’ (HpimoBÿ '4969) * I t ’can-therWoro b# concluded - that ":in/tho., edplt^ruminant 1 fa c t ,aM Slow' i # bçvo tho"- sààe\d istriW tion , ■' a#:- would ho 'e^octW ' on- the b#$ÙL-of th e ir ' moleml%$■ -i^ights #- . -/ " ...;#0n-the#e':roCultc'-for/Blow ahd’4 in ,older - ■■'■'animals# are compared.vât|vtho recuite-( In th lc \w atlon ' )..,-. ^ ''''for the' neonatal ca lf* -it i s .c le a r th a t .#mrè proportionately '’la rger .çKtravaûCular:pobii;l*a*Tah:P*V*/t.*V* ra tio ("ace .. . -„ÿ.Table.;2 ) of i#$ /i ':coA^#W\to' Mcnwm' ra tio 'o f 0*6Q/l 'for ’ 4. ,^-Slow- i0#/;Fbr,-the three mmdlarrhoolc: calves, the -ratic- */ ' : -■ w a o i # g / i # % e - f t l b n a l ' - c o t i c r a t o a - . - a n d . p i o c m e ' h o l f * ^ ■ ■ . :■■,-l&vee. 4-f (those ' col vos tmro: similar, to M nm m control’ values*' .■ ." y- ;Also î, th e mean F?C,R. for a l l : 12 cal ves $ o f 8 ,9# i s lower than the 14*1^ 1^ found by Hansen for Fast X 0 , In.'oneicowt-There I s evidence that th is a ltera tio n In the r e la t iv e .. si^e o f the extràyèscular pool# found in 'th e neonatal c a lf - ; also occiirs in man# In a group o f healthy nien and women# !16 to 56 years o ld # ^Cbhen, Freeman and McFatltahe (1961) , obtained the fo llo w in g .d is tr ib u tio n st( EyVi/l,% ) ; g.stlmatitinÆ: ■ calculation, , V : o a .7 / l ;, , 6 s te r lin g; '1 .0 7 d: O .io /l / Campbell1*30 * 0#T4/l . V : ' ' 6 T \ MatthewsCombining : th e ;r esu lts from,the throe methods, we obtain .a mean d istr ib u tio n Of 1*3/1 # vMch oorresponds to that obtained by th^ Matthevjs method #K rasiln ikoffI Anderson and R osslngÇ 1966) obtained an E/V./ivV. ra tio o f 2*6/1 in tm. babies# : 13 and 18 days bid*From t h is age# up to 8 years, they found a gradual change in d is tr ib u tio n / but from 3 to 8 years i t remained fa ir ly constant at i/o*96 :# Using thb ir -data for the, d istr ib u tio n s of; the six. children from IS’ days to 'S. y pars# a s ig n if ic a n t negative.* 93correlation : between albumin distribution arid age is''; found ( P " ^ 6*0i /From these results W find a decrease in thepercentage Ejctrayascuiar 4r6m 67^ to 50^ I .e . a fall of 17%. '‘ If we replacé thé ratio of l / l for the oldest children with that obtained bÿ jbhon et ai (1961) for.adults we.find e4 % -4:.. :. ;^ / A similar comparison of thé bovine aibumini results giyes ' ày,decreasé. from'AÔbÿ/td';4#'.;;(- in-yearlings .' )' arid/an overall !;■ decreepë to iT e ' fal l - . -of ,ll#v'/.- For IgG. the/ decrease -'/"is 'from'55/:t6'41 .^ i.'e./a fali/p'f'^ i4jû^ j.,..,vihèn ,we .dorry:),are^ the ■ distributions of calyes 4;» 4 und b (Table 2) -with; Nansens -'""results*::. Thus#'it\,cari\be'seen from: the charigeS ip albumin ; distribution in/mari#-^ -associated/ivith';. àgë and thù èimllar changes . for both, albimin and the neohatal calf# that in each-/ casethpro;;is,a/'similar age/ related,decrPasp in the relative size of the ektravascuiar cpmpUrtmeht* This odours even though albumin is being syrithesised ' in;: the neonatal calf v^ereas I-ga/is not.; / % -' //,: - ■ ;/■■,/- ■■/", 'A larger extrayascular/compartment can be explained in : terms of increase lary pé#eêbiiity or,.a low.raté of_ returh, or both# I t .has been kriOwn' for a long time;that young animals have : relétiyely 1 afger., quantities of lymph than oIder ■ 'oriésf (Fmmin0bus, :i&73y. Hoiman (1937) fbund;that lyn^h : flow was: doubled in growing dogs butr that once they were maturethere was no s ig n if ic a n t change#, even in very old dogs.Kruse (1970) found an increased perm eability to Evans Blue in the neonatal ca lf* Many o f the çalves in th is section also showed a s ig n if ic a n t lo s s o f la b elled m aterial (igG, or albumin ) oyer the f i r s t 10 minutes a fte r in je c tio n . This was vdiy i t was necessary to take several timed samples and extrapolate back.,to the plasma ra d io a c tiv ity at "t " ( see Section l ï .G 8â 1 ) .There i s thus evidence for increased ca p illa ry perm eability and increased lymph flow* Further in v estig a tio n o f both factors i s required, b e fo re . i t will;, be p o ssib le to assess the sig n ifica n ce o f the increased ca p illa ry perm eability in re la tio n to the. r e la t iv e ly larger extravascular compartment. Because o f the lack o f Steady State*- conditions# they cannot be determined by the method o f Matthews (1957). A related phenomenon i s the s ta te o f maturity o f the ground substance**. A lo t o f work needs to be done on the physical forces operating in the extravascular compartment so that th is p o s s ib i l i t y .can only be conjecture at the moment.in addition other factors w ill in fluence the situation*While not large enough to account for the to ta l change in d istrib ution # the d ecrease.in plasma volume (ml/Kg) with a g e and the to ta l body water as a fraction o f body weight are both w ell recognised. Changes, w ill also, occur in the re la tiv eamounts o f d ifferen t t is s u e s , but th is w il l b e .p a rtiy linked with the changes in body.flu ids with age.The p o s s ib i l i ty that for XgG ,^ the change i s as a r e su lt o f the lack o f sy n th es is , p a r t ic u la r ly \when the c lo se correlation between catabolism and .d istribution i s noted, can be discounted because o f the sim ilar change.in albumin d istr ib u tio n , which i s not correlated to catabolism but to plasma volume.The albumin d istribution , in adult c a t t le and the calves in th is study was determined by the equilibrium time method of Canïpbell et al and additional values determined by the S ter lin g method# , The IgG .d istribution o f ,Nansen and N ielsen (1966) and N ielsen and Nansen (1967), was determined, by an alysis o f the plasma curve, idilch canhot.be applied to the neonatal c a lf as i t i s completely dependent on "Steady State^ conditions* Other workers (Cohen, 1963, W etterfors, 1965 and P lan tin , i960) have shown that where the Campbell, S ter lin g and Matthews methods for determining the d istr ib u tio n have been compared, the S terlin g method tends to over-estim ate the Extravascular compartment, although the r e su lts are s t i l l quite c lo se .The other two methods show a good measure o f agreement under a variety o f d isease conditions* The r e su lts from the repeat determinations (Table 5) in d icate that there is, a change with age. T his, coupled with the agreement between the Campbell96' and ^ • $tàTl ing "dl strlb u tion a malcos I t u n lik e ly : that • the -1 arger' ■' •- , igG# GXtrQvascul&r pool -ip tho.nepnatdT calf§ can b& accounted .; for In te m s o f the d iffe r e n t ■■methods, o f measurement#. . ‘ ,,: : -.The corre la tion s hetween .thO/.Oc^hoil--and .S terling. 'd istr ib u tion s* , for -IgG'^ ■and albumin Indicate that the s te r lin g . 'method can''be used; içi...d©tëtmine th e .d is tr ib u tio n df.p lasm a ■ ■ -'. pro’teinsv-in th f .'.neonatal ca lf# . I t .does hot howeyercallow fo r - . ■"•■"any increased catabolism oyer ■the/pertod^of’d ir tr ib u tlo n » 'a s; ,■ ■,'a r e su lt o f diarrhoea;dr p a r tia l dénaturat&on o f itho Tabolled ■ ^, ; preparation and;:rnuét ; therefore' be applied with caution#; ■'J h e■ po'sltive corre la tion s betvveah-.the Campbell d istr ib u tio n ■: and--.th0':four'- measuresCof oà tà b o .li# o f 'T # . » may r e f le c t the .% lack nf'^synthesis * ,,vMch 'in - older' animais ;. # 1 1 bâlnhco : natabplisà. \.(un d ersteady .'stato' conditions)#, Sim ilar corre la tion s were not fouhd‘\for-\albumin*"y In’, addition'*'as'the''Cotobolism.,.'Of . ■"Tg0.- in the d ia r ih o é lc ca lves m s ;sig n ifIca n tly 'increased-. - '(Section IV D) the d istr ib u tio n I t s e l f may .a lso ‘^ have been' ■'-; ' a lto r è d -by-'the Incidence o f dlarrhoea#. /For t h i s ..reason*- ^it ; ' .. vas'-decided to uàé'^the- B.Vf/ij'V.# -ratio .'of; 1 #B/l'''of ''.the.three t 'non*^.diarrho0 lc -calves* fpr-'the subsequent;: Colo s tr a l A udi es./..,/-, ' . :{see :^eotion Vl)v A;;fur,th.er- four ;out' o fth e;rem ain in g 'n in e ,' caives* had-a S im ilar d istribution* . ‘ '''"The s ig n if ic a n t négative- correla tion between the albumin, ., . . . . . ^ .' .d istr ib u tio n (Campbell and S terlin g ) and the P.V* (mi/kg) can be understood Viàen i t i s r e a l i s e d ’that the plasma volume i s being expressed as a fraction o f the ..Total Body, pool i ,e * i t ; ■is. equivalent to t H*V*,’or l / ( l f E.V,/l«V*)#Thus i f the r e la t iv e s iz e o f the extravascuXar,pool in crea ses, th is fra ctio n must decrease i«e# the P*V, (ml/kg) w il l decrease* The .sam e:relationship does not e x is t for igO* , presumably because o f the c lo se relationsh ip botv/een the d istr ib u tio n o f IgCj and catabolism*The!very c lo se correlation found botwoeh the andthe Total B o d y i n Groups i and 2; and to a le s s e r extent in Group 3 has a bearing on thé v a r ia b ility or otherw ise o f the r e la t iv e s iz e o f the extravaseu lar compartment* Thus Anderson (1964) explained the poor rela tionsh ip betv/een these , ’ tvjo param eters.in human metabolic s tu d ie s , in terms o f the considerable .v a r ia t io n that e x is ts in the r e la t iv e ,s iz e o f the extrovascular p oo l. In the neonatal c a l f , the. amount o f variation m u stth erefore be sm all, p a rticu la r ly for igO*V This w ill ,b e important in co lo stra ! stud ies where the previously determined d istr ib u tio n Will be ; used to ca lcu la te the absorption e f f ic ie n c y * Also i t j u s t i f i e s the use o f the - ,Total Body, G.R.:'and the Total Body T§ as measures o f catabolism as w ell as the:F*R.C. and thé Plasma T t, in the neonatal ca lf*98Of the four measures o f catabolism , the plasma 1% m uid appear to he the le a s t sa tis fa c to ry (see Gestion IV D)# Other workers have found considerable varia tion s \^en so ca lled ; plasma %*s, are ;cp#ared and they thus prefer not to ,u se th is \ jpar#àatërrat;all% Tn th is section* i t has been referred to ; as the apparent plasma I t w ill have been influenced by changes In plasma;volume associated with growth and / or diarrhoea. . / ; %/ " ' ', To take fu ll account o f the changés in plasma volume over the 14 day experimental period* i t would have, been necessary to measure the plasma v o lp A e v e r y 24 to 48 hours*As the m ajority o f ca lves in a i l thes^ groups had diarrhoea at some p oin t during the ej^^erimental period* plasma volume flu ctu ation s as part o f the general disturbance o f body flu ids* w ill have occurred* These would not have been taken in to account i f the plasma volume had been measured tw5,co*-at the beginning and end o f the 14 day experimental period and a d a ily correlation factor then calculated* Thus in those ca lves iAere the p lam a volume was determined è t the end o f the e%erimeht no a#em pt W made to apply a correction to the^daily plasma radio a c t iv ity le v e ls i . e . to Qp*.- ■;The bio À agreement for I gG. and Albumin -between the S ter lin g and Gampbeli d istr ib u tio n s and for igG^? thé h ighly% ^sig n if ic a n t corre la tion s between thé four measures o f ■' catabolism , two _6f which; depend on appear to ju s t i f y.the o m io sio n o f the plasma yolW h ohanges in ;th ese studies*The plasma volijfies meaéùred M th ls o to p ic e l ly la b e lle d ;igG ^ and aibumin (ta b les: 0 and y ) were considerably lower V than the plasma volumes; o f approximately .90ml/kg* reported . by MoEwana"'fisher and:. Belman. (.1968) * Thése vmrkers used Evans Blue and calcu lated th e plasma volwae from a/single...10 minute ; sample* without making any : alioWànçë for lo sse s from the c ircu la tio n during ib is period#; $uch lo ss e s have .been .allow ed-for in"the;T sbtopic/studies# .■/'■ , As ■already.;indicated in the-Introduction* I t i s not p o ss ib le to apply a l l thé standard methods for evaluating ; the metabolism o f plasma protéins to. the neonatal ca lf* :; In thë healthy adult animal* t h e \ ^ cohoentration o f any ' p rotein i s m erely _a .'reflection .of-' the- ';dynamio equliibrium that e x is t s between catabolism and synthesls i* e »; the turnover o f the protein* ; thus I f the animal under^;A I s in asteady s ta te -to/the p rotein under study* themagnitude o f the turhover rate cah be determined from a measure o f e ith er sy n th e s is /o r ‘oatehoiisiB* Usually catabolism , the , amount\or fraction ,û-f th e 'protein ..brçÉem'dowh p"ef-'unit time*I s measured* . . . ..... ”• ' " ' / ’- 100In the neonatal c a lf we have ceveia i problems* hanieXy the lack o f syn thesis o f IgQ^, the"change In body weight - vMch* ih: these studies* ivas quite vérlàblo and the- p o ss ib le , in fluence o f diarrhoea* Any one o f these factors means'that steady s t a t e 'conditions do hot ex ist* . Taken togeth er , the s itu a tio n i s eVen further removed from that envisaged by Matthews* Reeve and others who hâve analysed plasma protein metabolism in very great d eta il* under stea d y ,sta te conditions# %.. ' ,Thus although the. model on vMçh the CanpbeiT method is.; based oversinp 1 i f i e s the situation: and i s thus, discarded by Tàkeda and Reeve' (.1963) ;as having ho p h ysio log ica l rea lity * for the purposes o f these, stud ies * i t had a nuniber o f advantages. Provided the change in the pool .sizes* as a r e su lt o f one or more o f the factors .mehtiened above i s hot too rapid or large* i t w ill determine the d is tr ib u tio n , While the accuracy o f th is determination may not'.bo as’ lilgli as that obtained by Mattheivs, Reeve, and others i t has, thé supreme advantage that i t can be used * vAereas the other methods asro completely .unacceptable#- -, / ' . ' -llie S ter lin g mùihod i s based on-the assumption that ; catqWiism takes p lace equally in both in tra and.extravascular pools* which i s knoml/hot to be the case# However*' for both1 0 1 -plasma proteins in vestiga ted i t g ives very sim ilar resu lts*agreement being c lo ser for albumin than for IgG ./ This' , . ’ ' . . . .i s possib ly-due to th e in fluence o f diarrhoea I n the f i r s t few days o f the experiment, t* ich would be allowed for, in the ; Matthews calcu lations* but would appear to g ive ,a larger extravascular pool by the S terlin g method:#Oyer and above these considerations, i t must be.borne in mind that the p.urpbso o f these stud ies i s to be able to ' quantitate the e f f ic ie n c y o f absorption o f co lo stra l XgG.#Thus although Matthews w ill not allow, for slow ly exchanging p arts o f the extravascular pool* what we want to know i s the d istr ib u tio n o f the la rg est part o f the IgG. absorbed* before catabolism and indeed variation s in catabolism s ta r t to s ig n if ic a n t ly a lte r the le v e ls o f îgG.> Thus any slow ly exchanging part o f the E.V» pool i s not as important and can probably be ignored#1 0 2In th lo Section a l l tho'inform ation roe^irecl fo r o ' study of the offioioaoy o f 'aboqsq^tion.of CQlootrol Fast igG was obtained# %©- d is tr ib u tio n ) o f F act.ïgâ (XgCl)-In the ca lf. IVÛD found to bo 1*2/1 $ For albumin* the ra tio was T"#9/i # Equilibrium '%%$ q% )lGte by 48''hovre*'' The o lder - the c a lf ; the lower the ra tio . Tor .$ A oloso rolationoM p mm found b a te o n the d is tr ib u tio n o f end tho, level of ■ cat aboil am* However, the increase in t l io 'ra t io .aacociated- with hvp.GrOatoiK?lism,âf' IçiG| in the diarrhobio ealveo mm email* and. not significant# '. TîiO re la tionsh ip betivcon the • Oat abolie Ratée, fa r W th pro téine çtùdiod* providod additional evidence th a t the d is tr ib u tio n - of. In the neonatal'calf* Id not subject, to much variation#- 1 0 3 -"%»SECriON-.VTHE METABOLISM OF BOVINE loM104 ; ^In Section I the gradual identification of a soporate 19s (IgM) class of "antibodies was traced* Although the, .early . &\K)rk of Rabat .and Pedersen had established that certain antibodies, in some s e c ie s were macroglobulins, i t was only 20 years la ter /ï>vith tho:'wrk bf\ Franklin*' Kunkel and others that tîüis 19$ fraction was recognised as o m m al constituent, of y globulin* In tm reviews, Kvakol (I960) and Kimkeiy Fudenberg-'and'Ovary' (i960) * Kunkel differentiated' between, ' .end macroglobulins* o f vMph tho y '^ grot^ ^ were closely . related to certain pathological Waldenstrom méoroglobulihs* > He concluded that 19$ 'maoroglobulins •constituted a distinct'- class-of.antibody and c la ssified them on the basis o f'th e ir high molecular weight * electrophoretic and antigenic properties 'carbdhydratO content; etcjln tl^x)d ies'ag'a inst -# o le bacteria and/red c e lls # r o identified in th is close* The development . o f iff^royed separation techniques* particularly molecular sieve chromatogrtphy. made..the preparation o f l9s fractions much .easier ( see .also Section III - Introduction).... , The preparation of 1 # from Gorum UBuaÛÿ requires, the . combination'-of-several .methods# ■'-ThusTvlullor*^ -Hborharared rabbit IgM# Bradley (1969) used the method o f ' . ;' t;/Barth' e t al; (1964). Jensen ( 1 9 6 # ) separated , lip op rote in depleted human, serum-oh; Sephàdex (^2ÜD and recycled the f i r s t peak on gepharose 4B# The remaining maoroglobulin was ■ removed on SHAE ce llu lo se# Jonas (1969) separated / Ovine /• IgM on Gophadex 0^200, Using sucrose density gradient u ltr a - ■ C entrifugation, followed by gel f i l t r a t io n àhd im nunogel-// f i l t r a t lb n , SoJomonK 1969). d istinguished à 7s p r# o in - .with y,:M:antigenip:. determinants#; / Johnston and M iller (1970); ’ sepatated pathplogicai "macroglobulins’on G-200, using a ' eugiobuiin fraction# F in a lly , recycling on Sepharpse 4B ; yie lded a preparation, 98$: pure# ' /,Most o f the; ea fiy -^mrk/.on. the catabolism o f f M was carried out using pathologica l m acfogiobulins, in human p a tien ts su ffer in g from Waldenstroms mecroglObUlinaemia# ïh e ré su lte therefore must be treated with caution# Truaz e t ai (1962) showed that: the same prêparatiôn/gâve plasma half: l i f e / o f rv 14 days in a pa t ie n t ^ compared # t h 6 - 7 .days in a normal su b ject. Thus the re su it s ’o f Driysholm ( 1961 ) , Oabuzda (1962), Oiesen ( 1963) and Wiikihson et a l (i966j must be treated v4th■/■ / , . ... ^ 108 ^ / : 'care# The general conclusion however, that th is plasma protein i s la rg e ly confined to the vascular^compartment and has a shorter plasma‘.half 1 i f eÿ associated vdth a high fraction a l catab olie rate ( compared to IgG), was supported by : the m rk of Cohen and Freeman (I960) and Barth e t a l (1964). /Barth o t / al found, that approximately 80)^ (65 to 100) o f the protein was in travascu lar, plasma h a lf l iv e s o f 3*8 to 6*o days and . F.R .G .'s o f iS5 to 14 %/day* The same ivorkers found that IgM y;as rapidly: catabolised ;.in p a tien ts with p ro te in ^ o sin g gastroenteropathy*Gtiehm, Vaerman and .Fundenberg (19.66) found a mean plasma T |-o f 9*6 days, (7 - l l j ;for IgM in agammaglobullnaemlc p a tien ts treated with whole serum# Subsequent s tu d ie s , Blrke, Norberg, Olhagen and P lantin (1967), Strober, Woohher, Carbone and Waldmann (1967), Jensen (1 9 6 8 ).and (1969a and b ) , Oiesen and Hippe (1968) and (1969).,/Bradley (1969) and Jensen, Goltermann, . Jarhumr Weeke and Westergaard (1976) have, produced further evidence that IgM i s largely confined to the vascular compartment in man* Thus Bradley (1969) obtained d istr ib u tio n s in /the range 65 to 70^ intravascular and Strober et àl (1967) o f 76 sb 12 Î?*//,Half l iv e s and F*R-0#* s have varied , probably depending, oh Wiat proportion o f the preparation was p artly .; denatured % On the one hand , Bradley obtained plasma :-, o f G*7 t o - 6*5 days and on tîié -other, Jensen (1969a) o f /' approximately 10' days* ' F«E*0* *s show an even greater v a r ia tio n ,/ the range o f 8; to 20^ $^ dete#*ined by Oiesen and Hippe (1969):/ Tepropenting both extremes* .# man i s well recognised a è .beingpr,edomina#y. an abnormal XgM conÿïonant/associa ted \dthv Ihieumatoid A # h # t ià (Edelman,. Kunkel and Franklin, 1958^Svartz, I960, Ki.ïlânder'and.ph.ilipson,/19.64s Squire, 1966 and - Bradley, 1969)# Bradley, in h is m etabolic stu d ies obtained : very 's im ila r ’'results. f o r , / l g M . a n d ' ■ B h e u m d t o i d ' F a c t o r * , ,/ Sim ilar '.distributions' have -'beeh ■. found ■ for Fibrinogen .'."//' (M cF ailàho,,'1^3vMoFarlane, fod4/àhd..''Cromwell, - 1964#:..,-/'' j Atencio and Réeve, 1965 and Tab,eda, . 1966 and 1970).* Worberg, ■/'Birlce,/H0 dfors/and;'Plantin, (1970) ..found. that--92:-t6 94-.% of; the macroglobdlin 1 s i at rave soul ér #. ; ; -;T|iere was no Inform ation.avaiiable .honc.erning the catabolism //'--of 'Boyihe. I # , On the b asls o f hUman/w , i t seemed reasonable .■ ■-'/to/e^ect/Bovine;;XgM'-to'be largely IntratasGUIar, in , 'd is tr ib u tio n * ''From 'thë/.réyiét?fof/the:H ifforant m ethods'^; p rep a ra tio n ,/it can bè seen; that most wrKers have used " mo 1 ecul ar e l eve chroma to graphy!'. with or without additional ' proèeedures^,to iso 1 ate 'thé:,. iTOuho.globu.lin,*• I t was therefore . " decided to si%)arate Bovine sim ilar manner using.■GephâdeX'(ÿ*200# ^ "/-'/■ !METHODS\ • A ll the , calW # Used in these / exp erimënts,, with the . -é'/-:/ y / /' ' : ' . ' ..y/'''"{çxcèptioh/b'f, C-;9, (un;:Ayrshire,.heifer/oaif) were Ayrshire / /'//: ;- ; î / i i > i v é s , / / ^ ; ; / : / , / .’ r -'î; Te ensure that the r e s u ltéi.ebtèined'',wlth-'; the Isc to p ie a lly ' / la b e lle d -preparatldh.'wèl^é rèpfoductblëj i t was necessary to use.r .. y/ - . ÿ / ^ ^ : / yseveral fra o tlo n sp rep a red .'sep a ra te ly , ■ and/then co#)are . ■ .;the/'results* /:Tp/reduce the_ le v e l to a 'minimum without haying to eii^loy an additional separatldn, ï'-colbëtral.'\éièy';%as/'used as sta rtin g m aterial# /■■bipo^/; p rotefo present would tend to p re c ip ita te o u t, duringA s"the'pppsib ility-'of -denatUratidn-:of therfgM' i t s o i f '■■■; ■-, mUld Have tp/be/\Oohsi&red,'/foU%^^^^^ ^ wero.ohosen. that ■, w u id enable qori^ailfons;'tO:b^ médé between preparatipns, based / on those rdcoimended by Bremah, (T97Ù), / / ' - /; / \:Thw.,wW: 'Y' ^'"VPl'asiaayfoiume (F*Vy) ■0eViation-''(%).,iëè* ■ the ■\ Y- y, "" : ' .Y //Y .-''' ^ ^ ' ' / /'.apparent/pefoehtag#:.: 'deprease in plasma volume .determined W th /th e iso to p io a iiy ia h e lle d compared toY/ YY/Y v' ' YY'^ /Y/Y.i -YY-fYY/ Y'Y/YY Y// : ' //..that measured y4th S im ilarly labelled- igG -^yOr th é dye Evans Blue*I l l. Plasma activ ity at 50 hours (%), to Indicate the rate at which the preparation disappeared from the circulation* , ;Excreted activ ity (%) for Pay.1 and Day 2 of the experiment to demonstrate any increase in the f ir s t 24 hours*Fluctuations.in the dally Fractional Catabolic Rate (see Section IX C Bb i i i ) .For the calculation of the number of atoms of Iodine/molecule of labelled protein, see Pargie, 1969*Figure 15Examination of Bovine loM prior to Isotopic Labelling1. ElectrophoresisIgM B.S.2. Double Diffusion IgMA. B. G.3. ImmunoelectrophoresisA. B .C .B.S.B. S. - Bovine SerumA. B.C. - Anti-bovine-globulin (with some anti-albumin activity)A single broad precipitin line was obtained on Double Diffusion (2)' /' 1 # ''- . ' ',A .'■In ik arioM :.S tu d y . ', i v gp'».tgs..af,..Qal,y:g,s’Thé calvos virore market ca lv es, purchased through a dealer* They were up to one week old#2# Source o f IgM50 ml o f co lo stra l whey was separated on Sephadex 6*200 (see Section i n A and B ), The bed volume vyas 90 x 4*5 cm and the flow rate approximately 40 ml/hour* Two. separations were carried out simultaneously* The break through peaks were concentrated with S'ephadex 6*25.: the IgM preparation was subjected to e lectrop h oresis ,double d iffu s io n , immuneeleptrophoresls and tested for a n titrypsin a c t iv ity (se e Section XV) as previously carried out for igO^ (see Fig* 15), '4* w k é l in q \(see Section II G 1) . - -,The number o f atoms o f iodine incorporated was approximately T$5/kolécUle o f IgM, ...5*This was carried out as described in Section IV for IgG, * F ig, 16 shows the chromatogram with thé p o sit io n o f the la b e lled .m aterial surperimposed*;Rim re,...16RECHROMATOGRAPHY OF IgM PREPARATION (0 - 0 . 1 m l IN 4 m l OF CALF SERUMON S E P H A D E X G 2002 ,o ABSORBANCE » RADIOACTIVITY1 .1.0.NUMBER OF TUBES4020 U■■■‘ ' : ' ■ The Di s tribe tiors ■ end ' Gat abo' 1 i sm'.o f Label led Iq?4 preparation ( i ) in 4 CalvesCalf I.V ./E .V . Ï .V ./.Ë .V . Apparent Total Body(s te r lin g ) ( Canpbeil ) Plaema T^ (days)(d@ys)h; " , l / i . ô 1 /0 .85 , 2 .1' ■ 2'S ;3 , ■ . 1 /1 .9 :' 1 /0 .97 3 .5 , 2 .4 'G 1 /0 .7 L 1 /0 .4 6 ; 2 .7 2 ,5!1t 4% ^1 /1 .$ ' 1 /0 .6 8 ' 8 .2 2 .8M AW 9HB kiv bM -M. AjM AM AW Iku k A MM rn.m A«a Mb .M&. ASt MM «W MJb A* Mb AM MM MM iMb AÛ ILtt M t AM .M* bAb .^Ui .jf , &ÙA MJk uka M£l mU biA k_k M*k Mb. MM mm mm mmMean 1/.1.5 1/0*80 :. 2 .9 2 .5\ / o . s i ■ ' - / o . s i 0*59 0 . 2 5 -. /0 -8 5 / o . i o 0 .30 0.12Figure 17TURNOVER o f Ig M PREPARATION^!) in CALF Etoo5 0Op,mOe,mRADIOACTIVITY QS % o f INJECTED DOSEO rQeOp100 150 200O m 5 0 2 5 0TIME AFTER INJECTIO N (Hr)Figure.-.18TU R N O V E R of IgM PR EPA RA TIO N fl")in CAI F C1 0 05 0R A D IO A C TIV ITY as % o f INJECTED DOSE Q e,mIQjOr5 0 100 150 2 0 0TIME AFTER IN J E C T IO N (H r )3 0 0;■ ' .. - 1 1 3 - ,6# Injection. _ Bach c a lf received approximately SOOpx o f - la b elled : IgM. ' ; ■' : ^7. For a l l other experimental d e ta ils ? see M aterials and Methods, Section IV.R esults(see Tables 9 and 10, and Figures 17, IB and 19),The apparent d istr ib u tio n s (i#V4./F,V, ) as determined by both Campbell and S terlin g methods, are shown in Table 9 , along with the Apparent Plasma T&*s# A marked discrepancy was found in the apparent d is tr ib u tio n , ca lcu lated by the two methods. Thus the Campbell d istr ib u tio n o f l/0 « 8 ^ 0*21 indicated a r e la t iv e ly larger intravasoular pool* The S ter lin g d istr ib u tio n o f l / l . 5 did not show t h is . Plasma and Total Body T^ ^s o f 2*9 ^ 0*59 days and 2*5 * 0*25 days re sp ec tiv e ly , were o f a sim ilar order* As the ca lves wore d iarrh oeic , th ese , are l ik e ly to represent about h a lf contrei values*There was an apparent increase in plasma volume o f 10.3 rb 4 .3 (fo), and only a quarter o f the. la b e lle d m aterial 6*0%) was s t i l l present in the c ircu la tio n at 50 hours*The le v e l o f excreted a c t iv ity on Day,1 -w as:sign ifican tly higher than on pay 2 ( p - < 0*001). ' Although the d a ilyTHE DAILY VARIATION OF THE FRACTIONAL CATABOLIC \ leM PREPARATION (i) IN 4 CALVESCALF E6^1 . 00.80.60 .40.200 .50.20 0 . 80.50. 20mCALF FCALF Gl-:aasBassi«&dsnT».-$3£amCALF Hm4 6 8 10TIME AFTER INJECTION (DAYS)12The Dénaturation Criteria applied to IcM preparation (1,VP*V^ Deviation (#)Plasma Activity at 50 hours/ m ■ ; ■Excreted Activity, Pay I Pay 2B F G HMoanS.P ,*13*514*28*15*310*34*3-2*1212 2 ,342425 6*0 3*027332429283*81*91314 16 14141*30*6. " ' ' . 114'- :Fractional catabolic rate ( F «H* CO varied considerably (see Fig 19), i t was r e la t iv e ly constant in ca lves G and H*I t increased in Calf F, but a fter an in i t ia l steady phase, it , started to fa ll. 4 days.before the c a lf died* .The Plasma Protein and P*V*C. figures for these calves . are in Table 10. - AThe apparent increase in plasma volume and the raised levolA of excreted a c t iv ity in the .f ir s t -24 hours vvould, seem to In d ica te a degree o f gross denaturationr The F.C.R**s ' decreased in only ohe c a lf , and then, prior to death, in d icating that the remainder of the preparation not irrjmediately excreted in th e urine was fa ir ly m etabolicaiiy homogenous* .The p o s s ib i l i ty that it . was uniformly denatured to an ex ten t, cannot be excluded# The discrepancy between the Canpbeli and S ter lin g distributions-..is ex p lica b le in, terms o f/th e high, in i t ia l . catabolism , which i s allowed for when ca lcu la tin g the Campbell d istribution*Figure %0RECHROMATOGRAPHY OF IgM PREPARATION (ii) -0 .1 m l IN 4nil OF CALF SERUMON SEPHADEX G2002.0■ O ABSORBANCE 0 RADIOACn\TTY1.040 5030 605025NUMBER OF TUBES' Table 11Preparation ( i l ) in 4 calvesCalf No. i.v ./E v :SterlingRatio, I.V./Ë.V. ' Plasma fe ’sCampbell . (days) Ratio Initial FinalInitialTotal Body T& (days)■■ ' 1/6.1 1/2.5 ' 2.7 4.3 4.61/4.3 1/ 2 .6 1.8 8.8 5 .81/6.7 1/ 5 .3 2.9 7.9 6.8n ' 1/3.5 1/ 5 .9 2 . 9 -, ■:,8.3, . 6.2Mean 1/5.2 1/ 4 . 1 2.6 7;3 ■; . 5 .9/1 .5 / I . 8 0.53 2,0 0.93■ s,E.± /0 .7 /0.9 0.26 1.0 0.46.ïâ tisjiiâ ilGalf P«V*/Deviation wn. :'Plasma A ctiv ity at SO hours .m . ÿ/ÿ Day 2, 8 9 10 1110114516718810 «5 18.0 11,3 11,51312791415 109MoanS fD.d:150371912,83,51,710.3 12,02,8 2.91,4 1,5T U R N O V E R of IgM P R E P A R A T I O N ( n ) in CALF 8_tooQe.mOer a d io a c t iv i t y os % of INJECTED DOSE Qp.m3 0 0250200150TIME AFTER IN JE C T IO N (H r)too5 0- 115B. IgMThe calve6 were purchased from Mr H# Guthrie*, Moss Side Farm, Kilmarnock* They whre.2 tc 5 days old*The method o f preparation was the same as for the in i t ia l study, (see above) except that i t was necessary to rechromatograph the preparation to remove contaminating IgG^, I t was stored at ^20^C* . - ■ ■"3* ■îaiâsM âa. ' ,Baeh c a lf received approximately Ipc o f ‘'"X la b e lledIgM, ‘ : ' y ■,4*. For a l l other proceedures, see Section V A above*( Conp ar ed ■ to the % ni t i a 1 at udy Tab 1 es 11 and 19. and Figures 21 ând 2 2 )« ,This time?, the apparent plasma volumes were very considerably increased i*e* 150 a 37 and the plasma a c t iv ity a t 50 hours correspondingly reduced to 12*8 & 3 ,5 There was however l i t t l e d ifferen ce between thé pay 1 and Day 2 excreted a ctiv ity * The F*HtCr.*s showed a. greater variation (see Fig 22) than in the i n i t i a l study* When the e f fe c ts o f the in i t ia lFiqixre 22THE DAIIiY VARIATION OF THE FRACTIONAL CATABOLIC RATE (F. C. R. ) O JfïiM T O Ë M R A T IO N l^ CALTOSCALF 8ICALF 9mCALF 10CALF 11: : : Ê ^142 10 124 6TIME AFTER INJECTION (DAYS)for Calves in Tables 11 & 12, ËlâlLfi BêX.7 Dav 1CaXf Moan , Total a/ g . Total A/G , Total A/Gm* p.c.y. Protein Ratio Protoitî Ratio Protein Ratio8 ^ 4 3 4.0 0.87 3.5 0.74 ■. 3,9 0.639 '' ■ ■ , 86 4 .4 0.94 4.0 Oî73 4,5 0.6910 81 4.8 , 0,70 4-4 0.75 4*4 0.8311 30 4 .4 0.80 4 .1 0.70 4*0 0.73Moan 35 4 ,4 0 ,83 4 , 0 0 .7 8 4 . 4 0.72'' ,5 ,9 ' 0.33 0.10 ’ 0 .37 0*02 0,31 0.08S.BU: 3.0 0.16 0,05 0.19 0*01 0.16 0.04116r is e s In, paives 8*, 10 and II ( p o ssib ly associated with diarrhoea) were removed,_ a l l four calves showed a fa ll in g F*C.E. 3 although in ,c a l f 9 i t was o n ly .s lig h t*■ The apparent d istr ib u tio n by both methods was very d iffer en t from that found in the in i t i a l study* This time there was an apparently,very large extravascular pool l .e *1 / 5 ^ 2 (.Sterling) -and l / 4 f I (Campbell)* The plasma disappearance curve ( # g , 2 1 ) consisted o f t w components with h a lf l iv e s of: 2>6;,=i:: 0*53 days end 7*3 2*0 days* The plasma p roteinand P«y«C. figu res fo r tîtese ca lves are in Table 13#%e plasma volume deviation and the low plasma a c t iv ity at 50 hours indicated .that a large, proportion o f the la b e lled Xgîi'I was grossly denatured* As the did not becomeconstant a fte r the removal of. the grossly denatured material? the remaining IgK!t was probably also damaged to an extent*The f in a l plasma % o f 7 ,3 iv 2.*0 days may however give somein d ica tio n o f the l ik e ly disappearance o f undenatured IgMfrom the circu lation* >,Ip viev/ o f the dénaturation # : i t i shardly surprising that the apparent d is tr ib u tio n ,o f the preparationwas very d iffer en t from the in i t ia l study* I t can be seenfrom Fig# 21 that even at the end o f the experiment, abouth a lf o f the iso top ic lab el i s s t i l l xetalndd extra-vascutariy(30 to 60 % of the injected dose)* . . ..■The oply.differences between the two preparations were' f i r s t ly that-tlie Ig# used for the repeat experiment had been = 'rechrqma to graphed; bn' 0^200 and secondly, i t had been storedo ' ' / . . .'a t .*20 G* I t may therefore foe concluded that one or both ofthese prpceècbres contributed to the increased dénaturationof the IgM used- .fpr.'-.thd f i r s t repeat'.study#, ^thus, while theresu lts from the in i t ia l e^^erimept; had been moderately encouraging,the repeat experiment, indicated thàt a considerable degree ofdenaturation had occurred.# Although'it might foe possibleto make valid deductions from m .e#erikent- in iMch only a :proportion of the.iafoolled material wore denatured, or In ''which''the general'level of dénaturation‘were slight^ neitherpreparations., .would be suitable for a study of the efficiencyof absorptibn of colostral^igMs /r. > ^. ^ \ ^ ' ' : : ' - ' yM . ^#ile''no.- infomatien^m s.aval fof Bovine' IgM, i\erk- ' with human IgM had indicated that the majority ^of it'w as retained .l'ntravaSGularly#:\:.Thus ' a preparation,suitable for colostra!/efficiency studies would not disappear - rapidly from the circulation i*e* . thé plasma activ ity muld fob a t least 50% of the in it ia l activ ity or even'.-.higher# Also, the'Plasma :. .Ti' ' mi ght, foe : é # acted to foe of the .order-of'10-days# An ,r Indication of the su itab ility of a preparétion could be-Obtained from the apparent plasma volume, plasma activ ity ^;-at 50 hours .end subsequent plam a\% :% ' \Both the .in it ia l and f ir s t repeat TgM experiments had been carried exit vâth IgM label led Mth 1*5 atoms df iodine per .molecule * = Freeman (1966) stated, that-for. human - 1 # , .the - ' level of; lodination should be kept'to 0*5 atoms p.er molecule* Although other f ho tors had already boon ir# Heated ,a.$ possible causes of. gross (Whaturation there ?;as s t i l l a degree of dénaturation in the in it ia l e%eriment- to be accounted for*Apai't from the method of preparation of,the IgM i t s e l f , the level. ■■ of' ibdihation X/l.#5' Ato.me- /Molecule); would'-appear to bo thenext -mg# lik e ly oauso#yl a b i# j 4The. PIasiiia. Dissppearancé. o f Lgbelisd IgM in 2 Calvevarying the le v e l o f Jod ioatlon■ Calf : No.. PoV*(mi/kgjPlasma A ctiv ity at 50 hrs..............Plasma Tvr*s I n it ia l Final1 .5 atoms 68,5 .. 34 .5 ■ 5 .0 19*6l/m olecu le ^13 64.9 28.0 , 5 .9 18,00*8 atoms 9 45 ,5 22 .5 ; 8 .4 17.6;l/moXecule ;•; ; 13 ; 61*7 27*2 6 .4 21.20*4 atoms 9 33.2 23 ,5 9 .1ï/m olecu îe ■■ H 3 52+7 30.2 7.1.■(varying th e .leve l :of Ipdination) iy ;■■• / ,.,I t •' '' '■;*/ \ ■ Tho tv^ ca lves used Were 22, weeks old (ca lf 9) and.19 ; week a old, ( cai 1 13), respéàtivHy di the start of the exp erlment, (see table 14),* They had been retained after the IgG-- - " ' . \ j - '' . - : ' *ex^eriRi^nta. * see- Bectioh T V * ' " % ■The itothod of ^preparation used and the proci-sdure followed were thd same as a llo y e d In the in it ia l experiment A#enable- a'^yalld/uo'uparisoh to h% made in these older calves ). the f ir s t preparation used was labelled with the original lèvel o f 1#5 atoms/raQlecule (reach ca lf receiving■ ' . . . ' loK , ■ ' . ' . , ' , rabout dOQûb of " I. labelled t 0 } * Three weeks later* the samecalves were injected with 10%g of I labelled I # ( 0*8atoms p # moiecdle) and ld%Lc of labelled IgM (0*4 atomspèrMïkïlecuie)*' : ' ' ; ,4:é % For 'other'proceedures* see Section' V A ^■ ' ■ (see ^Tables-14; and'15) _ / , :{ ’■ ,. ;y. Although :ther#' vas' anyinevitablè- delay bét%veen''thé, ti^ jo .Table 15Additional Information about t he 2 Calves -In the 2nd IgM Repeat StudyCalf\ ; NotMean■ p .c .v .Total protein (gïi/lOOml)A/0RatioAge(weeks)1 .5 atoms 9 : 'Wv , - V 6+3./' 0 .58 22ï/m olèbulè 13 ;v 30 ■ 7 .9 ' 0*39 190*8/0 *4 atoms 27 , ■ .. 6 .3 0c5B 2Ô ;I/m olecule , 13 28 7 .5 0.39 23......................................- 120 * ,parts o f the experiment, a d irect compafison between the resu lts , vms s t i l l possib le* Both calves showed apparent reductions ,:ln the plasma volume using the tm preparations w ith the lower number o f atoms o f Iodine -jper molecule*VJhen expressed as:: % increases over the lowest values they represented increases o f 66JS (1*5 atoms/molecule) and 19!^ ; (O.B. atoms/molecule) for C 9 , and sim ilar ly 23# and 11% for G 13* Conporisons on the. b asis o f the Plasma A ctiv ity at 50 hours and the in i t ia l plasma h a l f - l i f e are d i f f ic u l t because o f the differences,betw een the tw ocalV es* All the 50 hour plasma a c t iv i t ie s are c lo se to the.mean value obtained in the in i t i a l IgM; study o f 25?1 * 6/L The in i t i a l plasma show a tendency to be longer^ at the lower iod in atlon levels* This i s most marked in C 9 but v\hen taken in conjunction with the higher 50,hour plasma a c t iv ity , may be the r e su lt o f other, fa c to rs . All the in i t ia l plasma T-|-*$ are longer than those found in the ea r lier . Ic^i stud ies (2*9 0.59days and 2 .6 i 0 ,52 days, resp ectiv e ly ) and are c lo ser to the f in a l plasma Tÿ in the f i r s t repeat study o f 7 .3 ± 2 days*Thus only the plasma volume r e su lts g ive a c lear in d ication , o f the in fluence o f the higher le v e ls o f iodinatlon# The re su lt would sëem to be th a t ,in these p articu lar stu d ie s , the IgM m olecules thus denatured are very rapid ly removed from1 2 1 •the circu lation* The f in a l plasma T-|-*s are longer than might have been expected*. They may, at le a s t in p a rt, represent a . 19s form o f IgG, as described by Hammer, Kickhofen and Hemming (1968)* In view o f the ré su lté in th is in v estig a tio n o f the . in fluence o f d iffe r en t le v e ls o f iod in a tlon , i t was decided th a t , in ,fu ture, Bovine IgM, preparations m uld foe la b e lled with 0*5 or le s s atoms o f Iodine per molecule*1 2 20g- Third IoM Repeat Study . % ./JI fThe ca lves were purchased from Mr H. Guthrie, Moss Side Farm y Kilmarnock/" Th^Y were one week o ld at the s ta r t o f the ■experiment* ■■V- ' . , ■2* Sdtircë: of Isotoplcallv. labellod iqM +i tlô’ ( i l i )The startih g m aterial was co ld stra l Wiey (1 l i t r e )*An i n i t i a l fraction ation was carried put on DBAE h*W Sephadex* A fter-e lu tion with 0 .1 5M phosphate b u fferi the ^îgM enriched fraction was eluted with O è^ NaGi (i*e+ thé stefc' Wisè instead of. gradient elution)# The middle o f th is peak was concentrated in .an u ltr a f i ltr a t io n c e l l (M illipore U.K ., London) and then la b elled w i t h (0*5 atoms per molecule)* The’la b e lled frattiort was, then subjected to a f in a l separation on Sephadex G*^ 20D# The breakthrough peak, containing the la b e lled IgM was co llec te d in tubes containing povtfdered Bovine.Albumin (Fraction V, Armour, Eastbourne^ Sussex)*This separation also removed unbound a c t iv i ty .for both separations a Pharmacia KlOO/lOO column was used. ; The in i t i a l PBAE bed volume o f 72 x 10 cm shrank to 52 x 10 cm. the flow rate over the sème period increased from 125 Ml/kr %to 300 m l/hr. The second separation was carried put using an 82 X 10 cm bed o f Sephadex <^2pO. The flow rate was 120 ml/hr*Table 16I % u p > 0*001!.)* The low plasma volume deviation o f 3*5% in the rabbits must be.contrasted with that o f 50% ,in ca lves 17 and 18, using the same preparation# Thismay in part r e f le c t increased ca p illa ry perm eability o f the - c a p illa r ie s o f the two ca lv es . The moan 50 hour plasma a c t iv ity o f 7é3 * 0*97 % i s o f the same order as that o f the two calves»8 and 16 % resp ectively* These r e s u lts , while producing additional evidence that .the preparation, was p a r t ia lly denatured» when , compared with the r e su lts obtained with the same preparation in C 17 and C IS show that the short, piaenia h a lf l iv e s do in fact r e f le c t th is dénaturation» and not hypercatabolism associated vAth Neonatal Diarrhoea* ’ÀS w ell as avoiding problems associated with diarrhoea» the use o f rabbits to screen,a se r ie s o f IgM preparations, has other advantages* ^ An estim ate o f the excreted a c t iv ity can be read ily obtained#,. A lso, i t i s ,p ossib le to t e s t small q u an tities o f Ig?i preparations, id th a low sp e c if ic a c t iv ity i*e* preparations that could not bè examined in calves* To avoid problems associated w ith th e production by the rabbit, of. 'a n tib o d ie s to bovine,IgM, rabbits could be rendered to leran t to bovine plasma p rotein s at birth* However, in view o f the rapid catabolism o f the IgM preparations, so .fa r examined th is i s rnot a problem at the moment* : ,Figure 25RECHROMATOGRAPHY OF IgM PREPARATION (iv) - IN 2gm % BOVINE ALBUMIN - ON DEAE SEPHADEX A -50. USING A 0.15-0 . SM PHOSPHATE BUFFER GRADIENT (pH = 8 .0 )50 r© RADIOACTIVITY O ABSORBANCEwwgNUMBER OF TUBES2.01.51. 00.5Ft aAal^ '1 * ■ Experimental Animal ; ,Cai f 18 j now 3 months oId ÿ had hêen, r é ta lned ftom the earli'af. study (Section ,■s* ' .'■■■. ■'■'' ;.: The samè .foasié method'of piepas'tlo'n was followed as for .Section V P. Howeveri la b e llin g was delayed t i l l , a fte r the G-200 separation# - finbotmd a c t iv ity was ;;rempved by passage through a Sephadex 0*200 colimmÿ. àndtmore Bovine Albumin added* #ien ;reohromatbgraphed on PHAI A*50; Sephadexj the m ajority o f the la b e lled m aterial came through in the break, through peak (see pigure 28) in d ica tin g some change since' the f i r s t DEAE separation* Ih e-fr a c tio n s con stitu tin g th is peak were combined and/used for the experiment $ without furih or concentrations: 3 . V ; . i j 2 d â S | â M ; ■.■ ; , . ' , / ■ , ' 0 ■ ^The c a lf reoelW ii appïûxiBiateiy 1 1J.C oi' ^ ^ vl-labelled IgM*4# For other proceedures^soe Section V p* 'ums^ j :The d istribution^ .calculated by the S ter lin g method was l/b#75 (I*V*/E#VU# .The; c a lf had a plasma .volume o f 39 ml/Kg , and a mean p*G*V* o f 38# The plasma a o ily ity at ôO hoitrsFigure 26PLASMA DISAPPEARANCE of Ig M PREPARATION (BZ)in CALF 181 0 0INTERCEPT - 5 7 %5 0RADIOACTIVITYas % of INJECTED DOSEN . ,iO O50O 150TIME AFTER INJECTION (Hr)was 26% (see Fig* 26)• The in i t ia l and f in a l plasma T-g-*s were 43 hours and 65 hours, respectivoly*The plasma volume was sim ilar to that obtained for C 9 (0 .4 atoms/molecule) in Sectiori V C# The 50 hour plasma a c t iv ity was o f the same order as that found in the other studies# In view o f the fa ct that th is .ca lf was nonrdiarrhoeic» the short h a lf - l iv e s suggest that a l l the m olecules w ere ,p a rtia lly denatured, presumable a t some point prior to the f in a l DEAE separation.Other workers have considered thgt rechromatography o f IgM an Sephadex G-200 i s su ffic ien t' to show that the preparation i s undenaturedt The r e su lts here suggest that th is I s not the case and that ion-exchange chromatography i s m ore.sen sitive as was suggested by Cohen (1 9 # )# The d istr ib u tio n (S ter lin g ) - suggests that although the preparation .was p a r t ia lly denatured, the degree o f dénaturation was not su ff ic ie n t to cause the IgM \ to bo immediately removed from the circu lation* This i s further . supported by, the plasma volume.V , ■ - . - - ;' G. An: Boyi|ie^IhM# -Bovine IgM (contaminated with a trace o f macroglpfoulln)' was prepared by molecular sieve chrpmatography on Sephadox G- 2 0 0 V (see Section ¥ A 2 ) ♦■ iTOunoelectrophoresis o f the-antlserum raiseci against th is preparation ,(see Section IX H i ) showed antibodies against IgM, other immunoglobulins and Oy. macroglobulin. The antibodies I'Cross-reapting'* with the other immunoglobulins were absorbed with IgO^* To remove the anti macroglobulin component, i t was necessary to prepare : the niacroglobulin by fraction atin g adult bovine serum on Sephadex 0-20D and then Separating- the break through peak on ;gephade% DEAR A -50/: The f in a l anti serum was shoivn to be pure by double d iffu sio n against IgM fraction s and whole serum*2* . . .( see Section II E 5)Tv/o plasma sanples were examined from four ca lv e s , C/C3, O/OÔ3 C/ÇIO and C /C ll ( s e e Section VI)* The f i r s t sample had been taken four days post partum and the, second, 48 hours la te r . Adult bovine serum and 3 d ilu tio n s o f i t , were used as r e la t iv e . standards#X30 -' li'Jhen the standards {expressed as d ") were p lo tted against th erre la tiv e IgM concentration^ a stra igh t lin e was obtained.The apparent plasma Tf, for each p a lf , was then calcu lated from the change in d' .^ G/G3, G/G6 , ;Ç/GiO and .G/C1 2 had so f 7 i8 , 8 .4 , 6 * 6 and 5 .7 days re sp ec tiv e ly , g iving à mean plasma T# o f 7+2 1 . 2 days* :conclusions ■ ■When taken in conjunction M th the, apparent plasma h a lf - l iv e s o f the iso top iC a lly la b e lle d .IgM preparations, they suggest that the îÿ* s.obtained in the in i t i a l ,study (see Table 9 ) vare abnormally low and th at the I n it ia l T^*s found.for IgM la b elled with 0*8 and 0 .4 atoms o f 1/m olecule (see; Table 14) in older' ; ca lv e s , probably g ive a b etter in d ication o f the normal rate o f .disappearance o f co lostrab XgM from the c ircu la tio n . ‘» Mk'tW » * #** L m # MMPWW4 « *#% *#M w , Mpiir i»'W ..* p c m *»* iiliiiirlIIM-PiPreparationN o .'( i )( i i )( i i i )Calves ■ Rabbits( iv ) *P„V,'Deviation( f )10,3 dk 4 ,3150 ± 3750 :k 7o8 :3 .5 -t 13,6Plasma A ctivity: a t 50 hr s ,.12,8 *' 3 ,5Pay Excreted A ctiv ity D ifferences2 P.C.R.Variation25 * 6 ,0 S ig n ifica n t FairlyconstantNot DecreasingS ig n ifica n t12 * ;5.7. 7v3 ït: 0 ,97 ' S ig n ifica n t , 26* p lus abnormal behaviour on ion-exchange chromatography1 3 1 -There were certain differences in the methods of preparation of the four IgM fractions# Albumin was not used as a protective colloid in the preparation of ( i) and ( i i ) t i l l after labelling vhen i t was added to reduce the specific activity of the preparation# As already stated in Section V the only difference, between these two preparations was that no# (11) was re- chromatographed on G-200 and then stored at -20 C, prior to labelling. By comparison with ( i i ) , preparation ( i) was relatively undenatured# It may therefore be assumed that as both had been subjected to chromatography on Sephadex G-200, supposedly a mild proceeduro, that the cause of the very marked dénaturation of no#(11) was the storage of the IgM, unprotected by albumin# Although alterations were made in the method of preparation of ( i i i ) and (iv)* albumin being added Aftherever possible, and neither stored at -20^C, preparation (iv) did not bind to DEAE A-50 and neither pr^aration appeared to be an improvement on no# (1)#It can be seen from Table 9 that the results obtained for the dénaturation criteria using the four IgM preparations, varied considerably# In spite of the fact that preparation (1) was132labelled with 1*5 atoms of l/molecule, on the basis of the low P.V* deviation, 50 hoür plasma activity and F.C+R.'s* i t would appear to be the best preparation so far#Although many workers must have come up against the problem of dénaturation, i t has only been investigated and discussed by a few# Thus, protein dénaturation was considered at length by Mirsky and Pauling (1936) and by Neurath, Greenstein, Putman and Erickson (1944)* It consists of any non-?proteolytic modification of the unique structure of the native protein, giving rise to definite changes in its* chemical, physical or biological properties# It is an ambiguous term unless the conditions under which i t occurred are clearly established#The degree of dénaturation depends on the extent to which the structure is modified* It may result in aggregation, a process hot specific for dénaturation and i t is frequently followed by the related phenomenon of precipitation- However a denatured protein does not necessarily form a precipitate* Their results indicate that certain biological activities require the specific configuration of the whole molecule while others e*g* antibody activity, may not* Dénaturation is generally irreversible*Pirie (1940) considered, in relation to the modification of substances during purification, that d ifficu lties are primarily due to the absence of a satisfactory standard protein-133 -Colvin, Smith and Cook (1954) found that a relatively small physical difference associated with dénaturation could be associated with a profound difference in the biological effect of the protein, Cohen (1959) suggested that the behaviour of a labelled protein on ion-exchange chromatography, was the most sensitive physiochemical test available for dénaturation, such dénaturation having been ‘caused by self-irradiation, the method of fractionation or the technique of iodination.Freeman (1959) used two in vivo tests, in rats and in rabbits made tolerant to human plasma proteins, Kellar and Block (i960) pointed out that frequently, protein preparations that were believed to be undamaged, have subsequently been found to be at least slightly denatured* The risk of dénaturation may be increased by a low salt concentration, removal of protective substances, vigorous stirring, excess of surface active agents* amd, in some cases, freeze drying.Skvarii (1960) and James, Henney and Stanworth (1964) investigated dénaturation resulting from prolonged storage of y globulin* Andersen (1966) suggested that although a denatured protein may be metafoollcally heterogenous, this is not necessarly the case. Such an evaluation is more sensitive than physicochemical tests , of # ic h ion-exchange chromatography is probably the best. The metabolic studies may reveal an apparently increased plasma volume, increased excreted activity134in the first 24 hours or a variable Urlne/tlasma (ü/t) ratio i Freeman (1966) considered that the most common cause of dénaturation was the method of isolation# Thus while some of the Causes may be well recognisedthe corresponding molecular changes are uncertain* " .Schultze and Heremans (1966) suggested that any agency which interferes >^th the structure of the protein,' will cause dénaturation. Resistance to dénaturation will therefore depend on the stability of this structure# Clausen ( 1969) shov^ ed that Immune electrophoresis may detect dénaturation, e * g # from over iodination# Freeman and Smith (1970) working with Sephadex0-150, considered that some of their results could be explained: ‘ '' : r ; ,in terms of dénaturation caused by polymerisation, lo ss ofpeptides or change of configuration* Freeman (1970) consideredthat when a new protein was being examined, i t was d ifficu ltto be certain that the material under study was not all equallyaltered* Nielsenyand Nansen (1970) demonstrated the effectof selftirradiation on I-label led immunoglobulin in she^*While the risk of dénaturation has to be borne in mind for a ll metabolic plasma protein studies, i t can be seen from the work on human IgM that i t is a particular problems with th i$ immuneglpbulinT: Mul1er^Fherhard, Kunkel and Franklin (1956) found that they were never able to completely eliminate larger moi ecu! Us from their 19s pr^aration* This tendehcy to form aggregates when purified was also noted by Schultze, Haupt,,13b; Heldei' Moschlin, SchmWtberger and- $chwlck-'''(19'62)* ■"' After, repeated --u ltm cent#fu#tlon .they found that only a minor iOs component was present* Wilkinson et a l (19ô6) suggested ,-thàt" the in i t ia i rapW'decline^&n plama and # d y lev e ls o f ' -^ytheit-.lahellod^macpogiobuiln m lÿiVhe accounted' for .by an . aggreghtW fraction* ' -:, ■ ' '-'6outsoh'(l$#T)' fotm d$b'to\#5 $ o f h is 1 0 p^^aration - /.■ consistodypf polymers# jensen - ( l# 9 h ) - made a sW iar'.suggestion ■' ; wi#'' regard to{#mold#ous IgM;studies# --A lternatively/partial,^reduction o f '"1^ m i# t be the'cause* Becher and Btoriko/( 1970) ' Reived that standaidsprepared for radial diffusion m l c o n t a i n ' only 43ÿ o f 19S # le c u le s . ' ;. As w # l 'as ' th is tendency; to,-polynerlse, Mànnik (X96t) Stowed that # fom s complexes: tvlth albumih,' In' vivo# ' - ,- ,-■ vThero' (is. :evidence th a t some o f th e f i r s t - p r e p a r e t l o n s .■ V used for meteboliS, studies -mr#:- denatured, -'to variable degrees# ■'; |hus Oohen ato freeman (i960j obtained an in i t ia l Fractional■'-'’Oatatollc- Bate of'3b;,to' 3â f/day # ic h decreased.tolb; to IB'g/day after- B t o ,7-days# :p l# en (1963) found.;that ;.3'out |o f "-T of h is .1# pr^aratlons were, p artia lly denatured,-V in c^lte'-'o.f the. -Use o f mi.ld; mettodS ■ of p.r0 aration#. He..,showed, that a: single .euglobulin precipitation 'Way cause a gross',; '.denatUratlon .and,,.oveh^d cold agglutinl.n.preparatlon ' w asparti.ally '. 186 -denatured# Svartz, Sohatz and Madman (1965) found that although cold fractionhtion did not appear to alter Rheumatoid Factor ammonium sulphate precipitation dissociated i t and changed the characteristic Immunoelectrophoresis pattern# Wilkinson et hi(i960) considered that macroglobulins were easily denatured during purification, particularly, i f repeatedly precipitated and separated by ultracentrifugation*Birke et al (196?) could detect no physico-chemical changes in their IgM preparations, by electrophoresis, immune- electrophoresis or molecular sieve chromatography, as previously suggested by Plantin and Norberg(19ô5), They were unable to rule out partial dénaturation in a few cftheir patients, Ào showed an increased elimination in the first few days* They considered that there was some doubt about earlier studies* Jensen (1969a) concluded that his I # preparations were either partially denatured or heterogenous# Olesen and Hippo (1969) found that molecular f rainents were catabolised at a much higher rate# During purification IgM was relatively easily denatured, in part or total#Jensen (1969b) showed that thèiîmethod of Barth et al (1964) and the use of DEAE cellulose combined with Sepharose 4 B gel filtration both caused partial dénaturation of IgM.He found that in itia l lipo*pxoteih removal followed by molecularsiève chromatography on Sephaxose 4 B, heforo and after isotoplc labelling gave à süpèrlor aration# For the second separation the IgM was protected by donor serum# Norberg et ai (1970) had similar prèbiems vdth 0 ^ macrp|i|.obulin# Their labelled préparatif contained lb to';'20 % of 15s material, butfound that teoyclihg increased the risk of fre^entatiOn#It is thus hardly surprising that the first Bovine I # studies described here have run into the problem of dénâturation#In addition, no allowance was made for aggregation other than rechromatogr^hy on Bephade^ 0^200* %%ile aggregation does not In it s e lf necessarily constitute donaturatidn It is often associated with it* It Is ;One possible os^lanation of the dénaturation occurring durlhg the preparation of I # and may in part account for the abnormal behaviour of preparation (iv) on DEAE A-50 Eephadex* In future i t would be advlsable to examine a ll pr#arations by ultracenttllugatldn, before use*This tehdency for I # to aggregate has been noted before, by a number of workers (see Introduction)* The breakdown o f the I9s units to: smaller units would-have been detected by rechromatography* Thus fragmentation can be ruled out as a major sourc'e.-of '#ecdenatùmtion$' 'In the earlier work with IgM in man. i t was found, necessary to wait until the dally F.C*E* became constant* SUoh a preparationw hile y ie ld in g In fo ^ a tlo n about the metabolism o f IgM would s t i l l be uneatisfaotoxy for deteim lning the e f f ic ie n c y o f absorption o f co lo stra ! IgM# 'Various suggestions, have been made as to how the problem o f dénaturation may be lim ited or avoided# Alexander and Block(19 6 0 ) suggested that th e .r isk o f protein dénaturation could be /educed, by a :high concentration o f neutral s a l t s , a pH Glosé, to^'-the^isoelactrio p o in t, processing and storage a t low tonperatures and/the presence o f lip id s#K illander and philipson (1964) abandoned the use o f the Tris-NaGl buffer for molecular sieve chromatography o f Rheumatoid Factor, in favour o f a phosphate buffer# Porath and îli { 1964) prepared y g lobulins using g lycine rich solvent systems# th e use o f ,g ly c in e to s ta b i l is e so lu tion s o f Y globulin had been suggested previously by Kabat and Meyer( 1 9 6 1 ) # porath and lîi a lso Includedo^ amino caproic acid to prevent p r o te o ly tic digestion^ (as used by Skvarii and Grunbergef, 1 9 6 2 )* Other workers have suggested the addition o f albumin, dextran or vdiole serum to p u rified or p a r t ia lly p u rified preparations (Ahllnder e t a l , 1965, and p lan tin ahd\'-'' Norberg, 1965)# F in a lly , Jensen e t al (1976) suggested that some o f - the d i f f i c u l t i e s associated # t h IgM separation: ; proceadures, may be p a r t ia l ly overcome by the use o f serum_ ' ^^139 * - ^ ' ' :h igh content.of'-m onoclonai;;!#!, ,m o ta r tio g m aterial#- In view o f t W # suggestions an d 'o f the fin d in g s in these ■ :s tW io s , a_ n u # e r o f points', ohouid h#\ooheidoxed before fufëhex ■(investigations are imdertakon^ ' i f th e .b a s io # # 2 # . séparation :'in ,/et0 inod$ the follovAng; changes - # w M -be ooneidored# . .. :'A'.' ohahge o f b u ffer , 'from .|xio-H0T^to„;;a' Phosphate or, Giyol'no _.,:Wffer* %e U # o f # ih#ih ,' a# ‘a ^protective oolioid"'-''vM'oh - ..'was;. started-in'; these stu d ie s , or a lte r n a tiv e ly , -fibrinogen - .or.-a high m l ocular # 1 ght dextren#'. . '' 'â lternativo lY *‘O-2O0'‘.càuld,bë' rep.laoed' by B'^harose 4 B , '.’ as’- used by Jensen (19Ô9 ) # . Another ' method■ worth considering -would be large sca le is o ^ e ië c tr ic 'focusing Grpydon,-'v;Surrey)# Xbn^exchange chromatography -should probably not be 'used to .pr^ are .IgM;'for metabolic stud ies because'.-of a .risk o f .dénaturation resu ltin g from unfolding o f the .molecule* '' VJhatéVer 'Bàsi-o ■'method o f preparation i s used , lÀere ' '.'■ 'possible' fresh as opppsed to stored serum or co lo stra ! vAey '.. should bem used as 'startin g m aterial -and' 'during preparation, •Vstorage'-at ■ ■ stould" be ^aybided#In':: addition -to .rechromatography, o'n 0|AE A-SD and G 2^0'0 Sephadex'. the preparations should be examined by a n a ly tica l '■ ultraOent'rifMgation#;;-;Xn^'^dvo'--,.examination In rabbits made'-- - ■ to lera n t to bovine plasma proteins %'^ wld a lso bë very u se fu l#140Finally* with regard to the experimental animals, inhere possible It would be bettor to avoid the use of diarrhoeic calves, until control values have been established#141 -Four preparations o f Bovine XgjM (obtained by molecular siev e chromatography) were studied# Evidence was found viiich indicated that they were o i l p a r t ia lly denatured and thus not su ita b le for providing the infoxmatich required for a study o f the absorption o f co lo stra ! I # # ; Su itab le dénaturation c r ite r ia m vo investigated* the d istr ib u tio n o f two o f the preparations ind icated that Bovine 1^4 (a s In other sp ec ies)Is retained to a greater extent w ithin the c ircu la tio n ,.th a n IgG* Although apparent plasma h a lf - l iv e s varied considerably, ¥ B, C end.G a ll, ind icated à 1% o f between b and 10. days* ''- -:i43 -"... "tho Importonce of colostr# in'tho' transfer, of'.pasalve . - Immunity was recognised by I^rlloh (1092) along with placental transfer, in the mouse# Famulenor (1912) showed that in goats, the role of placenta was négligeable# He considered that the main difference betiveen colostrum and milk ms in the amount of serum proteins present, normal milk containing only a trace of globulin* The h i# antibody levels In colostram resulted from a storage of antibodies rcwved from the blood stream* ' These antibodies mre most readily absorbed immediotely after, birth, ago being critical .in. relation to ability to absorb# Subsequent work has expanded these ideas, and.applied thorn to other rmlnants#B om (1921) demonstrated that the c a lf rece iv es its* f i r s t supply o f g lob u lin s from the colostrum* A fter the in gestion o f colostrum r e ia t iv e ly large ç^ u n ts o f euglobulln and pseudoglobulin 1 were detected in the s a r ^ o f the Calf*Bmith and L it t le (195^) in a controlled éî^erim ont, demonstrated the importance o f colostrum in ensuring the survival o f the nevtorn c a l f * They concluded that the c a l f deprived o f colostrum lacks someW ng. ■ ib i s 's itu a tio n pexm its.-In testinal b acteria . to invade the body and m ultip ly in the various organs*144 *Aoehaffonburg, B a r lé t t , B riggs, Qotchin and to v e li(1949), shovved the p ro tectiv e .factor- in colostrum m s ' ■ prosént in tho aqueous phase* Thoy produced additional evidence fo r the re la tion sh ip between the in gestion o f colostrum by the nevAorn c a lf end subséquent survival#Dixon, Weîgio and Vazquez (1961) showed that the amount o f Y globulin in the colostrum corresponded approximately to the amount lo s t fYom the c ircu la tio n o f the cow* t i t t l e or no Y g lob u lin , was formed in the normal udder* The écinàt: q^itholium a c ts prim arily as a transporter o f serum p ro te in , y h ich i s transfered unchanged# Hammer, Kickhofen and Machov/ (196B) considered th a t th is tra n sfer , vdthout obvious degradation and resy n th esis , was one o f s e le c t iv e transport and secretion o f Fast XgG* The se le c tio n o f Faét rather than slow igG, they suggested, depended on o ith er a sp ec ia lised transport function o f th e ■ Fast ' IgG m olecules or ^ e c i f i c recep to r ,.s ite s on the surface o f the acinar c e l l s corresponding to s i t e s on the Fq fragment o f the Fast Ig0 molecule* They demon#ratod the s p e c lf l ty o f the process and found some evidence to support the concept o f s p e c if ic receptor "sites# Subsequently, Feÿ (1971) found evidence that the Fo p iec e does not p lay a d ec is iv eThe re la tion sh ip betm en hypogammaglobulinaemla in neonatal1 4 5 V*ca lves and.the absorption o f co lo stra ! immunoglobulins, has beon-'otudlod :by'O' number o f mrkors* ; /Belman '( 1 # 9 ) , demonstrated the importohce o f management fàotora* llhen those are oXimioated, the, le v e l of..Op.lostral Immunoglobulin becomes Irqpbrtant# ;.Out-■of 120 c a lv e s , not one- showed ûoormmoû absorptive .a b ility # o f th e 'fa c to rs , in fluencing the q u a lity and quantity o f bovine-colostrum ''were/investigated By KtUse (197%)* He found a vâdo varia tion in. th e to ta l y ie ld o f Inxnunoglobuiin (Ig ) ,'at''the f i r s t m ilk ing, a fte r calving# I t r e fle c te d the Ig _ concentration a t 'e a r lie r calvings* o lder cows g iv ing b etter -., ■■.. coI#t'rum , and-no'seasonal varia tion vas .apparent* He considered that; 12$ o f h i s . sample .pievided an' inadequato amount 'of Ig* From computer sirm lation stùdieB^XKruse, 1970c) ho ; ;concluded..that hypô.gm m gIobulinçW a "cannot be c w p l e t e l y , avoided under reasonable'farm ing con d ition s, because o f -variations In birth- a w u n t. o f colostrum - o ffered andthe. ago o f the c a lf a t f i r s t feeding#i'M le many vjorkors-heve measured the absorption o f sp ec if ic , co io .stre l .antibodies or .IBcv-^cclostral -immunoglobulins in terns o'f plasm a-le v e ls , I t : has not been possible,^ u n til the studios' . ,ih ; Section IV- vare-co% leted* to' .detorrdne .with any certainty* the tp lo l absorbed* Sc,me m rkers in fa c t have ignored the fa c t that a- tonsidorablo proportion,;'of the co lostra i; IgO.146absorbed by the neonatal c a lf w ill be d istr ib u ted extravascu larly l ie * in the extravascular compartment (see Section IV* Introduction) Other workers have recognised that I t i s e ssen tia l for the quantitation o f àbsorption*cpa% ticularly in terms o f the e ff ic ie n c y o f the mechanism* for the s iz e o f th is extra vascular compartment to be known# Once esta b lish ed , a v ariety o f methods can be applied to determine th e quantity o f a p articu lar antibody or Immunogldbulin In the colostrum and in the plasma (or serum) o f the neonatal ca lf» To complete the q uan titation , i t i s only then necessary to détermine the plasma volume at the time at which the plasma le v e l i s assessed# This may be performed with Evans Blue or with Albumin or IgG, la b e lled with the iso top es o f iodine» th e tim ing o f th is plasma volume measurement i s im ortant* Selman (19Ô9) based h is 43 hour plasma sampling time on the time taken for y g lobulin to eq u ilib ra te in the rabbit#Kruse (1969) on the other hand used a 24 hour sample, when plasma le v e ls may be maximal* In Section ÎV the/apparent equilibrium time for had boon found to be 45 ïfc. 16 hours* Thus, allovAng for the in terva l between p artu rition and feed in g , the delay between the in gestion o f colostrum and the appearance o f s ig n if ic a n t q u an tities o f IgG, in the plasma and a lso for the period o f absorption i t s e l f , the time for measuring the plasma volume and plasma le v e l was fixed at 48 to 72 hours#-■ 14? -■"Cal ves' .';■ - o n e , . o f t h e C a l v e s \ # r $ ' q W a l n $ d ; : f # m G a r t l o c h y'"'FaW* Gart4àsh/‘% illle s tç a ffoR|-|i(00dlleo. Fem* ■... Lenzla)# /;:%é''yGàlveë' we/e Tees thanythree;to old. and had hem } ' fWWvW ;Wm''their/dam :0t 'h ir # # - . W.l/hytheye;m##^^ ^ 'Q/bl ■ ' ; (an *: they 'ware'aUvAyrahiro. Wll:.Galyo$'# .. \. y--'All''but onè,.af^.,tihà éàlVeS;'raCalyad;;matarhàl colostrum* received'-''# coWstrwa -ës‘ C/O0";'vèichyhàd;Been stored ' \at;:^26^ë'^untll';/equi#^ tracer:''-#entity, (^ p w x # . 400 pc)'of la b e lled ' Fa at Igë# . pr^arod Jh^ advance 'aWy stored: a t : /*2 6 %'.%va$'-added "to,-$ knowri vçlume: 'of \ the' colostrum th a t/th e ' c a lf was ''..toyreceive#./,. 5 'or-15 ml ublobtral; stà'hdardç ware prepared 'BW'';20^ mB'of colostrum retained-;for ' further okamination^ The' temp'emture ,0 # .the- colostrum/.and g.f..thesmall'.'-.quahtltlos . '■of. milk y-gl Von 'before -arid. Immediately, a 'f t# It',- ' # e ' - approximately -,A . ; : : / : y . / :B#"-;:yy.^À - :rTyyy: -;4y, - \ ' , yA. / . ,»' . ( 3 ' tb y : d : 't o ç /ç \p O ç t . .p a - ' _ y. - ::'y 'y'..:'Cy%2'\ma % d 'U s in g '- a : :R o s e 4 A li le r the other ,.148oùlves m te fW o, û î colostn^m by 0tùmaÆ: ;tube* PTiot-.%Q feeing# the eelyee m m ûvi^û'mû weighed#- %he2ô urine mâ feee# vmm-lp he collected s 'brlÿ iet harness# "rubber ohute", and pblythéne bag attached to the calf i^Moh'VM then piaced in a metabolism'coge.. ( see, Section I Ï --È W * . / ,; ’ , / ,:■ ; :A stomach tube (Foal^ Portox Ltd^^.Hythe» Kent)with funnel attached#- vW passed via, the nose# so tha t the end of ' i t was opproximaieiy/'half way‘"down;the oesophagus# - Suckling rnovdmonta'TOro.-'tîxon stimulated"by'feeding th e 'e e l i .ÊO-ml'of / / milk from a syringe#, Pimaliy#. before' giving, the colostrum#,50 ml o f saline was-poured into the tube to-chock i t s position# so for m was. possible#’-‘With the .funnel raised - to. a suitable height'- the colostrum was slowly'odjninistored#. ; The . tube vao ■ _ - vjsshed out with 50 ml of -.milk'^before being remvcd# -UlovQ on attenpt mB made to measure the to ta l loss of ingested Ig0, necessitating-the col.leotion of o il excreted ac tiv ity over the f i r s t 4S to % hours* a balanced solution of Kl/tlal (containing # o equivalent'of 1 0a of I ) was slovÂy Injeoted:"Via the intravenous catheter (see Section-II C lb)# , Ati oral dose of 'Kl-.was' eéninistored a t .the samo time end , subaoquently every- 24 hours# - ^ 14’0 **■ £îa■ To', determine tho rato of appeorenco o f .103 i n the plasma» B#pW@, w re tokep; ewry^lO to 15 mlnutee over a 2 hour period* once signlfluent lev e ls had. been deteotod#I ,■ .The first-m llh,'-feed m s given 6 to 12 hours a fte r feeding . coXostrimi» Milk wao then fed tvdce daily* see 'S o stio n II A 2*4B to V2 hours a f te r feeding colostri^ * the plasms Volume was measured with ^^^I’^ labeilod îg 0 (epprox* I ^no/oslf)* Using # i s plasma' volume and the 'i^travàscular/lntravàsouiôr. ra tio ' o f i# 2 / l (from Section IV) the to ta l body a c t iv i t ie s .o f 'the - I 'IgG ^ vms'- oo lcu lated from a plasma oumple taken-immodiatoly p rior to in je c tio n o f the la b e lle d IgG, ' -ibia a c t iv ity was .then exi^resscd a s a % o f th o ’to to l a c t iv i ty , in tho colostrum fed# -Tlie .e ffio len o y o f éb so ïp tioh -o f 103 thus dotàrminod w ill be'-subsequently referred to', as the Iso topic, H fflcloncy#■ --iho %# concentration" in the pro^colostral# 48 to ■ 7a hours'serum samples and co lo stra l whéy’aaïfiples wore ^dptormioed by Radial D iffu sion (B oction .II B 5)# doing-an .anti“2x).vino Xg6 ant.lsorum (adsorbed vâth bovine 10 4 ), Standards- \vore ■prepared from Ig% (supplied a s ,7 s ^ Gama Globulin* pure by Immunoeloçtrophorèois* from f4ann Research baboratorioG, Nov;im ^thù to ta l ompunts o f oolostrai 10 Cgm) -In tho coiostsmm and ' in 'the ogII a t -48. to %' hours 4% # dotemlhod using a oorroation fàctoÿ o f 0#84i. for "tho, oolootr#! IgG». to allow for tho ooaoln c lo t (Salman* 1969)* Tlio Redial Diffusion Bffioiency viaa then ..obtainod.'.from -the 'ratio-.of 10 ab$orbed/lgG, fed and o?^roo$ed .as a 0# : - V , ' ■ ;■ ' ' in 12-o f ' # 0 calvos*'-'tho.-distribution (by one or-both ■ mothodo* aoQ'Seétion-XV) mû. the oata#ll$m of. the X03|.' * ■ wed for #o,. 48. to 72- hour 'plaarna volume wero' otudiodt - as in '■, Section IV*■I#Those have already been covored in-Boctloho, 11» III and1THE APPEARANCE OF I-L A B E L L E D FAST IgG IN THE PLASMA OF C /C 3 (EXPRESSED AS A % OF THE TOTAL ABSORBED)I■g031<75 r2520 40 60HOURS ( A FTER INGESTION OF COLOSTRUM)m■ ' ■ s m m m = .The appearahoe 6 f the tracer was followed for the f i r s t 48 to 72 hours9 a fte r the in gestion o f colostrum* In f iv e o f the ca lves s ig n if ic a n t le v e ls o f . i^ la b e lied igG were detected at 2*2 * 0*9 hours# As i t was not p o ss ib le to c o l le c t regular sarrples over the whole period in every ca se , th is may not represent the e a r lie s t time for a l l th è ,calves# For G/C3 and C/C4 the in i t i a l r is e was found to be gradual# This was followed in every case by. a steep r is e in thé le v e l o f a c t iv ity representing the predominance o f absorption over d istrib ution * I n it ia lly # in three o f the calves (C/C3, C/G5 and G/Cll) the • plasma a c t iv ity doubled every 0*40 * 0*11 hours# This then decreased to every 1*4 * 0*25 hours. (C/G3# C/C4# C/G5, C/C6 and G/G?)# The apparent rate o f absorption was thus changing a l l the time* As absorption decreased and/or d istr ib u tio n predominated, the rate o f Increase decreased s t i l l further (see Fig* 27)* In four calves (G/C3, G/04, C/06 and C/Cll) the h ighest plasma a c t iv ity was reached 18 ^ 5*4 hours a fter the in i t i a l in gestion o f colostrum* I t f in a l ly appeared to le v e l out at 29 * 11 hours, as catabolism becomes the over* ^rid ing processw I n it ia l ly # the plasma a c t iv ity representsEffioleney of AbeDiption -.of # W ; Fast -u/%,./C30/C9e/Gi:Volwe ù i Colostrum( .l it r e s )■Isatopic B fflc im cy : '; ( ; ) ■1 .00. a ,004 .0 04 1.004 .0 04 .0 0L o o-i'*1*00443165Radial ; D iffusion'■Bffioienoy ';■■ ' ('/>).5758 26 79. 40634 * 4* w * # .R # « # # # * * W # * » m # N, w W .# W w p à W. I N # * # # * * * & # * * . # * # # *45416 #5 ■ 4*616*54*6w. the intoraction-. absorption# d is tr ib u tio n àndcatabolism#, the xoXo'of. absorption cubsoquently déoreacing# 'fôllovvod by that of, d lc tr lb u tio h f . ,-,. {oeo'Table ' 19)'- '. ' tlie moan o fflc io n cy o f .obcorpilon'ior a l l ,1 0 m iv o p m n :46 6 16*5 0*.. l%eh ytho rocul'ts aro broken ,dovm on the h m iB o f tho volume .of 'colostrum ' fe d . ( caivoo ^'Q/G2 ond-Q/ci# oKcludod) ■ th is reproBontod 54 îiî 1? % for Group, i Calitro) and 36 à l4 $ for Group 2 .(4; l i t r e s ) ’#. The CalvoO-vmro o lso divided in to group0 o n 'th e 'b a sis o f yhich 'o fflo ien cy range tiiey . corrocpondod v-0 ^ 10.10 , ^v '20''20 - -30 ■• ' ■ 0/C5 ■ 0/0940 - 50 ,'■ G/gB .0/04 0/06 G/C12-.50 ^ 60 , ■ ; ' ■ c/07 . '( /to60 ' . 70 . / ( m70 " 00 ■ . Uùm90:' ..r /90 ^ .1 0 0 : . ■ --"'Mom' 'i£ È 4 iS 3 S lâ jja „ '^ lo s iî^ ^; o f XfiG fe d and:cibMr?>Q(JfiM JitâK tiiiâS -la ffl)in 13 oaIves) ' .Calf Nù.,, Colostra! IgG ., Level (mg/ml)Total IgG : V fed (gm)’ Plasma IgG ' Total IgG Level (mg/ml) Absorbed (gm)C /ei . ' ■ 72 V, ■' . 61 . : . ' 8/0'' ^ 42 .. . 'C/C2 ■ ■ 77 7 -7,' , :^ 130 ■' . : . ■ ;■' 18 . / / 79C/CSV : / .....88- / . 2 7 9 ; / ■ 30 159G/C4 142 285 7 32 ■7; 164,.C/C5 '''" iB T 460 . 25 118 ■-;./C/C6 83 70 ^ 7 - 11 à : ■ 55C/C 7 . 73 ■ - 'Oy-"; ■■'■ ' ■ 48 /c/cs 70 ^ 234' '. - 29 '''.. ■- 155G/Ç9 / ; :: % ' , . 291 2 6 / ■ ' . 117 „c/cio ' .'.'7X06':'^ , 89 . ,, li^O V :c / c u 83 279: . . : . /• ■' 10*6 55C/CJ.2 ■ 5 1 -/ . ,'' 45 25 7C/013#MI:,33,w M* *% 4M <#*> *>4 #4 *# fm #* **. w* #»# «rt, i2 7 , -' ; .' .: 3 .0 'M at tM Mb \ jte *W #M« Wm Kk* «M 0#* *•■« *ai M# M* «ua Wk' j*kà «a ,*L «■ aa j15i#É -7 ■■ . 8*3 : . ■% 8 7 . y / ; 2 .3 V". 15Ficaire 28THE CORRELATION BETWEEN THE ISOTOPIC EFFICIENCY AND THE RADIAL DIFFUSION EFFICIENCY= 0. 690U5oo5C /C 4 C / C 2 Group 2 Group 160 100RADIAL DIFFUSION EFFICIENCY ( % )153 -This shows that although the numbers were small ÿ* there : was a .tendency towards a normal d istribution» ■The to ta l amounts o f IgG absorbed are shown in Table 20, along with the concentrations (mg/ml) o f IgG in the colostrum and plasma at 48 hours to 72 hours> from which,the to ta l q u an tities were calculated*tow prerGOlostral le v e ls ( p “ 0*02) for the 13 ca lv esi S im ilarly when the Isot^^ic E fficiency was p lo tted against th d .to ta l amount absorbed (see Fig# 3 1 ), the régression l in e s ind icated an "ih itia r* E ffic ien cy o f 52$ and 56$ resp activ e lÿ $ Neither o f ; thé regression c o e ff ic ie n ts were s ig n if ic a n t* _ / k .: By comparisph with'^% ié V é ls (m g^ l) in colostrum 'found by other m rk ers, 43$3 4= 1 4 .0 (klaus et a l , 1969) anda S.E. o f 2 .?1 (Penhale and .C hristie , 1969) the mean le v e l '■ o f 85' * 30 mg/ml In.; these stud ies ( see ta b le 20) i s high*» The-v mean plasma IgG, le v e l o f 16#9 * 10$1 i s cphsiderabiy lower than .,31$6 ^ 1 3 * 6 : found at 48 hoUre,in suckled calves by Klaus .: et al (iW )' butr :^ -^ : - V v . - / : :mIs higher tfon 7*5^ à S*E* of 1,0# found by Penhale^ ^^ Christie, McMwpn^ Fisher and Selman (Î9Î0) in surviving market calves*Table 21 shows thq apparent distributions of the I IgG^ , in four of the calves These were calculated from the ratio of the total piisma activity to the apparent retained activity less the total plasma activity at 72 hours# At this time there was an apparent distribution (H. Vt/i>VO of 1 «7/1 ± 0*22•The same table showQ %ne brine protein bound activity (expressed as .a $ of thé activity invthe colostrum) Which gradually increased over .this'ptribd/ ;.,, ,, ;Tables 22 to 24 show the results obtained from the additional metabolic studies carried put vAtK the 1^$ used for the 48 to 72 hour plama volume*Four o f the ca lv e s , G/04V C/G5s C/C7 and 0 /C ll died a fter to 5 days diarrhoea ( tH ; ) i id , 10, 13 and 14 days post ‘partùiià, r e sp ec tiv e ly . Over the same period ( 17 days ) a further four ca lv es , 0 /01 , 0 /0 2 , O/OB and 0/09 were also d iarfhoeic ( t f t for .3 to 5 d ays), ^The Apparent D istribution o f Labelled Co.lostraX IgG^ CaS % o f the Total A ctiv ity in the Colostrum)Calf Mo. Calculated D istrib u tion . (E .V ./l .V .) Pay 1 Day 2 Day 3 MeanG/C£> . l / l i h ■ :: Ô.23 0 .62,0*43e/CT. ■ 1 /2 .0C/C9 ; 1 /1 .6 0.79 0,62 0-71c/cu ■ ' " t A .7 ' o a s ' ; , 0 .80 . 1 .6 0-68Mean 1 /1 .7 0 ,38 , 0,51 ' . 0.61/Ô .22 0 ,35 o a o • 0 .15 :-S.E.#: ' ' /Q ,W ■ V / V ':: : 0*20 0Û 0 ' 0.03 ;. . lA lr n .#Thg.MstrAbiitloo (by çàmobeXl...and S ter lin g mand theCalf'No* B*V. / l , : B,*V$/l*y, Apparent* .. Total Body FractionalSterling Gampheli Equilibrium Gatabolie CatabolicRatio , Ratio Time (hours) Rato Hatec/ 0 4 s . p / l 1 . 3 /1 ,4.8 ,0.061 0.131C/C5 1 . 5 /1 1 .2 /1 86 , - 0.061 0.089C/06 . 2 , 0 /1 1 . 2 / i . 48 : 0,047 0.114C/07 1 . 5 /1 \ 1 . 3 /1 60 , O.047 0,103C/C9 1 . 5 /1 1 .4 /1 , .6 0 / :. . 0,095 0.173G/Cli1 A# »* «1 M $W%- 1 . 8 / 1 ;ft}* &E% 1^1 . 8 /10»^ 4** 4f#> lint «ï* *4 p# it84ËP*# VJV «** «4* ^0.038 0.104^ xk ## *# ## ## "*lean 1 . 7 /1 ,1 .4 / 1 : 56" 0.056 0*119! É D« 0 .2 6 / <0 . 2 2 / 16.4 0.020 0-0290 .1 0 / 0 .0 9 / 6 .7 0,008 . 0*012m . .Half-Lives oind : additional dato for. Calves: in.-Tablo 22yiÆixMkt'üKWWr#w W»f'lh ' (1 »«mmiM w km-mWumM, iwm###,# wC a l fN o ,p . V o ( m l / k g )A p p a r e n tP l a s m a T - &T o t a l B o d y M e a nP X . V .P , V * / B o d y 1 i $ ) c h a n g e 1 4 d a y s ^C / C 4 6 8 5 5 . ■ ■■ m w 1* «1 iMiwi iilmii.u*i-iunf Mjj;*Jni.nm>iiiji»iix 1 2 * 2 2 4C / C 5 5 2 ■■ 3 5 1 4 * 4 . 3 3G /Ç Ô 6 8 : 2 2 ■ ' , . 1 6 , 0 • : 2 8 1 * 3 / 4 . 5C /C 7 5 9 1 3 , 1 , ' 1 9 * 0 ■ 3 3C /C 9 6 9 , 1 7 , 8 7 * 1 / ' 3 1 : 2 6 /C / G l l j 7 1 1 4 * 6 ; . . ■' ' '4 6M e a n / ;■" 6 5 2 6 * 8 - , : 1 4 , 6 , ; : 8 3 :S ^ D ê à [ 7 . 4 1 6 * 1 ' / - 4 * 5 / ■■ 7 . 53 * 0 6 , 6 3 . 0The D istribution (S terling) and Catabolism of Labelled IgG, in .a fu rther six Golostrum fed,Calvcs,(plus add itional data)CalfNo.EoV>/l*V.S terlin gApparent . Plasma T|- ,■P,V>ml/kgp.V. Body . WeightMean : P^G,V.C/Cl ; ''■1 . 2 / 1 .- 14$8 .66 / .'■^ 21.,,."; 24Ç/C2 1 , 4 /1 9 ,8 : ^ 64 . «, 6 31 ' ■ ' :1C/C3 1 . 1 /1 ; 17.8. , 58 . 2 '■■■■■'■.. - 36 _ : ..d/CS / , : 1 . 4 / r ■ M 4I, ■ 62 Ÿ - T-: Y ' : 34.c /e io . : ' / i , 4 / l ' , ,12*4 ; 60 IB 35'/..:;, - : ■C/C12 1 .5 /1 52 ' . . 4 ' ;■ .i" i ■ 30 '' \ \C/C13 ; 1 0 . 6 V 71 ; ' ; 4 i v . . .Uem 1 . 3/ 1 , 14,8 62 , 1 ,0 • , 33 :S.D,* 0>il5/ "3 , 0 / : ■' 1-6,1 14,5 : 5 .4 ■Q .05/ - , ;v v i . i : 2 .3 5,9 2 .0Table: 25 y- 'Plasma P rotë in. Result s , expressed.as.. Albumin ( IIP 3b)and -Globulin, concentra tio n s (gm/lOOml ) for a l l . 13 golostrum' isd -S à lïâ s ’: .'Calf No. :Alb, ■ Globk AibV Glob,(,ljp d&Ys),A lb .1 Glob.(I7_days)Alb. 1 Glob.C/Cl , 1-9 i 2-B . 1 .9 1.9 3 .9 1 2 ,2 ■ 4 .4 .0/02 1.7 1 .8 1.6 3 .6 1 .8 3 .4 1 .5 2 .8C/C3 1.8 2 .2 1.6 1-^ 7: , ■ 3 .3 à , 7 . l a a - ;G/C4 1.3 ; .. 2 .4 1 .7 4 .6 1 l .3 , 4 .6 1 ,5 ' 4 .9c/G5 1 .2 2 .6 '1.4':: 4 .5 :, 1 .6 5 .41 .7 1.1 3 .6 ■ 1 .2 3 .8 1 .2 3 .9 '1C/C7 1 .3 2 .7 1.6 3.1 1 .3 3 .5C/C8: 1.2 2.Ô 1.7 4 .1 1.4 1 '4 .4 .,, 1 .4 '.' 4 .3C/C9 1.1 2 .6 1 .2 3 .8 1 .2 : 4 .5 ' 1 .3 4 .2c /c io ■ la 2 .6 1 .7 3 .0 11.2. 3é6 ^/1>2; . 3 .8 ,c / c i i 1.1 '2471/ 1.1 :f . 3.1 .■:1,i>6 3#00/012 1 .0 1 2 .4 2 .8 1 .8 2 .8 1 ,9 3.1C/C13 1.9 : 2 ,4 a a -:; 2 .9 , 2 .0•* M M.m tn w MMi ##2 .7Mean 1.4 .2 .5 3.6., 1 .5 3 .9 . 1 .6 3 .70.34 0 .27 0.26 0.58 1.0.26 0.75 0.34 0.75■0.09 . 0 .07 0 .0 7 1 0.16 0 ,07 0.Q2 0,11 0.24156'DISCUSSIONThe in terva l o f 2 ,2 ± 0#9 hours> between the feeding o fcolb strum and the appearance o f the h b elled IgG, In the plasma(se e VI, R esults A) i s in c lo se agreement with th at found byother workers using s p e c if ic antibodies ( t i t t l e and O rcutt, 1922,Kerr and Robertson, 1946, McOiarmid, 1946 and Graves, 1963)$The same workers obtained a wide range ,of tim es for the pointat iÀ ich maximum plasma le v e ls are reached, 5 hours forB rucella agglu tin ins ( t i t t l e and O rcutt, 1922 and Graves, 1963),12 to 16 hours for anti*Trichomonas Foetus antibodies (Kerrand Robertson, 1946) to 24 hours also for Brucella agg lu tin in s(McDiarmid, 1946). By conparison thé mean time was 18 ± 5 .4hours in these s tu d ie s , for XgG^ *Lack o f information about the extravascular d is tr ib u tio n .o f immunoglobulins in the c a lf has not prevented some workersfrom attempting to determine r e la t iv e or absolute absorptione f f ic ie n c ie s , langham, Ingram, Roy, Shillam and terr y (1958) fed 131I- la b e lle d adult bovine serum and co lo stra l p rotein s to new-born calves# Although at 3 hours, 8 to 16 % o f the la b e lled protelhs were present in the c ircu la tio n , by 20 hours th is had decreased to 5 to 10 These workers recognised tbat th e ir r e su lts did not a llow for the extravascular d istr ib u tio n1 5 7 -o f the la b e lled proteins* Assuming the plasma volume to be 5/& o f the body w eight, P ierce (1961) estimated that 30^ o f the Diptheria an titox in fed to a c a l f , was present in the plasma 30 hour s. post partum* This represented a minimum valUe as no allowance was made for Extravascular lo s s .Hammer, Kickhofen and Henning (I960) Concluded from th e ir co lo stra ! stud ies that IgM is'tran sm itted le s s e f f ic ie n t ly than Y G* This m lÿit in d ica te some sort o f se lec tio n between the d ifferen t immunoglobulin classes# Subsequently, Klaus, Bennet and Jones (1968) claimed that both IgM and IgG were absorbed equally w ell from the g a stro -in te stih a l tract* Both groups o f workers fa ile d to consider the extravascular d istr ib u tio n o f these tm immunoglobulins* From the in i t i a l study in Section V i t can be seen that as in other sp ec ies , IgM i s present to a greater extent in the in travascular con^artment whereas in Section IV I t was found that the opposite was true for IgG.MeEwan (1968) obtained an e ff ic ie n c y o f absorption o f 46% in 13 bucket fed Ayrshire b u ll ca lv es , using an E.V/l*V* ra tio o f 0*65 (Nansen and N ielsen , I960)* The amount o f g lobulin absorbed by the c a lf was calculated by m ultip lying the concentration o f serum p rotein s before and a fter feeding colostrum, by the plasma volume. This quantity o f globulin expressed on a body weight b a sis was then conpared vjith the quantity ingested by the c a l f , ca lcu lated on the same basis*5'/ Subséquentl y 3 using the same r e su lts and applying the E* V , / l -V ;1 ra tio o f 1 *2 / 1 (o a lv e sv l, 6 and 7 in Section iV) McEvvan, Fisher ; and Selman (1970) obtained a value o f 65^# ■ t h is allowed for a 17# casein c lot* >- iKruse ( 1 9 fob) in h is abspiptlon stud ies in 14l ca lves ' (m ostly Black and I t i l t e Banish or Eéd Ba/hish) determined an absorption tb .e f f lo ient ^ assumihg a 40# tran sfer o f iimunoglobulin ; / to the extravascular pool over the; f i r s t 24 hours^//(T his # ” ' ■ ■transfer.: vvas. ca lcu lated from data for human XgO)V ' the h igh est ; value he obtained; was 43# assuming thé plasma volume to b é/5# o f Body W ei^t i#e* a.maximum of. 54# using the/mean plasma volume o f 63 ml/kg (Table S ), /.The.meah' c o e ff ic ie n t or,''■ - e f f ic ie n c y o f absorption ; for the 72* Black a n d c # lte ^Dai'nish.. calves,w as SO.-l'-.sfc S*3"(|Cruse,;-1 9 6 9 . ■■■ -t : Of th ese s tu d ie s , only the wpfk o f Me Ewan: e t a l (1970)%sed oh ;giobulin le v e ls # e work o f Kruse (1976) with*^iwunoglobulin" 1 evel s dériyed by olqctrophorests^ have r e a llymeasured. .effi.ciOncyi /; However.-neither 6f . these methods' includes ;- thé- dlréct,.measurément\:ofIgG and; both-depend on data derived from iso to p ic studies* Table 19 g ives th e e f f ic ie n c y o f , absorption by W th th e se m e#iodsy(Isotopic ;and Radial ■ D iffusion ) both o f vihich ■ are\ sp eclfic^ fo r /F a st igG; (IgG, )*The measurement o f : the B fficioncy o f absorption by both these , , methods 3 was the main o b ject o f th is Sect! on*, 159 " 'These r e su lts in d ica te an E ffic ien cy o f 50# ( vhen the mean values o f both methods are combined) which i s o f the sàme order, as the 65# value d f Mo Ewan e t a l (1970) and the maximum corrected., value o f 54# derived from the r e su lts o f Kruse (1970)*Although there was a wldq sca tter in the e f f ic ie n c y with which a l l 13 ca lves absorbed co lo stra l IgG (as determined by. both methods seé Section. VI^ R esu lts B arid Ç), the re la tion sh ip between the amount o f IgG (gm) fed and the amount absorbed (see Fig 29) strongly suggests that provided a certa in lim it (vMch may vary from c a lf to c a lf ) i s not exceeded, the absorption mechanism, under the Varied conditions in these experiments, absorbs a f a ir ly constant proportion o f the IgG presented to i t o f the order o f 50 to 60 The upper lim it iwuld appear to be reached at around 280gm o f IgG in 4 l i t r e s o f colostrum*Of the f iv e ca lves offered th is quantity, or more, three o f them absorbed 23, 27 and 31 # resp ec tiv e ly i#e# the e ff ic ie n c y was reduced by about half* Additional work w ill be required to in v estig a te th is lim it in re la tio n to the volume o f colostrum and the to ta l IgG (gm) fed* I f these three ca lves are a lso excluded from Table 19, the mean E ffic ie n c ie s become 52 it 14 (fo r the Iso to p ic method) and 62 4 7*6 (fo r the Radial D iffu sion method)* t h is vx)uld ra ise the E fficiency to 57#. vihen the r e su lts o f both methods are combined* Additional evidence concerning the160e ff ic ie n c y q f the absorption mechanism comes from conparisons between the iso top ic e ff ic ie n c y and the quantity o f IgG (gm) fed or absorbed (se e Figures 30 and 31)* In both graphs, the regression l in e s , p a r ticu la r ly for 10 o f the ca lves (i*e* excluding C/Ç5, C/C9 and c /G il) are nearly p a r a lle l to the abscissa and c lo se to the mean effic ien cy *: The oonsiderafale individual varia tion in the e f f ic ie n c yo f absorption found, may be the r e su lt o f a number o f factors* F ir s t ly , the tim in g 9 the feeding o f colostrum, i#e* from 3 to 6 hours and secondly, varia tion in the timing o f plasma sanple and plasma volume determination over a 24 hour period i*e* 48 to 72 hotars* Further, no attenpt was made to standardise the quantity o f IgG fed , on a body weight basis* In sp ite o f these v a r ia tio n s, the basic trend shovm by Figures 29 to 31, supports the finding o f Selman (1969) that under a p articu lar se t o f circum stances, a fixed proportion, o f the iïmnune lacto** globu lin presented to a c a lf may be absorbed* i*e* that Ayrshire ca lves are born with an eqUal absorptive, capacity»,The d istr ib u tio n o f IgG ^ , (see Table 22) in the colostrum ca lv e s , using both the S ter lin g and Ganpbell methods o f ca lcu la tion was sim ilar to thtt:found in the o r ig in a l stud ies o f the imetabolism o f IgG^, in Section IV (see Table 2)*The measures o f catabolism Were also sim ilar* When the_ ,'161 *apparent d istr ib u tio n was calcu lated from the co lo str a l date (see Table 21) the mean d istr ib u tio n (E .V ./l.V *) for four calves was the same as the, S terlin g d istr ib u tio n i*e* i . 7 /1 , but higher than the Gan^bell d istr ib u tio n o f l* 4 /l* We have therefore additional evidence that fob in the neonatalc a l f , the oxtravascular compartment i s proportionally larger , r e la t iv e to the in travascular compartment»À number o f workers have studied the proteinuria associated with the in gestion of, colostrum in neonatal calves* Smith and L it t le (1924) observed that i t developed soon a fter b irth and then disappeared a fte r the th ird day# The low le v e ls and the graduai increase In the protein bound a c t iv ity found in four calves (see Table 21) in these stud ies support the finding o f P ierce and Johnston (1960) that immune làctog lob u lin s are la rg e ly absent from the proteins (mostly o f low molecular weight) associated with the proteinuria#Other workers may b e ,c r it ic iz e d oh the tracers they used for the study o f absorption o f colostrum* Hardy (1969 a and b ) used a high molecular weight PYP (160,000) and an iso to p ic a lly la b e lled commercial bovine y g lobulin preparation* Finding that a fter absorption, the plasmaa c t iv ity o f both tracers was sim ilar he concluded that the■ : ■' ■ - . ■ 'PVP préparation ,was su ita b le for absorption studies# In the162f i r s t p la ce , although the molecular weights are very s im ila r , the two substances w ill have very d iffer en t d iffu sio n co e ffic ien ts* Thus, the rate and extent to/ which they eq u ilib rate with the extravascular compartment w ill be very d iffe r e n t . Secondly, the ïÿ 3 preparation i s open to several o b jectio n s . I t w ill contain a high proportion o f ïgG^ which i s almost completely absent from colostrum and neonatal c a lf sefum* The method o f preparation i s such that a probably s ig n if ica n t proportion o f the p rotein w ill be at le a s t p a r t ia lly denatured* In the stu d ies described here, every care was taken to prevent such damage to IgG^ preparations.iSelman (1969) found evidence o f a maternal factor that resu lted in very S ig n ifica n tly raised irmunoglobulin le v e ls in ca lves l e f t with th e ir dams, although not allowed to suckle them. These ca lves and the control group were fed from a colostrum p oo l, on a body weight basis* I t i s d i f f ic u l t to compare Selmans work with the stud ies in th is Section because o f a number o f d ifferen ces with regard to the experimental d e ta ils e .g . amount and quantity o f colosttum , method o f feeding etc* However, the E fficiency re su lts in Tables 19 and 20 in d ica te that a wide range o f e f f ic ie n c ie s occurred in those calves which were fed and absorbed r e la t iv e ly low q u an tities o f IgG, (i*e* the Group ; 1 ca lv e s , the range being: iô3'.r32^ to 81% )* Thus, while in no way accounting for the find ings o f Selman, these r e su lts in d ica te that there i s ample room within th e range o f individual e f f ic ie n c ie s for two groups o f ca lv es , the immunoglobulin le v e ls o f the "mothered" group being 70% higher than the "nonrmothered" group* Using the information concerning the d is tr ib u tio n o f IgG ,, and the iso to p ic method o f measuring E ffic ien cy , i t i s noiv p o ss ib le to in v estig a te more accurately the factors in fluencing the absorption mechanism I t s e lf* Whne many u sefu l stu d ies have been carried out in large.numbers o f calves using a variety o f estim ates o f immunoglobulin le v e ls , with the iso to p ic method for measuring the e ff ic ie n c y o f absorption in individual ca lv es , i t should be p o ss ib le to work with much sm aller groups o f animals e*g. ivhen in v estig a tin g the mechanism o f ^shut down^ ' in the neonatal c a lf i*e* the cessa tion o f the absorption o f co lo stra l immuh&'~ globulins*“ 164SUMMARYThe E fficiency with vihicH the hevpborn c a l f absorbs efelostrai Fast IgG was In vestigated by applying theE, V ./l • V* ra tio o f 1 #2/1, previously determined ( see Section IV) ^The mean "Isotopic" and "Radial Diffusion" E ffic ie n c ie s Wien combined, gave an E fficiency o f 50%* % o f1gG ( gm) fed , was re la ted to the amount absorbed, a fa ir ly constant E ffic ien cy o f 60 to 60% was found (up to an apparent lim it o f 250gm, in 4 l i t r e s o f colostrum)* Further evidence for th is was provided by thé re la tionsh ip between thé q u an tities o f IgG fed or absorbed and the "Isotcplc" Efficiency#Information was obtained about the in i t i a l appearance o f IgG ^in the plaWna and i t s rela tionsh ip to the proteinuria * The d istr ib u tio n o f the O olostral IgO, over the f i r s t 72 hours confirmed the presence o f a r e la t iv e ly larger extravascular compartment#165MI’ ■ ' ' , 166: . IMTROpUGTIOMMany workers,have demonstrated the presence o f plasma p rotein s in the lumen o f tKe alimentary tra ct- Using auto-V radiography, U llberg, B irke,Ew aIdsson, Hanson, U ljedahl,P iantin and Wetterfors (1960) found that albumin i s discharged in to the ,lumen.of the small in te s t in e , in ,addition to the stomach. The exact ro le o f the gut in normal plasma protein metabolism i s however, s t i l l debated. On the one hand,Armstrong, Margen and Tarver ( i 9 6 0 ) , .W etterfors, U llberg,Ulhedahl, P ian tin , Birke and Olhagen (1960), G lenert, Jarnum and Riemer (1962), Andersen, G lenert, and Wallervick (1963), Gan^bell, Guthber.tson, Mackie, McFarlane, P h ilip son and Sudsaneh (1963) and Vs/ettorfors, (1963), a l l found evidence that the alimentary tra c t accounts for a la rg e p a rt, i f not most o f the albumin catab olised . Their work has been c r it ic is e d on the .grounds that varying degrees o f surgery were involved .Franks, Mosser and Ansladt (1963) concluded that no more than h a lf o f albumin catabolism normally occurs in the gut- When they removed m ost,of the jejunum and ileum in ,ra b b its , the catabolism o f albumin was not retarded- Jarnum . (1963). considered that only 5 to 10 % o f albumin catabolism occured in the gut. Freeman (1964) using an improved resin167method { see Jeejeefohoy, 1964 ) obtained re su lts which supported the'yiew that only, a small % o f the d a ily albumin catabolism i s due to in te s t in a l leakage.,; In p ig s , Dich and N ielsen (1964) found that 10 to 30 % o f albumin and V globulin catàbollsm could be accounted for by in te s t in a l degradation. The same workers (N ielsen ahd Dich, 1965) found an enormous variation in calves in vMch albumin and y globulin were excreted in r e la t iv e ly s im i la r \ q u a n titie s , into the in te s t in e .Other workers, R othschild, Oratz and Schreibor, (1965) and Schultze and Heremans (1965) considered the evidence to be very c o n f lic t in g , a view which had previously been expressed by Freeman (1964). VValdmann (T966) f e l t that further work was necessary before the exact magnitude o f protein gut lo s s in normal subjects i s understood and the mechanism elucidated .R othschild, Oratz and Schreiber (1966) and (1970),Freeman (196?) and.McFariane (1969) a l l considered that catabolism occurred throughout the body, many t is s u e s possessing the p o te n tia l, the exact fraction lo s t in the gut being e ith er unknown; or sm all.In addition to the normal leakage o f plasma protein in to the alimentary tr a c t , i t has been known for some time that certa in diseases, are associated with an abnormally■ - 168 - , 'large lo s s o f plasma proteins into the gut. The discovery o f these p ro te in -lo sin g gastroéiateropathies ( a term introduced in an E ditorial in the Lancet, 1959), was largely due to the observation that I la b elled plasma proteins given in tra venously were rapidly excreted in the measurable q u an tities in to the alimentary tra c t o f p a tien ts with certa in d isea ses , but not to any extent in normal people. The assoc ia tion between hypoproteinaemia and protein lo s s in to the alimentary tra c t was in vestiga ted by S te in fe ld , Davidson and Gordon (1957), Holman, Nickel and S leisenger (1959), S to in fé ld , Davidson, Gordon and Greene (I9 6 0 ), Jarnum (1963), Jeejeobhoy {1964) and Jarnum, Jensen and Bro-Jorgensen (1966)* Waldmann and Schwab concluded th at i t was a bulk lo s s o f a l l plasma p rotein s associa ted with the hypercatabolism o f albumin and IgG. Waldmann (1966) in h is review, l i s t s over forty gastroin te s t in a l conditions in which excessive lo s s o f jplasma p rotein s has been demonstrated.Hypercatabolism associated with gut lo ss o f plasma p rotein s has been demonstrated in a number o f animal d isea ses . N ielsen (1966a) in vestiga ted O ster ta g ia sis , Johnes D isease and chronic catarrhal abom asoenteritis in the bovine, terminal i l e i t i s (N ie lsen , 1966b) and chronic e n te r o c o lit is (Nansen and-Nielsen, 1967) in the pig,' both being associated with ^ . - ' T / - .-;. d i a r r h o e a * ' ; . •-■ ' . ,3 Marsh; Mebus a h ^ found a s ig n if ic a n t ly: increased lo s s Of a l l seruan and unabsprbed milk p rotein s v ia the in te s t in a i tra c t in diarrhoeic neonatal ca lv es.They conolWed that the observed lo ss o f proteins via the faeces during diarrhoea could ser iou sly reduce the chances f : ; o f the ■calf surviving severe diarrhoea dr comp 11 eating ; secondary in fe c t io n s . _ ' /SufosQquentiy, de la Fuente (1970) showed a c lo se correlation between immunoglobulin le v e ls in ca lves and th e ir subséquent a b i l i t y toksurvive an attack o f neonatal diarrhoea.The preparatidn o f lab elled Polyvinylpyrrolidone (FVP) by Gordon (1959) marked the beginning o f a new ere in the in v estig a tio n o f alimentary d isea ses . Its* application was discussed by Holman, Nickei and S leisen ger (1959),B tein fo id , Oayidsoh5 Gordon and Greene (1960) found a raised PVP fa e c a l a c t iv ity in p a tien ts with a c tiv é regional e n te r it is o f u lcera tive , co litiSr . Jarnum (1961) considered that an ; abnormal PVP test;.wds the most x e lla b le in d ication o f a 1' g a stro -in te s tip a i leak and the only means o f estim ating th e ■ s iz e p f the leak . J e f f r ie s , Holman and S leisen ger { i96g )-in th e ir review o f the relatidhsh ip between the plasma p rotein s and the alimentary t fa c t considered the use o f la b e lled PVPU;;170to be o f proven q u a lita tiv e value, aifbpugh lim ited by a number o f fa c to rs . van Tongeren and Majoor (1966) stated that i t gave an approximate estim ate o f the lo s s o f protein through the in te s t in a l wall* %eh given o ra lly only 50% to 90% was recovered, due e ith er to absorption or breakdown.Waldmann (1966) was o f a sim ilar opinion, Pargie (1969) found a good correla tion between -the faecal excretion o f PVP and the in ferred increased plasma protein lo sses*Thus, bearing in mind the q uantitative lim ita tio n s o f the method, i t was decided to carry out an in i t i a l t r ia l with ‘ X, PVP in two diarrhoic calves and the tv;o older control calves* . ■' . y ; : : 51. - , . ■.Jh:e la b e ll in g o f plasma proteins with CrCl^ was f i r s t described, by Gray, and S terlin g (I960)* Ÿ/alcImann (1961) and Waldmann and Wdcher (1963) considered that the la b e l was o f considerable value in the study o f en teric protein lo s s .They recovered 93 to 98 % o f the label in the faeces follow ing oral adm inistratipn, and obtained a d irect correlation between Cr fqecal excretion and the fraction a l catab olic rate o f albumin. These findings were confirmed by.G uillen and Peterson (1964). van Tongeren and Majopr (1966) showed that there was no advantage in in v itro la b e llin g o f albumin with ■^ C^rCX^ as against in vivo lab ellin g* van Tongeren, and- 171Reichert (1966) obtained a normal moan clearance of 13 ml/day whereas patients with protein-losing^gastroenteropathy had much larger clearances- of up to 600 ml/hour. (sim ilar results had been obtained by Waldmann anci. Wochner, 1963) ^The technique has been further investigated, discussed and applied by many other Workers to the study of protein-losing ^ gastroenteropathiès* . . ,Other labels, Nb 1abelled ■ albumin, and Cu labelledceruloplasmin haye beep used; ( see Dargie, 1969)* Be labelled Dextran was described by Andorson and Jarnum? (1966)? Jarnura, Westergaard, Vssing and Jensen (1968)/ It gave similar results to the '^^CfCl ,^ and is not readily available.Thus, to quantitate the loss of IgG and IgM in the olimentary tract-in ca lf scours i t would be necessary toy ' s 4 ' | f s ip* 'use ^.Crpl^ simultaneously with labelled immunoglobulin s- ' 51 'and try and relate the increased excretion of the ,Gr,label in the faecps with increased immunoglobulin metabolism/172»miJRMLs„^ip_J5Emo§Three separate experimentis were carried out. An in itia l1 *'^ 1 ' 't r ia l with , I - PVP in four ca lv es , two older control calves and tvW> diarrhooic ca lv es , A comparison .was then made between the % recovery o f three isotopi'c, tracers given o r a lly ,- PVP (40,000 ay. iMol,¥ft.), - PVP (■.160,000 ay. tfol.wt.)and ’^ 'î'GrGl.s. Finally ‘^ ^CrGï ^ was used in five non-diarrhoeic ' ca lv es , to measure the leak Cf plasma proteins intO' the alimentary tr a c t .The presence or absence o f diarrhoea was determined by a d a ily v isual assessment o f the fa eces. Calves were considered to be diarrhoeic %hen they produced faeces corresponding to the :t f and/or + tt c la s s if ic a t io n of de la Fuente, 1970,173A-V ; ' y ." ■ " ; S : : y S a Û $ 3 ^ â ; i K ê | . ■ \ ' : / ' y "' Calves 15 and 16 viere purdhasecl ftoin Ite.A. Guthrie, =Mo BS Side F a iia K i Imarnock w h # '2 'to 5- days'raid.- : Calvos.,9' ;and 13 had been p reviously obtained from the ëame>source and wore 12 md JS old re sp èc tiv è ly . - \■ Each c^ ll/iëcé iy o d - s ;^khqwh quantity o f - PVP '( i .7 to roughly In proportion to body woigtit) byIntravenous. in jection* The Plasma Volume was determined. with" 2 6 /'%:-' '-V-. < ' -V " ' - . \aeoas . '■ ' / -^The .calves were km t;lh tubular cages'(see Section 11.A*2) A Fo latex 60 ba I loon : catheter ( si tâ_ Î2 FG - 5 to 15 oc ? . from Vicarey* Dayldson and Co.f Glasaow) was inserted Into the. bi adder’.pf-'G; 9, A ;hei f er/',palfand;/secured'. by # I ding a ^s trip :pf...eiastop.Xast round. I t ,, leaylhg a flap # lc h %ms then . sutured ' under jocal''anaethosla 'totthe sklh*.. A polythene .. ■tube;.; was',-'then attached to ■:the cathéter?' =■ the faeces ; vtëre ' collected oh%thO-'#idy8y%d.'#0' tray' beheath?- !,% collect the , : ' /uiTW: fromi'G.:l-3'%thû':^ ^ a dlsposabie p r lne '■■ Table 26Ih Dlarrhoelc and Oontyol Calves (rgi)Dlarrhoeic CalvesDay : No «9 f io a s No-',15':'' ■; . No.16/ ' 3' - : ’ . \.50 ,- 14,9 111 202 .i ' / -4 ; , :■ ,'TT ; 46:; - ' : 66. 5 ' 78;: 12,3 181. ; 5639 died died■ ■ Mean ' ' ' m ' . : ■ 28 . 119 . ; :86' '15.7 16.9 @8 104. S‘eE*«fc : ; ^ : 8 . 5 3 3 ' . 60 '174drainage bag (Portland P la s t ic s Ltd . 5 Hythe? Kent), the cut edge reinforced vdth e lastop last.an d then sutured under lo ca l,a n a esth esia round thé end o f the-prepuce. A polythene tube Was then attached to the outlet* The faeces o f C 13 were co llec ted as for C 9* The urine and faeces o f Calves 15: and 16 were co llec ted by the method o f de la Fuente (1970) ' see Section IX A 2 b*. .4V : :See Section I I . , . ' .Curnmulative fa eca l output ( % ) and FaeCal clearance (ml per day ) were calcu lated (see Section I I ) . To enable va lid comparisons to be made between the two groups, i t was necessary to ,co rrec t the cummulatlye a c t iv ity for the control calves to a 3 day period and both measures o f faecal excretion were adjusted for the d iffer e n ce s .in body weight and plasma volume, between the calves*.6 . Reâyliâ . .The re su lts are shown in Tables .26, 27 and 23 and the plasma disappearance curves o f one control and one dlarrhoeic c a lf are shown in figure 32* Although the apparent plasma T| . fpr both dlarrhoeic ca lvec was shorter than the of the1 O T ' •Faecal Excretion of ‘"^ I-PVP ip Dlarrhoeic and Control CalvesC0NTa0ICalfNo*I'-"-Clearance ml , ml/Kg / %'P.V'68 . 1.128 0,78.D,48280.900.28-1.81.11.5r0.5%/kg %/P.V.( l i tr e s )0.96 0.015 0.2500.51 0.013 0.1970.73 0.014 0.2230.31 0.001 0.037D.IA:lîR0EIC1516 \119 ’ 7,986 6.77.84,12,6 0.093 1.711.9 0.063 0.913MoanS.D,103 : 7.3 6.023, 0.84 ' 2.62.3 0.078 1.310.50 0.021 0.56"t" Test M.S p 0.01 M.S M.S. p 0.05 M.S.A d d i t i o n a l D a t a f o r C a l v e s i n T a b l e s 2 6 a n d 2 7Calf Tdtai;> \ Total ; /':#an. PVp 1P|- Injectad; P,V.Mo. ; Albumin > GloWlln ip.G.V, (hours) Activity '(ral/kg) , (gn4Q#T) (gm/lOWl) , ; , (me).9 : , - 2,2;' , , ; 3 l ' 75 " ■ ,:3.1:13 ■ , 1 . 9 - . , ''3,5 ' 28 ; : 66 . ■ 2.9-f# W Ml f#* ## W»** ** T*# It»* t* M **#*## W» I## *4 *## #15" :.' .1.3 ,:, 3.6 .V 4 4 ' -, .63 1.7 53-16-" ' 1.6 ' 3,6:.., ." ■;>■ 45 -;'2,: ’53',. , 1.8 ' 69^ »>*«# «à# m i ### *## # * # » # * w* W,*t* Test ' ■ . ■ N*S, ^F i a u r e _ _ 3 2PLASMA DISAPPEARANCE of - PVP ( 4 0 , 0 0 0 av. M o i.w t) in Q PIARRHOEIC and q CONTROL CALFiO O0— 0 DIARRHOEIC CALF *— o CONTROL CALF5 0RADIOACTIVITY os % o f INJECTED DOSE1505 0O 100TIME AFTER INJECTION (Hr)- 175control calvesÎ the difference was not significant* Because of the differences in Body Weight and Plasma Volume between the calves, the increases found in both measures of Faecal Excretion of ^ I - PVP were not marked* A greater increase noted in the % of the label excreted in the faeces, over 3 days, than in the mean daily clearance over the sane period (see Table 27)#When the results were expressed as a fraction of body weight, the faecal plasma clearance of 7 .3> 0#84 ml/ICg/day in the diarrhoeic calves was very significantly larger than the control level of 0*90 0*28,ml/iCg/day* Similarly the% excreted activity was 0*078 * 0.021 j^/Kg compared to0.014 ^ 0*001 /5/Kg for the control calves* The same measures when expressed as fractions of the plasma volume showed similarly elevated levels, which were not however statistica lly significant, tho; two groups being very small*The results clearly demonstrate an^increased loss of macromolecules into the gut of the diarrhoeic calves.nocomparison between thè Faecal Recovery (j^ ). of;^?-l'PVP(40,0W av.1%.PVP( 1 0 0 , 0 ^ 0 ) and'^^GrÇîlg, over 3 days.—X-PVP , ^^ I^fPVP66Mean ; ' 51,'. '■: 57 lOSScD^ it ■ ■ ig : ' 16 ' ' . ,33S.E.*;,V,\,- ^':\lb.4 6.6 ' 19- 176 -B. Faecal: RecQvery:Mi5l PVP (40.000 av. Mol, wt. ).'Qpyp (160,000 ày. Mol.:wt;) and ^^Crçi_, given orally.1. Source of CalvesSix market calves were purchased through a dealer*They were up to one week old*See Section II 0 4.' . ' ■ 5 1 . 'Three calves received lOOiic of CrCl^ and lOOpc of125j PVP. The other three received 15ppc of, PVP and1 OR’lOOiJic of I PVP. : All three isotopes had been diluted with saline so that each calf received approximately 10 ml of each label, in a weighed syringe.3^. ■' §.ami^Faeces were collected for 3 days following dosing, using long polythene bags attached to the calvo§- (see Section It A 2b). All of the calves had developed diarrhoea, the severity of which varied considerably. The total amount of each label in the faeces was determined and expressed as a $17 of the quantity administered.' / VThe rësults are shown in Table 29. It can be seen from the mean recoveries of 51 ^ 18 ^ and 57 *16 ÿ for the- 17? -two PVP labels that there is l i t t l e to choose between them*, ‘51The mean recovery of the GrClq label on the other handwas 108 ± 33 %, ; ,IsàLâ.3âRepeat Determinationsof the p Faecal Recoveryof i2ôj_pvp (lôOjOOO av,Mol.>t*) and ^^CrCl,, over 3 days ^ wii f Rli # 1(1 rti iihif I^ li* li> KifT r.WWMWU < *Calf No. 125%^ pvp , SlcrCL, ^ 2 .......'"I"" " .... .- 42 125■ ■ . 3 : 35 964 •125 ■.; ■■'• ■. 5"- ' ■■:. ■ ■•' 37 ' ■ d 9 k ^ - ■ ■ ^ ' 44 , : 114 : '■ '■: Meqn ■ ■' 49 . / 110-B.D.*'- 4*2 • , 17 :s . Be* ■ 2.1 7.4 ,^ . Xncojipiete collection of faeces“ 1780. Repeat determination of tho Faecal Recovery of 125j pyp and ®^ CrC),,1» •"■Gal.vaà,,,Five of the calves from the in itia l experiment were used ( see above )*2* Other,.See VIX B above*'Four calves received both labels, but the remaining■ '51.V cel:f P was only given ; CrCi,.^ . The interval between the experiments was one week* With the exception of Calf 5 which was s t i l l scouring profusely, the calves were no longer diarrhoeic. ^ Roguits . ■The results are shown in Table 30*110 * 17 % of the CrCl^ was recovered but only 40 *4*2 % of the PVP* Although this recovery of PVP is lower than in the first experiment, the difference is not statistica lly significant* From the results of both these oral dosing experiments, i t can be seen that whereas approximately 100$^ of the '^ C^rCio label is recovered only 40 to W % of either of the labelled PVP's Is present in the faeces. The rest179of the PVP wJ.il have broken down in the gut or been absorbedthus for the qhantitatibn of plasma pfotein loss into the' ' 51gut of the neonatal oalf, the use of ' ÇrGl» is indicated.Similar recoveries have been obtained in man and other specie ' xithe -fact that in most of the calves the recovery ofwas more than 100$, er^hasised the problems associated Mth obtaining a suitable aliguot of calf faeces for **counting^ *i In both these experiments the problem was increased by the bulking of 3 days faeces. The level of activity ; in the faeces will have varied; considerably overthis period. ' ■180 -D, Plasma loss in Non-^dlarrhoelc Calves measured with^CrClg1" Source of CalvesGalVes C^/l to Cr/4 were retained from the faecal recovery experiments* They were by,this time approximately 3 weeks old and no longer diarrhoeic* c/C 10 was one of the calves used in Section V vthlch had shown no; evidence of diarrhoea since birth* It was also 3 weeks old#Inject ion ,Each calf i was given a known quantity of CrCl^ by intravenous injection# At the same time, the plasma volume was measured with Evans Blue, see Section II F, except for C/Q 10. In this case, the plasma volume was measured with 125% labelled Fast IgG.3. - Collection of Faeces : See Section II A 2b.4 * Furtliè^ ex^ jjetail#See Section II.5. pqlcuiallon of ResultsCumulative faecal output (^) and faecal clearance ( ml/day ) were calculated.Daily Faecal Clearance of CrCl^ in Mon^Diarrhoeic Calves (ml)Day Cr/l Gr/2 Gr/3 ; Cr/4 C/CiO' 3 _ 44: 4 105 59 605 48 , 18 '''27 30& . ■ 50 v' 75 57 ' 44 86:7 49 / 25 . 15 : 31 '8 , 65■ . 'Mean. 74■59 \ 33 ' :S.D.6 22.8 ' ' ,11.3 20.8 14.6 : 12.4s . a . a ' 11.4 , ' 5.1 12.0 ' 8'4 \5.5r: t a b le , 32'fi ^ ■ ' 'Faecal Excrétion of ÇrCl^ in Non-piarrhoeic CalvesCalf NO. .. Mean dailyClearance(ml)ümulative Faecal Output($)Apparent Plasma TÿCr/l / ..H; - : 3.4 4.9Cr/2 59 - 5.1Cr/3 33 . 1#8 , 5.1Cr/$ 0,80 5.5c/cio 40 1^ 4 6.0Mean ' 4? - ; / 1*8 . ' 5.3,S*D,& 19 0.96 0.43S.E»* 8*5 0.42 0.17Calf No. TotalAlbumin(gm/lOQwl)TotalGlobulin(gm/lOOml)P.V.(ml/kg)InjectedActivity(lie)Cr/l 1.8 4,9 54 500Cr/2 1.6 ' 4,5 49 500Cr/3 1,7 4,0 55 800Cr/4 1.6 4,1 54 500c/cio 1,2 3,7 63 800Mean 1,6 4,2 55S,D.a 0*22 0,46 5,0S.a.a 0,10 0,20 2,3Figure 33DISAPPEARANCE of ^'CrCI; in o NON-DIARRHOEIC CALFJ Ç r /à L1005 0RADIOACTIVITYINJECTED DOSET V i (days) = 5-1O 50 100 150TIME AFTER INJECTION (Hr)Tho; roôiïXto nro: shovtf^ lo Tables' 31^; 32. and "33 and tho plasma disappearanco"’o f 'the activ ity In one o f -tho calvoG*In'Fig* 33 * ; The mean T-|* for the gronp 3*3 î?* 0.43 days* .A-'moan -daily qiearance o f 4T & T9, ml wa# fo'ond# which.'.represented, 2^2 & 0*90 % o f tho p lom a. volum e^ay*,1*8'- ^-0*95 ÿ o f .the Indeotod activity' was recoveted' In the faeces over. '#' 3 day - ‘ period# .1 . ' ■' ' ' ' ■ - - ’.. The control PVP, oloorànce as"a % o f P.V& of, 1*5 sî:0*5 (see Table 2?)#' previouo'ly-obtainedj^.thno' représente> ■ ' ï-\i • ■ \'. ' ■ /Gbent 2/2> o f the'. OrCl,^ _. clearance,- sim ilarly expressed*,A difference .of th is order.'v^nld have been e>pocted in view of-' the Tovar, recovery o f ,■ oraUy.; administered .labelled . P W » ■(Gee Section VIi, B end Gf i- X82DISCUSSIONOn the basis of 4he'è$ of the intravascularpooi of IgG . catabolised per day ise e Seotioh ly D - Resiiits ) the 2#2 * 0#98 pf the plasma vplme per dày, can only account for to l / z of the IgO catabollCed* As the oalyps used for the GrPi^ studies were 3 to 4 weeks Old, their catabdlio ;;. rate inK)uld have been hiÿier than 6^ # Ihus for fast IgG, i t would appear; that the gut of the neonatal; calf does not - "account for most pf the catabo 11 am, although mking a ; significant contribution*Exactly how a plasma prdtein loss o f 100 ml or more V pet day (see Table 27) occufs in diarrhoeic calves ié notknown* Jarnum (l%3) cohsidered that there were three possible mechanisms for the norme! loss into the gut, secretion, in : extracted epithetlal cells or, by- a pessîyo. leak# Where excessive . loss occurs in the presence of afe^rmal epithelium, hoconsidered i t to be due to abnq high capillary permeability*Schwartz, (19$3) suggested that such losses bccur between the ; ? cells* BierHrigV Jarnum and Schwartz (19Ô5) using the electron microscope found''that ,'the/inter dellulaf ' spaces may becomemarkedly dilated, containing a precipitated fiocular; material#'' ^ VÇ. V ' / . . ' I ' \ ..... ' : ::;3.Jarret, Miller and Murray (19T0) showed, in addition to this/ : : ;leak between the ce lls , a marked leak from the small blood vessels of the v i l l i , in their study of the response to Nippostrongylus infection in rats* The state of the intercellular junctions of the gut epithelium in the diarrhoeic calf has s t i l l to be investigated*While only a : lit t le is known about the mechanism of macromolecular leaks into the gut, i t has been known for a long time that neonatal diarrhoea in calves, as In other species, is usually associated vdth £> coli*Since the early work of Jensen (189i3), this relationship has been investigated and discussed by many workers* Wood (l95b) found that calves which had had colostrum died of a localised intestinal infection and not from coli-septicaemiat Dunne, Giantz, Hokanson and Bdrtree(1958) claimed to reproduce the disease experimentally and prevent i t with bacteriostatic agents* They emphasised the apparent sponta .iieous development of outbreaks* Smith (1962) found no excessive proliferation of the organism, f a i l t o transmit the disease and concluded that i t was probably not infectious* Attempts to prevent coiibaccillosis by vaccination were apparently unsuccessful (Gay, itcKay and Bar hum, 1964 a, b, arid c)* Loosmore, Anderson and Edgson (1964) described environmental build up* Roy (1964) while recognising doubt about the cause of death in-iêb' enteric co iib a cc illo s is , ''conoiuded;'t h a t : egIj, may' be of ' \ Considerable iR^ortanoe*- ' . ‘v C , ■..■,/ ' ' - ’ '/.'A:'''■■. -:Gay .^ ( 1965) .regardpd iC- m : only, part' o f . tho syndrome of ' the ■scouring Calf,: - He;-and ■|o3ka'-.(1965)' conslderèdl.'that .themC m s ' ;mom'0',e#denco.that ''c#tain .stmlhs.'could pmduoe scouring' '■'s.-.'-':. ' ■ 'pî calves ■Cnd\tha‘Ç'epc%iflç}.antibo'dy-might give' - protection # a.. So jW. while - stotihg the, générai ; ddnsOnsus 'that i s .'- Che-'argpnlm^most'; ^ isolated,'Chared,the' opinion; of' :Oey;that almost ^noth'ing',%le ': khpWÂabout) aetiology ; ,o f t h e A - A ' - - acondition^'''eaiiior'^werk? having'only'.oficphesiCed' Its'vCo^Mplexlty# lPenhale;ix965)-Was unable to\' d^^'hstÿato ahCunequivocal ; a ‘ ; " ■ ;. ' iClatlonsMp:''betWenL:scouring.and.;#"e;admlnlstratloh 'of- '■’ C/;- 'C■'\:experimental 'Cer0t^es#AA8oJkü-arri%d'atC; .similar-conoiusion^ - ' on the avaUabie ëvidenééè'- .. \ ' '■’•A;. ■ ' Amstutz ' (19'&5)'-Ain. a symposium on calf r'di'arrhoea : considered '■.that infèctîptm-Àdiarrhoeè-was thë-pàinv-.causa the high. _ ' ;'■ '/neonatal caif .wrtality# , AnotherCgntributgar,'Sacloatits ' 'C'■ '■ i 1965)' ' donsi#rOd A thé % 0 f obscure, and a. oausaT /:,/. ,reiatiomhiiiAdifficWit..'. to ■Establish* '.Smith and/Halls (196? a and b)VÀ:'studied th e ,p ossib le ,m le ofA''&^^li,#ntemtoxln*.' The ' ' r ,,,àa'A endotoxInA had - been:..fully ihvWtig'ated -;by Cenhala (1965) *. "-'Da .la'Tuentè,.il97ù) found'C'onsidemble-yerlation' in^ the /'■'.".àeÿarity ahd AduratlGn.'.oi'.h'epn# ^'diarrhoea in' oalvoe* He ■Goncludad'that-a high.ConophtratiOnCf ■ antibody waiild give186protection, over and above e ll other factors, including treatment* However, Smith (X971) concluded from bacteriological studies that neonatal diarrhoea is generally non-*infectious in origin, '' /While most workers accept that cgJl plays an important role in neonatal diarrhoea, other agents have been discovered and investigated* Baker (1943) and Brandly and McGlurkin (1956) described a virus enteritis, accompanied by pneumonia#The possible role of viruses was discussed by de la Puente (1970). Mebus, Kond, Underdahl and Wiehaus (1971) described experimental virus diarrhoea in neonatal calves. The presence or absence of viral agents in most outbreaks of "calf scours" has however yét to be demonstrated*In relation to neonatal calf diarrhoea, a number of points have been established in these studies, or are widely accepted* These are, the hypercatabolism of Fast XgO ( see Section ÏV), a macromolecular leak ( using^^^I PVP, 40,OOOMol.v;t.) and the association with an enteric form of Coli^baccillosis*The most likely cause of the hypercatabolism of IgG is thé increased plasma leak. Whether i t is sufficient to account for the total increase has yét to foe investigated.There may be an associated increase in endogenous catabolism ss well# Also unkhovm is whether this loss of plasma into187: the is a bulk process, unrelated to protein size* in inan, this mold generally appear to be the case (Strober,; Blaese and Wal#a#ÿ 1970, the relative increases in the Fractional Catabolic Ratés-of different plasma proteins being " similar# However in Çrohns Disease, the increase in the çataboiism of Ig@ is AbO# high# théh the albumin increase/'- a//(Jarnum, Behdixen, AJensen, A Solloft, Weeke and Westergaard, 1970)* Further investigation is therefore required# ,.. ,#ether'; or .\#t ls/aApr#érentiàl leak of a particular .iîmünoglobulin into the gut ih this enteric disease, the presence of l&unoglbboiins in thé alimentary ttact may influence the course bf # é disease# The presence of antibody ;in thaAf w#s fir st described by Davies (1922)Gillen, Young, Massey and Brande(1960) ahd Freter and Oangarosa (1963) found that circulating antibody had T itt le of fect on enteric infections* The latter #>rkers,found that:oral' vaccination w as/t# only method of maihtaining coproantibody aagainst choieta# Hooper and heelteman (19Ô6) showed that:A thé #ésencé of antibody in the; lumen of the gut was heoéssary : a; to'.^protect"pi9S:;a9ainst'Tran#issible Gastroente#tis-*;:,_This , '/Awas; later cortfixmedvby CartvwTÿït .(1969)*,. ', Schultze and; Herànaps (1966) concluded that coproantibody Was locally produced TgA and that i t pérfomçd a protective188 -role in the defence of the gut mucosa* Felsenfeld (1968) confirmed earlier doubts about the unreliability of serum antibody levels with regard to the actual immune status against enteric agents and the importance of IgÀ in man*According to Jarnum et al (1970), 90/à of this IgA is produced locally in the gut*There is conflicting evidence for the inportance of specific antibody In protecting calves against the enteric form of coli**baccillosis# McEwan ( 1950) considered lramu#ty to i t , to be independent of Specific antibody* Spain, Bradess and Greenblatt (1956) concluded that y globulin levels were not the determining factor with regard to death from neonatal diarrhoea, although an immuno mechanism might also be involved* Pénhalo (1965) showed that failure to demonstrate antibodies could often be accounted for in terms of the technique used to detect them* Thus earlier negative results, did not necessarily reflect the true position. More recently de la Fuento (1970) found that the calves which survived an attack of Neonatal Diarrhoea were those with the highest immunoglobulin levels, as measured with the Zinc St^phate turbidity test* However, Harwell and Fey (1970) were unable to show any correlation between low Y globulin levels ip market calves and diarrhoea*■do laFuente (1970) suggested that there must be an immunologicalmechanism, sufficient to neutralise the bacteria and their products, # th respect to diarrhoea* Such a mechanism might be accounted for simply in terns of a bulk loss of a ll plasma proteins into the gut, including immunoglobulins, or there may be an additional preferential secretion# This will have to be diff#entiatod Ttom increased endogenous catabolism, slnpiy associated with a bulk loss.Kramer (1963) and Penhale (1965) dmonstrated that in the bovine, as in other species, (Gitlin, Rosen and Michael,1968, Michael and ABosen5 1968, Bobbins, Kenny and Suter , 1965 and Hanson and Winberg, 1966), the main antibodies to E.coli are IgM Immunoglobulins# A solution to the problem of the presence, or absence of a preferential ieak of ohé or more immune globulins must await a satisfactory 1 ^ pr^aration (see Section V) suitable for metabolic studies. Miile there is l i t t le or no direct evidence’at the moment to suggest that IgA may also be important in this respect, its* relationship . to alimentary disease in other species (see above) suggests that i t should also be investigated. It Will thus be necessary to see i f the metabolism of one or a ll three iimnunoglobulins is influenced to a greater extent by diarrhoea than the metabolism of albumin (hwercatabolism of Fast IgG has already been daaonstrated see Section iV)*T, 1 9 0A sign ificantly Increased leak of macromoladulas Into the. alimentary tract of . neonatal .diarrhoorc calves .wad dmon&trated with PVPf ^ Subsequently, th is trader was. shorn . -to under estimate the.; plasma leak *. ' OrCl ^ was found to be a more suitably lab el .for ,quantitative studios # - ' I n an in it ia l ,study in non^dlarrhoelc calves, . a plasma Idss of approximately.50 mi/day ivOs demonstrated# This would account for le ss than .baif o f the. igGj. oatabolisod/day# . .SECTION VIIIGENERAL PISCUSSIONr‘:.A ' The 'iii r^tesanta;‘the f ir s t ^’■■■'dlréot' moesUrdfàorit,.'of vthe..BffiOiëhéy o f 'Absoiptioh',o f ' Gblostfmi: ■ '- ■■Fast' ih '# e Neonatal'-'Geif# ' : Boforo-.tho colostràl;.■' 'stWi # ■tphld. ho .loarried ' :oùt $■ .\lhfo'#atioh'\WO$;' reqoirod about '- • : the :di'stribution and m tab oils# 'of :'T0; * i- 'This ; Was/hbtalned . :' from-a"'study of/ th .e :'m ëta b o ll$ m ''''é f'U si# 6 purified ^,.. immhogiobuiin préparation, 'fraao-TabMiod w|th''-thd-'l'sotopasy:::of TodW# \ - 2 ' : ' V \-/v" 'ItA-hadAbeen.intondWjto■"oafry out a^similar investigation ''■ ihW'- tho,:.abso#tW^ % Ho,w#ver,' the woTfe ; -, ;with l(# (''' .see /Beotion V ).Tailed to . proddo#' undenatured ; ■ ’ .■ lgM..pr^©ratiôh,'. suitable;for' moWboiio:: md.toTostral studiesi ,- '■ This/w^tk'^wiifToquire’’further :- . \ / ..y :y, // ’ :'y\: =\ . , - : y. / \y\ m o6##IeaW .or' # 6 k ..o f p lom â'.pÿptéW ' I n tp -th e.plimentasy , ..y :/ /' "y/;' - traCt o f tlie 'd larrh oo lo oa lf# a Suoti’d 't e s s .moy'oonsidorably '■ -'. : /v y / ' / . t y -V. y y : # - :y y 'y .y y/' :' êadüee ' thé; .oT the' oa lf# ■ /.AqWidng ' - -, y y y . , : ,.:-T-^:yyy:y''/ y y / ÿ / ; y y y . y " ' : ' \ - . y : ' / y y . : l \ " / y : .y '/ythat , ' '-'y r-'r'tnm -loôtldiV rVIl) # 1 1 ;M W " #;. y - /y -y .P o M % /# d \Â a W S r(l970lyiWwW/##\l$À; In to s tlm ly /.y ' %' '%:''..'imunogldbuiin ih lth d /ThuR/'tW.MtfrholW)! ■ //'y ". ' " /. ' y . ' j y M x y.^-.■f ,o.f ' Wth ^ t#éB0 dmàumgiobulio#. #%l 1;. bavé y'to W s'LWicdyto ^ y./-. " - .y.:/ehoW 'lf..thapr0c#^:T é'.ôno/of::e^)0l leakage ù f - é l l p W a s ' ' .r -7^ '7 / '- / : ÿ' 'y yy-' ' \ y \ y y , . : ' \ 7 - , : : y : : - y / : y y ./ ^ y/ÿ yy .y ' : //y'ço0ôn%ts7#-#qtK0ryëome;ùry^^ - 'y\y;..y.;./y ' ' îà r # 0 é ^ i n # th e a lim e n ta # # p e t 7 ' . y /'-' y'--- %-;:/H #ë#K aW lim '0T lgGt4Wà 0ireBdÿWèO'absf.rb3d;lif 'Ihe aaif'iéy'nét; fyii^yuodérstood* ' ThdpoeelMo ’ ..■■.,.7/:7 y r o lo .o f W % 6 # % r /# h # W m o W y - # o W # / Ç, '- b f ' : ë b # r p t i o n ' W éh -m jgg$s#d'\W t'T équlr# -furth# stüdy -'y .cu% â-;#h# .y \#a - / -y-yy/y.,yÆ r0% ',m #$vrA eotyof tho BfflcW oyrdfyAbàW W n''^^^ oo. ' - - ' :y:.y y y y y y y 7 7 y ' y \ ^y ' : yyX .7^ y /v^ÿ /y -y ' -yy y y; ;;\''.y:yy-7n lréW ÿ 'lW Icatéd , .W m corr&W édt^T or..I^y*-. : ; ■'■■'' y^y'/7'yyy7 :y 7 /7 :^ ''3 '''/ .r .,. /7 y y '^ 7 y y _ ..'y yy : 7 7 " - - .„.y:' o f .7 # , # d . li&ve\ to.,W b efore y, ....' - : :y-y^yMe#%yo#ec#h^ -# sq 3 ^ )tlo n \o f.;# ir th # o ...# W ' '■ y/--y /y.yGWy&è^fuily.'.i^^ ^ Atythé'WmbntÿT^^ -"'y ...y '- '7/':../:.' /^'y/'y/' ' .'7,/ . y..-j<\;':.y-v. ^ ':7 ../: '' ■ .' ' y..' .'-A k.;vA'. 1- ' '7 /7 " y 7 '7'' ':y7/i93y-' y'pqssibïé■'fprIgGj '^7'''''/' ," -,7: 7'7" '/ . ;- , No, evidenceywa$/found in thq absoiptioa efficiencystudies (Section VI) o f premature shut dom*7 (l*e* an early loss of absorbing,cap.acxty)'* This Is lb keeping with the findings QÎ Kruse (1969) and Selmah (1969)* However,Kruse ( 1970c): shoyfed # under .normal farming conditions^ a certain level /o f hypogamaglobulinami^ is inevitable# . , , ;' Further, studies'^are''reqvdred'to-'find the'best way to-"'enable calves to make up th is deficiency# When more is knoivn. about . the factors inyoived in ’*shut downy, i t maÿ be possible to delay or even ; #nporariiy reverse -the’ process $ . A lternatively,; ' once. the relative importanae of the immunoglobulins has ■ : ■ - -beoh^fuiiy/wprked .out; Immunoglobulin preparations,or enriched fractions might be used as parenterally administered : colostrum substitutes# Some work along these l in e s ,has already ■been carried Out/(began and Pahhale, 1971)• Similar - preparation© might also be used ih the prophylaxis.and treatment of Neonatal Diarrhoea# ,The investigation of factor© involved in the absorption , ,of XgGj^ i i s nowrpossible*. Further ivork xvill be necessary - before sim ilar/studies can be. .carried out for IgA and IgM*V It w ill then be/possible to; investigate the role of a ll three Immunoglobulin© ih/Neonatal Diarrhoea#^ 1 9 4Aaiimdÿ-D, (1968), Thesist/Münksgaàrd, (hpmhagan#AhÿâmsoÀ, D ; I . (1967 ) # ”Ciroyla#on-'in the fixtrcmltioa^V, ' /-/'/'■■ âcàdemle'Press,■'London# ; '723#. -"\ y ;Ahlihdor^ 8#:,-;Birk#$ G., Norberg .H#, •Olhogon^.B*,■'Plentin.y''L*0*;:• ,:/'iM6taholismyjvHans Hubqr, Berne* .lib e rty A., Jphn#h, P* ( im iU ~■ ‘B#.cW%J#*_8Ï. 658* 77:7 / ■Alexander, P*t/BloÙh7 B*£#/-,{ 1960)* / "Â Lahbyatpry Manua 1 of. ‘Ahaiytieal/Methodo-of''Pîrotein'-Q^imistryS jL$"P0rgamen,‘7 ;■ london* ■' ' ' /y ; , " .7 ..7 .7 7 7 ' .7:- - .'-■7' -/■ 'Ai#,.â*S*l A* (19Ù2)*.-ÂmstütEÿ- m $ 1360,'Ahdersea,-.B*B#5 0lehort>7J*y7%it0vik, K* (1963)*'-.,7 7' .'Andèrseny $ .0*,. (190)7- '^Metoh&llsm o f 1lt«nan-;Garaa Giobullr , - '• : B l é c k m l !.. S o len tifie Publications, 'Ox&W*; _, ,■/„■■ '4jjder5cn,''S.B.'-:' E m M tiS S U m sJM s.» Aâ*;329A4lndersen>'S*B.*: Jarnums Sf (i960). Lance t , 3.,. 1080. Anderson» D*fc1*WV»-.AS,toddeH-s J.F. { 1 9 # ) . Amtotioa.j;J19S -Andrews, P. {1962). Nature. Lond. . 196. 36.Andrews. P .:‘(1964). BiochemI d .. 91, 222. -Andrews. P. (1966). Br. medk B iill... gg. 109.Armstrong» F.B., Margén» S.,;Tarver,-H. (i960). Proe., ; Soc. Biol. Med.Aschaffenborg. R.» Bartlettj s., Jon,’ S.K:.i'^- Briggs, ^ (1949). Br. J .: I96ÿy ;. : //; .> /k '" / ' ; / / ,Atenoio, AVC.'/, fteeVe, E.Bk ;(196S)• 3. Lab. ciin . Med.. 66. 20. Bailey,; J.L, ( 1967)/ "Techniques in Protein Chemistry",; Elsevier/ Amsterdam.■Baker. JIA. (lOd^V 3.;exp. M e d > L . 435. ■Balfodr,WiE., Online, R.S; (1962). J. Phvsiol.. 160. 234. Bangham, D.R,, Ingram, P.L., Roy, Shiilam,.K.W.G.,, Terry, R.J. (19S8). Proc. R.. See. B..- 149, 184.Barth, K'.M,, VbcHner, R;W.R, (1970). "The Transmission o f P assive Immunity - from Mpther. to 7oung" V NprtWlo 11 and/ Amsterdam. ; . . /Brandly f C . A. ^ McClurkih* A/W* (1956). Ann.. N * Y, i.Ac,ad *__8.ci.. #/ + "7 : :7 ' . \ ■B r id g esjR .A ., Condie, P.M*V Eak Good, R.A* (1959).• J,Labv cT ihl Med.# 53/ 381.:- 197 '.* ' ':;,,( 1 9 6 7 6 1 2 . .brovmyfH«D«. >- (l9S6).t ■ -,i m % ' R.'D,. 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' - ' - ' - Y ' Y Y \ " ' ' y:- / Y :Butterworth? London* : /yy_. ' . ': "Welch»:' G.S.,.: Adams,"N.Wakefield, E.C. (1937). J. c lin . . /' 'y -- . -■ / k:/ -'V ' . « ' - v . y. - , k ,::kyk, _7 l m : s s i ^ , : l 6 l . .^k/k: , ,kk-,.; .^ yy- ,;:" . :’ Weichselbàum. T;e. (1946). Amér. J . c l in . P a th .. iG. 40 . WëirtyD.M. (1967). "Handbdok o f Experimental Immunology", , k yk Blackwell S c ie n t if ic P u b lica tion s, M lnburgh. J: Wettèrfors, J . , Uliberg, P., W Plantin, L-0.,^y'yy-'::; 'k y : , - : k t y / . v ' Y' \ -%:, 7 - ' :k Y /Blrke,::G.>kOlhagen, B y ; ( l9 6 p ) . Actà med.kScend..; 168 . 347.kk'Wetterfors, J.:k (1963). , Z22. yk -k-‘ W ettëxforsj J . ■ ( 1965), Àota_.med, : Scand.. 177. 243» ^,. 7 -■ , '>'7 k ■ ■,■ ' - y^ ;.;Y 'W t ë k m ' (1 9 6 3 ).k AnalYt. Oiem.. 35. 1950. - ', - , ' 7/ ' / k' y . - / ' - '7ky: .k. . k y - k - ^ . 'k "k'. '-kyk/. . ' ' kWilkinson, P.? Dayidâdn, W*? Sonimaripû? A? 11966)* Ann#''yiP$H#jL.&d«.»k,6^,. 308:X ' 'î282G*A. ÿ (1968), "Methods In Immand Immunoohomistry" ? 2@ 72 $ Aoodoiilc Press $ London * YaguoM, M#, TorTossuk? W*P#* Hunzlk? J . (196%)#Yamamoto, I * , HaaMmoto? N#$ Yokoyoma? M# (1968)* SilBl.iââ /Ziff ? M'S,? Lo BpBl Into g Ja (1959)'^ 105 L/THE METABOLISM OF PLASMA PROTEINS IN THE YOmG CALFSummary of a thesis submitted for the degree of Doctor of Philosophy of the University of Glasgow by Du g a. Id F* Macdougall, V*S.The work described in this thesis represents the application of isotopic tracer techniques to a study of the metabolism of the imiïiunoglobulins absorbed from colostrum by the new-born c a lf . While previous studies? based on serum immunoglobulin levels? have greatly added to our knowledge of th is a b so rp tio n fo r precise quantitation i t i s necessary to know how the immunoglobulins are distributed between the intravascular and extravascular compartments ? the rates of equilibration between the coïïpartmonts and the rates of catabolism? in the neonatal calf* These measurements can only be made sa tisfactorily using isotop ica lly labelled proteins.Bovine Fast IgG (IgGj )^ was prepared from neonatal ca lf serum or colostra! whey by a combination of molecular sieve and ion-exchange chromatography? and trace labelled with radiO”iodineo The metabolism of IgG^ was then studied in Ayrshire calves? le ss than 1 week old. A distribution of 1 , 2/1 ( Extravascular/lntravascular) was obtained? equilibration being complete by 48 to 72 hours. The occurrence of diarrhoea in some of the calves had a marked effect on the catabolicrates but not on the distribution? although a close correlation was found between the distribution of IgG^ and a l l four measures of catabolism. The re la tive size of the extravascular compartment decreased with age* The albumin distribution was found to be 1 .9/1 ( Extravasoular/lntravascular).Bovine IgM v^ as prepared from colostral whey by molecular sieve chromatography and similarly labelled. In metabolic studies four separate preparations a l l showed evidence of dénaturation and were thus not suitable for providing the , information required for absorption studies. Dénaturation cr iter ia were investigated. The distribution of two of the preparations indicated that IgM (as in other species) is retained to a greater extent mthin the circulation? than IgG.Using the information obtained from the metabolic studies,i t was then possible to quantitate the effic iency with vhichthe new-born ca lf absorbs colostral IgG .^ Colostrum containing131a tracer quantity of I labelled IgG^ ^ v^ as fed by stomach tube to Ayrshire calves, 3 to 6 hours post partum. 48 to 72 hours la ter , the plasma volume was determined with ' "^ I^ labelled IgG ,^ The tota l absoi'bed activ ity was then calculated and expressed as a % of the act iv ity in the colostrum. IgG levels were also directly measured and the efficiency similarly calculated.When the mean resu lts from both methods were combined, an overall Efficiency of 50% was obtained. The close correlation betweenthe amount of IgG (gm) fed and the amount absorbed indicated a similar effic iency ( up to a limit of 250 gm, in 4 l i t r e s of colostrum). Information was also obtained about the in i t ia l appearance of IgG in the plasma and i t s relationship to the proteinuria.A sign if icantly increased leak of macroTholecules into the ,alimentary tract of neonatal diarrhoeic calves was demonstrated 1with I-PVP (40,000 avo Mol. w t.) . This tracer ivas subsequently51shown to be under-estimating the plasma leak and ”CrCl ^ wasfound to be more suitable as a label for quantitative studies.51 /Using ' CrCl^? a plasma loss of approximately 50 ml/day wasdemonstrated in non-diarrhoeic calves which would account forle ss than half of the IgG^ catabolised / day.The investigation of factors influencing the absorption ofIgG^ by the new-born calf? is now possible. Similar IgM studiesw ill have to wait u ntil the dénaturation problems can beovercome. In addition? a Bovine IgA is now recognised andw ill require study. It m i l eventually be possible to investigatethe role of a l l three immunoglobulins in relation to NeonatalDiarrhoea,GLASGOW UNIVERSITY LIüR/iRYThe copyright o f th i s th e s is belonv^s to the au tho r.Use made of i t must be p ro p e rly acknowledged, and in p a r t i c u la r any q u o ta tio n from i t in pub lished work re q u ire s the a u th o r 's p r io r w ritte n consent.Readers must s ig n th e i r name le g ib ly below to in d ic a te th a t they accept th e se c o n d itio n s , and must add th e i r permanent address and the name o f the i n s t i tu t io n o r o rg an isa tio n to which they belong.NAME ADDRESS AND AFFILIATION DATE■- . . . ............................ i ^ ' -------- --------- ---- •"'VGUL 75.354UNIVERSITY OF GLASGOWIn r e s p e c t o f my t h e s i s [ t i t l e to be in s e r te d 1 'The M e t a b o l i s m of Plasm a P ro te in s in th e Young C a l f1. I u n d ers tan d th a t no access t o i t w i l l be allow ed w ith o u t my p r io rp e rm iss io n u n t i l one y ea r has e la p se d from th e d a te o f i t s d e p o s itin th e U n iv e rs i ty L ib rary*2. T h e re a f te r* a* I g ive p e rm iss io n f o r i t to ,b e made a v a i la b le t o r e a d e rs in th eU n iv e rs ity L ib ra ry o r w ith in a n o th e r l i b r a r y .* b, I do n o t w ish i t t o be made a v a i la b le t o r e a d e r s t f o r a f u r th e rtwo y e a rs w ith o u t my w r i t te n c q u s o n ir i^ fT fa i i in g a r e p ly from me w ith in 3 months to a ^ e q u e s 'f f r o m th e U n iv e rs ity L ib ra ry ) th oconsen t o f^ h e - 'L llS ra ry Committee in c o n s u l ta t io n w ith th e H igher^^^Degree^""CommittGe o f th e Faculty*3. OncG any restrictions on access have expired:-a . I g iv e p e rm iss io n f o r a photocopy to be made by th e B r i t i s hL ib ra ry f o r le n d in g to o th e r l i b r a r i e s ,b . I A ^ iV e/d o n o t g iv e perm iss io n f o r th e U n iv e rs i ty L ib ra ry tomake p h o to co p ies f o r o th e r l i b r a r i e s o r in d iv id u a ls w ith o u t my s p e c i f i c a u th o r i s a t io n . Note : Any p r o h ib i t io n on photocopying w i l l la p s e a f t e r f iv e y e a rs from th e d a te o f d e p o s i t .D a t e /D. F. M A C D O U G A L LP;-..D., B. V. MG. , M. R. C. V P R O J E C T M A NA G E RS tr ik e ou t th e sen ten ce o r ph rase which does n o t a p p ly .* * i ^ t v a . . \ M L Ü IS I IUr U.F. MacUougall.Ph.D. T h e s is . * 2 n i+ - '2 _

m-aminocyclohexanol

Abstract

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m-aminocyclohexanol

Abstract

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