a wm m m m fob ts& anmranw of csoajssxxu mm m ^ fflica^ioh to w m m rs m T tm m m i mm < m « of jmjxamss fastis Sy Sdwari Otto Haeaai I l f mas is submitted to the Faculty of the Graduate ^ ehool of the University of Maryland in partial fulfillment of the MqulxtMtit for the degree of Doctor of Philosophy 1940 UMI Number: DP70377 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. U M I Disserta tion Publish ing UMI DP70377 Published by ProQuest LLC (2015). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code uest ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 AnnArhor, Ml 48106- 1346 Th® author «&prasa©a M s Binmm appreciation to Dr. I. B. Whit© fts4 Mr* H* A* Dapper of the food end Drug Adai&istratioii, United States Department of A^nouitur©, under i&©»© |®s#r®l supervision this work was undertaken. 1© 1© grateful to the latter ©loo for preparing the alimen­ tary paetee sod facilitating th© «©rfc in many waye* $he suggestion© aai criticism by Dr. It, L. Brsk© haw been of smeh help la the presentation* Hi© author thank© David Pauli of Antioch Collage for M o help as student assleteat In some of the routine analyses* The ©placid cooperation of fellow workers on the staff of the Food si4 Drug .MMmiatraMon., to© suftsroue to mention* is deeply appreciated* This dissertation is preee&ted mlth the permission of the Chief of th© Food and %ttis Admini strut ion. i i i TABUS, Of COSTS®**© mmmmim .. -------------------- x tax m m m M h n m o.f ce&u&kekbdl ♦*.*..„** 5 The Chole# of e Method « * « « . . 5 The CfceXeeterol Diferotiid® Method 10 !#vi«w of the Xdtevetare .... 10 The Preelpltstion of Cholesterol Dlhro&lde ..... IX The Xodaoetrle Estimation- of Cholesterol Based on the Cholesterol D&brQa&de""£eill\i&i Iodide Reeetiea •**••'•***«* XT The Interference of Meat Fhytoetevola is the Xfceter&i nation of Cholesterol • SX Preparation of Pure Cholesterol Dibimiid© and Fur® Cholesterol • *••••«*••*»••#••*«««'«*•*«•**•»*•*•*•••#*».•# 8© The Xo&onetrie Fetiiaetioa of Cholesterol bj the Tern dor MemXm Method a® Modified fey Kolthoff aid Yutsty 89 s istit tigm o? mi mi ccsthiy of Aumm^mt wastm m mmm- OF tiJk CdOLSaSXBOX. ZSBSXRkOKmCKr ............. .......... 56 The Cholesterol Coat eat of Hen*e Rgft* 96 The Cholesterol Content of %g® by the Preelpltatloa Method Beeed on the Cholesterol Difero®M#-*3©4iu£i Iodide Reaction ..................... ...... 36 The Cholesterol Contest of %ga by the Precipitation Method with the Oxidative Xodometrle Prooedure *.... . 59 The Cholesterol €oat@mt of Ccsaaerelel Frozen Eggs and Dried J3gga *«•«•«»••••.»*••.»«.•........ 41 The Sterol Content of Parisaoeott® Ingredients of Alimentary Peetee »•»...*»* *••*••****. ** ♦. * *........... * .„,.«*** 45 Review of the literature 45 The Sterol Content of Ferlaeeeeae Xagaredieate hjr th© Precipitation Method Baaed on the Cholesterol 2)lfero&id*~ SodiuEa Iodide Resetloa ............... 46 IT Pag® The Storol Content of F&ria&eeous Xnipredleats of Authentic Alimentary Pantos W the Freeipitetlea fcSethod Based on the Oxide tl 7* lo&oseirte Procedure ***** 40 The Feeevery of Cholesterol Mdfd to Flour toy the Beylsod Method .............. 50 Ike Sterol Content of Feriaaeeeu* Ingredient* Cmuoroially Used in Allme&t&ry Foste* fey the Revised Method ••••»•*• 50 The Sterol 'Contest of Alimentary Foot®* as aa Index of the Sgg Contest »»*•••••«*•*.•••••*»*•••,•*«*»*«***»••«•.»»»«,•»«»• 51 leview of the X*iter»tixFe .•*»••*«»**•#••••»*••*»*»#•.*.«•*•# §5 The Sterol Coat eat end % g Content of Authentic JUlstentefy Pastes by the Free!pitatloa Method Based os. the Cholesterol Dlbramlde«&alt&tio& of Cholesterol ®*sd Phytosterol Blbrosldes 24 XKTSt>IXJrCTXC9f la ik© Halted States, definitions and standards of identity for all- mmx%mrj past®® to serve a® a guide for official® charged with the ©nforc©- aaent of th© Food tint Brag® Act of 1906 first issued as Food Inspection Decisions Ho* 162* and Ho* 171g is 1916 and 191? respectively* They were « re~isaued in 1919 in 0* S* 13- A* Circular Mo# 136" * These advisory stand­ ard® established egg alimentary paste® as those pastes containing not less then 3 per cent of the solid® of ©hole ©gg and plain aliiaentary- past©® a® those containing lass than that amount* So moisture limit was set up* In 1927 ‘Food Inspection Decision Ho* 206* .provided revised standard® for ali­ mentary paste® which were re-issued in. 0 . B, D* A* Service and Beffolatary 5Announcements, Food and Drug Ho* 2 ' , in 1927 and appeared in th© a$m® fom 6 7 8 in Bevlsieas 1 , 2 tnd 3 of th® latter published in 19BS, 1931 and 1932 Q 10 respectively* Revision® 4" ©ad 3 In 1933 and 1926 respectively retained the 3m m limit® of composition as th© tojiediately preceding revisions hut changed th# foiu of the standards. The goners I terse ^alimentary pastes* wss dropped in favor of the elsesifieatlon ”mBC©roni and noodles*** Ttmmm advisory standard® are still guiding pending th® promulgation of legal definition® and standards of identity for such prodnets as authorised in the federal F#od, Drug, and Coes&eti© Act of 1938* Such legal standards are in the process of formulation at the present time* 10 The standards now applicable are set forth a® follows* E. Noodles, Kgg Noodles* The shaped and dried doughs prepared from wheat flour and eggs, with or without mter and with or without salt* Th© egg ingredient may b© whole c&gg and/or egg yolk* In th© finished product the moisture content does not ©xeeed 13 per­ cent and the egg-solida content upon the sioisture- free basis is not less than 5*5 percent* Noodles &r© commonly ribbon-shaped* 5* Plain Boodles. The shaped and dried doughs .pre­ pared from most flour and water, with or without salt, In the tinished product the moisture content does not exceed IS percent- Plain noodles are commonly ribbon- The problem of determining the egg content of alimentary pastes is os© is&ich has engaged the attention of food chemists for rany years. European workers, ©specially is Germany and Italy, vher© these products constitute a more significant part of the diet than in the united State#, have devoted particularly much time and effort to the problem- Stroheeker 11 and V&ubel' have published recently an excellent review of the problem, as a whole and of th© work of the European investigators- Tillmans, Biff art 12 ISand Kuhn also discuss the work- Buchanan.' *', in the only comprehensive report is English on th© analysis of alimentary pastes of know composition, has also briefly reviewed the field, including work done in this country* It is therefore unnecessary et this point to do more than stim&arlsMi briefly the deficiencies of the only .method which gained any significant attention {prior to the advent of the cholesterol method), namely, that based on the determination of the lipoid (lipid) phosphorus. This is the tern used to designate th© organically bound phosphorus extracted by lipid solvents* In the first place, referring to the basic values for th© lipoid phosphor- 14 us in flours and eggs reported by Mertwig even this average value for flour (0*055 per cent on the dry basis) constitutes a correction to be applied which approaches in magnitude th© amount of lipoid phosphorus con­ tributed by the egg component in a noodle containing 5*5 per cent of vsholo egg solids on th© dry basis. In view of th© fact that flours very consider­ ably in lipoid phosphorus content (0.041 per cent to 0.072 per cent accord­ ing to unpublished vork by V. E. Imnsey, 0. S* Food and Drug Admini strati on) and the chief objective is to detect noodles containing less than the 5*5 per cant of «gs solids specified in the standard, this la & serious. Head- v&ntfig© iis the method# Even more important is the feet that the lipoid phosphorus content of alimentary pastes frequently decreases .smr&elly ©a 15 gtara&e ea reported in the above reviews# Hcrtwlg also observed © de­ crease in lipoid phosphorus in th© course of the manufacturing process ©ad su;- tosted that the eapirieal fa ©tor 1*1 fee used to correct the detsr&iaed IS If' lipoid pho&pherus value for &uoh loss, Borzaaaaa sad Mitchell subse- ■cmefitly n m &ted that the lipoid phosphorus la egg© say decrease very rapid­ ly under certain conditions da® to »m eBxysaetl© hydrolysis# la view of the foregoing it is obvious that th© lipoid phoaptioyu© method has serious forea- sic disabilities and the enforcement of standards for ©g» alimentary pastes has boon greatly hampered a® a consequence* It was th© purpose of th® work here reported to develop & method for th® estimation of the egg content of alia@iiiary pastes wfclsh would he suitable for use in regulatory operations in on such products* AXfemd % «&o M b contributed much to the development of analytical methods in this field, suggested tfcet study of the sterol content of eggs, flour .and alimentary pastes bo undertaken# A© Tillmans# Hlffert IEand Kuhn had shown that th© sterol determination m s of value la this problem and that th# sterol content of alimentary pastes resaelned relative­ ly constant during storage, this investigation of the problem m s eede* for th© purpose of clarity in presentation, th© development of the method adopt ad for th© determination of cholesterol itself is discussed before consideration is ..-viven to its application to alimentary paste# and their ingredients# Actually the development of th® method &Ed its applica­ tion constituted concurrent studies, the experience gained in one pfceae of tbs problem leedlag to oodlflcations in the approach to the other* More­ over, though this investigation began in 1934, several unavoidable inter* ruptions, an© of two years’ duration, impeded progress and made i&poeeible ©moaiioa of v»xk tel been j&aansd* Keeping tl>me faef# is ntui will contribute to sa ii&6««vttinAi&$ of am» of tli« mfe* *&« yngpMNft of iflrtife nlgfet m % *fe«mUw to® ei«i?« iks m m m i m n o i i 01 c m u m m m L The C&ole© of 0 Method If & method is to b© of practical value in regulatory work it is soet important that it should yield re&eoaably ©©curat© and reproducible r©*ult*» hot milj in the hand# of different analyet# bat al#o ia diffeswt laboratori©#* Th© att<u&ettt of such m ©ad i« not to be expected if th© method require© undue attention to technique, too strict esstwl of cost- ditiona or a decree of experience ableh can be acquirod only by constant ub© of th.© procedure. fee food an&lyets en^&sed in regulatory work mat deal with a great variety of foods and noanselly will hava only occasional u«© for any one saethod. %©«# @r© ©eaential foot ora which must b© bom© In isind in cons! dor lag adoption of a surthod for th© pr®amt purpose* A aoo&i© eosttilsiag 5*5 per c©nt of ogg solids oa th® dry basis will have a sterol content of about 0.15 per cent to 0*£0 per east* Sine© th© need is for a method particularly designed to detect th© pr&senoe of lea© then 5*5 par eeat of eg,g solids* it is obvious that a ©ultshle Ciioloeteral deter&l&atlott on th© macro «cral© require© ©a inordinately l®rg© Mftple* As the eonte&t of uas&poaiflti&X© flatter (a gravimetric deter&ination} may fee expected to be of mm® interpretive value in eo&junction with th® storcl eeateat is th© detection of adulterated slimsatsry pastas, it is not desir­ able to tie© ® ismmple of loss then 10 grama* A smaller ©as&pl© ^ouM. also bo less representative* Th© procedure originally developed in th© pre#eut •^ ork was, in fact, bseed on th© uae of a BO grtssa sample* Th-t assount of cholesterol la suck mmplo# cowo# within the ssmt-mioro range. This mm.ua that © ssicr© method associated with a rather large aliquot factor or a emi-micro procedure asuat b« adopted* It is mil ksoffi tact ^usatiihtive organic microeaaXytlcel procedure® require considerable experience and unusuel attention to msatpal&tlv# tech­ nique ©a th® part of the sn&lyat* It is therefore sot artxrprlalsig to find such lack of jeeme&t mmmg various worker# regarding the reliability of if I the various mi eroaaclytiesl methods for cholesterol, Abelia sad fc&sltsky Hhave published reviews of th© methods and Buis sad Torres" have made a. critics! study ©f s. ssabw’ of ihesu The#® methods, particular­ ly th# very sensitive ecloristetri© ©aes, are, of course, aeeesn&rily used for clinical ©ad many other biological analyses# The colorimetric method# are based on unknown coacurraat reactions which aakt alight variation® la technique the source of eo&sidurable difference® in results# Such. method® require a close control of the time of reaction, temperature, concentra­ tion and purity of mgeat# and illumination, saoog other factor## They are, moreover, subject to interference by non-sterol substances# Lamport a©®4 ii colorimetric ssethod for «*ti&*tiii£ the #gg content of ice cream booed ©a the cholesterol present# Ee noted th® effects of interfering IBsubstance# in momm samples. Tillmans, Hiffari and 'Euim were unsuccessful la attempting to apply colorimetric method* to th# determination of sterols is alimentary paste# due to traces of interfering substances is the ex- tracts. Though the Sohonhelmer cad Sperry " procedure eliminated such, non- sterol interference by precipitation of th© sterols with digltonin before application of the color reaction, the other sources of error r«ia. The ,1?4method reported by Sobolt Dreicter and Eatelsom ' Involve# precipitation. of the sterol# urn pyridine sterol sulfates before application, of the color reaction* Riffart mad loller report th# application of a colorimetric method uai»$ a sj^ctrophotOEictrie ueosurfr-ent t© the dctoxsilnf.tiosi of sterol in alimentary pastes but again it is noted that ®nur #:oik&qo* Sim- fccltcn der eimmel smfgestelltea* Arbeitsvorschrift ergibt bei giddier Choi os t er inhonseut r®t i on den glelchen F&rbton und die gleich© Farbstnrke* * It has therefor© seeded highly improbable* that these ©olorisstrla jsetbods would, satisfy the re^uirosnents. The other type of micro method which has bean applied to a considerable extent depends upon precipitation of the sterols as aigitonides end oxidation of the letter under controlled con­ ditions with standard dichra&at©, the excess being determined iodisietri ©al­ ly. ka empirical factor is determined for th© relation of th© volume of standard thiosulf&te solution, used per milligram of cholesterol* The Mrthod is applicable to not more than two milligram©« Such procedures were described by Saeat-Gyorggri ^ and by Okey^ and modified by Turner^ £9 wand Yusada • The S'sent-Gyorgyi procedure was first adapted to the sterol determination in alimentary pastes and egg© by Tillmans, Biffart end ISKuhn . They again emphasise th® importance of the seat exact adherence to their procedure. It is noted that their duplicate determinations of cholesterol in eggs and noodles show differences of 10 per cent or more .25in go®© instances, ^iff&rt and Keller considerably improved the result® l>9 by application of fused©*e procedureJ but it is still subject to the general objections raised above. The mlero method® which depend on the precipitation of the sterols with digitoain are also subject to th# ob­ jections discussed below with respect to this reagent. Turning to a consideration of semi-micro methods for cholesterol the only available methods were found to be based on the precipitation SO of the sterol with digitonin, a method introduced by fini.au® in 1908 Tills depends upon the fact that d.igitosln combines &ole for -sole »ith cholesterol in alcoholic solution and forms a very sparingly soluble molecular compound, cholesterol dtgitonide, probably of the indicated formula: % # 4 6 ° * C56E92°29'^> C 8#136030* aThe high isolwmlsr weight of th© ©©©pound and its spar lag solubility in ;*muy lipid solvents smk® it a satisfactory substance for the gravimetric determination of cholesterol* It forms similar compounds with practically all naturally occurring sterols* Unfortunately digitanln is an extremely expensive reagent and os® of variable purity-. Is a study of the applies- tioa of th© digitonia method to th© deter&iaction of eholoeterol on m tk1 eetai-ttlero seele, item* found that th© weight of th® preoipitete depended upon the smmist of excess dlgibonin used, making nece»ry the use of a correction curt© to detem 1a« th© quantity of cholesterol* Then Schoa- S£hcimer and Das found that the amount of excess dlgltonim required is the precipitation varied sot only with different sterols hut with th® partiou- 33 lor ses&pl© of dlgitonla used# Xwrt*' confirmed this and showed that both the spsount and th# concentration of iigitosim affect tee weight of th® rs;re®ipit&te obtained* ffesae fasts xesn that th# analyst rust establish st correction curve .far each a w tmmpl® of digitonin \uxaci, a moat ungstin- 34. 3*«, factory procedure# Brsusch wore recently reaffirmed this* Terrlerw%' attempted to apply the digitomin method to- the present probl hut obtained uns&ti©factory result®* Is e later publication he described an incon­ venient procedure which depends on weighing the digitonids precipitate* dseasipoglKg it by boiling with, xylene, rewei^hing the insoluble residue and obtaining the cholesterol by difference* This method loses, th® chief advantage of application of the dlgltonia, thst is, the high gravimetric factor, end is baaed os th® unsound principle of asking the result depend on a sisell difference between two weights, each of which is determined by & procedure involving experimental error* Th.® fact that the expensive digitoaia is recovered is offset fey its failure to react quantitatively like th© original reagent # The recovered, .product 1® recdmended only for approximate determinations* 937 the report by $lndau8 In 190$ showed that cholesterol could he pre­ cipitated elmost quaatitat 1 vely fr® its ether solution as the Itferosld# by addition of a solution of bromine In gl&cl&l acetic &.ei&, wall© phyto- .sterol bromide precipitated only upon addition of considerable water* Is this brief report Wlndaus described several experl .r* at# of a #emi quantita­ tive character in which the procedure was .*; lied to relatively large 38 esaooats of cbolesterol-phytoeterol sslxtures* liolde in the s » year re­ ported briefly that an attempt to apply th© method to the detection of assail a&ounts of animal fete in ve$©iefele fats was uiuimccessfuX* Lew- 39fcowitsoh ' stated that th© method had served to his satisfaction for the sen© purpose, but neither of those investig&tors gave any dots on th# subject. It m s also found, that Popp^ had applied th# procedure to th# detection of egg in alimentary pastes apparently in & sore or loss qualita­ tive way* hut m#4© only brief mention of his work and submitted a© data. 1 survey of th# literature did sot reveal that assy adequate study of this method Lad been sod# with respect to its possibilities as a quantitative method* At th© same time th© labile oha.ru©tar of tie trealne in cholester­ ol dibrcmide (5-6 dihro^choloatanol) suggested that a seoi-oiero procedure based cm the titration of the bromine istgkt be developed* The necessary veegeut* are inexpensive and readily available* The extraordinary pre­ cautions as to purity of reagents specified In other available r*ethod# did not appear necessary and the use of special apparatus which cannot be readily assembled in any food analysis laboratory was not required. Moreover the possibility existed that the correctica factor necessary due to the phytosterois of the flour in other procedures might be issd* negligible, if not ellailiiBted* The investigation of th# possibilities of a cholesterol dlbvot&lde precipitation method mt& therefor# undertaken. 10 Ttm Cholesterol Oi&mwid® Method Mmimm of th# Literature* Cholesterol dibroisid® m.® pre>4r«4ifi~ 54 cation of th# Stepanov aotiiuia reduction method* /*& argsnto*3etrla detes&i* nation of the bromine in semi-silor© cuantitles of the dlbtoelde nee i©val~ ©pod involving reaowl of th# brosaine by evaporation with alcoholic potas­ sium hydroxide solution, alight acidification of the residue, and titration of the bromide ion with 0*01 H silver nitrate, using ©osin as indicator, a# reoo&c,ended by Kolthofr and Fursoan' v-,1 wine# tli# report of we faxi and Pi rro»e'“ suggested cholesterol of different origins sight behove differently in braminatios procedures, it was m£eIt necessary to demonstrate that ®gg cholesterol would rev.® A is th© present method just as did th® PfimatiehX cholesterol used to study the Accordingly th© ©jaolesteroX was isolated frcKi about a pound of r* J t f * fresh egg yolk. Moreover Bcfc©ohei?§®rw reported that cholesterol far®- gu®atly oontai&ed small e>sau»t® of saturated sterols and described a method of resaovl&g these and a procedure for determining th© oust of saturated sterele ia cholesterol* Th© purl float i as involTSS precipita­ tion ©£ ’the cholesterol ©s th® dibrosaid® by the Windaue aethod, rogeser®- tlon of the cholesterol by boiling th® dibnesaid© with sodium, iodide in alcohol and repetition of th© pros®#® on th.® regenerated product# Scaon- h®lm«r reported yield® of 10 per coat to IS? per ©eat# The Pfa&stiehl cholesterol warn *purified* fey this process, only on® precipitation being * mod© because of the .mall yield obtained* aafconhsiiaer*« method for determining th® saturated ©torola is cholesterol is baaed os th© feet that cholesterol dlbroold# does not precipitate with dlgitonln* Th® determine** tion intol®es precipitating th© cholesterol *» dlbroolde from sold alcohol, filtering,- tedding iigitoain to th© filtrate «sd allowing th® solution to stand in th® cold for 48 hours f whereupon any ®#turct®d sterol present precipltcte® as the dlgitonlde* %®r* this method m s applied to th® original Pfsnsti©hi cholesterol msd th® *purl£i©d*r product, the former its indicated to contain 2*3 per cent and tit# letter £*£ per cent of saturated sterol* At about this time the report of G&pSaer and Gaias- borough was noted in ^hieh it was stated tfcst cholesterol dtbaranld® Itself decomposed cm stsadiug In alcoholic solution under conditions of th® method, yielding B per mn% to 3 per cent of digitonixt-preelpitable E't sterol* They concluded that the wehonhelsier procedure was not accurate for aasll quantities of suture ted sterols* uTh® cholesterol dlbra&ld® precipitation method mis applied at -12® to 2$ mg* sempl®* of th® egg cholesterol, the original FtmmtXetl cholesterol © M the "purified* Pfaactlehl cholesterol. The results ah&m in Table X indicat® bo aigBifio&mt differences between thee® Sfsmpl©*. TAMS. I The Precipitation ©.f Various Cholesterol Sample* Description. of Sample Weight Egg cholesterol iSgg cholesterol dried in vacuo mt ISO® Ffsastte&L cholesterol P^urified** Ff&zuitie&l cholesterol dried is vecuo at 100® sg* 26*0 25.9 26*2 28.5 Bromine Coat©at per seat 28.1 28.1 £8.1 28.0 The theoretical yield im M*& mg. of cholosterol dlbrocslde contaia- lag 29.26 per oeat of bromine. Th© composition of th® precipitate will be die.sussed later. To deteziBtn® the effect of the bromine concentration, 20 Kg. samples of Pfnastic&l cholesterol w®dre braasia&ted cud precipitated *t 0®, varying only thM bromine content of the carbon tetrachloride* The results la Table IX show that the precipitation is not at all sensitive to the brotsia* concentration, tb® ©alp noticeable effect feoiag & ssmll decrease la the weight of precipitate with the higher concentretioa* of bromine* uTABLE II The &ffeet of Bromine Concentration on th# Precipitation mum * ! Precipitate, i # Weight Bromine Content * ♦ * 'mg* .por coat * 2 *10*4 27.9 0 £5*4 27*8 s £4.8 27.0 * • £4*8 28*0 s £4*8 £7*0 s Bromine Content of the g. 'per ml# O.X o.« 0*4 0*6 0*8 A concentration of 0.1 g* of bromine per ml* of carbon tetrachloride is ample for the purpose# for ehich the present method is designed* That th# dose control of temperature is not a critical factor in the brominatloa and precipitation is indicated by the result# in Table III obtained by applying the method to 2Q mg. efsmples of Ff&nstiehl cholesterol at different tempera twee* The only significant difference# represent the expected smell increase la the loss due to solubility with iacree.se in temperature* TASL2 III Th® Iffset of Temperature on the Precipitation Precipitate f eight Bromine Coateat per cent 28.2 £5.3 16 Sines a ttt&psYatur® of 0° is most eo&venlsxitly maintained this m s adopted as th© isssparatur© to 'lie niai* The procedure m s n©« applied to various ©mounts of cholesterol* Th® results ia Table X¥ s&ow tlie ©opposition of the prseipitate and tfee derifetion of th© individual results frmi ta« weight of the precipitate calculated from the linear function mg. of precipitate « 1*559 (;ag* of cholgsterol) -1*0 detoraified by th# least squares method* TABLE IV five Halation &®tweea tlx® J*r©cipliiate Obtslued and t&© h©igM of Cholesterol Used «© Cholesterol i Freetpitate i height „ deriatioa t Brm&Mt s Observed 0®JUml&t©& : Content * ♦* *-t *» 'K#* mg* ft f per cent 1 eft 10 l 12.2 if.Q *0.2 » «... to t £5*1 £5*0 -0.5 l .0 30 l tJ.-’S' . £ 39.2 0.0 *ft <> .9 40 l 53.4 51.8 *0 • 6 5 07.0 550 *ft se.o 06*5 -0.3 ftft 28*3 f *ft- Afmrmge deviation 0*3 The eeu&itiens of th® bremln&tlon and precipitation now having been ost&bllshed, n more rapid filtering device whloh could be ©o&vebl«atly used to maintain th.® low temperature during filtration '«« developed* la tlx® £ravt$etrie studios filtretlans on th© sintered glass sxeGibles wore ^uite tedious, ea a filter of fin© porosity (Jess 04) was required to retain the micro crystalline precipitate. The -method wa® applied to sesd-siicro quantities of egg cholesterol end the bromine is the precipitates titrated If with 0*01 M sliver nitrafc® according to the established proco&urs* "Si# gesult* &ave fcha relation mg, of cholesterol • 1*9 ♦ 2*0$ (ml* of 0*01 H 4glf0g)» Hi© arg@ntes®trie titration bad given sstisf&etory results la th# feramls® ietor&i»&ti©as, as it k&s a sharp sad point, -3owsv«r the end point in­ volves a ©hangs fro® sn orange-pink; to e rod-violet color, and couai&sr- able oa£porl«ajc© with the titration necessary to attain stood results* Mcor&iagly attention was given to the iodoswtric method based on tlx® re- action between alkali iodide and bhol«st«rol dibromide In $se*ton* ‘shioh E|0 gjt Pirromo" and Pe fanl end Ptrroao''" dad reported to bo fairly satisfactory- on th® macro seal#* The Xodoe^trle .-atluaIicag. of <&qlest«poi 0-" &nd on tho ChoXeatoyoX Bt bresaids-iSocliusi Iodide Sssisticm# In the &’*t tales last cited the Italian investigator* gave ao detail® of their procedure -other then the foot that tbey used a PS per cent aqueous solution of pot&salsea iodide and found £0 to 29 ml* of st&reh solution necessary to develop the stsreh-lodide color* It appears that the method wes applied to tbo analysis of only one cholester­ ol dibromids aasiple, & single result being reported* Thi® -value of £8*04 pQT cast bromine Is quite low compared with the theoretical bromine content (29*£S per cent) end the values reported by other etrfcdodi® (26*93 per eenh and 29*0® per cent)* Is the presence of soma organic solvents the steroh- iodl&e color is by far lass eeasitive thau the yellow color of the iodine R« Itself as indies.tad by Kolthoff and J U r « i f * Therefore* is the present work the iodise liberated in the ehoXesterol-sodium iodide reaction 1m acetone and &loohoi m s titrated: with sodium thloeulfete solution just to dlaeppeer&xice of th® iodine color* la th# eoneentretloii* of solutions used 0*03 ml* of 0*01 H iodine solution produced a perceptible yellow color ic&nd u*0S ml* a quit© distinct color, so that practically no eorre*- 18 iiosi for ifcts&i factor is neeeasciry. Xa ilia initial work aa iMa procedure tli© re^etloa between oftolesterol dibrmMc and sodium iodide mu tried in scetcne CS-.W.H3- ttJb * la eleobol* tb© ruction i so slow in tfee latter solvent that it# us© sot preetloeble* Attempts to obtain ebel©sterol dibroasid© containing the theoretical quantity of bnonliift had been unsuccessful. The aoenrsof of tbo method fii therefore by o;eess of sitosterol dlbromide tfould remain is solution up to th© point nth®?® its solubility as* also exceeded, accounting for th© deflection in the curves for mixtures containing, ss&ssll amounts of cholesterol* With an asacumt of cholesterol exceeding coiiaiderebly the amount of pbyto- st#i*©l which would, prod pltate is the ebsenee of cholesterol no de­ fleet ion in the curve for such cholesterol^pfcytoeterol mixtures would be expected* Although it was not believed desirable to introduce ®a aeetylatlon step into th© quantitative procedure, if this would have overcome th® eopreelpltatioa difficulty its feasibility would have been considered* However, a few experiments with ,-j.ixfttre« of cholesterol acetate and p&ytosterol acetate demonstrated that eoprscipitstion of the dlbnasldes also occurred with these compounds* from the standpoint of' th© cholesterol ds&erRtlafttian th© significant fact is that th® amount of sitosterol dlbrostide which pr&cipitates with cholesterol dibroaid© is independent of the quantity of cholesterol present so long as Mil® equals or exceeds th® sitosterol content of the sterol mixture* fiasco, in the anAlysls of flour, plain noodles or noodles of very low mgg cosiest sufficient cholesterol assist he added to the umsapOBiflsble miter to assure that an excess of cholesterol is present* Preparation of Fur© Cholesterol £>l'bromid.e &nd Purb Cholesterol* It was noted above that precipitates obtained in th© study of conditions for the cholesterol dlbxtxaid* precipitation iscrthod eonteincd consistently about SB par cent of bromine instead of the 19*26 per cent required by the pur© ilbromide, Cg^E^OBrg* The gss# composition of the precipitate wee found «hes «s ouch ss a ur&as of cholesterol mu? bro&lnsted and pro- eipltsted un&mr similar conditions* It .sdll be reeslied that these con­ ditions represeat a modification of th© Wiadaus szetbod to adapt it to th# .present purpose eh 11© ist the Bmm time ©feserYing, Insofar as possible? # the important factors noted fey Bells*'"'’ is studying tit® abnormal lodlm© ebsorptiom number of oh ole sterol, n&aely, kslogeaattoa nt 0®* us© of c®.rfeo» tetrachloride as solveat 3&d the ©beeac© of acetic acid during the halogamtion. It wee desired to obtain £*©&© of the fur© dlbra&ide for uaoqttivoesil testlag of the bromine doter&lns 11 on* 33aou#sh th# di~ hro&id© ia sot isolated an such is the cholesterol determination* orl- de&c# that it is ^osatltstitmXy forced under those e©Edition® #1X1 bo presented is the following section. Lifsehuts criticimed the «>iadauo saethod cm. the ground that th© precipitate %vea sot pur# but hold loosely bound ■acetic noid* He r©«* ported that fey krcstimstls^ cholesterol is ether ©ad preclpitetisg it with 00 per cost mlmhol pure cholesterol dibro^ide was obtel&sd* He also stated theVthis pura dlbromid©* reeryatcillised .from &l».ei&l acetic acid* had the properties of the Si&d&us product *u& on raery#tal* iiiatloa from alcohol reverted to th® pur© stste* <&© euslysis of th® fei&d&u® product is reported &ad a single analysis of Lif«chutx# product Indicated a fercsstlne coat eat of 38*64 per cost. In th# preeent work pure cholesterol dlbroslde m s sot obtalsal fey th® original lindens pro- 3s? ** 45ceduare* nor by a sosagrw&ot modified t o m described by &iad®us and Luders' ' * Difficulty in completely removing acetic as id from the preelpltste* mt» sis© noted* ffeo products ebon recrystalliseed from alcohol contained even. lee® ferossia© &&d the occurrence of dee&apositlQB. was apparent la the relations* Beerystelllssatlcm of th© Wixidmm product fross acetone as recommended by D© fasi sad Firrone*' also failed to yield th© pure dibrossid©* fh# sensitivity of th© compound to bent, moisture and sol­ vents readily attacked by the labile brastue causes th® difficulty ia obtaining th.© pure product. From, th® present work there 1© Yw&stm to believe that moleeulmr ©aiapoua&e of the cholesterol dihromid© with sms of its deeo&poai 4 i on products :say occur. This ^©uld aceount lor the observed formation of products having, different composit ions mid dif­ ferent melting points on reeraretalXisstioa of impure cholesterol 11- hremld© to constant melting point frost acetone, alcohol or ether* Qa th® basis of the experience with this compound it appeared that the preparation. should be obtained purs without the ne-e©ssity for recryetal­ ligation sad ia a dry state if possible* AQcordi&oly, a procedure m s developed involving brottiastioa with u slight excess of bromine ia carbon tetrachloride solution with chilling, precipitation of the itbromide with petroleum ether at -10® to -15°' and rapid filtration. end drying first by aspiration with dry air until room temperature is ettst&ed, then in vacuo over phosphorus pwntori&e to constant weight. Th® product melts to a clear colorless liquid at 114*4° - 114.6°, followed by effervescent deeompositiou with hrownlns* A H previously obtained preparations had exhibited such decomposition before ©r on melting* fhe pure dibromide con twined the theoretical bromine content {S0.B6 per coat) as iot@asii.ned 68by th® Volhard method ©ad by the lodcoetrle van der tS-cules' method, ©c~ 69eordlog to holthoff and Tutsy described Ik the following section. By the iodemetric method trnsed on the codim iodide reaction in acetone th© pure dibromide ecmteinod £8*6 per coat of bromine, which is the highest bromine content by this method observed in any of the previously obtained preparations. .Sims® en&lytiealiy par® cholesterol dlbraaide could be thus pro­ pared, cholesterol of uaque*tlonab I ® purity is obtainable therefrom by ** 4B&ebra»lB&tio& under mi14 conditions* Oehonheimer ” bad purified c.h©le#ie;*> ©X by preelpltstiag the dihreside {s'ladftu* method), reoryatslli sai&g it from alcohol srnd debroniaatiag by boiling it with ©odium iodii & Im sleohol* This pro cess ia repeated irith th® regenerated cholesterol* Th© yields r@- ported ere 10 par coat to 15 per cent* £vea lower yield* have been ob­ tained by this aethod ia the present work* Moreover, ia ?i» of th# in­ stability of cholesterol dibroctld© {noted also- by Bohoaheimer), it doe® not seem good practice to heat the grubfftsaca ia alcohol curing * purifica­ tion process* However, cholesterol dlbro&lde is debro&isstsd smoothly msd rapidly at rocaa temperature by th© setloa of sodium iodide in acetone* Accordingly, the pure cholesterol dlbrooide prepared a* described m a al­ lowed to stand over-night in seeion© solution with excess sodium iodide, the liberated iodine was reduced with ©odium ihioeulfat© solution and the preel pita ted cholesterol was recryst&llixed to ioasts&t melting point from alcohol (two crystallisations)* Th® yield was $3 per cent* It is emphasised that the ¥fa&stl®hl cholesterol used is th® puri­ fication process is evidently free of setttreted sterol* withia the limits of the Schoiifceli&sr determine, tl ©a, &© previously discussed* An opportun­ ity has not yet h&m afforded to test Aether th# preoipitctioa process will remove saturated sterols frost cholesterol, which was the primary purpose of AchSnheimer's process* However, it Is believed that the de~ bromlxisLtlaii is preferably carried out ia either process by ths< sodium iodide refaction at room temperature* The lodonetric Aatlmstlon. of Cholesterol by the van der Meulei* Afathod b& Modified by Eolthoff fand lutmy* oubseluent to the development of the iolomstrie method based on the cholesterol dibromlds-sodium iodide reaction and its application to the analysis of feliaumtary pmsfces, tboro 50 68 appeared th® rapid and ©©ettrst* modification of th® wan dor toulim method for determining m m l l gpi&ntltles of bromides described fcy Kelt- &Q hoft m & Tutzy * This method is 'based on the oxidation of th# brossld© ion to fcrosifete with 00d.i1® hypochlorite solution, reduction of th© ©at- sees hypochlorite with sodium formste sad lolometrle determination. of th© breast®. th© procedure »© published 1« applied to quantities up to 700 siilllgrems of bromine* Drake and Baaley had applied © simller pro­ cedure to the determination of methyl bromide la air* It is apparent %lmt tmob e sseikod would elimls&t© th© chief diea&veatagoe of th® ©ther lodometrl® method, finely, th® uncertain end point aosotiis#^ not ad due to colored extract© and th® effect of ^t©.r ©a the titration* Further- ssor#, ©Inc® th® vsn der tteolea method yield® for the titration six ®qulx®l«Bta of led la® for @©ch equivalent of bromine th© increased #©• curacy would permit us® of © smaller ss-.pl©* By some adjustment of the re&^e&t® it m e found that the Kolthoff and tut ay procedure gave very satisfactory results for th© determination of up to et least 40 milli­ gram® of bromine* The revised procedure «** the® applied to rumples of par© cholesterol dibroeaid© following ©vaporstloa of ethor^aleohol solutions of these with potassium hydroxide m d neutralization with hydrochloric aoid. Th© results are shown in Table VII* S I t m m ¥ii Aa&ly*®* of 0&olo#*«rol Dibrosild® by th® OxidotiT® Xodo»#tri® Hotbod Qioloftteroi Dlbrctssid# fibor Branla® o.oaose m h «2&$o$ (ac&?«et®d for blank)- Mg* ml* per e®at 5*65 6*06 29.57 10* IS 10*83 m *19 14*35 16*01 39*89 m*m 21*48 39*36 210*25 33*31 39*39 56*65 99*03 at* ax A®®**#® 19*27 Th® r*vl®®di prooodar# wao sow u&©4 to 4®t®iml&* t&® relation b®- twoea th® oholestoroi «ua& th* tit«r «&®a applied to th® ®&ol®®to:ral 41- bzanid® par#®tpit®tl©a m®tb®4 (Appoadi*, p®&® 86) * ¥h® purlfi®4 ob®I#«t«r- ol ®&® »a®&* Hit® result® ladloatod th® relation mg* of &h&l®*t®r®l •» 0*55 ♦ -0*68© (bX* of 0#Of H Is T*bl® ¥111 sro shown th® individual remits ®ad th# dovi&tl®®* fro® th® valu*® c&Xostlated fey th® sbov® relation. Th® ®orr*®tlo& for tfe® blank dtterKtlnfitlaa os th® ro&^eat* o^ouatod to 0*64 ml* 32 m^BLl VIII Xo&ometric Cholesterol Detemimticm. by th® Oxidative Method Cholesterol Titer Cholesterol Deriaticm Used 0.02 n Calculated (corrected for blank) ng. al. lag. sig* 3.40 7*24 5.50 -0.10 4*90 6*39 4*95 -0*05 5.20 4.92 5.30 -0.10 9*95 13*64 9.95 0.00 10.20 13.90 10.13 *0.10 9.75 15.35 9.70 *0.05 10.40 21.80 15.55 -0.15 14*SO 20*46 14*60 *0.20 15.10 21.02 15.00 *0.10 20*40 28.93 20.45 -0.05 20.00 28.32 20.D0 0,00 19.95 27.8© 19.70 *0.25 29.95 43.01 80*10 -0.15 30*30 43.43 50.20 *0.10 20.45 45.52 30.55 *0.10 39.95 57*01 59.73 *0.£0 39.80 57.10 59.80- 0.00 40.15 57.63 40.15 0.OB 50.10 72.20 50.20 -0.10 A r0 rm..m d m is. t i on 0 • 10 Turn theoretical factor In this doterminatiosi for converting the 0.02 M sodium thioaulf&t* titer to cholesterol 1 b 0*6444 instead of 0*688 ae found is. the above equation from the experimental data. Thin would indicate that only 93.7 per cent of the theoretical amount of cholesterol ctihromide m s actually foraed end precipitated, finee a aide-reaetioa involving oxidation of the secondary hydroxyl group might account for the discrepancy the aethod m # applied to ehaleeterol aeet&te* The vaults agree closely with the relation sg* of eholeaterol acetate • 0*85 * 0*757 (ml. of 0.Q2 M fegSgO^) mas shows la Table IS* The theoretical factor in till# deters&imatioa for cholesterol aeetet* 1m 0*7144, Indicating only 94.4 per o@n.t of the di~ bromide its a precipitated. This ia in very good agreement eith tiie value obtained sdtfe cholesterol, considering the asell number of sample© of cholesterol seekst# used* TA3LE II Application of th© Cholesterol Method to Cholesterol Acetate Cholesterol Acetate Titer Cholesterol Acetate D@Tlg.tioa Used 0.02 Ef IfojgSgOg Calculated fcorrected for^ blaiik) sag* 10*10 20. IS as. 95 39*9-0 ISiX 4 12*41 2S.25 38*52 51.74 mg. 10.20 19.95 30.00 40.00 i&ib * -0.15 +0.00 -0*05 -0.00 Is order to detexssi&e if the lacking brwsias was still preeeat or­ ganically. bound in the filtrates, c aeries of cholesterol eee&ple* m s brmiaetad end precipitated &a usual* the filtrates and waabiaga kept at 0°, being collected is 100 ml* volumetric fleeke. These aijctures were diluted to the mark with tat«? at 00# filter#! again -and th© broislm# determined la the second precipitate. Th® cholesterol vss calculated fro® the titer using th# theoretical conversion factor 0.5444* The re­ sult* in Table 'X show that th# theoretically required aaoirai of bxo&in* must still be organically bound, evidently a® cholesterol dlbrmide. The titer ha* been corrected for a blank on the reagent* of 0*54 sal* mTABLE X Total Cholesterol Becovery 1 t #ft Cholesterol *« free!.pltate *«- Precipitate * Total Used £ i from nitrate £ Cholesterol $ Titer Cholesterol t Tit or Cholesterol : B©covered •• 0.03 K C&lculated 5 0.02 M Calculated ftft I lirar *r 4? ft fls^SgO^ : JL 4 I i ft e• sg. I * nti* mg* 1 * si. mg. 3 s mg. par cent 10*05 ft *v 13,35 8*50 ft 1 2,46 1*58 * *• 10*18 101*5 1***8 *ft 27*85 17*94 f 3.46 a. 25 S 20*17 101*1 30*00 % 42*35 £7.29 : 4.5# 2,94 £ 30*25 100*8 39.fO « S5.84 £ 5*53 S. 43 S 40*06 100,4 £ £ t Xt was also found that tt th® precipitating sgeiat -#a« diluted to about 50 per ©eat acetic acid instead of the approxima. t©ly 80 per cent used morally in tie ssethod, sail but a few tenths of a of th# cholesterol was reoov©red in the precipitate* These results augment that an ©quilibriuK is established under the- bro&inatlen conditions between two fors# of cholesterol dlbrosid#, one of which is muck lee# soluble than the other. Sine© cpi8i®rt nation of the hydroxyl group is .not likely under ties# condition#* it seen® probable that th© formation of isomers aria lag froei cia and trass sd&itioa to the double bond ssy recount for 49, 50 the observed result©* Although the invest i g « r t l b y Pirron© und 0® F«i end firrone^ indicate th# existence of four cholesterol -&i~ brcsnide# waiting, et about 97**» 106s, 114° and 122° respectively, they have succeeded is obtaining only cholesterol by debrcmln&ti on of these products* Th© liaiit-atlcaa of tin# have not pomitted a further investl~ getios of the relation of the two form# evidently occurring la th© present ease. The iodotseirie method for the cholesterol determinetion based cm th© moxid&tiv® procedure in believed to bo th® mmt suitable os® from th# standpoint of th© objectives of th® present work* A« shows, th® method Is sufflolesLtlf' preel## and a#our*t#« Th® tit rati os end point 1® on© with which ©11 analyst* are familiar and th# titer is not ©ffeeted by th# presence of neater ia th# preetpltate** .A# eonpored with th® sodium iodide method th# chief disadvantage# are that th# oxidative method re­ quire# more tine and may b® exported to be effected more by cmy non- sterol bromides which might pvoeipltat#* tiae iisfi ;motf c:* op i U Z s si m m s oj Tin Choi * six^ol ujcrit xiiptioi; 'The Cholesterol Content of lien*® Is.-;® Itsyiew of tbs l4t,«r&turs* 2&e cholesterol of th© egg ©©eurs 71 yg paraetieally entirely is th® yolk. Dam and .lueui ' found only a trace XUIn the entire arihite of sm e£&* TiXlfsans, Riff&rt and iluJm "YrO&sluded .from s. lint tad asisffltjer of analyse* by their rut her urn eat i sfaetory method that all of the cholesterol mta preseat ia the free for®* However* n«a 74 7*% *£&, stu&im by teller' M* Thjamifcattser and Sehsber , Dan ' sand Eusut' in- die©te 10 per cent or more of the stesel ia fresh egg® ia esterified* DM?*’ foi&nd frosj 0*468 per-^ yam»*1Sp tf»$3LQ per ©©at of total ehole sterol / j g ia individual 0ggs# Tillmans, Bifrprt and &uhn* ’report an average of 1*49 per sent of free oftoleeterol im eight so&ple* of yolk* l*ae&~ yts port”' ' found an average of 1*3-6 per oout-of tot el. cholesterol in. th# 76yolk* Pertesn reported from 0*54. per ee&i to 0*7t p©r esst of free cholesterol in twelve individual eggs* <$&« aver®#;® being 0*60 per 8®t, fS£.*sd Kleiler’*' found th© sverage free cholesterol in three staple* of yelk to be 1*49 per seat ftltrlmetrie sethod) and 1*S0 per cent C coloris^etrie method)« The Cholesterol Coat eat of i&&* by the ffTeetpltetlon Method Based on the Cholesterol Plfrromid*^ odi. ^ ©a ©3 tfk £^& * # * O W O& o ft» ijz m ♦ * * O O <0 i«* ©S «5 » * * 4 • * * 4 | * » 4 4 * 4 4 4 4 4 50■s* #4a> 5 t© *0**S OS- Kj |>s Tfi. ?4* • * *c»*E3f **•H H* W &» W * # * W *O *3 ►«* ct ft H* ct «* C* £fe 0* ft H m m h ® a i H H H 0* O *k C l I I I I H *■ ***& R5> M ?'5v £ B Ss* cf & gr ft ft « & ©3 *•* * * 4 4 4 4 *•* r* i.| & w « £-* ** s~~ I a &H« 0 §3&r © H= » ►3»cf B cr «♦sr** tt |:3 Srf“|X a 5^» *a*oaap* H *5a05 <3 w Cr it13 fte %i «* © a 2a* or Mi f © £1 'J©© && ts

# *-■’s:f-* ftc+ ma &¥*© ©crr*- <-i& £3*H* fta < t. © 0 1 2 aefr 'r% % €S © © ISfa sa I & a ft ? «*cr €4*IT# «♦H»«t 3 ©s jt j is ms j. oo #* !: nm to^ a^ f f^iq »q % p3 &% nx © 5-O ti »© m t h ® chcissterol dotextinction is also sertosdly improved. The ebev* analyses were in the spring of 1036 # Th* fro&ea #ggts n«r@ kept at about -I#*3 C* , while the dried yolk m s stored at *nb©ut C. until subsequent snslyse# were am&e la the spring of XM0. Hie Cholesterol Content of £ta« by th® Pr^eipltstioE et .©d with the Oxidative lodosaetrie Srrocedure* Oa® of the purpose* ia the develop-•'I^it1'".1, f1 ‘i ‘ " '■ ~r ~ "T~'~i'n--~ '""" ~l~iTi'i|- ~i~ n’Tir ir ~ '~- if—r- v ~yr •|ir~HTJrni(iri*>iiiT)r~M. ifr" itfilfT#.iflW *' "*- m*nt of the axide.itv* iodoaaetrie procedure m e to elimiisat® the diffi­ culty experienced ia th# f t x m e r procedure due to the color is t hm acetone solutions of the precipitate#. The udeorptioa tmtmi%u® re­ duced this interference considerably but included certain feature# to 'litileh objection could he raided* The edeorptioa step m e therefore ellalneted. It we# noted {fable II) la the result# for the tiaeapaai- fiable TOtter by th# original method that the pair wbioh had been ob­ tained fey an acid'hydrolysis before eapoaification agreed perfectly, while the other result# varied considerably* The acid hydrolysis step was therefore included in applying th© new procedure to the egg# previous­ ly examined, with th® result # shown ia fable H I under wJ*el4 Hydrolysis Sethod* * Although th# result# were very good for the cholesterol determine** tioa, the uasaponlflefele waiter ssetl^ od a s .still unsatisfactory# In the ©ours© of these deiemiamtion# it ts§ noted thct la scc&e cess# fine­ ly divided calcium soaps suspended in the ether extract# weor# not tlrely removed fey the filtration through cotton* Taos® were racsoved fey filtration through anhydrous eodlu* sulfate, however, and the direct saponification procedure sac tried with thi# Bietbdd of filtration. The results obtained on th# »eaa* eggs used in the previous analyse# are shown in Table .XII under th© heeding Revised Method** between mch 40 series of analyse© the thawed egg® verm refross-en, bo the variations ia results as between series may be tabes as Including over all sampling errors. On, the oasis of these results this procedure (Appendix, pegs 83 ) was adopted for the det e&olns 11 on of the cholesterol content of egtgs* TASLiS X II The Cholesterol Content of Ifcgs 8sed in Authentic Alimentary Pastes by the Oxidetive Xetasetri© Procedure s #* #» Description « Total. * Acid -Dydrolysits Method I « _ ^ eTised Method of Ssnspl# s Solids Un&eponif 1~ Cholester­ ft«r U&s&po&lfi— Cholester­ ; * &hle at ter ol in *ft able Matter ol in J l is &ol&d* Solids 3 in Solids Solid® s : I : per cent ** per cent per cent ftft par cent per seat t • 3 frozen eV 26.91 3 2.66 1.27 «>ft shole eggs it 26.91 *-ft 2. 77 2.26 ; 0« 3 £#SjS 2.JB6 3 i t : 3 ft♦ *ft t I : £.86 2.EQ I 26.87 3 : 2*84 2.19 ♦ft 3 s 2.88 2.19 3 3 s I 3 : 1 ** # S. 7 8 2.28 ** 26.73 ft# ft 2.79 £.26 s 3 * 2.77 2*24 t •* tr t I I Frozen m■ft 46.11 I 3.56 2.94 * yolk 3 46.09 1 3.82 O OQ 4Um **0 3 S •* 3. 59 2*97 1 t *ft 3 *« I t : z ft 3*45 1*97 3 46.15 I t 3.45 2.97 3 $ : 2.80 2.96 3 I #ft ft 3 3 Chinese 3 96.94 ft• 3.62 2.34 : driod yolk:* 96.96 : 3*57 2.79 : 3 3 t 3 3 s 1 i s 3.45 2*81 3 96.97 e♦ ftft 3*89 2.80 41 T: a- Cholesterol Content, /;■.!•.;-rclsl ffroge-* ^ Pried. Jk^.* During XS36 su&plss of oaamerci&l froaen «g^s were collected at egg p®ok~ log pleats ia various parts -of th.® country by inspector# :&« original sample hs& been re-frozen following the first an#ly#ia* %«y are m % included ia the averages* temple Mo* 5 of fable XIII has also bean omitted frcmi. th© averages ia view of it# abaeraclly hi^* solid# content .for whole egg* This value (£9*4$ par cast I 1# far outside the rang© C25*9? per coat to 42 qjL 26.33 p#r cent ) observed fey Mitchell, AXf«M »ad h’efMXT' in seventy* four sample* of consrelally broken out who!© eggs* Otherwise the varia­ tion ia th# cholesterol content is essentially the sasse as was observed by tb* last-neuned worker# for other eg& yolic eonstituent#* ffeo aeeumi&tion of date on th# cholesterol content of eoaBsawpeial eg$ product# fee# sot been carried far ia the present work* partly fee* onus# it is only sines tbs 1939 season closed tfeet the legal definition# $ad standard# of identity for fcfe# various product# os promulgated last year feessat* effective* Operations under the## standard# ;.;ay significant- ly effect the composition of thm eosssercial #gg product# and it fee# therefore been considered inadvisable to eotaplet* analysis of nil the oil sample® os hand* ffee result# obtained, however, desnosuiirat© several important .fast®* It is evident that the efeolesteseX content of commercially broken out irihole eg£# and yolk# does not vary too sa&cfe to permit use of the cholesterol contest of foods as as lades: of the ®g4 cosrfcomt. Moreover, the cholesterol content of frozen eggs and dried eggs shows no- change on storage over long periods* It 1# likewise shown thct the cholesterol content of #&«■£# does not change when the egg* are dried* 43 TABLE XtlX Tim Cboleatarol Coat ©tit of Coasaftrelal Fr©*#ii lliola %ps *# 44 £ t a4 s # Ko. © » © Year sSo&s*©® of # Total iJtat by Acldt0a8tt|)Q&i«»:Cfcoleater*s Gbolaater* jPasteadsB&»11 %&»* Solid# si^ ydrolyal# % flabla s ol is s ol is % i (State) £ *« is Solid# »a .Matter 3 Gollda s Fat t f I * »•is Soiidai * t £ ipca? east* per mmt ♦ per ©eat t par oent 2 par oast s 1 l s s t s 1 » 1936 S Hafe*~»Ia* s £6.84 * 45.12 $ V 44 2*13 *© 4*72 £ ** w 1 ? s 27*26 S 45.6? #•* 5*17 S E.gS : 4*89 3 : m i Calif* 1 27*73 s 48*21 t 2*68 4* 2*15 •* 4*42 4 ** » s Mo*-*Kaaa., I £5*01 I 43.03 : 2*60 # 2.08 % 4*77 * £ .5 (28.65)t a* i— *© t (2.61) S (2.06) t s 1 I : n4 *•* t 5 * ft : Ida* I {E9.4S)s (47.16) s (S.01) $ {£.*?) £ (5.14) :. : 9• (St.lih 1 (3.04) £ (2.46) I ««MNt 4» a4 s > 4♦ 3 X 6 ♦ t* i U® *~>KmnM<,-a £6.85 S 45.51 3 2.77 : X * 13 t 4* 69 7 « tt s Calif. «'* £6*50 I 46*19 I S.60 *« sues $ 4.39 ** I *4 (26*63)2 . . **»W I (2.4B) t (1*99) S 5 X a : I S f 3 1 <* i Colo* s 26*26 t 45.54 4* 2.51 a4 2*02 ** 4*44 : s ** (£6. 51Is . * (2*55) 5 (2*01) ©* **'*m ♦* if* s s 3 : a• 9 t ; Knn»* ? 26*51 % 44.93 # 2*74 s a.go ©* 4*90 10 * *» : *&ak* ©* m * m s 45.15 S 2.69 t S. 06 *© 4.56 s *© : (26*11) * ~— I (2.52) *» (2*05) s a*© * i *9 «5. t : 11 i 1939 : ? t 27.a? s 44.30 * a.44 s £.05 ** 4.63 xt ©© ft * T i £■6.73 s 45*43 X 2*76 s £.10 s 4* 62 13 $ ** s T #m 25.46 £ 44*15 t 2.70 s £.14 2 4.85 : s t S % „ s JL t 3 t 3 • Marlaum ©* 27*73 * 48*21 t♦ 3*17 I SU23 s 4.90 Minimum *© «wtJ#46 * 45.63 $ 2.44: *•v 2* OS % 4 * 39 Average »4 26*61 * 45.32 I 2.71 $ £.11 • 4.66 I <#4 % s s 44 TJ3LB M W fh® Cholastarol Coata&t of Coa&aaroiai Frcrawsai % g Yolks * : : I 2 ** ♦•*. So,► 4 Yaar tiSoura* of #* Total iFfet by Ael6iUnsap«mi~8:C!ioiaator*»rCholeatasv S F&aked;£$i«ll %g#s Solids 2 Hydrolysia ♦ ftable a• ol ia ¥« ol ia * l (St&ta) 2 s ia Solids s 38&tter * Solids «•* 'Fat -a ....« JL. * . 1 iia Solid©i JL•* »# 2 pay coat par caat ♦apar cent 2 :,:#r ©eat T xmr cent 8 £ I f 0 «* i X : 1936 £ Y«X. f 43.54 ; 39*96 0 S,59 t 2«m a* 4*81 2 z w s flab* **Xa. 2 45*50 f 61,54 Z S.?? ** 2*93 a4 4.76 t m s Calif, S 45*80 J 61.69 «• 3.55 S £*8$ 1 4*6? 4 i n i Kans. £ 46.07 £ $1,33 2 4*00 : 3,1© $ 5.12 i £ a*(46*64h *-•**« £ (3.74) ** (3.11) 0 i %4 $ *• 2 ** 1 5 % « £ f •0 4.4*10 2 60*61 2 3*51 2 E.80 4* 4.62 6 i m I la. 00 45.51 2 61.16 a» 3.70 0 £,aa ; 4.71 ? z tf s 18o.-£a&*«■ Z 40*43 2 m * m t 3, Si 0# 2.7? i 4.78 9 I m £ Calif. 0f 46*49 2 59.54 i 3,49 «•a 2.69 * 4.52 «« £ 0*(■43*37)2 *i *1* '!*» 2 (3*26) ♦* (2*69) 4 % 1 1 1 I i 1 9 0 m i !£ash* *« 41,54 ? 59.76 % 3.59 £ £*§$ *.* 4.53 *# z «a(41*63)I t (3.9B) 1 I #* ¥#*•*■ *0 5,52 I 2.95 * «*«* 14 1 n £ f 2 46*11 s $1.05 ** 3*46 0a 2*96 I 4.83 IS t # 2 ? «. 4S,?S 2 $0,35 2 3*48 2 S.89 i 4.79 s 2 1 ; A JL JL 1 2 T «-a z Maxiaw 3 46*11 2 61.54 i 4*00 *•# 3*15 ,#♦ 5.12 &iniaKgni f 40*43 t 57.98 «B 5,39 2.66 * 4.52 Av«rag« t I 44*10 % 00 60.E5 2 ** 3.61 8 ♦ 2.88 1 2 4.77 45 t a b u xv The Cholesterol Content of Cera&erciel 5rled Eggs * * » » i * 2 I Wo** Description sYe&r :Source 5 Total s Fat by sCna&pen-tChoice-*Choice- s of iXsispl®** **• *Pro» t of : f pared u u o i l s l 5 : s i^tbte)* Bolide s Acid :ifiehle sHy&rely- ttt&tte? : sis in, : in sSolias tSolids s terol : 2 in : sSolids s % . s terol in Wm% *ft » i 2.per c#a.1itper cents per sent,sper cent per cea I i * : a* #* ftft s 1 sSrled Xnole s 1936*Wo*~&«iji, Wf+ 94 s 45*75 s 2* 66 s f*.ao s 4*81 * «6g* ** t t * *• ft t 2 :Orled yolk** «* * sEo.-kaa. 97.78 1 57. S3 * 3*48 *ft »o » 7 <5 f 4*IH 3 |. m m 2 1939 s *> 5 9?.SO. t 81*58 s 5.45 ft £.94 x 4*78 4 . w «# * « s ? 95*58 *e 60*99 : 3*48 ft« 2* 91 1 4.7? 5 j » j> X * s ? : 95*59 t 60*68 * 3.40 «e 2*84 t 4 . ® *-w * J t i ** ; . . t llftlXiMim 1 97.70 s 61 *.56 7 3*48 2.94 T M a i m s ® , i 95*88 «♦ 57.85 : 3*45 *ft 8*78 S 4.68 Average t f i . t t s 00.36 : S. 4,7 ft £.87 t 4.76 -p.-,..,. .T._r. „_rr,n._r_^_ 5 ?x* irf^Hi'.WwiWA+^'Jviu* (. M-m-'cS .W.»4v ,% ^ , r,, ,-l.,.,t>rfr fro® the s? & ot of liquid egg as 1« So. 4* Table XXXI **Prepared frcsa the a > to xot of liquid yolk *-a .£«:.ple So* ?, Table XI? The Sterol Coat oat of Faria&ceoue Inured!«nte of XI! moat «try Pas tea Review of the Mter&ture* There are but a few published investiga­ tions regarding the quantity of sterols ia flour oad similar wheat products* 12 Tlll&aae, Biff art end Sxtim. found frcss O.OlOf; per seat to O.OESi per ©eat of free sterol ealeuXetcd a®.cholesterol cm the dry besl® ia sssat flour and grit a {fmrina?K leal 1 or *a5 Crevai *' report 0.039 par cent of sterol in a s*ott sheet flour. Eeding®^ obtained 0.09-9 per cent of sterol by ether ex­ traction fead 0.115 per cent by hydrolysis and then extraction* Riffart and £».*>» Standard. Semolina: 85.55 2 0*137 0*025 i lpt> we Texas farlmm t 86*68 2 0.106 0*0-27 s Kansas Hard Hour; 86.95 •♦ 0*113 ■0.029 2 «.« Hard Spring Hour: *« S8.SS I *• 0*101 0.033 * *# 0.058 0.033 •flour used in the preparation of authentic alimentary paste® 48 The sterol Content of ffarla&ceous Ingredients ,of Autli.@atia.All- mentary Paatas by the Freeipitation Method Based oa the Oxidative Xod©~ metric Procedure* With the develoasent of the oxidative iodometric Tsethed which permits satisfactory cholesterol det ©rsiinati©as os. smaller samples the procedure was revised to spply to 10 g. of flour or alimen­ tary pastas instead of the 30 is* previously u*od# Us.® sample else «A@fet be reduced ©¥#.11 more *0 far as tbs cholesterol determineties, is concerned but til© weight of unsaponifl&ble asstter -would fe® too small t© permit reasonably accurst® dot©ruination# with am ordinary analytical balance* The unssponifiabl© matter determination should be retained ia this method since it is likely to bo of vela© ia detecting the adulteration of alimentary pastes* The enmples of the durum flour used in the authentic eltmeatary pastes sad previously analysed - ia 193$ as report®! above in Table M l bud been stored since that time ia tightly clewed Mason Jars at a t«§.*» peratur© of about 5° 0* The earlier analyse® indicated the sesrples to be identical. *h«a the new procedure was supplied to this flour In 1940 using 10 g# samples with the adsorption technique, slitf&tly lower re* suits were obtained than in 193$. That the. difference m s not due to the method of determining the bromine urns shown by applying the eholester* ©1 dlhromi&e-so&iwn iodide method# The seme result# were obtained when the new procedure erne applied without the adsorption step, with the ad­ dition of either 10 sag* or BO sag. of cholesterol to- the uns&pcttiiflehls matter before the brosain^tloa and precipitation* The results obtained in these studies axe shown in Table Xf'll# mTABLE Till The &terol Content of Flour After Storey® at 5° 0. fro* X936 to 1943 s i An&lytlG&l Method :Cholesterol: *Jn*» oaifiable : Sterol {as s Decrease x added to ; tter :Cholesterol}s ia sterol lUasapo&lfi-; In tne bolide t ia the « Content sable Metter:Adsorbed Total : Solids s1936*1940 i i s * Choi©sterol &ibromide-s sodium iodide : Gholseterol dlbrooide-s sodiust iodide t Oxidetire iodomotric mg. 10.4 9.9 10*0 10.4 10*1 to.o • *er east per sent: per sent s per ©ent I l ? 5 0.073 — s 0.031 s 0.006 s s s I 0.075 — I 0.034 I 0.003 8 t t * 0*073 « 0.075 JL * WMW ***"*** J 0 .US £ 0.114 * I 0.031 0.03£ O.0SS 0.030 0 • 006 0.003 0*005 0*007 The deerease is a very elicit one but still snay be significant* Par­ ticularly la view of the feet that & sli*$it decrease in the sterol content of tie elXneatary pee tee of about the earn# order m e observed under similar storage conditions, ia developing the revised procedur® for the cholester­ ol determination e study of the effect of the various conditions (acid hydrolysis, saponification, diyin^, ©to.) ©a cholesterol alone indicated that the presence of alcohol during the hydrolysis sad saponification as in the original rsethod led to sees© loss of the cholesterol* The eloohol was originally added to decrease the tendency of the flour t© lump on. ad­ dition of the acid. However the limps are readily broken up by occasional shaking of the mixture during the hydrolysis* Though the ©mission of al­ cohol did not produce any noticeable difference in the sterol content found by the method in cllsentfury pastes, it m s considered advisable to o m it it from the revised method. mThe Recovery of Qxolml&mX to. Flour fay the Rev,iced Method, The revised method (Appendix, page 89) was tested for the recovery of cholesterol added to flour. For this purpose the sterol found in ID g. ssnples of flour to which cholesterol had been added m s corrected for the sterol found ia the flour by the usual method of adding cholesterol to the unsapomiflatole matter extracted frossj. 10 g. of the flour. The results are shorn in Table X¥JXX* The slight amount of cholesterol not recovered is probably lost in the washing out of the soap® from the ether ©xtyeet of the saponifies ties, mixture. It was tmmA that the uasaponifiable matter in the sapoaificstion mixture Itself is completely extracted toy the pro­ cedure u@@4* TABLE XVIII Recovery of Wholestezol Added to Hour * » » Cholesterol Added ; Sterol (as : Sterol in : Cholesterol To Hour To Unsajxmi fishle : Cholesterol) i the Flour t Bo covered Matter t Found s t t t s mg* 0 0 10.0 80,0 The Sterol Content of Farinaceous ingredients Commercially Used in Alimentary Fastee toy the Revised Method. The revised anthod wee applied 'to the determination of the sterol content of samples of flour, durma flour end semolina actually la us© eoEsaereially in the manufacture of alimentary pastas, Thee© samples -ere collected at factories In various part® of the United States by inspectors on the field staff of the Food eiid .Drug A&min^ ffig* 5 m * * mg. x mg. per cent 1 «# t 10.0 *» IS.7 I *»Aj # f J 'Wl 1MB — 80.0 t SS.7 *•• 8.7 • nx|[ <]V 0 I 12 .3 * 8.? S 9.6 96 0 z 82.1 i 8.7 I l f * 4 97 % I I SI istvetiesu She results are tabulated im Table XIX* Tb© ©slues for the fat by aeld hydrolysis ©m these staple* are talea fro® report# by -analysts In tin© field stations ©f tbs Food and Brug Ad&tAlstrettoau The sterol eo&tegfc of tbs fsriasssous ingredients show® little terie- ti :t5 S3 H S3 M fit m fit w |faw* jfmi £*mI H |*~« l~*i H w m H O m •2D < fl» a» #► fit fit H O a> -4 e& ©» csi fit ** *-» *# *• »# fc* ♦* ** ft* *■» ** *« * m »!* »» ** *-« «« ft *(■ f t> # 3 * * 5 * © m • « 3 £ 3 « « 3 31 -S S IS €> *1 * £ « 3 3* H '* ** It m m m <©* 9 • « m f© & 0* #•% ** ** o o o• « * *^*1 H* O ♦ ♦ ** ** o 4#:^ c>• * o c ih& oJU, %*** £ zn f* ft* 3** ** tt ** 1>*t* • •o #•o 6*o #“» M *» H• *J^S om w ** 99 >* *• *« *• ft* ft* *■* ♦-t ** ** ** ** ft* »* ♦* ** ** ** ** cs CD CO M C6 05 ftft© ■03&yfsiiW© & fQ © m ffiu> m CD C$*© 0? &> SI W j" -irt Wise**3 «5 as t'Viw m m &$ s> >(0m W -5 C?i s*• * • * • # t * » * * # at * # * 0 ft * m • • * ft 03 « ?s3 #> CP <1 *© !» fllk© ©it H* iO <1 © m m © «D99 o *« fi®#* * m m «5 Cs3© ** ** *» *4 ** *•# »* ♦<* ** ** »* «r* « H* •« ft* *« *» ft* ** ■»* O o o o © © O © O o © © o o © O o © O* » * * • * * • » • 0 « * « • « • * * • # * • •|nwt1^*4H |l«*t H M H H H H* H H H |m* a o o O o o O W H O © © fit© © 1® 0 © |ni 0 ** V* ** - ft* ** 0* •* ** ** •* ft* ft* •t ** O Q o o o Q o o © o O © © © © O © © O 0 0 o © o* • * • # • * ft * 9 * * * • • » * * • ft * »fit©3 H H 90 & p M *4 fit f t * •"* He# *•* w *C- 0w| m & «* >.* f t* * » f t * * * f t* « * f t* f t* f t # f t* f t* f t * f t* f t* f t* * » <*» f t* f t* f t* f t * f t * • • H H M H H H ?$ * • * * • # * « ;; j ju « |* x t * im * JP .J j - s i |>«4 f r t • * * «' t 5 O ^ M I » « £ * « & € # { £ H» fi® fit p* fit * * * 05 #» €| fit -a Q * > # ♦ * * jH* fi* • * * * • > « * *>* * * * « » • i » > * * » * ♦ * » * » • • ♦ * * * # * * * * * # # * * |M H m H* h" • H H h * H H H H H H H ft • * ft * • t * fit * * • * • » * i » « « ft • 1® Z M o ID Eft ts 1 €& "*3 1 fit If m © m t fit P 0 © fi© m fit at 0> P fit H © o # m <3% © 0 ©» «B ei*^• ♦* *♦ S1fn ht 1 fde» gj tr#|ftp O 53 O ♦♦ *» C3 ^ 4 0.0H* efl«A»jEft^4 tt *# *» H 99 C2 t f i p P **■ r + i t 9? 0 • t a * © M 0 )H , H m « f 1*4) m • * 99 t *•* a te** fx et* £ 0 0 H *jp ® o 0 • H | wm* 4j*$ «#•**** &J 0 H * * »» t* ♦■* •* r* 22 feg 0 P» ft* It? h S3 2« 6 0 5f fit i'i h*1 H 1 *S fie t , PS fb ® > ft ® H* O « Hk ps ♦* ♦* «!« *-* ».# hri ffi # (4* %sf «+ 1^1 © H *# #4* © © **s hi © a t*** Q i| €5 ^ fi# Phi «+■ © © M* S fi* ft h-8 © S’* h* U m 0 H P *-* «*■ St $ & © ^ # ©hi c ts © % w © IS 0 m P13 «+ *•••5 mThe 3terol Content of AXimomt&yy jreetes as aa Indev of the Igg Content in Review of the Literature* Pops* reported briefly on the possibility of uaisg lindam** cholesterol &ibrosid© reaction for estimating the ®sg content of alissat&ry pastes but |tf® no data* Cappiif® any date* 30 further worn on this subject appeared Mat 11 1930 when TlUaen*, IBliffsrt s M &uha published %he jsioro method based on sa adaptation of m pA the Sssent-Cyorgyi procedure* This ir*ethod was discussed shove* Soldi £ya sad T®stoap#v " reported ® colorimetric method applicable to approxiaat© |H£, estimation of the egg sonttat of »lim«ntary pastes* Mffart and loll or"4-' considerably improved the oxidative ligitoaia procedure a® applied to B9noodle* by adopting the fused®'" modification to those product®* They also devised a oolorl&etrls procedure which ggf© good results ia their hands, hut is head1capped by am inconvenient col or Aeaeuressent sfcioh must be ised© at froauamt interval* over period# up to as hour to determine the ■aa «4ssxiinm* Kluge" sad Costs" te?t applied a corMnatlon of the sterol mud lipoid phosphorus deterslaatioms to alimentary pa@t@# for detesting adul- ieratlon of the latter with plant lecithins* Terrier discussed diffi­ culties encountered is, applying a gravimetric dlgitonin. method to the 56estimation of tkm cholesterol ia egg pastes* Subsequently Terrier described tbs method previously cosroeated upon which depends os the loss in weight of the digitoni&e precipitate observed os decomposing the molecu­ lar compound with boiling xylene, thin loss representing cholesterol* The Sterol Coateat and Igg Contest of Authentic Alimentary Pastes by the Precipitation Method Based on the Cholesterol SibrQride-&odlum Iodide Reaction* In 193$ H. A* l»epper of the Tool and Drug Administra­ tion personally supsrvised the preparation of m series of -'llBsntery 54 p-aates of various kaaowa ©gg contents by asiil ooscaercial sisthed# at thm plant of A, Zsregs*# Soaa, Inc# v Brooklyn, low York. A su ry of his r©~ port of tie preparation of thou® noodle* is given ia the Appendix, page 91. # The actual egg solids content of tbs noodle# was calculated from the solid# content of the egg product# and flour used es liwa above is Table# XI and XfX respectively# Tfe® various product# war© identified by batch numbers# The liquid eggs used sere freshly broken* Samples of the#©- for analysis «#r* frosea for preservation and arc so designated la Table XI# The sterol content of the authentic alimentary pastes m s determined by the original method {Appendix, page 801 and for a few samples by the adsorption method (Appendix, page 81 )» The results of the analysis are $kmm in fable XX. In Table XXI are the percentage* of ®§g solids found as calculated fro* the general formula * * e - f in sfctlch £ •* the percentage of egg solid® in the total solids of the staple S » the percentage of sterol in the total solids of the aasaple e * the average percentage ©f cholesterol ia the egg solids used f * the sYcra«ie percentage of sterol ia the total solids of flour. The value* used la the present calculations are the average values found by the respective methods ia the egg* flour, Tables XX and Xfl respectively# 55 ?a&ue xx The Sterol Content of Authentic Mimentery Peartm by tbm C&©l©st©ral 0ibr©mX4©~S©&i^ Xodtd# Procedure : s I Batch: Total i Original Method I iuttorptiam Method i . Ho* : Solid® lUaftepoai-* 2Sterol (es i X S S 5 5 ^ « ^ S ¥ 3 i 3 S 5 ^ ' ^ s 5 fiabl® :Cholesterol) t fi&bl© Matter :Choi©sterol) 5 i Matter i la. Sol I sis s in. Solid® t in ■Solids - : aim Solids a t iioii .ti..LLitULllHraTMi"i1iii.'j)iti||i'[>wijj,iL*OTmrTjTmnjiai-imiii*iwwuriiinrr.iinu'Myru.uricii>>’»irt~tr»~'f~it‘niri iri",u-'i*ir"~‘"'"fi:ir"Ti‘['*~“,J T^f'n'nr •i"*~r ~J‘ *TT—-; i ,i,v ir,(C.""T,“-tYr''t-t * - * * 1-— r-~—- ——-■ — ■*"■*■ ■ ■ ■ ■ ■»■ - -nn-i. .mi.i.-iimninin h ii.mmi. «■ t I 1 5 % I 3 * 4 2 5 2 5* % 6 : 7 2 8 * * 2 9**2 10***: 11 2 _ _____ _ _ ~&l,0 ^ & r » ® aaj&pl® used ia ©%b«r caeee **OontaiB® added ear®tome in oil ***Ccntains &M«d Ylt*«Fro £ aostaeareiitl oarotm® ia oil preparation) ir sent: ft per cant * is' % »ft #r seat 87.79 ft 3 0.13 %j » 035 .32 «e 0.23 «ft 0.123 &&*68 : 0*90 *ft 0*197 ae.fi s 0.45 5 0.531 89.21 i 0.50 *• 0.3?? 89# 21 i 0*88 ♦ 0.5?? 88.88 i 0*27 2 0*160 88*19 t 0.39 ♦ft 0.276 88*89 t 0 * 31 $ 0*191 39.33 # 0.14 3 0*040 89* 89 * 0.15 ft* 0.036 90.7? ft t 0.29 ftft 1 0.186 ft 2 per eoat * i 4 per ee&t S i» *ft» %<> ft -** 2 m» ftu 2 0*84.9 : 0.19? ft•ft 0.389 2 0*390 ft 2 t ftft ****** *♦ o.as ftw O.160 ftft 0.329 2 0.270 ftft 1 ****** 1 *»•*- I **nmm S. 2 3 ft o + n m I. ■5 0.185 56 TMIM III Igg Content of ■Authentic Alteeatery Parte® bj the Cfeoleeterel BihK*alde~Sodi«® tod 14.0 Procedure Bat«&r;D©s«yiptieaj JE^ g soI M jsu . „ . Ho* i of Mm* i Proseat * igg Bolide t Used :in Total ; la Total i s Solid# : Solids wngnthodl t Maorptlea, 3Egg 0©iid»:lgg Bolide Egg Solid® - * • " * -" *• * ^eooveredRecovered :is Total ‘ solid® : : per cent ■i per ©eat per ©set * per ©eat. per cent I i t i I 0*0 i 0*0 «... «e «* 2 sfrsife y o l k i 3,1 : M 94 « 5 * #9 tt f• * 5*6 ■*■* 3*4 m # 5.4 96 4 * v* n . 10-* 3 t 9 . 0 m t 10.0 Wf 0 * « « • 12*1 l U*» 9 4 ; •mm*. m *m 6 slfiwh whole: 5,6 £ 5 . 5 9 S s 5.6 100 : @ g g t I « ? :7reah wholes 11.1 t 10.? 95 s 1:0*0 9$ * egg 5 ft€ I 8* sfyeeh y o l k i 5.2 t 5.2 X00 s •*mtm ftftwW* 9 * 0,0 i 0 * 1 « ,« ► 5 — J w*® 2 0.0 +e 0*0 * • t * * • X X sChixteee t IE $£ w-e *5 + S.3 it I 3*5 m t dried yelks *# 1 : : ,«L JL * Contains -idol carotene ia oil ** Oontaiae j*dded Tltr-Pro {cmsaierolfil eareie&e la oil prepereiica) It ia ©wldeai that the method yiaUe -quit# satisfactory remits for the detemiaatioa of the ®§g solid® ©oat eat of cliaeatary past## over a «lde t&.eg# of composition* The pro««sfc teatativ© saethod for detexnl&la® Aether m egg needle coataiae wtoi© egg solid* or yolk ©elide 1® gitren is the Method# of Analyst* 90 of the Asaeeetlon of Official Agricultural Ch©e-l®taf Third Mitioa' • fliia method ha© mot beau entirely satisfactory and progress has been made oa the improvement of the method* However this work ha® aot been completed and 1® © subject .for later report lug* the Sterol. Content and Cgptsnt, of Authentic Alimentary Paata# by the Revised lethod. The authentic slliaeatary pastes prepared in 1936 have- been stored sine© that tin® in cardboard cartons ia a constant tem­ perature ro«a at 5° 0. Although it will he necessary eventually to test tlis revised method (Appendix, page 89 ) using a series of freshly pre­ pared noodle# of Itnowa ©oaaposJtlon, It ha© mot been considered advisable to §© to the necessary expense n% this time, in view of the fact that new legal standards for noodles are now under considerstion. It is also de­ sirable that work: he completed on other determinations * such a# the crude albumen nitrogen method* before such a series of authentic noodles is pre­ pared. However the application of the revised ssthed to the authentic alimentary past©# prepared in 1936 can be used to test its accuracy ia the estimation of the ®gg solids, The method w&s* applied to these sample#, with the restilts shown in Table XXZX* It was shown above that a alight decrees# in the sterol content of the flour used la these samples had oc­ curred under lifce conditions of storage. J*or the purpose of determining the recovery of egg solids by the revised method, the calculitions by the general formula given above were made, tajfciag the sterol eoatmt of 0,031 per ©eat for th© flour as determined by the revised method end the average sterol content of the eggs used as determined by the same method* The re­ covery of egg solid# appear# to b® senswhat better by the revised method. In an alimentary paste of low egg cant eat prepared with a flour of higher than average sterol content (the case in the present series) the cholester­ ol content may approach the mlnlisam one to one ratio of cholesterol to sitosterol required according to the copreoiplt&tlon studies. It is there­ for® advisable in the erne© of samples below the standard in content to add 10 mg, of cholesterol to the uns&ponlfiable matter before the dibrcea- 58 Id® precipitation* Xa the ^ppliection of the netho& practically to B&mptm of uajenaan the advisability of adding the cholesterol can fee judged f m the content o f uasaponlflshle natter* The presence of less than the 0*2? per emit of u©#ajwXfimfeIe Ratter found ia. a. sissadard whole egg noodle wm.y fee taken as a guide* XX a sterol content obtained im the ease of Batch Husber 2 ia this aerie# is noted to be sosiwhat higher she* cholesterol is added before the precipitation, though the effeet on the indicated egg content is not of serious consequence from a practical stendpoint* TABLE HII the Sterol Content and % g Coutent of Authentic Alia.eate.ry Fastes fey' the Bcriscd Method • * •> 4 « 4* •4 ftft ftft Be tabs De-script ions Total *mEgg SolidstUasapoai--sSterol(a# sEgg x Eicfs*^C93Sp Ho* 1 of %gs tSolid# *Present Iflsbl® :Cholester-s3 lidsxSolid# ; Used : *ia To'tel ft*Mctter **©1} ia # in *.Beeoversd : t 3Solids S ia *Total :Total s % % 1 wTotal *Solids sSolIds: t i * 4ftSolids ♦ ,ifound)si ;i?er cent' *F per cent £ ■y«r cent 5per cent 1percent per cent i % I i s t ? 1 i — : 87*65 J 0*0 * 0.12 ft 0.029 »« 0 * 0 » •*. & s .fresh yolk : §7.3? 3 3*1 * 0.32 -ft.4 0,1X2 I 2*8 5 90 2* j «r * » 87*57 * 3*1 *♦ 0*32 4* 0*1X8 ft 5* 0 x 97%Hi? * « w .* * 87*83 4* 5*6 A* 0.51 ft4 0.X92 i 5*5 x 98 4 ♦ a t* j. 87*62 ft* 10.5 fft 0.4? 4ft 0*331 *•10*2 ; S9 5 . it ,t j 88.07 X IS. I * 0.55 »-4 0*406 ft12.8 s 105 6 s fresh arhole 87*06 % 5.6 4ft 0.07 $ 0*154 * 5*6 t 100 ^ egg * I 4 * ftft : 7 * m # it * 4 * 87.13 t IX. 3 # 0.40 •* 0.267 ;10*7 1 95 S'**$fresh yolk s 86.93 ft 5.3 I 0*52 « 0.191 ft 5*5 s 106 $** : ■— j 87.08 J 0.0 *■ 0,15 •kj * 040 «* D.3 * *»«* 10***;: — x 87.30 ft■e 0.0 # •0*13 : 0*033 # 0.1 : —- n :Chinese dried 87,44 ■» 5.5 0.30 s 0*181 m« 5.4 t 98 : yolk _ s, * * ; 5 fta * 10 gig* of cholesterol a id e d to the ttaeeponifieble mitten* before p r e c ip ita ­ t io n ** Contain® added carotene in oil *** Contain# added Vita-Pro (eeosserelal carotene in oil preparation) 59 Hi© above results srt sufficient to ikw th&t the re?U©d aetbod :,;ltii a Igood recovery of the egg solids ia *XiBMMitary pastes ever a wide n&Bg# of soakpoaitioiu This method does mot require any re^&eat® whlan are unduly difficult or expensive to obtain ia e ©atlef©story state of purity nor *my apparatus not aawttiiy available la the .food laboratory# 'Moreover f sines the determination 1© not sensitive to slight variations in condition* and involve* a® unusual manipulative operatleas, it is believed that the netted mill give good results mot only ia the hands of analysts iaexperteaoed la it® use but also 1® different laboratories* Tout®.tiro formula® for the calculation of the egg content from the sterol content of nli&e&tar/ p^etea ere given ia the Appendix* page 92 * Hi.® £ff«ct of Storage on the &terel Ceateai end Xaftleated l®g Content of Alimentary Pastes It ff&0 -poiated out above tMt oa® ©f the chief disadvantages in the me# of the lipoid phosphor a ®s an index of the egg content of noodle® tma the decrease ia the lipoid phosphorus content os &toxrttt£6m The results gl w m In Tables XX &nd XXII nr® m indication th&t the sterol content of all- F*«atary pastes do#s not deoare&se to a serious extent on four pira* itortg# at 5^ C. An even mor# severe tost «&iafc m y b* expect ad to tout® a for skmto drastic effect cm tbs noodle® than any storage oeadttioas conceivably to &©• encountered in practice has aloo been sfede* The grouad scaaples of noodle® pr«imr#4 for analysts la April, 1956, ®«re fcspt ia closed Meson Jars under laboratory conditions until Murafe, 1940, during vhleh time they had been exposed to the high temperatures prevailing in tor four su&ners* These samples had developed a strong, musty odor* % * m analysed at the end of this time by the revised ssethod, the showed the sterol con­ tent® indieetod is Table XI.III* so TiiBIj: XXIII The Sterol Content end Indicated Xgp Content of Authentic Ali&sntary Paste® After Four Year®’ Storage at Boca Tespear&ture s t i t s ffetehxBe^eriptien: Total i % g sOasapoai-s Ho. x of %g» X Bolid# s Solids i f table t 3 Used t sPresent* Matter 5 s 2 * ia 2 la Total ; i : 2 Total * Solid# x t t sBolide t $ Sterol(a® 2 % g J l&g Cholester-sSolid# s Solid# ol) in g ia 2 B#~ Total 4 Total 3 covered Solid.® :Solid® s I sp&r centspercent: par cents per cent 2percent»sper c 3 S 3 s I I i 1 | *•#» | 90,53 2 0,0 A4 0.18 3 0*020 t 0,0 ? ***** 2 :Fresh yolk#: 90.49 »* 5*1 *« 8.22 ; 0,107 e# 2.4 ? 7? 2* g « : 90.49 i 5.1 ** 0*22 3 0,116 s 1.7 s 87 5 » 9# » * 90.5? 5 5.6 3 0.52 #•# 0.190 3 5*2 4© 93 4 • « « * ♦ * 89* It • 10.3 ** 0.4? 0.520 3 9.9 e* 96 5 g « « 91.04 2 18*8 e.« 0.53 i 0.391 ** 18.1 I 99 6 :J¥eah whole: 90*80 * 8.6 a* 0*27 •4 0,151 S 5.2 99 93 3 egg t * * 3 3 •* ? sBreah ©holes St.19 5 11.5 s 0*40 S 0.267 3 10. 5 •a 93 2 egg * 2 1 s ** 3 8** sJfceeb yolk i 90*86 2 5.2 2 0.52 *• 0.189 3 *9% 1 ** * X #* 98 9** J .aw wfc J •59.90 2 0*0 « 0*14 s 0*056 3 0.0 2 10*** ^ IWWIM J 89.88 2 0*0 ** 0.13 2 0.029 3 0.0 «♦ IX tChineee dried 91*04 » 5,5 : 0,29 ** 0,177 3 5.1 *« 93 2 yolk 2 * i £ 1 f * ID ®g. of cholesterol sided to the uaaaponiYlabl© setter before pre* eipltation ** Contain# ©Mod carotene in oil #13Mt Contains add®! Yita-Pro (cohere! al carotene in oil preparation) The indicated egg content has been calculated by th.® gMirti formula, taking the sffra*# sterol content of id® agg# used as determined by the re­ vised method but taking for the sterol coat ©at of the .flour the original value of 0*03? per cost found ia 1936. This giTO# the asinimajs egg solid® content on the basis of the sterol contest ♦ It is noted that ©too under these eitr«a0 conditions practically all of the sterol originally present can be recovered* It is worthy of note fctmt ia both series of determina­ tion® s&suSa in 1940 the chief loe# of sterol se©a to be due to the sterol® S I of til# flour* It was ahawn sfeoT# tfemt th© sterol #©&*«&£ of f*os«a 9| p So** sot «ba*go on proloagoS B t o m m a M that at oral mnt®ut of liquid «f$e do#* sot oteag® on toymg, $ro®. tfe# *t**4p*l&t of reg*iH#tory ©pwstisa©* therefore, It ##n b# o*tt*lud*4 tb*t tb© sterol east sat of a sample of ©llKMNktftgy ps*t© is ©#s«atl©ll)r lttd©p«ad**t of th# typ# of ##t# used, fresh * frosts or dried* ® M of th# ©eaditletts of stomg#* mw m o a m A L m m The Procedure 0©®d ia the (fcavimetrle and irgeatoaetria Sttt'iitf of tli© Cholesterol ftlbromide Prwoipitatietti 111# Broalxiation mud Freci pltatl on Procedure* Th© cholesterol ia a g$ x 150 mm, toot tub© was dissolved la t ml* of anhydrous other end th® solution aaa cooled la a hath to -th# desired t«pwetur«# T© th# soln~ tioa was added 0*S si* of a solution of bromine la ®®ii>©a. tetrachloride• After ib® solution hsd beea ia tha bath 10 mia* there m o added 10 ml* of aceti© solution (4 vol* of glacial coetls sold * X ¥©1* of vatsr}* ale® cooled to the hmth tesapeaMsture, and the mixture wan vigorously stirred* After 5 sain* th© preeipiteta wee filtered with auction, isiliel slt Heed ia the Construction of Jfigur® 1 The Phytosterols Peed in the Copreglal.tatioa Studies. The phyto- sterol# wort; isolated frm. wheat gem oil kindly rural shed hy Br* George if Jamieson, of the United states BapartBsent of culture, using es- Sisentially tbs procedure of Anderson* S&rlaer end Burr" * Two pv+perattoft* had respectively melting points of 137°-139® and 138®-159®, specific rota-* tiens (chloroform* 20®} of -I?0 find ->27*?®* and contained 2+50 per cent and 3*07 par cent of water. Ia spit® of the slight difference# ia properties these two phytosterol preparations sheared mo significant difference# in the eopreelpltetlon studies* Assuming the validity of the Anderson end 3 abeohauer*C> formula* tb.m& phytosterol# contained approximately 15 per 64 emnt of 4ihy&rosi teat «rol* frit# Coixrooipit ati on of Qholootogol ami. Pfeytostorol PitoroalAoo. Mixtum of ehoXeatorol oad phytoiitorQl wore &xo®isa*#f, tin* dibrosaido proelpltatoA aa& t&« broata® in ttm yxwftlpltat** 4«tw&lA«d by ttoo pro-' eotero d«ae?lb«d ia the App*&&ix» page 76. $&• weight of tho sterol pre~ olpitalo ssi m ax©#®® of ©*£ ml* over the ©aleulated eaoust theoretically required tes beta added* Tb© clear solution is cooled la lee water for 15 ain* and 70 to 80 ml* of petroleum ether at -10° to -IS® is added. The fixture is vigorously stirred for ©bout 3 min. while kept sit -10®, filtered rapidly with suction on a Jen* 03 filter end the precipitate washed until white with petrol#®® ether at about. -I©1®* The precipitate is pressed down bard to r*®v® excess solvent © M dry air is drawn through until the filter and precipitate hare attained approximately room temperature* The precipitate is broltea up end dried to constant weight in & vacuum over phosphorus pemtoxlde* It ia kept over phosphorus pentexld© in a .’iesieestar protected frosi the light* On the above seal# the yield ia about 1*5 g* of cholesterol dlbrwid© salting clear at 114*4°-114*.8® (corrected) followed by decomposition* Ibis pro­ duct was found to contain £9+26 per cent* 39.26- per cent, SS*£9 per ©eat of bromine in triplicate deteminniicac on Mere quantities by aabing with potassium hydroxide and applying th® Volhaxd method * By the oxida­ tive iodocsetri© metbod on the macro seal© quadruplicate determinations ladlentil 29*1£ per cent* 29*15 per cent* IS.25 per cent and 29.S© per eeot of bromine. Keeult* obtained on sei&i-micro quantities were given in Table ¥11* Pr#pa.rtiti0u of Bug# Cholesterol* !*uro ©holeeterol m u prepared frcss IS g. of the pur# cholesterol dibremlde* the dibromide was dissolved in & liter of acetone at room temperature, 50 g- of sodium iodide was added the solution m s allowed to stand evedr-alght« Th® iodine m s reduced eitfe & ©light excess of so Hum thlesulphete solution and the mixture w®s diluted mt® whmt 2*8 I* with wrnto** © whit® prooipitat® w®& filtered ©ft ©ad eopiouslp with suter. fh® pareduet iss r®®ry»t«lli&®d from alcohol (twin®) to ooawtont aoltiag point «»d dried to oonstwat w®ight ovor pho®** phcmis poatoxido itt «n Abdorhaiden drier with boiling t®lw«a® in tho heat­ ing ®han&©r* Th® jrield m s 8*0 g, siting at ld&«0o«14&*8° {©orrooted)* corresponding to 87 per seat ©a th® bonio of tho dibromide uaod and 6$ par ®m% os the bafti* of tfco original cholootoxol 115 g*) u*®6 la preparing the dibronid®* 68 mmrnmi fhe nmd for a suttfeed for tba detorcii&etlon of t&« content ot mXi&mfmy pmstm k m k&m discussed. Th% f&otora 1 avoXvod la the »4©pti«a ot a aatbod b»®«4 m, m. ®J*©X®*t*r» oX dfttonalna tlon k m * boon stated and airaiiabl# method# brre b o m oo&aldox**' ®4 1» the light of ouoii footer#. a &tudy of tb# condition® aooa#»*ry for tbo brosti&iitioB, of ©fe©Ie®t#r«* ol and pvoolpitatloa of eholoatovol dibromldo m tfe# basis of a $iMjatlto~ tlv® dotomin&tioii of eholosteral h m boon mm&*» Am ledoesotrle mothod for tho determination of eiloXoirtoaeoX boaodi * oX dibrosaldo and sodium tod14® in seotone baa boon dovolopod and applied t o tb® aatimatiom of tho sterol eoBteat of ® g g s » f l o o r s a d allnoatary past®#* ffce interference of vrfheat phytoaterola in th® determination of «hole#te*el by the okeloatorel dibrottide praeipttstien method baa bos® studied. A method for the preparation of ^art cholesterol dlbremld* baa boon developed mud cholesterol boa been purified thereby* Am iodoaetrie method for the determination of ahoXoatazoX basad on tli® cholesterol dlbrmide precipitation and the application of the iran dor Keulem oxidative bromine determination baa boon developed* fhla method ha® bam appliad to the debemi-antipa of the etexol eenteat of «££#» flour and alimentary It has boon shorn that the sterol content of the solid# la not changed by long aterete of ®g&# In the frcmea condition nor by the «<*&•• maarela 1 prooeaa of jxroparing drlod agg** Uta atarol oomtemt or a aaafoa? of aaaplaa- of CKBasaore tally prapsrad fzasaot agga «usul dried eggs s»l of fariaaeaous iagradla&ta uaad oaMaareial- If to aliaaatavy paataa lisa baaa dataratoad. ffee ataxoX aoateat of alls&antaxy pmatoa M l baaa @bom to bo a- aaaful tatos of too @sg oomtmt* It h m b o m aatafeXltffead that tha «ter«& coataat of allaaatarsr pnatea doaa mot daavaaaa awtariallf om 1m® atoaaga at or&lmary tasparsturaa* 70 m m m i x Th© Xtotoxmlafttion ©f tb© 5l&«roX 0©it&@at of l$gs B&aod on ill© Gfeoli*#t©t©X B;ibr©siid#-*00diim i#41d# Origin©! Motnod. Oaoapoaifiabl© M©tt«r Bo&gonta Coaeentratod potcaslm feydroadUt© ©olattoa* Dimsolv© 60 f* of r#~ &£©nt~quality ‘p m t m m m i t m bydroxid# is 40 g» of m , t or* Aleo&ol# l#ag#at~-fiiaXiiy otbyl ©loefeol* ©pproximatoly tS p«r ©«at fey wlnmo* .It&oar* Stb#r P* S* JP* Dilut© potaoalun hydroxide solution* Pi.««©iv« 1 1 .t g* of rooga&t*- §uallty pet*a#in& kydxcncid© ia I 1. of mt«?» Dtlmt© bydxoohlorlo sold* Bilui© «eisfred tost tube using four 10 Ml.* portions of ether. (far satisfactory results tfa.ct dry­ ing mad n©igMng of ih® tub© suet b® don# as follows dry the ©lean test tub# and a sisailiar on® to b® -,ts#d a# © counterpoise at !OO0-lO5y for 1 boori T m m % from tbs ©van plae© near tbs balane© with the balance ©a#© ©pern for 30 mi&»; ©sigh the tabs using tin# counterpoise.) Fla®# tin# tub# la a 300 ml* &rl©m©y©r flask full of mt«r at an initial teraperatere of 450-5®0 on the ©team tmtl while dtreating a stream of ©lean air upon the surf©©© of the ether so that it is rapidly rotated a d ©vapor©t« to apparent dryn©##. Mmmm the tub© fron the tattr, alp© #ith a ©lean towel and place in ib® ©van with tlx© ©ou&terpols© at 1OO0-1O0° for 1 hour* Cool and woigli in the east© as before* Bedust fros fch® woight of the ma&poaifl&bX© i&isbter any blank ofet&ined from the reagent® used determined by the ©a?s® procedure described la the ®«rth©&* Determine the sterol is the uasaposifiafcl® matter ©« described under "Cholesterol*, P*#9 76 * (b) liquid %gs* loi^ s fessmratsly into a IIS ml. JEriensseyer flask approximately 4 &* of •sfeol# egg or 2 g* of yolk; add to the whole egg 10 ml#, to the yolk 5 ml., of concentrated potassium hydroxid© solution and proceed as described under -*Bried %g«*, page 71 * Adsorption Method* Adsorbed Unsaponiflsbl© Matter Ssagsttts Msmisam oxide adsorbent* Prepare as follows from basic aluotisutt fa &estate ponder {apjaroxi&urteiy to per eesrfc ©f thi* ^eli should paes & 100- i&esh sieve) s hast slowly at first, then igaits in a platinum. dish et •« bright red beat over a Mecar burner, stirring occasionally to facilitate oxidation, until the powder i# pur* i^iit#* About 3 hours* beating is required* As soon as tbs material b&s cooled ©mougb to handle eomrea- lently, pass the powder through a IOOhmm&i sieve and ln&ediately transfer to a oomtalaer having a tight eover* Juabestos. Digest asbestos of the aa&phibole variety with hydrochloric a eld solution i95 M mMxm. hypochlorite* Sodium .formate solution* As aqueous solution of reaa«»t-qunlity sodium, formate containing 0*5 ,g* per ml. Hydrochloric acid solution* An approximately 6 M solution of hydro­ chloric sold 0 * 8 * ?* Sodium chloride* Reegerit-quality sodium chloride* Methyl red indicator* ^>iasolT« 0*5 g* of methyl red in 50 ml* of alcohol* dilute to 100 ml* with water saS filter* 0*01 II sodium thiosulpat® solution* Prepare from ro&geat-quality sodium thiosulphate and freshly boiled and cooled iistilled lattr, to which 1 per cent of amyl alcohol has been added* Standardise against pur# pot&«~ si urn iodate* This solution, if protected from, evaporation and light, will e? retain Its titer elmost indefinitely* starch eolutlem* A X per sent solution or soluble atansk* Cenoemtrwted potassium hydroxide solution* A §0 .per cent solution of T0®&mnt~qu&llty potassium hydroxide* Potassium iodide 'solution* A 20 per sent solution of rsagemt quality potassium iodide* Ammoalira aelybdate solution* A 8 per seat solution of ©isesonium molyb&ete* f!i@ other reagents used era those described under "Cholesterol" , peg# 76 9 omitting tie acetone, sodium Iodide and sodium thiosulp^ate solution* Apparatus Tbs apparatus used is the same as described under "Cholesterol* * page 76* DetexniaetlcsL ti&h the exception that the tl < of stirring the precipitation mixture is reduced from 5 aim# to 3 aim*,, tbs procedure is mm described under "Cholesterol** page 76, down to the point where the precipitate on the filter has been washed f»® of acid* Place a 300 ml* Irlenmeyer flask under the filter so that the stem projacts well into the flask* Ieoh the test tube and filter with 10 si* of alcohol, 10, 5, 8 ml* portions of other sad finally sltb 10 ml* of alcohol, stirring up the s^ sae with each portion of solvent cad allowing, the mixture to stead for about a minute before applying the suction* lash down the stem of the filter with 2 nl* of ether, «/M 1 ml* of concentrated potassium hydroxide solution, mix mud wash Iowa the ©Ides of the flask with 5 .ml* of ether* Evaporate Si. the ®th*r aleohoi oomplatwiy as tli® at*«M bath, usirng & atrmm ©f eltsm air to r»oig the final eloohol vapors* Add 40 ml• of hot wator to tho r^aidua* .six, &»d cumtralissa th* alkali with $ M hydroohlorle sold, using 1 drop of the mmtHay.1 red Indle&tor. M d 10 g* of sodium chloride, 3 g* of monobasic sofiisa phosphate and SO ml* of sodium hygKNshlerlte solution. Bring the solution just to vigorous boiling, wsott from the heat and mid immediately, with ears, 5 ml* of sodium formate solution* Cool and dilute to mbmzt ISO ml* with water* Add 5 ml. of potasstom iodide aolutiom. & drop or two of &Ecaoslusa molybd&t# solution end S'S ml« of 6 1 hydrochloric sold. Tltrata at os®# with 0«0k 1 sadism thiosulphato solution.f a#log stareh solution a* indicator* Correct the titer for & blank detezmlnatlon os tho mpat®. mg* of cholesterol ** 0*$S ♦ 0*683 (ml* of Q* 02 N Ka..SgOsi. 8V Tse Determination of the sterol Content of Alinentary Fast## and f&rinsseoue Ingredients Based oa the QxldatlTe lodoBielri© Proee&ure Ifaaaponif table Mutter Heagents Choleaterol* Higbett quality oholeBterol obtainable of melting point &©t lee# than X4?6* foot for parity by the cholesterol method given ©st p®m 86. Potassium hydroxide* 'Pellets of reagent-^uallty hydro** side* Concentrated fey&roohlorlo a© id* BiXute. by&roahloric aeid 0 # 8 . P* isrith an equal volume of water* *Ph© other reagents used ers those dsseribed under *Uaeapoalftable Matter*, page 83 , omitting the ©oaeeijtrated potassium hydroxide solu­ tion* Apparatus the apparatus used is that deserlbed under *Bnsa?oiiifiable Matter*, peg© 83» In addition to si BOO mi* Srleinseyer flask# determination '^ elgh c 10 g* sample (ground to pa*» a BQ-metih sieve) iat© a 500 ml# ^rXejnseyer flask and add *ith shaking 00 mi* of the ©oneeatrated hydro- ehlorlo solution* Seat oa the steam bath SO sin* with frequent shaking to disintegrate any lumps. Khli* eooXing tbs IneXined flask under the tap 90 add carefully SO g* of potassium hydroxide pellets at suck a rate that the liquid may boil but not m violently at to cause loss by spurt lag. Ifcile it in still hot place the flask a© the steam bath, cover with a saall watch glass »a.4 beat for 3 bourn with occasional swirling of the mixture to carry down any material adhering. to the aides* Cool to shout 30°, add 30 si* of alcohol and -8*0 ml* of water and mix well* M l 100 ml* of ether, swirl vigorously for a minute and transfer the mixture to the separatory funnel, mashing, the flash with $0 ml* mn& 28 ml. portions of ether* Wash the saponification flask with 20 ml* of dilute potassium hydroxide solution, pouring this into the funnel in a alow stream while the mixture is gently rotated. After the ether layer hue asperated sharply at the upper liquid iaterfaee (about 10 mia*} draw off the lower layers into a nursing bottle, including any small quantity of emulsion* Proceed from this point as described under "^termination1*, page 87 * beginning with the direction* "vines down the sides of the funnel with 10 ml* of dilute potassium 'hydro­ xide *** *. If the uasapoaifiable matter amounts to lose than Q*W per sent on the dry basis add 10 stg. of cholesterol to it before applying the cholesterol .method and correct the result accordingly* Tha Preparation of the Authentic Alimentary Paste© Th& aliiaentary pastes mar© prepared dnrtn& ardh,X93&, at the plant of Am Seregaf© Sons, X»c«* Brooklyn, Kew York, u:.-d«r the personal super­ vision ®T f£* A. Lepper of the Food &nd Brug Administration, United &tatea Department of Agriculture, by the usual camKsroi^X process. The flour used is all batches was from the same carload lot of Durum Fancy Patent, H. H. Slag Hour Mills, Miimoapoli#, Minn* Tiio liquid egg yolk: and whole egg u&ed were freshly prepared fi*o®. Missouri shell eggs of Tory good quality at th© egg breaking establish- sent of Marshall end Kirby* Heir York, M* IT* Samples of the churned eggs for analytical purposes war© immediately placed in a sharp freezer and maintained in the frozen conditio©, until analyzed* Th© dried egg yolk used wa# fra® China. Th© "Fita-Fro* was a eowaerctal ©aroteaized flour' preparation ob­ tained from the national Krmm Company, Brooklyn, Hew York* Caret sue mix *A" «a# saade fro®. 5 lbs. of Ceresota flour and £93 g* of oil containing fOOQ p*p*au of earot©nelds* The oil m s obtained trmt the Amerieaa Chlorophyll Company, ffashingten, B* C*. Carotene mix *BW sms prepared from 5 lbs* of Ceresota flour and £90 g. of the nmm oil* Samples of other farinaceous products taken included So* 1 semolina* standard scsaollaa, Texas farina and Kansas hard flour# Th© weighings were nade m. a .seal® mmmitiwe to oae-half ounce* The composition of the mixes used in preparing the alimentary past©® is shown la Table XX?* 32 TABLE 3EX? The ingredients Used in the ibroparation of the Authentic Alimentary Pastas t t Batdh* Flours. So, tUa«d s m Oaed Kind Weight lUsed i Si ad -'eight * *.. . * . . * a* * *M* . .. S lbs* s •Mi » lbs* : lbs.t 8 S *49.701 lbs* 1 i1 * «S e ee s i m o tfroafe yolk 11*69 8'42*? 9 8 —» ww> 3 i SOQ * w w 22*00 138*9 1 'JW*P'*W 4 1 200 I * ** 4S.3? *88.9 l 5 l 200 s « * 51*66 123* 0 1 «_• 6 i 200 iFresm whole 37*50 t£&*?$* 1— «». ? 2 1 t too if^esb whole 80*00 * * «— 8 2 2 Ogg ilt4,4iFrasb yolk 20 *53 J 1 :2? *?SiCarotene mix *BW 5* S0 9 * 194.4# * 52.26fCarotene mix WA" 5,5# 10 i188*41 •M* 131*6 :«¥X4a~Pro* 12.00 11 : 200 5Chinese dried 10*50 :55*0 fBalt 4*00 t 8 yolk S 8 * * i... * Tentative Formulas for the Calculation of the Contest of I%g Yolk and of lliol^ /g# in Alimentary Pastes fro® the Steml Content a for tli# Coat @st of as. The following Yormla is proposed tentatively, pending th® accumulation of more data, for t!i# ealeul&tloa of th# amount of eowiereial egg yolk solids la an alimentary pastes Y (0 «■ OtO^i) 100 -n (ft r% *»*■ 1 - j E ^ r o j & S T (0 ~ «•***>** I' * the percentage of es®iterci&l egg yolk solids in the sample {moisture^free basis) * the percentage of sterol (eelaulated sample (moisture^free basis} - cholesterol) in the §3 Tli# follow* lag forn&la is proposed tmtativf&j# poadlag tho ©©©tamilfttioB of mmrm data» for tii# ealsulaiion of t&© waavat of eoaBnorolal ulieX# •&& solid# la aa allaoatazy poatmt m m C— * (C - 0*034)4® 1 » tis# pwNmUiti* of ommmoi&l wholo «gg mli&o in tb* mmplo (m®i»twr&**frw- basis) 0 ** ill© pmmmtmgo of otor&l (MUnaXatod ms ©holosterol} ia thm emapX® (aoi3tur©»:fr*© baeie)* 94 LIXSmmiEE CXf® 1* S. 4* 6* limited Stmt®a Dep&rt&e&t of ^ 162, WsaWlngton, B. 0* agriculture, Food Inspection Decision January 6, 1916* Food Insrectlaa. Dealstea 1?1, Bcsbiagton, B. D^fr October 14, 191?* Circular 136. Iftshingtcm, B* C. Juta®, 191.9. Food Inspection Decision S§, Ws.Siimgtoa, B* 0. February, 192?. Service and Beguletory iimoumcoatomta, Fool Dseastber, 1929* end Drug Mo* 2, ifs@h.iiigtom, B* G* Service end Bajsuletonr A^ouaeoaeats, .food "nisA Drug So. £ (.First Berislon), Washington, D. C* Sooembor, 1928* f * Sorsrio# «&& Eagulaiory Asmmmtm&ntB« Food a i Iteug. Ho* B (Socoiid. Boriston) y lasfeimgtoa, 2>* 0* 0#pterabsr, 1931. B, _______ _ _ Sorvic© a.a.4 Begolatory Aaaouaossasats, food and Bxug Ho* 2 (Third Bertsion) , B. C. Jus®, 1932. 9. Service amd l&togy AxmcRmoeHMRitA* 'food and Drug Ho* 2 (fourth Berlsloa), Wash lug ton, D, 0, August, 1933* 10* Service fesd Begul&tery Afuseuaesne&ts, food rsad Brag Mo* I (Fifth Berisioa), ft&aki>on, B* C* Borember, 1936. 11* Stroteecfcer, H. H«y end B. Tsubel, is t b n & b u o h ster Lebems&ittel- chorale» edited by a* JuGkenaek, £. Bames, B* Bleyer suad J* Groesfold. Band ?. Berlins Julius spriogor, 1939* F* 261* w 12* Tillssca®| J*t H* ftiff&rt and A* luBm, 2* Untersuelu Lebensa* 60» 361 (1930). 13* Buchanan, B . , J* Aaaoo* Official Agr. Gbwau ?.# 40? (1924). 14. Sort wig, 1?*# J. Assoc. Official Agr* Cbesu 8, 10? (1924). 15* 1. Assoe* Official Agr. Chc*u 91 (1923). m16* Bvrmmm, J”. K,t J* A^ioe* Qfflalal %r* C&a&u 14, 417 (if51). 17* Eitahell, I** C*, 3*. A»»oe* Offielil Agr* C&ass. 15, 282 (1953). IS* Al£«s*d, a*, I. Aaao*. Official Agr. Cham* 1^, 500 (im), 19* Amalia, I«» &* m&tm Cbam* j^, 439 (1955)« SO'* Wasitakjr, A .f Mifcrocftaad.e 14* 889 (1934)* 21# Buis, A, 8,, «m& I* ttarraa, Aaalas* aoc* mp®&* fia* *pti». M * 6135 '(1935)* 88. i*a»p«vt# L* M*, Xnd* lag. Chao#, Agptl. 14* 8, 159 (1950)* 83. SohSsahaiimr, t*f and 1* M* Sparry, J# Biol* Cfetfft* 104,. 745 {1954} 84* Bafeal, A* 1., I* J. Brsikti&r and a* Katalsoa, 1* Biol* Ofea®i* 115.* 381 C19365. 15* Rlffart, S#> and H* Kallar, 2* ttataarsuifo* Let-anao# 68, 115 {1954}. It* Ssaat-43fS*gfl, A. ▼*, Bioefeam. 2. 156* 107 (1933}* 37* Okay, R*, J* Biol* Chav* 88, 367 (1990)* 18* Ttaraar, 1., J. Biol. &MMU .98. 499 (1931). 89* YUsada, M*, J. Biol* %.*», jjg, 503 (1981). 30* Wladaoa, A*, Bar. 41, 25m (190x8). 31* Baa, H*t Blotimu 2. 194. ITT (192S). 38. MuSgfealmar, 1., and H. Xtaa, 2. physiol. 0h«* 815, 59, (1933). 53. Ewart, 8*, Bloa&aa* 2* 845. 149 (1953). 34* BrauMb, P. L., J* Biol. Otuau 184. 151 (1938). 35* farrier, 1*, Mitt* laBanAn. Hyg» 88. 154 (1937). 56# Mitt. Hyg* 29, 15 (192©). 37* Vixtdaaa, A., Bar. 39, 518 (1906). 38. Solda, II., 2. ftitgatr# Ghaa» 19, 1609 (1906). 39* Lewkowitaok, J*, Jahr. Ctaam. 16. 406 {190$}. 40. Popp, G*, 2. offanti# Chmn* 14. 459 {1908). 96 41* ftlelieesgae, I. 8., sad P. Moldenbe.uert Ann* 148 a If5 (1868)* 42* Liafeeimama* C.f Ber* IB* 1805 (1885). 43* Cloes, Ch* * CoRpt* read. 114* 864- (1B97). 44* illideas, A*f end Bauth, Ber. 3 9 , 4378 {1906}* 45* ttisdaus, A*, end E. Ltiders* 3* pbyviol* Cheat. 109.* IBS (1930) * 46* Hfftehttta, X*, £* physiol. Ohae# 114. 186 {X921). 47* _______ physiol* Cheeu 106* 371 (1919) . 4S* BehSsihelmer, R*,. £* physiol* Obtest# Ifg, 86 {1930}* 49. Xlrroae, P., Oassz* ohlm. Ital* 62, 1101 (X9SB)* ®°* Industrie eMmies £, 131 (1932). 51. Re Jtai, 1., and -S’* Pirrottet $e«z. cMm* itsl. 61, 735 (1931). 58. BmlX#, J. 0„, J* Jte, €&«&• See* 55, 2003 (1933). 55* Bulm, 0. f* w a # loo* trev* ofeim. 45, 363 (1936)* 54* Stepwiowt A., Ber* 39, 4056 (1906). 55* Eolffeoff* X. H*» *&& 1* E, Ruxmxt, Volumefrle Aa&lyai®, felome II, Pr&otio&l Telenet?!e Analysis. Mew forks Xoim ®il@y ®ad Son®, Xae« 1929* 56* Gardner* 1. A*# sm4 H* C&isMberou&fe» Bioeheou 1* 88* 1631 (1934)* 57. SehooBelffier, R., J. Biol. Chest* 110. 461 {1955}, 58. A&deyeea* 1* X.# and P. P. 22afeeafemi*ey, J. Abu Cheau Soe# 46, If If (1924). 5®. Bell, O* B.* Jr. Tfeeeig: A Study of Sfeo&t Oil* Baiveralty of Sinnesoiis * 19 24* 60* Anderson, R« J*f end P. P. M&beaJiauer, X* An* Chea. Boo* 46, £115 (1924)* 61, Asdereen, B. X*, R* L. Bhrl&er* & M 0. 0, Bair, X* As.. Chera. Boo* 48, 2987 (1926), 62. Wallis, 1. S., and 1. PsrakoIsL, X* A*. Ohm. Soo. 58* 2446 (1936). 65. Beagteaoa, B. 1., 1* physiol* Che®. 237. 46 (1935). 64* Bernstein, S., sad S. S. Ball!©, J. 0rg* Cbessu 2^ M l {193?}. 85* leliiba, A.g Sci. Paper® Inst, Pfeys. Cfeem. Reeeerdb (Toityo) IB, 113(1955). 3? $$u Kajprer, P., and M. Salsaon, Belv. OBIeu /vats. Jj0, 424 (131$?)* 6?* Wfrtri, H*, Ana* 495, 41 (1938)* 08* Meulen, J* H* van tear, Cfcem. *s#®i£Bl&d j|8, S3 (1331). 09 . golthoff, I* II., «&& H* T u tay * la d . ®a«. 0&«bu» Anal. * d . , 9* 75(193?)# ?0 . Suebey, 1*. L., cad H* X*. Brtttee* lad. lug* Ch«eu, Anal. Id*,. 10, 590(1938). 71# Dam* E*, fiioehen. Z. 191., 186 (1918). 78. Xuettl, 11*, 2* s&yelol. €&«&* 181, 121 (1989)* 73* HueXler, S» H*., 1. Biol. 21, S3 (1915). 74. T&asxi&aea©!?, 0* l.f and B. Se&aber, 1* phyalol# Clam. 127, 278 (1923} * 75. Dw e* H., Btoehem. g. SIS, 475 (1983), 76* Perl-van, J. I**, Annual Report of t&e Ztoparta&eat of Agriculture ®nfl Marfcete for fc&© Tear 1938. State of low Toric. Legislative .Bocusient (1332) Ho. 37* p* 111* 77* Bam, H*, ©iee&eia. 2* SIS, 468 (1983)* 78# Kerr, R# M*, and D* 0* Softar, I* Abbog. Official Agr* Chesa. 8, 90 <1984). 7 9 * H a rtw tg , R . f 0 * S.* Jgsat®sont 1 * P* Battghjft&a and I*# 11. B a i le y , J« Aeeoe. Official %r. Cheeu 8, 439 (1914). 80* Association of Official Agriculture! Ciienlats, Official ant Tentative ^t&o&a of Aaalyeie. Iwarth Mltica. Waehingtottt Association of Official Agricultural Ghmil&ta. 1935. p, 399* 81. Ml t oh ell, 1, C., 8, Alfeod ant f* #T. J* Assoc. Official Agr* OBess« Ii>, 24? (1933) * 82. Lculier, A*, ant H* Crevat, J. phaxra. Chlm, 14, 214 (1931). 82. Kediag, S., Bieefeeou 2. 254. 374 (1952)* 84* Costa, 0*, Ana* eMin* applicate 25, 355 (1955)# 85. Hageeaim, cited In food, MarcB, 1938, p. 232. 86. Sullivan, B*, and 1* Emm, Cereal Ohm*. 15, 716 (1933). 87* Cappeabea^, H., Cham. £tg. 33, 985 (1909). 8-8* Soldi, A*, and 8. Teetori, Ann. efclm. appliesta 21, 338 (1931)* 98 89* Mm z* Uaiarsuofc* St* 9 {1998}* fO. JL'sool«tlmi at Official %rlciiX*ua?®tl Chmi»tst Official m & T*&t&tire Kftt&ods of Aaalaraiju $&ir& Miftosu Sftafeiagtttu Association of Qfflol&l Agricultural Ohcmifft#. 1930* p* IBM