— —— —— —— —— _— —— _—— _— —— ————- —— .— —— _——— — 110 590 THS AN IN VITRO STUDY or THE HYDROLYS!S or SEVERAL FATS AND OILS BY PANCREATICV LIPASE Thai: for the Dog“. of M. S. ‘ MICHIGAN STATE COLLEGE Duward D. Harbaugh 1954 .- - ~--.!.s.*:r¥.-:~‘=' Michigan State C 0119 0 East I g- 351 I?! VIJLi‘ FSCJ;XKSE 33;; JV vaIF’SIS. Oi" imVrm'AL bA'L‘fl AM) GIL"; 13‘." E) TSP-2'6 n .Lfi. L113 LI 9.3313 b V Julqu 9. Eqrbqugh A PH3SIS oubmittei to the “Dailqto f5 chool of Niohifiqn state Jollege of Ag? 311t1re nni ” 331101 Science 131 3xrtizl tuli ilmuxrt of the 1 331 rem(x1t tor the iezroe 3f 1 ."‘f"1 1,, ' r‘ 1 ‘\ V 1 A 31...“; I.) -.}‘J Lle'JJ—‘J Department Cfi‘ffiumniqtrv 1954 .‘r mint (3 )5) .v‘ ‘\,-‘ "‘1AL L.} A)KNO“LuQ3E31-IT 333339 tiaheq to qcknovlelqn “he 33313 assistance given by DP. “ “ Pt in the ulanninz of the 9x313:- ‘Elll in 13:16 ’);_’ef)r{1°s1tion 3,? t‘qg . '- )1- ‘L 0 344094 CONTENTS HISTCBICAL REVIEW INTRODUCTION TO PROCEDURE PROCEDURE DISCUSSION SUMMARY DATA BIBLIOGRAPHI assyompg HISTORICAL RSVP"?! Ptohobly tho moot obviouo ond ooomingly tho mot importont of tho intootinol lipoooo io that ouppliod by tho poncrooo. No doubt it no for that moo that Eborlo, in 1831;, firot oboorvod tho notion of poncrootio lipooo upon noutrol tot. Notwithstanding, tho firot worker to nako progrooo in tho otudy of ponorootio lipooo woo Cloudo Bormrd (1856) , who found thot ponorootio Juioo both omni- fiod ond hydrolyood tho tat. In thooo dayo tho notion of tho pon- orootio Juioo no oboorvod noinly by who of fiotnloo, oithor noturolhor ortifioiol, oo in um otudioo hod not yot boon undo. In 1903 Pottovin roported thot poncrootio lipooo hydrolyood Into to fatty ooid ond 111ml. he no oloo oblo to show that tho reaction woo rovoroiblo. By ooing oloio ooid homogeniood with glycorol ond mixing it with o glyoorol oxtroot of tho poncroao ho woo oblo to dononotroto tho ootorifiootion of ohoot 33% of tho oloio ooid with glycorol in 50 houro at 38°C. In 19lh Arnotrong ond Ooonoy found oquilihriun no roochod vhon hm: of tho ooid no oombinod, and in 1936 ”to: and Roalo ropoftod our 501 ootorii’iootion. Hooovor, noot of tho mo rooont otudioo on poncrootio lipooo hon boon dono in on offal-t to loan ooro obout tho hydrolytio ootion of tho onoyno ond tho 'foota'o that favor or hindor thot ootion. |' Two of tho tootoro thot hovo roooivod o oonoidoroblo olount of ottontion oro tho optimum pH for lipooo ootivity ond tho pH ot Ihioh it io loot otoblo to hoot. HoGillivny (1) roportod tho optiIIl pH to be 8.0, and tho option thonol otobility in tho rogion of pH 6.0. Bo aloo oboorvod that an unpurifiod oxtract no more heat otablo than a purifiod, which ho oXplainod .wao duo to tho protoction tho impurity gavo to tho onzyno. Flatt and Dawoon (2) phood tho optimal pH in tho region of 7.0 to 8.1:, with tho oXplanation that it dopondod upon tho buffor nood. Noyco, Siognira, and Folk (3) mo or loco confined tho findingo of Platt and honor: by placing tho optimal pH at 7.0. Sabatka and (flick (h) in quito an oxtonoivo otuchr on tho onbjoct found that tho optimal. pH lioo botwoon 6.1 and 8.2, and that tho type and natnro of tho buffor, ao woll ao tho onbotrato and tho onzyno proparation hawo such to do in dotoroining tho optinol pH. Bloor (S) plaood tho optical tonporatnro for tho 0!!sz activity at that 10°C. Ho found that tho activity woo ro- dnood by ton minutoo boating at 16°C. and dootroyod at 55°C. Flatt and Dawoon (2) holiovod that tho optilal pH any to loohod upon ao tho outcono of two oppooing tondoncioot with an incroaoo in alkalinity on incroaoo in hydrolytio activity takco plaoo, howowor, at tho highor pH tho otability of tho onsyno docroaooo. click and King (6) woro of tho opinion thoro are at loact two oppooing tond- oncioo oporating whon foroign oubotancoo m added to a oolntion containing onzyno and oubotrato. Ono tondoncy io to inhibit tho onwno, duo to a oonbimtion of tho foroign compound with tho any. to produoo an inaotiwo couploxy tho othcr, io to offoct an activation of tho onayno by acting tho only-o and aubatrato holoculo acro ao- ccociblo to oach othor. 'rhoy oitod ao an onnplo of tho activation of an onsymo tho loworinc of tho intorfaoial tension botwoon tho onoyno and ouhotroto, an offoct producod by bilo oalta !‘hoy oain- tainod that in tho one of pancroatic lipaoo tharo are very fow inactivating oubotanooa. In fact, it wao loarnod that tho hoot inhibitcro for livor ootoraoo aro not only tho hoot activatoro for pancroatic lipaoo but aloo lowor tho ourfaoo tonoion tho loot. In addition, thoy loarnod that activation oocuro only in an alhlino nodiun. Conooquontly, ao hydrolyoio proooodod, tho p8 tondod to fall ao a roonlt of accumulating fatty acido with tho oonooqooat alowins of tho prooooo. Hm, Voraci- and Kathy (7) oxplaiaoa that bilo oalto offor an onooption to tho rnlo in that tho pa dooo not go down ao Inch ao would to apootod. Thoy oxprooood tho via! thatthioiodnotoatondoncyofthobilo aaltotocoabinowiththo fatty acida and honoo to town than, which would pan-it tudrolyoio to go on. Thio viow wao onbotantiatod by hoinotoin and Wynno (8) in thoir otndioo on panoroatio lipaoo. In 1907 Loovorhart and Poiroo (9) advanood tho idoa that though tho pancroaao oontaino nootly lipaoo and tho livor aootly ootoraoo, it in poooiblo that tho pancroao also contain ootoraoo and tho livor aloo lipaoo. In fact, lalvorkanp and Griffioon (10) in 19310, ropa'todtwotypooofonq-oiathopanoroaowhiohconldhodoaoa- otratod ao ooparato ontitioo. Thoy alao ouggootod that thoro fly bo oovoral additional only.» in tho panoroaa oach opooific for a dofinito poop of «too. in antithooio of thio idoa wao advanood by Platt and Dawoon (2). Thoy holiovod that tho protoin otrncttn'o which hao boon aooribod to lipaoo io charactoriotic of tho activating protoin aooociatod with tho unm- rathor than that'of tho anayna itoclf. Thoy woro ablo to altar tho ootor hydrolyaing proporty of tho onayao by changing tho aooonpawing protoin. They ozplainod tho apparont daatruotion of lipaoo by trypoin ao being duo, in part, to tho removal by rwdrolyoio of the protein which activatoo tho lipaoo. The work of Folk (12) rather otrongly oupporto tho belief of Platt and Dawson, for Falk wan able to ohow that a pancreatic extract having definite hydro- lytic action on ethyl butyrato and olive oil had ito action modified by the addition of albumin. The. addition of thio protein resulted in an increased rate of ivdrolyoio of olive oil. “mover, when odcotin an added to tho lane oyoten the action of tho enoyae no incroaood for both ethyl butyrato and olive oil. He concluded that lipaoo, or ootor-hydrolyoing action, io due in all probability to a definite ohonical grouping, and that thio action can be altered by changing tho accompanying protein. Sabatka and click (1}) oboorvod that in tho one of pamtio lipase only a few percent of the total poooiblo hydrolyoio io effected when the reaction between the enzyme and oubotrato ooaooo. They found that it no not due to low affinity for onnylo to oub- otrato, neither one it due to the producto of hydrolyoio, ac fatty acido and glycerol, for addition of more emu did not produce aw more action. Thio would excludo enzyme destruction; homer, when an additional amount of oubotrato no added new action ensued, and they wore ablo to repeat the phenomenon. They believed thio to ohow that a great part of the oubotrato disappears from oolution without being Ivdrolyood. Their explanation wao that tho oubotrato io bound to inactive aroao of colloidal pancroae globulin particloo in ouch a aannor that it io not acceoeiblo to the active group of the onoyao. If thio hypothoeio io correct, the combination of large aaounto of oubotrato with inactive areao on tho lipaoo particle or with altogether inactive globulin particleo would eventually dininieh the actual concentration of the oubotrato until action would ceaoe. In contract to thio, they obaorved that the action between liver ooteraoo and oubotrato gooo on until at leaot 90$ of the oubotrato hoe been hydro- lyoed which they think indicateo that there ia little, if any, inhib- itory effect by cleavage producto. The findinge of Murry (lb) aupport those of Babette and click. However, hurry reaooned that if oubotancea pcoeoooing a particular molecular atructure, other than that of the utoral oubotrato, are adoorbed at the ourface of the enayne but not activated they will compete for the active center with thoee which are activated, and hence bring about a reduction in tho velocity of reaction of the natural oubotrato. He learned that a oubotance containing the ear- boxyl group competeo with the oubotrato for the oncyeo. Bo woo of the opinion that it io by thio group that the oubotrato ie normally attacked, which nokeo the relative non-opecificity of the enoyle more underotandablo. ' The ability of lipaoe to twdrolyae fate hao been acre or leeo taken for granted. In fact, pancreatic oxtracto are known to moire- lyoe all ordinry zlycoridoo except poeoibly oteerina. Ball and Iatlock (15) believe that the atearino are no exception in principle, but merely are ludrolyoed acre olowly. They were able to ohow that triatearin woo aplit oo poorly by otherwioe powerful preparation of pancreatic lipaee that ito digeoticn, when ebeerved at all, no entirely out of line with what night be expected. However, they were able to carry out the ludrolyoio of Iono-di-cr triotearine at a rate quite comparable to that found for other fate, provided high temperature and good dispereion were maintained. They learned that pancreatic lipase hydrolyoed all the triglycerideo of noraal aaturated fatty acido, but that they were oplit at different rateo. They found that the fate which are eplit hoot rapidly are the once that have fatty aoido occurring aidway between acetic acid and otearic acid. Aloe, there wao noted a ourprioing indifference on the part of the enayne to the conetitution of the alcohol ao long ao it no of the when variety. However, the length of the carbon chain, the occur- rence of free hydroxyl me, the preaenoe of a double bond, or of eecondary or tertiary carbon atone may all affect the rate of oplit- ting but do not change the qualitative or quantitative rooulto . ‘rhe reoulto obtained with oocondery propyl otearate, oecondary butyl otearate and tertiary butyl etearato were in oarked contract to thooe of the correeponding eetero of n-propyl, n-butyl and n—awl alcohol. In fact, they believed that the aplitting of aecondary eater lining takeo place at an alaoat negligible velocity in comariaon with the aplitting of a print-y eater linkage. I Bell, lath and Tucker (16) found that the hydrolyoio of the higher but not the lower aaturatod triglyceridea ie dependent on temperature and that at a moderate temperature the nation- roto of aplitting 1. exhibited by glyceridea containing the otraight chain oaturatod acido of approximately 1 to 10 carbon atone. However, they explained thio deal not apply to unaatarated glyceridea, for eloin behaves ao if it contained a c rather than a on acid. 9 The work of Ball and Hatlock (l?) elaborated on thio point by chewing that the rate of hydrolyoio at ho°c. io independent of the oubotrato concentration over a wide range. However, only the ootoro of lower or unsaturated fatty acids are hydrolyzed in the cold. In fact, triotearin undergoes aluoot no hydrolyeio at 20°C., whereas bonzyl etearnto hydrolyoea at thio temperature with fair rapidity. The butyrnto continueo to hydrolyoe rapidly even when the oycton io frooen. Bell, hatlock and Tucker (16) carried the hydrolyaio of triatearin to practical completion. They chewed that what remained after inter- rupting the hydrolyaio of the oubotrato woo almost entirely triotoarin. They concluded therefore, that the green chemical compooition of the oubotrato romaine practically unchanged throughout digeotion. weinotoin and hynno (18) obeorved that the rate of liberation of acid from diacetin woo exactly twice that from nonoacotin. However, the rate of liberation of acid tron triacetin woo only 2.66 tineo that of nonoacotin, ouggeoting that the middle eater linkage io loco readily attached than the two end linkages. They aecunod that the two end linkegoo in triacetin, ao in diaoetin, are hydrolyood at the oaae rate . ~8- Sinoe pancreatic lipaeo bringa about the twdrolyaia of fate into fatty aoido and glycerol, and oinoo bile oalto are neceeoary to lower the ourface tenoion and co aid in emloification and in turn accelerate the action of lipaee, a digeot conoioting of a fat, pancreatin and oodiua glycocholate wee prepared, and digeotion carried out at 31°C. The object woe to etudy the effect of continued hydrolyoia on varioue fate. Therefore, at intervalo of progreeoively greater length a portion of the digeot wao renoved and the undigeoted fat extracted with other, weighed and characteriaed.‘ Becauee of the general occurrence of unaaturated fat acida in natural fate and oill, the Iodine Nuaber woo aloe doterninod on the reoidual fat. Il‘ho Reichert-Heiool Nuaber, which giveo a eoaaaro of the volatile aolublo fat acido,‘ waa .1» dotcrained in order to follow the hydroly- oio of glycerideo containing the chart chain acida, particularly in theicaoe of Iilk fat. In the preliainary otudioe, doteroinationo were aloe node of the fne acido fer-ed at variouo otagoo by titrating with otandard alkali. Theoe reoulto,ao well ao theee obtained in a otudy of the changeo in melting point, proved to be of little valne in revealing the eouru of lipolyoio ac that thoeo doteroinotiono were diocontinuod. The firot digest consisted of 200 m1. of 301 milk fat, 2 gram of pancreatin (0.11%), l gran of eodiua glycocholato (0.2%), and enough water to give a total volume of 500 ml. The control used for comparison concicted of 200 ml. of 30% milk fat and 300 al. of water. Digestion woo carried out at 37°C. At intervalc of l, 2, h, and 2h houro, S n1. portione were transferred to erlenacyer flaeko, to each of which no added 10 cl. of ethyl alcohol and 5 drape of phonolphthalcin. Titration no then carried out with 0.1 N. codiul hydroxide, giving a neaouro of the amount of fat acido liberated. it the cane intervalc, portionc of the digeot and control were transferred to 500 ml. ceparatory funnelc to which were added 5 al. of 201. codiuo hydrczdde and 20 al. of 95% ethyl alcohol. After choking the contente thoroughly, the unhydrolyced fat can then extracted with one 100 :1. portion of other and cubeequently with two additional 50 ll. portione of other. The combined ether extract one then wached with water until free from alkali (phonolphthaleia no need co the indicator). The alkali-free ether extract wac' dried by adding 20-30 grace of anhydroua codiua culfate, clowly agitating for 15 ninuteo and then decanting the ether extract. The codiul culfate wee washed with 50 ll. of other and the other each added to the min ether extract. The dry other colution woo then placed under reduced preecure to renove the other, and leave the extracted fat. Both extracted fat canplcc were weighed and the Iodine Runbor and Roichert-heicel hunt-r deternined. ' The name lethod need to determine the Iodine Number wac carried out ac follow” about 0.5 grano of the fat woo diccolved in 10 al. 0 of chlorefcrn and placed in an iodine flack. Then 25 al. of hanuc iodine colution wao added, the flack atoPpcrcd and allowed to ctand for 30 ninutec. The ctopper woo then removed and 20 al. of potacoiun iodide colution (3.0 gram of potaeciun iodide) poured into it, followed with 100 ll. of water and titrated immediately with otanderdioed eodiuo thioculfate colution (approxinotely 0.1 13.). then the colution becane pale yellow, 2 al. of ctarch colution (1-200) wao added and titration continued until the diuppearenoe of the blue cola. By cubctituting the alount of thioculfate colution uced limo the moat need for the blank, into the following rm, the Iodine Runber can be calculated: ml. of thioeulfote X normality of thioculfate X a.e. 0M . Iodiu m, weight of couple The procedure need for the Reichert-lleicel Huber wee ac follow" to S grace of fat in a fleet 2 al. of potaociua hydroxide (1-1) and 10 ml. of 95$ alcohol were added. The contentc were refluxed on a etede bath for 25 mt», after which the alcohol wee evaporated off. Then 10!; ml. of recently boiled dictilled water cooled to 50°C. wac added to the flack and warned until a clear colution of coop wao obtained. The colution wao cooled to 60°C., 8 al. of culfuric acid (1.4.) added and refluxing continued until the fat acido hed cowlotoly ceperated ac an oily layer. Subcequontly 110 el. wee dictillod in ac nearly 30 nimtec ac pocoible and the dictillate filtered and 100 ll. of it titrated with 0.1 N. codiun hydroxide ucing phenolphthaloin ac the indicatc. The Roichort-heiccl Nuinr ic calculated by ucing the following formula! m1- of 0-1 F- "<11“ mom- 1: 5 1g " Rolchcrt-irelscl umber weight of canplc X 10 The results of the first digestion may be found in Table I. For the second series, four different digests were set up, each being the same as the first except that the amount of pancreatin used was respectively 0.11%, 0.8%; 1.6%, end 3.2%. Digestion, sampling and extraction of samples were carried out in the same manner as before. The Iodine Number and Reichert-Eeiesl Number were determined as previously outlined. In addition, the melting point of the ex- tracted fat was determined as follows: a small sample of the fat was melted at as low a temperature as possible, and drawn up into several thin-walled capillary tubes about 3 cm. long. They were then placed in a refrigerator for not less than 12 hours. One of the tubes was attached to a thermometer in such a unner that the fat was as close as possible to the bulb of the thermometer. The thermometer was supported so that its bulb I. immersed in water in a wide mouth test tube, which in turn rested in the neck of a round-bottomed flask also containing water. IThe water was heated mdually until the fat ‘ eelted. The temperature at mich the fat becane transparent was taken as the final melting point of the fat. The results of the second digestion may be found in Table II. In a third series, four different digests were set up, each being the same as the first, except that the amount of sodium glycocholate used was reapectively 0.21, 0.14%, 0.8%, and 1.6%. Digestion, sampling, extraction and analysis were carried out in the same manner as before. ‘ The results of the third digestion study may be found in Table III. Since milk fat, a fat of animal origin, had been used for the digestions carried out so far, three fate of vegetable origin, .12- eorn oil. olive oil. and orisoo. a hydrogenated vegetable oil. were selected for further study. latter fat being included in this group for comparison. in each case. the digestion was carried out in the presence of O.“ pancreatin and 0.4$ sodiu- clyoooholate. It was desirable to have the fats in a well dispersed state to facilitate digestion. This was accoupliined by the following leans: two grass of gun arabic and two grass of sodiu- ¢lyoocholate were added to 275 al. of water-and warned. To this were solution were added 2 ll. of eleis acid. 5 ll. of 0.1 I. sodinl hydroxide and 60 crane ef fat. ihis was well sized and repeatedly passed through a Gunfield emlsifier until a good emulsion was obtained. To the emulsion was added 4 grass of pancreatin with stirring and enougm water to give a total volune of 500 el. i’hs emulsified fats were annealed and the residual fat extracted in the usual nanner. lbs pH of the digest was obtained each tine Just before sampling. The Iodine Dunbar and Reichert-leissl lunber were deterIined on each of the extracted fat residues. the results of this digestion study nay be found in Table 1?. It is apparent fron the results shown in Table 1' that there were fairly unifor- conditions with respect to the p3 of the digests ex- cept in the case of butter fat. The low pH of the butter fat was obviously due to the rapid release of soluble acids and could be expected to affect the rate of hydrolysis of butter fat. it would therefore be of interact to know how the digestion would be changed if the digests were held at a p8 of 7.0. Accordingly the anus fats were used (corn oil. olive oil, crieco, 1'. n‘. butter) altho it should be noted that they were of different origin than the ones used in the previous experinents. The digests were set up in the same manner except that the pi! was kept at 7.0 throughout the digestion. This was accomplished by regularly checking the pH of the digest with s pH meter end sdding 1.0 N. sodium hydroxide as needed. It might be noted that very little slkali was required after the first two hours. Sampling and the extraction of samples were carried out in the naval mnner and the Iodine lumber and Reichert-taoissl timber determined es before. The results of this portion of the study new be round in Tabb Vs ' ITISCUT'SSION Fran Table I it will be noted that during the first hour hy- drolysis of the fat took place at a rapid rate, but as time increased the rate of hydrolysis decreased. It is also evident that as 1v- drolysis increased there was a concomitant imrease in the amount of frae fatty acid formed as indicated by titration with 0.1 N. sodiua hydroxide. There was a slight increase in the Iodine Hunter as digestion processed. This indicates that the hydrolysis of unsaturated fatty acids did not keep space with that of the saturated fatty acids. The relative high content of saturated acids of. low nelso- ular weight in butter fat would make such a result reasonable. Nevertheless, in subsequent studies both slight increases and de- mases were observed. it the end of the tint hour of digestion the Reichert-Heissl Number of the fat sample was found to be much lower than in the control and continued to decrease progressively as digestion con- tinned. It is apparent that the esters of the volatile fatty acids were more rapidly twdrolyssd than those of the non-volatile acids- as found by Ball, Hatlock, and Tucker (16). The results sumarissd in Table II indicate that the saount of wdrolysis increased with increasing anounts of pancreatic. Enninatien of the Iodine Rushers again scene to indicate that the propctien of maturated fatty acids in the residual fat re- mained fairly uniforn throughout the 214 hour period regardless of 1 the ascent of panes-satin used. i . rmMLaé-v’rwm" 1;- ' m The Reichertnmeiesl Numbers show that the hydrolysis of the esters of the more volatile fatty acids increased cost with in— ‘ creasing amounts of pancreatin during the first hour. The variations although slight in the melting point of the rcsidual.fat at various stages indicates a consistent increase due to the preferential hydrolysis of the glyceriden containing the short chain fatty acids. The increases are also consistent with the more rapid hydrolysis at the higher concentrations of pancreatin. With increasing amounts of sodiun.glycocholate, the rate of hydrolysis was increased. The Iodine Number‘values indicate that at the higher concen- trations of sodius glycocholate there was increased liberation of unsaturated fatty acids. A similar result was observed in the case of the hydrolysis of the glycerides containing the lower saturated fatty acids. From‘Table IV one can see that the fat least readily hydrolyzed under the conditions prevailing in this part of the study was butter. This can easily be emplained on the basis of the rapid decrease in pH as soon as pancreatin was added to the digest. In the case of corn oil, olive oil, and crisco, the pH remained appreciably higher throughout the 2h hour digestion period. Consequently conditions were more favorable for the hydrolysis of these fats. Only in the case of corn oil did the Iodine Number decrease as hydrolysis progressed. This would indicate a somewhat sore rapid liberation of linoleio acid which is the nest abundant fatty acid in this oil. Butter does not follow the pattern of either corn oil, olive oil, or crisco in that the Iodine Number seems to change but ~166- little, if eny, with continued hydrolysis. In contraet, the Iodine Number nan found to increase throughout the éigeation period in the case of olive oil end crieco. A more critical etndy of the digeetion products would be necessary to eXplein thoee differences. Although smell changes were observed in the Reiohert-Meieel numbers of corn oil, olive oil, end crieco, the veluee ere perhepe too smell to have reel signifioence. The date in‘reble V ehowe clearly thet maintaining e pH of 7.0 during the digestion effected the hydrolysis of earn oil, olive oil, and crieco but elightly during the 2b hour period, but eerkedly in- fluenced hydrolyeie of'butter tut. It is of interest to note that butter fet ehowed the greatest enount of digestion, followed by orieco, olive oil, end corn oil. Slim-EAR! A etudy of the couree of fat digestion was carried out in Which reeiduel fet wee extrected end partially cherecterised et verioue etegee of the digestion period. The following conclusione my be node: 1. With increased emanate of pencreetin, both the rate and extent of hydrolyeie were increased during e 2h hour period. 2. A einiler effect elthough of eneller negnitude we noted when the concentration of Iodin- glycocholete wee increeeed. 3. In a comparative digeetion study with uncontrolled pH the following order no establiohed with respect to the maxim rete and extent of hydrolyeie: crieco, olive oil, corn oil, end butter let. 1.. lhen e of] of 7.0 wee ninteined throughout the diaeetion period, butter fat wee found to head the list followed by crieco, olive oil, end corn oil. 5. The Iodine Nunbere ehow in the case of milk fet um with increased enounte of eodiun glycocholete the rete of hydrolyeie of the unsaturated fatty ecide no increased, but little change was noted when the mount of Pancreetin me increeeed. 6. The Reichert-leieel Numbere ehow in the ceee of milk fet thet the ivdrolqeie of the eetere of the more volatile fetty ecide increeeed vith increasing eeounte or pencreetin ee well ee eodiue glycocholete. 7. There ”1:. noticeable drop in tho Iodine Number ee corn oil 1e tydrolyeed which would indioete en increeeed rete in the liberetion of linoleic ecid. -18- RT'SHLTS 09‘ HF»??? 1'IGELSTION TABLE I CONTROL (he Pancreatin or Sodium Olycocholetel rm 1! HOURS 0 1 2 1. at Wt. 12 .00 ll .98 11.97 11.95 11.93 Neon USFD 0.50 0.149 0.50 0.1.9 0.52 I. a. 38.10 38.00 38.20 38.30 38.60 a. M. 26.00 25.90 26.30 26.00 25.60 DIGEST mm 13 HOURS 0 1 2 1. 2h 31'. 12.00 9.9!. 9.51: 6.7h 6.53 11.0}: mm 0.50 5.130 9.90 12.70 1h.h0 I. N. 38.10 38.70 39.50 110.10 10.90 R. m. 26.00 16.65 1h.SO 1b.05 11.80 RESULTS OF DIG-FSTION B! INCREASING THE P378017??? OF“ PANCR't'ATIN TABLE II 00150. 01? PANCRTTATIN 6041.211 rm IN Hams 0 1 2 h 21. 821'. 12.000 9.168 8.377 7.963 7.566 n. P. 32.00 32.0 3h.o 35.0 36.0 I. 11. 38.10 39.60 1.0.10 1.1.10 1.1.80 11. '1. 26.00 17.80 9.80 9.10 8.50 cone. or am...“ (0.85) 1 7an IN moms o 1 2 6 2h 73. 12.000 8.970 8.021 6.225 6.530 1:. P. 32.00 32.0 35.00 36.0 36.00 I. n. 38.10 39.30 39.30 10.50 110.20 R. M. 26.00 16.50 9.00 8.h6 8.00 c0130. 0? 1111;025:1711! (1.65:) 7181‘ IN HOURS 0 1 2 I. 21. 7.7. 12.000 7.298 7.123 5.330 5.011. 1:. P. 32.00 3h.o ' 35.0 36.0 36.0 I. N. 38.10 39.10 39.110 110.90 39.10 R. 2.5. 26.00 13.10 7.22 8.02 7.22 807:0. OF mma'smn Q21) TIME IN nouns 0 #1 2 h 27. Wt. 12 .000 7.035 6.053 11.293 3.813 M. "P. 32.00 30.0 35.0 36.0 36.0 I. N. 38.10 39.00 39.00 39.130 38.50 a. M. 26.00 13.00 8.55 8.35 7.02 -20 RFSHLTS 08 ‘sIGF-‘STION BY IiJ-‘tfiT'ASIWG THE PERCENT OF EDIE-UM GLYCOCHOLATE TABLE III who. or 501mm 0130001201173 (0.2;) _ 2m: IN HOURS 0 1 2 h zh ‘1. 12.000 9.82}. 9.868 6.550 6.120 I. 8. 38.10 83.60 83.80 83.10 h2.50 R. 2:... 26.00 19.00 15.75 11.60 11.30 c0110. 0? 8.02118; GLYCOGMLATHOJQ 71:15: 18 HOURS 0 1 2 h 2!: we. 12.000 ‘ 8.272 8.103 6.511 6.081 I. .8. 38.10 111.60 111.110 39.10 38.60 a. 17. 26.00 16.75 13.110 9.35 9.00 0088. 0? SODIUN GLYCOCHOLATE (0.871.) .1 TIME IN HC'URS o 1 2 1. 28 7.1-. 12.000 7.806 7.1.171: 5.8219 5.803 I. N. 38.10 111.20 110.60 37.80 37.20 R. H. 26.00 111.15 12.60 9.00 8.85 0080. or 507713]! GIXCOUPOLAT}! (1.651 mm IN Home 0 1 2 1. 2|. 0:. 12.000 7.168 6.281. 5.1490 5.231 I. N. 38.10 39.90 10.50 37.30 37.00 R. M. 26.00 13.00 12.00 8.20 8.20 ~21- EIGVSTIQN RESULTS OF A F"W FATS TABLE IV corn: on. 71128 18 HOURS 0 1 h 27. 1:2. 12 .000 7.871: ' 5.030 1.176 I. N. - 126.50 126.50 118.10 R. v. - 0.98 0.95 1.1.9 pH of Digeet 5.8 5.8 6.0 6.0 01113011. 712138 {LU-5.13 0 1 1. 2h 11'. 12.000 7.037 5.509 1.008 I. 1:. - 80.01 86.50 87.50 8.. n. - 0.9!. 1.09 1.73 ph of tigeet 5.8 5.8 5.7 5.7 087550 mu: 1! HOURS 0 1 1. 2k 731'. 12 .000 5.6146 b .582 3.630 1. N. - 70.02 76.20 78.10 a. 8. - 1.25 0.95 0.76 pH of Digeet 6.0 5.9 5.7 5.7 BUTT‘P. 7123 118 30883 0 1 I. 21 82. 12.000 7.977 7.596 6.608 I. u. - 37.50 38.00 38.90 a. u. - 19.60 18.50 12.90 pH of Digeet 8.6 h.h h.h h.h ~22- DIGESTION RESULTS OF 1 ram FATS mu pn 8118711810 M 1.0 TABLE 7 com 011. mm: In HOURS 0 1 a 2!: 81‘. 12.000 7.2113 11.982 11.123 1. a. 128.00 126.00 125.00 116.50 a. n. 0.9!. 0.96 0.97 1.65 01-1?an mm D! nouns 0 1 h 21 81'. 12.000 6.616 8.213 3.871 I. N. 75.00 81.80 87.50 89.80 R. u. 0.71 0.95 1.58 1.92 _ _ CRISCO TIME IN 30053 0 1 h 2!. 70'. 12.000 5.201 1.192 3.172 I. N. 78.78 811.02 88.21 90.31: a. u. 1.02 0.92 0.8!. 0.72 81mm 7128 IN HOURS T 0 1 h 21 WT. 12.000 32.168 3.291 2.13h I. N. 35.70 38.10 38.90 39.50 a. H. 26.80 18.10 16.80 11.20 (1) (2) (3) (h) (5) (6) (7) (3) (9) (10) (11) (12) (13) (1h) (15) ~23- Franc-71:113.: heb‘illivray, J. H. - The Inactivation of Pancreatic Lipase by Heat. BiOChEMe Jo, Eli, 3?]. (19B). Flatt, B. 3., end Environ, F... R. - Factors Influencing the Action of Pancreatic Lipase. Eiochen. J. , 32, 860 (1925). bloyee, .‘E‘. 38., Siegnire, K., end ”8.112, K. 0. - Studies of Enzyme Action. Je Diflle Cheae. £2, 653 (1933)e Sabetke, 14., end Click, 0. - Lipelytic Pnzymee. J. Biol. Chem, 105, 221 (1931:). Bloor, '2. R. - Biochmeietry of the Petty Acids. (F'ninhold PUblishing C00, ”0' York, 19143.) Click, 0., end King, 0. 0. - Reletionehipe Betmn the Action of Pancreatic Lipeee end the Surface Mfeote of the Compounds Involved. J. Biol. Chem, if, 675 (1932). 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