LIBRARY HidxiganSu. U' . ABSTRACT THE EFFECT OF SHORT-TERM.FEEDING OF CATHODE RAY- AND W-IRRADIATED WHEAT UPON FAT DEPOSITION AI-ED EI‘SZYIE SYSTEMS IN THE RAT LIVER by NermaIMagnus Gilmore Ionizing radiation has been hailed as a most effective means of decreasing food storage losses through better insect control. Although small chemical changes are known to occur with radiation, foods so pre- served have yet to be shown detrimental to animals with regard to re- production, longevity, and growth. Wheat is one of the basic grains capable of long term.storage and irradiation could conceivably increase its storage ability. Any process which may affect the future utiliza- tion of such wheat needs investigation. This nutritional study was un- dertaken to further evaluate the effects of feeding four levels each of cathode rayb and gamma-irradiated wheat to experimental animals for 28 days. Growth measurements and protein efficiency ratios were examined. To investigate possible micro alterations which the metabolizing of an irradiated food may produce, liver enzyme systems xanthine oxidase and cytochrome oxidase were measured and histological sections were made of the livers. Total liver nitrogen and fat were also measured. There appeared to be no difference in the feed intakes and weight gains due to the type of irradiation, but there was indication Korma Kagnus Gilmore that the level of irradiation had some effect. The animals tended to grow at the same rate except for those on the highest level of irradia- tion who appeared to grow somewhat more slowly. The same trend persist- ed when the average weight gains per gram of diet eaten were calculated. Protein efficiency ratios were found to be similar in animals fed the lower levels of irradiation but again, the highest level of irradiation, regardless of the type incorporated into diets, resulted in lower pro- tein efficiency ratios. The activities of xanthine oxidase and cytochrome oxidase meas- ured in the livers of animals fed irradiated diets were consistently lower than those obtained from.the livers of control wheat-fed animals. The depression of activity of both enzyme systems was most pronounced in livers from gamma-irradiated wheat-fed animals. Histological examina- tion of fat deposition within the cell and lobule of the individual livers presented a picture of possible intensified dietary unbalance, especially in the game-irradiated wheat-fed animals. No significant alterations were found in the total liver nitrogen or fat determinations. The findings suggest that the type of radiation.may have more significant influence than.the level of irradiation. THE EFFECT OF SHORT-TERM FEEDING OF CATHGDE RAI- AND GAMM-IRRADIATED WHEAT UPON FAT DEPOSITION AND mm SYSTEIB IN THE RAT LIVER By Norma Magnus Gilmore ATHESIS Submitted to the School of Graduate Studies of Dfichigan State University of Agriculture and Applied Science in partial fulfillment of requirements for the degree of MASTER OF SCIENCE Department of Foods and Nutrition College of Home Economics 1960 16mm heathergretemuyaehesledgestheessistsneeendeeepm- ties gives her Wt this study by Dr. B. Elsi-s Rutherford. Marsalseexteadedtothestsffeumustedeetsinthe DWefPeedssnlletritien for their help, interest, shim- egueet. Speedsltheskssreuteededtenr.leisCs1heneeIm-.H. Biehsrdees for theirhelp in «fleeting the histological slides, end to the pm of Agriculture]. mm, mm sue. University, mmmmmmnm, Udvsrsityeflfimaln “hemismthefiest. ii TALLE OF CONTENTS INTRODUCTION .................................................... REVIEW OF LITERATURE ............................................ Ionizing Radiation ............................................ Radiation Effects upon Protein and Amino Acid Composition ..... Radiation Effects on Nutritive value of Foods ................. Wheat Protein ................................................. Irradiated “1103.1; PrOtein eeeeeeseeeeeeeseseeeeseeseeeeeeeeeeeee METHODS OF EVALUATING RADIATION DAIAGE TO NUTRITIVE VALUE OF FOODS ........................................................ Protein Quality ............................................... Enzymes and Liver Fat ......................................... iii-stelow 0.0.0.0000...00.0.0.0...0.0.0.0....0000000000.0...0.. EXPERIHENTAL PROCEDURE .....................o.................... RESULTS AND DISCUSSIONi.......................................... SUEEARY AED CONCLUSIONS ......................................... LITERATURE CITED ................................................ APPEI‘JDIXOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. iii 2h 2h 27 32 16 67 7O 77 ' LIST OF TABLES TABLE PAGE 1. compOSj-tion Of dj.et .00...OOOOOOOOOOOOO...OIOOOOOOCOOOOOOO. 1-d- 2. Average food intake and weight gain for four week periOd 0....OOOOOOOOOOOOOOOOOOOO00......OOOOOOOOOOOOOOOOO us 3. Average protein efficiency ratios in grams per gram of protein eaten for four'week period ...................... h9 h. Average of the liver nitrogen, xanthine oxidase, and cytochrome oxidase activities in the four week study .... 51 5. Comparison between averages of xanthine oxidase and per cent of liver fat in the four week and the ten week Stlldies eeeeeeeseeeeeeeeeeoeeeeeeeeeoeeeeeeeeeeseeeeeeeeee Sh iv LIST OF FIGURES FIGURE PAGE 1. Average weight gain in grams per gram of food eaten ...... 1L7 LIST OF ILATES PLATE I. Histological sections of liver from animals fed 12 per cent and 7 per cent wheat diets ................ Figure 1. Twelve per cent protein diet ............ Figure 2. Seven per cent protein diet ............. II. Histological sections of liver from animals fed gemma— and cathode ray— irradiated wheat diets ........ Figure l. Gamma-irradiated wheat, 9.3 x 106 rad ... Figure 2. Cathode ray-irradiated wheat, 2.8 x 10 rad eeeeeeeeeeeeseeeeeeeeeeeeeeeeeseeo III. Iflstological sections of liver from animals fed gamma- and cathode ray- irradiated wheat diets ........ Figure l. Gamma-irradiated Whed3, .93 x 106 rad ... Figure 2. Cathode ray-irradiated wheat, 9.3 x 10 rad eeeseeeeeeeeeeeeeseeeeeeeeeeeeeees vi PAGE 57 58 58 S9 61 62 62 LIST OF ESTEIODS - APPLIED IETIIOD l. LID..L‘CLOGICIAL .00...OOOOOOOOOOOOOOOOO00.00.0000...0.....00.... vii AGE 78 Xe LIST OF TABLES - APPENDIX .OOOOOOOOOOOOOOCOOOOO0.0.0.0000....0. Height gain in grams Feed intake in grams ..................................... Weight gain per gram of food eaten ....................... Protein efficiency ratios, expressed as weight gain in grams per gram of protein consumed .................. Percentage of nitrogen in liver (dry weight) ............. Percentage of fat in liver (dry weight ) ................. Liver xanthine or'dase activity, expressed as micro— moles of xanthine disappearing per hour per gram Of liver (I'Ie'b Ifeight) eeeeeeeeeeeeeeeseoeeeeeeeeeeeeoes. , . . . . -l Cytoenrome oxidase actiVity, eXpressed in seconds as first order velocity constant per gram of liver (VIC-b ~[4'63i3ht) eee'eeeeeeeeeeeeeeeeeeseeeeeeeOeeeeeeee Scoring of histological sections for fat and possible amino acid unbalance 000......0.0...OOOOOOOOOOOCOOOOOOC Data compiled for the 7 per cent wheat diet ............. PAGE 80 82 83 8h 85 86 INTRODUCTION Preservation of food has been an age-old problem for man and until recently has been limited more or less to the following uthods as given by Tanner ('51:): 1. Asepeis. 2. Addition of chemical substances: sodium benzoate, salt or sugar, or both, spices. 3. Low temperatures: refrigeration, slow-freezing, and quick freesiug. h. Hi9 temperatures: pasteurization, boiling, and We 5. Permutation: sauerkraut, pickles, an! fenented flCe 6. Abstraction of moisture: neats , vegetables , and {the fiththeadventofatedepererandthehugestookpilisgefatods netsproduets, great iatereethasbeengeneratediausingato-iousste products for feed preservation. Ionisilg radiation has been hailed as a not effective leans ef sterilisation, a means of deereasihg food lessee because of better insect control, a lease of “proving flavor and texture of nests, fruits, and vegetables siaoe no heat 1. required, a means of pending increased variety of food selestion and distribu- tion of perishables, and as a means of attending shelf-life. (Petunia, '56; m. '56). ' a .4 «m1. quality afforded by irndiatien, upon-11; withtheeefeodseapableoflengteresterege, istheeentrelofiaseet aaineldiafestatien. Thegreatest proble-iathe storage ofgrain is lineal growth whieh is the nJor source of respiration and heating. In tine, respiration and heating an lead to eh-ieal deterioration of the grain. fen, Miller, and Ward ('56) noted that when the noisture sentent of fleet was nintained at 121 or 201, can dosages of 250,000 and 625,000 rep, respectively, would elininste grain spoilage magi but below these levels nelds would proliferate. The effect of irradiatien upon heeterial. eouteflntion has been studied also, end rediesensitivity appears tevary free speodes to speeies. lunerous investigations have generally suggested the following net desirable levels of irradiation applicable to ee-sredal use: 12,000 rep, potatoes and onions will be inhibited from sprouting: 10,” to 20,000 rep, adult ineeets will he sterilised; 25,m0repwi11hereqeiredtokinwerns, fluhes,andnw inseete; m0,w0npdnbethepasteurisatienlevelatwhiohmst dens-organisnswillhehflled; 250,000 to 625,000 rep, grain will he preserved free fungal 0"“! and 2,000,000 rep the sterilisation level at whieh the nest resistant of bacteria will he killed.* In 1951., Lehnn and Lang nde the first state-Int eenoeroing ienisingrediatieninfeod preservationasviewedbytheroedandnrag Wration. The authors advocated a eonservative approach beeanse ef the inadequate nsens of testing safety. It was suggested that the nini-en dosage of cathede ray-and gem-irradiation neeeseary to effect *repz roentgen egivelent phyeieal. end is equivalent to the ah- eerptienef93ergsperunitgrenefdensity. I‘ {O es-reielly satisfactory sterilisation he deterninsd first; then, the nini-In dosage which would produce definite deterioration of a product. Andfiully, consterebetwoen thssotwo extremes, theninimndossge for pocossing food finish would be acceptable in texture, color, odor, can, taste, nntritive value, keeping quality, etc., not be detonined. u the prosonttine, 2.02106rsd**hasbeeneonsiderodthonoetlikely hulofienisingredietienetiehtdnbeuedec-rually, alleuing a safety tolerance above the 2 x 106 rep level established for steril- isation. but since there isnewwofhnovdnghowanirradiated product will he used at sens future date, deliberate enaggeraticn of dosage was suggested, also, as a noose of acquiring inferntien necessary for future use. Under the plan suggested, irrediation studies not detenine nutritional adequacy and possible tensity. Since this is difficult to do in the sane onperinsntel situation, nutritional adequacy has re- eeived nsre attention, with prinry emphasis upon longevity, repro- duction, and growth. lo significant adverse effects have been feud in those gross neesureuuts, but odor and flavor changes in irradiated foods have been widely noted. lslehy ('50) in using cathode rev-irradiated wheat in dosages free 5 x 10" to .l x 106 rep found the diets mtritienenyadoquato for growthandthoproteinresdilyavaflnhlotothorat atthose levels. Canon (’59) using both cathode rey- end ”radiated wheat varying “redisoquivalenttothosbsorptiencflmergsperunitgren ofdensity. mendsnitisoquelto93/100rop. ammoearm‘ to 9.3 x 106 red has suggested that neither the type of radiation nor the level of irradiation affects the value ofedeostprotoinoxoept atthoveryhiglestga-levol, andthat the effect of the two types of radiation appears to be slightly different. Cannon (’59) nintained rats for ten weeks on the various irred- uue um. 1. addition to growth neasurensnts, protein we, ens assessed using the enzyme systu, liver xanthine enclose. Per cent of fat was deternined in the livers, also. An interesting trend was noted in eni-ls consoling the cathode ray diets finish was not observed in the gene dicts. As the level of irradiation increased, there was an increaseinliverfetecoonpaniedbyasnlldsereasoinnnnthineoni- dase activity. Carroll ('60) working with threonine deficient casein am has run on. murmur. to be a “mum adaptation nsehanisn. Possibly the altered fat levels found in the rat livers as part of Canaan's ('59) study my point to a noro specific mesh-able side-effect of irradiated feedings. fishes and lob-n ('56) discussing the food additives situation declared that for all practical purposes, ionising radiation is a food additive and that the fate of this ingested food additive not be under- stood in nor-l as well as in subclinicsl plusical conditions. The safety probl. is nest euplen and difficult because the flavor and odor changes point to chedoal reactions within the food which in turn sug- gest nutrient changes with new and possibly tonic end-products. he authors pointed out that: Sole reliance on findings of orthodox toncological study of irradiated foods the-selves, in ignorance of both the identity end quantity of substances therein that any influence such findings, invites valid reservations to aw finel conclusions. ‘fhe desirable advantages of irradiation preservation of foods neeessitates oontimod research in this field. If the high levels of irradiationdeproduceohengesinthe chdcalconpounds offoodseo preservedwhiohmuldbetodotoanilals, thenthosenstbecarehny studied. And in turn, aninl findings net be evaluated with reference to the heterogenous nature of nan and every assurance given to nan that irradiated preservation ef foods will be beneficial to his. his Mwasundertakeninaaatteupt to furtherevaluate the effects of feeding catheda ray- and ”irradiated meat protein to enperinentalaninls. Itwasdesignedasanutritionaletudyandno attqthasbeen-detodeternine shuical ehengesinthe wheat. In addition te growth of the rat, the sore sensitive techniques of only-e syst. and histological evaluations of liver tissue have been used. WWW W Ionisationisanlocularchangoinnhcodbyanenergysourcothat increases the ch-ical reactivity within the noloculo. There are cor- taintyposofradiatien energytdxich-yblasoatrailthroughanterial. here are those Idlich are active agents th-solvos colliding with solo- cules, ad there are those that can cause the ejection of electrons fron atom of the ntorial. is a result of this process the ronaindor of theatenaequirosapooitive charge, aflapairof “ions“ hasboon fornodeutofanolectrieallyneutralaton. This-ybeoallodan'iou- pair" fornation, and is a fundsnental process which not be understood in considering the biological effects of any radiation. When a nlocule isienisod, itgonorsllybroaksupbutsiace the piocosareunablo teoscape, theyroactandre-roactuntilthonoststahleoodination can be found. (nuns-r, '57). Too 1mm source used, a. neohanisu of its offset, and the appliention it finds m be considered sono‘tat newbut the appearanse ofnowandhrgolyunhnonohodoal substances inapreductaronot. Thisisanoldproblenenoounterodwithallnothods of food preservation. there are two basis problens to consider mu choosing the energ source of ionisation for food preservation. One of those is the source itself, whether .shins or isotope, and the other is the type of radia- tiouwhiohnstbo suohthataomcloartransfomtionswilleocar. The sources of irradiation for food preservation are chiefly electron accelerating mchines and radioactive isotopes; the possible types of radiation are x-rays, game waves, cathode rays, and beta rays. The electron accelerating mchinos in oomon use are basically of four types: resonant transformrs, Van do Graaff, linear, and the Capacitron. All of thm operate on the principle of projecting arti- ficially produced electrons (co-only designated cathode rays) directly at a mterial as it passes through the field. (Evans, '55). he also- trcnaceeloratingscurcoaareadvantagoousbesausethaymybestoppad or started at will, do not require excessive precautionary masures, and are available at outputs sufficient for large scale processing. The isotopic scones my be of soverhl kinds. here are gs.- dttingradisaetive isotopes auchascebalt-éomiohmyboprodneod in ate-is reactors. There are fission isotopes such as (lesion-137 (gem-muting) mich my be separated fron the caminatiens of radio— active elamntaramindngaftartheumsoduraniuniarmovedfren apmtreactcrfuol. fherearethocanpletolyspanternoarlyecnplotoly apantfuelelutsavailablefronreactorsandstoredaswasteproducta untiltheycanboreturnadteprocessingplantafarrooovoryoffissian- able mterial. Probably the greatest advantage efisotopic sources is thatthayuncmstantlymitathommusandmaruesbutsinoo they are comtantly anitting, extra-e precautionary noasures mat be used to protect personnel. (Bremen and o'o-authors, '57). Thetmmeteamanfeimefradiantanargyoonaideradsuitablo for bodarflng feed mterial in food preservation are eathode rays produced by mohino sourees, and ge- waves anittad fron isotopic sources. hogs—wavosarefaundintheupporfnmnaies of the Win spectrul and consist of electric and mgnetic vibrations miehtnvelinstraightlinesthroughamterialbutperpendicularte one another. The term cathode ray and beta-irradiation are used inter- changably because they are ”anally identical although true beta par- tielos are adtted only by radioisotopes. The ability of those two sources of radiation to penetrate and therefore produce ionisation throughout a mterial varies. Goldblith and Proctor ('51.) clain the cathode ray distribution of energy within a mterial is nan-uniform and that with a 0.55 as per a2 thickness of mterial, the variation in dosage received fron the top to the botton ofthemtorialmyboas mohaswpercent. herofore, itishighly inpertant in using this source that this lack of unifonity be ninidsed as moh as possible; for instance, with wheat, only one layer thickness of grain kernel is passed under the electron generating been at a tine. The gan- mitting sources, however, penetrate deeply into a mterial giving a sore uniforn effect. This source itself has high activity and its location in relation to the mterial absorbing is ilportant. The energy introduced via cathode ray- and gmirradiation is thought to ionise a given mterial either directly or indirectly or through . embinatien of both. The biological action of irradiation, regardless of the approach, my have a fours-fold effect: a primry physical process, a secondary plvsical process, a primry biological process, an! a secondary biologieel process. The direct approach or the target theory as it is called suggests that energy is released directly in the nolecular structure at the noet sensitive area with the primry plusical reaction and the primry biological reaction taking place at the sane location within the mterial or cell. The extent of damgo will depend upon the degree of radicsensitivity within the mterial, with dryonic and i-aturo cells undergoing the not eaten- sive changes. Bacteria,mterials with low noisturo content, and fats are thought to be affected in this way. (Hunger, '57). Ileannder and co-authors (€60) have recently postulated that the m fiysical process is the biologically inportant stage in direct ionisation. After subjecting crystalline serun bovine albumin to anor- gies obtained fron a Van de Graaff generator, and subsequently staking solubility, Ivdrolysis, and ultracantrifugation results, the authors concluded that the first effect of a direct ”hit" in the protein was to alter the shape of the nolecule. Then with . mom ”hit,“ the nolecule appeared to opm out still further until one-half of the disulfide bonds were accessible. And fimlly, with a third “hit,“ the chdeal changes in the nolecule becano sinifieant with losses of carbonyl, anino, and disulfido groups. the indirect theory proposes that irradiation creates free radi- cals inthobcdyefthemterialwhichdiffucetothe surface ofthe nearest nolecule and than react. The primxy phaioal process my take placoinenoaroa, thenthe energydiffusionof eitheraplvsiealor ohenical nature brings about the biological changes in another area. The production of these free radicals in contrast to organic radicals depandeuponanaqueousnadiun, \nichiereadilyavailabloinfecd mterials. The primry msiml reaction has been described by O'lIaara ('52) as follows: . .. v . . . . , . . . l I . . . . . . . . . . . v . , . , . o . a . O 1 . ._ . . b7 . ‘ 1 ' I . . a 820 -9 H2014 e" 320+; 3“" + mi" H20 += e"-—-7 H + 03‘ These short-lived deco-position products of water initiate the production of long-lived radicals and peroxide-like derivatives mich in turn effect oxidatiens and reductions throughout the mterial, particularly anong the pretains andlanino acids which are so closely associated with water mlecules. Barron ('51.) has suggested that the ohior oxidising radicals moon by ionising'radiations are an, 302, and 3202, the 1120, having a negligible role, and the chief reducing radical is n“. no total effect will vary with the quantity of various radicals present, their active life, and the presence in the solution of others conpeting for then. If the mterial has a high misture content, it is nore apt to undergo irradiation damge, and the over-all chedcal effects have been feum to be reduced whm mterial is irradiated in the frosen state. (O'lbare. ‘52). ' I-rays,gannawaves,andcathoderaysareccmonlynoasurodin roentgen units, mnod z... Konrad Roentgen mo discovered in 1395 tho first of the artificially produced radiations, I-rays. The International Maia Recs-andations for Radiological units (1951.) defined the roentgen as the unit or dose of radiation “such that the associated corpuscular fission per 0.001293 gram of air produces, in air, ions carrying one electrostatic unit of (pentity of electricity of either one.“ In ionisation terns, one roentgen produces about two ionisations per cubic nicron of tissue. The "absorbing dose of any ionising radia- tion is the amunt of ensrg imarted to nutter by tho ionising particles per unit nos of irradiated naterial at the place of interest,“ and in the elder literature, the torn ”rep" (roentgen equivalent physical) was used, usually refering to the unit of energy absorption equal to 93 ergs per gran of tissue. (Swanson, '57). The official ten in use not is ”rut," and one red is equivalent to the absorption of 100 ergs per gran of time, so one ”red“ unit is equivalent to 0.93 rep. Eff t 0 Prote and A Go on It is generally agreed that the irradiation effect is not greater in individual, isolated eta-pounds than it is when the anino acids, sugars, an! fatty acids are within the franework of a whole food. The density of the naterial nay be great enough to slow down or nininize the ionis- ing action. This protection which is afforded by intact food nterials, my be of value in food preservation but individual results suggest that subtle irradiation effects can lend to declination, decarbonyla- tion, all polylsriution of proteins, carbolqdrates, an! fats, am one of which could be responsible for denage to nutritive value and palotahility. Barron and coworkers ('55) treated simle ulna acids with low levels of Lirradiation and m...“ thot tho initial reaction was one of oxidativs damnation. The authors postulated the following re- actions in the promos of the outlining radicals, 0H and 302: cazmzoom +03 ficmzoow + 302. mzoom + no, ~> Hc=m00m+3202 non if natal ions were present, H202 could react with amino acids, eventually giving 002, E3, aldehdes or acids with one less carbon aton. a-uia for-tion the found to be highest in acid and alkaline solu- tions indicating that pH influences the irradiation effect. Barren ('50.) and Dale and Davies ('51) have both than that thiolsand sulfur-containinganinoaeids are ch-ically altorodIhon irradiated in an... aqueous solutions. Anbe and oo-vorkors ('60) in irradiating tho anins acids cysteine, cystine, and nothionine, gluta- thions, andothor salfyhydrylprotoins foundonlytracos ofizso (body and co-anthors ('55) have suggested that tho notation of sulfur anino acids probably prevents and dininishos irradiation dongs by supplying electrons to danagod protein nolocules in a mterial. Dale (mo, m2) in lurking with oaay-o protein, carbozypeptidase and d-aflno acid oxidaso, found that the noro dilute the onsync concen- tration, the nsro inactivation Liz-radiation could produce. Glucose, fructose, and nucleic acids were found to be offectivo protective agents. to protein portion of the onsyns ns noro sensitive to irradiation than the phosthetic group. Lokon and coworkers ('59) observed that albunin, cystoanino, cysteine, glucose or sucrose would protect the only-s pepsin if one of then was in solution during irradiation. Food naterials have been used to study the effect of irradiation on adno acids. Proctor and Bhatia ('50) irradiated haddock fillets with 9 x 105, 2.7 x 106, 5.7 x 106 pop halo of oathoa rays aha observed no significant destruction of arginino, valine, histidino, louciao, lysine, nethioaino, phewlalanine, tryptophane, throoninc, or cystine. lysine, nethioninc, and tryptophano exhibited at tho hoot only 9.3 per 13 ‘l'soin and Johnson ('59s) prepared protein hydrolysatos of non- irradiated and irradiated beef and milk and studied tho changes in alias acid conposition after 2.3 x 106, 5.6 x 105, and 9.3 x 106 red gafi-irradiation. Both products were frozen prior to irradiation since this as. considered a desirable way to minimize the effect of free radical fonation in water. Stein-Moore colum chromtographic tech- niquo was hood for anins acid dotsrninations. it tho 2.3 x 106 red level, the following amino acids were found significantly reduced in milk: aspartic acid, serinc, nethionine, am isoloucino. it the 9.3 x lo‘ rad level; glutanic acid, glycine, leucinc, phowlalahiao, and histidino were found significantly reduced also. In the beef, the folloning order of anino acids were seriously reduced: glutanio acid, serinc, aspartie acid, throonino, glycine, lysine, nethionine, arginino, histidine, and proline. And in both the beef and the nilk, the greatest reduction was found in glutanic acid, aspartic acid, serinc, and glycine. Bf t t Value of 1' Various studies of irradiation effects in food natcrials have so far indicated no traces of radioactivity produced in the food. lleinke (' 51.) tested for radioactivity in 21; olsnsntal food constitu- ohto ordinarily found in goat and found no detectable radioactivity following low levels of irradiation. However, Itch higher onerg levels of irradiation need to be tested further before the possibility of in- duced radioactivity is comlctoly discounted. The threshold energy level for the production of induced radioactivity is thought to be about ten nillion electron volts. (Hannah, '56). ll. Irradiation studies have for the most part been directed toward the nutritive value and possible toxicity in foods. Richardson and Brock ('53) reported, that in general, tho data obtained in an 80-week feeding study with rats indicated the nutritive value of an irradiated diet was slightly less than that of a non-irradiated diet but the dif- ferences were so snall as to be of questionable significance. Four generations of rats were followed and fed a synthetic diet based on soybean protein irradiated with 2.79 x lo6 rad gm... Life spans of both the cultrol-fod and irradiation dict-fed aninals were essentially the sans; of the 80 females of each group who reproduced, the irradiated diet group produced 31.67 young and weaned 81.3! per cent of the 2993 allowed to rennin with then, while the control group produced 3290 young and weaned 90.0 per cent of the 2923 allowed to rennin with then. Fats and water are thought to be inortant targets for radiation dosage in food mterials. Since pork is a high lipid and high noisturo content food mterial, it has been considered an excellent mterial for etching possible radiation danage. Bubl and Butts ('60) fed gan- irradiatod pork, which had been stored at roon temperature for three to amt nonths prior to diet nizing, to rats through four generations. The pork was incorporated as 35 per cent of the dry weight of the diet. lo statistically significant differences were obtained in growth, brood- ing, and longevity in the four generations. Road and lraybdll ('58a) selected fourteen food itsns representa- tivo of different ch-icalsystsns and fed the. g m to rats for eight to twelvo weeks. The foods were stored frosen following ga-a- irradiation at 3 x 106 rep and 6 x 106 rep, prior to incorporation into 15 diets as 35 per cent dry weight. The following foods were investigated: ground beef, fresh hen, haddock fillets, boned turkey, sliced bacon, whole kernel corn, loaf spinach, sliced peeled boots, green snap beans, sliced peaches, whole strawberries, canned bread, military cereal bare, and viola powdered nilk. Decreased growth was found in animals fed diets containing 6 x 106 rep irradiated sliced peaches, which any indi- cate that repeated ingestion of low level radiation Ivy-products night establish a “toxic condition characterised by depressed growth." Sig- nificant weight gains were found anong the rats fed irradiated cereal bar and corn, and this was attributed to the breakdown of cellular walls Idling it possible for the aninale to utilise the foods better. Very little work has been done in investigating the nutritive value of wheat subjected to irradiation. Since irradiation of wheat suggests inprovod insect infestation control, a stow directed toward uncovering the effect of irradiation upon the nutritive value of idioat can be considered practical and desirable. W heat is a basic farn crop in the United States and a staple in the diet, and it affords an excellent notorial for irradiation studies. It oedines the advantages of studying direct and indirect effects of irradiation upon the onbryo and storage reserves as well as continued study of seedling growth and second generation properties. The unique constitution of its protein deem that it be understood before such use is ids, however. Since the beginning work of Osborne and Mendel, wheat has been know as an inadequate protein, not biologically couplets in its anins acid content. 16 Osborne and Mendel ( '12) , characterising the gliadin portion of wheat protein, ta carefully attracted glisdin which has been dried to afinopowdortorats forvaryingpariods of tine. The gliadinvnsin- corporated as 18 per cent of the diet with starch, sucrose, agar, salt lixturo, and lead selling up~ the balance. Long tern feeding trials with young an old aninaIs were done, and the older aninls varied in their ability to rennin on the diet without undue loss of weight fron 79 to 290 days. The young annals were found to ronin more or less at status quo, an indication that the glisdin was able to naintain the aui-l but not able to pro-eta growth. (aims at that tino was generally considered to be different fruaninslproteins because it containednonooralso avory snail aneunt of the anino acid lysine. Since growth response was so pronpt when even a lidted smut of aninal protein (which was thought to be high in lysine) was added to a gliadin am, Osborne and Mendel ('11.) undertook manna-.1 feeding trials using mm supplements. AnlB percontlevolofgliedinwasagainusodinthebaaaldietandreduced enlyenoughtoaddOJk paroontlysineintho supple-outed diet. The aninlswerofoundtorespondpro-ptlytothe additioneflysinewgrowo ing rapidly. than the lysine supple-ant was renoved, youth stopped and did not begin again until ”supplementation no started. The authors felt this gave conclusive evidence that lysine was indispensable for growth in the rat and apparatly the rat was not able to aynthosise lysine. with the w in nethods of deter-lining exact motitios of amino acids present in proteins throughout the years, the 3.1.0. 17 lutritional Studies No. 16 ( '5?) established a “reference protein“ as a guide for supplolelrting ma wheat diets. m. ”reference protein" established that the amino acids which will require supplementation in order to bring a basal wheat diet to a couplets protein basis will be lysine, try-poem, nethionine, isoleucine, valino, and throonine. lysine, the not linting anino acid, has received attention, since it can not undergo reversible daanination nor can the carbon skeleton of the d-anino acid be used for the l-forn synthesis, and all aninels studied need lysine as a conponent part of the foods being taken into the body. Lawrence and oc-workere ('58) carried out nierobiologieal assays for lysine on 236 samples of meet, representing durun, triticun, and hybrids. The various varieties had a dnimn of 2.1.6 per cent lysine to a naxinn of 3.8!. per cent per unit of protein, and the over-all average was 2.89 per cent for wheat of about 13.5 per cent or nere protein. The authors reported no signficant differences in lysine con- tent aneng the oping vdlaate, red swing vhoets, and winter \daeats, and no alterations due to year of growth or locatien enept wherein the total protein level was affected. Below the protein level of 13.5 per cent, however, an inverse relationship in lysine content was found. It appeared that nera lysine was available in the bran and gorn and there wasaleoahigherlysinacententpreoontintheendosperninlew—protoin enact. Perue and co-workare ( ' 50) also showed the gluten-lysine content of 17 different flours derived 5.. wheats of all the major types rang- ing fren 5.7 to no.2 per cent nitrogen to be consistently higher in the low-protein flour sanplos. Aninals fed with a diet containing null anount of lysine my not show outstanding symptom of a deficiency, and Harris and coworkers 01.3) havo suggested that for the young rat, lysine nay be required purely as a ”passive ntorial" for synthesis of proteins. Using an 18 per cent level of corn or wheat and starting with three week old rats, rats were fed for five, six, and seven weeks on supplennited and non- npplanmtsd diets. Those diets with the sullest amounts of lysine (controls) resulted in cessation of growth as. Oshawa and Mendel had shown and hypoprotoinaemia as well. It was felt that the blood picture obtained indicated not so much an anemia as a retarded developent of the has-stopoietic system. Radiological examinations showed decreased subcutaneous fat, waste of msclo, and reduction of bone calcification. Apparently the animal body in detecting the absence of lysine reduced growth of ones organs and transfered the available anino acids to organs with higher growth priorities so that status quo could be main- tained. , Since wheat is so inportant in the scenery and diet of the people of the United States and the world, any process applied to it which nay affect the liniting amino acid lysine needs further investi- gation. Barcroft ('39) in arranging foods into three groups basedupon the tine they could be preserved satisfactorily in storage for emergency purposes classified wheat among those that my be kept for ten years or longer. when it is considered that irradiation would greatly in- sroaso this projected ostints, aw nutritive value study utilising irradiated meat nest attsnpt to investigate aw affect on lysine. 19 W Irradiation in low doses does appear to affect the physical and ahenical properties of wheat in ways which alter its storage and baking qualities. Grains are solemnly thought to be rare affected by direct ionisation than by indirect since better quality wheat tends to have a law noisturo content. Conger and Randolph ('59) observed wheat seed embryos, before, and after irradiation and storage for free radical fonation. Ground that gene was used because the najority of the prinary nutrients of the seed are located in the snbryo and biological dangs is nest apt to soar there. Both x-rays and Cobalt-60 gem rays were used, varying fr- lxlo5to6xlo5 rcentgenunits, andneasuruentswere nads fron three trustee to 30 days following irradiation. The misture content was carefully controlled by liniting the dry gsrn to 1.5 per cent by weight and the wet gern to 8.5 per cent by weight. One hundred silli- gran samples were used to detect and neasurs electron resonances with each resonance prasuned to rswosent a free radical. The non-irradiated vddeat airyos whether wet or dry gave no detectable yield of free or- ganic radicals but the irradiated vdieat gorn gave a broad radical sig- nal, vary sinilar to those obtained frcn other irradiated proteins (Grady and co-workors, '55). ‘l‘wo to three tines as m radicals were .deteeted in the dry wheat gorn after irradiation as were found in the wet, epprexieeteiymnrediceie per-filigrancfdrywsight of gen irradiated dry in air per one-kilc-roentgen of Cobalt—60. Sinilar results were obtained whether the irradiation took place in air, or in an atnssphere of ongen or nitrogen. The authors crudely fractionated the wheat gern into oonponent parts in an effort to detereins in which area the largest share of radicals resided. 'lhe fats and oils gave no detectable signal even after doses 100 tines as large as that required for whole gern. The carboludrats fraction gave a slightly different signal, as yet morphined, but for equal dry weights and doses the water-soluble protein-molds acid fraction gave a signal averaging 2.8 tins as great as the dry whole gore. Although the post-irradiation radicals were not identified, it us found that radicals decayed rapidly within the first ten flutes and then progressively less rapidly as storage tins increased. It was suggested by the authors that the wide difference in radical, quan- tity noted between the dry and wet teat ger- night be due to the acre rapid decay of the radicals noted in the wet germ and that biological differences noted later in the seeds naybe due to this decay rate. Ion, Hilnsr, end Ward ( '56) in addition to storage studies found after using ga- radiaticn at cm, 2.5 x 10", ,6.25 x 105, 1.875 x 106. end 3.75 r 106 rep levels that gsrdnaticn could be clininstsd at 1.25 2105 rspifthsncisturswasnintainsdatm percent but 6.25:105 rep Ins necessary for annuating gar-inatien in a 12 per cent noisturs wheat. Pat-splitting did not occur with dosages as high as 3.75 x 10‘ rap but there as nrksd increase in fluorescence of the grain extracts after irradiation at high levels. This was an indication of the inter- action sf carbohydrate and protein and suggests gern damage. his two 310”“ dosages produced a snall but definite loss in protein solubil- ity as nsasursd by turbidity of saline extracts and this was found strongly intensified by storage, much greater than was found in the non—irradiated inset storage. In those instances in which the wheat was wettsd prior to irradiation, the wet grain was apparntly less resistant to all the above changes. mlner and Ian ('56) also studied the baking qualities of wheat gene-irradiated et eere, 1.25 x 10“, 2.5 x 10", 5 x 105, end 1 x 1.06 rep levels. In investigating the meat prior to filling, decreased gar-ination but no imsdiatc changes in protein solubility and fluorcssncs at these lswer levels was found. But the flour nillsd fro. the cans wheat showed a decreasing ability to hold water as irradiation levels increased, and drastic reduction in viscosity. 'l’nese findings agree with work reported by Bremen end co-authors ('55) in uhdeh bread flour was found to be norc starch-like, drier, and less able to bind nicturc then subjected to dosages of game-irradiation above 5 x 105 1'09- ‘ Alsup ('59) reported definite changes in the protein structure at dosages above 3 x 106 rep when crude gluten was extracted fren irradiated flours. Up to 1 x 106 rep. the would of emdo sluts: was increased indicating increasing water retention but after the 3 x 106 rep level yields were very mall. Electrophoretic analysis by the nov- ing boundary nethod Ins attempted in order to study changes in the pro- tein structure, end it was found thet above 1 x 10‘ rep, the six sep- arate protein component peaks noted in the nan-irradiated wheat controls gradually changed, until only two nain peaks were distinguishable at 1 x 107 rep. The total nitrogm levels remained the cans so one could suspect that there not have beat a change or alteration within the 22 structure of the protein itself. All of these investigators have re- ported eff-flavors and palatability changes in the irradiated wheat prsdncts. It is possible that the physical alterations in the wheat pro- tein nay be due to partial destruction of essential amino acids or the binding of then so they are no longer available to the animal. Tsien and Johnson 05%) observed changes in the amino acid content of line beans and garden peas due to irradiation. The authors used hydrolysatee of gem-irradiated and non-irradiated peas and beans and the Stein- lloorc chromatographic technique. Anino acid assays showed marked destruction of the lysine and arginine with destruction increasing with increasing irradiation. Control 2.8 x 106 rad 9.3 x 106 red lysine 10.Mi.05 9.21: .09 5.83: .07 Arginine 7.57: .01 6.05 t .08 A.23 if .06 Further work by Hstta and Johnson ('59) in studying the affect of gens-irradiation and heat sterilisation upon the nutritional value of con protein and wheat gluten indicated no changes in the lysine content of the corn or the wheat gluten. A high protein corn and con- nercial wheat gluten were used, suspended in water, frozen, than irradiated, 2.79 x 106 red gene for wheat and 2.79 and 9.3 x 106 red gala for the corn. The samples were dried, ground, and proxinate analysis redone prior to diet preparation, providing 10 per cent pro- tein, for rat growth studies. The aninals on irradiated wheat diets averaged nine grams of food intake per day, growing at a rate of 0.55 grans per day. Prodnats analysis showed that neither the lysine 23 content of the wheat or the corn was affected by heat processing or by the level of irradiation used, and the urinals accepted the diets readily. In outlining the general course to pursue in establishing the feasibility of ioni sing radiation for food preservation, Lehman and Lang ('59.) suggested that the amount of research would have to be sonsflat proportional to the scope of application in preservation. If there are purposes for which irradiation preservation is better suited than an other method of preservation, then more risk can be Jastifiably accepted. Heat processing has been criticised for its al- teration of physical and chemical properties of food and if irradia- tion sterilisation could alleviate this, be proven nutritionally adequate , and non-toxic , it could become a valuable preservation nethod. mums OF EVAIIJATING RADIATION DAME TO NUTRITIVE VALUE (1‘ F00) Prote t ‘lhs prinary aphasis in studies of the nutritional adequacy of irradiated food products has been directed toward reproduction, longev- ity, and growth. by food product when fed to an aninal not provide to the cells of the animal body protein adequate in amino acid content so that tissue nay be built, nintainsd, and repaired. Two nethods of assaying protein quality, nitrogen balance and protein efficiency ratio, have been used. Nitrogen balance, detenining the nitrogen in the food consuned and the nitrogen of the excreta under controlled conditions, is a quan- titative nsasure of the gain or loss of protein fron the animal body. If the sninal body is found to be in positive nitrogen balance or in a state of having sufficient protein for naintenance, repair, and growth, a norc exact neasure of the effect of protein quality upon growth is possible than is provided by silplc weight increase (Maynard and Lcosli, '56). lielchy (’58), feeding rats diets conposed of 11. per cent wheat protein and irradiated with cathode ray at 5 x 10“, l x 105, 5 z 105, and l x 106 rep levels, determined nitrogen balance. All the diets wercequallyacceptablstothcaninals; allaninelswere foundtobc in positive nitrogen balance, and no significance was found among the diets in utilising this ncasurc of protein quality. Cannon ('59) also 25 detcrline nitrogen balance of aninals nintained on cathode ray- and ga-irradiatsd theet diets varying in dosage fron 0.23 x 106 to 9.3 x 106 red. Only in results obtained fron feeding the galls-irradiated fleet diet of 9.3 x 106 rad was my decrease in nitrogen balance ob- served and found statistically significant. Apparently neither cathode m- rer ga-a-irradiation up to the level of 2.8 x 106 rad affects the ntritive value of wheat protein when neasured in this way. Another assure-at of protein quality is given ty neasuring protein efficiency ratios. Protein efficiency ratios in terns of gain amnigbt pcrgranof proteinornitrsgsn fed «press the growth- prenoting ability of a protein, and the greater the ratio,— the better the aninal cell is able to utilise the protein provided for necessary body functions. However, this nsasurcnsnt has its linitations since thsprotcincontsnt ofthe actualgaininbodyweightnybevariable. Protein efficiency ratio received its initial usefulness fron work of Osborne and Mendel he felt that prolonged growth was a good criterion for protein synthesis. In 1915, these authors reported attsnpts to establish the absolute protein intahc at which exact Iain- tsnsnos of be” weight could be found, the intake at which slight de- clinecouldbefound,andthsintaheatwhich slightgainosuldbs found. Gliadin ins anong the proteins used in the study but the results obtained were so variable that high value could not be placed upon thu. However, they did show that when gliadin was the source of proteinintherat diet therewasaslightdsclinsinwcightona? per cent protein level and a slight increase in weight with a 10.5 per ccntproteinlevcl,§Mfseding. Itwasnotsdthatinyonng 26 ani-ls the protein intake had to be decreased beyond the point where growth Just ceased since there appeared to be a considerable range below this at which naintenance was still. possible. The nxilln efficiency of the different proteins was found to vary with the type ef protein supplied by the diet. Barnes and coauthors (-1.5) in investigating the various nethods of studying protein quality suggested that the efficiencies of mind proteinsarehighestwhenfedatlowlevels suchatholZpercent, but that efficiencies of cereal proteins inprove as the level approaches 20 per cent of the diet. On the basis of protein efficiency, it would appear that a dietary level of protein thich can givenan efficiency ratio of 2.0 or greater would need to supply only 10 per cent of the diet. The authors suggested that no utter what experimental approach was used, paired-feeding «5M feeding, if the protein level of the diet was adjusted so that about 0.9 gram of protein could be retained each day, then the efficiency of the protein would be close to its nan-In. Recent research reported by Canpbell ('60) has attempted to clarify use of the variables associated with protein efficiency ratio deteninations. Using a basic diet furnishing 10 per cent protein and 10 per cent fat and correcting back to a casein reference standard, the author found that narked differences in protein efficiency ratios result fron differences in age of the ani-ls. The protein efficiency ratio derived fron casein decreased as the age and tine on the test increased. then casein ins oo-pared with plant protein sources, the differences noted in protein efficiency ratios were greatest during the early part 27 ef a four week emperinsntal period. When a 10 per cent level of either protein source was fed, the protein efficiency ratios were near nan-In values. The author concluded that if only one level of protein could be used in an expend-ental situation, then the 10 per cent level of protein would be satisfactory regardless of the type of protein, if all variables were carefully controlled. Before irradiated foods can be conercially feasible for hunn eonsunptien, work nust be done at the nicro level also. It is know: that only a shell percentage of the ecnpcunds in a food are i-edintely affected by irradiation (Goldblith and Proctor, '55). But often a tiny ansunt of an product or chemical reaction can bring about extensive biological and/or physiological changes. The final determination of suitability will have to rest with a large body of biological data nore extansive than afforded by growth and protein efficiency studies. m and gver Fat Lehman and long ('51.) suggested that enzyme systems which are such important unite of animal metabolisn should be studied as a means of exploring every possible facet of the metabolism of irradiated foods and their possible damage to the animal body. One of the most sensitive enzyme system for indicating quality of protein. being fed into a system is liver xanthine oxidase. Litwack and co-authors ('52; '53s) in studies of the liver xan- thine oxidase activity of rats fed casein, whole gliadin, and gliadin supplemented with various amino acids found that this enzyme system is especially sensitive to the amino acid availability in the dietary 28 proteins. Any measurement of its activity actually measures the forea- tion of that ensyne protein, and this has been found to correlate well with growth results without the added interference of homonal state, water intake, and balance, etc. When the gliadin was supplemented with tryptophane to bring it to the level present in casein, greater activ- ity was noted than with whole gliadin alone. When lysine and trypto- phano supplemts were added to gliadin, a still greater response as found Meeting that the lysine was the nest inportant factor lacking in the gliadin to stinulats the processes of growth as well as snsyne function. Bothwell and williene ('51.) studied the effects of a lysine-free ration upon the liver xanthine endsee activity in weanling rats fed either forcibly or 99. 13311.32. The rats were not started on the puri- fied anino acid retion until between 55 and 60 grams in weight and then naintained for ten to fourteen days. Rats force-fed the lysine defi- cient ration began to die earlier but no natter how the ration was fed, the lysine deficient diet resulted in a small but definite reduc- tion in liver nitrogen content and reduction of the liver xanthine oxi- dase activity to sonowhat ever one-half that of the controls. The lysine-free ration as found to give a conpletely different ensyne pic- ture in comparison to a histidine-fres ration which had no effect and a nothionino-froe ration Idiich completely depressed ensyne activity. Uhsre living tissues are concerned, radiant energy invariably produces injurious effects. Besides the mtations which nay occur, nitetie activity nay be depressed, the synthesis of DNA nay be slowed down, chi-ping and fragnentation of chronosones nay occur, as well as 29 various cellular changes such as vacuclisation, increased fatty changes, and cell death (Swanson, '57). The action of ionising radiation is thouylt to be endetive in nature and possibly directed at the oxida- tive activities of the cell. A study of the enzyme system directly involved in the electron transport could possible shed additional light on the adequacy of irradiated foods. Benditt and co-workers (Us?) fed a non-irradiated protein- deficient diet to rats and used cytochrons maidens activity as an in- dication of protein quality. The authors found that the cytochrene oxidase activity decreased progressively with tins and emit faster than the liver protein decreased. Cytochrome ozidase would provide a nsans of “Wing a dtochondrial ensyne systen of the cell while liver xanthine oxidase would provide an excellut neans of studying a soluble cytoplasnic ensyne systen. Read and coworkers ('58b) are the only investigators who have incluled evaluations of both "m systens in irradiated feed studies. The authors have reported that no changes were found in the xanthine oridase systen but that the cytochrome oxidase system increased in ac- tivity throughout a four generation study of rats. The diets used were conpoeites of nine frosen-stored 6 x 106 rep galls it-s: beef, pork, bacon, haddock, greenbeens, bests, peaches, powdered nilk, and nilitary cereal bar. Work has been done in the Feeds and nutrition Departnent, Inchi- gen State University, concerning the ability of an aninal systen to adapt to or adjust itself to the dietary protein fed. ' Carroll ('60) 30 worldng with 9 per cent casein diets deficient in threonine found the liver xanthine endase system depressed to a manila point in 19 days after which it began to recover. Along with this decrease, the per cent of liver fat increased, reaching its highest point five days after the xanthine oxidese nod-In depression. Then the fat began to nove out of the liver, and the fat returned to its initial level following the lead of the recovering xanthine eddase systems. In addition, to the xanthine oxideee systen, eytoohreee oxidase was studied. Cytochrone endase, however, was found to be depressed throughout the emeri- nsntal periods and did not tend to recover. Since cytochrone enidase is so intintely associated with the oxidative processes of the cells, an depression here would have wide effect in all the cell's activities. Canon ('59) in studying the nutritive value of cathode raw-- and gala-irradiated wheat fron are to 9.3 x 106 red levels found that as the per cent of liver fat on a dry weight basis increased the liver xanthine endase activity decreased. The progressive increase noted in the liver fat levels with increasing radiation levels was signifi- cant. This could suggest a possible alteratiu in the irradiated teat wish the aninal was capeble of detecting. The liver nitrogen levels in this work and that of Carroll ('60) showed no significant increase or decrease with the various diets used. Since the aninal liver is vital to the well-being of the aninl, rapid changes in its sise and conpesition my be due to the protein and/or glycogen being quickly added er withdravn. And either of these will carry with it a nltiple of its weidat in water. The anount of protein present in the average liver cell nay vary with the availability, 31 quantity of, and.the aldno acid conposition of the dietary protein. is the aninal body adjusts to the dietary intake, fat nay be called upon to nova also. In starvation, for instance, as the liver is de- pleted of its proteins, fat will nove quickly into the liver cells unp til the depots are emptye Fat infiltration of the liver under nunerous conditions my be thought of as an alteration in the nor-Bl processes governing the fat content of the liver. An excess of fat nay be due to increased fene- tion of fat, decreased oxidation of fat, increased flow of fat fron the depots, and inpaired removal fron the liver. And any one of these nay be the secondary result of a primary factor. These basic prinary factors have been catalogued‘by Popper and Sohaffner ('57). 1. Imbalance . ' A. Nutritional factors 1. Starvation 2. Low-protein diet: Methionine deficiency; cystine deficiency 3e High-fCt d1.t A. High carbohydrate diet 5. Lipotropio deficiency 6. Vitamin idnlance: thianine excess; biotin .13...e B. Hstabolic factors 1. Pituitary honones 2. Adrenal cortical hornones 3. Mid deficiency or encess h. Insulin deficiency 32 5. Sex hormones 6. Central nervous system influence 7. Obesity. 2. Toxic factors A. Chemical poisons: carbon tetrachloride; chloroform; phosphorus; trinitrotoluene. B. Bacterial toxins C. Anoxic factors; anemia; congestion 3. Codined factors A. Alcoholic fatty liver In studying this complicated interaction of dietary protein-, liver-, and fat netabolisn, growth periods my be of great help. During growth, the aninal bow is called upon to utilise everything at its sound to develop the uiimal. If there is any deficiency apparent in the dietary protein, the general metabolic stimulation created by the growth process can aggrevate the deficiency. Histology is another research tool which nay be used for studying the aggravation at the ce'Ilanar level. tin-2.1m There is such a wide variety of biological events which may be attributed to irradiation that no single response has been found to be unique for radiation damage (Putt , '53) . Likewise, the feeding of irradiated foods and their subsequent nstabolisn in the shin; cell night be suspected of as yet undetected effects. According to EJJJnger ('57) , there is disagreement on the effect irradiation has on liver tissue proper, probably due to the cyclic changes in cells following ,0 33 irradiation and the enormous repair power of the liver. But cellular and functional charges do occur as well as reactions to toxic sub- stances and an intimate examination of liver tissue such as histology affords could possibly indicate effects attributable to the feeding of irradiated foods. The liver is one of the largest organs in the animal body, and it is one of the most homogenous of organs regarding its primary cell type. The classic lobule of the liver is established by the location of central veins and portal areas. The portal areas are composed of bile ducts, lymphf channels, and blood vessels. They are the gateway for nutrients to the liver cells. The liver cells are thought to be no are than two cell thicknesses and are arranged in cords surrounded by continuous ducts called sinusoids. Host of the notabolic products of the cells are emptied into the sinusoids and gradually are pooled in the central vein areas from which they will find their way out of the liver and into the body as a whole. Using this concept of the liver lobule, there not be a large number of portal areas in compari- son to the central vein areas. This gives the classic concept of the liver lobule a characteristic hexagon appearance. (Copenhaver and ~ Johnson, '58). The average liver cell of the rat may contain one or two nuclei which nay have one, two, or four nucleoli in them. This can give the rat liver cell a high degree of DNA. Mitochondria are also prominent in the liver cell, and it is here that the oxidation-reduction activ- ities of the cell are thought to be carried out as well as fat metabo- lien. 3h In the normal liver, fat is found in the hepatic cells and the Kupffer cells in the for: of small droplets. This can be demonstrated by sensitive histological techniques. The rat liver, however, ordin- arily does not show fat deposition by any of the routine methods. Pepper and Schaffner ('57) have attempted to classify the various patterns of fat deposition which have become known through research studies, first, according to its position in the liver cell itself, and secondly, according to its lobular location. Cellular classification: 1. 2. 3. A. 5. Perisinnsoidal pattern. Fine droplets, like beads on a string, lie at the edge of the liver cell wall next to the sinusoids. Under abnormal conditions they grow in sise. Perinuclear pattern. Fat droplets will. be found the mcleus or located in the center of the hepatic cells. Diffuse snall droplet pattern. Fat droplets will be found surrounding the nucleus or located in the outer of the hepatic cells. Large droplet deposition. 3‘11 fat droplets will coalesce to fern one or two large droplets which become one huge droplet. It will replace the entire cytoplasm in tins and push the nucleus to the side. As this fat droplet continues to grow, it will break the cellular neabrane and two huge fat droplets will coalesce to form a fatty cyst, enabling greater fat storage. Diffuse fat. Grossly the liver will be enlarged, yellow, and even doughy in consistmcy. Every cell will contain excess fat, readily shown chemically, and sinusoids will be narrow. Since this class is actually an exaggeration of the central and peri- pheral foras, it is usually nutritional,toxic, or endochrins in origin. The classification on the lobular level is independent of the type of fat deposited within the cell itself except the perinuclear fat which is usually centrolobularly located. 35 1. Scattered fat. Isolated fat rich cells can be found throughout the normal liver lobule and will be seen in w “mute d1“, e 2. Controlobular fat. This occurs readily with choline deficiency. The fat rich cells will appear first and disappear last from the central vein area. The condi- tion will be noted on gross examination because the liver will show definite yellow areas. 3. Internediary fat. whenever the central cone area can no longer function, the intemdiary none will take over. It will be active in severe passive congestion and carbon tetrachloride poisoning. 5. Peripheral fat. This will be found nest co-only in toxemias and as result of other nisfuncticns such as altered protein notabolisn. Since the fat deposition of the liver can be correlated with various nutritional conditions and the type of protein being eaten, there is a wide body of histological literature which has accu-lated. larch work has been done using a 9 per cent casein as the protein source of the diet stuhing the variance of individla‘L anino acid levels and choline in relation to the fat picture. lino-Herrera and co-workers ('51.), varying the choline in the diets of male weanling rats on a 9 per cent casein diet, reported that histological findings could differ- entiate the choline deficient urinals fron the low protein plus choline diet eninals. The choline deficient aninals in this study showed dif- fuse fatty infiltraticn nost severe in the antral vein area. Whereas the low protein diet plus choline mowed fatty cells distributed in sense interspersed anong cones of noranl cells with only rare fat cells being found in the central vein area. The 9 per cent casein plus cholirn aninals had a per cut dry weight liver fat of 8.7 but the choline deficient aninals averaged 27.3 per cent. It was noted that there existed a close correlation between the extent of a fat deposit as ... 36 determine chancelly and the severity of the fat infiltration noted histologically. Phenylalanine, leucine, histidine, tryptophane, methionine, as well as threonino and lysine have been individually studied. Ada-stems ed Specter ('50) fed casein hydrolysates to show tryptophane influence upon fat deposition. The animals were on the diet for three weeks and then killed at approximately seven, fourteen, and twenty-one day inter- vals. A heavy accumlation of fat was found in the liver. During the first week the fat was primarily peripheral and periportsl with noderate sised droplets. The second week, larger droplets appeared, and within nine to fifteen days, an increasing amount of fat was found around the central vein area with eventual complete infiltration of the lobule. In addition, a change ems noted in the nucleus as the fat became more centrally located. Instead of retaining two to four small nucleoli, a single large nucleolus appeared in the nucleus. Dick and co-workers ('52) showed fatty liver developnent by feed- ing diets deficient in threonine or lysine. The rats were started on the respective diets at 2? to 28 days of age and fed for 28 to 63 days. Although the chemical fat levels determined were not high, a fatty change was noted on histological examination. The threonine deficient diet aninl livers showed small to medium sized fat droplets which distended the cytOplasm. Wherever there were fat rich cells, they compressed the sinusoids although the nuclei were usually still centrally located in the cells. One large nucleolus was prominent in the nucleus rather than the usual two to four mller ones. The majority of the livers showed fat around the central vein area. When lysine deficient diets were fed, the histological exandnation revealed a much slower develop- 37 lent of similar fatty movement into the cells, and the nuclear changes were less prominent. Another report on the suboptimal intake of lysine and threonine reported by Singel and co-authors ('53) showed that no large excess of fat would develop on diets completely deficient in threonine or lysine. But if graded levels of each were fed, fatty livers did develop. It was felt that in order to overcome the requirements of growth, the diets Inst be adequate for at least 80 per cent of the optimal growth rate. Plant protein diets have been studied in relation to their effect upon fat deposit in the liver. Shils and eo-aworkers ('51.) showed that a portal type of fatty liver can be rapidly and consis- tently produced in weanling rats by feeding diets in which protein source as eorn, rice, wheat or cassava. In one week on the corn ual diet excess fat appeared in the portal region of the liver lobule and dur- ing the twelve weeks of the study, Just kept piling up. Even with very high fat levels, the portal areas were nest seriously involved. When an unenriched vheat flour diet was fed, adjusted to 7.8 per cent pro- tein level, the same number of portal placements as diffuse place-ants was found. mes. diet aninals were killed after 21 to 28 days on the diet and although the fat levels were not as high as others fron a chen- ieal standpoint, it was felt there was enough evidence to indicate that the fat weuld Just keep piling up on this diet also. In work reported by Vennart and ee-mthors ('58) the addition of tryptophane and lysine to a diet based solely on corn reversed the portal fatty livers observed. The aninsls on the supplenented diets . v D O . . 0 ‘ ‘ \ , . I . ‘ I A " e ‘ . , . ‘ ' . , Q .. , . - ' e ‘ o - I ' - v, - . . e ‘ ' . ' O a e . u , a > e “‘v - e '- ‘ A s - O ‘ . . . . A . , o v . ‘ ' I , , ‘ . . .- . _ , ' ' ‘ ‘ q ‘ 38 showed nor-a1 or near normal levels of fat when killed at 28 days, but the control group showed portal fat with small droplets visable. These studies point to a readily discernable picture of fat with- in the cells and lobules of the liver. When choline is deficient in the diet, a central displacement of the fat in the lobule my be found. With low protein levels or diets based on low biological value proteins or with amino acid deficient diets, a portal type of fat can be observed in the lobule. Now how do these patterns change when the protein of the diet has been subjected to ionizing radiation; There have been few studies involving the histological differ- entiation of fat in liver tissue derived fron aninals fed irradiated diets. The naJority of the work has been directed toward the detection of nalignsnt and benign conditions which are found in aninals sub- Jected to external radiation. Polling and co-authors ('55) conducted a long term extensive study feeding albino rats a diet composed of raw ground beef irradiated with 2 x 106 rep cathode ray. The feeding continued through three genera- tions with a total ef 2685 aninale. The nest was equivalent to about 1.5 per cent of the diet solids and results regarding growth, adult sise, efficiency of food utilisation, reproduction, hcatology, sur- vival, pathology, and neoplasms were reported. Host of the histologi- calworkwasdirectedtowardthe presence ofnsJignantandbenign neoplasm of which the authors found equal distribution betweu the experimtal and control groups. Histologically, nest of the tissues maindwere nor-l, W117? percent inboth groups. 0f the slight to quite severe hietologieal deviations noted, there were 18 classes according to nature and location. The nest frequent was 39 henosiderosis ef the spleen found in nearly all adult aninals, both control and experinental, which is of obscure significance. The fifth most frequent occurence was fatty livers but here again there was equal distribution between control and eXperinental groups. Nearly all the histological abnornalities observed in both groups were those of heter- ogenous, spontaneous changes which one would upset with aging rats. Growing chicks have also been used for irradiation studies. Waite leghorn chickens were raised and maintained for thirteen nonths on a wet-lash diet irradiated with 3 x 1.06 rep genre. The proper Ralston-Purina Conpaw diets for each growth level were used but con- plete vitamin nix was added to each one. Bums and co-werkers ('56) reported the study and gross and hietologieal findings were observed. he pathology of the birds showed no obvious abnonalities as being attributable to one group or another. Extensive fat infiltration in the livers of sons of the aninls fed irradiated diets was noted, however. (be other stw, Ousterhout ('60) has been reported with rats naintained through three generations one diet conposed of 1.68 x lO6 rep ga-a—irradiated butter-fat , along with skin milk powder, ground isle that, salt, and vitanin A and D supplements. Extensive pathology was done but not reported in detail. All changes found were essen- tially the sane in the control and «perinatal animals with equal fre- meney in both groups. No evidence of nalignant tuners was found. Although histological work has been done using varims plant protein sources, none have been published in which irradiated wheat was the print-y dietary source. LO Cannon ('59) suggested a possible adaptive «chanisn oh the pert of the rat in the conplicated picture of irradiated protein, liver, nay-e, and fat deposition. Although experimental fatty infiltration had not given any convincing indication of significant inpairnent of liver function, this does not rule out the possibility of damage yet undetected. It is known that the liver is nore susceptible to injury when fatty, that detoxification of injurious substances nay be faulty, and that the liver nay be nore sensitive to toxins. If the picture previously reported was an adaptive mechanism, possibly the initial point of adaption could be detected with a shorter grewth and feeding trial. If there were alterations in the irradiated protein nolecule, possibly the aninal liver cell could detect these enough to bring about changes in the ensyns systens. And if fat is being noved into the liver cells as a secondary result of protein al- teration, histological sections could give indications as to its nature and newer. This study was undertaken to deternine the effect of 28 days of feeding cathode~cray- and galls-irradiated wheat, at levels of 0.28 x 106, 0.93 x 106, 2.8 x 106, 9.3 x lo6 rad, upon the liver xanthine oxidase and cytochrome oxidase systems, and the deposition of fat in the rat liver. mu PROCEDURE 1 15 per cent protein wheat obtained from the Agricultural College of Michigan State University was used in this study. It was divided into nine lots, one of thich was not eXposed to radiation and served as a control for the entire study. Of the remaining eight lots, four were exposed to cathode ray-radiation fron a high energy electron l bean generator and the other four were exposed to gains-radiation Citted by Cobalt-602. The four levels of irradiation used with each type were 0.281: 106 rad, 0.93 2:2106 rad, 2.8 x 106 red, and 9.3 x 106 red. Each of the nine samples of wheat was ground and incorporated into diets to provide 12 per cent protein. The composition of each diet was as follows: TABLE I Composition of diet % Wheat 75 Corn oil ' 5 Mineral Mix” A Vitadn W 2 Sucrose 1h * wesson's Salts were obtained fron the Nutritional Biochemicals Corpor- ation of Cleveland, Ohio. *1? The vita-in nix provided the following per kilogren of diet: 1 mgn of thinnin hydrochloride, 2.1» Isl of riboflavin, 1 age: of pyridoxine hydrochloride, 3000 LU. of vitamin D, and 298 LU. of vitamin A. J‘Dept. of Agricultural hgineering, Michigan State University, East lensing, Michigan. 2Phoenix laboratory, University of Michigan, Ann Arbor, Michigan. 1.2 Male weanling albino rats weighing between 1.5 and 55 grams were allotted at random to the experimental diets so that each diet was fed to four rats. The same procedure was followed during a second eXperi- nental period two months later. A third group of four animals, a supple- nentary control for the histological part of the study, was fed a non- irradiated wheat diet whose protein content was 7 per cent. The urinals were placed at randon into individual wire-bottomed cages and food and water were given ’3 gym- for a four week period. Records of weekly weight changes and daily records of food consumption were kept. On the 25th, 26th, 27th, and 28th days of each feeding period, one rat fron each diet group was sacrificed by decapitation. The animals of each diet were sacrificed at varying times within the four days in order to minimize this source of bias as much as possible. The four animals of the low protein diet group were all sacrificed on the 27th day of that period. After the aninals were decapitated, livers were removed and xanthine oxidase and cytochrome oxidase activities were determined the same day. One lobe of each liver was placed in 10 per cent formaldehyde for later histological emanation, and the remainder of the liver was frosen and stored for subsequent. nitrogen and m. determinations. Ianthine oddase activity, eXpressed as nicronoles of xanthine disappearing in one hour per unit weight of liver, was determined by the colorinetric assay of Litwack ani co-workers ('53b). One nodifi- eatien (increased observation tine) suggested by the procedure was utilised in this stub in order to minimise the variability introchiced by the i-aturity of the aninals. Although a long induction period h3 we noted in nearly all aninals, first order reaction rates were even- tuallly obtained. The cytochrome oxidase activity was determined spec- trophotometrically as described by Smith ('55) using a reduced cyto- ‘ ohrc-e oxidase solution and was expressed as a first order velocity constant 1- seconds-4' per unit weight of liver. Difficulty was found in keeping the cytochrome endase solution sufficiently reduced for deter-instien of an entire day's samples. mintaining the solution in a frosen state and re-reducing it mid-way through each day yielded sonewhat more stability, however. During the reduction process, the nitrogen was bubbled through a solution of potassiu- wrogallste as an eddit ional precaution against oxidation. The dried livers were «ahead for total nitrogen by the boric- acid modification of the (Jeldahl—Gmming method, (11.0.1.0. , '57), fat was deteradned by other extraction in the Goldfisch apparatus, and misture was detemined by difference after oven-drying. Since the liver weights were snll in all the experinental groups and large anounts . of wet weight were needed for the ensyne studies, it was necessary to do the other attractions on extremely snll events of dried liver. Therefore, it was deened advisable to use a histological techniqae which could give an inti-te picture of as within the «11 with regard to distribution and quantity. The liver lobe placed in 10 per cent fonsldehyde at the tine of sacrifice was allowed to rn'uutn. .11 the «perinatal groups were conpleted. Sections were prepared using the carbon dioxide freezing technique with some mdifications. (See Appendix for nethod). M The data was evaluated by analysis of variance and by the T-test (Snedecor, '56) using the average of the irradiated diets as a unit against that of the control diet. 1' - egg!“ of thg cgtrol - average 2f the mom gets Standard deviation of the difference (derived from the remainder term of the analysis of variance) Use of the T-test was considered because there'was a consistent trend of depression noted in the xanthine oxidase and cytochroee oxidase neasurelents obtained free these animals fed the game-irradiated diets. The additional analysis was incorporated as a neans of detaining a possible significance of this trend. (See Appendix, Tables vii and viii) . RESULTS AND DISCUSSION Osborncand Mendel ('15) pointed out that when the protein level of the diet is low an animal may try to eat more liberally in attenuat- ing to make-up the percentage deficiency of the ration by increased intake. The protein levels of all diets in this study varied from 11.8 per cent to 12.2 per cent which suggests little difference for practical considerations. The animals accepted the diets readily and at the time they were sacrificed had doubled their initial weight. No animals were lost during the study. The weather was extremely hot in the first experimental period, and although the animals were maintaied in an air conditioned laboratory throughout, additional stress was undoubtedly placed upon them by some variation in temperatures. Food intake records were kept on all animals during each experimental period. Food intake of animals receiving the irradiated diets tended to be greater than that of animals on the control wheat diets. (Table 2). TABLE 2 Average food intake and weight gain for four week period ( Control Cathode m Gena ation Feed Weight Feed Weight eed Weight ~ I: r 1.: L. in - , :-i « .tc- ' '(, inn-n. ~. lone 191 gm 30 gm 0.28::106 2039! 31:91 2139:: 385: 0.93 x 105 205 35 205 36 2.3 x m6 212 37 210 37 2,1; 106 201 32 202 3: m 191 30 202 21!. 5 208 26 L6 There appeared to be no difference in the feed intakes and weight gains due to the type of radiation. However, there is some indication that the level of irradiation had some effect. The animals tersied to grow at the same rate except for those on the highest level of irradia- tion who appeared to grow sonewhat more slowly. The three lowest levels of irradiation neasurenents tended to be similar and greater than the control diet measurements. But the animals fed the highest level of irradiation, both cathode ray and gamna, showed weight gain averages more closely aligned with those found for aninals on the control diets. Cannon ('59) noted this sane trend in the growth neasuremnts of that study is which the animals were fed irradiated wheat diets containing 15.2 per cent protein and growth curves showed sinilar growth rates in animals fed diets made from wheat irradiated to the level of 2.8 x 106 red. However, at the highest level of both cathode ray- and gem-irradiation, 9.3 x 106 red, a slower growth rate was noted. The author suggested that the animals on the highest level of irradiation night possibly have reached the sane level of growth as the other aninals if the study had not tensinated at ten weeks. Since the same pattern of growth was found in this study when animals were under in- tensive growth stress, it may nore logically suggest an alteration in the protein which the animl was not able to utilise as well. As Reed and Krsybill ('58) suggested, it is possible that irradiation broke down the cellular walls of the wheat so that the animls found the wheat easier to eat and accept. However, at the higher level some other undeterdned factors may have opposed this beneficial effect. h? 0.0 Batu 80L .50 2‘80 .011 so: ZQPSQCE (go 90.0 8.0 :2. 52»: 33.9.4 .._ mane; «01¢ o0: 20533. (PHI #6 Cu . . .. _ . o £00.. #3.. l d... ids... 000.! do «was 8“ "IV. MIDI“ wuss L8 The average weight gains per gram of diet eaten (figure 1) were calculated also in order to determine whether the trend noted he. the growth results would be altered when weight gains were corrected for feed intake. The trend persisted, appearing to be inversely related to the level of irradiation rather than to the type of radiation, as the aninls fed the highest level of irradiation, both cathode ray and gene, showed weight gain averages similar to those found for the animls on the control diets. The results were subjected to analysis of variance but were not found statistically significant. (See Appendix, Table iii). Although weight gains and feed intakes do give a relative unsure of growth and in turn indicate the protein quality of the feed, another measurement is that of protein efficiency ratio. These ratios in terns of gain in body weidit per gram of protein or nitro- gen fed, express the growth-pronoting ability of a protein, and the greater the ratio, the better the aninal cell is able to utilise the protein provided for necessary body functions. The protein efficiencies of the various irradiated diets used in this study were calculated and expressed as the weight gain per gram of protein eaten during the four week tudy. (Table 3) . Very little difference was found in the average efficiency ratios obtained among the control and the irradiated diets. This suggests that the type of radiation had no measurable effect upon the nutritious). quality of the protein. 1»9 man: 3 Average protein efficiency ratios in grams per gram of protein eaten for four week period Radiation ) Cosine]..— CW £m_.t____ None 1.39 0.28 x 106 1.1.1 1&8 0.93 x 106 h 1.39 1.1.1. 2.3 x 10‘ 1.1.5 1.1.5 9.3 x 106 1.32 1.36 Average 1.39 1.39 1.1.3 ltandard error 2f m 0,1,2 These results compare favorably with those reported by Cannon ('59) in the ten week study. The results obtained in this study aver- age slightly higher, in agreement with the tine variable suggested by findings of Campbell ('60). Since the protein value of the diets was 12 per cent and a level of 5 per cent fat was used, and the ratios were obtained during a four week study, the variables suggested by Campbell as mst desirable for obtaining reliable protein efficiency ratios were apparently adequately controlled in this study. The ratios compare favorably with those obtained by Hove and co-workers ('hS) who in assay- ing protein quality fed a 10 per cent level of protein supplied by con- nercially blended hard spring wheat, milled and unnilled, to 35 to 1.5 gran weanling rats for six weeks. The authors found that the protein efficiency values ranged from 0.85 for patent flour to 2.86 for wheat gerl. The protein efficiency ratio of whole wheat was 1.1.0. ...»—_ 50 It should be noted in reviewing Table 3 that the same trend is observed with regard to the level of irradiation as was suggested in the results of weight gain per gram of food eaten. The lower levels of irradiation when incorporated in diets and fed to animals have re- sulted in sinilar protein efficiency ratios. The highest level of ir- radiation, 9.3 x 106 red, whether cathode ray or game, when incorpor- ated in diets and fed to animals, has resulted in apparently lower protein efficiency ratios. However, the results were not found statis- tically significant. Although growth and protein efficiency ratios may give a rela- tive picture of the quality of irradiated foods fed to animals, it was considered advisable to investigate more sensitive neaeurenents of dietary quality. Thus, liver xanthine caddase, liver cytochrome oxidase aotivifigwere determined and the results are presented in Table It. The quantities of these components measured in the livers of aninls fed irradiated diets were consistently lower than those ob- tained from the livers of the control wheat-fed animals, and the depression was most pronounced in those livers from gums-irradiated wheat-fed animals. The depression was observed in the activities of both the liver xanthine oxidase and the cytochrome oxidase enzyme system. The level of irradiation appeared to have little effect upon the activities measured. Cannon ('59) assayed only liver xanthine oxidase. The ten week study suggested depression of the liver xanthine oxidase activity also, but the slight depression noted was with both types of' radiation and varied inversely with the level of irradiation. The results of this study do not agree with those reported by Read and Kraybill ('58) who Sl L.~..~ «A $.86 sees no segue sundown» 8:: n.“ “80.0 ”8...de «m5 8.? En m... .886 .386 eases: +3.0.” .32.: 06 , mi «30.0 080.0 00a N «.0 when.” $33..” n.n «.4 0.80.0 38.0 00H N a.“ 8.3 «E3 a...” .3 38.0 386 .03 w 86 mas—H 3.9” m.m 4.4 v «80.0 «90.0 00H N 3.0 assoc hen one no «snow evonoso «anew hwmwueonaeo can e coon c333 nemmmeumwxu 4mmnmfl4mflnmmmqammmmmo LHMNdflJHHMfiw in eunooeeeeeeofixo eaonnoophw, evade. .eeeefiwo enananmm. somehow: no>dq Has 83. see: .88 e5 5 83:33 eeeoflno eaonnoonho and .3358 cannon anemone? no.5...” on» no emshebd 4 Hum: ‘IU -e. 52 found the liver xanthine oxidase activity to be unchanged and the cyto- chrons oxidase activity increased upon feeding game-irradiated diets to rats for four generations. The levels of irradiation reported, however, were not so high as those reported here. Pirie ('56) has suggested that the effect of irradiation upon living cells may be to disturb the internal barrier of the cell so that enzymes may move into sites where they are normally excluded. Thus the immediate activity night appear to increase but as the enzyme was influenced by the unnatural surrounding nedia, its activity would decrease. Whether this is also possible when cells are notabolising irradiated foods rennin to be seen. The den-ession of activity suggested by the results of this study were not found statistically significant then subjected to analysis of variance, and therefore, the T-test was utilised in an effort to eonpare the over-all effect of feeding cathode ray- and game-irradiated meat diets with that found in the non-irradiated wheat diet-fed aninals. (See Appendix, Tables vii and viii). Significance was found by the T-test between the control and the average obtained fren the gan-irradiated wheat-fed animals for both systems, but no significance was found for either ensyne system between the control and the average obtained from animals fed cathode ray-irradiated wheat diets. Although the T-test can only be considered a supplementary appraisal of the trem noted, it was interesting to observe that the average obtained fre- asssy of the livers of animals fed gonna-irradiated wheat diets was responsible for the significance found in the T-test in both enzyme ”We 53 Table 1; also contains the average liver nitrogen measurements which were not found significantly different by either statistical procedure. (See Appendix, Table v). Miller ('h8) and Younathan and oo-authors ('56) have both suggested that when the organism is sub- Jected to an unusual physiological condition, such as protein de- ficiency, that it may tend to first sacrifice those enzymes which will noble the organism to conserve its metabolic econony under the stated conditions. Since analysis of variance showed no significance in the results obtained in either enzyme systen, and only questionable signif- icance nay be attached to the trend observed until additional data is obtained from further studies, possibly the conservation of metabolic econow noted here is one of depressed activity rather than reduction of actual encyne protein. The growth, protein efficiency ratios, and measurements obtained fron the enzyne studies of both this stub ‘deannon's ('59) seen to suggest that there is a difference in wheat quality due to the type of radiation and the level of irradiation used. Variability of results, however, possibly due to the maturity of the animals has node the trans noted difficult to prove statistically. Amlysis. of variance of the liver fat data showed no signifi- cause. The liver fat levels (Table 5) obtaimd were not high in either the aninals on the control or the irradiated meat diets, and they corresponded to those for normal rats reported by Elvehjen ('56). This authorusedweanlingratsnintainedonadiet composed of88percent inset supplying 9.1 per cent protein and after two weeks on the diet, the liver fat was found to be 12 per cent. The fat content of the livers .1 5h 0w an N .NH Hmdm New H2930 Immfi no.0 mac." 3.9” 00$ .8.” Nun m3 encased ~06 mmé «.3 «A. ou.o Sé ca m3 «3 w m.m 3.0 $6 4.9” H5." «.10 00.0.” n.n «.4 o3 N m.~ mod 3% dm 0.3 23 86 5n 4.4 no." N $6 an «.4 H25” $22“ .36 3.0 o.n 4.4 03 H 3.0 Eco. Hen _ infidel: Hep oregano 5.. has 335 on e358 use o deco avocado evonpeo ~333on harem .35." 5E . . and. .3st 3% u... 1 Im\psu M sodas qcanons agenda am\asu m codes .3353 sagas as x02» pooh seduces ass: so» 05 one goes noon on... 5 new used.” no «use hen use sedans e553 no semsuebo cosh-en gauge—on n “Han. If I an. a I s I a. .e n .I. I . a‘|l. .I‘ 55 in this study averaged between 8.12 per cent and 10.09 per cent (Stan- dard error of the mean, 2.8). The high average noted in the 9.3 x 106 rad cathode my diet animals was due to one animal in this diet group who showed 20.79 Par cent liver fat. The high percentage found upon ether extraction was substantiated upon examination of the histological sections of this liver. (See Appendix, Tables vi and 1):). In comparing the liver fat obtained in this four week study with that found by Cannon ('59) in which progressively increasing liver fat levels varied directly with increasing irradiation levels, it should be noted that the results reported here compare with the highest level reported by Cannon. Also, the relationship between the liver xanthine oxidase activity and the per cent of liver fat observed by Cannon was not found in this study. Although the data obtained does not bear out the type of adapta- tion to an amino acid imbalance reported by Carroll ('60) and suggested by Cannon ('59) as a possible factor in that study, adaptation still can not be completely ruled out on the basis of the present findings. It is possible that the adaptation is of another nature. In the involved picture of dietary protein, the liver and fat netabolisn, the accumula- tion of fat tends to follow the removal of protein from the liver cell. It is possible that irradiation of the wheat slightly altered the protein structure and in turn altered the netsbolisn of it by the liver cell. Whereas the aninls upon the control diet were able to adjust to the limitations of wheat protein, it is suggested that the aninls on the irradiated wheat protein diets were not able to do this as readily. It would be advisable to undertake a long tern study, the nature of which could attempt to determine at regular intervals over 56 a long period of tine, the various changes noted in these two studies. Not until adaptation of some nature is clearly ruled out can it be taken from the realm of possibility. It was hoped in using the 12 per cent level of dietary protein that the wheat protein and its limiting amino acid lysine would be sufficiently borderline so that am damage to lysine induced by the irradiation would be detected by the animals. The histological study was undertaken to see if the aninl was able to detect am difference in the protein which would result in the indirect effect of altered fat deposit within the cell and lobule of the liver itself. In reading the histological slides, some point of reference had to be established. Two were chosen from non-irradiated wheat diets, both representative of the average, one caning fron the 12 per cent protein diet and the other from the 7 per cent protein diet. Each of the individual animal tissues was compared with both of these in regard to the pattern of fat deposition. In addition, each of the animal tissues was compared with the 12 per cent wheat control animal tissue for the amount of fat present, and this was reported as one, two, three, and four plus. (See Appendix, Table 1:). The 12 per cent con- trol was ranked as sens. In general, the individual histological estimate of amount of fat agreed with the ether extractions. In comparing the fat deposition of each against the 12 per cent and 7 per cent control wheat-fed animal tissues, a picture of a possible intensified dietary unbalance, partic- ularly in the galls-irradiated wheat diet aninls evolved. Photonicrographe of representatiee tissues are presented in fletea 1.5 :II: and III- PLATE I Histological sections of liver from animals fed 12 per cent and 7 per cent wheat diets. Oil Rod 0 Fat stain Figure 1. Twelve per cent protein diet The tissue exhibited predominately portal deposition of the fat. Only fine drOplets of fat were observed and these tended to be located at the ends of the portal areas. Kagnification, 1000 X Figure 2. Seven per cent protein diet Fine to medium droplets of fat were observed in the cells of the portal areas and in the intermediate zones. Fat-rich cells were scattered throughout the areas. magnification, 370 X PLATE II Ifistological sections of liver from animals fed gamma— and cathode ray— irradiated wheat diets. Oil Red 0 Fat stain Figure 1. Gamma—irradiated wheat, 9.3 x 106 rad Fine droplets of fat were observed in individual cells scattered throughout the lobules. Ho definite pattern could be established. kagnification, 390 X Figure 2. Cathode ray-irradiated wheat, 2.8 X 106 rad Eedium to large drOpletS of fat were observed in cells of the portal areas and intermediate zone. The fat-rich cells were scattered throughout the areas and some large single nucleoli were noted. Eagnification, 360 X PLATE II PLATE III Histological sections of liver from animals fed gamma— and Cathode ray—iirradiated wheat diets. Oil Red 0 Fat stain Figure l. Gamma-irradiated wheat, .93 x 106 rad Fine to large drOplets of fat were observed in cells surrounding the portal areas. Some fat-rich cells were scattered into the intermediate zone and the central vein area. kagnification, 365 X Figure 2. Cathode ray-irradiated wheat, 9.3 x 106 rad Fine to large droplets of fat-rich cells were massed in the portal areas, intermediate zone, and the central vein area. Heavy fat deposition was noted throughout the tissue. Hagnification, 330 X .63 There appeared to be a difference in the way in which fat was deposited within the liver lobule between the tissues obtained from feeding the cathode ray- and game-irradiated diets and the 12 per cent control wheat diet. All the tissues exhibited predominately portal deposition of the fat in agreement with Shils and co-workers ('50 and Mustone and Specter ('50). As more fat was moved into the lobule, rather tho being found at one end of a portal area as the con- trol showed, the fat became periportal. Then from the indicts peri- portal deposit, it fanned out around and away from the portal areas and along the periphery of the lobule. As more and more fat arrived in the lobule, the heaviest deposits were still seen in the portal areas but now individual fat cells were found in the intermediate sons and on rare occasion in the central vein area as well. The fat droplets thaselves varied from a fine peppery sise to large dreplets. The nuc- lei did not appear to undergo am changes other than being pushed to the side of the cell. There were one to tour nucleoli present in the nuclei with occasional large ones as Adamstone and Specter ('50) showed. The above changes noted in the fat pattern have been suggested in studies discussed earlier in which phemlalanine, leucine, histidine, tryptophane, nethionine, threonine, and lysine deficiencies have been individual]: studied. 01 all the amino acid deficiencies, lysine appears to be the nest passive in nature and slowest to show tat-pattern changes. It has been well established as the under liliting amino acid for wheat. In the study reported by Harris and Burress ('59), fatty livers were found in annals saintained on an 8 per cent cereal protein diet fruutozsdays. Theanimlsmaintainedonals percentpro- tein diet for the sane period of tine did not show fatty liver. When 151; the 8 per cent diet was supplemented with varying levels of lysine, the fatty livers were prevented. It follows that the 7 per cent level of wheat protein used in this study might be expected to show fatty livers induced by the limiting amino acid, lysine. The fat cells found in the sections made from the liver of the animal fed the 7 per cent non-irradiated wheat diet had a charac- teristic inpact upon the observer. Fine to medium-sized droplets were found in individual cells following the portal areas, fanning around then and over the periphery. Individual cells were found in the intermediate sons and occasionally in the central vein area. There was a clumped scattered appearance to the fat-rich cells in contrast to the smooth steady movement of fat cells observed in agreement with the 12 per cent control. Dick and co-workers (' 52) and Vennart and co-workers ('58) have reported changes similar to these when diets deficimt in lysine were fed. When the tissues derived from the animals fed various levels of cathode ray- and game-irradiated wheat were compared with the 7 per cent control, the gamma-irradiated tissues seemed to have a gener- alised scattering of fat-rich cells with 1.2 out of 31 tissues compar- able with the 7 per cent control. And of the 12 tissues, ten were from 6 the two lowest levels of games-irradiated diets, 0.28 x 10 and 0.93 x 106 rad. The upper levels, 2.3 x 106 red and 9.3 x 106 red had a scattered appearance but were so diffuse that no definite pattern could be discerned. The cathode ray-irradiated diet-derived-tissues, however, compared more closely with the 12 per cent control tissue and on]: eight of their total mmber of 32 showed any characteristics of the 7 per cent 65 control. The eight tissues were evenly distributed among the various levels. These tissues do not represent a majority of the tissues exam- ined. However, this does not preclude the possibility of an amino acid unbalance intensified by irradiation. No explanation for the majority of changes in the tissues de- rived from feeding the lower levels of game-irradiated wheat diets can be made since the source of irradiation was the same. A more uni- form pattern of fat deposition as was found in the tissues derived from cathode ray-irradiated wheat diet animals was to be enacted. The tissues from the game-irradiated wheat-fed animals as a whole had a more scattered clumped appearance. The tissues from the cathode ray- irradiated wheat-fed animals gave a clearer, smoother picture of fat deposition. It is possible that a long term feeding study would clar- ify the reason for this, and whether the trend in fat deposition noted here is transitory or characteristic. There was a consistent trend throughout the results reported in this study and in the findings of Cannon ('59) indicating a possible protein alteration of some nature. The structural damage which may be induced by irradiation and the slowness of response of an animal to lysine deficiency as noted earlier may well be an explanation for this consistent trend. It would appear from the findings reported here that the type of radiation had more influence than the level of irradiation used. Since only the initial attack of cathode-ray- and gamma-irradia- tion is thought to be different, the nature of this difference should be further investigated. In general, the animals liked the irradiated diets, ate them, and gave no outward signs of being other than healthy, growing young animals. It remains to be determined whether the changes observed in the liver tissues are detrimental to the well-being of the whole animal. SUMMARX AND CONCLUSIONS Seventybsix male weanling albino rats were allotted at random.to individual wireébottomed cages and fed food and water‘gg libitum for a four week period. Wheat was divided into nine lots, one of which was not eXposed to radiation and served as a control. 0f the remaining eight lots, four were exposed to cathode ray and the other four to gamma-irra- diation, each at the following levels: 0.28 x 106 rad, 0.93 x 106 rad, 2.8 x 106 rad, and 9.3 x 106 rad. The wheat was ground following irra- diation, incorporated into diets to provide a total of 12 per cent pro- tein, and eight animals were maintained on each of the nine diets. A third group of four animals was fed a non-irradiated wheat diet of 7 per cent protein as a supplementary control. Records of weekly weight chan- ges and daily records of food consumption were kept. On the last four days of the experimental period, one rat from.each diet group was sacri- ficed by decapitation, the liver removed, and xanthine oxidase and cyto- chrome oxidase activities determined. One lobe of each liver was placed in.lO per cent formaldehyde for histological examination, and the remain- der of each liver was saved for nitrogen and fat determinations. Protein efficiency ratios and growth measurements were evaluated. All findings were subjected to analysis of variance, and in addition, measurements of the enzyme system activity were subjected to T-testing. There appeared to be no difference in the feed intakes and weight gains due to the type of radiation but there was indication that the level of irradiation.had some effect. The animals tended to grow at the same rate except for those on the highest level of irradiation who appear- ed to grow somewhat more slowly. The same trend persisted when the aver- age weight gains per gram of diet eaten were calculated. Protein effi- ciency ratios were found to be similar in animals fed the lower levels of irradiation but again, the highest level of irradiation, regardless of the type incorporated into diets, resulted in lower protein efficiency ratios. The activities of xanthine oxidase and cytochrome oxidase meas— ?‘ ured in the livers of animals fed irradiated diets were consistently lower than those obtained from.the livers of control wheat-fed animals. a» The depression of activity of both enzyme systems was most pronounced in livers from gamma-irradiated wheat-fed animals, and was found statisti- cally significant at the 5 per cent level with the T-test but not with ' analysis of variance. Histological examination of fat deposition within the cell and lobule of the individual livers presented a picture of possi- ble intensified dietary unbalance, especially in the gamma—irradiated wheat-fed animals. The level of irradiation appeared to have little effect upon the degree of depression noted in the enzyme system activi- ties and upon the fat deposition noted histologically. The findings suggest that the type of radiation.may have more significant influence than the level of irradiation.with regard to the animal's ability to metabolize the food presented. There are indica- tions in the findings of this study that some type of alteration has been produced in the nutritional value of the wheat by the irradiation pro- cess. Since wheat protein is nutritionally incomplete, assay of the irra- diated wheat for the limiting amino acids might clarify one possible as- pect of the alteration. In addition, a long term feeding study, with ani- mals being sacrificed at regular intervals, might clarify whether the fat 69 deposition noted was transitory or characteristic and potentially detri- mental to the well-being of the animals. LI'I‘MTURE CITED Adamstone, F. B., and H. 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Johnson 1959b The effect of radiation steril- isation on the nutritive value of foods. IV. On the amino acid composition of garden peas and lima beans. J. Nutrition, 68: 1119. O O ' . \ O I 1 . e e . o I s o I O . O . . C e O I I . . l I \ . O 76 Vennart, G. P., V. P. Perna and‘W. B. Steward 1958 Fatty liver of ports: type: cured by Lysine plus tryptophane. J. Nutrition, 611-: 350 Vorhes, F. A., Jr., and A. J. Lehman 1956 New Problems of Food Safety. U. S. Public Health Service, Public Health Reports, 71: 57le Yen,'Iin-Chao, M; Milner and H. T. ward 1956 Treatment of wheat with ionizing radiations. II. Effect on respiration and other indices of storage deterioration. Food Tech., 10: All. Younathan, E. S., E. Frieden and K; Dittmer 1956 Sensitivity of rat liver.xanthine oxidase to amino acid analogues. J. Biol. Chem., 219: 531. APPHDII HISTOLWICAL METER]! The liver lobe used for histological examination was cut from the intact liver before taking the amount necessary for the shame and chemical studies. The same lobe was taken from all animals. It was the null tear-drop caudate lobe situated to the left beneath the two upper large lobes, and varied in weight from 0.32 to 0.80 grams. Each lobe was quickly placed in ten volumes of 10 per cent fonnalin neutra- lised with marble chips. They were allowed to fix for a shim of two months and were cream white in color. Tissue was prepared for cutting by gelatin infiltration (Gm-r, '56). The lobe was cut into two or three lengthwise pieces depending upon its over-all sise. These in turn were cut in half, crosswise. An attempt was ude in every liver to secure the same middle, lower- half of the lobe for infiltration. The tissues were rinsed in running water and innersed in 5 per cent gelatin solution for 21. hours is a 37° incubator. Following this, they were immersed in 10 per cent gelatin for 12 hours, 15 per cent 3.1.11.1 for 6 hours, both in 37° incubator, and than embedded in 20 per cent gelatin and allowed to set overnight in the refrigerator. The next day the tissues were trined from the gelatin blocks and imersed in 10 per cent fonalin for 21. hours, after inch they were ready for cutting. Although the time involved in gelatin infiltration was considerable, sections could be cut at five microns and handled with each throughout the staining and mounting techniques. 79 The tissues were washed in running water for ten minutes prior to freezing. They were frozen with carbon dioxide and cut at five microns on a Spencer Automatic Clinical Mlcrotone with freesing attach- nent. The sections were stained with 011 Red 0 following the technique of Bell, ('59). Since the gelatin no not removed prior to staining, the free sections were allowed to remain in the Oil Red 0 from six to sixteen hours. No appreciable less of fat was noted and an intense stain was obtained. The free sect ions were counter-stained one minute with Groat's hastoqlin and blued for ten minutes with l per cent sod- inn bicarbonate. After rinsing in distilled water, they were fixed to the slides with Mayer's albumin and mounted with glycerine Jelly. 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