'\ ' “F .m"§"‘-""u “3".“ 9‘3: A ’ y ‘(u (2.: \f‘f“: EL»; '.S'urcv 9’? E:&.°s an. , ”‘.“’"‘I. (“.56 f .. .. . . z . Efifmtahfkhc‘ &: :V: .‘Jx 95"" ‘0‘.“ £0 a 5:! " 0V. i:;:\.‘ .1: @a-Ji ‘ ' fib‘lvcr‘ : ‘ n to“. .t..v¢'\:t . ‘3 talk”: nus! ‘1‘}?{3‘5 : .1, Thesis far $210 Dagmar of M. S MCHSGAN STATE COLLEGE :a “1 ;.‘..‘; *0 ’r ‘5’“, ._".1 0- i‘ M ‘ ' V. 3. I" < s ‘ (I! 1" 3 XEC‘HIés 4' -. .sx‘a’ubdw . w - I) “153 3 «.2 *1 can" THESiS H. W ‘I f i -‘ .\ i 1 . l-'.I}' 3.“! ".""1‘ v “-\1"-‘l \/_. University M.chigan State College E58? 14..-}--- ‘Ic u . A STUDY OF THE NFLUEECE OF WATER IETAKE 03 THE COEDITIOH OF RATS MAIHTAIHED ON A MARS HAL DIET WITH AND WITHOUT HE ADDITION OF SODIUM DIEYDE ”EH PHOSPHATE BY Priscilla F. Iledan An Abstract Submitted to the School of Graduate Studies of Eichigan State College of Agriculture and Applied Science in partial fulfillment of the require- ments for the degree of MASTER OF SCIEECE Department of Chemistry 1954 Approved It has been skan in ”“6’1013 cv‘v"1~“.us t at 11.13:- tion of. or ~xcosaivc onsamc*1oa of water a 3333 and rato itcroased "P303 3? us excretion. this study was an attomyt to domonotrztc t? 3 effect of water consuwptlon on the oomfiition of rats given a m r33133113t with 311 without tic aifilticn of sodium dihylrogon QLOSnxnte. Pats were given unll3itr31 aflonnto o arimhing water for the first two weeks, 333 restricte? aflounts 31r1n3 the next two wcczs. Erowth gains and food and/or phoc3hor33 1rt-l -:oa were recorflei. 30331181033 were cot 01 by means of analyses of variances, with the an ant o” urinkln; wa or. food and/or phoophoruo consumed, and sex as variablco. rho results indicated that zato giv- 1111.1t91 amounts 0 :3 of water grow mars than those wit 1: restrictafl amounts, where tno 3333 kinfi of diet was cmployad.fho males, on an averago, ha1 a m~cat 3r growth gain than the {033138. “hon the 6333 amount of 801113 dihylro; m: Loo“ ate was 31131 to the 11f~ repeat dicta, he rats fed éioto cofltainirg more milk and loss rice 331931 more than those £31 333-3113a111 more rico..dii- tion of sodium dihy1r0393 phoaonate to these expor 13 ontal dicta resulted in a decrease in growth. Bhia 13 croa 53 was in proportion to he amount of 3103313 3 31131. ftor the rats had been subjected to a limited water in age for trzroe to twenty-six we 333, tr oy more 33 icrlficed at wocxly intervals. and the blood incrja Mn 3 pi cscnarua canton dotcrminoz. tosults showed that the typa of diet fei and th Ad 45 .‘3 .4. ' . L ‘3‘: U 0).. ’ c ..... “ 1" -13313 1 110 81‘1"; ‘ \‘1;. ¢_.*-A. 111 1 tal 3311 «J-.. ("I ‘3‘ I «.711 M, 4 ‘4‘- .Aho o tho 0 .0 0! 503‘: ‘13“: x)..." r. - v ‘1 'w ‘.‘ 0:. . I ; C ‘.U 1113‘: \f' r 7 “u; 1;. bl~ -\ .4 I ~ . L“ o? t” A. \I conten- . 01“.} 3'3 .11 0 32-5. "'- v 11 n “I :1 O '3 a. rut A ‘I ‘ 1" .35 kg} 4 hi .. 18: 11'}! Q ‘ t; 1.... 0" ’\ '1 zt“ ey \lA ‘r .a '22". O I‘LL 3 l- . 31:03: ~02” ..v_.. - 3“ k - 1 .5. q 1 a h 1fi .~ v yr. - ' l I .1 A ‘K ’c' 4-: 8 1 4. F3143 . F 0:11:38 Q :2 ,.‘ ‘5‘- than the ol; - «9' 1411.51 cont A STUDY 07 TH? UTLUITC" O? VATEH ICTKKE ON THE COEDITIOH OF RATS HKINPKITED ON A NAHGITAL LI ’HITi AID WITHOUB HE ADDITION OF 30? JIU3 DIE IYD O"'" FHQSDLAIE By Priscilla F. Ileaan A TLESIS mitted to the Sch 01 of Gre d11ate Studies of filchi; an State Collc e of Agriculture and ipplied Science in partial fulfillment of the require- ments for the degree of If (7U “17?, '1 Hf! 71'- 111-. ii¢k13PLAK 04“ UV “-4 iU-lb Department of Chemistry an '7! PJ'Ofi, 91 mm awry-T i \l ;‘o nu: . " J—J ,iJ‘JLI-.‘..JJ.M The author wishes to express her sincer appreciation to Dr. Carl A. Hoppert for his interest,counsel,and guidance throuxfiout the experimental work and with the preparation of the manuscript. Special gratitude is due to Mr. Leo J. Klever of the Jitamin Assay Laboratory who has been exceedingly helpful sod has provided excellent instruction in the care and handling of animals. Finally, the author wishes to thank Dr. William D.Baten of the fiathematics :epsrtmcnt for much advice and assistance in the statistical analysis. TA“? ‘1? « fi‘““fl”'\ “##pr (fig—J5:— Vat . am... 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L .L.l.-.-L 1.;I‘TI'VIC, ‘J ,,_ . , :. -41....vu 3 V'- ‘!-' .. * r 9 F1? ‘ 13 {1 ‘T I 'T.) ' "7‘! 31“" 1'.“ ‘ h l .flk::“‘ J.) I 2 O G . ‘u‘hl: . G3". ‘ ‘- .l .3. -..l ....‘ .14 Gr ’Ffif‘r", :5 I“ “'r‘1':“r‘" .-‘ L‘v‘LJ'J 3-..“...D o C o o o 0 o o o o o o t o o o C-V 1"! "f'f‘y “1 I-ffifi- 7:. v'wr-“rI-r.‘ «WV-"1“? \v ‘3" :9 ‘ . 2 Id .1 .1). -LLJQI'. I-.-.‘.. ”a fik'-J’J-.L\Jr." D .’L 1. ’ ' A. IJL-‘Jar ‘L? I v - t.“ v”.- wf“ .( (n— .1 fl ‘- 71! r.:a:) ‘ . »_ v.31", ‘3' =1 :AL. .3.) '. P: G .1» 4.4- 5.7-1.1...“ DIE . j -33. PHOS" - "’“Yrr. "'t’.‘ fx‘Y-11vv'fv“ .‘fi 29 :5‘&‘V-‘\JK) Vu‘-..)\JL. ~40: . ‘ . . . . . . . . . . . . . Bhoseherus was discovered by Erandt in L 39. It is one “3 cf } .‘ 0.: (D e nest important inorganic elenents involved in animal 1...: lie. he other one enters into such a diversity of conpounds and plays an in portar nt part in so many fuzi ctions. It is a constituent of cell nuclei and all cell structure; it is also prominent in the skeleton, in mil:, in glandular tissue and tiie nervous s3stem. Through the interqedisry fornati on ’7! of lecithins, it is concerned with the metabolism o~ fats. hrougi the fernatien of hexosephosphates, of seenylic acid, adenosine dichosphate, and afienosiae tr L hosphate, it plays primary role in carbohydrate metabolism. fhere have been some suggestions that it is involved in protein metabolisn(l). Phosphates also play an inpertant role in the pH regulation of the erzanisn. These and many more n::e nhes twhortzs i.' dis- pensable for life Although the cone. oxnds of phosnhorus eartici nets in numerous important functions of the body it is significant that the litei eture nrovifies very litt e infornatiee scout the influence of water eonsu;ption in relation to the intake of “hos Hthu s in the eiet. In 1913 Osborne a d ”0 del (2 ) restric tei rats to a diet soneviat deficient in p:hosohorus but unfortun fiely their only observations were an growth. Since that tine there ha e con nu erous in es tigaz.iens of phosphorus in relation to rickets but the principal emphasis has been on the Caz? ratio. r f 1 1 study the in? mono: of water intak; on 'he conditiov -1 ine' on a diet with an; without the {D .4- ’LJ rail 4‘ 1‘61 us 129. g 3 of sodium di1ydrogen ohospnate. fl 1 J. a A q“ ‘- ”-0- L‘~ °- 4 P-—‘. ;9 purpose of bLO pTCSugu Stuo« UMCFC;UL h) P4 3) i .. C‘ L13 1—! C J '5.” I?" The necessitv of ph:)sohorus for 'hc grost; of plants, bacteria, and animals has been known since the very b0313- niz‘13 of tizo biochemical era initiatei by 1.1.313 (3). s13ho- rue was incluaed in the list of inor3 snic elements which Liebig stated were necessary for ple.=- b 3r .Ith and as early as 135’? Jille (4) 1m ,uorrirentally proved the re c1uire- ment of pie nts for phos:horus in tr e form of 3hos >hete 3. However, the early investi3stors considered phosghates to .0 function chiefly as a structural element of livin3 tissues. The catalytic and regulatory functions of eh sesstes n mots‘solic processc s were also rve sti3stei d1rin3 the letter pa irt of the 19th century, but isrien and Y011213 in 1905(5,E) were the first to csrry out i more detailed s11 critical stud;. In 1394, Lsndeuer found that deprivation of water after resch1n3 a certain limit, the 3hosoh rue eli instion returned to normal (7). trsub (3) studied the ef?ects of thirst in do3s. Es fourd out that phosehor 3 e113 mtion in the urine during periods of 3-4 days ”it out water intel c has 12M103803,th0u3h only to a slight extent, about 5-10 In connection with his thorough L we tigstion o: the efzects of water consifiotion 0‘.1 "ets‘olisw Saw: (1905) 4 '4. ' 'L .' ' r' " -v- . V 1 - ' \fi'r’ as r. . - '1” c- ‘r -. - foand at that COleQS water drinian 1;: creased the eicret on of phosphorus in the urine, this incx e39 e eel“ ascaned to increased cellular activity and the acco vindin catabolis.s‘ of phosphorus~contsinin3 ccmooisis. In v~ry instance, the excretion of oi. mo fziorus was iLCPGESOa score the normal level, the nszcinun excretion occurrin; on the second day of the increased water in .‘3estion. from this e"i m1 e, they su33ested that ei tin or 3rc at thirst, or unusual consunption of water, will inm: ass the phosphorus excretion, hou3h further work is re 1uired to estaelisn the source of thi phosonorus, and therefore, the signific nee of this increws d excretion (9). During the ?irst orld a1, Embdon et a1 (10) conducted experiments on aercsi sold iers Lith acid-shes _hato drinks. ‘ith the introductioz1of the nhosphate drink, the require~ ment of drinking water by minors b care much smaller. Vnen the use of tnc ones Late drini: has liscozzti-1acd,a neightenea thirst immediately manifested itself. They su33en the reduced need for water durin3 a rio d of nhosonate supplementation can be attributed to decreased 3ers:.iration. ins fortsulat fro: natural foodstuffs of a suitable d et deficient in ,“oseuoras presents some difficulties '4. M3 cause alnos all so mrrcs of protein conta L1 relatifely large amounts of Inios fiber 3 (ll). Schneidc3 and Steenbock (1:) god rats a purifief diet very low in r1031uar‘s a zd lackin; in Vitamin u, but adequate in all other nntritiona requirements (P,0.04 ; Ca, O.Dfi). 5 ifter 12 to 1’: £153 th e rats defeloeefl ricxets, and within (.21 six wee;s sevex e shelets 30:: ort1it 5.03 set in. Growth sto3pe after 4 to 5 weeks, followed by 3 3031111013 weijht, sad c1estl1. usually occurred by the end of the sixth week. However, when the low 3hosph3rus-diet was supeleeented with sfiesuste Uitsnin 3, e1 immediate weight loss cccurrej in the :roui-. rats, which was not regs sea for more than three weeks. From calcius and phosphorus balances and tissue analyses the atztho re (13 ) corcluded the 'itsmin D induces the utilize- tion of phosphorus by bone, thereey deer'ving the soft tissues 1 their sup 111g of 13-hosphorus, wlmi in turn inhibits growth. H, 0 These siii1s ls survi ed for as long as t1e3ty weeks, 3 003-. trast to those not receiving Vitamin D, sad cousi Wst =3tly developefl urinary calculi 213.e efzfect of oncs¢1 m1s eeficicnc1 on growinf rats was studied by :or >es (14 ) in s 270—3-1 body balance enl_3etsbo- 118:1 exp riment conductea with psiree—feeiing controls. In the first estpe r=i1e mt, t1.e phosc 1erzs-3cficrcnt and phosphorus- sueplenentei diets contained 0.1373 and 0,51 3 of phosphorus respectively. The corresponding a erase P content of their bodies after 70 day s was 0.0 ' -and 1.033 resoeetively, a A, Q ‘ .- differeece of lLfl. There was no eif fer: see in t3 gr w h or O utilisation of food eiergy or protein. In the second exveri- meet the l w and itigh phos1horus diets containefl .ljjfis ald 0.653} of P respectively. ifter 70 days, the 11hos¢no‘us con- tent of their belies was 0.93) asd l. l a res _JcetivelJ, a Eif- ferencc of 13}. Here they observed a slight but significsLt 6 depression in the digestahility of protein. This was attri- butei to the effect of N3H2?04 in the high phosphorus-diet. There was no effect on the utilization of protein or food energy or growth. Hhe P content of the low-phosphorus diet was as low as could be fade and yet sufficient for some growth. flash the P content was further decreased, no growth was obtainable. Day and thollun (15) prepare; a diet containing 0.0175 phosphorus but adequate amounts of all other nutrients(Ca,O.4 é). Young rats restricted to this ration grew slowly for 5 to 3 weeks, and then declined in weight and died 2 or 3 weeks later. Extreme rarefaction of the skeleton quickly occurred, accompanied by progressive disability in walking, standing, and breathing. Eitrogen retention was positive for 5 weeks and then became negative simultaneously with the onset of loss of weight. The mobilization of calcium and phOSphorus from the bones led to a tremendous loss of calcium and a considerable loss of phosphorus. The greater loss of calcium than of phosphorus can be attributed to the fact that part of the phosphorus mobilized from the bones is taken up by the soft tissues to permit their growth. Then the phosphorus was increased without the addition of calcium, or when the cal» cium was reduced in their diet, rickets did not develop(16). Bherman and Booker (17) found that with diets of cons- tant phosphorus content (O.#2§) increasing the percentage of calcium progressively from 0.13 to 0.503 permitted more rapid calcification of the growi.: body and earlier maturity and the apnea rance of senescence. Phosphorus must be available in the animal diet not only in adequate amounts but also in the preper balance with cal- cium. The ratio of calcium to phosphorus in the diet e: :erts a marked influence upon growth, reproduction, -sh content of the bones, and the calcium and inorganic phosehorus content of the blood serun. Shohl and Wolbach (13) concluded that, in the absence of Vitamin D, ricLets may ‘ee produced with not only hi3h-calciun,low-phosphorus and low-calciui,hi3h-phosphoe rus diets, but also with low~calc§un, low-phosphorus rations. n Phis last grou occuns‘ in a zoneAwhich the ratios of Caz? have been called normal, thus demonstratin3 that rickets may be produced with any ratio of C :3) in the diet. hethke,nick, and‘ .iilder (19) have demonstrated that increasi1': the Caz? ratio from 1: l to 5:1 caused a pro3re ssive decrease in growth, bone ash, and the percenta3c of inor3anic phosphorus in the blood serul. Decreasing the Caz? ratio from 1:1 to 0.25:1 decreased the growth and the percentage of calcium in the blood serum but had only a sli3htef1ect up on the bone ash. According to their data, the ratio of Caz? in the ration is more important in determining the 3rowth and calcification than the actual concentration of the elements in the food. They conclude d that the most favorable Caz? ratio for growth and bone formation lies be can 2:1 and 1:1 (9,0.53 to 0.59§). Cox and Imboden (20) found that successful reproduction and lactation in the ra at were dependent upon both the actual level and the Caz? ratio of the diet. The best results were obtained on a diet containing 0.49; each of calcium and phosphorus, r) k) correspondin; to a daily intake of am) out 42 an. of each ele- ment. hubhell, Handel, and Vatenan (21) devised a new salt mixture for use in experinental diets. Durin3 a selected period of very rapid 3rowth (from 60 to 200 3ran body wei3ht at a rate in excess of 5 grams per day ) on a diet containing this mixture, adequate calci?ication occurred with an average daily intake of 50 mf3. calcium said 35 m3 of phosnhorus. In 3roxin3 animals the amount of phosphate in the diet (required for bone farnation, etc.) is larser than in the mature animal where only sufficient phosphate must be taken up for that ex reted. This can be fully explained in the study of the normal phosphorus content for each sex of rats at various stag es of 3rowth and development by Sherman and guinn (22). Their results showed an avera3e of 0. 345 phosphorus in the body at birth to :1: t 23 lava; 0.57-0.55; at 51 0.49 A at 15 days; 0.53 ~0.5€j a days; 0.62-0.63} at 3 months; 0.63-0.19$ at 4 months; 0.73 7s, in adult life. After 15 days of life, the total wci3i;t of phosphorus was higher in males than in the females, due to their 3reater avera3e body we 3hts only. P1 0 female that had not borne young showed a hi3her percenta3e of phosphoru ha males of the same age, heredity, and dietary history. A diet h 3h in beryllium and strontium hindered phosphorus absorp- tion,a and lib:e calcium, gave rise to a form of ric :ets. Absorption of phosphorus is enhanced by an acid reaction within the intestines. Cohn and Greenherg 23) have recently used radioactive phosphorus in aosorrcion studies. They showed 9 that the absorption of phosphates by racbitic rats was in- creased but little by Vitamin D, but the inorganic phosphorus uptake in the bone increased 25 to 50;. They suggested that Vitamin D may act to aid the conversion of organic to inor- ganic phosphorus. ilkalies, as a rule, decreased phosphorus absorption. ‘he effect of fat is not clearly established. Some believe that fatty acids combine with calcium and thus favor phosphorus absorption, whereas others have stated that the use of high-fat diets causes an excessive loss of both cal- ciun and phosphorus in the feces. The deficiency o? phosphorus in the food only gradually changes the relative phOSphorus content in the organs, and only in the case of poor growth does it become noticeable, especially in the bones (24). Txcessive excretion of phOSphates has been reported in certain bone diseases like osteomalacia (25). diffuse perios~ tools and rickets, in the early stages of pulmonary tuber- culosis, in acute yellow atrophy of the liver, in disorders associated with marked breakdown of nerve tissue, and after sleep caused by administration of chloral hydrates or bro~ sides. It is also increased after cepious water drinking, as was previously mentioned. A decrease in the excretion of phosphates is noted in acute infectious diseases accompanied by fever, in pregnancy during the period of fetal bone formation, in diabetes after insulin administration, and in kidney diseases due to failure 10 of elimination. The fern in which _hosphorus must be taken in the body has been the subject of considerable speculation, and ideas which we now know to be erroneous have been widely held. Of the various classes of phOSphorus compounds present in food, Sherman, et al (11) found out that organic combina- tions appeared to have greater nutritive value than the in— organic forms, and he gave this as a reason for the varying amounts of phosphorus reported necessary for the maintenance of equilibrium in man. Gregersen (26) found out that rats fed a diet of edestin, lard, sugar, cellulose, and salts, with hahgtoy as the sole source of phosphorus, were able to maintain equilibrium over considerable periods of time, or even exhibited a positive balance. rem this he concluded that the organism can build inorganic phosphate into organic phosphate. According to present-day concepts, the form of phospho- rus in the diet is of little importance, since in any case, as a result of the digestive processes, phosphorus is largely absorbed in the inorgan c form. This is not surprising when we reflect that in all the complex tissue constituents such as phospholipids, nucleoproteins, and probably phosphooroteins also, the phosphorus is present as phosphoric acid esters. It is to be expected that the organism would be able to build up those esters when provided with a source 0? inorganic phosphorus. It is generally agreed that inorganic salts o? phosphorus 11 are readily utilized by the animal when included in the normal ration. However, Eddy, Nuller, and Heft (27) hare shown that the phosphorus of phytin, in contrast to inorganic phospho- rus, is unable to protect rats receiving a high-calcium, low-phosphorus diet from rickets. truce aid Callow (23) have confirmed this finding and have shown that treatment of the phvtin-containing material with 1} H31 renders the phOSpho- rus available for the cure of rickets, presumably because of the hydrolysis of the inositol phosphoric acid. Inasmuch as a large part of the phosphorus of grains and seeds is in the form of phytin, these findings are of importance in consider- ing the phosphorus content of the ration. Probably all of the phosphorus of the leaves and stems of plants is available to the organism for these contain very little, it any, phytin. It may be noted that the requirement 0? phosphorus varies, depending on the diet, the age and s x or the animal, and other factors. In View of the importance 0? phOSphorus to the health and well-being of all animals, as well as human beings, it was of interest to study the influence of water consumption on the condition 0? rats maintained on a marginal diet with and without the addition 0” sodium dihydrogen phosphate. . Q' . l u Animals and Diets: Pale and female albino rats of a strain aeveloped in the Kichigan State College Chemistry Department w-re mostly 21 to 25 days old when selected for these or eri ants. A few rate, between two to three months of age, were also set up to observe differences in growth, if any, due to age. Tr its rats were selected as the experimem1t l miials for this study because of ti leir omnivorOLxs food habits, the close resemblance of the chemical prose as: s of human anCL rat me tasolisn, th convenient size of the rat for investigation of this charac- ter, and the feet that many features of the nutrition and life histories of the rat have been extensively studied by nu serous investi3ators. A total of 112 ats xere SttldilCd. In I each set, 16 animals were divided into groups of 4, with each 5roup having an equa l n mher of males and females weighing mostly from 45 to CO grams. The initial weights, teta l wei3 t gains, and total dietary intake are listed in Table II. Table III shows their average 3t 3ains, dietary intake, and phosphorus intake per day. A percentage comparison of 5rowth gain to food andxor phosphorus censumt tion is i: clad-e d in Table IV. Different experimental diets were preeared as shown in Table I. One—half 5 and 1 fl NaH9204 were addre d to Diets 2 and 5 reapeetively using Diet I as the control. Diets 5 and 6 we 1e made up of 0.3; a d l; lahéiO4 reseectivels', with Diet 4 13 as the control. "he different components were finely ground and thoroughly mixed so as to ensure uniform composition of he diets. These were stored in dark bottles at room temp— 3". Luring 'he first two weeks 0; each execrinent, the ani— mals were given ‘nlinited food and drinnin; water. After that period, they had free access to food, but their drinking water was limited to 15 milliliters per day (20 ml. per day for the older rats). a careful record of their weekly food consumption was made. The food intake was calculated from the differenc- ( between the total amount supplied and the tare weight cor— resoonding to the food spilled. The animals we‘e kept in individual, raised cages, and the room temperature was main- tained between 75 and 73°F. After two weeks of limited water supply, and weekly intervals thereafter, the animals were sacrificed, and the blood phosphorus determined. Analytical Method for Blood Inorganic Phosphorus: After sacrificing the animal using ether, blood was quickly removed and placed in beaaers having a thin dry film of anticoagulant. The method used for analysis was essentially that of Fiske and Subbarow (29). It is based on the fact that when trichlor-acetic acid is added to blood, the proteins are pre- cipitated; treated with an acid molybdate solution, this acid- U4 4:. U1 (1\ 1" .-“Q I U011 uI‘Ol 10 10 10 3‘. \J A U 13 U1 Ui U} U": '1 t U] f‘ " L4.) 25 HO U: h .. , ., c- ' were calculated. .- _..:I}li " 57.5111. 3 :‘1 '3) “0133) f Unlinited Drinkinj “ator ‘r I. 0 DJ. -p 11 final blit 7’ ,_,‘ .cignt Fetal Total -i 1; "1 "1 * mi. .’ ("r . . l ‘- -.. '1- , _. balm 1n taio [Ci 1 fo. 3.) (3.) “ ) L). 2? 112 112 15? 103 1%} 97 30 be 93 _ 101 _ 152 195 e: 110 135 121 51 55 97 42 HHHHHH .waa amoew MMRJN HI—‘l—‘l—‘l—‘H L] O \ L x.» 1 H k. 1...; b 123 “5- 23 13; 133 101 \q-J L4 4 10‘ 1 9 6 1.3 173 193 4 V3 123 7 let 15) l}; A 31 115 L 146 115 15; a 5% 110 23 110 5c .p #- (e 10 25 I I“ 1 n N 19 57 9 4'1 1', 129 Y” 3 £9 12? 137 55 102 47 g 102 9% ' 43 101 51 132 10; sex anunLn #- 11; 5 153 5 c 54 103 1 14. 20 152 OLDK .lf”: 1 203 223 22 2“5 253 262 3 203 225 20 224 935 245 r‘ I. 7:"? r ," "1"“? 2 3%1 £41 A} is; $31 QLA U CLD 64:) (.1, A! LDJ (.1 J nhir13 iota Voight Gain OLIa \Ju‘ PO h) .J O f.» L; -Q , 41“ h.) R.) 437‘03 N LU L. h) R) h) N) U'} \3.‘ 24 10 3:”) 6-.) ‘- 111401" Total Food I 11 03.1-6 l_‘\ . 10v 1 4 ”RESP“ EH <3~36-efiim O . ’w. I! JHHH P Q It) FD. t‘ f 5“. R) 1‘.) U4 P H .V H E? 14 214 134 1+; AVERAGE HEIGHT 05.1333 AID N-‘i‘lffiiGIfiI DIETUZY IEITAE’CS Growth Gain Food Consumed P Intake Sumner For Day 2er Day Per Day * of Rats Hale Female dale Tenale Male Female Diet to. (5.) (5.) (5.) 5.) (03.) has) I~A. Unlimited rater Supply (Young flats): 1 2099 2.80 11000 1.1.1.0 £19.50 29.16 24 2 3.07 2.70 11.00 10.20 41.3) 33.67 15 3 3.95 2.70 14.55 11.55 72 57.44 8 4 4.37 3.87 10.70 9.67 34.3 31.22 24 5 4025 3063 8095 8.63 38093 3705L 15 6 4.45 2.70 l2.L5 10.70 70.42 53.63 8 I-B. Limited tater Supply: 1 2.“3 1.93 12.»2 11.47 34.30 30.73 2 2. 03 2.03 10.81 10.52 41.09 30.24 3 2.75 2.65 13.40 12.30 63.16 03.11 4 2.72 2.12 11.92 11.00 37.49 30.03 5 2.33 1.70 10.11 9.63 43.99 42.00 6 2.95 1.80 3.10 11.25 71.79 61.65 II-i. Unlimited Drinking water (Older Hats): 1 2.50 1.90 17.10 14.20 19.03 33.03 4 3 1.70 1.40 15.30 15.50 75.45 73.91 4 4 1.30 1.70 14.20 13.80 45.37 '-74 .57 4 6 1.10 1.40 15.30 13.50 83.34 73:93 4 11-3. Limited Drinking Water: 1 1.80 1.50 18.40 13.60 39.31 36.45 3 0.70 1.60 . 15.50 13.80 81.?4 57.33 4 2.00 1.70 17.30 ” 13.60 55.‘3 43.93 6 2.00 1.70 3.00 14.30 93.54 73.36 * Calculatedfimmlastual food consumption 17 m TAEL IV Cht'fiqMIC-“w :- fl'finm-n" G *n Val-1L .".LI 0Q1~ ';.t’\.llv -‘bb; ‘ o1i~i . fan/x" ‘ ' ' ‘ y f\rffl‘jf¥’\f}"9fl nan-111.”. ‘3111?r‘.,‘r.* TO M‘ \J‘b’a‘) ‘ahv DI/OR de3— L.b<'l."‘-J '3 CL’Vn-QU‘f-L Jack-U“ Growth Gain Per Day(3.) Growth Gain Per Day (5.) Food Consumed Pe? Day (5.) Diet Hales Females Eales females 10. I-A. Unlimited Drinking water (for 95 Young Hats): 1 0.27 0.25 0.101 0.096 2 0.28 0.27 0.074 0.070 3 0.27 0.23 0.055 0.047 5 0.49 0.43 0.112 0.093 6 0.36 0.26 0.065 .Cfi? I—B. Limited Drinking Eater: 1 0.19 0.17 0.072 0.064 2 0.19 0.21 0.051 0.055 3 0.21 0.21 0.042 0.041 4 0.23 0.20 0.071 0.062 5 0.24 0.19 0.035 0.043 6 0.22 0.13 0.041 0.029 II-A. Unlimited Drinking water (for 13 Older Rate): 1 0.15 0.13 0.137 0.050 3 0.11 0.09 0.024 0.013 4 0.13 0.12 0.039 0.038 6 0.07 0.10 0.013 0.019 II-B. Limited Drinking Tater: l 0.10 0.11 0.046 0.041 3 0.04 0.11 0.009 0.024 4 0.11 0.12 0.056 0.039 6 0.11 0.12 0.020 0.022 13 free filtrate forms phosphomolybdic acid with any phosphate present. Ehe phOSJhomolybdic acid is reduced by the addition of l,2,4-aminonaphtholsulfonic acid reagent, to produce a blue color whose intensity is proportional to the amount of phosphate present. Inasmuch as inorganic phosphate is slowly liberated from phosphorus compounds on standing (30,31),the blood phosphorus was anlysed almost immediately after it was drawn. T10 amount of anticoagulant used affected the color tre- mendously. tormal color deveIOpment was obtained by using about 2-3 milli;rams of lithium or potassium oxalate per milliliter of blood. Excessive use of anticoagulant resulted in a diminished Color intensity, sometimes to the extent that no color was produced at all (Table V). The color intensities of the solutions were ueasured exactly five minutes after the addition of the aninonashthol- sulfonic as d reagent, using the Hellige Jhromatron” photo- electric coloriweter. There was little change in readings at the end of 10, 15 and 20 minutes. rho concentration 0“ inorganic phosphorus in the aliquot was calculated using the density values of the blood sample and the standard phosphate solution; the milligrams of in- organic phOSphorus per 100 ml. of blood is equal to density of sample ~-—-”~~~*~---~-—--- :: 0.0}? :< 100. density of standard analytical Xethod ”or Inorganic Phosphorus in Tood Samples: For the dotcrninatlon of inorganic phosphoric in the diet, 1” TISO m Lil-"1‘ 5.33 130.2(1)"? D ELI) ‘I‘ For lithius eralste ( 2-? ng. per n of blool F"““”C“1Cl)( 2) Amount used: Color into: sity: 2 m;. cl. 0? blood KOFTRI color de elonment ._ - _ , u u n 5.3 nQ. ml. of blood n n n n m . 8.0 pininishcd color csuclosscnt n u n v a . 11.0 ' tracticall" no color us elo‘owet '20 5:. n u n n n n n ' n JVOJ ‘ .I‘ r? ~ V ‘- v 4. c. .- f‘ V '3 ’ .I '. \‘r‘ _. v“ 1' \ --V O - r soulun citrate ( ) mg. 70? ml. 0a eloei recovlenled)( a) Amount used: Color intensity: 5 mg./ml. of blood Normal color deucloooest 8 N H H H H I! I! 1;: II N H H H H H '25 " " " " K;Iy sl-;;t color " 4O " " " " Sractically no color de‘welo nent 20 a weighed sample was placed in a porcelain dish, e; rrei slowly over a free flags, 31‘.i finally burn-d at a dull red heat in a muffle furnace to a white ash. The ash was dis- 1 solved with hydrochloric as d, the solution filter ed, and made up to 200 ml. A 50 n . aliquot of this solution was then withdrawn, diluted and heated on a steam bath for seve- ral hours until the solution hai e accreted to ancroxlratelj 40 ml. fhis solution was then e "lo ed for the determination of phosphorus, by the metr ed 0” double precipita tion, first as annonium phosnhomolyhdate and then as ammonium-magnesium- ‘ 1. a - m- -.~. .. ,...,. . - - , ., u ‘14. - phospi ate, with final hul Ling as manesiln oyropnos hate, all “I '\ essentially as des crihed in the methods 0? analysis 0: the Association of the “fficial agriculture 1 Chemists (33 . rhe figures found in Table II are the averages of a group of four young rats, each containing two males and two females. Sixteen rats between 2 to 3 months of age were set up for Eiets l,3,4,and L at the earlier part of the exocri- mcnt. 30 rats of the latter age group were fed Diets 2 and 5. A comparison was made between the growth rate of the rats and the various factors involxed in the experiment. The variables considered were the amount of drinking water sup- plied, the type of diet employed, and/or the amount of phos- phorus consumed from said diet, and the sex of the exocri- mental animal. The conclusions were obtained bi means of analyses of variances, based on he figures found in Table 61. A two factor analysis was made on the blood inorganic pHOSphorus content of rats which received a limited amount of drinking water. The type of diet used (for source of phosphorus) and the length of time when drinking water was restricted were the two variables considered. The results found in Table IX were obtained from rats which were 21 to R) U—n days old at the outset of the experiment. Those included in Table KI involves both the young male rats (21 to 25 days old) as well as the older ones (3 months). Explanation 0? symbols used: D - type of diet 31 Control 71 32 " " plus 0.53 DI n n n l 3 oh Control j2 H H d - amount of drinking water Y1 Unlimited water K2 Limited water 01 fl 91 hale rat 32 Female rat T - time period when rats were given limited anounts of drinking water ' i "‘ H r" I! T4 :3 I.» I! :9 7 tr 8 H 10 h .112 1 3 N T 24 " T 26 " - sex of the experimental animal d) A. U) 51 El 32 S1 2 B. Ratios of Growth Gain Divided by S1 17:, 1 32 81 We 8 0.27 0.25 0.17 D1 0.101 0.096 0.072 0.054 0.19 32 0.074 0.070 0.05 0.055 TQULE VI 0.21 0.21 0.055 0.047 0.042 O . O‘Z‘Ll FhOSphorus Consumed L”) 4: 0.123 0.129 0.071 ”A 0.0}. I: Ratios of Growth Gain Divided by Food Consumed 0.19 0.112 0.093 0.055 O o O'Q‘B 0.16 H O\ 'XZIALY )T Source of Eotel X 3 error Total EPPOP "Ill! TATLE If‘ ‘V 0 ‘ ".X Mm” 0“ TEES DIVID1J BY T003 Variance Degree of 25 5 l l 5 3 f it 8:11: 11 S 1 5 ’3 t ”-12: ll 5 l 5 Fisher ratio 11 H II N H signifiioant at the 3' 24 0 3 Q'T" GXIE #827 119,321 1+",- .4121; 740 'K‘ '3" 5,392 1,770 2,030 2,036 3 level n l 3 H "0.1 3 "I PO U1 “ABLE VII-A .— P Overall Buss and Averages for Variables firowth Gain Divided by Tood Consumed “ I ‘ variables X K Variables X Di 0.33 .22 02 0.95 0.24 31 3.41 0.23 03 0.92 0.23 32 3.01 0.25 t) p- 1.36 0.34 1055 0.31“ 1‘: 4.02 0.331 U m D , 1.00 0.25 1,12 2.40 0.20 O". 31 0.52 0.25 0.36 0.13 (:1 H a. 0.50 0.25 03 0.42 0.21 Du 0.93 09"“? T14 0043 0022 Um 0.92 0.45 D O.h 0.22 3 .2 05 0.62 0.31 0 0.: 0.19 2.13 .36 (d H 22 1.91 .32 CC! PO G.) ( DIVIDED BY FOOD CONSUMED F WEIGHT GAIN (6.) .50‘ .45‘ ,4 0“ 30' 25‘ ,20‘ CHART 1 FIRST ORDER INTERACTION L‘» AT LEVELS Wan-wl awe .UNLMTID warea- ‘. Rat K0. Diet E0. Eoginnlng of 1:“fi. *1 u’. of Becks on m3.P/lOO K20 Supply ml.Slood 0 ID \2.) 3 months 24 K)! m -1 u l :5 I! I! 22‘1‘ 4 O [‘3 O ##f-a- C‘\ u) 4:- U“: (“x O ' 24 I ‘1 (Tx 17 1 21-25 days m C \ .1:- O K] m 70 H K14 R) 0\ U1 0 D O 25 4 n n n “1‘ 4.43 '1 {I t! n t h) to O\ m 0' \ 0 VJ AIALYS‘S O’ Source of Variance Total D T Error wwww A f" 1“ ~‘ .2 f\_ -‘ . . ' ;- I - .3‘»/ mi .) .V pp - J v v n. 1' '* —.- u _. L L . “.LJ.» A - Freedom (1 1‘ $" (1’7“.13'“ '...“\.a].x.L ‘ J. u)-- ‘9 (“1- scan squarew .4 14.090 2.271, 5.993“ 427 Overall Sums and Averages Vor Variables Variables U 22%. Dz v, .‘ '15" (.1) O >4 L43 w L») H H KID-4 OCDKOV.) FHA O O cps o mus >3 owom kkkw kw mph? 0 «wk (2-) k): Hithin the limits of this exoerimeat, the amount of drinl'.in3 water suoeliei, and the type of diet used and or he amour at of phosohorus free the different diets have a very marked influence on t2'1e growth of alb Lno rats. The factor with the least significance is the ser. 0.‘ .._....__1 is indicated in Table $11, the three variable evolved exerted a marked influence on the growth rate of the rats. Of these factors, the amount 0? dri :ir v.ater supplied hai the greatest effect, and the sex of the animal, the least effect. tith an unlinited water intake, 3r wth rate of the rats :as 0.34 compared to 0.20 at a limitea water intake. Lats 3iven Diets 4 er d 5 3a"e the hi3:nest 3ain weight (0.34 and O.;# Ies11ecti1ely), followed by those t11at had Diet 6 (0.25). These given Diets 1,2,and 3 had the lowest evera3e growth rate (0.22, 0.24, and 0.23). This is as expected inasmuch as Biets 4, 5, and 5 contained more milk. Ehe sex of the animal also hai an effect on growth. fiales had a greater weight gain (0.23) as compared to the females (0.25). Of the interactions indicated by tLis three factor nalysis, the only $1311 ficant one was D ( .'. T1118 1? dicates that thee ffe ct of 1.1ater snooly on growth is deo. mnd1t on the 35 type c: diet encloJea, and v ce verse. A clear picture of his interactions 093 ha seen on Chart I, On the other hand, the eff cect of sex is 1r ede 9e endent of lay othe main effect. The 3rowth ate of rats 3e net on ax‘x unlir.ite1 water supply is 3reatly affected by the tyre o? diet 0 toloyed. Fats fed Diets 4 and 5 Save an appre M01 hly hi3her wci‘ht 3ain (0.47 and 0.46 ) than those fed Diet 5 (0.31). Diets l, 2, and 3 gave the lowest 3rowth (0.26, 0.23 and 0.25 reseectively). The sex of the animal did not affect the rate 0? growth. 11th a limited water intake, the type of diet employed and sex did not affect the rate of 3rowth. Phoaehorus Intaka,versu0 gravih-:a391 "F From Table VIII, it is apparen 1t that tte amount of drink- in; water 3iven, the amount of phosoherus consumed Tron the different diets, and the sex 00 the exocrinelcal anir al are the important factors affecting 3rowth. The interaction involvin3 the sac cunt of phospr ores consumed and the water sup— plied is also significant, whic dimpiies that these two factors are dependent upon each other where 3rowth is concerned. A graphical interpretation of this interaction is shown in Chart 2. The most significant factor is th is ar cunt of supplied. Thendrin31n3 water was uulinited, 3rowth rate was 0.09 compared to 0.05 when water was limited. The phosphorus intake is also of inportance. Based on the same amount of phosphorus, rats fed Diet A 3ave the great- est 3rowth (0.10), followed by those given: ets 1 awid (0. 08 36 and 0.08). Growth rate was lowest for those given Diets 2,3, and 6 (0.07, 0.05 and 0.05 respectively). As shown in Chart 2. an increase in phosphorus content by the addition of sodium dihydrogen phosphate resulted in a slower rate of growth. This was true whether drinking water was limited or not.The deleterious effect on growth was greater on Diets 4.5, and 6 than on Diets 1,2, and 3. then drinking water was unlimited the type or diet from which phosphorus was acquired was or significance. The sex of the rat did not affect the rate of growth. The sex characteristic of the experimental animal was not important when water supply was limited. Phosphorus con- sumed from the different diets. however, was significant. Rats given Diets 1 and 4 had the greatest growth (0.07 and 0.07) followed by those which had Diets 2, 3, 5, and 6 (0.05, 0.04. 0.05 and 0.04 respectively). filgod inorganic PhosphoggshContent: As seen in the two factor analysis of variance on Table X, the type of diet and sex of the experimental animal did not affect the blood inorganic phosphorus content. The only fac- tor influencing the phosphorus content was the time that the rats had been subjected to a limited amount of water. Blood phosphorus content was about the same during a period of 3 to 8 weeks of limited amounts of drinking water (T136.64mg./ 100 m1. of blood, $236.22. T3=6.88, s4=6.27.T5=7.21)3 1t 37 decreased when water was restricted for a longer period of time (T5=S.SE. T735.63. Ta=5.18). When young and older rats were given a limited water supply, the blood phosphorus content was not affected by the type of diet used (Table XI). However, it was greatly influenced by the length of time when the rats were given restricted amounts of drinking water. After 25 weeks of limited water in- take, young rats had a blood inorganic phosphorus content of 4.98 mg./1OO ml. of blood. Older rats had 4.45 mg. of phospho~ rus after 23 weeks of restricted amounts of drinking water. l lync-oa‘~, gpm 1: 1'? L.,. 38 SUE?! ARI 0n the basis of weight gain per food and/or phosphorus intake, the following conclusions were reached: 1. 2. 3. 5. 6. 7. 8. 9. Rate of growth was influenced by the type of diet fed, the amount of drinking water supplied, and the sex of the experimental animal. Rats kept on an unlimited water supply had a greater growth rate than those with restricted amounts. The males, on an average, had a greater growth gain than the females. When the same amount of sodium dihydrogen phosphate was added to the different diets. the rats fed diets contain- ing more milk and less rice gained more than those fed diets containing less milk and more rice. than sodium dihydrogen phosphate was added to these diets, growth decreased in preportion to the amount of phosphates added. With an unlimited water supply, the type of diet had a marked effect on the growth rate of young rats. Sex did not affect growth when water supply was unlimited. On a limited water intake, the amount of phosphorus con- sumed exerted an influence on the rate of growth. Male rats kept on a limited water supply had almost the same growth gain as compared to the female ones. 39 On the basis of blood inorganic phosphorus content, the following conclusions were reached: 1. 2. 3. The type of diet fed to the rats did not affect the inorganic phosphorus content of the blood. Sex of the experimental animal had no significant effect on the blood phosphorus content. The level of blood inorganic phosphorus was influenced by the length of time to which the rats were subjected to a limited amount of drinking water. Longer periods of res- tricted water intake resulted in a smaller amount of phosphorus in the blood. The age of the rat was important in relation to blood inorganic phosphorus content. Younger rate had a higher phosphorus content than the older ones. J A 1' I‘ll-1:5 G A. u T. L . J . E ”ILLIUJ‘ADHY 1. Lonningor, A.L. and E.P.Kennedy, "The Requirement of the Letty Acid Oxidase Complex of Rat Liver” - J.Biol. Chem., liLaTBB (1933) 2. Ce corne ,T.:)., L.T.Tcn e1, a: d E.A.Park, Proc.300.3xp.Biol. 8.1“” 3911:. £43., u] (1929-2; r) 3. Liohi3,J., "“ie C};on 110 in Ihrcr Anwen1n3 au:€ Agrihultur und .nysiologie" ,it11 ed., “ra‘nochJC (l...) 4. Villo,£.}., 'Lcc horchos Experimentales sur la Vegetation", Paris (1353-57) 5. Hardcn,A. and Young,fl., J. Chem. 800.. 3;, 139 (1905) 6. Ha rdozi,a. and Yozzg. '., Proc. Boy. Soc. 3., 11,405 (1905) 7. Landauor,A., "Ungar. Arch. iiod., i, 133» (199+) 8. Straub, n., Zeit. 3101., 2:. 537 (1399) 9. anh.3.3.. "On he Influence of Copious Tater DTiNKiF ", niv. Penn. Led. Bul., Larch,l905,53 pp. 10. In.odon, 3., L.Grafe, and LH.30hmlts, "About the Incroa so in Azroductivity lnrough Phosphate -)upply" , ~anslatio:1from hoove~4c110r s Leitschrift foe: th. C.emie, 113th vol., p. C7 (1321) ll. Sherman,H.C.. A.J.Eetlcr, and J.E.Sinclair, "Calcium, Magnesium, and Pi loophorus in Foods and Lutrition", v.3. Dept. Agri., Off. nxpt. Stas. 3u1., 222. 70 (1911) 12. Schno der,h. and H. Steezibock, "A Low Fhosphorus Diet and the hesponse of nets to ’v'itanin 32", J.Riol.Chen., 133. 139 (1939) 13. Schneider,H. and H. Steonbock, "Low Zhos horas Diets and Urinary Lithiasis", 3.3101. Chem., lxrvvii (193 9) ate of Deficiency of Phosphorus on the 14. ,Forbes,3.8., "Lffe Food -nergy and frotoin", J.Lut., ;fi, Utilization of 419 (1937) 15. hDay,H.3., and 32.chollum, "”inernl ”etabolism, Glowth, and 83? ptorxalogy of Q;ats on a Diet thronely Doficio:1t in Phosphorus”, J.Biol.0hem., 130, 2L9 9(193 9) 18. 19. 20. 21. 23. 24. 29. 30. 41 Glanzmann,E. "Disturbances in Calcification in Richets", ‘JvoIEGQ. Assno’ L353 857 (1926) Sherman,H.C. and L.E.Booker, "The Calcium Content of 'the Body in Relation to That of Food", J.Biol.Chem.. 21.93 (1931) Shch1,A.and 3.3.Volbach, "Hickets in Rots.XV «The Effect of Low Ca-H 13h P Diets at Various Levels and Ratios Upon he Production of hickets and Tetsny" ,J.E Mt..l1. 275 (1936) Bethke , £1. 11' o ,CLHLKicli, and VLLilder," he Effect of the Ca-P Feletionsixip on Growth, Calcification and Blood Composition of the Eat”, J. Biol. Chem.,9_,3 3‘9, (19% Cox,V.H. and M.Imboden,"”he hole of Calcium and Phos» phorus in Determining Reproductive Success", JLHutL, ll: 147 (1930) Hubbell,R.B.,L.B.Vendel, and ALJLVaAemah, "A how Salt Mixture for Use in Experimental Diets", J. HutL, ;&, 273 (1937) Sheman,LI.C. and SJ. Zuinn,"1"fl e Phosphorus Content of the Body in Relation to Age, Growth, aim Food", J.Biol. Chem., _1, 5C7 (1925) Cohn, 3.3. and D.H.Greenberg "Studies in E~'ineralI eta- bolism with the Aid of Artificial Ladioactive Isotopes" , J031Olo Chemo. lfiog 2i 5 (1929 Euebner,h’., "The Phos chorus Content of Deficient Orzo n8 Under L>iffereht Dietary Conditions" Arca.Lip.Laun. Fharn., 1.: 24 (1914) Kilcs,L.H. "Eetabolism and Chin-Tuna Fens, "Calcium and FhOSphorus in Osteomalecia",J.Expt1.Hed.,fi;,137 (1925) Gregersen,J.P.,"Heseerchos on Phosphorus fietabolism", L.Physiol.Chem., 12, 49 (1911) .. :ddy,V.n., H.n.Hu11er, and? .L. Heft, "ihgtin as a Source of {hos phorus in the f—revontion of Lickets" ,J.Biol.Chem., r’Ofiili (1922) Bruce, II.H. of Inositol~zexaehoschoric Acid” ,Diocth. Jour.,2 (I934) Fishe,C.A. Role 1 517 and E.X.Callow,"Cereels and RicketszT i. :30 and Y. E}ubherow, "The Colorimetric Determina- tion of I’hOSphorus' ,J.Biol.Chem.. §§. 375 (1925) and FLHobinson,i Biochem.Jour.,;§, 755 (1924) l 33- 34. 42 Burkens,J.C., "necessary Precautions in the Determination of Phosphorus in the Blood", J.Netherlan. Tijds'chr. Gonoeskunde, 12, 4944 (1934) Hawh,P.B., B.L.Oser and W.H.Summorson, "Practical Physiological Chemistry", Blakiston 00., flew York, 12th ed., p.491 (1951) "Official and Tentative Eethods of Analysis of the Assn. of org. Agri. Chemists", A.O.A.C., Hash.,D.C., 5th ed., (1940 . tioal Tables for Biolo- gical, Azri search", Oliver and Boyd, 3rd 63. (19433 Department of Chemistry Michigan State College East hunting, Michigan -459. Date Due Demco-293