UNIVERSITY OF MISSOURI COLLEGE OF AGRICULTURE

AGRICULTURAL EXPERIMENT STATION

l�egenrrb �1I[(ctill 190

GROWTH AND DEVELOPMENT pPilh Special Reference 10 Domeslic Animals

XXVIII. Decline of Endogenous Nitrogen Excretion per Unit Weight with Increasing Weight in Growing Rats, and Its Relation to the Decline in Basal Metabolism. Decline in Live \/ITeight, Nitrogen and Energy Metabolism with the Advance of the Period of Nitrogen Starvation and the Influence of Live vVeight and of Pre­ceding Level of Protein Intake on These De­clines and on the Survival Periods.

URAL S. ASI-IWOJ(TH ANU SAMUEL BRODY

(Publication Authorized JUlie I, 1933)

COLUMBIA, MISSOURI

JUNE, 1933

UNIVERSITY OF MISSOURl COLLEGE OF AGRlCULTURE

Agricultural Experiment Station EXIiCOTlVE nOARD OF CURATORS.-MERCEIt ARNOLD. Joplin: F. M. McDAVID,

Springfield; H. J. BLANTON, i'uil

ADVISORY COUNeIL.-TIlE MISSOURI STATE BOARD OF AGRICULTURE

STATION STAFF, JUNE 1033

WALTER WILLIAMS, Llh D., P,uident

f II. MUMfORD. M. S .• D. Arl., Direclor S. R. SHiRKY, A. 1\1., AliI. 10 Director

MISS El.LA PAIH.IEIER, Secretary

flGRI CULTUltAI. ell EI\·IISTll Y

A. G. 1I0GAt., Ph.D. f .. I). !IAlG". I'h.n, W. S. ItncUlP-, Ph. D. I�. W. CO'VAtI, A.M.

ROUr:I\T BO\lCII"II, J ..... A.M. !,uTI.r; .. R. RICII.UIIIIOtl, I'h.D. U. S. ASII"'OaTll. A.B.

AGIUCULTURAL ECONQfo,·IICS

O. 1t' loIINION, A.M. Ur:" 1- . FII ...... r:, A.M. F. L. T"o"'I':I<, I'h.O. C. I-I. HU.N ...... Ph.D.

AGRICULTURAL ENGINEERING

L C. \VOOL�Y, M.S. t.IACo; !\1. JONU, M.S. It. It. !'.un. 1'.,11.1. I). I). SOllnr. A.M.

ANIMAL IIUSBANDIU'

E. A. Tro.ownroo1'!. B.S. in Agr. I.. A. Wuvu. U.S. in A8r. fl. G. 1I0c ... I<, Ph.D. f. U. i\·lu�'l'"oro.n. M.S .• D. fI�,. D. W. CnrTTEI<UItIl. ,\.M. F. f. !\lcKuIl;'It. 1'10.1) .•

I. E. COlll'"oaT. A.M.-1. C. MOFfItTT. ,\.M.

R"'Lru \Y. I'IIILLlrs. fI.t-1. S. R. JOIII<�ON. A.M. C. E. To; .... ".L. B.S.

BOTANY fiND PATHOLOGY

\Y, J. RObO',.", Ph.D C. M. "l'uerr:""'. I'h.V.

DAIRY IIUSB,\NDRY

A. C. R"'C!O"'LI':, M.S. WlI. II. E. RE.n. A.M. S""VI!L Brt.ooy, Ph.D C. W. TUI\lurt.. rh.D. W ... ro.ro.\:N G'I'"I'"O.II. A.M. E. It. G ... UrsON. A M. II. A. H ......... N. 1'..,..1. M. N. IhLU. B.S. W .... U:N C. H ..... L, A.M. H .... oI.o ALLEY. U.S. \V".I.I"" E. Ecltl. It •• B.S.

ENTOMOI.OG\'

1.1':0/'1 ... ,,0 HAlUI ... /'I. I'h.D T. E. B,UETT. A.M

fiELD CROPS W. C. ETnUIOGII. I'h.D. C. A. I,IEL .... A.M.-

tin cooperative lerv;ce ... ith .he U. 5 I)ep�rt'nenl of AKficullllfe.

L. J. ST ... nl.E". Ph.D.· R. T. KUU"T"'Crr:, A.M. Il. M. KrflG, A.M." F.. M ... ro.IOfl Ihowfl". A.M.­"'11" eLA"'" FUlu. M.S.-

1I0ME ECONOMICS

r-.1AU:L C ..... 'BIII.t.. A.M. J ...... .: ALICE CLI NI!. A.M. AUP.:Lt." Errl!l. Glfl"TU. M.S. SYLVI ... eovn, A.M.

�:��I7 ... nn.��·:T�"

I�h. 'f:;�. hUIII V. COl.l!f. I'h.D. f.,'irNI!U'" V. GUClt. M.S. FII. ... fl"cU SUD', M.S. IlKn"" K. Wlurnl!. M.S.

HOltTrCULTUJtI� '1'./. T ... LIIEII.T. A.M. A. �. MUII.fl"I!I!I:. I'h.D. II. G. S ...... II.T"\1'OVT. A.M. GEO. C ... II.L VIIIIO". Ph.D.

l'OUtTRY IIUSIJANDRY

II. L. KIIMrlTU, M.S, E. M. fUfl"Ii:. A.M.

RURAL SOCIOLOGY E. I.. MOlI.o ... N. A.M. W"'.TEa BUII.It. A.M. 1I1tfl"II.1' J. BUIlT, A.M. Aro.Tnu" S. E"IU, Ph.D.

SOILS

M. F. MILLItrt.. M.S.A. H. II. K"Ulnor,. A.M. W. A. Aurt.ECIIT, Ph.D. II ... ", JIIII><1'. Ph.D. I� D. lJ ... \·u, Ph.D. 1I .... oL .. f. Rno ... oll8, A.M.

VETEIlI NAR Y SCI ENCE

A. J. DU""'"T, A.M., D.V.M. J. W. COIIII ... W ... ' . D.V.M .. M. D . el!CIL El.ol!It. A.M . • D.V.M. O. S. C"I!tRl<. D. V.M. AIIIJIlP.W Urt.P.N. D.V.M. A. M. McC ... ru, I).V.M. 1·I ... ro.o •. o C. Mcl)ovor.lI. A.M.

OTIIER OFFICERS

R. B. I'",CP.:, B.L .. Trc�.ureT LI!SLI� COW ... II. B.S., Sec'y of Univenity A. II. JEH.U. A.B .. A8riclIltllui Editor J. F. U .... nul. PhOl08Uphcr J ... NII Fltoosnul. Librarian

CONTENTS

Abstract and SUlllmary 4 Illtroduction and General Discussion of Problem

Results 8 Detailed Discussion of Results .10

Endogenous nitrogen excretion as fUllction of body weight .1.0 Decline in total nitrogen excretion, food intake, and ratios

of fecal nitrogen to food intake. ."11

Relation bctween b"lsal metabolism and cndogenolls unn-ary-nitrogen excretion .......... 12

Decline in energy metabolism on the N-free diet

Innucnce of the preceding \evci of protcin intake on the limc reqllired to reach the endogcnous level of nitro-

.10

gen excretion ............................ 14 Decline in body weight ....................... .......... 16

Survival period .... . ............... .................... 17

Conclusions . ........ I 7

FOREWORD

t > �J'It.e. s�cci<l! ,investigation on the gTowlh and development is a cooncrativc

clI.c �prlse III Whlc1� the departments of Animal Husbandry, Dairy Husbandr �.grl�.llltllral C}�cIIILstry. Ollie! Poultry HI1:;bandry have each contribl1\ed a <;ul;� � ,11111,11 ]lar�. II,H! Illans for Ihe inveslit-;'alion ill the beginning were inallg�lratcd /j' [ cf..'llmllt�� 1IlCludiug'" F. B. �rl1111 ford, A. C. [�agsdalc. E. A. Trowbri�!gc

: ". ,,-emIlS!c l , A. G., Hogan. Sallluc.i Brody served as Chairman f 1', � n ,HltcC and has been ducHy responsible for the eXI..'Culioll of the )hll� i[�t�: l��:� :;��c of '�fSll�IS all�1 �h� preparation of the puhlications resulting ll;' on� this �lIte'r= r . - Ie Ilivesllg.ll1on has been Ill<ldc possible through a - . I 1 I H 'ra�cJl FOImd;ltioll represented hy PI', R Vi '1'1 I '

, . Igr'I

II�. )y.t le erman

:HlvICC from the be<Yinning o[ ,I " . ,'.. latc 11;1 , W 10 I"� g"IVCll valu .. bll; '" . Ie lIlyesltgallOn.

F, n. MUMFO�..n, IJiralDr AgricuftHml EX/ll'I'illleJlI S/llli()n

.,., 'ABSTRACT AND SUMMA,RY

�} 111��'I�� /i� '�O�II���I�:l ll il�l:t-\�e�I���i�il�;;

t6�o

l����'

e:ft i

;:�'� e (�s '��'h:�'is ��

etl;�c��!;�)

incre;;inhll\

i��lelYt�ital �ndogellolls urinary-nitrogen coefficient decli.nes will:

ralio of 1�1.sal CIlC�' �bl .15 .doc� the ba�;�I.-en�rgy metalJolisUl c.ocrficie.n l . The

(aboul 0.7 Calorie;�):�l�:;�}Q!ls�n to b,I:;:il 1!lIrogcll cxcr�tlOll IS thlls constant

inine-N to cllIlo'cnous . . ' Cll Og�1I01lS IInna.ry-N), while the ratio of creat­

lila! (this percel�lage ra�i�Ilf:Y5�n '�lc.35��es with ilH:rea,se .in size of the ani-

1\ IS sll�gested that the [nlensit of ha��fn

� r,at; al:d .2 III a 340-gra,.n ral).

me.tahol!slll, is limited by the ". �. . . I ncr9y, .IS als

.o of basal nllrOgen

A prolong-e(1 period 0 . NC lve Pt�top <lsmlC mass" III tbe body . I!le same minimulll level a

l� �;uri�

:eer (It!! rc ,h.'tes the energy, meta hoi is III \0

Calories per S(tllare meter e I gp o onge( .1 solule starvation, namely 400

'TI [ I f p r (<ly.

Ie eve 0 protein in lake. pre 'cdi I ' I f . has a profound influence 011 Ihe "

C Ilff. tIe peno( 0 nllrogen starvation

imlllll) level of nitrogen exc 'cti lint; r�qull.ed to reach the cndo�ellolls (mill_

levcl for "!ow-protein" nts Ian

�1;8(!j. lcql[me��I� clays to. reach Ihe minimulil

required 10 rC'lch the .,:10' ( :- I (tlylS [or llgh-protem" rats). The tilllc

f ' ' ( genou:; eve 0 N c.xcl't.;tio . II I

unctlon of the level of Ihe pr�cc]' . . k'.n IS HIS an un {nown

poriallt influcncing factor I ' , ,'llIlg protetn

llllia c. Llvc weight is not an ill1-

I ' " III lere IllIlSI )C great indi . I I . . t IC "nullals laken fronl Ollr reg I . I .1 l"

. . VI( un varlatlOns as reach the lllinilllUlll level

U OIl S oc .. (let n:qulred from <I to 26 days 10 The fecal nitrogen 01\'" N-free d' -'I . '1

roral nitrogen excretion (aho I 20c/ )IC .15 a ne�t y constant percenlage of the

advance of the pcriod on' II �I N' [/C wl,llh a ,sltght tendency to increase with

'k . Ie - rec (Il't rhe ralio of fe I N [d' a .� lIlcreascs very markedly with th ' 1 ;' [ .

, ca - . to 00 111-V<Itlon,

t; .1( \anee 0 the penod of nitrogen star-

Depending on Ihe initial livc " d 1" I . ' survive up 10 six I110nlhs N f

WCIf: 1 S ,Ill( ag{:? of Ihc allllll;tis, rats In;\y

duritlg this time ahoul 601j{°'�f II r.eel·(hlll o.tl,crwlse. complete) diets, losillg

percenlage loss as was pre\�ousl 1�lr I�e

f weiR II. which is the. s,nlle order of

of live weight 011 the N-frec diZr �lll1t or?11 absolute fast. The time cmvc

successivc usc of lissu' [. S l�WS st;urcas� phenomena slIggcstinf{ the I

. C.<; 1"1 successIvely gre'ltcI' sl'll Tt '1'1 .

P lenOtliena arc also exhibit' I' � " . • • ,JI I y, lese slalrC;lse

e( !11 t Ie lillIe curves of mtrogcn cxcret:oll.

GROWTH AND DEVELOPMENT H/ilh Special Reference 10 DOlneSli( Ani.lIlllls

XXVIII. Decline of Endogenous Nitrogen Excretion

per Unit ,,yeight with Increasing Weight in

Growing Rats, and Its Relation to the Decline

in Basal Metabolism. Decline in Live vVeight,

Nitrogen and Energy Metabolism with the

Advance of the Period of Nitrogen Starvation

and the Influence of Live Weight and of Pre­

ceding Level of Protein Intake on These De­

clines anrl on the Survival Periods.

UllAL S. ASHwonTH ANI) SAMUEL BRODY

INTRODUCTION AND GENERAL DISCUSSION

It is generally known that the basal eJlergy melabolism pCI'

unit weight declines with increasing \",eight of animals. Little,

however, is known concerning the manner of change of basal nitro­

gen metabolism per unit weight with increasing weight, and of

tht ration between basal energy and basal nitrogen rnetaho1islll.

The m.ajor object of this research is to contribute some data and

discussion towards the solution of these prohlems.

VYe have demonstrated with considerable definiteness that the

total creatinine-N excretion per unit live weight in the rat is I'd a­

tively constant between the age limits �f �� and GOO days alld

weight limits of 50 and :1flO grams (MissollriRes. fiul. IS9), 'fIle

basal energy metabolism per unit weight, on the other hand, de­

clines between these limits from about 200 to about 90 Calories

per 1.:ilo Jive weight ("rvlissollri Res. Bills. H16 and ·l'7G). The oh­

violls conclusions, therefore, is that there is no direct proportional­

ity between energy met<lbolisll1 and creatinine excretion; or th;lt

the functional relation between basal energy metabolism and creat­

inine excretion is the same as between basal energy metabolism

a nel body weight,

These results on t.he rat were suhstantiated all dairy cattle

(Missouri Res. Buls. I (Hi, 1'76, and 191 ).V\'hlle the ahsolute values

uf the creatinine cpefficients in cattle differ from those in rats (the

t(;tal creatinine-N coefficient in male rats is of the order of 15, while

III female cattle the total creatinine-N coefficient is of the order of

P,a.per No. 60 in the Herman Frasch Foundation Series.

G MISSOURI AGRICULTURAL Ex I'ERI M.ENT STATION

17, and the preforllled crcatillinc·N coefficiellt is of the order of

n.v), they appear to remain constallt at all ages and weights (if the

conditions of fatness relllai1l the same), and they arc lI ot influenced

by the dietary protein level (at least not in the rat), while the basal

energy metauol islll coerricients decline rapidly with increasing

weighl. If, as is generally believed, the crcatininc-N is an iudex of

l otal cndogcnous-N excretion, then the endogenous-N excretion

per ul1it live weight should likewise remain ... -0115tal1t at al: ages and

weight. Thi� gcncr:t1izatinll would seem to be 5ubstal1ti;'llCd by

TVrilchell's cO lllpikl. lion (B\I\. Nat. He�. COllncil, No. G7, VVilshing­

tCll, ]929) indicating that the urinary endogellous-N is of the order

of 2!J-3ii mg. N. per kilo live weight flOr such unrelated species as

pigs, caltle. sheep, humans. VVe have :tltclllptcd to find out whether 01' not this conslancy

for the endogenolls urin:lry-N excretion hohls true for rats v;}rying widely in live weight; th;}t is. whether or not the creatinine-N is a constant fraction of the total urinary cndogeuous-N at various live ,,,'eights. The results were l'epor ted ill 'Missouri Res. Bul. :IS!), but tlley were not �atisf:lctory, because ot iler difTictlltics camc up, as for examp le the question concerning the enormoliS variations of the till1e required to reach the minimu111 coefficicnt, and the factors causing these variations. The preceding report, thus, ",·hile soh'­iug' the total creatinine coejficient problem. leaving no dOllbl' that it is const::lllt-that i�, it is not afTecled by live weight, age . or dietary proteil1 level-, has Idt the total endogellous-N problem . ilnd its relation to hasal energy rnelabolisrn in an unsatisfactory �Iate.

l'vlitchell and Halllillon in their Biochemistry of Amilia Acids (New YoQrk, 192il), while they pub lished a tablc giving the endog­enous nitrogen excretion in rats of diITerent ages, cle-a rly illdicat­ing a decline ill the endogenolls nitrogen excrction per unit wcight

with incrca.5ing weight, did not discuss the relation of this decline tn thc declinc in basal energy metabolislll per unit ' .... eio:rht with in­<.:rcasing weight. This lllonograph seellis to give the

O impression

also that the creatininc�N is a constant fractioll of the total endorY-o <':1I01lS nitrogen.

The tahle in 1'\11 itchell and I I amilton 's book is based 011 (jnta ubtained incidentally by Mitchell and Carman in cOllnection with a rC.5earch 011 hiologi<.:al values of proteins. They assullled that four days 011 a N�free diet sufficed to reach the endogenous level of urillary-N excretion. \,Ve, in this work, kcpt the rats on a )J-

RESE/lI�CII DULLETIN·190 i

free diet until the lime curves of N-excretioll showed an irreducible lllinilllUlIl.

More recently Sllluts, Illlder Mitchell's direc tion (Smuts, An Abstract of a Thesis, Urbana, 111., lUa2), studicd this problem in mature animals of different species (mOllse, rat, guinea pig, rabbit, pig). The [ollowing quotations [rolll Smuts' abstract summarize his findings:

"The <l.vcr<lge icrcatinine coefficient!' for thc different species are: for malc r<lts. 41; .for felll�l; rats. 40; f�r �uinea. I)igs, 3<1: .for mice, 81 � for r;lhbits. 46; and for p gs, 19. J hc temlency IS. 111 the smaller :1111111<1.15. for the creatinine nitro­gcn to represcnt <I smallcr. P:Cfccll

.tagc o f. the tol:11 endogcnous nitrogcn. The �vcragc perccntage of Crcalltlll1e IlItrogcn III ICI"IIiS of 101:11 clidogCIIOliS lIitro�cl1 15' for 111ICe,

:1.9 pc,r celli ; for m;dc r:1ls. 7.3 pCI' cenl; for female! rats, 0.7 pCI'

cent; for gIlIlH':1 pigs. 7.2 per ccnl: for 1':100;15. \'1.3 per cenl; ;lIld £01' pigs , 18.2 per cent.

"A COllSt�l1t relationship cxisis betwecn the basal metabolism am\ Ihe Intal cn�logcnolls 1� lt!Og CIl of mature .individuals of diffcrent spccies, thc average ratio bemg 1.99 mllhgram s per CalOriC of basal heat for Ihe species studied."

Smuts' results thus conlradict the conclusion that mi ght be drawn fmm Mitchell's compilation (Bul. 67, Nat. Res. Council) that the endogenous nitrogen coefficient is thc same in the several species there listed.

Smuts' conclusion that ";1 consl'ant reb tionship exists hetw('cn the basal mel<lbolislll and the total endogcnous nitrogen of mature �ndi�idllals of d' ilTcrcnt spccies" is oft course of vcry great theorct-1ca.1 lilt crest, and if true needs to be cxtended to include IIIcmbers of the S:l1l1e species of different live weight and <lgcs. and tlris is the major object of research presented in this bulletin. Smuts con­cludes that "the validity of lhe surface area as the best I1nit of ref­crencc both for the basal metabolism and [or cndoO"enous nitru-

. . . gen, IS substantiated by these data." ""e believe, 011 the other hand, UJ'at these dat<l substantiate the "act ive prOloplasmic mass" theory of Benedict and of Schaerfer and Le Breton. <lnd we feel that Ollr N-fasting data presented ill this bulletin support Our belief.

The general pla.ll of this research was to pillce on N-frce hut otherwise complete diets rats 1-anging in age from :}O to about GOO days, keep tllelll Ull this diet until death, while lllea�l1ring the nitro­gen and energy metabolism. Preceding" the placement 011 the N­free diet·, it \'I<lS planned to fecdl some of the rats with a very lIiO"I1-prot ein stock diet (100 Illg. N per gm.). and others on a low-

'prot: in

stock diet (IO mg. N per gm.) iri order to determine the innuence of the prcceding- dietary protein level 011 the time required to reach the endogenolls, or basal. level of nitrogen excretion. Since this research, unfortunately. had to be abbreviated, some modifications had to be introduced as d'escribcd ill the detailed discussion below.

8 NIISSOUHI AGIUCIII.TUH,\L Ex l'ERll'II ENT ST/HION

RESULTS The results arc given in Tables I and 2, and in a series of

charts. Further details arc given in the following disCllssions.

Methods ,of urine collection and analysis. as also the detailed litera­ture, are given in the preceding repon (Missouri Res. Bill. 189).

T"I",1l I.-Tul!: II'fHUr./i'(;E Of '1',,,1[ ON P."CTICAI.I.V N-VlIII': DU:T ON '"UI: AVIt .... CE Lu',: Wa:WIIT, DA'l,.y FKI!l> COI<Su ... TIOlf, UIlllu.ay.N Exc aETION

j TOTAl. lIotlY.N LOii.u o�

I.,"CI!: RAn (Nul. 2116, 2213, 3287, 3289) ANIl S .... '.I. t " " i1 (Nos. 7528, 752<), 75JO 75JI 7SJ2 7533 7SJ� 7S35 7SJIi 7537) >.

feed Urinarr',N Toul-N lJody-N Crulininc-N Av. live WI.. COIIIUml'l;On CICrctlOll CICtclion lo .. u Urinc-N

Day. on II"", IIOII.-kllo_day mg.-1r1C -d�y mg.-kK.-day mll.-kt;.·d�y % % N·free -- -- ---- -- -- -- -- -- -- -- --

.liet Lart;e Sonall LarKe SIIla11 I.arge Small I.uge Small I.uge Small I.�rge Small ---- -- -- ---- --- -- -- --- --0 363 57 35 101 883 1927 lOW 2HO -39� i iSii 1.71 0.8 2 l5l '0 35

. \'05 30S 1140 m 1463 <.90 I' 4 3H " JO " '" 390 300 -f82 28-f '89 '.06 3. , , 352 '0 1 I 17 21' )91 270 m lH J7) 6.89 3. ,

8 350 .. 11 OS 199 J7J '" HI 231 379 7 .59 .1.1 to H8 " H 61 166 J7) 20S HS 190 J7J 9.10 LO J2 H' <5 " 60 179 "0 220 m 201 )61 8.H U 14 'H H 35 62 164 327 213 -113 1 95 JJ7 9.1 J U 16 3H " H 61 1<5 J2S 182 406 165 "6 lD.H U 18 H8 43 " 57 110 30� IH 382 126 30S 11 . 7 , .0 20 ", " 31 58 130 lI' 164 399 W J12 1 1.6 '.7 22 J l8 " '0 H 12S 29 1 164 J78 1-17 ,OJ II.S , .1 " JJJ .1 27 " 132 293 175 368 159 289 ]I .� '.2 26 JJ2 41 25 61 129 292 160 '" 1<5 290 II. 7 '.2 28 J2S '" 21 H '47 314 182 401 168 J2S 10.3 U 30 321 40 2J 6J 142 297 170 36S 156 29' 10 .6 ,. I J2 31S J9 22 H 159 J26 188 .., 174 J-I1 9.' •. 6 J.! 312 J8 " SS 1<5 '" 170 m ISS H6 10.4 4 7 J6 307 37 2J SS 14' ll3 ]72 m 157 '59 10.1 U J8 300 37 - '17 57 158 JSJ 182 H5 170 m 9.' u .0 29' 16 " 161 H2 188 4010 175 '" , .. U " 294 )6 22 52 135 36. 176 m ,<5 40' 11.2 .1. I H 291 J6 26 " ]21 J2S ,(,() ·,03 121 162 12. J . . , " 290 37 '0 47 125 28' 157 36J 117 m 12. I S.2 .. 292 40 II SO lIS 29' 149 192 109 3J8 11. I , .1 SO 291 )9 H 43 III '" 1S4 368 III 320 12.5 s.s 5 2 292 .. JO " 112 260 153 '66 III 3 11 11.5 5 . 8 H 290 40 27 H 116 276 161 389 125 335 12.0 , . , 56 288 J9 2< <5 12S 265 162 161 I" , ., 11.8 '.7 " 282 J8 H " 14' 289 18' 382 148 JJ.j 10.1 , .1 60 178 J8 21 " lSI 263 194 H' ISS 29� 10.0 5.7 62 27J " 148 18' 162 10.2 .. 27J " 1 37 '74 ISO 11.0 66 264 16 157 188 16' , . , 68 2S8 " ISS 178 1)9 <).6 70 261 21 161 212 172 '.4 72 257 2J 143 187 116 10.6 " 2SS ""2i 17-1 22-1 ' 92 8.7 76 251 16' 220 183 '.1 78 251 22 153 '" 185 ,., SO 2-17 11 153 194 152 9.9 82 m 20 ISS 21l 171 9.' " 2<0 " 174 197 IH 8.7 86 138 21 160 198 H2 9." 88 '" 20 "4 1lJ 182 8.2 <J() 1lJ 22 16(1 209 161 9.4 n 230 25 152 106 186 9.' 94 '" 19 lSI 198 181 tOO 96 22' 20 165 194 176 9.2 98 222 22 167 116 "8 9 0

100 220 " 171 217 180 8.8 102 214 13 174 224 18" 8.7 10' 111 " 178 230 209 8.' 106 111 29 164 110 187 '.2 108 109 ,. 159 206 193 9.' 110 206 11 168 2J7 198 9.0 112 206 11 IS2 137 217 8 3 114 203 " 189 250 m 8 0 116 196 26 161 220 1')7 9.4 118 19. 14 198 1 38 2J7 7.6 IlO 190 2J 199 ,,. 2JJ 7.6 1 22 1S8 19 108 263 W ,. J 1201 186 14 179 146 m 8. I 126 181 26 176 2J7 19 1 86 128 182 25 183 201 8. ) IlO lSI "

1.0'1 l'llil!"'\\ ,(IlO1I , oo�"':-: : ... �o-. �-:�,,!r!o-. ':''': ':'':'''�';'''! __ li01 N"(POU :i��� :����::::-'::::�2::r.::;��:-.::::�

�'1U"1 1'00:1 '����������:;�������;:: :D - 1 _,-�'�'��I�N�··7'P�O;.;+:.":· ����-��=:-�����"�.�" _'"������::��>'������:;� , " "D/ lI pJ , ." ... '- .... "'O ... ' ''- ..... . �. ' � ... .... ..,��R;;.og.>

'>'1C1ul [l00"" -O"'�.':":"':��":�'':��� . . . . . . N'" .�U!Jn ...;,..: -0"'." ..... 0- ",-0." ..... " ..... -0""-0-0-0..:1."

; �I , '" N. ' � I O.l. ' ..... "':':'"":�.':�:"':.':":"':';�-:�a-.�.':o. ; N ' , ",>A � : ;:; �;-:?;:; � � �!::!:: g � � �:t::;::g� : � : --L-.:::..:..,:,:.t=, =00> �. ooooo:-=-.. o _--.:-:_�'"=_�_:::::::o_ 00

liOI

1t1�� ;; � ;;�g :����ci����si��:i�:�

10 MISSUURI AGHICULTURAI, EXPEIUt.1ENT STATION

DETAILED DISCUSSION OF RESULTS

Each o( the resulls is discussed' in the order of its imporl-allcc. Basal (Endogenous) Nitrogen Excretion as Function of Body

Weight.-

To simplify matters, the data were divided into lwo groups, Olle representing the average of the small raLs, and lhe olh­er representing the large rats. J 11 Fig. ] the average nitrogen cx-

MG;tG MG/DAY 'OO'l!c-<-r_--_r,.-•• ,-r,-.-, r.-_,_r".-,r-�-,--- ".J-r_-r,�-.-.r.-.-"r, .---r.---_ ,r.-,���-,I__--,::

4W ! � :£.!!! -' _1 __ ------I----- ,-- ,-- , --• 1-----'\

• I-)� H_I_-,�,:-_I__t_ --1--- ---------f � " ,J \ '" *' '" ""- -\;,,""I---1---l--j---1-+-1 � >- \' § � '.-I \-+--1--'+-+-+ ----J--I---1

, "" IU' ''' . /1 ...

'M O

-- \--1--+--1---1---1--- 1--1-+-� ,-�----I-- z ,',-h -f-----I-- w:!-tJ" i ;-- IV" "\ 1v11V1. 0 !I - lO

�',,""I__+-I-t------ -1-- 10 I"'::: "'=-�- I--I---I---I---I-- <

fig. I.-The declin� of nitrogen excretion with adv"nce of the Ilefiod Oil the N-free diet. Right lide e�Cfe,io" 1'''' "n;null'�r 011)': left .id�, ucretion per kilo 1i\'� ",�ight per da)'. Total N refers to urin�r)'· N plu, fCC31-N; bod)'-N IOlt refcfI to ur;nary-N plul rec�I-N leu dietary_No

RESEARCH BULLETIN 190 II

cretion of each of these two groups ,of rats is plotted against the time On N-free diet. On the left side the data are represented in terms of mg. N excreted by kilo live weight (coefficients); on the right side, tbe excretion per animal. The same dal'a are given in Ilumericai form in Table]. The total nitrogen is lhe SUIll of urill­Jry pluS' fecal N; body N lost is the sum -of the tOlal nitrogen less food nitrogen. The indicated changes for vitamin supplements (ti1<itiki plus Armour's liver extract) relate Lo the large rats only, the small rats were given a uniform dose o( 0.4 cc. o( this supple­meut per (by. The CtlrVCS showi11g somcthing o( a "sta'ircase" phcnomellion may be due in part, in the case o{ the larger rats t o the changes ill vitamin supplement and' food intake. This will be discllssed in greater delail in connection wilh the time curves of losses in body weight.

The absolute amount of nitrogen excreted per d:ly is of course greater for the large than for the small rats.

But as regards the nitroge1l excreted PCI- kilo Jive weighlJ (N­coeiTiciellt), it is decidedly greater for the small thall for the large animals. The average minimum urinary-N excretion per kilo live weight is seen to be 1]0 mg. for the'large rats as compared to 2!)2 for the small rats; for the fecal-N (in Fig. 2) this is seen to be about 7[' mg. for the small rat as comp'ared to 25 for the large rats; the h)tal nitrogen excretion is about 3�'0 mg-. for the small rats as COIl1-pared to 150 for the large rats. The average minimum\ total N­loss by the body (N of urine plus N of feces less food N)' is about 290 mg. for the 5111<111 rat's as compared to JtiO (or possibly ll3) for the large rats.

It is thus evident th�lt unlike in the c�l.se of creatil1ine excre­tion, the endogenous urinary and total N excretion per unit live weight decrease with the increase ... ill size of the animals.

Dec1ine in Total Nitrogen E*cretion, Food Intake. and Ratios of Fecal Nitrogen to Food Intake:-These are illustrated in Fig. 2. The food intake per unit live weight declines more rapidly in the small than in the large rats. This may exphill in parli the more rapid decline in live weight of the small than large rats to be dis­cussed presently. The fecal nitroge1l, which is, of course, almost entirely of body origin (sillc� the diet w:ts practical1y N-free), tends to fol1ow the same course <\5 l'he urinary nitrogen. The up­per-right curves show plainly that lhe ratio of fecal to total nitrogen i� nearly constant during the entire period; about 20% (±7%) of

1 2 J\'I I S:>IILF1H ACIUCULTUI�:\L Ex 1' lilU M ENT STATION

t h e total endogenolls nitrogen excretion is by way of the intestlllt wilh a tendency to increase \,v ith the t ime on the N-free diet. The ratio of fecal nitrog-en to food i n take is seen to increa!le decidedly w i t h the time on t h e N-free diet .

PlJI(IUI ""'n-:-.::T:.-:":T�=,,TTT-,,-nT---'

, " , , , ..

.... " , '" • , '" • , = "" "" "" "" "

'-. '" .,

TIME ON N-FkEE DIET

0

"";bn "

'" z , • .-

0 � , ,

,.., " 0

" liiH· ... . , '" ., � "" ,., ,'" ,.,

t'il. 2.-f'ood irnakc, fecal.N. and raliol of fcnl-N 10 boJy weicht, food inukc and lotal-N ex­cretion wilh .. dunu o' period of N "uyalioll.

The Relation Between Basal Metabolism and Endogenous Urinary Nitrogen Excretion.-This is i l l llstrated in Fig. 3. The hroken �lIrVC�j represent t h e basal e nergy mctabc , i i s l 1 l per u n it live

weight. These curves wcre copied from pp. 72-7-J , Missollri Res. Bu l. ]66. The circles represent data on endogeno11s urinary n i t ro­

gen excretioll, a s ohtained n n smaIJ and large rats (011 the left side,

data arc from M i tchell and Hamil toll, Biochemistry of Amino Ac£ds, New York, ]f12!J ; on the right side, !vIi SS() uri d a tn ) . T t is seen that

RESE/\]: C I I DULLETIN 190

the ratios of basal metabolism to endogenous 11rinary ni trogen cretion arc practically t h e sa me for sllIall and large rats.

c ... t� II o.oG H,/lG ·.to" \ _ 0, CN/�'fII "I-"·,�,,��-no��_�,,�_�oo�'�-�·_I

'o�--I---1---1---1---1

o .. 1"'10 Of I AI HII Gl "" .... 01.1\ 1-..,b-rU,�'H\l.� � .J.!Qluh!.!m! I .. 10 " ! HOOG /IOU! 1110" f�C lll�. : ) ...... u ,,1\ - 0,61 ( t;\!G-

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aopv �£1G11l

1 3

ex-

Fii!' J.-I�eb'io" betw"�" Laul energy and baul nitrogen rneuboliun with j"cr�aolll!' live wetHh, of auimal •.

The Decline in Energy Metabolism on the N-Free Diet.-F ig. ., shows the course of change of heat production per u n i t we igh t

a n d per unit area i n one rat w i t h the advance of the period of N

" , "". ,. 1- . -- , -- ..

,, -�-- --' - .p , '� IL- --'.

" I-=-r-----r---I-I---- � -

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l'iH . .... -ll�lal ",�t�l>oli"" per �ilo anJ pcr 14"ar� meter with �dvA'''c ol lltc pe,;od of N lIuv�ti""

MISSOURI AGRICUI�TURAL EXPERIMENT STATION

st<lrvalion. The rat "':lS not in posl-absorptive condition, having f(lod i n frolll of h i m ; It all times. The data were secured by the closed-circuit oxygen consumptioll method.

The energy metabolism is seen to decl i n e fronl 8;:;0 Cals/S(I · l 1 1ctcr before placing on the N-frec d i e t to about 400 Calories 011 the �tith day, then i t rises to about 500 o n the l OOth day,

This rat. 011 a N-frce diet, reached lhe S:llTle level of energy Il1ctabolisJ11 (400 Calories per sq. meter) a s Benedict'� rat. which W:lS fasted completely (Benedict, I-Iorst a n d Mendel, J. N u t r i t ion, 1 !l:-!2, ti , ti7.1 ; sec also addenda Oil p. 28, M issollri Res. Hill . 1 8 0 ) . Is i t possible that the same energy-metabol ism level W:lS reached by

Belledict's :lnd ollr l(lts because the "protoplasmic mass," or "ac­t i ve mass" was re(l'tlced to the same level by the two k i nds of fast­i n g ? If so. should not then these results be considered as sub­slrtnti:l t i ng t h e active-protoplasmic Illass theory :lS previollsly ex­

plained. rather than the �urface area theory as asslIl11ed by S m u t s ? 1 t would seelll reasonable tn assume that t h e intensity o f both types nf rnetabolislll (energy <l11c1 n itrogen) are actuated by the salllc ulti-11 1ate mechanislll. namely t h e "active protoplasmic mass."

InHuence of the Preceding Level of Protein Intake on the Time

Required to Reach the Endogenous Level of N Excretion.-The results of t h i s i 1 l vestigation are SUl1Hllrtrizcd i n Fig. 5. Fr01l1 the upper left-ha11d quadrant we see that not until a fter 28 days o f spe­cific n i trogen st�lrvatioll does the urinary n itrogen cocrficient of the ,high protein a n imals re:\ch a Icvel similar to that which the low protein rats reached after about S days. The fecal n itrogen excre­t ; 0 1 l of the high protein rats is also derlllilcly higher. in fact the ratio of fec;'tl-N to urin;lry-N curvl"S show that tlie i n crease is pro­portional to the urinary n i trogen increase. The food i n take fl'T tltc two groups was approximately the same. but the body weight loss i ... morc rapid for the h ig'h protein group. This is no doubt caused IJY their more intcnse n i t rogeJl catabolism. From the lower Icft­hand quadrant one may observe that the high protein rats excrete 1II0re fecal nitrogen per u n i t food intake than the low protein rats.

All these observations poin t to the fact that the previous level (if dietary proteill has a marked i n Auence all the t i m e necessary to reach the endogenous level of n i trogen excretion i n the rat. This 11\;IY accOll n t for the d i fYerences observed 1 11 the t ime previously notcd 'at this laboratory and tha t given by M i tchell.

lO< z 'i' , '" > -� 3: , z > _ 0 � g

RESEARCH BULLETIN 190 1 5

� -

\ , -. 1 1 I-H--I--I-I--1--+-1

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... -e . LfZ'" .... TIt" ' � 60 AV ( II N AUf-

... " �0-�'--;!�;--!";-:;!.20':--;;'�' --:)01:--,1,'---;40-0 � 10 I) 20 1) jO H 40 TIME ON N-fREE �ET

16 rvllssouRI !\GIHCULTURAL EX PEIU M I::NT STATiON

The Decline i n Body Weight:.-T h i s is illustrated in Fig. Ii for individllal rats of various Jive weights. 1\ striking feature of the Cllrves, especially of "he larger a n i mals, is a peclIliilr staircase e ffect, especially during the fi rst (iO d'ays. This phenollienon might i n d i cate I he presence i n the body of various tissues d i ffering' i n sta­l)ility or in what might be termed as " lytic potential." Some tiSStH!S or constituents, sHeh for example, .<\s what is termed " deposit pro­tein," succumb most easily ; this is fpllowccJ by the " lysis" of llIore totable tissues, etc. Or this phenomenon ( i i pl'esent) may be l i m i t­ed not so- much by the stability of die several tissue systems as hy changing enzyme cOlllpltxes.

JIM! OIl tHlf! DIET Fig_ 6.-Dccti,,� in body weight of indi"idual rarl wid, the time nn d,e N-fr�e dict. Th� inlctl

rCl'rC\eUI plotl 0 " �rid,log paper, wit I, the 1.:'. rCl'rclcnti"ll d,e .Iopel 0/ thc curve • .

On the other hand, some of the irreg-u !arities may be t h e reslllt of fluctuations in d i c tal'y intake ; for, as noted, one of the dittary cullslilllenls, the vitamin supplement (mixtl1rc of t ikit iki and Arm-0l1l-S' l iver extract), was not kept constant in the diet of t h e large

RESEf\RCI i BULLETIN 190 1 7

rats, I t Ill"Y b e seen from the chart" that i n lhe case o f t h e large rats, �It about 42 days, t h e tikitiki was increased frolll 0.'1 1 0 ] .[i cc. in oreler to stimulate the a n i ma l s to take Illore of the l i N -free" diet. This dietary change coincides ,,,, i t h a fl a t tening of t h e curve. Bill this docs not e n t er a s a faclor i ll chang'c o f slope at aboLlt 2� days ; nor t h e cha nges ill slopes of the smaller rats which received it COIl­�lan t amount o f this supplement.

The insets represent the Same data plotted on a r i t h log paper On a smaller scale. The values o f k represent the average slopes of t h e curves ( the relative, or w h e n multiplied b y '100 t h e percentage, d e ­c l i n e ) , A comparison of the I lumerical values o f k shows clearly t h a t the small a n i mals decline in live weight more I"flpidly than t h e large onego T h u s , r : l t II declines i n l ive weight about 2.4 t i m <.: s a s rapidly a s r a t 2 (.01 2G/-OOfi2=2A) ; a n d the s m a l l e r rats als,! silccllmb sooner than the large rats. ft would t h u s appear that since the energy needs were met by the N-free diet, the more rapid decl ine in l i v e weight of the sn�all animals. must be due to their 1l101-e in­tense n i t rogcn metabolism, which is indeed the case as demonstrat­ed by the nitrogen excretion curves i n Fig, ] .

Survival Peri od.-Some of the rats died a s reslllt of i n fect ion or intest inal disturbances. 'J.�hc greatest survival periods of thcse n i t rogen-starved rats, however, seem to OCCllr when the a n i mals lose about 00%· of their live weight, ,vhich is the same order of loss i n l ive \-\leight of sevCI"al species o f a n imals that are starved absolutely (water given), as described i n -rvrissouri Hes. B u ! . "1 7!L The u l t i m a te cause of death may b e the sa m e i n absolute or n i t ro­gen starvation, namely, t h e stepping over a critical point in loss of "active protoplasmic mass." A possible d i fference in the sur­vival periods on the two types of starvation is that in the case of speciflc l I i tTogen starvation t h e " active protoplasmic mass" is pro­tected by the N-free d ietary i n take. This reasoning, i f correct, would seem to furnish further substantiating ,evidence concerning the l imit ing nature o f the "active protoplasmic mass" 011 the mela­bolie processes. 1 t would then be more correct to say that dealh

Cccllrs not ",hen a certain percentage of body weight is lost , bl l t when a certain percentage of '''active protoplasmic mass" i s lost ; 01' at least when a certain percentage of body n i t rogen is lost.

CONCLUSIONS

The principal contribution a n d concliisioll of this reseilrch i s t h a t the basal (endogenolls) ni trogen a n d t h e basal energy metah-

)

I S MISSOURI AGRICULTURAL EXP£RIMENT STATION

o l i sll l follow the sall lc cuurse with i 1 1creasing l ive weight, age, o r conditiolls o f starvation.

The theoretical implications. as the)' seem to LIS, arc first, that there is a very intimate functional relation between basal energy and endogenolls n itrogcl I met;!.boiislIl ; and second, that this rela­t i on is ult imately caused by the sallic mechanism, nalllely, by the "aclive protoplasmic mass" i n the body.

T t docs not seem reasonable to assume that surface area is a l i m i t i ng faclor under conditions of thermal neutrality. The bet t hat the basal mct;'\ \ )o\ islll (energy or n i trogen ) tends to be pro­portional to su rface area is probably c lue to a more or less constant relationship between .surface area and "<1ctive mass" (see Fig. Ie, p. S. M issouri Res. Bul. .I '/G ) . It is the l iving, continuously active. cataboliz.ing- tissues o f the body·-thc "active protoplasmic l11as<;"­that expends cnergy and nitrogen for its l i f<;: processes, and not area. 1 t is necessary to specify "active protoplasmic mass" rather than l 1 lerely "pr()toplasmic 1l'Iassll because 110t n I l protoplasmic tissues arc equally active. Thlls, IInder conditions of absolute rest, the skelelnl Hl l1scles ;Ire relatively inactive alltl stable ill cO lll par ison to the vis­cel'al organs Stich as kidney. heart, l iver, d igestive tract i n which catabolic processes arc continuous. The skeletal and similar tis­slles while relatively i n;:\ctive are yet. o f course, not inert ; so that t h e active mass is a term representing in a s yet vague manner the statisticnl average o r the catabolic act ivities o f the protoplasmic mass. The vagueness o f the concept is i n itself no argument <lga i l lst i t . and ;"\ concrete equivale 1 l t may be discovered in due lime. In­deed, i f a satisfactory method 'were available for measuring arca, then ; 1.I·ca might be fou1Id to be the pr<1clical i f 110t concrete equiv­alcnt of active protoplasmic mass. Unfortunately, as previously noted (Missouri Res. Bul. 1 G6, p. 1 1 ) . the measuring of "active" area. o f l iv ing <1l1 imals ofTers "pparently unsurmountable d i lTicul­ties. (Professor Max Rubner, lndeed, told' one o f the writers that hy area hc meant not so much measurable area as "physiological area" ; this seems to liS to approach in vagueness the "active m;\ss" concept.)

As a corollary o f til(' principal contribution. it follows that crea tinine n i trogen is a decreasing fUllclion of the total endogenous ni trogen with decreasing live weight of the animal (sec the last two colu1l1ns i n Table 1 ) .

A practical implication o f t h e principal contribution i s that the d i ctilry protein needs for maintenance per unit l ive weight probably

RESE/\RCII i3ULLETIN 190 19

decreas.e with increasing live weight i n the same manlier as the energy needs.

A number o f incitlental contributions have been made which are listed i n the abstract at the beginning of this report.