27 I. A. PREECE AND M. SHADAKSHARASWAMY I99 2. With fi: a ratis varying frm : 1 t 1:, the actin f the tw enzymes is truly additive up t 2-25 % hydrlysis; beynd this pint it may be equal t, greater than r less than the sum f American Sciety f Brewing Chemists (19). Methd8 f Analy8i8, Malt-6, th ed. Wiscnsin: A.S.B.C. Blm, J. & Rsted, C.. (197). Acta chem. Scand. 1, 32. Claus, W. D. (197). Cereal Chem. 2, 59. Freeman, G. G. & Hpkins, R. H. (1936). Bichem. J. 3, 6. Graesser, F. R. & Dax, P. J. (196). Wallerstein Lab. Cmm. 9, 3. Hpkins, R. H., Murray, R. H. & Lckwd, A. R. (196). Bichem. J., 57. REFERENCES individual actins, depending n substrate cnditins. 3. The significance f these facts in the assessment f amyllytic activity is discussed. Institute f Brewing (1933). J. Inst. Brew. 39, 517. Kneen, E. & Sandstedt, R. M. (191). Cereal Chem. 18, 237. Kneen, E., Sandstedt, R. M. & Hllenbeck, C. M. (193). Cereal Chem. 2, 399. Lampitt, L. H., Fuller, C. H. F., Gldenberg, N. & Green, G. H. (197). J. Sc.,chem. Ind., Lnd., 66, 68. Preece, I. A. (197). J. In8t. Brew. 53, 15. Preece, I. A. (198). J. Inst. Brew. 5, 11. Changes in the Extracellular and Intracellular Fluid Phases f Muscle During Starvatin and Dehydratin in Adult Rats BY S. E. DICKER (Beit Memrial Fellw), Department f Pharmaclgy, University f Bristl It has been shwn in a previus paper (Dicker, 198) that rats fed pn a prtein-deficient diet develp tissue edema very rapidly, i.e. the extracellular fluid phase f muscle and liver, as estimated in terms f chlride space, increases. The edema starts at a time when the plasma prtein cncentratin and the plasma cllid smtic pressure are still nrmal. It seemed, therefre, f interest t investigate changes in the extracellular fluid phase f muscle in rats underging inanitin r dehydratin ver shrt perids in which the plasma prtein cncentratin was nt decreased. METHODS (Received 28 July 198) Experimental animals. Adult male and female albin rats were used, f bdy wt. 25-32 g. Diet. Sme weeks befre the experiment the animals were fed n a cmmercially prepared diet cntaining wheat ffal 17-7, grund barley 8-8, white-fish meal -5, meat and bne meal 8-8, dried skimmed milk 1-, dried yeast 1-2, salt -, and cd-liver il.%. The ttal N cntent f the diet amunted t 3-26 %, and its water cntent t 1-5 %. The mineral cntent by analysis was -3 g. Cl, -21 g. Na, -1 g. K/l g. and the calrific value 36- cal./ 1 g. During the perid f experimentatin the same type f fd, but cmpletely dehydrated, was given t ne series f rats. Analytical prcedures. The fllwing data were determined in each rat: (a) the cntent f water, chlride, sdium, ptassium and nitrgen f heparinized plasma (in sme cases, the urea cncentratin f plasma was als estimated); (b) the cntent f water, chlride, sdium and ptassium f muscle samples (the muscle used was the rectus abdminis); (c) the cncentratin f C1-, Na, K and N in the urine; urea and NH3 in the urine were estimated in sme cases. Water cntent, C1-, Na and K cncentratins were estimated in tissue and plasma samples, in the same manner and by the same methds as described in a previus cmmunicatin (Dicker, 198). Urea in plasma was estimated clrimetrically accrding t the methd flee & Widdwsn (1937), and the N cntent f plasma was determined by a micr-kjeldahl methd. In the urine, Cl- was estimated accrding t Vlhard (1878), and Na and K by the methd f McCance & Shipp (1933). NH, was adsrbed n permutit and estimated after nesslerizatin (Flin & Bell, 1917), and the urea determinatin fllwed the methd f Sctt (19). The specific gravity f urine was determined in the apparatus f Heller (19), using a mixture f carbn tetrachlride and light petrleum. All data fr muscle are expressed per 1 g. fat-free tissue (see Hastings & Eichelberger, 1937). Estimatin f the extracellular and intraceuularfluid phases f muscle. The extracellular and intracellular fluid phases f muscle were calculated n the assumptin that all the C1- is extracellular, and that its cncentratin is that f an ultraffitrate f serum (Fultn, 197). The vlume f the extracellular fluid phase was determined by calculatin (Hastings & Eichelberger, 1937). The amunt f ptassium in the intracellular fluid phase was calculated as fllws: m-equiv.intracellulark/kg. muscle= (K)M - (H2),'x (K)p, where (K)M=m-equiv. K/kg. muscle, (K)p=m.equiv.
VI. TISSUE-FLUID CHANGES IN STARVATION 275 K/kg. extracellular water=-95 x (K)8, where (K),=mequiv. K/kg. plasma water, (H2)F=g. extracellular water/ fallen frm.5 ml./1 g. bdy weight in the cntrls average amunt f water drunk ver 2 hr. had kg. muscle = -99 x F, where F = extracellular fluid phase as t 3.3 ml./1 g. bdy weight. This finding agrees calculated frm the chlride space. The amunt f Na in the intracellular fluid phase was similarly calculated with that f Adlph (197). (Fultn, 197). Table 1 shws a typical experiment n tw rats Respnse t water administratin (water diuresis). Starved starved fr 6 days. It will be nted that the amunts and dehydrated rats were given an amunt f water equal f water drunk and the amunts f urine excreted t 5% f their bdy wt. by stmach tube, and their urinary were nt nly irregular, but did nt seem t bear excretin was cmpared with that f nrmal rats. The any relatin t each ther. It will als be nted that evening befre the tests the nrmal rats were deprived f fd and water. was cllected in graduated cylinders; the urinary vlumes were recrded at 15 min. intervals and expressed as percentages f the amunt f water administered. The urine cllectin was cntinued ver a perid f 2 hr. General prcedure. Tw series f rats were investigated: (a) rats fed n a dry diet, withut any supply f water; (b) rats which were allwed a free supply f water, but n fd. N experiment lasted mre than 6 days. During the perid f bservatin rats were kept in individual metablism cages. was cllected under paraffin, and the cntaminatin f urine by faeces was avided by using glass separatrs. At the end f the perid f bservatin, the animals were either killed under ether anaesthesia, and bld and tissue samples were taken and analyzed, r they were given a standard amunt f water t drink and killed after their water diuresis had been fllwed fr 2 hr. Statistical treatment. Results are given as means and standard errr. Student's 't' test (Fisher, 19) fr small samples was used fr estimating the significance f means. The prbability P fr t was btained frm the Tables f Fisher & Yates (193). RESULTS Rats withut fd, but with free access t water The intake f water and the urinary excretin were measured daily. The amunt f water drunk varied frm day t day and frm animal t animal, but in spite f imprtant individual variatins (Tables 1 and 3), at the end f the 6 days f bservatin, the Rat n. 23 Day 1 2 3 5 6 7 Table 1. Wt. f rat (g-) 272 268 26 231 228 22 21 in spite f the water drunk, the urine remained mre cncentrated than in the cntrls where the sp.gr. was fund t be 1-17 + -16 (Heller, 199). Twards the end f the experiment there was a fall in the urinary cncentratin f sdium, chlride and ptassium, but nt in that f nitrgen. After 3 days withut fd, but with free access t water, the plasma prtein cncentratin and plasma water cntent, as well as the cncentratin f chlride, sdium, and ptassium in plasma, were cmparable with thse f cntrls. The ttal muscle water cntent was nrmal, but the cncentratins f chlride and sdium in muscle were increased (t=-761, P< 1 and t = 2-2, P < -1 > -5 respectively); this resulted in an increase f the extracellular fluid phase; it amunted t 2-3 + 1-1 ml./1 g. fat-free tissue, as cmpared with 16-7 + -5 ml./1 g. in cntrls (t=2.978, P<.1). Chlride and sdium space, hwever, remained cmparable in size (t=-585, P>-5), as in nrmal animals. Cncurrent with the increase f the extracellular fluid phase f muscle, there was a decrease fthe intracellular fluid phase (t = 2-3, P <.5), thugh its cncentratin f water remained unchanged (Table 2). After 6 days with water, but withut fd, the plasma cncentratins f prteins, chlride, and sdium were still in the nrmal range (Table 2), but there was an increase in the plasma cncentratin f ptassium as cmpared with that f The effect f withdrawing fd frm rats with free access t water n bdy weight and vlume and cmpsitin f urine Water drunk (ml./1 g./ 2 hr.) 2-61 - 3- -2 5-5 2-86 2 1 355 2 3-88 3 315-297 1-68 5 286 1-71 6 262 1-1 7 25-81 excreted (ml./1 g./ 2 hr.) 1-86 2-2-16 1-91 2-7 1-52 1-76 2-6 1-69 2-5 3* 2-98 (sp-gr-) 1-9 1-7 1-6 1-1- 1-38 1-56 1-57 1-51 1-6 1-9 1-3 (g./1 ml.) Cl- Na K N -29-272 -187-9 -5-11 -39-256 -32-129 -12-187 -15-131 -11-3 -5-79 -196-158 -125-122 -11-1 1-56 1-6 1-2 -9 1-1 -718 1-3 1-23 1-6 1-52 -9-73 -55-56 -75-5 -59-8 -6-77 1-25 -82-53 18-2
276 S. E. ]DICKER I99 t > - - -C _ c pcv GP )~ * -* c8 eo O CO chr ( (."tb G CA) '- 1* 1 1 -H G iq Hc C) ~ ' -H-H -H -H G p _ O- O- C- O- -H -H -H H DEs- e C - S 9 OO1 1 i -H---Hq O -HS -H _H W - - - -H - - - -: C* l-, C) :e *t C), pe - -_q H q C-- C3 a. r e l CO CO t-. q t c C ' le.e C ~-H -H c s O az m O > _ O.. O -H 1 -i: -H ~ -H C C-. * S C--+ _St -H -Hc -Hcqi -HlOch H - ch - a- Cdb-e > Eq Ci3 _ -HO- e X ~~~~d O s > s L CCCb. C)~~~~~~~~~~C r O - r 5 ~ 1X z ;Xs BsgP I Pa E e Ca
Vl. TISSUE-FLUID CHAN cntrls (t = 2-5, P =.5). The ttal amunt f muscle water was nt significantly different frm that f nrmal animals, but the extracellular fluid phase, expressed in terms f chlride space, was markedly increased; it amunted t 22-3 + 3- ml./1 g. fat-free tissue instead f 16-7 ml./1 g. in cntrls. In cntrast with nrmal rats, in which chlride and sdium ccupied a cmparable space (Dicker, 198), the sdium space in this series f rats was greater than that f chlride (t= 2-28, P<-1>-5), indicating that sdium had penetrated int the muscle cells. The amunt f muscle ptassium decreased significantly as cmpared with cntrls (t = 3.23, P < -1). There was thus clear evidence f tissue edema in the starved rats with access t water. It will be nted, hwever, that the standard errr f the calculated mean value fr the extracellular fluid phase was much greater than that f cntrls (Table 2): the cefficient f variatin f the extracellular fluid phase in the series f rats starved fr 6 days amunted t 52-8 + 1-8 %, as cmpared with 15- ± 2- % in nrmal rats (standard errr Rat n. Day 17 1 23 56 18 1 23 56 Time after water administratin (min.) 15 3 5 6 75 9 15 12 Table 3. Decrease in bdy wt. (%) 7.5 6-5 6-6- 7-6-5 8-2 6-1 2-3.5 3-3 GES IN STARVATION 277 f difference = 3-). This significant increase f the cefficient f variatin was crrelated with the variability in the amunt f water drunk by these rats. Table 3 shws changes in the bdy weight f tw rats cmpared with the amunt f water drunk and that f urine excreted per 1 g./2 hr. during 6 days f starvatin. It will be seen that the amunts f urine/1 g. bdy weight/2 hr. were cmparable, but that the amunts f water drunk were very different: they averaged 3-1 ml./1 g./2 hr. in ne rat, and nly -6 ml./1 g./2 hr. in the ther. This resulted in a discrepancy in the decrease f bdy weights. The lss f bdy weight amunted t 2-8 and 33-% respectively. Cncurrently with the discrepancy in the water lad, the plasma inic cncentratin, the muscle water cntent and the extracellular fluid phases f these tw rats were markedly different (Table 3). These differences suggested that there was a failure in the mechanism f water excretin. T investigate this hypthesis, a standard amunt f water was administered t rats which, thugh starved, had free access t water. Table shws the renal respnse t water The effect f withdrawing fd frm rats with free access t water n bdy weight, plabma and muscle cmpsitin (All values fr muscle are expressed/1 g. fat-free tissue.) Water drunk (ml./1 g./ 2 hr.) 1- -8-7 -5-6 3-3.9 5.5 3-.3 Table. excreted (ml./1 g./ 2 hr.) 2-2 1.8 2.3 2-2-7 2-3.- 2-2 2-1 2-2-7 1-5 2-1 Plasma cncentratin (m-equiv./l.) C1- Na K Water cntent Muscle (ml./1 g.) Extracellular fluid phase Intracellular fluid phase 112-7 16-5 6-3 75-6 17-3 58*3 92-13- 5-1 77 23.7 53.3 Water diure8i8 in 8tarved and dehydrated rat8 (Each dse f water was 5% f the bdy wt. and was administered by stmach tube.) utput (% f administered dse) 6 nrmal rats 1-9±-5 1-±-82 22-± 1-5 37-± 1-83 52-±2-69-± 1-81 81-9±-9 12 rats dehydrated fr 6 days A 1 starved rats with water ad lib.,_ A I First dse Secnd dse Third dse After 3 days After 6 days f water f water f water 1.9 t-71 7*±2*59 17-±- 29-±7-2 37-9±7-13 -A-(! 8-2,, 6*± 1-31 15-2+2-5 22-1±3.55 3 3±5 7 37*5±7-9,,,,9 8-+2-53,15,,,9 16-±-36,, 9,, 19-±5-2,,,,11126-+5-2,, 9,, 9 3 ±7 57 I
278 S. E. DICKER I99 administratin in rats starved fr 3 and 6 days: after The urinary vlume excreted/2 hr. decreased nly 3 days f starvatin, and in spite f the fact prgressively frm the first t the sixth day. that the animals had water ad lib., there was a Table 5 shws a typical experiment n tw rats. delay in the nset f the diuresis, and a very marked The analyses f the urine samples reprted in decrease fthe urinary vlume. Ninety minutes after Table 5 are thse f the secnd, furth and sixtth water administratin the amunt f urine excreted days. The mst striking feature was the disappearance f chlride in the urine, in spite f amunted t 29X + 7X2 / f the water given as cmpared with 52- + 2- % in cntrls (Table ). the fact that the specific gravity remained high This finding and the fact that the samples f (Table 6). This disappearance f urinary chlride urine excreted during the perid f experimentatin cannt be explained by a decrease in the cncentratin f plasma chlride (Table 5), nr is it had a relatively high specific gravity (Table 1) the suggest that the water retentin, and hence the tissue edema might be, directly r indirectly, f renal rigin. Rats fed n a dry diet, withut access t water During the first 2 hr. the intake f fd was nrmal, i.e. between 15 and 2 g./animal, yielding between 5 and 6 cal./day, which cmpared well with cntrls; but frm the'secnd day, the amunt f fd eaten fell sharply, and frm the third t the sixth day all the animals refused t eat. During the last 2 days the rats became extremely nervus and restless, biting the wiring f their cage and trying t escape. result f a failure in the ability f the kidney t cncentrate (Tables 5 and 6). Hwever, frm the fact that n the sixth day the cncentratins f chlride, sdium, ptassium and urea were higher in the plasma f these rats than in cntrls, it may be assumed that the glmerular filtratin rate f the kidneys was decreased. After 6 days withut water, the plasma water cntent f rats amunted t 91 1 ± 1% (Table 2), and their chlride, sdium and ptassium cncentratins were significantly higher than in cntrls (t = 7.63, P < 1; t = 2-37, P = -5and t= 6-738, P < *1, respectively). The ttal water cntent f muscles in this series f thirsting rats amnunted t 73&5 ml./1 g. fat-free tissue, which was Table 5. Effects f withdrawal f water n the cmpsitin f urine and plasma f rats (On the secnd, third and furth days the urine was cllected ver a perid f 2 hr.) Vl. C1- Na K NH3 Urea (ml.) (g./1 ml.) (g./1 ml.) (g./1 ml.) (g./l ml.) (g./1 ml.) Male rat (initial wt. 298-5 g., final wt. 26- g.) 6-65 -283-332 1-23 ()-889 2*75 2-9 -6-115 1-21 )*37 9-35 1-8.11-86 1-591 5-785 9 75 Plasma -327 *318-28 -18 Day f bservatin Day 1 2 3 5 67 Secnd Furth Sixth Sixth Secnd Furth Sixth Sixth Plasma Male rat (initial wt. 272-5 g., final wt. 19- g.) -5.76-29 -98 1-36 2-6 -29 7 1*635 1-58 2-5.15-19 1-787 -35-312 37-3 Effects f dehydratin and f subsequent administratin f water Table 6. n the bdy weight and vlume and cmpsitin f urine f rats 2-857 1175 8*325-129 (The rats were deprived f water frm day 1 t day 6, and free access t water was allwed n day 7.) Average Average.tv;rage Aviaapn.f. WW+r. t. I nf vunl VOl. OI nf A_ fd water Average vl. eaten drunk excreted Cl- Na K (g.) (ml.) (ml./2 hr.) Sp.gr. (g./1 ml.) (g./1 ml.) (g./1 ml.) bdy wt. (g.) 2 181 171 159 15 19 16 19-6-5 J,, 21-8-8 1-6 2-5 1-6 2-6 1-6 1-2 1-6.8 1-59 3-5 1-2 -39-22 -171-5 3-21 -3-21 -13 *36-2 -17 79-86 -826 9-713 -72
TISSUE-FLUID CHANGES IN STARVATION significantly lwer than that f cntrls (t= 3-818, P < 1). The lss f muscle water affected bth the extracellular and the intracellular fluid phases, but nt in the same manner. InI spite f an increase in its inic cncentratin (Table 2), the extracellular fluid phase, expressed in terms f chlride space, decreased frm an average f 16-7 t 15-6 ml./1 g. fat-free tissue (t = 2-26, P < -5). The hypertnicity f the extracellular fluid was checked by the cncurrent lss f water frm the intracellular fluid phase, which fell frm an average f 59-3 ml. in cntrl animals t 57-5 ml./1 g. fatfree tissue (t=2-15, P=-5). The magnitude f this water lss can be best estimated by cmparing the cncentratin f water f the intracellular phase in the present series f rats with that f nrmal rats; in nrmal animals, the intracellular water cncentratin amunted t 71-2 ml./1 g. but was nly 68-2 ml./1 g. in dehydrated rats (t = -225, P < -1). The cncentratin f intracellular ptassium fllwed that f water and decreased frm 97-2 t 87-3 m-equiv./kg. muscle (Table 2). It is thus likely that while the extracellular fluid phase cnstituted the first line f defence against dehydratin, intracellular water must have been made available t avid an increase in the inic cncentratin f the bdy fluid which wuld have been fatal t the animal. The extent f the state f dehydratin in this series f rats culd be indirectly assessed by bserving the amunt f water needed t induce a water diuresis. Table shws that in a series f 12 rats, which had been dehydrated fr 6 days, the administratin f 5 % f their bdy weight f water failed t prduce a water diuresis. Three hurs later a secnd administratin f the same amunt f water als failed. A third administratin f the standard amunt f water, hwever, prduced a mderate urinary excretin. This finding led t the questin: Was all that water (15% f their bdy weight) used t reduce the increased smtic pressure f the bdy fluids, r was its retentin in the tissues partly the result f the failure f the kidneys t excrete it? In the fllwing experiment a grup f six rats, each weighing abut 2 g., was kept in a metablism cage fr 6 days. During the first 5 days they were given dry fd but n water; n the sixth day fd was withheld but water allwed. During the first 2 hr. f bservatin, they ate an average f 19- g. f dry fd/rat; n the secnd day, the average quantity f fd eaten amunted t 6-5 g. nly; frm then n, all the animals refused t eat (Table 6). The urine was cllected daily. Table 6 shws the average vlume f urine excreted/2 hr./ animal, its specific gravity and the cncentratin f chlride, sdium and ptassium; it gives als the average amunt f water drunk/rat during the last 2 hr. In spite f an average amunt f 21- ml. being drunk (representing 13 % f the bdy weight) the average urinary vlume excreted did nt exceed 3-5 ml., i.e. 2-1 % f the bdy weight (Table 6). Furthermre, there were n signs f increased excretin f chlride, sdium r ptassium. The sudden increase f the average bdy weight indicated clearly that mst f the water drunk had been retained by the tissues (Table 6). When killed, a grss pst-mrtem examinatin shwed that the intestinal tract was edematus, and that the muscles and liver were abnrmally 'wet '. Further investigatins shwed that the plasma water cntent and plasma prtein cncentratin were nrmal (Table 2), but that the plasma cncentratin f chlride and f sdium were significantly decreased when cmpared with nrmal (t = 2-6, P<-5 and t=2-988, P<-1), while the plasma cncentratin f ptassium had returned t nrmal values (Table 2). The ttal water cntent f liver and f muscle was markedly increased and exceeded significantly that fnrmal rats (t = 5-16, P < -1 and t = 6-533, P< -1). Examining the partitin f the water in muscle, it culd be shwn (Table 2) that the increase f the ttal muscle water was mainly the result f an increase f its extracellular fluid phase. The intracellular fluid phase, hwever, remained cmparable t that f dehydrated animals, thugh its cncentratin f water returned t nrmal values (Table 2). It can thus be cncluded that the increased smtic pressure f the bdy fluid in dehydrated rats was nt the nly factr ppsing further lss f bdy water; when these animals were allwed water, the amunt f water drunk exceeded that required t bring the enhanced inic cncentratin back t nrmal values. As this excess f water lad was nt excreted by the kidneys it resulted in a marked tissue edema. It is, therefre, likely that during dehydratin fanimals the renal mechanism fwater excretin intervenes directly r indirectly in the preservatin f bdy water. DISCUSSION 279 In the tw series f rats investigated, thse which were starved but had ample supplies f water, and thse allwed a dry diet but n water, the first develped an early tissue edema, the thers did nt. Thugh sme fd was eaten during the 2 first days, the rats f the secnd series sn refused t eat and starved fr the last 3- days f the experiment. It may, therefre, be assumed that bth series suffered frm a cmparable degree f starvatin, but that they differed in that ne was allwed access t water and the ther nt. Accrding t accepted theries, the mechanism f water preservatin in dehydrated animals can be
This hypthesis f a renal interventin is supprted by the results f the present experiments where water was allwed t rats which had been s deprived fr 5 days. Adlph (197) claimed that, when water was again ffered t rats which had been entirely deprived f it fr several days, nly a ' small excess' f water was ingested. In cntrast with these findings it culd be shwn that the amunt f water drunk in 2 hr. by rats which had been previusly dehydrated fr 5 days amunted t 13 % f their bdy weight. (Cntrls drink n the average -5 % f their bdy weight f water in 2 hr.) The crrespnding urinary vlume, hwever, amunted nly t just ver 2% f their bdy weight as against 5 % in nrmals. The marked discrepancy between the amunt f water drunk and that f urine excreted resulted in a sudden increase f bdy weight f nearly 1% (Table 6). Further- 28 S. E. DICKER I99 represented as fllws. At the beginning, the extracellular fluid phase is kept nrmal by transfer f f the liver and muscle f the test rats exceeded mre, it culd be shwn that the ttal water cntent water made available frm the intracellular phase, significantly that f nrmal rats, and that there was where it has been released by the cnsumptin f a marked increase f the extracellular fluid f prtplasm incidental t fasting (Gamble, 197). muscle, i.e. clear symptms f tissue edema This shift f water t the extracellular fluid phase is (Table 2). accmpanied by an extrusin f the intracellular Had the smtic pressure been the nly factr base, ptassium (Elkintn & Winkler, 19; Heller, respnsible fr the regulatin f the vlume f bdy 199). As the dehydratin prceeds, water is fluid in dehydrated animals, it wuld be difficult t drained frm the extracellular fluid phase, with the understand why the regulatin failed when the rats result that the latter tends t becme hypertnic were, allwed t drink. Besides the mechanism f (Elkintn & Taffel, 192; Winkler, Elkintn, hypertnicity, there must have been ne which Hpper & Hff, 19). This in turn prduces mre ppsed the renal excretin f the excess water lad. transfer f water frm the intracellular int the Frm the results presented there is evidence that, extracellular fluid phase. The maintenance f the bth the rate f glmerular filtratin, and that f extracellular fluid phase, within limits cmpatible tubular water reabsrptin were affected. It was, with the survival f the rganism, is thus ultimately hwever, utside the scpe f this wrk t determine the factr that prduced bth a decrease f prvided at the expense f the intracellular phase (Table 2). This interpretatin, derived frm the the rate f glmerular filtratin and an increase f estimatin f bdy fluid lst, and calculated frm that f tubular water reabsrptin. changes in bdy weight and frm the urinary In cntrast with dehydrated animals, rats which excretin f sdium, ptassium and nitrgen, is were starved, but allwed free access t water, based entirely n the assumptin that the renal develped signs f tissue edema. As early as 3 days functin remains nrmal. after the beginning f the experiment, the chlride It has repeatedly been demnstrated that during space f muscle was significantly greater than that advanced dehydratin there is a functinal renal f nrmal rats (Table 2). The bjectin might be failure (see McCance, 1936) leading t retentin f raised that t equate chlride space with extracellular fluid phase hlds nly fr nrmal rats, crystallids. The results f the present series f experiments shw that dehydratin in rats prduced ultimately a retentin f urea, ptassium, vlume f distributin (Dicker, 198), and that in where chlride and sdium ccupy a cmparable chlride and sdium in the bld, suggesting im- starved animals there might be changes in the cell pairment f renal functin. Furthermre, Gilman & Gdman (1937) have shwn that the urine f dehydrated rats cntains significant amunts f an antidiuretic substance, which they assumed t be similar t 'vaspressin'. It seems likely, therefre, that the attempt t explain the shifts f water during dehydratin by a purely physical mechanism, like smtic pressure, mits ne imprtant factr, viz. the influence f the kidney. permeability which wuld accunt fr changes in the chlride distributin. Hwever, the fact that chlride ccupied a cmparable fractin f the muscle water in nrmal and starved rats suggests that chlride space can be assumed t give sme measure f the extracellular fluid phase in muscle. It must be remembered, hwever, that chlride space is likely t be smewhat larger than the true value f the extracellular space, even in nrmal animals (Fultn, 197). As these rats were deprived f fd, it culd be assumed that the decrease f the intracellular fluid phase bserved in this series (Table 2) was the result f a lss f water, released fryn the cells as a cnsequence f the degradatin f prteins incidental t fasting. This lss f intracellular water was accmpanied by a lss f cell ptassium, which prceeded in spite f the fact that these animals culd, by drinking, maintain an apparently nrmal state f hydratin. Furthermre the lss f intracellular water seemed t be independent f the amunt drunk. The extracellular fluid phase, n the ther hand, was nt nly increased, but the magnitude f the increase was directly crrelated with the amunt f water drunk (Table 3). This suggests at nce a failure in the regulatin f water excretin in these
Vl. TISSUE-FLUID CHANGES IN STARVATION 281 rats, as is als indicated by the fllwing facts: (a) the urinary vlume did nt bear any relatin t the amunt f water drunk; (b) in spite f the fact that water was freely btainable, the specific gravity f the urines remained much higher than in nrmal rats which had free access t fd and water; (c) fllwing the administratin f a standard amunt f water, the nset f urinary excretin was delayed and its ttal vlume diminished. Cmparing the results btained in starved rats having free access t water with thse in rats withut water the fllwing cnclusins culd be reached: (a) prlnged starvatin, up t 6 days, leads t tissue edema when water is drunk; (b) in starved animals, allwed free access t water, there is a direct relatin between the amunt f water drunk and the magnitude f the extracellular fluid phase, expressed in terms f chlride space; (c) the nset ftissue edema in starved rats with access t water des nt bear any relatin t the plasma prtein cncentratin, and seems mainly t be the result f a failure in the mechanism f water excretin. SUMMARY 1. Ttal water cntent f plasma, muscle and liver and the chlride and the sdium space f muscle were estimated in tw series frats: (a) thse kept fr 3 r 6 days withut fd but with free access t water; (b) thse allwed dry fd but n water fr 6 days. 2. Grup (a) develped an early tissue edema, and after nly 3 days the extracellular fluid phase f their muscle, expressed in terms f chlride space, was significantly increased. When water equal t 5 % f their bdy weight was administered, the nset f the water diuresis was delayed, and the vlume f urine excreted in 2 hr. was lwer than in nrmal rats. 3. Rats allwed a dry diet but n water fr 6 days (grup (b)) shwed retentin f chlride, sdium, ptassium and urea in the plasma, accmpanied by a decrease f the intracellular and the extracellular fluid phase f the muscles. Administratin f water (as abve) failed t prduce a water diuresis. When ffered water ad lib. after 5 days f water deprivatin, an amunt equal t 13 % f their bdy weight was drunk and the animals develped tissue edema; the ttal water cntent f the liver and muscle was significantly increased ver that f nrmal animals, and s was the extracellular fluid phase f muscle. The plasma prtein cncentratin, hwever, remained nrmal. The expenses f this investigatin were partly defrayed by a grant frm the C(lstn Research Cmmittee, whse help is gratefully acknwledged. The authr expresses his appreciatin f the technical assistance f Miss P. A. Ashby. REFERENCES Adlph, E. F. (197). Amer. J. Physil. 151, 11. Dicker, S. E. (198). Bichem. J. 3,. Elkintn, J. R. & Taffel, M. (192). J. clin. Invest. 21, 787. Elkintn, J. R. & Winkler, A. W. (19). J. clin. Invest. 23, 93. Fisher, R. A. (19). Statistical Methds fr Research Wrkers, 9th ed., p. 11. Lndn: Oliver and Byd. Fisher, R. A. & Yates, F. (193). Statistical Tables fr Bilgical, Agricultural and Medical Research, 2nd ed., p. 3. Lndn: Oliver and Byd. Flin,. & Bell, R. D. (1917). J. bil. Chem. 29, 329. Fultn, J. F. (197). Hwell's Textbk f Physilgy, 15th ed., p. 9. Lndn: W. B. Saunders. Gamble, J. L. (197). C1semical Anatmy, Physilgy and Pathlgy f Extracellular Fluid. Cambridge, Mass.: Harvard University Press. Gilman, A. & Gdman, L. (1937). J. Phy8il. 9, 113. Hastings, A. B. & Eichelberger, L. (1937). J. bil. Chem. 117, 73. Heller, H. (19). J. Physil. 98, 1 P. Heller, H. (199). J. Phy8il. In the Press. Lee, M. H. & Widdwsn, E. M. (1937). Bichem. J. 31, 235. McCance, R. A. (1936). Lancet, i, 37. McCance, R. A. & Shipp, H. L. (1933). Spec. Rep. Ser. med. Res. Cun., Lnd., 187, 35. Sctt, L, D. (19). Brit. J. exp. Path. 21, 93. Vlhard, J. (1878). Z. anal. Chem. 17, 82. Winkler, A. W., Elkintn, J. R., Hpper, J. & Hff, H. E. (19). J. clin. Invest. 23, 13.