SEeffen, J. M., G. L. Rigler, A. K. Moore and M. L. Riedesel. 1980. Urea recycling in active golden-mantled ground squirrels (Spermophilus laleralis).

An. J. Physiol. 2392 R16B-R173 (Regulatory Integrative Comp. Physiol. B)

Urea recyclittg in active golden-mantled ground squirrels

(Sp

ermophilus later atis)

J. M. STEFFEN, G. L. RIGLER, A. K. MOORE, AND M. L. RIEDESEL Department of Biology, (Jniuersity of New Mexico, Albuquerque, New Mexico gz1sl

fromfastedorfastedwater-deprivedgroundsquirrelsihatwere tative evaluation of the role'of protein ir"tuUl1r,,' o, urea loaded and injected with a tracer dose oi taC-tagged urea urea recycling during hibernation. However, observations revealed a 1'000- to 1'500-fold gteater 'nco, ex.pitaiion than u"trr hibernating marmots (1) and (peni- "" jrsl reveat th-at urea excretionarctic ground squirdurin! intertorpor iiill,i"li;,lliilfi:::.":"1?H;HiliiJi:lantibiotics '"i. no^rm'othermia underestimates loss of lean body mass by croflora reduced expired ,,co, to control t"""t.. i;?:!|!.f*nrels, drug tr"ut'''"nt also enhanced wine ;;;;;;;;;ilir",h a factor of seven. ".", values approaching those of controls. The results Urea nitrogen recycling via bacterial urease is well "i tt """ experiments point to the possibility of urea nitrogen recycling documented in ruminants (6). Urea and ammonia nitroin nutritiona,v srressed ground

urine urea excretiin decline"d .nutpry i" no;ilffiil *.;;; squirrels (Spermophilus tateralisf *n"" .o-fu."J ;rtf,-hb;: ratory-rats. Intraperitoneal injection of urea to o.iZ "qui"J""t of body weight failed to increase blood urea in l2-h fasted ground squirrels, and led to littie urine urea excretion when compared with similarly tieated rats. Collection.of expired Co2

l1|T"JT?:l"il:3,:.tdl:111":! *,1."--*:li"JixhT

srrrrnN, J. M., G' L' Rrcr,rn, A. K. Moonn, aNo M. L. squirrel differ liom the bear in a number of respects, Rrnoosnr,. (Jrea recycling in actiue golden-mantled ground such as energy reserves and depth of torpor. The depth squirrels (spermophilus lateralis)' Am' J' Phvli-o-I 2l?ll"ryoilo.por in the ground squirrel permits little or no renal
urea cannot be excreted and must be stored or recycled. Fasted and fasted water-deprived normothermic lquirrels likewise exhibit limited urine excretion (3). Conflicting reports concerning plasma urea levels in a number of hibernating sciurids [f ia, f e , 26) do not permil quanti-

fo, p",ioaJoi6;" 3 wk (21, lt;q;" argued that in the hibernating 28). rt,therefore, may be ground squirrel,

squirrers. "

urea metabolism; nitrogen ba-lance; intestinal microflora;

rng;

n)?ophagra

marked increase in protein catabolism (2i, which,

with reduced urine volume, enhanced production of ni. Laboratory rats of both sexes were obtained from the trogenous wastes such as urea, and with only moderate Holtzman Clmpany, Madison, WI. Ground squirrels of urine-concent-rating abilities (5),the ground squirrel must both sexes were captured with Sh"t;;;;;upi1"t*""r, exhibit a mechanism(s) for ameliorating the iubsequent 2,550 and 3,300 m i.r tn" Jemez mountains of norther' uremia which would be expected to develop. New Mexico, and experiments conducted S-24 mo after , UIgu recycling has been reported in one hibernator, capture. All animals were caged singly, provided with the.black bear (Ursus ame1icanu:), by Nelson and co- *ut"r, and fed commercial rat chow (2I7o protein) supworkers (see Ref. 23fo.t review). Theyobserved no loss plemented with fresh vegetables. Animal quarters were of .lean body mass during the period of hibernation de- maintained at 28 + B'C ;ith a natural tigh; ctcle. spite the continued production of urea (24) . ln addition The first series of experiments examined urea excretion they noted l) urea was reabsorbed from the bladder at a after deprivation of iood, water, or both in different rate equivalent to its rate of excretion by the kidney (24); Rats were fasted and water (FWD) for ii) blood urea levels remained unchanged during rrin"r- ""u"o.,". December. Ground squirrelsdeprived 6 days in *ere pWn in both nation (25); and jjl) intestinal storage 6f urea- cJuld not Ma5iand December. A third group of squirrels was fasted be demonstrated (25)' Hibernators such as the ground for ? auyr during fufuv tr,ro"sh Julv. Urine was collected
Rl68
0363-6119/80/0000-0000901.25 cop5a'ight

coupled flora-reducing antibiotics. with reduced evaporative (4) and urinary water ioss, serves to maintain positive water balan"". Aa a result, MATERIALS AND METHODS

WHEN CONFRONTED with the reduced food and free water nitrogen balance. availability of the winter season, the golden-mantled This study was underbaken to i) monitor urine urea ground squirrel(Spermophilus lateralis) enters a period excretion in fasted and fasted water-deprived ground of hibernation during which energy is supplied from squirrels, il) determine the response to urla loadiirg, lil) stored triglycerides (15,22). Summei-active animals, in quantitate the metabolism of [1aC]urea, and iu) iete/contrast, respond to food and water deprivation with a mine urea excretion following administration of micro-

fast- rabbits (27), and swine (10). Conflicting reports have appeared concerning recycling ofurea carbon (18,27). In both normothermic and hibernating anirnals the intestinal flora may make a significant contribution to overall

i:ffi::::'Jg"itr#il""hruffi,:li:Tl"S,:Tl"A"riT*:

1980 the American physiological Society

UREA RECYCLING

IN ACTIVE GROUND

SQUIRRELS

R169

well as FWD and drug-treated FWD animals, had an average 25Vo weight loss prior to urea administration. For comparison of tnCO2 production with urine 1aC elimination in ground squirrels, a separate experiment involved animals fasted for 4 days with water withheld
for the final day of the fast. Groups of these animals were drug treated (FDT) according to one of the following protocols; i) 500,000 IU procaine penicillin G intramuscularly 36 h prior to administration of urea, ii) 0.027o sulfamethazine in the drinking water for 4 days prior to urea administration; iii) 5 ml tetracycline (1.0 g/100 rnl water) by gavage given in two equal doses 24 h and 8 h prior to urea administration.

CO2Tmp

Liquid
Cochtoil

NoOH

Tmp

Scintilhioa

rrc.

1. Chamber for collection of urine and expired 'aCOz.

An additional experiment involved monitoring

urea

under oil in large watch glasses placed beneath wire mesh cages. Once daily, urine was pipetted from beneath the oil and refrigerated (1-5 days) in tightly capped vials. Duplicate urea analyses on fresh and stored samples were similar. Urea analyses were made by a diacetylmonoxime and thiosemicarbazide colorimetric method (8), which is not sensitive to ammonia, creatinine, or amino acids. Blood and urine urea were measured after urea loading in a second series of experiments. Ground squirrels (166222 d and rats (366-466 g) received an intraperitoneal (ip) injection of urea equal to 0.37o of body weight. Urea was delivered, in all cases, in 2 ml of aqueous solution after the animals had been fasted for 12 h. An objective of this experiment was to test the effects of urea loading on fasted animals with water available. Because leakage

excretion following drug treatment. Control animals received food and water ad libitum. Fasted and FDT squirrels received water ad libitum. The FDT animals received
0.027o sulfamethazine in the drinking water. Urine collection and urea analyses were as above. The ['4C]urea (60 mCi/mmol), PPO, toluene, and NCS solubilizer were obtained from Amersham/Searle; ethylene glycol monomethyl ether and ethanolamine were from Sigma; sodium sulfamethazine and tetracycline-HCl were from American Cyanamid; procaine penicillin G (Streptillin) was from Pfrzer. Data were analyzed by the Student's f test and the null hypothesis was rejected at the SToIevel ofconfidence.
RESI,JLTS

from water bottles into the cage and urine-collecting vessels can give elroneous measurements of urine volume, raw potato ad libitum was the single source of water throughout the experiment. The nutrient content of the potato consumed represented 9-237o of the resting metabolic rate of the animals and therefore the animals were considered to be in a fasting condition. The mean water intake was 4.7 and 9.4 g per 36 h by ground squirrels and rats, respectively. Blood samples were obtained by tail clipping. Urine was collected and urea content of blood and urine was analyzed as above. Metabolism of [lnC]urea was investigated in animals following a single 2-ml ip injection of an aqueous solution containing 0.3 g unlabeled urea per 100 g body weight and 10 pCi ['nC]urea. After injection, animals weie plaied in a 1.25-liter chamber (Fig. 1). Air, 100-200 ml/min, entered the chamber after passing through a COz trap of 2.5 N NaOH. Upon exit from the chamber, the air passed through two solutions containing a COz-trapping scintilIation cocktail and a final 2.5 N NaOH trap to prevent contamination of laboratory air. The COz- trapping scintillation cocktail consisted of 0.5Vo (wtlvol) 2,S-diphen-

The excretion of urea is altered following water deprivation and./ or fasting of laboratory rats and ground squirrels. Within 3 days, FWD rats had reduced urea excretion by 46Vo (Fig. 2). Extension of the period of deprivation to 6 days was accompanied by increased daily urea output. The FWD ground squirrels during both May and December, as well as summer-fasted squirrels, also had an initial
(5

= O

600

soo

ff ooo F
uJ

Q
IJJ

soo

yloxazole (PPO), 55Vo (vol/vol) toluene, 39Vo (vol/vol)

ethylene glycol monomethyl ether, and, 5.5Vo (vollvol) ethanolamine. Aliquots of each CO2 trap and urine samples were removed after 24 h. A 0.25-ml aliquot of urine was transfened to a counting vial along with 3 ml of NCS solubilizer, and the total volume increased to 18 ml by the addition of 0.5Vo PPO-toluene cocktail in preparation for counting. The CO2 trap and urine samples were counted in a Beckman LS-100 liquid scintillation counter. Control animals were allowed free access to food and water prior to urea administration. Fasted animals, as

:)

fi E

2oo

o123456
DAYS
Seasonai effects of fasting or fasting and water deprivation (FWD) on daily urea excretion. FWD rats (Dec.) H; FWD ground fasted squirrels (May) fH; FWD ground squirrels (Dec.) H; ground squirrels (May-July) H). Vertical /izes represent SEM.

rrc. 2.

R170

STEFFEN, RIGLER, MOORE, AND RIEDESEL

depression in urea excretion, but to a greater extent (62, 72, and 62Vo, respectively) than did the rats. Reduced urea excretion was maintained in ground squirrels even

with extension of deprivation to 6 days. trt should be noted that simultaneous food and water deprivation, or
fasting alone, induces similar trends in urea excretion. In an attempt to elucidate the mechanism underlying these differences in urea excretion, blood and urine urea were monitored in rats and ground squirrels subjected to an exogenous urea load. Prior to urea loading, ground squirrels had a higher blood urea concentration than rats (Fig. 3). Within 6 h of the injection, blood urea levels in the rat had risen threefold, whereas the ground squirrels responded with little or no change in blood urea. In the rat, blood urea levels fell to base line within 24 h. In contrast, values for the ground squirrels were maintained below those at time 0 for the remainder of the experiment. Thus, an ip urea load in the squirrel does not lead to short-term accumulation of urea in the blood. A greater renal efficiency in the removal of urea from the plasma may have accounted for the ground squirrel's Iack of short-term blood urea accumulation. Urine urea levels presented in Fig. 4 show that this was not the case. Ground squirrels excreted urea at or below the levels of the rats on a body weight basis for 48 h following urea loading. The results presented in Figs. 3 and 4 lead to the conclusion that, in S. lateralis, not all urea produced or injected may actually be excreted; and, therefore, a low rate of urea excretion in the squirrel (Fig. 2) rnay not necessarily imply a low rate of urea production. A tracer dose of [tnC]urea was administered to urealoaded animals to investigate the possibility that species differences in the capability for either tissue storage, metabolism, or both may account for the failure of the exogenous urea load to appear to a similar extent in the

E = .9

c,

E n (t

g
trJ

E
l

z_

lrj

5
-)

F
].torJRs

course of urine urea excretion following urea loading Vertical lines represent SEM.

rrc. 4. Time

rABLE 1. Twenty-four hour laCOz (dpm) expired by control, fasted, and FWD rats and ground squirrels
Laboratory Rat Ground Squinel

col
rrol

Fasted

Fasted waterde-

coltrol
262

Fasted

Fasted water-de-

prived

prived

Mean -fsE n7775

103 183 273 +14 +74 +22

!42

280,000 400,000 +10,000 +15,000

55

FWD, fasted water-deprived.

ffiUND

TA8 RAT

flJlffiL

blood and urine ofthe ground squirrels and rats. Fasted or FWD rats responded with nearly two- to threefold increases in tnCOz collected (Table 1), indicating only slight catabolism ofthe injected tracer. In contrast, fasted and FWD ground squirrels increased laCOz production
1,000-

3 ol

to 1,500-fold. Thus, the capability for increased

s
fl E.
o I
CD

urea catabolism would appear to be a component of the metabolic response to reduced food and water intake in the ground squirrel. The extent of catabolism of urea in the ground squirrel is demonstrated in Table 2. Control squirrels excreted the great majority of the injected 14C in the urine within 24 h. Elimination of the tracer via catabolism is slight. Approximately 20Vo of the label is retained for at least 24 h. A 4-day fast coupled with 24 h of water deprivation resulted in an 8-fold reduction in urine volume whereas 14C elimination in the urine was decreased 30-fold. This was accompanied by a l0-fold increase in the amount of urea carbon in expired air. Whole body retention of the tracer also increased to approximately TOVo of the injected dose. Treatment with penicillin, sulfamethazine, or tetFIOIJRS

course of blood urea changes following urea loading. Vertical lizes represent SEM.

rrc. 3. Time

racycline resulted in decreased urea catabolism with values approximating those of controls (Table 2). In an effort to describe a potential direct relationship

UREA RECYCLING IN ACTIVE GROUND SQUIRRELS

R171

rABLE 2. Percentage of injectedraC collected in urine and expired air in control, FWD, and

that urea is treated primarily as a waste product in the

laboratory rat.
Fasted and FWD ground squirrels, like the laboratory rat, exhibit an initial decrease in urea excretion, but with two important distinctions, namely: i) the depression of urea excretion is more pronounced (62-72Vo vs. 467o fot

FDT ground squirrels

Twentyfour-hour
ljrine Vol
ume, ml
COz trap trC Percentage of Injected Collected in 24 h

Control (n : 9) Fasted water-deprived (n + peniciliin (n : 5) + sulfa (z = 4) + tetracycline (z:3)

8)

8.9 r 0.7 79 ! 4.4 2.4 + 0.15 1.1 + 0.7 2.6 -f 0.80 26.5 + 0.80 7.3 4.4 1.4

-r

0.9
1.2

0.6

Values ale mean t SE. FWD, fasted water-deprived; FDT, fasted water-deprived, drug-treated.

between urea recycling and intestinal microflora, urea excretion was monitored in control, fasted, and FDT squinels receiving water ad libitum. Urea excretion in the fasted animals declined progressively throughout the 7-day fast (Fig. 5). The FDT squi-rrels, on the other hand, maintained urea excretion above the levels of the fasted animals, being2.7 times greater by the 7th day' These same data are expressed on the basis of body weight in Fig. 6. Fasted squirrels reveal a progressive decline in urea excretion over the length ofthe fast. Urea elimination by the FDT squirrels remains close to that of the fasted squirrels for the first 4 days of the fast. The following 3 days reveal an increase (P < 0.001 for day 7, P < 0.01 for days 5 and 6) in urea excretion by the FDT squirrels over that of the fasted animals.
DISCUSSION

the rat), and li) there is no follow-up increase in urea excretion upon extension of the duration of the fast. Bintz et aI. (2) reported S. laterolis preferentially catabolizes protein under the same conditions of stress. These conflicting findings may be accounted for, in part, by the response of the ground squirrel to urea loading. It is evident that the movement of urea into the plasma in the squirrel nearly balances its removal in view of the stability of plasma urea concentrations (Fig. 3) following urea loading. In fact, plasma urea concentrations actually
5 loterolis CmrolElf
Foded

FDI @

6
!
o)

o
o

UJ

o o
o'

Mobilization of lipid reserves as a fueI source, with a consequent sparing or conseryation of protein, has been recognized as a nearly universal response to fasting. This adaptation is enhanced by the high caloric value of fat per unit mass and the ability of considerable portions of the nervous system to convert from a carbohydrate-based metabolism to the utilization of lipid intermediates. This selective lipid catabolism significantly prolongs the du-

34567
Doys

of Fostirg

5. Effect of fasting or fasting combined with drug treatment lFDT) on daily urea excretion.

rrc.

ration of fasting which an organism may tolerate. The initial depression of urinary urea excretion by FWD rats (Fig. 2) is consistent with the above proposal. Bintz et al' (2) have reported respiratory quotients indicative oflipid metabolism in similarly stressed rats. Millward et al. (19) report a decrease in muscle protein catabolism in similar sized rats aftet 2 days c;f fasting though there is an increased protein fractional turnover following 4 days of fasting. There are indications that less mature rats may not show a like response (17). The marked increase in urea excretion seen after 3 days of fasting may reflect a substantial depletion of lipid
reserves and a greater dependence on protein as a source of energy. 'ftre 20-25Vo body weight loss seen in these rats over the fust 3 days of fasting may represent catabolism of most of the lipid reserves. The work of Cuendet et al.

S lolerclis Cmtrol o
Fosted

.9 q)

= E

co

o a
E

ol

o o
d)
(D

u
UJ

(9) on lean and obese mice would suggest that body protein is spared until triglyceride stores have been seriously depleted. The buildup of urea in the blood following urea loading (Fig. 3), its excretion (Fig. 4), and lack of
significant release of
laCOz (Table 1) clearly indicates

) or
nrc. 6. Effect offasting or fasting combined with drug treatment on daily weight-specific urea excretion. Vertical llnes represent SEM'

o o

Rr72

STEFFEN, RIGLER, MOORE, AND RIEDESEL

decrease in the final half of the experiment. Lack of the urine following arousals from hibernation is \hth the quantitative recovery of injected urea in the urine (Fig. amount that would be expected on 4) coupled with the above demonstration that plasm"a lean_ body mass. The existence of the basis of loss of an operative recycling urea concentration does not increase in response to ip mechanism during hibernation is dependent upon bott ure_a loading implies that, in the ground squirrel, not ail the identification of cryoduric microoiganisms capable of endogenously produced urea may actually be excreted. urease activity at low temperatures and the activity of Therefore, a low level of urea elimination by the squirrel the appropriate amino acid-synthesizing enzymur. Thu (Fig. 2) does not necessarily correlate wiih a rehuced urea recycling capacity of the hibernator mav be a critical degree of protein and amino acid metabolism. factor determining the onset of hibernationl Montova et The high metabolic rate and limited fueI reserves of al. (?0) report a protein-free diet was nearly as effeltive small mammals necessitate rapid adjustment to condi- as starvation in inducing torpor in the garden dormouse. tions of fasting or water deprivation. fhe capacity of the Thus, the nitrogen-sparing effect of urea recycling may ground squirrel to make these rapid adjustmentsls dem- permit some hibernators to delay the onset of hibernation onstrated by the absence of change in blood urea follow- induced by starvation or protein deficiency. ing urea loading. Isotopic studies have demonstrated re-cycling of 15The reduced urea elimination discussed above must 357o of thgwrea synthesized in the normal human subject elicit either increased storage or induce metabolism of (18, 29). The role of urea hydrolysis and nitrogen incorthe retained urea. Urea hydrolysis in mammals is re- poration into nonessential amino acids is particularly garded as being of bacterial origin (11, 31). There is no important in uremic patients maintained on iow protein evidence in the literature to warrant suspicion of a sep- diets (32). Recent studies have described the benefit of arate mechanism unique to the ground squirrel. The supplementation of low protein diets with ketoacid anaresults depicted in Figs. 5 and 6 substantiate this claim. logues of the essential amino acids (80). Through transAntibiotic treatment has proven effective in a number of amination of these analogues, nitrogen released from urea may be incorporated into essential amino acids. The 9f1d1es in reducing urea hydrolysis in mamrnals (12,29, 32). Drug treatment of fasted squirrels results in a reduc_ present study clearly supports the hypothesis that urea tion of urea catabolism (Table 2) and leads to enhanced recycling in response to nutritional stress is well develexcretion of urea (Figs. 5 and 6). Colonic or gastric flora oped in the golden-mantled ground squirrel. are.most likely to be the source of the endogenous urease The authors are indebted to the R. D. Boom family for collecting activity, though the exact site of urea hydrolysis is unground squirrels. known at present (14, 81). This research was supported in part by funds from the Univ. of New The existence of urea recycling as a nitrogen-conservMexico Research Allocations Committee and Graduate Student Assoing mechanisn in rodent hibernitors is unk"nown; nor is ciation. Present addresses: G. L. Rigler, 919 Goff Blvd. S.W., Albuquerque, it known whether this apparent recycling is operative in S. krteralis d,uring hibernation. Ceitainty the reports of NM 87f 05; A. K. Moore, School of Natural and Mathematical Sciences. Benedict and Lee (1) and Galster and Morrlson (13) Seattle Pacific University, Seattle, WA 981f9. Address reprint requests to: M. L. Riedesel, provide support for such a mechanism operating during University of New Mexico, Albuquerque, New Department of Biology, Mexico, 87181. deep hibernation. They indicate that recoverv oi urea ii Received 24 September 1979; accepted in final form 16 January 19g0.
_

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