323

J. Physiol. (I958) IVI, 323-331

RENAL FUNCTION IN THE FOETAL AND NEW-BORNGUINEA-PIG

By J. W. BOYLAN*, ELIZABETH P. COLBOURNAND R. A. McCANCE

From the Medical Research Council Department of ExperimentalMedicine, University of Cambridge

(Received 29 November 1957)

Renal function before birth has been studied in comparatively few species,and observations on the lamb, rat and human infant have contributed thegreater part of our knowledge. When Jacque (1905-06) was investigating theorigin of amniotic and allantoic fluids in the sheep, he determined the osmolarconcentrations of these fluids and compared them with those of the foetalurine and plasma. The foetal urine was hypotonic (average 138 m-osmole/l.)and the changes in the osmolar and chloride concentrations of allantoic fluidsuggested to Jacque that it was largely the product of the foetal kidney. Thistheory was supported by Alexander, Nixon, Widdas & Wohlzogen (1955a, b),who described changes in the composition of the foetal fluids and urine of thesheep during gestation. It has also been applied to rabbits (Davies & Routh,1957).

Wells (1946) ligated the urogenital papilla of rat foetuses shortly before termand measured the volume of urine found in the bladder 2 hr later. If thevolume found in the bladders of untouched litter-mates was deducted, thefigures indicated that urine accumulated at the rate of 0.015 ml./g body wt./hr.Urines collected in this way over a 24 hr period (Daly, Wells & Evans, 1947)contained an average concentration of 195 mg urea N/100 ml. and, in oneobservation, a creatinine concentration of 42 mg/lOOml. Although theseurines probably had a higher osmolar concentration than plasma, they had alower concentration of urea and chloride than the maternal urine formed in thelast 2 hr of the experiment.McCance & Widdowson (1953) summarized the knowledge then available

about urine formed by the human foetus and added a detailed study of twelve* Special Research Fellow of the National Heart Institute, U.S.P.H.S. Present address:

Department of Physiology, University of Buffalo, School of Medicine, Buffalo N.Y.21-2

J. W. BOYLAN AND OTHERS

urines collected at delivery. These urines were invariably hypotonic, with anaverage concentration of 137 m-osmole/l., and the urea and creatinineurine: plasma ratios seemed to indicate that the glomerular filtrates had passedrapidly through the tubules without much concentration having taken placein the distal portions. Hanon, Coquoin-Carnot & Pignard (1955, 1957a, b)have more recently analysed twelve more samples of human urine formed inutero, and their results are in general agreement, particularly as regardstonicity. They consider, however, that the products of the kidney have nosignificant excretory function before birth.More data are at hand about the development of renal function after than

before birth in experimental animals and man (McCance, 1950; Heller, 1951;Smith, 1951; McCance & Widdowson, 1954 a). Evidence cited in these writingsand supported by more recently published material (McCance & Widdowson,1954b, 1955; McCance, Naylor & Widdowson, 1954; Falk, 1955) indicates thatthe kidney of the new-born mammal has a smaller concentrating capacity thanthat of an adult, a much lower rate of glomerular filtration per unit of bodyweight or of surface area, a smaller and usually a delayed excretion of waterafter a standard dose, and lower sodium and chloride clearances. These functionshave been shown to mature at characteristically different rates and there areknown to be considerable differences in this and other respects between rat,dog and man.In the present study some observations have been made of the renal function

of foetal guinea-pigs and these have been compared with similar observationsin new-born and older animals.

METHODS

Guinea-pigs of the usual laboratory type were employed. The age of foetuses was in most in-stances known to within one week from the time of exposure of a group of does to a buck. Theperiod of gestation in this species varies from 65 to 72 days depending upon whether conceptionoccurs in a virgin or lactating doe, for in the latter implantation of the fertilized ovum may bedelayed.The urethral orifice of the female guinea-pig is accessible and a catheter of appropriate size can

readily be introduced. In foetal and new-born animals a short length of polythene tubing was usedfor this purpose. At the beginning and end of each period over which urine was being collected thebladder was filled two or three times with air, and gentle supra-pubic pressure was applied. Thisenabled the bladder to be emptied. Blood was drawn into a heparinized syringe at the end of theperiod over which urine had been collected, in foetal animals from the umbilical vein, in the new-born from the bifurcation of the inferior vena cava or the aorta, and in adults from the heart.Urine was collected from the pregnant does by catheterizing them at the beginning of theexperiment.

Foetal urine was collected in the following manner. The gravid doe was restrained on a smallanimal board with the hind legs crossed to give a semi-lateral presentation of the uppermost side.After infiltrating the abdominal wall with 2% procaine, a horizontal incision 6-7 cm in length wasmade across the exposed flank and abdomen 2-3 cm below the superior limit of the uterus asdetermined by palpation. The incision was carried through the subcutaneous tissue throughout itslength and opened into the peritoneal cavity at its lateral extremity. The uterus was then attached

324

RENAL FUNCTION OF FOETAL GUINEA-PIGSto the upper edge of the peritoneal incision by a running suture which was continued medially asthe incision into the peritoneal cavity was extended. The suture was then extended around thelower border of the incision to make a window of uterine wall sealed from the body cavity. Theuterus was opened, and the hind quarters of the presenting foetus delivered. Ifnecessary a versionwas performed. If the foetus was a female it was catheterized and the urine present in the bladdercollected into a weighed tube. If the foetus was a male it was delivered and the second foetus, iffemale, was catheterized. The term urine 'found' has been applied to the urine found in andremoved from the bladder on passing the catheter, and urine 'formed' to the urine which collectedin the bladder during the subsequent period of observation. After the urine 'found' had beencollected the external portion of the catheter was reduced to approximately 1 cm in length, its tipsealed with Plasticine and the hind quarters of the foetus returned to the uterus with the catheterin place. It was not found necessary to use an antispasmodic drug, and the uterine cavity wasclosed with silk thread and the overlying skin with suture clips. The doe was released and placedin a comfortable box with access to food and water. Three to five hours later the doe was againplaced on the board, the wound re-opened and the foetus delivered. The doe was then bled fromthe heart and killed by injecting air into the heart. The occluded tip of the catheter in the foetalbladder was snipped off and any urine which had been formed was collected and weighed, and theresults have been expressed as ml. without applying any correction for specific gravity. The slighterror introduced in this way does not appear in clearance values, for these are computed from thetotal excretion of the solute measured.New-born and young animals were either held in the hand with the catheter in place while the

bladder was being allowed to fill, or were catheterized at 20-30 min intervals; adults were restrainedon an animal board with an indwelling catheter. All studies were carried out with the animal in itsnatural state of hydration.

Urea and endogenous creatinine clearances were measured. The freezing-points of the urinesand plasmas were determined before they were diluted for the analysis of urea and creatinine(Ramsay, 1949; Hervey, 1955), and the total osmolar concentrations calculated by dividing thedepressions of the freezing-points by 0-00186. Urea in plasma and urine was determined by themethod of Lee & Widdowson (1937), creatinine by a modification of the Jaffe reaction as describedby Hawk, Oser & Summerson (1947), and chloride by the method of Sendroy (1937) or by that ofSchales & Schales (1941).

RESULTS

A comparison of maternal and foetal plasmaTable 1 shows the average total osmolar, chloride, urea and creatinine con-centrations in the plasma of maternal and foetal guinea-pigs at or veryshortly before term. The differences were not significant, but differences in thesame direction have been found in other species for osmolar and creatinineconcentrations (McCance & Widdowson, 1953).

Comparison of maternal urine with the urine found inthe foetal bladder

Table 2 contains comparisons of the total osmolar, chloride, urea and creati-nine concentrations in maternal urine and in foetal urine found in the bladderat or shortly before term. Five other foetal urines have also been analysedwithout the corresponding maternal urines. The results have been given foreach individual animal because the wide variation from one litter to anothermakes average figures of lesser value. There was considerably less variation

325

326 J. W. BOYLAN AND OTHERS

between the individual foetuses within one litter. The osmolar concentrationswere always much higher in the maternal than they were in the foetal urine,but the latter were generally hypertonic to serum, and much more con-centrated than those of the human foetus or of the foetal lamb.The concentration of chloride in both the maternal and foetal urines was

very variable, but there was evidently a relation between the two urines, forthey both tended to have a high or a low concentration together. In fiveinstances when the maternal concentration was low, the foetus had a higherconcentration of chloride in its urine than the mother.The concentration of urea was always higher in the maternal urine, and this

was largely responsible for the differences in total osmolar concentration. Innine out of the fifteen comparisons the concentration of creatinine was higher

Total osmcChloride (rUrea (mg/]Creatinine

TABLE 1. The total osmolar, chloride, urea and creatinine concentrationsin the plasma of foetal guinea-pigs and their mothers

Foetus McNo. of A

r

comparisons Average S.D. Averag()lar concentration (m-osmole/l.) 12 292 12-3 297a-equiv/l.) 8 99 7-1 102100 ml.) 13 30 4 6-97 33.7(mg/100 ml.) 16 1-6 0-52 1-4

ithere S.D.

10.94-18-08

5 0-48

TABLE 2. A comparison of the total osmolar, chloride, urea and creatinine concentrations in theurine found in the bladders of foetal guinea-pigs and of their mothers

Total osmolarconcentration

State of* (m-osmole/1.)Litter maturity A

no. of foetus Foetus Mother1 M. (a) 338

(b) 376(c) 564

2 M. (a) 371(b) 402

3 M. (a) 328(b) 339(c) 360

4 M. 3875 M. 2466 M. (a) 336

(b) 348(c) 393

7 M. 2428 S.I. 3049 S.I. -10 S.I.11 8.I. 34412 S.1. 32013 S.1. (a) 252

(b) 302

Chloride U(m.-equiv/l.) (mg/l

Foetus Mother Foetus1354 149

130123

1306 158188

912 642839

1157 136946 611114 44

5869107

1602 16

- 134

80- 35

240 187228138

310 244208

25 354638

517 29042 66138 195

130300

- 35841 -- 100113 220- 365- 226- 291- 131

,ea Creatinine)0 ml.) (mg/100 ml.)

Mother Foetus Mother587 114

100122

1950 2238

1315 230276236

500 32688380 81

162144106195

1000 28760 38- 68

773639

Urea:creatinine

ratio

Foetus Mother92 1-64

2-281-14

168 11105-47

138 0-150-160-16

75 9.05

98 2-410-802-08

- 3-388363 3-5765 5-80- 5-38- 2-94- 8-13

3-38

11-60

9.55

7-15

3-85

15-811-7

*M., mature, approx. 70 days foetal age; S.I., slightly immature, approx. 60 days foetal age.

I

RENAL FUNCTION OF FOETAL GUINEA-PIGS

in the foetal than in the maternal urine. The most striking thing about theseresults for the urine found in the bladders of foetal guinea-pigs, however, wasthe variability of the concentration of urea and creatinine in them. Unlikethe chloride, there seemed to be no relation whatever between the compositionof the maternal and the foetal urine in this respect. In the urines from fourof the fourteen full-term foetuses the concentration of creatinine was higherthan that of urea; in the other ten it was lower, and the urea: creatinine ratiosvaried from 0-16 to 11 1. In the slightly immature animals the urea was alwayshigher, as though this rising concentration of creatinine was something thathappened at or about term. Samples of urine found in the bladders of full-term foetal guinea-pigs have been treated with Lloyd's reagent (Hare, 1950;Owen, Iggo, Scandrett & Stewart, 1954), and this removed all the chromogenwhich gave a colour within 20 min with the Jaff6 reagents. In this respect,therefore, the chromogen behaved like 'true' creatinine.

Rates of urine formation and urea and creatinine clearancesVery often no measurable amounts of urine could be collected from the

foetus during the 3-5 hr after catheterization. Measurements of urine flowwere made in ten foetuses and these were of three degrees of maturity. Theirvolumes, separated into three age groups, are presented in Table 3. The foetalurine flow tended to decrease as gestation advanced, and at or about term itappeared to be about one-tenth the rate it had been four weeks earlier. Thehigher figure is of the order of magnitude obtained by Wells (1946) in thefoetal rat at term (0-0025 ml./100 g body wt./min), but considerably less thanthe lowest rate reported by Alexander et al. (1955b) for the foetal sheep(0 004 ml./100 g body wt./min). A fall in the rate of urine flow during foetallife was evident in Alexander's studies.

In the first hour after birth the average urine flow was much higher thanthat of the foetus at term in spite ofthe fact that no fluid had been administeredand that water was then being lost by the respiratory tract and skin. In thesecond and third hours after birth the flow of urine fell unless supported bythe administration of oral fluids. This is not shown in the table. A furtherincrease in urine flow took place during the first week of life and this wasfollowed by a slower rise to adult levels. In the adult urine flow was oftenhigher in the first half hour after catheterization than subsequently, and mightthen fall to a third or a quarter of its initial value.From Table 3 it would also appear that the average concentration of urea

in the plasma rose during the first week of life and then fell slowly towardsthe level characteristic of the normal adult (see also Table 1). A similarphenomenon has been observed in human infants, rats and pigs (McCance &Widdowson, 1947; Schultze, 1949; Halvorson & Schultze, 1950; McCance &Otley, 1951).

327

328 J. W. BOYLAN AND OTHERS

The urea and creatinine clearances were variable during foetal life and therewas no definite trend in one direction or the other. The urea clearance increasedmore rapidly than the creatinine clearance during the first week -of extra-uterine life with the result that the urea: creatinine clearance ratio rose toabout 1-0 between the fourth and seventh days. After the first week the ratiofell to the level usually found during adult life. The creatinine clearance of theadults amounted to 0-25 ml./100 g/min, which is very close to the figureobtained by Dicker & Heller (1951) for the inulin clearance (0.23 ml./100 g/min).

TABLE 3. Development of renal finction in the guinea-pig

ClearancesPlasma -' A _

-A ~ Urine flow Urea CreatinineUrea Creatinine (ml./] 00 g (ml./100 g (ml./100 g Clearance

No. of (mg/ (mg/ body body bodv ratios,Age animals 100 ml.) 100 ml.) wt./min) wt.!min) wt./min) urea: creatinine

Before birth40 days approx. 1 29 1-72 0-002 0-003 0-015 0-20gestation 1 50 1-48 0-0016 0-002 0-006 0-33

60 days approx. 1 45 0-00036 0-0004 - -gestation 1 32 1-80 0-00047 0-0008 0-011 0-07

1 35 1-25 0-00040 0-0019 0-010 0-191 30 1-19 0-00229 0-0051 0-017 0-301 44 1-38 0-00222 0-0124 0-069 0-18

70 days approx. 1 20-5 1-38 0-0003 - 0-004gestation 1 40 1-40 0-0002 0-003

1 32 1-20 0-00085 0-00265 0-018 0-15After birthNew-born 7 35 1-40 0-002 0-006 0-042 0-130-4 days 5 43 1-20 0-005 0-096 0-125 0-774-7 days 7 46 0-85 0-01 0-20 0-18 1-117-19 days 5 41 0-85 0-015 0-16 0-23 0-70Adult 7 39 0-80 0-03 0-16 0-25 0-63

Urine 'found' and 'formed'The urine 'found' in the full-term foetal bladder at the beginning of an

experiment has already been described (see Table 2). It differed in appearancefrom the urine 'formed' in utero during the experimental periods. The urine'found' at term had a dark amber colour, whereas that freshly 'formed' inutero was colourless or a very pale amber colour. Analysis (Table 4) revealedthat this difference probably extended to chemical composition as well as tototal osmolar concentration. Both physical and chemical differences were lessobvious in younger foetuses. At 60 days the concentration of creatinine inurine 'found' in the bladder was somewhat greater than that 'formed' inutero during observation while at term the concentration was a great dealhigher. The highest concentration of creatinine in a urine 'found' at term was320 mg/100 ml. and the urine 'formed' immediately afterwards in utero con-tained only 19 mg/100 ml. The concentration of creatinine in the urine 'found'

RENAL FUNCTION OF FOETAL GUINEA-PIGS

in the bladder increased with foetal maturity, but there seemed to be nocorrelation with age in 'formed' urine.TABLE 4. Urea and creatinine concentrations and the total osmolar concentration of (a) urine

found at the first catheterization in 40, 60 and 70 day foetuses, and (b) urine formed duringthe experiments by foetuses of the same ages

Total osmolarUrine 'found' Urea Creatinine Urea: creatinine concentrationin foetuses of (mg/100 ml.) (mg/100 ml.) ratio (m-osmole/l.)

40 days approx. 88 (1) 17 (1) 5a2 (1)60 days approx. 222 (6) 48 (6) 4-6 (6) 304 (5)70 days approx. 216 (20) 140 (20) 1-5 (20) 368 (13)Urine 'formed'

during theexperiments

40 days 57 (2) 9-4 (2) 6.1 (2)60 days 134 (4) 32 (4) 4*2 (4) 256 (2)70 days 100 (1) 25 (1) 4-0 (1) 250 (1)

The number of observations is shown in parentheses.

DISCUSSION

The plasmaIf the chromogen in foetal plasma is all creatinine, this substance may be one

of those which does not equilibrate freely across the placental membranes. Thepresent work suggests that if there is some other chromogen in the plasma it isnot excreted in the urine. Further study is required.The rise in the concentration of urea in the body fluids after birth, and its

subsequent fall, would appear to be similar to the findings which have beenestablished in other animals and in man. It is due to the rate of urea produc-tion, from tissue destruction or from the metabolism of ingested amino acidsnot used for growth, temporarily exceeding the amount of urea which theimmature kidney excretes. With so many variables it is not surprising thatthere should be considerable differences between species and individuals inthe extent to which the blood urea may rise. The matter has been investigatedin some detail in pigs (McCance & Widdowson, 1956).

The urineThe urine found in the bladder of the foetal guinea-pig differed from the

urine formed subsequently, and also from the urine which has been taken fromthe bladder of the foetal lamb or human being, in that it was usually hyper-tonic to the serum, and also in the very high concentrations of creatininesometimes found in it and the correspondingly low urea: creatinine ratios.Indeed, these latter findings differentiate it from any other urines previouslytaken from the mammalian bladder or passed spontaneously by such ananimal. They will be of value in future considerations of renal function in uteroand they add interest now to the observations already made on the foetal rat(Wells, 1946). The composition of the urine formed immediately after birth,

329

J. W. BOYLAN AND OTHERS

and of that formed after catheterization, suggests that the urine found in thebladder may have been secreted very slowly in the foetal guinea-pig, especiallynear term. If the rate of formation of urine were extremely slow near termand the tubules and collecting apparatus, including perhaps even the bladder,impermeable to creatinine but permeable, as at all ages to some extent, tourea, the findings could be explained; to postulate that creatinine was excretedinto the urine seems less reasonable. If this suggestion is correct; theremust also be considerable reabsorption of chlorides from these glomerularfiltrates, but most very young animals have low chloride clearances and thereis nothing inherently impossible in this part of the theory. The way in whichthe chlorides in the maternal and foetal urines varied in parallel with eachother may be a point of some significance.

It is impossible to say at present whether the urine formed after catheterizingthe foetuses or any of these guinea-pigs is a strictly normal product or not. Therate of formation may have been changed by the operative interference,although as a rule it is thought not, but further work on the young guinea-pigboth before and after birth is in progress, and it is hoped to resolve some of thedifficulties which have been discussed.

SUMMARY

1. The uterus of the pregnant guinea-pig was incised and female foetusesat three stages of maturity were catheterized and returned to the uterus for3-5 hr. Urine flow, and urea and creatinine clearances were measured.

2. The rate of urine flow, expressed per unit ofbody weight, declined between40 days of gestational age and term, and rose rapidly immediately after birth.

3. The urine found in the bladder of the foetus at or near full term wasgenerally hypertonic, with an average total osmolar concentration of 368 m-osmole/l. The concentration of chloride in the foetal urine was very variableand tended to rise and fall with the concentration in the urine of the mother.The concentration of creatinine was sometimes extremely high, greater eventhan the concentration of urea.

4. These unusual urea: creatinine ratios were not found in the urine formedafter catheterization, or in new-born, growing or adult animals.

Professor E. C. Amoroso, Dr E. M. Widdowson and Dr M. W. Stanier have been a great helpto us and we take this opportunity of thanking them. Some of the animals were obtained fromthe Imperial Cancer Research Fund Laboratories, Mill Hill.

REFERENCESALEXANDER, D. P., NIxoN, D. A., WIDDAS, W. F. & WOHLZOGEN, F. X. (1955a). Changes in

composition of the foetal fluids of the sheep during gestation. J. Physiol. 129, 66P.ALEXANDER, D. P., NIXoN, D. A., WIDDAS, W. F. & WOHLZOGEN, F. X. (1955 b). Urine production

in the foetal sheep. J. Physiol. 130, 13-14P.DALY, H., WELLS, L. J. & EVANS, G. (1947). Experimental evidence of the secretion of urine by

the fetal kidney. Proc. Soc. exp. Biol., N. Y., 64, 78-80.

330

RENAL FUNCTION OF FOETAL GUINEA-PIGS 331DAVIES, J. & ROUTH, J. I. (1957). Composition of the foetal fluids of the rabbit. J. Embryol. exp.

Morph. 5, 32-39.DICKER, S. E. & HELLER, H. (1951). The mechanism of water diuresis in adult and newborn

guinea-pigs. J. Physiol. 112, 149-155.FALK, G. (1955). Maturation of renal function in infant rats. Amer. J. Physiol. 181, 157-170.HALVORSON, H. 0. & SCHULTZE, M. 0. (1950). Nutritional value of plant materials. III. Acute

uremia of newborn rats from mothers fed plant rations. J. Nutr. 42, 227-237.HANON, F., COQUOIN-CARNOT, M. & PIGNARD, P. (1955). 1tude physico-chimique de la premiere

miction du nouveau-ne normal. Bull. Acad. nat. Med. 139, 272-275.HANON, F., COQUOIN-CARNOT, M. & PIGNARD, P. (1957 a). Etude de la fonction renale chez le

foetus et le nouveau-ne normal. J9tud. neonatales, 6, 97-109.HANON, F., COQUOIN-CARNOT, M. & PIGNARD, P. (1957 b). Etude de la fonction exer6toire du rein

ant6-natal par ingestion d'acide para-amino-hippurique per partum. Gynruc. et Obstgt. 56, 64-73.HARE, R. S. (1950). Endogenous creatinine in serum and urine. Proc. Soc. exp. Biol., N. Y., 74,

148-151.HAWK, P. B., OSER, B. L. & SUMMERSON, W. H. (1947). Practical Physiological Chemistry, 12th ed.

Philadelphia: Blakiston Co.HELLER, H. (1951). The water metabolism of newborn infants and animals. Arch. Dis. Childh. 26,

195-204.HERVEY, G. R. (1955). A method for determining the freezing-points of biological fluids. Analyst,

80, 284-289.JACQUP, (1905-06). De la genese des liquides amniotique et allantoidien: cryoscopie et analyses

chimique. Arch. int. Physiol. 3, 463-469.LEE, M. H. & WIDDowsoN, E. M. (1937). Micro-determination of urea in blood and other fluids.

Biochem. J. 31, 2035-2045.MCCANCE, R. A. (1950). Renal physiology in infancy. Amer. J. Med. 9, 229-241.MCCANCE, R. A., NAYLOR, N. J. B. & WIDDOWSON, E. M. (1954). The response of infants to a

large dose of water. Arch. Dis. Childh. 29, 104-109.MCCANCE, R. A. & OTLEY, M. (1951). Course of the blood urea in newborn rats, pigs and kittens.

J. Physiol. 113, 18-22.MCCANCE, R. A. & WIDDowsoN, E. M. (1947). Blood-urea in the first nine days of life. Lancet,

252, 787-788.MCCANCE, R. A. & WIDDOWSON, E. M. (1953). Renal function before birth. Proc. Roy. Soc. B,

141, 488-497.MCCANCE, R. A. & WIDDowsoN, E. M. (1954 a). Normal and abnormal aspects of renal function

in early life. Ber. phys.-med. Ges. Wurzburg, 66, 115-135.MCCANCE, R. A. & WIDDOWSON, E. M. (1954b). Normal renal function in the first two days of life.

Arch. Dis. Childh. 29, 488-494.MCCANCE, R. A. & WIDDowsoN, E. M. (1955). The response of puppies to a large dose of water.

J. Physiol. 129, 628-635.MCCANCE, R. A. & WIDDOWSON, E. M. (1956). Metabolism, growth and renal function of piglets,

in the first days of life. J. Physiol. 133, 373-384.OWEN, J. A., IGGO, B., SCANDRETT, F. J. & STEWART, C. P. (1954). The determination of creatinine

in plasma or serum, and in urine; a critical examination. Biochem. J. 58, 426-436.RAMSAY, J. A. (1949). A new method of freezing-point determination for small quantities. J. exp.

Biol. 26, 57-64.SCIIALES, 0. & SCHALES, S. S. (1941). A simple and accurate method for the determination of

chloride in biological fluids. J. biol. Chem. 140, 879-884.SCHULTZE, M. 0. (1949). Nutritional value of plant materials; 2. Prevention of acute uremia of

the newborn rat by vitamin B12. Proc. Soc. exp. Biol., N. Y., 72, 613-616.SENDROY, J. (1937). Microdetermination of chloride in biological fluids, with solid silver iodate.

II. Titrimetric analysis. J. biol. Chem. 120, 405-417.SMrI, H. W. (1951). The Kidney: Structure and Function in Health and Disease. New York:

Oxford University Press.WELLS, L. J. (1946). Experimental acceleration of secretion of urine in fetal rats. Proc. Soc. exp.

Biol., N. Y., 62, 287-289.