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INFLUENCE OF A LIQUID DIET ON WATER AND ELECTROLYTE EQUILIBRIUM AND NITROGEN EXCRETION IN THE ALBINO RAT
A Thesis Presented to the Faculty of the Department of Zoology The University of Southern California
In Partial Fulfillment of the Requirements for the Degree Master of Science in Zoology
by Irwin Cooper August 1950
UMI Number: EP67189
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z 's-i c-im T his thesis, w ritten by
I R m T COOPER under the guidance of h~..V?. F a c u lty Com m ittee, and app ro ved by a l l its members, has been presented to and accepted by the C o u n cil on G raduate Study and Research in p a r t ia l f u l f i l l ment of the requirements f o r the degree of
f.f. J&£JL Chairman
TABLE OF CONTENTS CHAPTER
Statement of the problem .....................
REVIEW OF THE L I T E R A T U R E .......................
MATERIALS AND M E T H O D S .........................
Animals, cages and equipment used, method of u r i n a l y s i s ..................................
Preparation and composition of the liquid-milk
d i e t ........................................
Weight studies ................................
Water balance s t u d i e s .......................
Electrolyte balance studies
Nitrogen partition studies ...................
R E S U L T S ......................................
Observations on weight experiments ...........
Observations on water balance experiments
Observations on urinary electrolyte concentra tions
Observations on nitrogen excretion ...........
Summarization of results .....................
DISCUSSION OF R E S U L T S .........................
SUMMARY AND C O N C L U S I O N S .......................
A P P E N D I X ...............................................
LIST OF TABLES TABLE I.
PAGE Modifications of Standard Methods of Quantitative Urinalysis . • • • • • • • • • •
Use of Liquid Diet Const i t u e n t s.......... ..
Composition of Solid Constituents in Liquid D i e t s ..........
Formulae for Stock and Experimental Liquid D i e t s ....................................
Nutritional Composition of the Various Solid and Liquid D i e t s .......................
Vitamin Content per 100 c.c. of Liquid Diets •
Water Balance and Weight Gain in Normal Hats •
Water Balance and W e i g h t ................ Urinary Chlorides, Inorganic Phosphates and Total S u l p h u r ...........................
Urinary Sodium, Potassium and Calcium Values of 5 Separate Samples from 11 Normal Hats (5 Male and 6 Female) Fed 50 c.c. of Liquid Diet D a i l y .............................
Comparison of Water and Electrolyte Output During Changes in the Fluid Exchange of Hats
Concentration of Urinary Electrolytes ........
Nitrogen Partition Determinations . . . . . . .
Nitrogen Partition Determinations ............
LIST OF FIGURES FIGURE 1.
The Effect of Addition of a 1% Dried Gelatin Solution on Milk Diet " S " .....................
Effect of Growth on Feeding 25, 50 and 75 cc of Diet A to Rats Weighing Between 170-220 Grams
The Average Daily Weight Gain and Urine Output of 11 Rats Given Liquid Diet A for a 30 Day P e r i o d ........................................
The Effect of Liquid Diets A, B, C, and D on Weight Gain or Loss and Urea Nitrogen Excretion
Comparison of Effects on Body Weight of Rats by Feeding Increased Amounts of Diet B over a 5-day P e r i o d ..................................
6 . The Effect of Dietary Water Content in Liquid Diets A, B, C, and D on the Urinary Output of 11 Normal R a t s .................................... 7.
The Effect of Varying Water Content in Liquid Diets A, B, C, and D on the Specific Gravity of U r i n e ......................................
8 * Cubic Centimeters of Urine Excreted at Various Intervals after the Administration of Diet as a Function of cc of Diet Consumed During the Same Time I n t e r v a l s ................................
The Rate of Urinary Flow after the Intake of 75 cc of Liquid Diet A ........................
Urinary Rate of Flow after Intake of $0 cc of Liquid Diets A, B, C, and D and Solid Diet S
The Effect of Free Water Intake (in Excess of Diet) on the Concentration of Urinary Na, K, and C l .....................................
Changes in Urinary Concentration and Output of Electrolytes in Rats in Response to Administra tion of Liquid Diets A, B, C, and D ........
Variations in Retent of Urinary Electrolytes, Expressed as mgs of Dietary Electrolytes Minus mgs of Urinary Electrolytes, in Response to Administration of Liquid Diets A, B,
C,and D .
The Excretion of Urinary Electrolytes in Normal Rats During a High Fluid Exchange . . .
The Retention of Dietary Electrolytes in Normal Rats During a High Fluid E x c h a n g e .........
LIST OF PLATES PLATE I,
PAGE External View of Metabolism Cage Used for Liquid Diet Experiment
. . . . . . . . . . .
Internal View of Metabolism Cages Used in Liquid and Solid Diet E x p e r i m e n t a t i o n .............
Reproduction of Liquid Diet Drinkers Tested During Liquid Diet E x p e r i m e n t a t i o n .........
Surgical Approach to Basisphenoid Synchondrosis
CHAPTER I INTRODUCTION The extensive work done during the last twenty-five years on the "modus operandi" of water balance in animals has involved many separate phases of research.
Where cause and
effect relationships are masked by overall physiological pro cesses the problem has been approached in such a manner as to reveal only temporary effects or partial explanations of the phenomenon.
Investigators have been unable to fully correlate
experimental conditions and results with mechanisms operating to produce a known effect in the intact animal.
of compounds effective in producing water diuresis or elec trolyte shifts between the fluid compartments of the body, may be above (or below) the physiological level.
Again, the dis
turbance involved during experimentation may result in false negative or false positive reactions on the part of the animal. This study proposes to Investigate water and electrolyte equilibrium associated with a high fluid exchange in the rat placed on a liquid diet regime adequate to maintain normal growth rates (compared to rats on solid food). It is clear that for an accurate analysis of urinary components during long or short term metabolism experiments, the urine must be free of food contaminants.
of collection of urine from animals on solid food diets can
2 not completely prevent such contamination.
At the same time,
amounts of urine collected from these solid diet animals are relatively small in relation to the needs of volumetric assay and chemical analysis.
Utilization of the liquid diet can
overcome these difficulties and special cages can be constructed to permit its administration without uncalculated loss while catching the larger amounts of urine excreted. While the work contained in this report is more directly concerned with the influences of liquid diet on water and elec trolyte equilibrium, it serves as a tool designed to simplify future research on fluid exchange.
Recent research relating
the role of the adrenal cortex to water metabolism has dis closed the actions of the various adrenal cortical fractions (in vivo and in vitro)
on water and electrolytic shifts and
has resulted in a new theory based on a physiological antago nism between diuretic and salt-conserving effects. et al 1949).
However, an accurate description of these re
flexive actions involved between the adrenal gland and posterior pituitary remains to be seen. An ideal method for studying this antagonism and the water imbalances it involves may be through the use of an ex perimentally-induced polyuria that can be found in experimental Diabetes insipidus.
The onset and development of such a poly
uria is well defined in the literature as is the site and for mation of the antidiuretic hormone of the posterior pituitary.
3 A number of small animals, such as the laboratory white rat, induced into this condition can be studied over long periods of time for the effects of pituitrin and adrenal cortical principles (as well as other compounds).
Other workers have
done experimentation along these lines, but their results are not always coincident and are based mainly on short-term periods. It is at this point that the use of a liquid diet, pre viously studied for its influences in normal rats, may be im portant.
Its effect on an experimental polyuria and its role
during the administration of posterior pituitary and adrenal cortical principles can be studied.
The results of this long
term investigation may possibly reveal more conclusive evidence pertaining to the basic mechanism controlling water and elec trolyte balance in the body. Statement of the problem.
Experiments on the use of
liquid diets as a medium for a quantitative study of a normal high fluid exchange in the rat are presented.
The findings of
this work will serve as a basis for future research on the role of adrenal cortical principles and posterior pituitary hormone in the”modus operandi” of water and electrolyte equilibrium in the body.
CHAPTER II REVIEW OF THE LITERATURE The background material for this study was widespread in its scope and implications.
In order to present a compre
hensive view of the previous investigations that were perti nent to this study, the survey of the literature was limited to the use of liquid diets in metabolic studies. A search of the literature revealed no intensive re search primarily concerned with the influence of a liquid diet on fluid balance.
A few isolated sources disclosed diets of
this nature used for short term water studies with no inten tion of maintaining animals in normal states over considerable lengths of time.
Nor was special attention directed to the
development of a standard or stock liquid diet for these pur poses.
Many workers utilized some form of liquid diet from
time to time but largely as a tool to study nutritional prob lems, enzymatic reactions and the like. The earliest work involving a milk-type diet was used for the investigation of nutritional anemia and the factors involved in its correction.
Schmidt (1912) fed a low iron
ration of rice and whole milk to white mice to secure an ane mia .
The production of anemia in the rat by feeding a whole
(cow!s) milk diet was also studied (Happ 1922) (Waddell et al 1928).
The effectiveness of iron and copper and the
5 ineffectiveness of manganese, cobalt, nickel, zinc, as well as some of the essential amino acids, in correcting nutritional anemia (in rats) produced by milk diets, was studied by several workers (Krauss 1931) (Drabkin and Miller 1931) (Elvehjim, Steenbock and Hart 1931) (Orten, Underhill and Lewis 1932) (Orten et al 1932). The first note of interest to this problem came in 1933, when Orten et al studied the effects of prolonged feeding of a milk-iron-copper diet to rats.
Although their efforts were
not directed to a study of water balance, the use of such a diet showed some interesting results: 1.
Body weights of the liquid-diet animals averaged slightly lower than the stock-diet controls,
Total blood volume determinations made on this group at about 300 days of age, averaged 5-5S c.c./ 100 grams of body weight (a normal value).
pathological abnormalities were observed. 3.
Generally, all animals exhibited an average amount of reserve fat and appeared to be in good nutritive condition.
The comparatively low body weights may
have been due to the low caloric intake resulting from the exclusive liquid diet. In 1943 Winter, Ingram, and Eaton concluded that in Diabetes insipidus there is a deficiency in the ability to concentrate NaCl in the urine and that a similar deficiency
6 exists with regard to N compounds.
The N compounds were more
important because N substances form the major part of the total solids in the urine.
Polyuria was markedly reduced in dogs
and cats with experimental Diabetes insipidus on a constant chloride intake by reducing the N intake, and these effects were independent of the caloric intake of the diet.
was parallel to NaCl intake even on a low protein diet if NaCl was fed in amounts over dietary salt.
These workers also ob
served that the polyuria varied directly with the sum of os motic activities of salt and N compounds in the urine and that in order to reduce polyuria of Diabetes insipidus dietarily, both N and salt intakes must be low.
Nitrogen partition de
terminations showed no evidence of any abnormality in Diabetes insipidus.
Creatinine clearance tests gave evidence that
changes in urine volume accompanying alterations in protein intake extended into the post-absorptive period, only after glomerular filtration had returned to control post absorptive levels.
On a high N diet there were more osmotically active
particles present in the tubular fluid to hold water and less with a low N diet.
(Effect was independent of the antidiuretic
hormone (ADH) titer of the blood). Adolph (1948) furnished rats diluted whole milk (2.6% of solids) and in this manner was able to assay atropine, pilocarpine or posterior-pituitary hormones without administer ing water by stomach tube or needle.
In the same year Adolph
7 and Parmington used this dietary regime to provide an easy method for passing excess water through rats.
drinking cylinders in metabolism cages containing 250 c.c. of dilute milk: 2.6 per cent solids, made up every 24 hours by mixing 25 c.c. of Formulae (canned fortified milk) with 0.5 grams of benzoic acid plus 225 milliters of distilled water. Rats were placed on a 3-10 day pre-diet experience period be fore experimentation and were observed to drink this liquid quantitatively in amounts up to their body weights.
tempts were made to continue this regimen for long term trials. Also the authors noted disparities between fluid intake and urine collected, and explained them as due chiefly to incom plete catchment of urine resulting from evaporation on funnels beneath the cages. A high carbohydrate liquid diet was used by Ingle (1946) in studying the effect of adrenal cortical extracts on body weight and N, Na and Cl excretion in the rat.
diet was force-fed to male rats weighing about 300 grams. Preparations of corticosterone and 17-hydroxy corticosterone were tested using 5,
1 and 0.5 mgm. doses.
The results of
this short term experiment (taken at two 24 hour intervals for 1 week) were as follows: 1.
Only high level doses (5 and 2 mgm.) caused weight loss or inhibited weight gain.
Greater weight loss occurred with 17-hydroxycorticosterone.
Urinary N, Na and Cl were excreted proportional to the doses.
Urinary K levels were not affected except "by high dosage (17-hydroxycortieosterone was most effective).
In summary, the following points may he stated about the role of liquid diets in metabolism studies: a.
A milk-iron-copper diet was adequate to prevent a nutritional anemia in the rat.
Prolonged feeding of this milk-iron-copper liquid diet did not change the blood volumes or general nutritive condition of rats.
Stock solid-diet rats showed slightly higher growth rates than did rats on a milk-type diet over the same period of time.
The assay of certain drugs and hormones in relation to water shifts during short-term trials was facili tated by using diluted whole milk as a diet instead of forced water administration in the rat.
Dilute canned fortified milk, used as a dietary regime, was employed as a method of passing excess water through rats over short periods.
Further use of a liquid diet was evidenced by the forced-feeding of a high carbohydrate liquid diet in studies related to certain effects of some adrenal cortical extracts.
CHAPTER III MATERIALS AND METHODS The various procedures used during this study were closely interrelated.
The first step dealt with the prepara
tion and composition of the liquid diet to be used in experi mentation.
The influences of this diet were then studied in
relation to weight gain or loss and to changes in water and electrolyte balance. performed.
Nitrogen partition studies were also
In all cases, rats on this diet were compared to
those on stock solid-diet. Operative experimentation on the production of polyuria in the rat was undertaken during the early phases of this re search.
A surgical method to induce Diabetes insipidus was
devised after trial surgery and is included in an appendix to this thesis.
Further work along these lines was discontinued
as attention was directed to the dietary phases of the problem and their influence on fluid exchange.
Results of posterior-
pituitary lesions will be embodied in future research reports. The organization of this section of the report follows the schema outlined below: I.
Experimental animals, cages and equipment used, methods of urinalysis.
Preparation and composition of the liquid-milk diet.
III. IV. V. VI.
Weight studies. Water balance studies. Electrolyte balance studies. Nitrogen partition studies.
Experimental animals used, cages and equipment used, method of urinalysis. 1.
Animals used for experimentation.
Male and female
rats of the Sprague-Dawley strain ranging from 100300 grams body weight were chosen for this work and were divided equally as controls and experimentals. 2.
Construction and use of metabolism-type cages.
accurate measurement of urine outflow and quantita tive analysis of this urinary output was an essen tial requirement.
There was a prime need for a
metabolism cage and urine catchment system that would result in a minimal error in volumetric esti mation and also permit collection of urine relatively free of contaminants.
The following factors had to
be considered: a.
Time elements and available facilities limited the construction of such systems used by large research laboratories.
the same time cages and catchment designs as the Hendryx or Hopkins type were believed to complicate methods of urine collection to a
point beyond careful measurement. Straining or filtration of the urine (after being voided) to prevent contamination with solid food particles (when solid food was used), feces, and hair had to be of simpli fied design; otherwise urine retent on the strainer would be too high.
paratus itself could not contaminate the urine with metallic elements, nitrogen con taining substances, nor could it extract any of the substances or products present in the urine. Passage of the urine from the floor of the cage to a receiver had to be rapid and over the least surface area possible.
funnels used by most workers offered an in creased surface area despite their channel ing effect. Cages had to be large enough for rats ranging from 100-300 grams and yet small enough to restrict the voiding of urine to the least area of a wire mesh floor. Finally, the cages had to accomodate liquid diet drinkers in such a manner as to prevent any diet overflow into the urine and also to
12 permit the measurement of unused diet* Cages had to be equipped to hold scatterproof food cups so they could be used for either solid or liquid dietary regime. Designs of various cages were considered and a basic type was constructed.
After subjecting this type to a trial
of approximately two months (during which time a selection of an efficient liquid drinker was made) it was further modified (Plate I,A, page 13).
A description of these cages follows:
The tops and bottoms of cylindrical one-gallon cans were removed and at one end 1/2 inch wire mesh was cut to circular size and soldered firmly in place.
At the other
end a door top was made from a metal ring, filled in with 1/2 inch wire mesh, which was hinged and latched to the body of the cage.
Two holes approximately 1-1/2 inches in diameter
were cut into one side of the cage (spaced 5 inches apart). Cylindrical tin housings for the drinkers with circular open ings on top were fitted from the inside into these holes and soldered in place.
A curved tin drain was made continuous
from this housing to the outside of the cage.
shell vials were used as overflow catchers. Liquid plastic was mixed and poured along the insides of the housings and drains and permitted to dry under an infra red heat lamp for 12 hours.
This offered a smooth glass-like
surface and allowed overflowed liquid diet to be drained off
PLATE I External view of metabolism cage used for liquid diet experiment. A —
Metabolism cage with liquid drinker in cage rack.
Metabolism cage rack with cages and funnels in place.
14 into the catch vials immediately without drying in the hous ing.
Plate II, A and B (page 15), clearly shows the construc
tion of these cages. All cages were painted inside and out with 2 coats of aluminum paint and placed in a specially-built wood rack (Plate I, B, page 13).
Cages were mounted individually in
9-inch pyrex glass funnels with shortened stems which were carefully coated with Drifilm on their inside surface.
adaptation markedly facilitated the urine flow along the glass surface and voided urine coursed directly down the fun nel with minimum adherence or wetting.
Excess evaporation and
retent was therefore avoided. Fifty-c.c. graduated centrifuge tubes were fitted around the funnel stems with drilled rubber stoppers and served to catch the urine. To prevent contamination of the urine by solid food particles, rat feces and hair, various contrivances were em ployed during early trials.
At first, stainless steel wire
screening of relatively fine mesh was cut into circles of 3 inch diameter and placed in the funnels.
Coarse filter paper,
5.5 cms. in diameter, was placed in the pit of the funnels and tested.
An effective valve and contaminant catch was finally
devised by dipping ping-pong balls in melted paraffin, letting them dry and placing them in the funnel pit. Twelve rats were placed in the metabolism cages and
PLATE II Internal view of metabolism cages used in liquid and solid diet experimenta tion. A —
Top view of metabolism cage showing liquid drinker and overflow catch.
Top view of metabolism cage adapted for solid diet, show ing the position of the scatterproof-food cup.
16 fed measured amounts from
of liquid diet
(see below). Two rats
this population were placed on
each of the following
strainer combinationsr 1.
Wire mesh only.
Filter paper and wire mesh.
Filter paper (with bottom of cone removed) plus wire mesh.
Filter paper plus wire mesh plus ping-pong balls.
After a seven
days* trial it
fifth system was most effective in straining contaminants and obtaining the highest per cent of urinary return from the v/ater intake.
This contrivance was used exclusively throughout sub
sequent experiments. The collection systems used were corrected for evapora tion and retention of urine.
All systems were cleaned; urine
was collected from all the animals in the rack for 12 hours and mixed together in a flask.
Animals were returned to stock
cages and each system left uncleaned.
Two and one-half c.c.
of this mixed urine (warmed to 32 degrees centigrade) was pipetted into the system from various points along the cage floor at one hour intervals for 12 hour periods.
and per cent humidity changes were recorded during the period. Calculations, made on the basis of ten separate determinations,
17 showed that during this period an average of 8.5 per cent of the pipetted urine was lost through evaporation and retent. Repeating this experiment during the cool of the evening and with the rack area heated by two infra-red lamps, there were no significant changes due to temperature or humidity.
factor of 8.5 per cent was adopted and check periodically. 3*
Original liquid drinkers designed and used in trial of this work proved ineffective (see Plate III, A, page 18). Clotting of the milk, breakage of the glass feeding heads oc curred too often.
A second type (see Plates II and III, B,
pages 15 and 18) designed as a water drinker by Emil Greiner for the Biochemistry Department, The University of Southern California, was tested and found to be efficient, although its maximum capacity was 75-80 c.c. of fluid.
This drinker was
also used for the administration of free water both with the liquid and with the solid diets. Water and diet evaporation from the feeders were cal culated by the change in volume of 50 c.c. of diet or water in their approximate drinkers exposed to the atmosphere for a period of 24 hours.
Evaporation of water from the feeders
amounted to 0.3-0.4 c.c./50 c.c. of water, while the liquid diet value amounted to 0.6-0.8 c.c./50 c.c. of liquid diet A. Liquid retent of the drinkers was evaluated from differences between wet and dry weight after emptying.
On the basis of
PLATE III Reproduction of liquid diet drinkers tested during liquid diet experimentation. A —
100 c.c. prescription bottle
equipped with glass delivery head and number one (one-hole) rubber stopper. This type proved inadequate for use. B —
Greiner-type pyrex glass
liquid drinker. is 6 1/2 inches.