inorganic salts in nutrition - the journal of … salts in nutrition vi. the mineral metabolism of...

INORGANIC SALTS IN NUTRITION

VI. THE MINERAL METABOLISM OF RATS RECEIVING A DIET LOW IN INORGANIC CONSTITUENTS*

BY RICHARD 0. BROOKE AND ARTHUR H. SMITH

(From the Department of Physiological Chemistry, Yale University, New Haven)

(Received for publication, November 30, 1932)

INTRODUCTION

Recent studies have emphasized the importance of the inorganic constituents of food, not only in a general way as sources either of necessary base for the neutralization of acid produced in metabolism or of essential factors in the maintenance of electrolyte con- centrations of the body fluids, but also as determinants of certain specific functions now attributable to individual elements. The controversy occurring during the last 2 years in regard to the role of copper in the remission of nutritional anemia indicates the modern tendency in this connection.

Previous studies from this laboratory have been reported in which the intake of the inorganic constituents of the diet have been reduced to a minimum. It has been shown that the weight of young rats (40 gm. body weight) fed under such conditions does not change materially (Winters, Smith, and Mendel, 1927). The same general method of feeding will also restrict the develop- ment of larger animals (Smith and Swanson, 1929). A condition of retarded growth under such conditions is not so remarkable as the ability to exist at all. Animals restricted in this manner present an opportunity of investigating many physiological processes.

It has been observed that, although very young rats on a ration

* The data reported in this paper are taken from a dissertation presented by Richard 0. Brooke in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Yale University, 1932.

A part of the expenses of this investigation were defrayed by a grant from the Sigma Xi Alumni Research Fund.

A preliminary report was presented before the meeting of the American Society of Biological Chemists at Philadelphia, April, 1932.

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106 Mineral Metabolism

poor in ash constituents fail to increase in weight to any extent, the long bones continue to grow and the kidneys become enlarged to a marked degree (Winters, Smith, and Mendel, 1927). Hydra- tion of the tissues has also been demonstrated as a result of a dietary deficiency in mineral salts (Forster, 1873; Smith and Schultz, 1930; Swanson, 1930).

Smith and Schultz observed a pronounced increase in the num- ber of erythrocytes in the blood of rats receiving a diet poor in ash constituents-a marked though not a typical polycythemia. This observation has since been confirmed and studied in detail (Smith and Swanson, 1929).

The present investigation deals with a study of the mineral metabolism of rats subjected to a strict limitation of inorganic salts in the diet. Complete balance experiments were carried out on nitrogen, phosphorus and calcium, and chlorine, and determina- tions of the acidity of the urine and the excretion of urinary ammonia were made.

A group of rats was given the ration poor in inorganic salts and collections of excreta were made at intervals over a period of 90 days. The results obtained in this way were compared to those secured from control animals of two kinds: (1) a group of normal animals of the same age receiving an adequate diet; and (2) others also of the same age but fed limited amounts of a special ration so adjusted that although the caloric value of the food eaten would be restricted to that of the group of rats fed the low salt diet, yet the protein and mineral intake would approximate that of the normal group.

Procedure

Animals-All the rats used in this experiment were born in the rat colony of the Connecticut Agricultural Experiment Station, New Haven, and were of the same vigorous strain described by Anderson and Smith (1932). Male rats weighing 45 to 50 gm. at weaning were placed by pairs in suitable metal cages. Water and food were available ad lib&m. Until the rats were 28 days of age, the food consisted of modified calf meal and paste food.’

1 The calf meal was that manufactured by Cooperative G. L. F. Ex- change, Inc., Buffalo, in which was incorporated 3 per cent of cod liver oil (see Maynard, L. M., Science, 71, 192 (1932)). The paste food had the


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R. 0. Brooke and A. H. Smith 107

From the age of 28 days, only modified calf meal was fed. To secure a satisfactory condition of retarded growth on a diet low in mineral constituents, a certain rate of increase in weight in young rats has been found requisite from statistical studies (Swanson and Smith, 1932). At 28 days they should weigh from 78 to 88 gm. and they should reach 120 gm. in about 9 days from this time. Only rats that satisfied these requirements were selected.

The rats were then divided into the three groups and the experimental rations given.

Individual cages constructed of wire cloth with a mesh of at least 3 inch were employed. The bottom of the cage was elevated so that access to the excreta was impossible. Until the rats were placed on the experimental diets they were weighed at frequent intervals in order to make selection possible, but after a weight of 120 gm. had been reached, the weight was recorded only once a week. Food intake was also noted.

Collection and Separation of Excreta-The method adopted for the collection of feces and urine was essentially the one advocated by Mitchell (1923-24), except that in place of the crystallizing dishes a glass cage, specially designed for studies of this kind, was employed (Smith and Brooke, 1931). However, this cage presented an unexpected difficulty when used in connection with rats on the low salt diet. Maintenance of retarded growth on such a ration is only possible if extreme care is taken to clean and remove all traces of excreta from the cage at frequent intervals. The urine of the rat is so viscid that the glass rods composing the bottom grid become soiled and salts from the urine then become available for the rat. In this connection, it may be mentioned that Skinner, Steenbock, and Peterson (1932) observed a delay in the onset of nutritional anemia due to the greater adhesion of excreta to the tubes which formed the bottoms of the glass cages used by them. In the present investigation animals were therefore left in individual metal cages except during the collection periods. At such times the glass cage as described was employed with the modifica- tion that the bottom grid was replaced by one of Q inch wire cloth.

The filter paper used to absorb the urine was Durieux No. 121 (Palo Company, New York). The circles were large enough to

following composition: whole milk powder 25 per cent, casein 25 per cent, wheat embryo 20 per cent, lard 30 per cent.


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rest on the flange of the plate where they were held in place by the weight of the cage. Before use, the papers were subjected to the following treatment.

Bundles of 100 were first soaked in 2 N acetic acid for 24 hours, then placed in a large Buchner funnel and subjected to strong suction. After removal and soaking in 2 N acetic acid for 5 minutes, they were sucked dry on the Buchner funnel as before. This process was repeated ten times. Finally they were soaked overnight in a 1 per cent solution of thymol in alcohol, to which:12 cc. of glacial acetic acid had been added for every 100 cc. of solution, and dried at room temperature. In this way a filter paper retaining a distinct odor of thymol and acetic acid and free of extractable salts was obtained.

14 days after the change of diet, each rat (120 gm. in body weight) was placed in a glass cage to accustom it to the new environment. On the 17th day, the cage was placed over two filter papers prepared as described above. Each day the feces were removed and preserved in a 1 ounce bottle containing 95 per cent alcohol acidi- fied with a few drops of glacial acetic acid. At the same time any food which had been spilled was brushed into another 1 ounce bottle and kept for future weighing. Every 2nd day a clean cage was provided. The top filter paper was removed and a clean one placed below the one remaining.

The following procedure was found satisfactory in rinsing off the cage: The glass cylinder was placed in the supporting plate and the inside rinsed with 2 N acetic acid, the washings being poured into a wide mouth 500 cc. jar in which was placed the removed filter paper. The metal grid was cleaned by removing it to a circular photographic dish. A small amount of water was poured into the dish so that an oscillating motion would force the liquid to and fro over the grid. This was repeated three times. Each portion of wash water was added to the jar and the jar was stored in the ice box.

In the extraction of the urine from the filter papers, small Buch- ner funnels fitted to large suction flasks were used. Acid-washed filter papers were fitted to the funnels. The acetic acid in the jar containing the filter papers to be extracted was poured into the Buchner funnels and the papers themselves were placed in 400 cc. beakers. They were thoroughly macerated with 2 N acetic acid,


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transferred to the Buchner funnel, and again sucked dry. The pack was removed to the beaker and the process repeated. Six such washings proved to be sufficient, but washing was continued until the filtrate amounted to 1500 cc.

The feces were transferred to tared crucibles and evaporated three times with 95 per cent alcohol on a steam bath and dried in an electric oven at 100’ for an hour. After the weight had been recorded, the sample was ground in a mortar as iine as possible and preserved in tightly stoppered vials.

Each collection period was of 6 days duration except Period III of the rats fed the low salt diet, which was extended to 12 days. In the case of the normal rats, four such periods were studied; but subsequently Period III was omitted. The gain in weight during the collection period was recorded and the food intake weighed to the nearest 0.1 gm.

For the estimation of the amount of urine excreted in 24 hours, individual rats were confined in special cages. The cages consisted of a grid made of Q inch mesh wire cloth with a low wall formed by a strip of galvanized iron extending 2 inches above the grid. An inverted funnel was supported above this. The cage formed in this way was made as small as was compatible with a reasonable amount of freedom for the animal, and rested over a glass funnel. Urine was collected in a graduated cylinder beneath a layer of mineral oil and toluene. A wire grid, situated low down in the funnel, served to hold back the feces and a 5 cm. filter paper, moistened with a 1 per cent solution of thymol in alcohol, placed in the apex, insured a clear and uncontaminated urine. Water was supplied by means of a glass fountain.

Analytical Methods-The diets and vitamin adjuvants were analyzed by the standard procedures of the Association of Official Agricultural Chemists. Analysis of the excreta was subjected to considerable study and the adequacy of the technique in each case established by recovery experiments. The urine was analyzed according to the following scheme.

Nitrogen and ammonia were determined on the dilute extract by a modified Kjeldahl procedure (Cohn, 1923) and the Van Slyke and Cullen (1914) aeration method, respectively. The dilute extract was then concentrated to a volume of 500 cc. and kept in Pyrex flasks until the fixed base had been estimated by the method of Stadie and Ross (1925).


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Calcium, phosphorus, and chlorine were estimated in this concentrated urine extract. For the estimation of calcium an aliquot of 100 cc. was taken, evaporated to dryness, and ashed at a red heat. The residue was taken up in dilute nitric acid and diluted to a suitable volume. Calcium oxalate was precipitated according to the technique of Van Slyke and Sendroy (1929), the precipitate washed by the technique of Clark and Collip (1925), and finally Gtrated with 0.01 N potassium permanganate.

Phosphorus was estimated calorimetrically by the method of Fiske and Subbarow (1925). The ashing was accomplished with magnesium nitrate as described by Berggren (1932).

Chlorine was determined according to the blood chloride determination of Van Slyke (1923-24).

To determine the pH of Dhe urine, 1 cc. of urine collected as described under the method for securing a 24 hour sample, was pipetted into a clean test-tube and diluted to 5 cc. with distilled water. A suitable indicator was added and the resulting color compared to standards of known pH, in a comparator. A study of the efficiency of the method for the collection of urine and determination of its constituents indicated that an accuracy of &4.0 per cent was attained.

Diets-Three experimental rations were used in this investigation. They were prepared as described by Swanson and Smith (1932), and may be designated as follows: the adequate diet (Diet II), the low salt diet (Diet III), and the special diet (Diet IV) which was fed in limited amounts to the controls with restricted intake.

The vitamin adjuvants to all diet,s were provided by the following additions given to each rat apart from the ration: cod liver oil, 5 drops; ether extract of wheat germ, 3 drops; yeast powder, 200 mg.; and alcoholic extract of wheat, 1 cc. Dried yeast? com- monly contains about 14 per cent of ash. For this reason only 200 mg. were fed per day, but as this amount provided too little vitamin B, a supplementary addition of wheat germ extract was made. Both the ether extract and the alcoholic extract were prepared according to the methods given by Swanson and Smith (1932).

2 Northwestern Yeast Company, Chicago.


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The composition of the three experimental diets is given in Table I.

The low ash diet (Diet III) is practically devoid of calcium, but because casein was used as a source of protein, the phosphorus content is fairly high. Chlorine is present in appreciable amounts. Since the average food intake of the normal controls is 10 gm. per day and that of the other groups only 6 gm., the daily amounts of nitrogen, phosphorus, calcium, and chlorine ingested respectively

TABLE I

Composition of Experimental Diets

Ingredients Diet II

per cent Low ash casein.. Dextrin............................ Hydrogenated fat (Crisco). Salts (Osborne and Mendel, 1918). _. Vitamins as described in text

18 51 27

4

TABLE II

Diet III Diet IV

per cent per cent

18 25.7 55 41.6 27 27.0

0 5.7

Analyses of Experimental Rations and Vitamin Adjuvants

Diet No.

II III IV

Vitamin adjuvants, 6 day portion.. . . .

I Nitrogen

per cent 2.75 2.75 3.74

ml.

143

Phosphom

pm cent 0.43 0.16 0.50

36

Calcium Chlorine

per cent per cent 0.52 0.21 0.0018 0.06 0.72 0.27

%?. m!J.

3 8

Sulfur

per cent

1.75 1.40

were as follows: normal controls, 0.280, 0.043, 0.052, and 0.021 gm.; restricted intake controls, 0.220, 0.030, 0.043, and 0.016 gm.; rats fed the low salt diet, 0.170, 0.010, 0.000, and 0.004 gm.

It is apparent that, although the controls with restricted intake received the same caloric value as the rats fed the low salt diet, their intake of these elements approached that of the normal controls.

The analysis of the three experimental diets and the vitamin adjuvants are presented in Table II. Diet III, the low ash ration,


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is practically devoid of calcium but would appear to contain ample phosphorus. The importance of this fact is emphasized later. Fixed base values could not be determined with any certainty and are therefore not included, but it would appear from the low percentage of total ash in the ration, from the phosphorus content of the ash of the casein, and from the urine analyses, that the amount in the low ash diet is negligible.

It is apparent that the main source of inorganic constituents in the low salt ration (Diet III) is the vitamin supplement, especially the source of vitamins B and G.

CHART I. Typical growth curves for rats

Results

Growth-Growth response of the different groups is presented in Chart I. Conditions of retarded growth were developed in both the controls with restricted intake and in the rats receiving the diet deficient in inorganic constituents. The former group showed a rate of growth intermediate between the animals fed the low salt diet and the normal controls. Comparison of the normal controls with rats described by Smith and Bing (1928) indicates that Diet II was adequate, as far as growth is concerned, over the period of time required in our experiments. In regard to the two groups showing retarded growth, the appearance of the animals


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cannot be overemphasized. At the end of 90 days, the restricted controls were in excellent general condition and in appearance

TABLE III

Balance Experiments*

Element

Nitrogen

Chlorine

Calcium

Phospho- rus

Normal controls Restricted intake controls Rats on low salt diet

output output output

i; 2 2 2 ‘g i I 9 z ; J 0 4 ,8

2 SFr

I B 5 0 i

i

j .E

p: 2s;EZ --- -~-___----_-

mg. mg. mg. nag. mg. mg. mg. mg. mg. mg. mg. mg.

14 2197 1360 281 556 50 1259 1092 95 72 34 2148 1697 191 260 1519531167243 543 5112591101 84 74 4317271479265 -17 2418401197217 426 521259 953116 190 4413781294122 -38 2518481249188 411 5512331009113 111 4716781164165 349 2717111018176 517 5612591226 77 -44 4819551512207 236

581259 1155 90 14 49 1800 1273240 287

14 165 163 2 50 89 77 12 34 57 73 -16 15 146 148 -2 51 89 74 15 43 47 59 -12 24 138 144 -6 52 89 76 13 44 52 50 -2 25 138 139 -1 55 87 66 21 47 46 42 4 27 128 122 6 56 89 84 5485254 -2

58 89 75 14 49 49 52 -3

14 291 10302 21 50 219 4 173 42 34 7 10 3 -6 15 345 12308 25 51 219 5 155 59 43 7 6 4-3 24 324 14261 49 52 219 6 172 41 44 7 7 3-3 25 325 17232 76 55 214 5 135 74 47 7 14 6-13 27 299 9228 62 56 219 5 148 66 48 7 15 2 -10

58 219 5 138 76 49 7 10 3 -6

14 357 134137 86 50 186 85 98 3 34 154 150 12 -8 15 319 134133 52 51 186 72 76 38 43 120 117 22 -19 24 301 123101 77 52 186 81 91 14 44 100 88 11 1 25 303 128104 71 55 183 81 63 39 47 117 159 17 -59 27 281 113104 64 56 186 72 72 42 48 133 130 16 -13

58 186 88 65 33 49 125 134 18 -27

* Only Period IV has been included in this table.

were superior to the normal rats of the same age; their coats were sleek, the animals were active and alert. On the contrary, the rats receiving the low ash diet were evidently on the verge of


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collapse at the finish of the experimental period. Their fur was ragged and “staring,” with baldness frequently present, and their movements though nervously active, were uncertain. The fore- legs of most of these animals were bowed. A bloody nose was a

TABLE IV

Averaged Balances for Periods I, II, and III

Element

Nitrogen

Chlorine

Calcium

Phosphorus

Period No.

I II

III

I II

III

I II

III

I II

III

-

I

-

-

\Tormal controls

,mf;

644 566

19 3

-1

130 108 52

130 55 19

TABLE V

b&rioted intake controls

wl. m!J .

685 590 374 285

21 19

183 129

131 113

21 7

-4 -7

43 20

Digestion of Protein Expressed As Coeficients of Apparent Digestibility

Group Period I

Normal controls.. . . 91 Restricted controls.. 94 Rats on low salt diet.. _. 89

Period II Period III Period IV

90 90 89 94 92 92 89

consistent phenomenon in this group, though abnormality of the lungs was very infrequent at autopsy.

Nitrogen Balance-Typical nitrogen balance figures are given in Table III which, however, presents only values obtained in Period IV. The remaining periods are summarized in Table IV.

On averaging the coefficients of apparent digestibility of protein for the three groups of animals, the figures presented in Table V


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were secured. There is, therefore, no doubt that the rats receiving a diet low in mineral matter maintain their power of digesting protein practically unchanged throughout the 90 day period, and at a level equal to or better than the control animals.

Chlorine Balance-The fecal chlorine was not estimated in our experiments. Typical figures for the intake and urinary output are presented in Table III. A consideration of the excretion of chlorine was complicated by the discovery that the salt mixture used in making up the adequate diet contained less than half the correct amount. Such a shortage would not appear to be significant in view of the powers of retention for chlorine possessed by the animal body. There was, however, a slight negative balance indicated in some of the normal rats although in no case was there any appreciable effect on the growth of the animals. The controls on restricted intake all show a positive balance. Diet III, although extremely low in mineral matter, nevertheless contained 0.06 per cent of chlorine, an amount well above the minimum requirement of rats as defined by Osborne and Mendel(l918).

From the apparent utilization of protein it seems likely that, even in the rats on the low ash diet, the diminished chlorine intake did not interfere markedly with the production of gastric juice, although it is recognized that vigorous proteolysis occurs in the intestine also. Furthermore, the continued loss in the urine indicates that the threshold value of excretion had not been reached in these animals.

Calcium Balance-The absorption of calcium ingested with the food is influenced to a great extent by factors affecting it,s solubility in the intestine. An abundance of phosphates in the diet increases the fecal loss of calcium because of the formation of insoluble phosphates. The presence of unabsorbed fatty acids in the digestive tract tends to a formation of insoluble calcium soaps which are excreted in the stools (Telpher, 1926). A potentially acid-producing diet by inducing an acid condition in the intestines improves absorption because it tends to prevent the formation of insoluble calcium compounds. In precisely the same way the ingestion of hydrochloric acid (Givens and Mendel, 1917) and of ammonium chloride (Stewart and Haldane, 1924) increases the urinary calcium at the expense of the feces.

Typical values secured in balance experiments with our experi-


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mental animals are given in Table III. A consideration of the normal control calcium balances indicates that the retention of calcium varies with the rate of growth. That is to say, as the growth curve flattens out in Periods III and IV, the retention of calcium becomes correspondingly less (see Table VI). The re- lationship is more striking in the case of the control animals with restricted intake, as one would expect, because the growth of these animals is retarded by the limited caloric intake. In connection with the balance experiments with the rats receiving the low salt diet, there is evidently a decided contrast in comparison with either of the groups of controls. The low salt diet is practically devoid of this element so that the vitamin adjuvants are by

TABLE VI

Retention of Calcium by Normal Rats in Relation to Rate of Growth*

Rat 14

Body Ca Body weight retained weight

om.

170 231 309 356 378

per cent

45 33 18 20

om.

160 190 262 292 314

-

Rat 15

Ca retained

per cent

40 32 20

7

_

. _

-

Rat 24 T Body CS

weight retained

om.

126 200 253 280 318

per cent

38 28 15 15

--

1

-

Rat 25

Body CS weight retained

om.

136 191 244 278 314

per cent

45 36 12 23

-

7

-

Rat 27

Body Care- eight tained ____

om. per cent 126 184 47 222 45 272 17 294 21

* Body weights were taken at intervals of 21 days.

far the greater source of supply. There is in general a small but persistent drain of calcium from the organism, the average loss for Period I being 4 mg., for Period II 7 mg., for Period IV 7 mg., and it is conceivable that if a small supply of calcium were added to the low ash diet at the beginning of Period IV; that is to say, after the animals have received the low ash diet for about 60 days, a condition of retarded growth might be maintained for far longer periods of time than has yet been attempted.

Phosphorus Balance-The absorption of phosphorus by the organism like the absorption of calcium is augmented by any factor that influences the solubility of phosphates in the alimentary tract. Each of these two elements influences the metabolism of the other. A diet high in calcium and low in phosphorus will tend


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to produce a high fecal loss of both because of the precipitation of insoluble calcium phosphate in the intestine. The ingestion of acid increases absorption of phosphorus; alkalies have the opposite effect. Fatty acids by the formation of calcium soaps may pos- sibly induce greater absorption of phosphorus by diverting calcium. Under ordinary conditions, fecal phosphorus consists almost entirely of unabsorbed phosphorus.

In fasting rabbits 93 to 99 per cent of the excreted phosphorus is found in the urine (Wellman, 1907). The magnitude of phosphorus absorption and excretion has an interesting significance in the present investigation, because, with the possible exception of sulfur, it is the one potentially inorganic constituent of the low ash diet of which there appears to be an ample supply. In Table III will be found the results of the phosphorus balance experiments with all three groups of rats.

In spite of the distorted calcium-phosphorus ratio, evident in the low salt diet, due to the use of casein in a ration practically devoid of calcium, no pathological condition such as rickets develops in the animals fed the low salt diet. There is instead a condition of extreme osteoporosis (Swanson, 1930).

It is an interesting fact that if the phosphorus ingested with the vitamin adjuvants by rats fed the low salt diet be subtracted from the total intake of this element, the urinary phosphorus, in most cases, is roughly equivalent to the phosphorus combined in the casein eaten by these rats (see Table VII).

Excretory Acid-What is the physiological mechanism that enables rats receiving a diet low in inorganic constituents to metab- olize a ration so potentially acid? The fluids bathing the cells of the body possess a certain electrolyte concentration. This concentration is one of the most carefully guarded constants within the organism. Under various conditions the anions may change considerably; vomiting may cause a replacement of the lost Cl by HCOB; hyperventilation reverses this adjustment and in fasting organic acids replace both Cl and HCOZ. There is, however, little or no lowering of the concentration of total fixed base except under the severest pathological conditions such as may occur in the terminal stages of nephritis.

Conservation or control of base in the organism is a major func- tion of the kidneys. The mechanism of this regulatory power


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involves two factors, base economy and ammonia production. Base economy arises from the ability of the kidney to secrete a urine of greater acidity than the blood. Phosphoric acid and organic acids can enter the urine with less base than they require in the body fluids. On the other hand, at the usual pH of urine, carbonic acid cannot be considered as a cause of loss of base.

TABLE VII

Comparison oj Urinary Phosphorus of Rats Fed Low Salt Diet, Intake of Phosphorus Deriveti rom Casein of Diet

Period No. P intake less P in vitamin sdjuvants

I (6 days)

II (6 days)

IV (12 days)

P output in urine Rat No.

34 43 44 45 47 48 49

34 43 44 45 47 48 49

34 43 44 47 48 49

m7. WJ. 70 66 58 52 57 47 58 58 84 62 61 56 50 49

38 102 64 64 57 59 53 47 59 50 63 56 46 71

154 150 120 117 100 88 117 159 133 130 125 134

Henderson (1907) has pointed out that although phosphates at the pH of the body fluids are excellent buffers, yet the concentration of inorganic phosphorus in the blood is quite low so that it is chiefly in the urinary excretion that phosphates contribute to the acid-base economy in the organism. Conditions tending to produce an acidosis increase t,he total excretion of phosphates in the urine.


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The strong acids, hydrochloric and sulfuric, cannot be present in urine unbound. They require their full equivalence of base even in the most acid urine.

The second factor in the conservation of base is perhaps more important than the first. Ammonia occurs normally in the urine in appreciable amounts, but is is found in the blood in such minute concentration that it is difficult to estimate. Ingested ammonium salts are removed with extreme rapidity even when the renal cir- culation has been cut off; it is therefore not directly excreted by the kidneys. Benedict and Nash (1929) concluded from their experiments that urinary ammonia was produced in the kidneys and there is much clinical evidence to support this theory. Pre- sumably a reversible reaction takes place in the kidney between urea and ammonia, depending on the requirement of the body for the excretion of acid. It is not primarily a mechanism for the control of the normal H ion concentration in the blood and the tissues, but is more directly concerned in the conservation of base by substitution, and seems to be responsive to the level or avail- ability of base irrespective of the presence of acidosis (Gamble, Ross, and Tisdall, 1923).

The principles discussed above underlie the ability of the experimental rats in the present study to exist for considerable periods on a diet extremely poor in base and possessing a high potential acidity. A camparison of the pH of the urine excreted by normal rats fed the adequate Diet II with that of the animals fed the low salt diet (Table VIII) makes evident a marked increase in the urinary acidity in the case of the rats subjected to the low salt acidogenic diet. This indicates an improvement in the base economy because of a more effective removal of acid from the blood by the kidneys. Since the ratio BH2P04:B2HP0, in the plasma of these rats is approximately 1: 4, the base bound in terms of normality by HsP04 while being conveyed for excretion is 1.8 times the molecular concentration of the acid. There is of course some conservation of base in the case of the normal animals which have a urinary pH of about 6.7 but the renal eiliciency of the rats fed the low salt diet is remarkably increased, as indicated by a urinary pH of about 6.0. At this pH the base equivalence is 1.12 which represents a saving or conservation of base for the organism of nearly 38 per cent.


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In Table IX are given the percentages of absorbed phosphorus excreted in the urine of all three groups of rats, together with the values representing urinary total fixed base and ammonia. The low figures for fixed base in the urine of the rats fed the low salt diet indicate the marked power of conservation. Furthermore, these animals increased their excretion of urinary phosphates, a result of the acidogenic nature of the diet. Therefore, the quan- tity factor was altered to assist in the maintenance of the acid-base equilibrium within the organism. However, a significant increase in the excretion of ammonia is perhaps the most effective factor in the mechanism of acid excretion in these rats. The extent and a

TABLE VIII

Comparison of pH of Urine of Normal Controls and Rats Fed Low Salt Diet

Normal controls* Experimental rats

Rat No. Urinary pH Rat No.

61 6.7 34 62 6.7 43 63 6.7 44 64 6.8 47 65 6.7 48

Urinary pH

Period II Period IV

6.1 5.9 5.8 5.9 5.9 6.0 6.0 6.0 6.2 6.1

* It will be observed that these control animals were not the same animals used in the mineral balance studies. They were, however, subjected to precisely the same treatment.

comparison of the ammonia production of all three groups of animals are presented in Table IX.

In so far as is warranted by the present investigation, an analysis of the acid excretion of the rats receiving the low salt diet is given in Table X. The method of collection of feces and urine, involv- ing as it did the use of acetic acid, made it difficult to estimate the titratable acidity and the determination was not accomplished. In this connection, on the basis of the values given for sulfur, an assumption is made that 88 per cent of the total urinary excretion is in the form of inorganic sulfate (Folin, 1905).

It would appear that despite a limitation of inorganic constituents of the diet severe enough to retard growth and despite an


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unbalance in the ration between base and phosphorus, there is an excess of base available for neutralization of metabolic acids excreted in the urine in all except the final experimental period. This is made possible by the high proportion of ammonia in the urine. It seems likely that one of the causes of death in these

TABLE IX

Conservation of Base by Rats Receiving Diet Low in Inorganic Constituents

‘F-c” .*

- I

-

-_ Excretion of ammonia and iixed base in urine expressed

as cc. of 0.1 N NaOH Percentage of absorbed P

excreted in urine

Re- stricted intake

:ontrolr

31 31 32 40 38

40 44 50 42 60

66 65 68 63 73

-

Restricted intak controls

- 1

4mmo- nia

AmUD nia

1 4mmo- nis

55 62 88 13 122 18 6 54 45 53 65 13 147 10 7 47 41 53 75 13 138 12 6 47 42 62 89 11 150 9 11 48 37 64 80 13 148 15 5 43

39 68 78 12 162 55 73 91 1 147 66 68 86 15 88 32 62 78 15 121 57 97 94 12 133

61 89 86 11 194 72 87 88 9 202 62 117t 75 18 199 64 85 75 13 185 64 93 81 12 166

15 12 15 10 14

15 9

15 14 14

5 37 11 43 6 33

10 47 14 47

6 94 5 61

24 45 18 64 4 76

- -

I PI CA

3

-

c --

-

I

II

IV

* Periods were 6 days in duration except Period IV, experimental rats, which was 12 days.

t Negative balance.

animals is the failure of ammonia production, inasmuch as they usually fail to survive much beyond the 90 day experimental period. During Periods I and II the potential acidity derived from the metabolism of the dietary casein was well within the control of the animal, because the intake of acidogenic food was voluntarily reduced. However, the tolerance in this respect had


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evidently reached its limit in Period IV, a fact reflected in the poor condition of these rats at the end of the 90 day period.

Unfortunately the intake of fixed base was not accomplished satisfactorily; however, from a consideration of the authentic analyses of yeast (Leach, 1920), it is evident that even though the

TABLE X

Analysis of Process of Acid Excretion by Rats Fed Low Salt Diet

I

II

IV

Rat No.

34 43 44 47 48 49

cc. cc. cc. cc. cc. cc. cc. cc.

24 11 4 39 54 6 60 21 19 9 5 33 47 7 54 21 17 9 4 30 47 6 53 23 23 7 5 35 49 11 60 25 21 9 4 34 43 5 48 14 18 12 4 34 39 6 45 11

34 38 13 10 61 70 5 80 19 43 23 7 5 35 39 11 50 15 44 22 10 3 35 33 6 39 4 47 18 11 6 35 47 10 57 22 48 26 5 7 38 47 14 61 23 49 30 9 5 44 40 8 48 4

34 53 23 21 97 94 6 100 3 43 42 15 17 74 68 5 73 0 44 32 19 14 65 41 24 65 0 47 56 14 12 82 64 18 82 0 48 46 20 15 81 76 4 80 0

HaPOa H&O4 HCl Total NHrN _-

0.1 N acid excretion 0.1 N base excretion

Fixed base Total

* Period IV for rats fed the low salt diet was 12 days.

rats on a low salt diet continued to excrete fixed base, the balances in this respect were not negative.

SUMMARY

At the outset it appears remarkable that rats receiving the experimental ration extremely low in inorganic residue can survive for 90 days. The present study of the mineral metabolism during maintenance on the salt-poor regime has emphasized the


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flexibility and efficiency of the biochemical mechanisms whereby the organism adjusts itself to the physiological emergency im- posed by the deficiency of the diet employed.

The utilization of protein was unimpaired, the retention of nitrogen being roughly proportional to the gain in weight. The chlorine in the diet was apparently ample for the continued secre- tion of gastric juice. Chlorine was excreted into the urine in amounts that indicated that the renal threshold value for this element was not reached.

The pH of the urine of these rats and of the normal animals fed the adequate diet was 6.0 and 6.7 respectively. There was an increase of phosphorus excreted in the urine of the experimental animals as compared to that of the controls. A marked increase in the production of ammonia by the kidneys of the rats fed the low salt diet was the most important factor in facilitating the excretion of acid.

There was a significant drain of calcium from the rats fed the low salt diet. It seems probable that this loss of calcium is an important cause of the ultimate death of these animals. The fact that tetany frequently develops in rats receiving a diet low in mineral matter gives some support to this idea.

BIBLIOGRAPHY

Anderson, W. E., and Smith, A. H., Am. J. Physiol., 196, 511 (1932). Benedict, S. R., and Nash, T. P., Jr., J. Biol. Chem., 82, 673 (1929). Berggren, R. E. L., J. Biol. Chem., 96, 461 (1932). Clark, E. P., and Collip, J. B., J. Biol. Chem., 63, 461 (1925). Cohn, E. J., J. Gen. Physiol., 4, 713 (1923). Fiske, C. H., and Subbarow, Y., J. BioZ. Chem., 66,375 (1925). Folin, O., Am. J. Physiol., 13, 66 (1905). Forster, J., 2. Biol., 9, 297 (1873). Gamble, J. L., Ross, G. S., and Tisdall, F. F., J. BioZ. Chem., 67,633 (1923). Givens, M. H., and Mendel, L. B., J. BioZ. C’hem., 31,421 (1917). Henderson, L. J., Am. J. Physiol., 21, 427 (1907). Leach, A. E., Food inspection and analysis, New York (1920). Mitchell, H. H., J. BioZ. Chem., 68, 873 (1923-24). Osborne, T. B., and Mendel, L. B., J. BioZ. Chem., 34, 131 (1918). Smith, A. H., and Bing, F. C., J. Nutrition, 1, 179 (1928). Smith, A. H., and Brooke, R. O., Proc. Sot. Exp. BioZ. and Med., 28, 854

(1931). Smith, A. H., and Schultz, R. V., Am. J. Physiol., 94, 107 (1930). Smith, A. H., and Swanson, P. P., Am. J. Physiol., 90, 517 (1929).


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Mineral Metabolism

Skinner, J. T., Steenbock, H., and Peterson, W. H., J. Biol. Chem., 97, 227 (1932).

Stadie, W. C., and Ross, E. C., J. Biol. Chem., 66, 735 (1925). Stewart, C., and Haldane, J. B. S., Biochem. J., 18, 855 (1924). Swanson, P. P., Dissertation, Yale University, New Haven (1930). Swanson, P. P., and Smith, A. H., J. Biol. Chem., 98, 479 (1932). Telpher, S. V., Quart. J. Med., 20, 1 (1926). Van Slyke, D. D., J. Biol. Chem., 68, 523 (1923-24). Van Slyke, D. D., and Cullen, G. E., J. Biol. Chem., 19, 211 (1914). Van Slyke, D. D., and Sendroy, J., Jr., J. Biol. Chem., 84, 217 (1929). Wellman, O., Arch. ges. Physiol., 121, 508 (1907). Winters, J. C., Smith, A. H., and hlendel, L. B., Am. J. Physiol., 80, 576

(1927).


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Richard O. Brooke and Arthur H. SmithINORGANIC CONSTITUENTS

RATS RECEIVING A DIET LOW INTHE MINERAL METABOLISM OF

INORGANIC SALTS IN NUTRITION: VI.

1933, 100:105-124.J. Biol. Chem.

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CORRECTIONS

On page 113, Vol. 100, No. 1, March, 1933, Table III, column 3, fifth and eleventh figures, read 1716 and 691 for 1711 and 991, respectively; column 6, eleventh figure, read 79 for 91; column 13, last group of figures, read 180, 166, 166, 159, 169, and 161 for 154, 110, 100, 117, 166, and 186, respectively; last column, ninth figure, read 9 for -9 and in the last group of figures, read 18, 17, 67, -96, 89, and 9 for -8, -19, 1, -59, -16, and -.W, respectively.

On page 114, Table IV, column 3, last figure, read 109 for 19; column 4, first figure, read 90% for 685; last column, second, third, and fourth figures, read 8.81, 16, and 4 for 886, 81, and 7, respectively.

On page 121, Table IX, column 2, eleventh figure, read 97 for 66.

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