PROTEIN METABOLISM, PROTEIN INTERCHANGE, AND UTILIZATION IN PHLORHIZINIZED DOGS

BY J. W. HOWLAND AND W. B. HAWKINS (From the Department of Pathology, The University of Rochester School of

Medicine and Dentistry, Rochester, New York)

(Received for publication, November 26, 1937)

Can the body utilize plasma proteins in its metabolic processes when there is a demand for protein material? Previous reports from this laboratory have shown that dog plasma given intrave- nously to a protein-fasting dog will maintain the dog approxi- mately in nitrogen equilibrium. No surplus nitrogen elimina- tion is found in subsequent periods. Such evidence points to an efficient utilization of the introduced plasma protein either to replace or repair tissue protein (2, 9, 3).

Further knowledge as to the fate of this injected plasma protein is desirable. Is it metabolized in the same manner as it would be if it were fed to the dog? It seemed possible that additional evidence as to the mode of utilization might be obtained by in- troducing the plasma protein into a dog rendered diabetic by phlorhizin. If it should be catabolized in the usual manner, then there should be an increased amount of nitrogen and sugar eliminated in the urine. Lusk (6) has shown that dogs rendered completely diabetic by phlorhizin convert 58 per cent of fed proteins to glucose and Janney (4) has demonstrated that 55 per cent of fed serum protein is converted into glucose.

The metabolism of injected plasma protein apparently is different. It promptly disappears from the blood stream and yet no excess nitrogen or sugar is recovered from the urine. Either the protein is removed from the blood stream and stored in body tissues in its original form or else it is only partially broken down and then rebuilt into tissue proteins. It is obvious that it is not catabolized to amino acids before being utilized, since no excess nitrogen or sugar is found.

This suggests an interesting method of protein exchange within 99

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100 Protein Metabolism

the body-not a complete protein catabolism as in digestion but a slight catabolic change of serum protein into large aggregates of amino acids with subsequent reassembly into the protein peculiar to the cell of the particular organ or tissue.

Methods

The plan of the individual experiment was simple. The dog was fasted for 24 hours and then with continued fasting, phlorhizin in olive oil in 1 gm. doses was injected subcutaneously daily throughout the experimental period. On the 3rd or 4th day after phlorhizin injection was begun, when the D:N ratio indicated the dog had been rendered completely diabetic, urine collected over a 12 hour basal period was taken and determina- tions carried out. Following the basal period a known amount of plasma protein was injected intravenously and the animal followed through an adequate number of periods all 12 hours in length. Control experiments were conducted under identical conditions, a similar amount of Locke’s solution (300 cc.) contain- ing a known amount of anhydrous glucose (300 mg.) being used. The dogs were kept in metabolism cages with access to water. At the end of each period the dog was catheterized and all urine removed with washing out of the bladder with water until the return fluid was found to be water-clear. Toluene (5 cc.) was used as preservative.

Total blood plasma nitrogen, albumin and globulin, non-pro- tein nitrogen, sugar, and acetone bodies were determined. Total urinary nitrogen, urea, ammonia, acetone bodies, and sugar were determined.

Blood for analysis was drawn from the jugular vein into hem- atocrit tubes containing a 1.4 per cent solution of sodium oxalate and centrifuged for 35 minutes. Total nitrogen of the plasma obtained was determined by the macro-Kjeldahl method, KZS04 and selenious acid being used as the oxidizing agent. 1 cc. of plasma was used and determinations were run in triplicate. The determination of albumin and globulin was carried out according to Howe’s method, as described by Peters and Van Slyke, with 22 per cent sodium sulfate at 37”; triplicate determinations were carried out.

Urine was analyzed for total nitrogen on 1 cc. aliquots by the macro-Kjeldahl method.

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J. W. Howland and W. B. Hawkins 101

The urease method with aeration and titration for both urea and ammonia, according to Van Slyke and Cullen, was utilized in determining urine urea and ammonia.

Blood filtrate was obtained by the Folin and Wu tungstic acid precipitation method as modified by Van Slyke and Hawkins. Non-protein nitrogen was determined by nesslerization.

The calorimetric copper method of Benedict was used for blood sugar and Shaffer and Hartmann’s copper titration method for urine sugar.

Titrimetric determination of the acetone bodies in blood and urine with Deniges’ reagent were made as described by Van Slyke and Fitz. The Folin-Wu blood precipitation method was used in place of the mercuric sulfate after careful check determinations.

Blood volumes were determined by the brilliant vital red dye method.

Plasma for injection was obtained from donor dogs, heparin being used as anticoagulent, with centrifugation of blood for 35 minutes. Total plasma nitrogen and sugar were determined. The plasma was given intravenously from a gravity bottle.

The phlorhizin utilized was a preparation of either Merck or Schering-Kahlbaum. It was first recrystallized from an alcohol- water solution, filtered, and dried over HzS04 in a vacuum desic- cator. With sterile technique this was ground in a mortar with olive oil which had previously been immersed in a boiling water bath for 30 minutes. 1 gm. of phlorhizin mixed in 7 cc. of olive oil was the daily dose injected at 24 hour intervals.

The animals used in the experiments were mongrel dogs of ap- proximately 15 kilos weight. Between experiments these dogs were on a kennel diet of hospital scraps for at least 6 weeks, during which time their former weight was completely regained.

Experimental Observations

Table I (Dog 35-13) illustrates the results obtained from both the feeding and injection of plasma protein. As soon as the dog had been made diabetic by phlorhizin, a 12 hour basal period collection was made and analysis performed. At the beginning of the next collection period the dog was given 302 cc. of dog plasma by stomach tube. This plasma contained 3.36 gm. of nitrogen, 20.84 gm. of protein, and 0.25 gm. of sugar. At the end of this period 1.97 gm. of extra nitrogen and 9.48 gm. of extra

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102 Protein Metabolism

sugar were present in the urine above the control basal level. The extra nitrogen amounts to 58 per cent of the fed nitrogen. On the basis of this extra nitrogen one would expect an increase of 7.2 gm. of sugar, whereas 9.48 gm. were recovered. Accom- panying the presence of the excess sugar the acetone bodies of both blood and urine drop markedly. These data show that plasma protein when fed is digested and partially converted into sugar just as other proteins are. Since the D :N ratio had re-

TABLE I

Dog 35-13.

Plasma by Stomach and Vein

Urine Acetone bodies

pF:d Plasma PhSIN3 D:N

Total N “pHE.$- Sugar protein volume ratio

Blood Urine ~__ --

gm. per cent gn. “:tinY om. gm. Pm cc cent 1 6.14 705 2 5.82 60 22.03 103 2.95 5.27 3.78

Plasma by stomach tube 302 cc. = 3.36 gm. N = 20.84 gm. protein

: 1 ::I”, 1 ;; 1 fi:: 1 8: j ;:ii 1 ii::“9 1 641 / ::::

Plasma by vein 278 cc. = 3.08 gm. N = 19.0 gm. protein

5 4.96 76 19.77 49 0.86 5.57 3.98 6 4.47 84 19.01 58 0.72 6.01 4.26 7 3.31 76 17.18 67 0.40 5.82 5.19 8 3.73 87 15.15 46 0.26 6.12 638 4.06

turned to 3.6 at the end of the subsequent basal period, 278 cc. of dog plasma were injected into the jugular vein at the beginning of the next period.

The plasma was given in two doses with an hour elapsing between the first and second injections. It contained 3.08 gm. of nitrogen, 19.00 gm. of protein, and 0.26 gm. of sugar. In the urine collected at the end of this period there was no excess nitrogen or sugar. In subsequent basal periods the total nitrogen excreted decreased and the sugar diminished slightly. Blood and

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J. W. Howland and W. B. Hawkins 103

urine acetone bodies are low as compared with the original basal period. Owing to lack of space all data cannot be shown in Table I. The weight at the beginning was 14.7 kilos and there was a gradual daily loss to 13.4 kilos. The red cell hematocrit also decreased from 48.9 to 39.8 per cent. Approximately 25 cc. of blood were removed daily for analysis. The albumin to globu- lin ratio determined daily showed no significant alterations. The blood sugar varied from 24 to 30 mg. per cent.

As the experiment progressed the dog became lethargic, but shortly after the injection of the plasma it became more alert and active. At the site of injection of one of the doses of phlorhizin a small abscess developed on the next to the last day of the experi- ment. It was lanced and 15 cc. of reddish semifluid material were obtained. On the morning of the last day, while being catheter- ized, the dog had some convulsions with loss of sphincter control. This is no doubt related to the hypoglycemia. The dog promptly returned to normal upon feeding.

This experiment illustrates two phenomena. By calculation the total circulating plasma proteins were determined before and after injection. By addition of the amount injected (19 gm.) and deduction of the amount removed in sampling (3.7 gm.) it is found that a total of 10.78 gm. of the injected protein has disappeared from the blood stream without any evidence of its having been catabolized. Also following the injection of the pro- tein the total urine nitrogen decreases in each basal period. This suggests conservation of nitrogen on the part of the dog.

After 2 months rest a repeat experiment (Table II) was performed on this dog, except that feeding of plasma protein was excluded. After three 12 hour basal periods 340 cc. of plasma were injected intravenously in two divided doses early during Period 4. This plasma contained 3.88 gm. of nitrogen, 24 gm. of protein, and 0.31 gm. of sugar. Following the injection the dog became more ac- tive and alert. At the end of this 12 hour period the plasma protein percentage was elevated from the control level of 5.94 gm. to 6.55 gm. The urine nitrogen shows a distinct decrease as compared with the control levels and there is a slight decrease in the urine sugar. At the end of the subsequent basal period there is a very marked decrease in both urine nitrogen and sugar. This marked reduction in the amount of nitrogen and

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104 Protein Metabolism

sugar seemed unlikely and loss of some urine was considered. The volume of urine, however, was approximately the same as during other collection periods and there was no evidence of urine loss in the region of the cage. Calculations were checked and found to be correct, so we have to accept the figures as given although we do so with some reservations. During the last two basal periods the urine sugar and nitrogen rise but are still much below the control basal levels. The acetone bodies of the blood decreased slightly, while those in the urine are much lower than the control levels. There was gradual progressive weight loss

TABLE II

Plasma by Vein Dog 35-13.

I 2 3

Urine

Total N “&v.f 3

Sugar

!?m. per cent 9772.

4.71 78 18.30 5.17 78 17.88 5.09 78 / 17.64

Plasma by vein 340 cc. = 3.88 gm. N = 24 gm. protein

4 3.74 76 15.74 47 0.92 6.55 4.21 5 1.22 80 4.54 18 0.47 6.42 641 3.70 6 2.92 83 12.28 60 0.36 6.29 4.21 7 3.44 84 13.05 67 0.47 6.21 3.79

from 15.2 to 14.1 kilos. Blood sugar levels ranged between 19 and 22 mg. per cent. Non-protein N is present in normal amount.

In this experiment the entire amount of injected plasma protein disappeared from the blood stream within 24 hours, and there is indication of nitrogen conservation.

Dog 35-15 (Table III) was phlorhizinized in a similar manner and then given 355 cc. of plasma which contained 4.01 gm. of nitrogen, 24.74 gm. of protein, and 0.316 gm. of sugar. Non- protein N = 0.017 gm. At the end of the period in which the injection was made the plasma protein circulating was but slightly elevated. In the urine 0.414 gm. of nitrogen in excess was ob-

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J. W. Howland and W. B. Hawkins 105

tained but only 1.14 gm. of sugar, part of which can be accounted for by the 0.316 gm. given in the plasma. In subsequent periods the amount of nitrogen and sugar eliminated is definitely below the control values. 48 hours after the plasma was injected the total circulating protein is 37.29 gm. which is 5.74 gm. less than that circulating just previous to injection. 3.92 gm. of protein were removed in the samples taken. Again there has been com- plete disappearance of all of the injected protein with no excess of nitrogen or sugar in the urine. Blood and urine acetone bodies were decreased after injection but rose in subsequent periods.

Dog 35-15.

TABLE III

Plasma by Vein

Period NO.

Urine Acetone bodies 1 I I PhlIla Plasma D:N

Total N “j$$.j- protein VOlUWV3 ratio

3 Sugar Blood Urine

1 2

gm. per cent gm. mg. per cent gm. gm. P@ cc

cent

6.08 643 4.59 76 17.85 89 1.50 6.40 673 3.88

Plasma by vein 355 cc. = 4.01 gm. N = 24.74 gm. protein

3 5.01 1 77 18.99 56 0.99 6.55 3.79 4 3.11 78 13.75 91 0.49 6.99 4.41 5 3.05 73 11.21 105 1 .Ol 3.67 6 3.22 76 12.38 99 1.28 6.64 562 3.84

Non-protein N was not elevated following injection. The weight dropped from 14.95 to 14.15 kilos and the red cell hematocrit from 46.2 to 43 per cent. The albumin to globulin ratio showed no significant change. The blood sugar ranged from 22 to 32 mg. per cent.

Table IV (Dog 35-127) gives results on another dog that weighed 10.5 kilos as compared with about 14.5 kilos for the other dogs. Only 260 cc. of plasma were injected and it contained 3.18 gm. of nitrogen, 19.08 gm. of protein, and 0.23 gm. of sugar. The plasma was injected in one dose over a 15 minute period with no untoward effects, but an hour later the dog vomited and def-

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106 Protein Metabolism

ecated and appeared inactive. Recovery was prompt and there was no other disturbance. Following the injection there is no excess nitrogen or sugar in the urine and in subsequent basal periods the nitrogen and sugar levels gradually decrease in amount. There is no striking change in the blood and urine ace- tone bodies. Non-protein N remained at normal levels and the blood sugar ranged from 10 to 32 mg. per cent. The weight fell gradually from 10.7 to 9.64 kilos.

Within 48 hours of the injection practically all the injected plasma protein has disappeared from the blood stream. 3.09 gm. of protein were removed in samples taken for analysis. Again

Dog 35-127.

TABLE IV

Plasma by Vein

Period NO.

1

Uliine Acetone bodies

Total N u;r;“3:,j- Sllgitr Blood Urine

gm. per cent gm. “%?” gm.

3.49 77 11.11 59 0.59

PhSUla Pb8lllS3 D:N protein VOlUUle ratio

g%nY 5.58

___-

cc.

432 3.18

Plasma by vein 260 cc. = 3.18 gm. N = 19.08 gm. protein

2 3.77 76 11.69 58 0.52 5.96 3.10 3 3.28 77 9.55 47 0.46 6.18 2.91 4 2.76 75 7.24 55 0.29 2.62 5 2.39 75 7.46 89 0.50 6.42 395 3.12

there is no evidence that this protein has been catabolized in the usual manner, since no excess nitrogen or sugar is recovered.

Control experiments were conducted in which 300 mg. of an- hydrous glucose in 300 cc. of Locke’s solution were injected. These amounts approximate the amount of blood sugar and the volume of plasma that had been injected. The experiments were performed in order to learn what effects the small amount of sugar and the volume of injected fluid might have on the acetone bodies.

Dog 35-15 had been used in the previous experiments and Table V records the data obtained. There is a slight decrease in

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J. W. Howland and W. B. Hawkins 107

protein percentage in the circulating plasma, as would be expected in a fasting animal. The urine nitrogen and sugar progressively decrease. Following the injection of the sugar and

TABLE V

Glucose and Locke’s Solution by Vein

Dog 35-15.

Period No.

1 2 3

Urine Acetone bodies

Total N Sugar Blood Urine

gm. gm. mg. per cent gm.

4.54 19.86 101 3.34 4.59 16.73 101 3.77 4.40 16.02 130 3.76

PlllSllla protein

gm. per cent

6.34

Locke’s

D: N ratio

4.38 3.64 3.63

By vein 300 mg. anhydrous glucose in 300 cc. Locke’s solution

4 3.07 14.23 71 1.65 4.64 5 2.83 9.57 78 0.21 3.38 6 3.05 9.4 91 0.55 5.75 3.12

TABLE VI

Glucose and Locke’s Solution by Vein

Dog 35-13.

Period No.

-7

-

Urine

Total N Sugar Blood Urine

f7m. gm. mg. per cent gm.

4.46 17.39 57 2.78 4.52 17.71 82 2.63 4.31 17.62 79 2.66 4.38 16.42 74 1.98

-7

Acetone bodies

-

Plasma protein

gnz. per cent

5.99

D: N ratio

3.89 3.91 4.08 3.74

By vein 300 mg. anhydrous glucose in 300 cc. Locke’s solution

4.41 3.82

solution the acetone bodies of both blood and urine decrease and remain low, particularly in the urine. The weight fell from 15.1 to 14.3 kilos.

A control experiment was performed on Dog 35-13 also, and

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108 Protein Metabolism

there was but little change in the circulating plasma protein percent.age (Table VI). The urine nitrogen following the injec- tion of sugar in the Locke’s solution is slightly less in amount than during the fore-basal periods, but the urine sugar is on the same general level. The increase in urine acetone bodies at the end of the injection period may be the result of a flushing out by the fluid. The weight decreased from 15.2 to 14.5 kilos.

DISCUSSION

Previous experiments in this laboratory have shown that the dog has protein stores that it can draw upon when there is a de- mand for plasma protein formation. These stores become de- pleted upon fasting of the animal (2, 7-9). Luck (5) has given evidence for liver storage of proteins related to the diet and Addis (1) and his associates have reported that when rats are fasted there is prompt decrease in the protein content of the liver. In our experiments the dogs were fasting and also were under the additional strain of being diabetic owing to phlorhizin. Dur- ing the 4 or 5 days prior to the injection of the plasma protein there undoubtedly was utilization of reserve protein stores with partial depletion. Consequently the body was in a state where it would welcome any protein introduced and utilize it to the best of its ability. The data indicate that the injected protein very promptly disappeared from the blood stream. No protein was lost in the urine. However, there was no increase in urinary nitrogen or sugar as there was when the plasma protein was fed.

What happens to this protein when it leaves the blood stream? It might be argued that it was removed as such and stored in the body without alteration. The body cells vary as regards their protein make-up and it seems unlikely that plasma protein could be taken in by the cells to form their particular types of protein without its first undergoing alteration.

In these experiments there is evidence of nitrogen conservation following the injection of the plasma protein. It seems that the body economy is altered as the result of receiving this protein material. In other experiments performed in this laboratory the evidence all points to actual utilization of the protein (2, 9, 3). When dogs are fed a protein-free diet and are given plasma by vein over a 2 week period, it is possible to maintain them

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J. W. Howland and W. B. Hawkins 109

approximately in a state of nitrogen equilibrium. There is no loss of protein in the urine and no excess nitrogen is eliminated subsequently.

This utilization of the injected plasma protein without the elimination of any excess nitrogen or sugar points to a different metabolic mechanism on the part of the body. When the pro- tein is fed, amino acids result from the complete digestion of the injected material. However, when injected, such breaking down of the protein does not occur, as no sugar is formed. We suggest the following mechanism occurs. The plasma protein is removed from the blood and then undergoes slight catabolic change with formation of large aggregates of amino acids and these are then reassembled by the cells to form their own particular protein matrix.

It is reasonable to believe that it is in this manner that protein exchange occurs within the body rather than that protein must be always completely catabolized and the desired type of protein then be elaborated from the amino acids.

SUMMARY

When phlorhizinized dogs are fed plasma protein, it is digested with conversion of part of it to sugar. Ketosis decreases as the result of the sugar formation.

When phlorhizinized dogs receive plasma protein by vein, the injected protein promptly disappears from the blood stream. No protein is lost and there is no excess elimination of nitrogen or sugar in the urine.

There is some decrease in the ketosis following injection of plasma protein and the dogs are clinically improved.

There is evidence of nitrogen conservation by the body follow- ing the injection of plasma protein.

The metabolism of protein when fed is different than when it is injected. It is suggested that there is partial catabolism of the injected protein with reassembly of the large aggregates formed by the cells to form their own peculiar type of protein.

This partial catabolism with reassembly of the large aggregates may be the method of protein interchange within the body rather than a complete catabolism to amino acids with subsequent re- synthesis to protein.

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110 Protein Metabolism

BIBLIOGRAPHY

1. Addis, T., Poo, L. J., and Lew, W., J. Biol. Chem., 116, 111, 117 (1936); 116, 343 (1936).

2. Holman, R. L., Mahoney, E. B., and Whipple, G. H., J. Exp. Med., 69, 251, 269 (1934).

3. Daft, F. S., Robscheit-Robbins, F. S., and Whipple, G. H., J. Biol. Chem., 133, 87 (1938).

4. Janney, N. W., J. Biol. Chem., 20, 321 (1915). Janney, N. W., and Csonka, F. A., J. Biol. Chem., 22, 203 (1915).

5. Luck, J. M., J. Biol. Chem., 116, 491 (1936). 6. Lusk, G., The science of nutrition, Philadelphia, 3rd edition (1921). 7. Madden, S. C., Winslow, P. M., Howland, J. W., and Whipple, G. H.,

J. Exp. Med., 66, 431 (1937). 8. McNaught, J. B., Scott, V. C., Woods, F. M., and Whipple, G. H.,

J. Exp. Med., 63,277 (1936). 9. Pommerenke, W. T., Slavin, H. B., Kariher, D. H., and Whipple, G. H.,

J. Exp. Med., 61,261, 283 (1935).

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J. W. Howland and W. B. HawkinsPHLORHIZINIZED DOGS

INTERCHANGE, AND UTILIZATION IN PROTEIN METABOLISM, PROTEIN

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