PROLINE AND HYDROXYPROLINE IN NUTRITION*

BY R. REDER ST. JULIANt AND WILLIAM C. ROSE

(From the Laboratory of Physiological Chemistry, University of Illinois, Urbana)

(Received for publication, August 1, 1932)

Of the twenty amino acids generally recognized as components of proteins only proline manifests the property of being appreciably soluble in absolute ethyl alcohol. According to Kapfhammer and Eck (1927), 100 gm. of a saturated solution of proline in absolute alcohol at 19” contains 1.5 gm. of the amino acid. At 40’ the solubility is approximately 2.8 per cent (172.6 mg. in 6 cc. of alco- hol). Furthermore, the same authors call attention to the fact that a solution of proline in absolute alcohol dissolves hydroxy- proline. Thus 6 cc. of the solvent saturated with proline at 40’ are said to dissolve 9.6 mg. of hydroxyproline.

The above observations justify the assumption that proline may be completely removed from a hydrolyzed protein by a suffi- ciently large number of extractions with absolute ethyl alcohol. How successfully hydroxyproline can be separated by this method is uncertain. So long as proline is being extracted, hydroxy- proline would dissolve in the solvent. In the case of casein, which is believed to have a very low hydroxyproline content (0.23 per cent; Foreman, 1919), and a moderately high proline content (Dakin, 191S), there seems little reason to doubt that the former would be satisfactorily removed during the extraction of the latter. With edestin, however, which according to Abderhalden (1902-03) contains 2 per cent of hydroxyproline, the completeness of ex- traction would be open to question.

* This communication was presented before the meeting of the American Society of Biological Chemists at Philadelphia, April, 1932.

t The experimental data in this paper are taken from a thesis submitted by R. Reder St. Julian in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Physiological Chemistry in the Graduate School of the University of Illinois.

445

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446 Proline and Hydroxyproline

Inasmuch as the extraction method appears to be the best avail- able for the removal of proline and at least part of the hydroxy- proline, it was employed for the preparation of diets deficient in these amino acids. In so doing, we are fully aware of the possible limitations of the procedure. On the other hand, the extracted hydrolyzed proteins certainly contained greatly diminished amounts of the alcohol-soluble amino acids. Evidence for this fact will be presented below.

For a review of the inadequate and conflicting data already re- corded in the literature concerning the nutritive importance of pro- line and hydroxyproline, the reader is referred to a recent paper by the senior author (Rose, 1932).

TABLE I

Proline and Hydroxyproline Present in Proteins Employed

Proline Hydroxyproline

per cent per cent Casein...................................... 7.63 0.23 Lactalbumin................................... 3.76 ? Edestin........................................ 4.10 2.00

EXPERIMENTAL

Three proteins, namely casein, lactalbumin, and edestin, were made use of in the preparation of the proline-deficient diets. The casein was a commercial product. The lactalbumin and edestin were prepared in this laboratory from skim milk and hemp seeds respectively. The lactalbumin may have contained traces of cas- ein. The proportions of proline and hydroxyproline said to be present in the proteins in question are reproduced in Table I. We know of no value for the hydroxyproline content of lactalbumin. Most analyses of edestin represent the amount of this amino acid with a question mark. The value given in Table I is that reported by Abderhalden (1902-03).

The proteins were hydrolyzed with sulfuric acid in the usual manner (Berg and Rose, 1929). After careful precipitation of the sulfuric acid with barium hydroxide, and thorough washing of the barium sulfate, the combined filtrate and washings from each prot,ein were evaporated to dryness, first by means of a vacuum

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R. R. St. Julian and W. C. Rose

still, and finally in a vacuum oven. Each of the resulting materials was then subjected to forty extractions with absolute ethyl alcohol. The proportions of the latter varied somewhat with the quantities of proline and hydroxyproline present in the proteins. Thus for each kilo of original casein 1 liter of the solvent was used in each extraction. With lactalbumin, having a lower proline content, about 950 cc. of alcohol per kilo were employed each time. In order to afford a better opportunity for the removal of hydroxypro- line from edestin, each extraction involved the use of approxi- mately 1300 cc. of the solvent per kilo of the protein.

In making the extractions, the finely ground hydrolyzed protein was suspended in alcohol, and the mixture was heated to boiling, with frequent shaking, on a steam cone. The solution was then cooled in an ice bath to about lo’, and immediately filtered. This process was repeated forty times. The alcoholic filtrates were combined into groups of five, Filtrates 1 to 5, 6 to 10, 11 to 15, etc., and evaporated to dryness. The non-amino nitrogen was then determined in each of the eight fractions obtained from each pro- tein in order to secure a rough measure of the rapidity of removal of the soluble material. These data are not reproduced in full inasmuch as they cannot be taken as accurate estimates of proline and hydroxyproline, since traces of other forms of non-amino nitro- gen (arginine and histidine?) doubtless dissolved in the alcohol. However, it is significant that with each protein the first three frac- tions (fifteen extracts) carried most of the non-amino nitrogen, while the last five fractions showed small, approximately constant amounts. This may be illustrated with casein. The first three fractions from 1 kilo of the protein showed 6.6, 2.4, and 1.3 gm. of non-amino nitrogen respectively. The remaining five fractions varied from a maximum of 0.73 gm. to a minimum of 0.37 gm. of non-amino nitrogen. Similar figures were obtained with the la&albumin and edestin extracts. It seems probable, therefore, that most of the proline, and possibly a large part of the hydroxy- proline, had been removed in the first fifteen extracts. With each protein the non-amino nitrogen recovered from the forty extracts was considerably larger than that necessary to account for the proline and hydroxyproline present, the figures recorded in Table I being assumed correct.

Finally, the dried residues remaining after the extractions were

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448 Proline and Hydroxyproline

analyzed for non-amino nitrogen. The results are recorded Table II. Figures are also shown representing the analyses of 1: original hydrolysates of casein and lactalbumin. The hyd lyzed edestin was not analyzed prior to the alcohol treatmen The data show that the non-amino nitrogen of casein was reduce from 4.55 to 2.26 per cent. Original and final figures for lactal-

TABLE II

Effect of Forty Absolute Alcohol Extractions upon Non-Amino Nitrogen Content of Hydrolyzed Proteins

Total N in original hydrolysate. . Amino “ “ “ “ . ., Son-amino r\; in original hydroly-

sate............................. Total N in residue after forty ex-

tractions........................ Amino N in residue after forty ex-

tractions........................ Non-amino N in residue after forty

extractions. . . . . . Correction for non-amino N of ar-

ginine, histidine, and 5 per cent of lysine*. . . . . . . . . .

Non-amino N unaccounted for. . .

Casein Lactalbumin

per cent per cent

13.29 12.30 8.74 8.05

4.55 4.25

12.42 11.94

10.16 10.30

2.26 1.64

Ed&in

per cent

14.07

9.12

4.95

1.78t 1.07t 4.21t 0.48 0.57 0.74

* Under the conditions ordinarily employed in the Van Slyke method (5 minutes reaction time) only 95 per cent of the lysine nitrogen is liberated (Van Slyke, 1912). Therefore, the remaining 5 per cent appears as non- amino nitrogen.

i These values were calculated from the analyses of the proteins as given by Mitchell and Hamilton (1929).

bumin were 4.25 and 1.64 per cent respectively. The non-amino nitrogen content of the extracted edestin amounted to 4.95 per cent. The seventh horizontal row of figures in Table II represents the non-amino nitrogen present in the form of arginine, histidine, and 5 per cent of the lysine. These values were calculated on the assumption that the proportions of the three amino acids in the extracted materials were the same as in the original proteins (cf. Mitchell and Hamilton, 1929). Perhaps somewhat larger relative

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R. R. St. Julian and W. C. Rose 449

(mnts were present because of the smaller quantities of the pro- s; but in any event, the values tabulated probably are not too il. The data in Table II show, after the corrections indicated )ve are made, that the non-amino nitrogen unaccounted for lounted to approximately 0.5 per cent for casein, 0.6 per cent

for la&albumin, and 0.7 per cent for edestin. When one recalls hat these figures possibly include traces of peptide nitrogen, and ?rtainly some nitrogen in the form of diketopiperazines produced

TABLE III

Composition of Diets*

Proline-free casein ................. I‘ lactalbumin ........... ‘I edestin ................

Tryptophane ...................... Cystine ........................... Lysine ............................. Dextrin ........................... Sucrose ............................ Salt mixture?. ..................... Agar .............................. Lard ............................... Cod liver oil .......................

- Diet 1

_ gm.

8.4

0.2 0.4

46.0 15.0 4.0 2.0

19.0 5.0

100.0

-

_

-

Diet 2

gm.

Diet 3

!?m.

8.6

0.2 0.2

42.0 15.0 4.0 2.0

23.0 5.0

_

100.0 100.0

8.0 0.2 0.4 0.4

42.0 15.0 4.0 2.0

23.0 5.0

* Vitamin B factors were supplied in the form of two pills daily each containing 50 mg. of Harris yeast extract. Each of the control rats re- ceived 50 mg. of proline daily admixed with the vitamin extract.

t Osborne and Mendel (1919).

by the repeated hot alcohol treatments, it appears that the quanti- ties of proline (and hydroxyproline?) present must have been quite small.

By use of the materials prepared as indicated above, three diets were formulated, as represented in Table III. These were em- ployed in the growth studies. Young white rats served as the experimental animals. The total nitrogenous components in the basal diets amounted to 9 per cent (cf- Osborne and Mendel, 1915). The hydrolyzed proteins were supplemented in each case with

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450 Proline and Hydroxyproline

128?(57) -/ I 1

&ART I. Litter F. The numbers in parentheses signify the initial and final weights of the rats. The arrows show the points at which the diets were begun, while the numbers just above the arrows correspond to the diet numbers. When proline was fed, this fact is indicated by the symbol P. following the diet number. Thus 1 f P. signifies that Diet 1 was supple- mented with 50 mg. of proline daily. The supplement was incorporated in the vitamin pills.

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R. R. St. Julian and W. C. Rose 451

cystine and tryptophane. Lysine also was added to the edestin ration (Diet 3) because of the low lysine content of this protein. Vitamin B factors were supplied in the form of two pills daily

t 20 gm.

1

CHART II. Litter G. The figures and symbols have the same meaning as in Chart I.

each containing 50 mg. of Harris yeast extract. The control rats received 50 mg. of proline daily admixed with the vitamin extract. The basal diets were kept before the animals at all times.

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452 Proline and Hydroxyproline

The growth curves are reproduced in Charts I to III inclusive. Chart I shows the results with the casein hydrolysate. It is evi- dent that the animals succeeded in growing at fairly rapid rates,

:/;

119Sl (57) J

1 :<@*) 1979 (53)

1194 * (55)

735

(77)

1195 9 (55) T a0 gm-

4

a0 days .

CHART III. Litter H. The figures and symbols have the same meaning as in Chart I.

and that those which received proline (Rats 1128 and 1129) did not make greater gains than did their litter mates upon the basal ration alone. Similar data were obtained in the lactalbumin (Chart II)

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R. R. St. Julian and W. C. Rose 453

experiments. The animals upon the hydrolyzed edestin (Chart III) grew less satisfactorily than did those in the other two groups; but evidently the deficiency is not to be accounted for by the ab- sence of proline inasmuch as the administration of the latter failed to exert a detectable improvement.

In Table IV are exhibited figures representing the average daily gain in weight and average daily food consumption of each animal. It is obvious that the variations in each group are well within the

TABLE IV

Food Consumption and Daily Gain in Body Weight of Experimental Animals

Rat No. and sex Diet No.

1130, Q 1131, Q 1132, Q 1128, Q 1129, Q 1177, Q 1178, Q 1179, Q 1175, Q 1176, Q 1196, Q 1197, Q 1194, Q 1195, Q

1

1 1 1 + proline 1+ “ 2 2 2 2 + proline 2+ “ 3 3 3 + proline 3+ “

T -

Average daily gain in Average daily food body weight consumption

am. gm. 0.81 5.7 0.93 5.8 0.93 6.1 0.81 5.4 0.90 6.2 0.79 6.2 0.80 5.0 0.85 5.6 0.84 5.8 0.68 4.5 0.37 4.2 0.46 4.2 0.51 5.4 0.33 4.1

limit’s of experimental error, and are not associated with the pres- ence or absence of proline.

The above results appear to indicate that proline is not a neces- sary dietary constituent. We refrain from making a similar de- duction concerning hydroxyproline since we are not certain as to the completeness of its removal by the procedure employed. Furthermore, the supplementing effect of hydroxyproline was not t’ested inasmuch as this amino acid was not available to us at the time these experiments were undertaken. Unquestionably, most of the hydroxyproline was extracted, at least from casein, by the forty alcohol treatments. Under the circumstances we are in-

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454 Proline and Hydroxyproline

clined to believe that this amino acid also is dispensable. After the above experiments had been completed, Adeline (1931) reported that rats upon diets containing 6 per cent of edestin cease growing after approximately 12 weeks, but that the addition of either pro- line or hydroxyproline remedies the nutritive deficiency and in- duces growth. The author concludes that the two compounds are interchangeable in metabolism, but that one must be present in the diet. Unfortunately, her data are not as clear-cut as one would wish. As will be discussed in the following paper, we have consid- ered the possibility of several 5-carbon amino acids being inter- changeable with proline, but have obtained no evidence for such a relationship. In our opinion, further and more convincing proof must be presented before the mutual interchangeability of proline and hydroxyproline can be accepted as an established fact. In the present paper we are particularly concerned with the fact that pro- line is a dispensable dietary constituent.

SUMMARY

The dry mixtures of amino acids obtained by the acid hydrolysis of casein, la&albumin, and edestin, have been subjected to forty extractions each with absolute ethyl alcohol for the removal of proline and an unknown part of the hydroxyproline. The resulting -materials, when incorporated in diets at 9 per cent levels (including suitable amino acid supplements) enable rats to grow at moderately rapid rates. The administration of 50 mg. of proline daily fails to accelerate the increase in body weight. It appears, therefore, that proline is a dispensa.ble amino acid.

In view of the uncertainty as to the completeness of removal of hydroxyproline by the method employed, no positive statement can be made concerning its nutritive importance. We are inclined to the belief that it also is an unnecessary dietary component, but realize that final conclusions must await the results of further investigations.

BIBLIOGRAPHY

Abderhalden, E., 2. physiol. Chem., 37, 499 (1902-03). Adeline, M., 2. physiol. Chem., 199, 184 (1931). Berg, C. P., and Rose, W. C., J. Biol. Chem., 82, 479 (1929). Dakin, H. D., Biochem. J., 12, 290 (1918). Foreman, F. W., Biochem. J., 13, 378 (1919).

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R. R. St. Julian and W. C. Rose 455

Kapfhammer, J., and Eck, R., 2. physiol. Chem., 170, 294 (1927). Mitchell, H. H., and Hamilton, T. S., The biochemistry of the amino acids,

New York, 191 (1929). Osborne, T. B., and Mendel, L. B., J. Biol. Chem., 20, 351 (1915); 37, 572

(1919). Rose, W. C., Yale J. Bid. and Med., 4, 519 (1932). Van Slyke, D. D., J. BioZ. Chem., 12, 275 (1912).

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R. Reder St. Julian and William C. RoseNUTRITION

PROLINE AND HYDROXYPROLINE IN

1932, 98:445-455.J. Biol. Chem. 

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