THE CYSTINE CONTENT OF HAIR AND OTHER EPIDERMAL TISSUES.

BY ROBERT H. WILSON AND HOWARD B. LEWIS.

(From the Laboratory of Physiological Chemistry, Medical School, University of Michigan, Ann Arbor.)

(Received for publication, March 21, 1927.)

There have appeared in the literature of recent years occasional reports concerned with the relation of cystine to the growth of hair and wool. Evvard, Dox, and Guernsey (1) in experiments with sows, and Sjollema (2), with rabbits, have suggested that the feeding of cystine or proteins high in their content of this amino acid was a factor in promoting an increased growth of hair. Cer- tain German investigators (3, 4), who employed a commercial product (Humagsolan), a hydrolysate of keratin, reported similar results, which were not, however, confirmed by Fuhs (5). In con- nection with a study of factors concerned in the growth of hair in progress in this laboratory, we have had occasion to review the data concerning the sulfur and cystine content of the more com- mon types of keratins. The data are open to the criticism that the degree of purification of the keratins has varied in the hands of different observers, no large comparative series, in which the methods of preliminary treatment and determination of cystine have been uniform, being available. We have accordingly con- sidered it necessary for our purposes to determine by a uniform procedure the cystine and sulfur content of a considerable number of keratins. The results, while they may not be of abso- lute accuracy in view of the lack of an entirely satisfactory method for the determination of cystine, afford a more exten- sive comparative study of the keratins than has heretofore been available.

The cystine content of the keratins has usually been deter- mined by isolation of the impure cystine from the protein hydroly- sate and subsequent determination of the sulfur content of this

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impure cystine. From the sulfur content, the cystine has been calculated. This method must give low results, since cystine, although precipitated almost quantitatively from pure solutions, is not precipitated completely in the presence of organic substances, such as would occur in a protein hydrolysate. We have shown previously (6) that this is true when cystine is added to urine, and will present in the present communication further evidence of the presence of cystine in the filtrate, after the precipitation of cystine from protein hydrolysates by the usual methods. A few determinations of the cystine content of keratins are available from the analyses by the Van Slyke method (7) of character- ization of proteins by the determination of specific groups, the cystine being calculated from the sulfur content of the solution of the bases precipitated by phosphotungstic acid. Sources of error are a considerable correction for the solubility of the cystine phosphotungstate, as well as an alteration of a portion of the cystine by prolonged boiling with acid so that it is no longer pl;e- cipitable by phosphotungstic acid (8). Van Slyke has estimated that the amount of cystine obtained by this method represents less than half of that actually present in the protein.’ Analyses by the method of Folin and Looney, which we have employed in the present series, have been relatively few. This method, which depends upon the fact that, while oystine itself does not, react with the uric acid reagent of Folin and Denis, in the presence of sodium sulfite it is reduced to cysteine, which gives a deep blue color with the reagent, has been criticized by Okuda (9) on the basis of non-specificity, since in his experiments chemical groups present in proteins, other than cystine, gave the same color as cystine. No further details are cited in the publications available to us, nor have we any experience with the iodometric method proposed by him (9).

In Table I are summarized the analytical data available for the cystine content of the tissues studied in the present investigation. Unless otherwise noted, the cystine determinations have been made by the older method which involves precipitation of the crude cystine from the hydrolysate and calculation of the cystine from the total sulfur content of the precipitate.

1 Van Slyke (7), p. 28-29.

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R. H. Wilson and H. B. Lewis

EXPERIMENTAL.

545

The keratins used in these experiments had been thoroughly ex- tracted with organic solvents by Dr. Henry C. Eckstein, who has reported a study of their lipid content. The extraction was in four steps over a total period of 96 hours: treatment with boiling vapors of absolute alcohol, chloroform, ether, and again with absolute

TABLE I.

Cystine Content of Epithelial Tissues.

SOUrCt3.

. . . Hair, human.. “ “ I‘ “ ‘I ‘I ‘I “

Wool, sheep..

“ “ . . . . . . . . Goose feathers (quills). Hair, dog.. Tortoise shell (Chelone

imbricata).

C _

-

>ystinecontent.

-

.- Observer.

pm cent 6.5-13.92

11.5-14.53 9. IO-9.90*

16.50t 13.2-17.4t

6.9-12.5

7.8t 6.30 8.49*

Mijrner (10). Buchtala (11). Sammartino (12). Folin and Looney (13). Klinke (14). Abderhalden and Voitin-

ovici (15). Folin and Looney (13). Buchtala (16). Van Slyke (7).

5.19 Buchtala (17).

* Value calculated from the analysis by the Van Slyke method (7). t Folin and Looney calorimetric method.

alcohol (118). The keratins were then dried for several days at 100” and placed in tightly stoppered bottles.

1 gm. of the sample was usually used for the analysis. It was found advintageous, particularly in the case of the rabbit and rat hair and the fan portion of the feathers, to use a weighing tube, open at each end, about 1 cm. in diameter, into which the sample was packed as tightly as possible. After weighing, the sample could be transferred to the flask, in which the hydrolysis was car- ried out easily and without loss by pushing it out of the tube with a glass rod. The hydrolysates were tested by the biuret test, which was uniformly negative except in two cases, in which a faintly posi- tive test was obtained.

Total sulfur was determined by the Denis modification of the Benedict method. Trotman and Bell (19), who made a compara- tive study of the methods of sulfur analysis in wool, consider this

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546 Cystine Content of Hair

method satisfactory. The hair or other tissue was heated over a low flame with 20 cc. of concentrated nitric acid in a long necked 500 cc. Kjeldahl flask for 4 hours to dissolve the material and per- mit of partial oxidation. Evaporation was prevented by inserting an inverted 50 cc. Erlenmeyer flask in the mouth-of the Kjeldahl flask. The solution was made up to a volume of 105 cc. and ali- quots of 50 cc. were used for the sulfur determination. These were evaporated to dryness in small evaporating dishes, about 20 cc. of water were added, and the solutions were again evaporated to dryness. This treatment was to remove the excess acid as com-

TABLE II.

Cystine Content of Hair and Wool of Various Species.

SOWCe.

Human. Children (If-5 yrs.) .

“ (7 yrs.), “ (9-10 yrs.). I‘ (12-13 “ ). “ (All ages).

Adult.. Rabbit. Rat, white (normal).

“ “ (rachitic). Cat., . Dog., Sheep, wool..

-

I

-

-

No. of samples

rnalyaed.

7 5 5 6

23 6 4 1 1 1 1 9

-

-

Cystine.

Range. 4verage.

per cen2 per cent

15.6-19.9 18.7 18.0-19.4 18.9 16.5-21.2 19.4 15.6-18.4 17.3 15.6-21.2 18.9 16.8-18.4 17.6 11.9-14.0 13.0 14.1 14.1 14.5 14.5 13.1 13.1 19.0 19.0

8.0-10.9 9.5

pletely as possible. The subsequent procedure was the same as in the sulfur determination by the Benedict-Denis method, 5 cc. of the reagent usually being required for the oxidation. Nitrogen was determined by the Kjeldahl method.

DISCUSSION.

In Table II are summarized the results of the analyses of twenty- three samples of the hair of children of various ages. Frequently the sample obtained was so small that it was not possible to de- termine total nitrogen and sulfur in addition to the cystine. The

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R. H. Wilson and H. B. Lewis 547

cystine values showed considerable variation, ranging from the minimum figure of 15.6 to a maximum of 21.2 per cent. The con- tent of nitrogen was more constant than the cystine in all cases, varying only within narrow limits (15.25 to 15.50 per cent). The nitrogen to cystine sulfur ratio (2.7 to 3.2) and nitrogen to sulfur (3.0 to 3.2) ratio were relatively constant and approximated the ratios found for human hair by Rutherford and Hawk (20).

The high cystine figures obtained are striking, three samples containing more than 20 per cent of cystine. Two samples showed a cystine content as low as 15.6 per cent, but none approached the lower values (13.2, 13.3, and 14.5 per cent) found by Klinke (14). There seems to be no relation between the cystine content of the hair and the color of the hair, age, or sex. The nitrogen to sulfur ratios also fail to show any significant variations, so that while the ratios are similar to those of Rutherford and Hawk, it does not seem possible to draw the conclusions they suggest. Despite the fact that no treatment with gastric juice or any other enzyme prep- aration was made in our experiments, it may be noted that the nitrogen content of the hair of our series compared well with the values of Rutherford and Hawk, in whose experiments the hairs were treated successively with artificial gastric and pancreatic juice.

The values obtained for the hair of adults (Table II) also fail to demonstrate any of the above mentioned relationships. As a rule, the cystine sulfur was nearly equal to the total sulfur. Two specimens of adult red hair did not differ materially from the hairs of other color in their content of sulfur (5.05, 5.12 per cent, as compared with a range of 4.32 to 5.08 per cent and an average value of 4.84 per cent for hair of other colors), nor was the pro- portion of total sulfur not cystine sulfur as marked as in the red hair studied by Klinke (14). Although we recognize the dangers of reasoning from too small a series, we cannot fail to be impressed by the apparent tendency of the values for cystine in adult hair to be slightly lower than those in children’s hair.

In Table II, the results obtained with four samples of rabbit hair are listed. The cystine values ranged from 11.9 to 14.0 per cent. The number of samples is too small to permit of considera- tion of individual variations, but is large enough to show that rabbit hair contains a greater amount of cystine than either wool

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or feathers, the content of cystine approaching that of human hair. The samples of rat hair were mixtures of hair from several animals. There was no significant difference between the two sam- ples, although one was from rachitic rats.2 These samples and that from a silver Persian cat had a cpstine content resembling that of rabbit hair. The one sample of dog hair analyzed is inter- esting since it contained as much cystine as human hair, a value much greater than that obtained by Van Slyke’s partition method (7).

Considerable difficulty was experienced in the analyses of wool (Table II), due to the presence of dirt in the wool, which, even though removed as completely as possible, interfered with the determinations somewhat. Of the nine samples, only one ap- proached the low value of 7.8 per cent of Folin and Looney (13) and none approximated the higher value of 12.5 per cent of Abder- halden and Voitinovici (15).

The scutes of several species of turtles3 were analyzed (Table III) The cystine contents of these keratins was lower than that of any of the types previously studied. The value obtained by Mulder (21) for sulfur (2.22 per cent) is comparable to the two values for total sulfur found in our series.

The most striking feature of the analyses of feathers (Table III) was the high cystine content of the fan portions of the goose feathers (10.7 per cent) and especially of the duck feathers (12.2 per cent). The other values were fairly uniform. The variation in the nitrogen to sulfur ratios was greater in this case than in any of the other keratins studied.

The three samples of skin analyzed (Table III) were all low in their cystine content and resembled the common types of proteins (22) in this respect more than the keratins. These values and that found for a sample of connective tissue (the residue obtained after extraction of the lipids from human adipose tissue) agreed fairly closely. Although the variations in the percentages of cystine and nitrogen were considerable, the ratios of nitrogen to cystine sulfur were very constant.

2 We are indebted to Prof. Amy L. Daniels of the University of Iowa for the samples of hair of rachitic rats.

J For the samples of tortoise shell and the identification of species we are indebted to Prof. A. G. Ruthven of the University Museum.

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R. H. Wilson and H. B. Lewis 549

In general, throughout the series of analyses, the differences between the cystine sulfur and total sulfur were not large, and can usually be exp.lained by experimental error. In the few cases where the difference was more marked, we may have indications of the exis%ence of sulfur compounds other than cystine. In only two instances was there obtained a value for cystine sulfur which

TABLE III.

Cystine Content of Tortoise Shell, Feathers, and Skin.

Material. Cystine.

per cent Tortoise shell.

Pseudemys elegans .................... Graptemys pseudogeographica .......... Chrysemys marginata .................. Testudo pardalis. ..................... Kinosternon odoratum ................. Chelydra serpentina ................... Emys blandingii ......................

Feathers. Turkey 1. Fan .......................

Quill ...................... Turkey 2. Fan .......................

Quill ...................... Goose. Fan ..........................

Quill ......................... Duck. Fan ...........................

Quill .......................... Skin, human.

No. 1.. ............................... “ 2 ................................. “ 3 .................................

Connective tissue.*. ....................

7.0 7.5 7.3 6.4 7.9 7.3 8.1

7.1 8.9 7.7 7.7

10.7 9.1

12.2 8.8

1.8 2.1 2.3 2.3

- Total

sulfur.

per cent

2.12 2.44

2.44 2.40

3.00 2.53 2.90

0.70

Nitrogen.

per cent

15.1

14.4 15.3

15.1 15.8 14.8 15.5 15.1

15.5 15.9

11.3 12.4 15.3 15.0

* The residue from human adipose tissue after extraction with fat sol- vents.

was considerably in excess of the value for total sulfur. It is not our purpose in this paper to discuss the question of the types of sulfur compounds present in the keratins, but we believe there is little evidence to be obtained from our data in support of the contention that in these proteins there exists any considerable amount of non-cystine sulfur. The high cystine values obtained

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by us cannot be due to the presence of sulfides, or mercapto com- pounds, such as thiolactic acid, since we have tested the reaction of our hydrolysates with the Folin-Denis uric acid reagent without the addition of sodium sulfite and have never observed a reduction of the reagent in the absence of the sulfite, which could influence significantly our calorimetric readings in the cystine determination.

We have also made a comparative study of the results of the Folin-Looney method and the older isolation method for cystine. 7 gm. of rabbit hair (Rabbit 177) were dissolved on the steam bath in a solution of 25 cc. of hydrochloric acid (sp. gr. 1.18) and the same volume of water, and the heating was continued for 100 hours, after which the biuret test was negative. The hydroly- sate was transferred quantitatively to a flask, and the combined hydrolysate and washings were evaporated to 50 cc. under di- minished pressure. The liquid was cooled, made slightly alkaline with 50 per cent sodium hydroxide and immediately 3 cc. of glacial acetic acid were added. An equal volume of acetone was added to increase the insolubility of the cystine. The mixture was placed in the ice box for 2 days, the precipitate filtered off, and the filtrate allowed to stand in the cold. After several days, an additional precipitate appeared, which was also removed by filtration. A small amount of material was precipitated during the next week. The combined weight of the precipitates after drying was 1.012 gm. They were dissolved in dilute hydrochloric acid and the cystine content was determined calorimetrically according to Folin and Looney. The three precipitates contained 403, 190, and less than 1 mg. of cystine, or a total amount equal to 8.5 per cent of the original hair. The sulfur contents of the first two precipitates were then determined (the usual procedure in gravimetric cystine determinations) and found to be equivalent to 402 and 183 mg. of cystine, respectively, a total sulfur content which corresponded to 8.4 per cent of cystine in the hair.

The acetone in the filtrate after the precipitation of the crude cystine was removed by distillation, and the cystine content of the filtrate as determined calorimetrically was found to be 266 mg. or 3.8 per cent of the original hair. This figure added to the value calculated from the sulfur content of the crude precipitated cystine gives a value of 12.2 per cent of cystine in comparison with 12.3 per cent as determined directly by the Folin-Looney method.

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R. H. Wilson and H. B. Lewis 551

The sulfur content of the filtrate was 80 mg., a value corresponding to 300 mg. of cystine, or somewhat more than that found colori- metrically.

A summation of the sulfur content of the precipitate and filtrate gives a total of 0.2364 gm. of sulfur, or 3.38 per cent of the original hair. The value as determined directly was 3.88 per cent.

A similar experiment was carried out with human hair. 23 gm. of hair were hydrolyzed with 75 cc. of concentrated hydrochloric acid and 75 cc. of water on the steam bath for 102 hours. The hydrolysate, which gave no biuret test, was treated as described above, except that alcohol was added to depress the solubility of the cystine instead of acetone. After standing in the ice box for 14 days and at room temperature for 10 days, a precipitate, which weighed 3.237 gm. after drying, was filtered off and carefully washed. This precipitate was dissolved in dilute hydrochloric acid. Calorimetric determination showed the presence of 2.78 gm. of cystine, corresponding to 11.6 per cent of the hair, or about 68 per cent of the amount of cystine found directly by the regular Folin-Looney procedure. The sulfur analysis of the precipitate corresponded to 2.75 gm. of cystine, a value in good agreement with the calorimetric determination.

The filtrate, after the removal of the impure cystine, was an- alyzed as in the first experiment, and contained 867 mg. of cystine by the colorimetriemethod. This gave a total cystine content of the hair of 3.62 gm., or 15.1 per cent of the original sample, a considerable reduction of the value found by direct analysis (17.0 per cent). This difference is to be explained in part, at least, by the fact that a fairly strong test for hydrogen sulfide was given by the vapors above the filtrate after the alcohol had been distilled, prior to the calorimetric determination. The solution did not contain sulfides or mercapto compounds, how- ever, as there was a negligible amount of color developed with the uric acid reagent in the absence of the sulfite.

The sulfur content of the filtrate was 304 mg., which corre- sponds to 810 mg. of cystine. A summation of all the sulfur values gives a total of 1.039 gm. of sulfur, or 4.33 per cent of the original sample. The value originally found was 5.05 per cent.

We believe that the correspondence between the cystine con- tents of the filtrates as determined calorimetrically and as cal-

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culated from the analyses of the sulfur content is too close to be accidental. In our opinion, these experiments indicate that the precipitation of cystine is incomplete and that a considerable proportion of the cystine remains in the filtrate. m’hile we cannot of course, be certain that cystine is the only substance in the filtrate which reacts with the Folin-Looney reagent in the presence of sulfite, it seems hardly possible that so close a correspondence between the cystine content of the filtrate as determined by the two methods could exist, if any considerable amounts of sulfur compounds other than cystine were present. Further work on the application of the calorimetric method of analysis of the cystine content of proteins is in progress.

SUMMARY.

1. The cystine content of various types of keratins has been determined calorimetrically by the method of Folin and Looney. Human hair gave values from 15.6 to 21.2 per cent; sheep wool, 8.0 to 10.9 per cent; feathers, 7.05 to 12.2 per cent; rabbit hair, 11.9 to 14.0 per cent; tortoise shell, 6.4 to 8.1 per cent; rat hair, 14.1 per cent; cat hair, 13.1 per cent; dog hair, 19.0 per cent.

2. Although the sulfur and nitrogen values of keratins from individuals of a species varied rather widely, the ratio of nitrogen to sulfur was relatively constant for that keratin.

3. Three samples of human skin, and one of human connective tissue, were analyzed. The cystine content was lower (1.82 to 2.34 per cent) and the nitrogen to sulfur ratio (23 to 25) higher than those of human hair.

4. No relationship was evident between the cystine content of human hair and the color of the hair, age, or sex of the individual.

5. Little evidence was obtained of the existence of significant amounts of non-cystine sulfur in the keratins studied.

6. The cystine contents of two samples of hair were determined by the older method of precipitation of the impure cystine and gravimetric determination of the sulfur content of the precipitate. It was found that the difference between the calorimetric value and the value calculated from the sulfur content of the precipitate could be accounted for almost completely by the amount of cystine (and sulfur) remaining in the filtrate after the removal of the precipitate.

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R. H. Wilson and H. B. Lewis 553

BIBLIOGRAPHY.

1. Evvard, J. M., DOX, A. W., and Guernsey, S. C., Am. J. Physiol., 1914, xxxiv, 312.

2. Sjollema, B., J. Biol. Chem., 1923, Ivii, 271. 3. Zuntz, N., Deutsch. med. Woch., 1920, xlvi, 145; Deutsch. landwirtsch.

Presse, 1919, xxix, abstr. in Jahresber. Fortschr. Thierchem., 1919, xlix, 594.

4. Blaschko, A., Deutsch. med. Woch., 1920, xlvi, 512. 5. Fuhs, H., Wien. ?&in. Woch., 1920, xxxiii, 707. 6. Lewis, H. B., and Wilson, R. H., J. Biol. Chem., 1926, lxix, 125. 7. Van Slyke, D. D., J. Biol. Chem., 1911-12, 15. x, 8. Hoffman, W. F., and Gortner, R. A., J. Am. Chem. SOL, 1922, xliv, 341. 9. Okuda, Y., J. Biochem., Japan, 1925, 217. v,

10. Morner, K. A. H., 2. physiol. Chem., 1901, xxxiv, 207. 11. Buchtala, H., 2. physiol. Chem., 1907, lii, 474. 12. Sammartino, U., Biochem. Z., 1922, cxxxiii, 476. 13. Folin, O., and Looney, J. M., J. Biol. Chem., 1922, li, 421. 14. Klinke, K., Biochem. Z., 1925, clx, 28. 15. Abderhalden, E., andvoitinovici, A., 2. physiol. Chem., 1907, lii, 348. 16. Buchtala, H., 2. physiol. Chem., 1910, lxix, 310. 17. Buchtala, H., 2. physiol. Chem., 1911, lxxiv, 212. 18. Eckstein, H. C., and Wile, U. J., J. Biol. Chem., 1926, lxix, 181. 19. Trotman, S. R., and Bell, H. S., J. Sot. Chem. Ind., 1925, xliv, 1116. 20. Rutherford, T. A., andHawk, P. B., J. Biol. Chem., 1907, iii, 459. 21. Mulder, cited by Drechsel, E., Eiweisskdrper, Ladenburg, Handwbrter-

buch der Chemie, Breslau, 1885, iii, 534. 22. Jones, D. B., Gersdorff, C. E. F., and Moeller, O., J. Biol. Chem., 1924-

25, lxii, 183.

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Robert H. Wilson and Howard B. LewisOTHER EPIDERMAL TISSUES

THE CYSTINE CONTENT OF HAIR AND

1927, 73:543-553.J. Biol. Chem. 

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