The Spermine Bases of Ocular Tissues*

The Spermine Bases of Ocular Tissues*

THE SPERMINE BASES OF OCULAR TISSUES* ARLINGTON C. KRAUSE, M.D. Chicago The life of the ocular tissues is obviously too complex to be centered ar...

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THE SPERMINE BASES OF OCULAR TISSUES* ARLINGTON

C.

KRAUSE,

M.D.

Chicago

The life of the ocular tissues is obviously too complex to be centered around a few simple substances. Many chemical compounds occur in the water-soluble extracts of the various tissues, although frequently the reports on the general chemistry of the eye apparently overlook this fact. In the soluble extracts, the nitrogenous bases are an important fraction. Of the twenty or more bases occurring in the ocular tissues, this communication is concerned with two, spermine and spermidine, which occur in relatively small quantities. Crystals of spermine phosphate in human semen were seen and well described in 1678 by Leeuwenhoek! in his famous communication on the discovery of spermatozoa to the Royal Society in London. In 1791 Vauquelin, at the time of his work on the analysis of tears, attempted to investigate the nature of spermine phosphate. Subsequently, in 1833 Berzelius, who was then curious about the chemistry of the eye, believed that the crystals consisted of an inorganic phosphate. In 1865 Bottcher rediscovered the crystals, which were then named after him. Schreiner in 1878 noted that Bottcher's crystals were formed on old pathological specimens which had been kept in alcohol. It became gradually established from this time on that the substance was a phosphate of an organic base. It is only recently that Rosenheim" and his coworkers determined the chemical structure of spermine and spermidine and isolated these substances from various organs.

METHOD

Fresh ocular tissue of the ox was ground, weighed, and then dried on a water bath. Alcohol was occasionally added to hasten the drying. After it had been weighed the dried material was extracted with ether. To the extracted tissue was added nine volumes of 2.5 percent trichloracetic acid. The mixture was shaken and then filtered. A mixture of equal volumes of filtrate and of a saturated solution of picric acid was heated on a water bath until clear, and then cooled slowly. After twelve hours the precipitate was removed quantitatively, dried in air, and then dissolved in a minimal quantity of boiling alcohol acidified with hydrochloric acid. Ten volumes of acetone was added. After maximal precipitation had occurred, the picric acid was removed with acetone, and the crystals were dissolved in a minimal quantity of boiling water, and an aqueous solution of saturated diammonium phosphate was added until the pH of the solution was 7.1. Alcohol was added until the solution was 25 percent alcohol by volume. After 24 hours the precipitate of spermine phosphate was removed and washed with a solution of 25 percent alcohol. The crystals were dried and weighed. To the filtrate sufficient alcohol was added to make the volume 50 percent alcohol. The spermidine phosphate which then was precipitated was tested in the same manner as the spermine phosphate. The concentration of spermine bases was similar whether the usual 50-gram or lOoo-gram samples of tissue were used. Extraction with chloroform of strongly alkaline aqueous extracts of tis* From the Division of Ophthalmology, De- sues gave similar yields in percentage of spermine bases." partment of Surgery, University of Chicago. 508

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THE SPERMINE BASES OF OCULAR TISSUES

For ultimate analysis each substance was recrystallized to remove any impurities. From 15.5 to 15.7 percent phosphorus, 14.2 percent nitrogen, and 20.6 percent water were found in the spermine phosphate obtained from choroid, iris, retina, corneal epithelium, and brain. The

theoretical 15.9 percent phosphorus and 14.4 percent nitrogen. The melting point of the phosphate isolated from iris and brain was 205 °C. (cor.) The sample of recrystallized spermidine phosphate which was obtained from Hoffmann-La Roche was 206-207°C. (cor.)

TABLE 1 100 GRAMS

SPERMINE AND SPERMIDINE IN MG. PER

OF BOVINE OCULAR TISSUE

Conjunctiva

Sclera

Corneal Epithelium

Corneal Stroma

Whole Cornea

Choroid

Spermine

Spermine

Spermine

Spermine

Spermine

Spermine

Dry

Wet

Wet

Dry

Wet

Dry

Wet

Dry

Wet

Dry

Wet

Dry

--- ---- ---- ------ ------ - - ------ --0.1 47.7 211.4 <0.2 2.3 7.9 0.34 24.2 9.7 3.5 6.8 2.1 0.3 1.2 40.1 202.6 <0.3 38.5 4.2 14.1 8.4 8.8 0.1 0.48 20.4 3.1 10.4 2.9 9.6 4.9 <0.1 12.6 3.7 Iris

Optic Nerve

Retina

Lens

Vitreous Humor

Brain

Spermine

Spermine

Spermine

Spermine

Spermine

Spermine

Wet

Dry

Wet

Dry

Wet

Dry

Wet

Dry

Wet

Dry

Wet

Dry

--- ---- ---- --- --- ------- ------- --5.8 18.0 31.8 246 18.9 35.0 184 3.0 7.6 0 4.4 26.4 219 208 35.5 4.3 17.2 4.3 4.6 16.7 11.7 0 34.6 5.8 18.0 0 38.1 226 5.5 19.4 226 4.6 14.7 30.6 36.8 184 235 3.5 9.2 0 26.2 36.3 198 261 2.9 7.7 Spermidine Wet

Spermidine

Dry

Wet

-----6.6 1.30 7.4 1.67 1.54 8.3

Dry

-----0.06 0.31

NOTE: Spermidine was found only in the tissues as recorded. The retinal pigment epithelium was removed with the choroid. The cornea without epithelium was recorded as stroma.

theoretical analysis is 15.6 percent phosphorus and 14.1 percent nitrogen. The melting point of spermine phosphate of each tissue was 228-233° C. ( cor.) as compared with the melting point of 230234°C. (cor.) of a sample of recrystallized spermine phosphate which was obtained from Hoffrnan-La Roche, Switzerland. The spermidine from the iris contained 15.8 percent phosphorus and 14.2 percent nitrogen, corresponding well with the

The crystals of the spermine bases of the ocular tissues showed the same physical and optical properties as those obtained from Hoffmann-La Roche for comparison. DISCUSSION

The results of the analysis for spermine and spermidine are given in table 1 in mg. per 100 grams of fresh and dry ocular tissues. Only in the brain and iris could spermidine be found, although the

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ARLINGTON C. KRAUSE

combined extracts of the tissues of several thousand eyes were used. There seems to be no relation of spermidine to spermine which depends upon the structure or function of these two issues. No spermine was found in many kilograms of vitreous humor. Evidently the substance is retained in the living tissue and is not diffusible. Only small quantities of spermine were obtained from conjunctiva, choroid, sclera, lens, optic nerve, and brain. Practically none was found in the corneal stroma. The spermine of the stroma as recorded in table 1 is evidently contained in the corneal endothelium. Large amounts were obtained from corneal epithelium, iris, and retina, which are tissues showing high metabolic activity. Spermine, ll-a ('(-amino-propyl-amino) butane, is apparently a common constituent of animal tissues. A similar compound, spermidine ('(-amino-propyl-amino) -'( amino-butane is commonly associated in small quantities with spermine and, as may be noted from the above formula, it is structurally related to spermme. Indirectly it is probable that these two substances like the other basic organic compounds are metabolic products that arise from the breaking down of amino acids in the tissue or that are synthetized from amino acids through some vital

process. It is likely that the diarninobutane group may be derived from the decomposition of the amino acid, arginine, into urea and ornithine. The latter substance is decarboxylated and forms putresine (diaminobutane). The amino propane group may result from the loss of carbon dioxide from the amino acid, c-amino-n-butyric acid. Thus spermine and spermidine may be formed from the conjugation of the two groups resulting from the decomposition of two amino acids. It appears probable that spermine phosphate exists as such in the tissues in an undiffusible form. Since it occurs in such varying amounts it must play some important role in metabolism, although as yet no demonstrable function has been discovered. SUMMARY

A quantitative analysis of the spermine and spermidine in bovine conjunctiva, sclera, corneal epithelium and stroma, choroid, iris, vitreous humor, retina, optic nerve, and brain was made. There is reason to believe that these substances take a part in some unknown important physiological function of active metabolic tisues. The technical procedures were performed with the aid of Mr. W. Tauber. 950 East Fifty-Ninth Street.

REFERENCES

Rosenheim, O. The isolation of spermine phosphate from semen and testis. Biochern. j our., 1924, v. 18, p. 1253. . • Dudley, H. W., Rosenheim, M. C, and Rosenheim, O. The chemical constitution of spermine. 1. The isolation of spermine from animal tissues and the preparation of its salts. Biochem. jour., 1924, v. 18, p. 1264. Dudley, H. W., Rosenheim, 0., and Starling, W. W. The chemical constitution of spermine. II. Structure and synthesis. Biochem. Jour. 1926, v. 20, p. 1082. Dudley, H. W., Rosenheim, 0., and Starling, W. W. The constitution and synthesis of spermidine, a newly discovered base isolated from animal tissues. Biochem. Jour., 1927, v. 21, p, 97. • Harrison, G. A. The approximate determination of spermine in single human organs. Biochem. Jour., 1933, v. 27, p. 1151. 1