CHAPTER XXIX.

LEUCINE AND TYROSINE.

LEUCINE.

THE elementary composition of leucine is expressed in the formula CHNO,. It is homologous with glycocine or glycocoll and alanine, and has the same relation to valerianic acid and its aldehyde, as they have to formic and acetic acids and their aldehydes. When leucine is heated with potash, or allowed to putrefy, it yields up valerianic acid and some other products.

Leucine appears to be so frequent a product of diseased action in the human body, that I have thought it necessary to enter fully into the description of all its properties.

It was first (1818) discovered in rotten cheese by Proust,1 who named it oxyde of cheese. Braconnot, in 1820, obtained leucine as the product of decomposition of animal matter by oil of vitriol. The identity of oxyde of cheese and leucine was recognised by Mulder,3 in 1838.

It has since been ascertained by a series of observers to be an almost constant product of the treatment, by concentrated or dilute sulphuric acid, and by caustic alkalies, of albumen, fibrine, caseine, muscle, glue, gluten, legumen, wool, and horn; it is also produced by the putrefaction of these substances, mostly together with some glycocine.

Frerichs and Städeler have proved the occurrence of leucine and tyrosine in certain pathological conditions of the organism, such as typhus, and exanthematic fevers, as variola.

Wien. Med. Wochenschr.,' iv, No. 30. Müller's Archiv,' 1854, p. 387. Pharm. Centralbl.,' 1854, p. 861.

These substances were found in the urine, blood, and bile of the patients. In the urine the leucine seemed to have been partly transformed into valerianate of ammonia. The liver seems to be the organ in which leucine is formed during life, and where it is found most abundantly after the death of such patients. In the liver of healthy human subjects and of animals Frerichs and Städeler could not find any leucine. In a case of acute atrophy of the liver, these observers found the finer branches of the hepatic veins filled with zeolithic efflorescences, and firm yellowish-gray strings of matter containing crystals in the shape of balls with a radiary arrangement, and bundles and sheaves of needles. The balls were proved to be leucine by an elementary analysis; and the needles were tyrosine, forming a combination with sulphuric acid, and this combination forming salts with bases.

Leucine has been detected in the liver of the calf.1 Dr. A. Cloetta2 found it in the tissue of the lungs of the ox, together with inosite, uric acid, and taurine.

I found leucine in the urine of a man whose liver yielded a large amount of it. Goessmann found leucine in the charcoal, which is left in the retort when the residue from the evaporation of urine is subjected to dry distillation.

There is at present no very characteristic test for leucine; its diagnosis in urine, therefore, mainly depends upon the exclusion of all known normal and abnormal ingredients of that fluid, and upon the coincidence of several or all of the tests of leucine.

Production of Leucine.

According to Braconnot, leucine is obtained in the following manner. A piece of beef is chopped, washed with water, and subjected to strong pressure; the dry residue is warmed gently with its equal weight of sulphuric acid, until complete solution has taken place. After cooling, the fat collected on the surface is removed, and the solution is mixed with a quantity of water, amounting to three and a half times the weight of the meat originally employed. The mixture is kept boiling during nine hours, the water which evaporates being replaced from time to time. The sulphuric acid is then removed by carbonate of lime, the filtrate is evaporated to the

Gregory's Handbook of Organ. Chem.,' 1852, p. 422.

2 Verhandl. d. Naturf. Gesellschaft zu Zürich,' Bd. iv. Journ. für Pract. Chem.,' lxvi, p. 211, 1855.

consistence of a syrup, and the latter is repeatedly extracted by boiling alcohol. From this alcoholic solution leucine in a crystalline form is obtained by evaporation; the product of impure leucine amounts to about one thirtieth part of the muscular fibre used. It yet contains some animal matter, which may be precipitated from the watery solution by tannic acid. According to Mulder, it also contains some glycocine.

Hinterberger1 adopts the following proceeding. One part of horn turnings and filings is mixed with four parts of sulphuric acid and twelve parts of water, and boiled during thirty-six hours, the water being replaced from time to time. The sulphuric acid is next removed by an excess of milk of lime; the fluid with the precipitate is boiled for twenty-four hours longer, and strained. The filtrate is neutralized by means of sulphuric acid, and filtered again; acetate of lead is now added until all sulphuric acid is removed. The excess of lead is removed by means of sulphuretted hydrogen, and the fluid is boiled with the sulphuret of lead. On filtering (which should be done when the fluid is cold, and has again been saturated with sulphuretted hydrogen, Th.), a scarcely coloured fluid is obtained, from which, on concentration, tyrosine crystallizes. From the mother-liquor leucine is obtained (mixed with a great deal of acetate of lime, from which it must be separated by extraction with boiling alcohol. Th.) It may be further decolorized by treating it with hydrated oxyde of lead, sulphuretted hydrogen, and animal charcoal.

Zollikofer2 found that the elastic tissue of the animal body yields leucine as the sole crystalline product of decomposition by sulphuric acid. The nuchal ligament of horses or of oxen is particularly suitable. It is first freed from fat and fibrous (cellular) tissue by mechanical means, and then (Goessmann's improved proceeding) dissolved in its equal weight of concentrated sulphuric acid at the ordinary temperature of the air. This solution is then mixed with its own bulk of water, and boiled from ten to twelve hours, during which time the loss of water by evaporation is being frequently made up. After cooling, and removal of the fat floating on the surface, the solution is diluted and neutralized by milk of lime. The excess of lime is precipitated by carbonic acid, and the filtrate is evaporated, when leucine crystallizes. It is taken up with

1 (1849) Ann. d. Chem. und Pharm.,' lxxi, p. 70. Pharm. Centralbl.,' 1850, p. 213. Liebig and Kopp's Jahresber.,' 1849, p. 502.

* (1852) Ann. d. Chem. und Pharm.,' lxxxii, p. 162. Liebig and Kopp's 'Jahresber.,' 1852, p. 696.

boiling alcohol; its watery solution may be further decolorized by animal charcoal.

Method of Bopp.'-One part of dry powdered albumen, fibrine, or caseine, free from fat, is mixed with one part of caustic potash, while fusing on the fire in an iron pot, of about twenty-five times the capacity of space taken up by the quantities of albuminous matter and potash. The mixture froths up, by the evolution of hydrogen and ammonia; and after half an hour, when the brown colour of the mixture has passed into yellow, water is cautiously added, the alkali is neutralized with acetic acid, and the fluid filtered while hot. The filtrate on cooling deposits groups of needles of tyrosine. (In case the operation has well succeeded, these crystals fill the entire filtrate, but their amount is the smaller, the longer the melting process has been continued.) The fluid decanted from these crystals is then again evaporated, until a crystalline pellicle begins to form on its surface; it is then allowed to repose for twenty-four hours, and is then extracted with alcohol, which leaves a residue consisting of leucine and some tyrosine. A certain quantity of leucine remains in solution in the alcohol; to this solution sulphuric acid diluted with alcohol is now added as long as sulphate of potash is thereby precipitated. From the filtrate the alcohol is evaporated, the sulphuric acid removed by acetate of lead, and the excess of lead precipitated by sulphuretted hydrogen; the filtrate on evaporation now yields crystals of leucine, and an uncrystallizable syrup, the quantity of which is the smaller, the longer the melting process has been continued. In order to free the leucine from tyrosine and a peculiar colouring matter, it is dissolved in such a quantity of hot water, that on cooling only little leucine crystallizes, together with most of the tyrosine; the mother-liquor is digested with hydrated oxyde of lead, which removes the colouring matter and some leucine; the filtrate is treated with sulphuretted hydrogen, and, being now of a faint-yellowish hue, is evaporated in a balloon until a crystalline pellicle begins to form on the surface, when, on cooling, leucine crystallizes, which may be further purified by washing with cold water and spirits of wine, and by treating it with animal charcoal and recrystallizing it.

If it is intended to have leucine alone, the mixture of the albuminous substance with potash requires only to be heated until the strongest frothing up has passed; the same quantity of leucine is then formed, but no tyrosine.

Ann. der Chem. und Pharm.,' Ixix, p. 20.

As a product of putrefaction, leucine may be obtained by allowing one part of cheese, flesh, or albumen, with fifty parts of water, to decompose at a temperature somewhat above 20° C. (68° F.), for about six weeks. The dirty fluid is then boiled with milk of lime; the lime is precipitated by a slight excess of sulphuric acid; the filtrate is evaporated, and precipitated with acetate of lead; the filtrate from this is evaporated to a syrupy consistence; and the leucine which now crystallizes is freed mechanically and by the assistance of alcohol from the syrup; it is then dissolved in water, treated with sulphuretted hydrogen, and is obtained pure by crystallization from alcohol and water.

When the solution, at the stage where it contains gypsum and a slight excess of sulphuric acid, is evaporated slowly in an open china dish, the gypsum crystallizing acts as a very potent decolorizer. From the crystalline and black sediment of gypsum the fluid may be poured off while hot. The decanted fluid should then be evaporated to dryness and extracted with alcohol, whereby a solution is obtained which contains no lime, and may easily be freed from sulphuric acid by acetate of lead. Another mode of removing the lime and sulphuric acid is by treating the decanted solution with oxalic acid, until no more precipitate of oxalate of lime is produced. The excess of oxalic acid, together with the sulphuric acid, is removed by acetate of lead; the excess of lead from the filtrate by sulphuretted hydrogen; and the filtrate from this precipitation will be almost colourless, and on evaporation will yield leucine, which may be purified of the last traces of admixed foreign matter by alcohol and crystallization.

Chemical and Physical properties of Leucine.

The sublimate of pure, dry leucine appears under the microscope in strings of rhombic plates, as represented in fig. 5, pl. VI.

When crystallized from an alcoholic solution, leucine represents white scales, of the lustre of mother-of-pearl, much like cholesterine, floating on water, and imparting the sensation of an unctuous matter to the finger. Under the microscope, it has the appearance of minute larch-agarics, with which it was compared by Proust. In the liver, and the extract therefrom, it crystallizes in balls, with a radiary arrangement of particles.

It dissolves in 27 parts of water at the ordinary temperature of the air, and in 658 parts of alcohol of specific gravity 0.828 (Mulder), or in 1040 parts of alcohol, of 96 per cent., at the ordinary temperature; and in 800 parts of warm al