THE

VOL. CXVII., No. 3061.

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ON NUCLEIC ACID AND ITS ANALYTICAL EXAMINATION.

By A. CHASTON CHAPMAN, F.I.C.

DURING recent years nucleic acid and certain of the metallic nucleates have found somewhat wide and increasing application in medicine, and particularly in connection with surgical practice. At the outbreak of war these substances were obtained chiefly from Germany and America, and so far as I am aware were not manufactured at all in this country. A number of urgent inquiries having been received from France and elsewhere, the Pharmaco-Chemical Products Company, Ltd., suggested to me that I should undertake the investigation of this matter with the object of devising methods for the manufacture of pure nucleic acid and its derivatives on a large scale. Yeast was obviously the most convenient raw material, and for more than a year the above-mentioned company have been manufacturing considerable quantities of pure yeast-nucleic acid and its compounds.

It will be obvious that, in the prosecution of this work, it was necessary to have methods for examining the products obtained and to establish analytical criteria of purity. The production of pure nucleic acid on a large scale presents many difficulties, the two chief ones being the complete removal of protein and the prevention of the contamination of the acid with the products of its own decomposition. This will be at once apparent when it is remembered that nucleic acid results from the breaking down of the nucleo-proteins of the cell nuclei on the one hand, and that, on the other, it yields, as the result of further hydrolytic change, a number of complex nucleosides and bases, together with phosphoric acid and the carbohydrate d-ribose. The conditions for hydrolysis have therefore to be very exactly determined and very strictly observed in practice.

Up to the present two nucleic acids have been very fully studied, the one derived from yeast, the other from the thymus gland. Whether these two acids represent typical members of two sharply defined groups, or whether all nucleic acids are identical with one or the other, is at present a little doubtful. The latter view is, however, the

nucleic acid." It is with this plant nucleic acid that the present paper chiefly deals.

The following statement shows at a glance the chief products of hydrolysis of these two nucleic acids, and at the same time affords an insight into their respective chemical constitutions:

Plant Origin.-Guanine, adenine, cytosine, uracil, d-ribose (pentose), phosphoric acid.

Animal Origin.-Guanine, adenine, cytosine, thymine, lævulinic acid (from a hexose), phosphoric acid.

Plant nucleic acid is a white friable substance devoid of odour or taste, and having the formula C38H50029N15 P4. It is practically insoluble in water, but dissolves readily in solutions of alkaline acetates. It is also readily soluble in From these solutions a few drops of hydrochloric acid solutions of the alkalis forming the soluble alkaline salts. precipitate the nucleic acid in a dense curdy form, which dissolves completely on the addition of a large excess of

the acid.

When acetic acid is added to a solution of the sodium

salt the acid is partly precipitated, but no precipitate is formed on the addition of any acid in the presence of a sufficient quantity of alkaline acetate.

When added to a solution of sodium nucleate a solution of copper acetate acidified with acetic acid gives a bulky greenish blue precipitate.

Calcium chloride in excess in the presence of a few drops of acetic acid gives a white precipitate at first flocculent, quickly becoming more granular.

Silver nitrate, when added in considerable excess to a fairly strong and neutral solution of sodium nucleate, gives a white gelatinous precipitate. On the addition of a little sodium chloride this dissolves, forming an opaque colloidal solution. One drop of hydrochloric acid added to this precipitates the silver as chloride.

Solutions of sodium nucleate in water exhibit a marked tendency to gelatinise, and, if sufficiently strong, set to a jelly.

The above reactions, coupled with the recognition of guanine and adenine (which are the most easily isolated and best defined of the bases formed on hydrolysis), suffice for the identification of nucleic acid, when it exists in a fairly pure condition.

For the hydrolysis of nucleic acid 10 grms. of the acid (or a larger quantity if available) are heated in a boiling water-bath for two hours with 40 cc. of 10 per cent sulphuric acid (or a proportionately larger quantity if more than 10 grms. are taken) in a small flask fitted with an air condenser. At the end of this time strong ammonia is added to the hot solution in the flask until the liquid contains an excess of about 2 per cent. The guanine is precipitated in a granular form, the adenine remaining in solution. After filtering and washing with 1 per cent ammonia the guanine is dissolved in the smallest possible quantity of dilute sulphuric acid, decolorised if necessary by means of a little animal charcoal, and the base precipitated again from the colourless solution by the addition of an excess of ammonia. The purified base may then be converted into the hydrochloride, which crystallises very readily and may be easily identified by the application of any of the well known tests. The ammoniacal filtrate from the guanine, together with the ammoniacal washings, is acidified with sulphuric acid, heated to boiling, and the adenine precipitated as a cuprous compound by the addition of a 10 per cent solution of copper sulphate. Inasmuch as the solution contains the carbohydrate ribose it is not necessary to add sodium bisulphite for the purpose of reducing the cupric compound. The adenine-copper compound is suspended in hot water, decomposed with sulphuretted hydrogen, and the filtrate from the copper sulphide evaporated to dryness on the water-bath. The residue, consisting of nearly pure adenine, is dissolved in 5 per cent of sulphuric acid, and the adenine sulphate, which is readily soluble in hot, but very slightly soluble

in cold water, allowed to crystallise. can then be applied.

The following is an outline of the procedure to be adopted for the examination of nucleic acid, with the object of ascertaining its purity and quality.

The acid should be white or at the most have a very faint buff colour. It should be completely soluble in an aqueous solution of sodium acetate or in dilute solutions of ammonia or of sodium or potassium hydroxides. The solutions in these reagents should be bright and almost colourless. When a few drops of hydrochloric acid are added to a solution of the sodium salt in water the acid should be precipitated as a white curdy substance, and on the further addition of a large excess of the hydrochloric acid this should dissolve completely. An aqueous solution of the sodium salt should give with cupric acetate, calcium chloride, and silver nitrate the reactions described above.

Tests for the Absence of Protein. Biuret Reaction.-A solution of the acid in an excess of caustic soda should give, on the addition of one or two drops of dilute copper sulphate solution, a greenish blue colour with at the most a faint tinge of purple. The test is quite sufficient, but occasionally the following test is applied :

A 5 per cent solution of nucleic acid in a small excess of ammonia, when heated in a bath at 105° C., should give at the most a slight turbidity.

In connection with these tests it may be pointed out that the removal of the last traces of protein matter from nucleic acid involves a very troublesome series of treatments, and that acid of high commercial quality will usually give an indication of the presence of traces of protein when subjected to the above tests. The second of the above tests is the more severe, inasmuch as an amount of protein equal to not more than o'r or 0.2 per cent (expressed on the acid) gives a very appreciable

volume of precipitate.

Inorganic Phosphate.-To a solution of the acid in excess of ammonium or sodium acetate a few drops of acetic acid are added, and then a little uranium acetate. A flocculent precipitate is formed, which in the absence of more than a trace of inorganic phosphate should dissolve completely on boiling. A considerable excess of alkaline acetate is necessary in this test.

Estimation of Nitrogen and Organic Phosphorus. Nitrogen.-This is estimated by the Kjeldahl method in the ordinary way.

Phosphorus.-A weighed quantity of the acid is fused with six times its weight of sodium carbonate containing 10 per cent of potassium nitrate. The fused mass is taken up with water, and in this solution the phosphoric acid is estimated either by the molybdate method or by direct precipitation with magnesia mixture in the presence of a little ammonium citrate.

It will be seen that the formula given above corresponds with 161 per cent of nitrogen and 95 per cent of phosphorus.

Commercial nucleic acid of good quality ought to contain not much less than 15 per cent of nitrogen and 9 per cent of organic phosphorus (expressed on the moisturefree sample). The examination of nucleic acid should always include these two items, and the ratio of the percentage of organic phosphorus to that of nitrogen is one of the best criteria of the purity of the acid, since, if this ratio (117) is approximately correct, the presence of any appreciable proportion of the products of hydrolysis is excluded.

NEWS

Dr. WYNTER BLYTH, referring to the identification of the nucleic acid by the production of adenine and guanine, asked whether there was any other method which would require smaller quantities than 9 or to grms., and also whether Mr. Chapman could say how the nucleic acid was secreted, and in what manner it affected the urine. Presumably the phosphates would be increased, and possibly the amino-acids.

Capt. R. H. A. PLIMMER asked whether Mr. Chapman had any further information on the subject of the increase of leucocytosis by nucleic acid used in surgical injections. It had been shown that meat extract caused an increase of uric acid in the urine, and meat extract contained purine and pyrimidine bases, which were constituents of nucleic acid. This suggested that the uric acid might be produced from the pyrimidine bases, but it might be the result of some synthetic process in the body. He had been hoping that Mr. Chapman would have described some method for the quantitative chemical analysis of the decomposition products--for the estimation, for instance, of the quantity of guanine yielded. Such quantitative analysis was a difficult matter, and Mr. Chapman's paper gave an increased power to deal with it. Some years ago Professor detecting the minutest quantities of inorganic phosphorus Scott and he (the speaker) had discovered a method of or phosphate mixed with organic phosphorus compounds, using an ammonium molybdate reagent made with hydrochloric acid and containing some potassium persulphate. A mixture containing organic but no inorganic phosphorus compounds gave merely a yellow coloration, but if inorganic phosphate was present a yellow precipitate was produced.

Mr. H. F. E. HULTON asked whether Mr. Chapman would include absence of the biuret among his criteria of

the purity of nucleic acid.

the nucleic acid was readily obtained by the Kjeldahl Mr. CHAPMAN, in reply, said that the total nitrogen in process. As to the question of obtaining enough guanine

and adenine for purposes of identification, there was no difficulty nowadays in obtaining sufficient nucleic acid to enable one to work with the 10 grms. required for this purpose. He was sorry to say that he had no information as to the form in which nucleic acid was excreted. A great deal of work had been done on the action of various enzymes on nucleic acid, but precisely what happened to the phosphates or to the other constituents he was not able to say.

Capt. PLIMMER said that the phosphates were increased in the urine, and to a probably greater extent in the fæces.

Mr. CHAPMAN, Continuing, said that he did not know precisely what happened in connection with the increased leucocytosis which resulted from the use of nucleic acid, nor did he know the reason for the increase. That it did occur there could be no reasonable doubt, for the German literature on the subject was specially voluminous, and numerous experiments on this property of nucleic acid had also been made in France. Nucleic acid was, in fact, being used in considerable quantities for this particular purpose, and appeared to afford material help. He had not attempted the estimation of guanine, which would be a troublesome process and beyond the scope of his immediate object. The production of guanine and adenine by hydrolysis had been recommended merely for the purpose of effecting some more complete identification of the nucleic acid itself. He was much obliged to Captain Plimmer for the reference to his special molybdate reagent, and should certainly try it. He was obliged also to Mr. Hulton for his reminder about the b'uret reaction, which was, however, dealt with in the paper. This reaction should be practically absent. On dissolving the acid in a slight excess of caustic potash or soda and a few drops of copper sulphate there should be not more than the faintest possible tinge of purple; more than that would, of course, indicate the presence of protein.-The Analyst, July 1918.

DURING a silver recovery from residues the following perhaps unusual change took place. The silver chloride was converted to the oxide, Ag2O, in the usual way, and thoroughly washed.

To show its reducing power formaldehyde 60 per cent was used for the ultimate reduction. This operation was carried out in a vessel containing a mechanical stirrer. At room temperature, 14° C., reduction was slow, and nothing out of the ordinary happened. On raising the temperature to 35° C. reduction was accelerated, but the liquid was found to be acquiring a pale lilac tint increasing in intensity as reduction proceeded. This solution passed unchanged through ordinary filter-papers, and has been preserved since the experiment-June, 1917. The colour of the solution is now a rich ruby-red.

The experiment was repeated using acetaldehyde, but no similar result was obtained. Possibly the acetic acid produced, less volatile than the formic acid produced above, was sufficiently ionised to ensure immediate coagulation of the colloidal particles formed.

Further experiments dispensing with continuous stirring and elevation of temperature gave no result in several cases, and in others only after several days, the colour produced being only slight.

There seemed reason for suspecting colloidal silver. The following experiments gave some support to the idea :

1. The colour was discharged by salt solutions, though only slowly. Nitric acid was most effective especially on warming.

2. Hydrogen peroxide was slowly decomposed on slightly warming. The peroxide solution was, however, very dilute-4 volume.

Summary of a Paper in The Analyst, October, 1918.

NEWS

ENEMY METHODS OF GAS WARFARE.

IN April, 1915, a German deserter in the Ypres salient gave warning of the first attempt to use poison gas in modern warfare. No one believed him; but a week later the enemy launched his first attack with chlorine gas against our unprepared and unprotected troops, and claimed subsequently to have killed 6000 men and to have taken an equal number of prisoners. Since that time gas warfare has become a combination of a science and an art, and its development shows the usual struggle between attack and defence, and in this case between poison and antidote. The first method used took the form of a gas cloud. The cylinder was covered with a layer of moss containing potassium carbonate solution, and surrounded by sand bags. When the attack was made the protective covers were removed, and the cylinder connected with a lead pipe bent over the top of the trench. The success of such an attack depends largely on physical conditions. The wind direction must lie between two straight lines which make angles of 40° with the neighbouring sections of the front, and its velocity must be within the limits of four and twelve miles an hour. An upward current is the worst foe of gas, and the ground should slope gently away from the point of emission. Above all, the element of surprise plays a very important rôle. The gas used must be easily compressible, easily made on the large scale, of high density and toxicity, and preferably of low chemical activity. Thus the choice is practically limited to two gases, chlorine and phosgene.

Chlorine suffers from the defect of being too chemically active and therefore too easily absorbed. The first protection against it consisted of pads of cotton-wool soaked in solutions of sodium thiosulphate and carbonate. The type changed every week, till finally a helmet was introduced, consisting of a flannel bag soaked in the above reagents and containing a mica window. This proved an efficient protection.

Phosgene was first used in December, 1915, by the Germans, but, thanks to our Intelligence Department, the danger was foreseen and provided against by the issue of helmets containing sodium phenate. These gave adequate protection when the gas concentration did not exceed I: 10,000, but as the enemy was soon able to increase the concentration a more efficient absorbent had to be found The Russian suggestion of using hexamethylenetetramine, (CH2)6N4, was adopted. In conjunction with sodium phenate it gave protection against gas concentrations of I: 1000 for a considerable time. The type of respirator was changed to the box type, which strikes a useful balance between the efficiency of a larger apparatus and the lightness and convenience so essential in a gas mask. Thus an oxygen apparatus would be useless on account of its weight and short life. "The side that can first force the other to use oxygen respirators for protection has probably won the war."

Besides the antidotes already mentioned, activated charcoal, another Russian suggestion, is much used. Charcoal and alkaline permanganate will protect against nearly every gas, even up to concentrations of 10 per cent for short periods.

The German apparatus, which is not so efficient as ours, consists of a small drum attached to the face piece of the respirator and containing three layers of materials, viz., pumice soaked in hexamethylenetetramine, charcoal, and on the outside baked earth soaked in potassium carbonate solution and coated with charcoal.

In all probability the method of attack by gas cloud has bad its day, everything pointing to the gas shell as being the more deadly weapon and the more capable of development. The term "gas shell" is somewhat misleading, as the contents are generally liquid or solid, but the materials used are vaporised or atomised by the explosion, a proper adjustment between bursting charge and poison being necessary. The chief advantages of a gas shell are:-It is not dependent on the wind, it can be fired with all the accuracy of modern gunfire, and it does not require a