dissolved in absolute alcohol and filtered; the filtrate from this is again evaporated to dryness, and again dissolved in absolute alcohol, and this should be again repeated. To the final alcoholic solution, an alcoholic solution of platinum chloride is added, and the precipitate so formed is allowed to settle and is washed with absolute alcohol by decantation; the precipitate is then dissolved in 15-per-cent. alcohol, filtered, and the filtrate is allowed to slowly evaporate in a watch-glass at 40° C. The crystals can then be seen with the microscope. They are recognised not only by their yellow colour and octahedral form, and by their solubility in water and 15-per cent. alcohol, but also by the fact that on incineration they yield 31 per-cent. of platinum and give off the odour of trimethylamine. There is a danger of mistaking such crystals for those obtained from the chlorides of potassium and ammonium; but the presence of such contaminations may be minimised by the use of alcohol as water-free as possible. (2) The following test, however, is entirely distinctive of choline and leads to no risk of confusion with other substances. The final alcoholic solution prepared as above is evaporated to dryness, and the residue taken up with water, to this is added a strong solution of iodine (2 grammes of iodine and 6 grammes of potassium iodide in 100 c.c. of water). In a few minutes dark-brown prisms of choline periodide are formed. These look very like hæmin crystals. If the slide is allowed to stand so that the liquid gradually evaporates, the crystals slowly disappear, and their place is taken by brown oily droplets, but if a fresh drop of the iodine solution is added the crystals slowly form once more. (3) A physiological test, namely the lowering of arterial bloodpressure (partly cardiac in origin, and partly due to dilatation of peripheral vessels) which a saline solution of the residue of the alcoholic extract produces this fall is abolished, or even replaced by a rise of arterial pressure, if the animal has been atropinised. Such tests have already been shown to be of diagnostic value in the distinction between organic and so-called functional diseases of the nervous system.

A similar condition can be produced artificially in animals by a division of large nerve-trunks; and is most marked in those animals in which the degenerative process is at its height as tested histologically by the Marchi reaction.1 A chemical analysis of the nerves themselves was also made. A series of cats was taken, both sciatic nerves divided, and the animals subsequently killed at intervals varying from 1 to 106 days. The nerves remain practically normal as long as they remain irritable: that is, up to about three days after the operation. They then show a progressive increase in the percentage of water, and a progressive decrease in the percentage of phosphorus until degeneration is complete. When regeneration occurs, the nerves return approximately to their previous chemical condition. One chemical feature of degeneration is the replacement of phosphorised by non-phosphorised fat. When the Marchi reaction disappears in the later stages of degeneration, the

The Marchi reaction is the black staining that the medullary sheath of degenerated nerve fibres shows when, after being hardened in Müller's fluid, they are treated with Marchi's reagent, a mixture of Müller's fluid and osmic acid. Healthy nerve fibres are but little affected by the reagent, but degenerated myelin is blackened like the fat of normal adipose tissue.

non-phosphorised fat has been absorbed. This absorption occurs earlier in the peripheral nerves than in the central nervous system. The non-phosphorised fat of degenerated myelin is also either richer in olein, or the olein is more loosely combined than in the healthy medullary sheath; hence the deeper reaction with osmic acid even in the presence of chromic acid as in the Marchi test. The following table gives details of these experi

Noll

The foregoing figures relate to the peripheral portions of the nerves. has also shown that the phosphorised fat diminishes somewhat in the central ends of cut nerves due to 'disuse atrophy.'

Further, it has been found that in human spinal cords in which a unilateral degeneration of the pyramidal tract has been produced by a lesion in the opposite hemisphere, and which gives the Marchi reaction, there is a similar increase of water and diminution of phosphorus on the degenerated side.

Cerebro-spinal Fluid. This plays the part of the lymph of the central nervous system, but differs considerably from all other forms of lymph. It is a very watery fluid, containing, besides some inorganic salts similar to those of the blood, a trace of protein matter (globulin) and a small amount of a reducing substance, the nature of which was for a long time uncertain but which seems now to have been proved to be sugar. It contains the merest trace of choline; but this is not devoid of significance, for this fact taken in conjunction with another-namely, that physiological saline solution will extract from perfectly fresh nervous matter a small quantity of cholineshows us that lecithin is not a stable substance, but is constantly breaking down and building itself up afresh; in fact, undergoing the process called metabolism. This is most marked in the most active region of the brainviz. the grey matter.

LESSON XXIII

UREA AND CHLORIDES IN URINE

ESTIMATION OF UREA

If albumin is present it must be first separated by boiling after acidulation with acetic acid if necessary, and filtering off the flakes of coagulated protein. The hypobromite method of estimation (see p. 141) holds its own, as it is easy and sufficiently exact for clinical purposes. It has entirely replaced the older method of Liebig (titration with mercuric nitrate), which is now of purely historical interest.

When absolute accuracy is necessary, one or other of many recently introduced methods must be employed. We shall be content with describing two of these.

(a) Folin's Method. This depends on the fact that urea is decomposed quantitatively into ammonia and carbonic acid by boiling with magnesium chloride solution. The ammonia is estimated by distillation into standard acid and subsequent titration.

Analysis. Three c.c. of urine, 20 grammes of magnesium chloride and 2 c.c. of concentrated hydrochloric acid are boiled in a flask, closed by a cork through which a glass tube 20 centimetres in height passes. This acts as a reflux condenser. The boiling is continued for 25 to 30 minutes. After diluting with water the mixture is then transferred to a litre flask, 7 c.c. of 20-percent. caustic soda are added, and the ammonia is distilled off and estimated as described in the final operation in Kjeldahl's method (see Appendix, p. 235). Every c.c. of decinormal ammonia in the distillate corresponds to 3 milligrammes of urea. A small correction has to be made for the ammonia present as such in the original urine.

(b) Method of Mörner and Sjöqvist.---The following reagents are necessary: i. A saturated solution of barium chloride containing 5 per cent. of barium hydrate.

ii. A mixture of ether and alcohol in proportion 1: 2.

iii. The apparatus, &c., necessary for carrying out Kjeldahl's method of estimating nitrogen (see p. 235).

Analysis.-Five c.c. of urine are mixed with 5 c.c. of the barium mixture and 100 c.c. of the mixture of ether and alcohol. By this means all nitrogenous substances except urea are precipitated. Twenty-four hours later this is filtered off, and the precipitate is washed with 50 c.c. of the ether-alcohol mixture, the filter-pump being used to accelerate the process. The washings are added to the filtrate; a little magnesia is added to this to drive off ammonia.

The alcohol and ether are then driven off at a temperature of 55° C., and evaporation is continued at this temperature until the volume of the residue is 10-15 c.c. The nitrogen in this is estimated by Kjeldahl's method. The nitrogen found is multiplied by 2.143, and the result is the amount of urea.

PREPARATION OF UREA FROM URINE

(1) Evaporate the urine to a small bulk. Add strong pure nitric acid in excess, keeping the mixture cool during the addition of the acid. Pour off the excess of fluid from the crystals of urea nitrate which are formed; strain through muslin and press between filter paper. Add to the dry product barium carbonate in large excess. This forms barium nitrate and sets the urea free. Mix thoroughly with sufficient methylated spirit to form a paste. Dry on a water-bath and extract with alcohol; filter; evaporate the filtrate on a water-bath and set aside. The urea crystallises out, and may be decolourised by animal charcoal and purified by recrystallisation.

(2) The following method is well adapted for the preparation of microscopic specimens of urea and urea nitrate: Take 20 c.c. of urine; add baryta mixture (see footnote 3, p. 5) until no further precipitate is produced; filter, evaporate the filtrate to a thick syrup on the water-bath, and extract with alcohol; pour off and filter the alcoholic extract; evaporate it to dry. ness on the water-bath and take up the residue with water. Place a drop of the aqueous solution on a slide and allow it to crystallise; crystals of urea separate out. Place another drop on another slide and add a drop of nitric acid; crystals of urea nitrate separate out.

ESTIMATION OF CHLORIDES

The chlorides in the urine consist of those of sodium and potassium, the latter only in small quantities. The method adopted for the determination of the total chlorides consists in their precipitation by a standard solution of silver nitrate (Mohr's method).

The following solutions must be prepared :—

Standard silver nitrate solution. Dissolve 29.075 grammes of fused nitrate of silver in a litre (1,000 c.c.) of distilled water: 1 c.c. = 0·01_gramme of sodium chloride.

(a) Saturated solution of neutral potassium chromate.

Analysis.-Take 10 c.c. of urine; dilute with 100 c.c. of distilled water. Add to this a few drops of the potassium chromate solution.

Drop into this mixture from a burette the standard silver nitrate solution; the chlorine combines with the silver to form silver chloride, a white precipitate. When all the chlorides are so precipitated, silver chromate (red in colour) goes down, but not while any chloride remains in solution. The silver nitrate must therefore be added until the precipitate has a pink tinge. From the amount of standard solution used, the quantity of sodium chloride in 10 c.c. of urine, and thence the percentage, may be calculated.

Sources of Error and Corrections.—A high-coloured urine may give rise to difficulty in seeing the pink tinge of the silver chromate: this is overcome by diluting the urine more than stated in the preceding paragraph.

One c.c. should always be subtracted from the total number of c.c. of the

silver nitrate solution used, as the urine contains small quantities of certain compounds more easily precipitable than the chromate.

To obviate such sources of error the following modification of the test, as described by Sutton, may be used: 10 c.c. of urine are measured into a thin porcelain capsule and 1 gramme of pure ammonium nitrate added; the whole is then evaporated to dryness, and gradually heated over a small spirit lamp to low redness till all vapours are dissipated and the residue becomes white. It is then dissolved in a small quantity of water, and the carbonates produced by the combustion of the organic matter neutralised by dilute acetic acid; a few grains of pure calcium carbonate to remove all free acid are then added, and one or two drops of potassium chromate. The mixture is then titrated with decinormal silver solution (16.966 gr. of silver nitrate per litre) until the end reaction, a pink colour, appears. Each c.c. of silver solution represents 0·005837 gr. of salt; consequently, if 12.5 c.c. have been used, the weight of salt in the 10 c.c. of urine is 0.07296 gr., or 0.7296 per cent. If 5'9 c.c. of urine are taken for titration, the number of c.c. of silver solution used will represent the number of parts of salt per 1,000 parts of urine.