When bilirubin is treated with oxidising agents a series of coloured products are successively formed. This constitutes Gmelin's test for the bile-pigments. If a drop of bile be spread in a thin film on a porcelain plate, and a drop of nitric acid containing nitrous acid in solution be placed in the centre of it, the drop of acid becomes surrounded by rings of colours, green, blue, violet, red, and yellow. The green colour is the first stage, the yellow the last stage in oxidation. The green pigment is biliverdin; the blue or violet product is called bilicyanin 1; its composition is unknown; it shows certain absorptionbands. The red product has also not been further investigated. The end or yellow product was called choletelin by Maly, whose formula for it is CHINO. It is soluble in water, alcohol, acids, and alkalis, and is amorphous. MacMunn 3 describes the spectroscopic, changes that occur as follows: As the blue colour appears a broad shading composed of two bands appears at D, then a black band close to F. The two bands first mentioned are separated by a narrow interval in which the D line is seen (fig. 88, spectrum 1). As the colour changes progress the band after D fades away, then that before D; and when the yellow stage is reached one band, that at F, is alone visible (fig. 88, spectrum 2).

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2

Biliverdin has the formula C,H,NO, (Thudichum). It may occur as such in the bile; it may be formed by simply exposing red bile to the oxidising action of the atmosphere; or it may be formed, as in Gmelin's test, by the more vigorous oxidation produced by fuming nitric acid. It gives the remaining colours of Gmelin's test quite well.

Maly obtained biliverdin by the action of acetic acid or monochloracetic acid on bilirubin. It differs from bilirubin considerably in its solubilities, being soluble in alcohol, insoluble in chloroform and in water, almost insoluble in ether. It can be precipitated from bile by means of hydrochloric acid. It has never been obtained in a crystalline form.

Haycraft and Scofield have recently shown that not only may the bilepigment undergo changes of an oxidative nature, but that reduction processes may occur also; for instance, placing the positive pole of a battery in bile, and then completing the circuit, will cause a series of colour-changes to occur, indicating oxidation; if, now, the negative pole be substituted for this the reverse series of colour-changes occurs, indicating reduction. They also show that under certain other circumstances, especially in the presence of putrefactive organisms,. reduction may occur in the bile.

1 Heynsius and Campbell, Pflüger's Archiv, v. 497.

2 Sitz. Wien. Akad. lix. Abth. ii.

5 Clinical Chemistry of Urine, London, 1889, p. 170.

4 Zeit. physiol. Chem. xiv. 173.

Hydro-bilirubin.--If a solution of bilirubin or biliverdin in dilute alkali be treated with sodium amalgam, or allowed to putrefy, a rosered or brown-red pigment is formed which is slightly soluble in water, -easily soluble in alcohol, ether, chloroform, salt solutions, or alkaline fluids. Maly investigated this substance, and gave it the name of hydro-bilirubin, and assigned to it the formula C32H,,N,O,; it thus contains less hydrogen, and rather more oxygen, than bilirubin.

Its spectroscopic appearances are as follows: A dark band between band F, and a fainter band in the region of the D line (fig. 88, spectrum 3).

The ammoniacal solution of this pigment gives on the addition of zinc-chloride a well-marked green fluorescence, and then shows three bands instead of two (fig. 88, spectrum 4).

The interest of this substance arises from the fact that many physiologists believe it is identical with the substance called stercobilin by Vaulair and Masius,2 which is the colouring matter of the fæces, and according to some also with urobilin, the chief pigment of the urine. We shall see, when discussing those pigments, that hydro-bilirubin is not absolutely identical with either. MacMunn3 and Disque1 both regard hydro-bilirubin as an impure product.

Bilifuscin (CHNO,) is a pigment which has been obtained from brown gallstones. The gall-stones are powdered and thoroughly extracted with a mixture of ether and alcohol to remove the cholesterin, then with dilute hydrochloric acid to remove calcium salts; the acid is washed away with hot water and the residue shaken with alcohol. On distilling off the alcohol from the extract, a reddish-brown amorphous pigment is left, which is bilifuscin. It is insoluble in water, chloroform, or ether; soluble in alcohol. It shows no absorption bands. It does not give Gmelin's test (Städeler).

5

Biliprasin (CHNO) is the name given by Städeler to a green pigment which he separated from gall-stones. Maly considers it is identical with biliverdin.

Bilihumin is the humous-like residue left after extracting gall-stones with water, alcohol, ether, chloroform, and dilute acid successively (Städeler). It is probably an impure substance.

Cholohematin--This pigment occurs in the bile of the ox and sheep, and gives a three-banded spectrum (fig. 88, spectrum 5). An ethereal extract of the residue -obtained by agitating the acidulated bile with chloroform and evaporating this--is evaporated, and the residue again taken up with chloroform, which is washed in a separating funnel with water. On evaporating the chloroform a dark green pigment with a musky smell is left. It is considered by MacMunn, who first described it, to be a derivative of hæmatin, probably an intermediate stage in the formation of biliverdin.

1 Centralbl. med. Wiss. 1871, No. 54.

3 Loc. cit. p. 107.

5 Ann. Chem. Pharm, clxxv. 76.

2 Ibid. No. 24.

4 Zeit. physiol. Chem. ii, 259. Journ. of Physiol. vi. 22.

Hæmoglobin itself and a substance like methæmoglobin have been described in the bile of animals killed by freezing, or after the injection of aniline,. toluidine, and other substances that destroy the red blood-corpuscles (Wertheimer and Meyer').

Biliary urobilin is a substance like urobilin, which has been sometimes found by Mac Munn in the bile of man, pig, ox, and sheep. The bile was treated with alcohol and acetic acid, and filtered; the filtrate was diluted with water and

FIG. 88.-Spectrum 1 represents the bilicyanin stage of Gmelin's reaction; spectrum z represents the final (choletelin) stage of Gmelin's reaction; spectrum 3 is the absorption-spectrum of hydrobilirubin; spectrum 4, the same after treatment with zinc-chloride and ammonia; spectrum 5 is the absorption-spectrum of cholo-hæmatin; spectrum 6 is the absorption-spectrum of the colour formed in Pettenkofer's reaction. The faint band near D fades as the colour is developed (after MacMunn).

agitated with chloroform. The chloroform became orarge; this extract was evaporated on the water-bath, and the pigment extracted from the residue with rectified spirit. This solution showed two bands very like those of hydrobilirubin. With ammonia and zinc chloride it gave a red, which on exposure to the air became a green fluorescence, and by further oxidation it was made to resemble choletelin. The probable origin of this pigment in the bile is as

2 Proc. Roy. Soc. No. 208, 1880; Journ. of Physiol. x. 108.

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follows: The bile is poured into the intestine; the pigment in the intestine is changed into a substance like hydro-bilirubin; this is absorbed and carried back by the portal circulation to the liver, and then excreted in the bile.

3. THE USES AND FATE OF BILE IN THE INTESTINE

One of the most remarkable facts concerning the bile is its apparently small importance in the digestion of food. It is doubtless to a large extent excretory, but it is probable that it may in the future be found that bile is a more valuable digestant than is at present supposed. It has no action on proteids, except to precipitate the undigested albumin, as has been already described (p. 652). It has probably slight actions on the fats and carbohydrates, but appears to be rather a coadjutor to the pancreatic juice than to have an inde pendent digestive activity of its own.

Action on carbohydrates.--Although some observers have stated that the bile of herbivora has a slight diastatic action, bile, as a rule, has no power whatever in this direction by itself. If, however, bile or bile-salts be added to pancreatic juice, that juice will convert starch into dextrin and maltose more quickly than a control specimen containing no bile (S. Martin and D. Williams 1). How bile favours the action of pancreatic juice it is at present impossible to say.

Action of fats.-It is found in cases of jaundice, when no bile enters the intestine, and in cases of biliary fistula also, that the fæces contain a large amount of undigested fat. In the dog, 40 to 50 per cent. of the fat in the food is found in the fæces. Bile is therefore important in the digestion of fat. Here again, however, it is the combined action of the bile, with the pancreatic juice, that is important. Although the bile is by some said to have a slight emulsifying action, There is, however, no doubt that it is if present at all very slight. pancreatic juice plus bile act on fats better than pancreatic juice alone.2 Bile is said also to aid in the absorption of fats by lubricating the If an animal membrane, such as a mucous membrane of the bowel. piece of bladder, or even a filter paper, be moistened with bile, fat will pass through it under less pressure than if they are moistened with water.3 It is a little dangerous to draw positive conclusions from such We shall see that absorption is not simply a an experiment as this.

More recently these observers have shown that bile also 1 Proc. Roy. Soc. xlv. 358. favours the action of pancreatic juice on proteids (Ibid. xlviii. 160).

p. 782.

3

A recent paper on this subject is one by A. Dastre, Compt. rend. Soc. biol. 1887,

v. Wistinghausen, Diss. Dorpat, 1851; J. Steiner, Arch. f. Anat. u. Physiol. 1873, p. 137; 1874, p. 286.

matter of diffusion or filtration, and is a very different matter from what occurs in dead membranes; and there is perhaps no substance in which the living activity of the cells is so much needed for absorption as fat.

Bile as a laxative and an antiseptic.-The faces in animals or human beings who suffer from jaundice or a biliary fistula are extremely hard, and have an intense putrescent odour. Administration of bile relieves this condition; it is also known that a large increase in the flow of bile has a purgative effect as in bilious diarrhoea. The bile itself is readily putrescible, and the power it has of diminishing putrescence in the fæces is due chiefly to the fact that by increasing peristalsis it hastens the passage of putrescible matters through the bowel. Copeman and Winston2 performed a number of cultivation experiments with bacteria of different kinds, and found that, though bile is able to a small extent to control putrefactive changes, the bacteria grew almost as readily in the tubes to which bile had been added as in those to which no bile had been added. Limbourg 3 made similar experiments, and estimated certain products of putrefaction (amido-acids and ammonia) in artificial pancreatic digestions with and without the addition of bile-salts. In the specimens where the bilesalt was present, these products were somewhat lessened.

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The fate of the constituents of the bile.- -We have seen that fistulabile is poor in solids as compared with normal bile, and this is explained on the grounds that the normal bile-circulation is not occurring, and hence the liver cannot excrete what it does not receive back from the intestine. Schiff was the first to show that if the bile be led back into the duodenum, or even if the animal be fed on bile, the percentage of solids in the bile secreted is at once raised. It is on these experiments that the theory of a bile-circulation is chiefly founded. The bilecirculation relates, however, chiefly, if not entirely, to the bile-salts; they are found but sparingly in the fæces; they are only represented to a slight extent in the urine; hence it is calculated that seven-eighths of them are reabsorbed from the intestine, especially the large intestine. This is by no means the least curious of the phenomena of bile-secretion. The bile is a most elaborate secretion; it is poured into the intestine, and finds apparently little to do; it is split into simpler constituents, which then hurry back by the portal vessels to the liver again, when once more they unite to form bile-salts. It is stated that of the two bile-salts, the taurocholate is the more easily decomposed. Small quantities of cholalic acid, taurine, and glycocine are found in the fæces; some of the taurine

1 McKendrick, Physiology, ii. 122.
3 Zeit. physiol. Chem. xiii. 196.

2 Journ. Physiol. x. 213. Pflüger's Archiv, iii. 598.