sulphate or ammonium sulphate. Like the gelatin from which they are derived they give the xanthoproteic reaction very faintly.

Hofmeister distinguishes two of these peptone-like substances; one he terms semi-glutin is insoluble in 70-80 per cent. alcohol, and is precipitated by platinum tetrachloride; the other, called hemi-collin, is soluble in 70-80 per cent. alcohol, and is not precipitable by platinum tetrachloride.

The following equation exhibits the relationship of these substances to collagen :

C10H119N1O38+3H,O=CHNO+C1HNO19

[collagen]

17

47 70 14

[semi-glutin] [hemi-collin]

Chemical constitution of gelatin. The formulæ that have been given to gelatin by different observers are purely empirical; as in the case of proteids, though there are several theories as to the constitution of the molecule, we are practically entirely ignorant on this point.

Strong agents, like sulphuric acid or putrefaction, decompose the substance, forming glycocine, leucine, various fatty acids, carbon dioxide, and ammonia. Schützenberger heated gelatin in sealed tubes with baryta-water to 200° C. The products he obtained were ammonia, carbonic acid, and oxalic acid, these compounds being in the ratio of the products of decomposition of urea. The other products were amido-acids of the acetic series, the most abundant in the order of their importance being glycocine (amido-acetic acid), alanine (amidopropionic acid), amido-butyric acid, and leucine (amido-caproic acid). The general conclusion is drawn that gelatin, like a proteid, is a compound of urea with certain amido-acids.

It will be noticed in all these decompositions no substances like tyrosine containing aromatic radicles are obtained as they are from proteids.

THE ELASTIC FIBRES OF CONNECTIVE TISSUE

The elastic or yellow fibres, the microscopical characters of which have been already described, consist chemically of the very insoluble albuminoid which is called elastin. During development, elastic granules are deposited either in rows, which subsequently become fused together end to end to form fibres, or they may be deposited in patches, and by their fusion form an elastic membrane, as in the elastic (fenestrated) membrane of arteries. The elastin granules make their appearance first in the neighbourhood of the cells; this renders it probable that the deposition of the granules is influenced by the cells, but it does not prove that they are formed by a direct conversion of the cell-protoplasm.

Elastin

Preparation.-As usually described elastin is an albuminoid sub-. stance which, like gelatin, is free from sulphur, but which offers great resistance to the action of reagents. The method of preparing this

1 Comptes rendus, cii. 1296. For some work on the decomposition of gelatin see also Buchner and Curtius, Berichte d. chem. Gesellsch. Berlin, xix. 850.

material from tissues which contain a large quantity of it (such as the ligamentum nucha of the ox, horse, or giraffe), consists in treating the finely divided ligaments successively with reagents in which it is insoluble, and in which adherent collagenous and proteid substances readily dissolve. The ligaments are treated first with boiling water, then with 1 per cent. potassium hydroxide solution, then in 10 per cent. acetic acid, then in 5 per cent. hydrochloric acid, and lastly with alcohol and ether. This method of purification takes several days, large excess of each of the reagents mentioned must be employed, and each fluid must be changed two or three times.

Composition. By this means a substance free from sulphur is obtained. Chittenden and Hart in some of their preparations of elastin omitted the extraction with potash, and in these a small per centage of sulphur (0-3 per cent) was found; it is, however, doubtful whether this is really in the elastin molecule or in proteid matter which is present as an impurity.

The following table shows the results in percentages of elementary analysis by different observers :

The higher content of carbon in the preparations of Müller and Tilanus is doubtless due to the presence of more or less fat, not completely extracted with ether.

Properties.-Elastin is not soluble in any liquid that does not decompose it. It is soluble in hot concentrated caustic potash, in cold concentrated sulphuric acid, and also in cold concentrated nitric acid. When boiled with sulphuric acid elastin yields leucine, but no tyrosine.

When digested with pepsin or trypsin, elastin is gradually but slowly dissolved. The older writers looked upon it as being almost insoluble in the digestive juices. Kühne and Ewald appear to be the first who obtained a solution of elastin by digestive agents, and these

1 Chittenden and Hart, Zeit. Biol. xxv. 368; Studies from the Lab. of Physiol. Chem. Yale University, iii. 19.

Müller, Zeitsch. f. rat. Med. 3rd series, vol. x. part ii.

3 Tilanus, Gorup-Besanez' Physiol. Chem. 3te Aufl. p. 148.

4 Horbaczewski, Zeit. physiol. Chem. vi. 330.

5 Ewald and Kühne, Die Verdauung als histol. Methode.

observers noticed that pepsin was more active than trypsin. More recently Etzinger,' Horbaczewski, and Morochowetz 3 have shown that finely divided ligamentum nucha, or powdered purified elastin, are fairly soluble in artificial digestive juices, and Horbaczewski was able to verify this with natural gastric juice obtained from a man with a gastric fistula.

Horbaczewski named the two products of digestion hemi-elastin and elastinpeptone. Chittenden and Hart,' who repeated these experiments, using the methods adopted by Kühne and Chittenden in the examination of the digestion products of proteids, found that both these substances are analogous rather to the proteoses, the intermediate stages in the formation of peptone, than to true peptone; they are both, for instance, precipitable from their solutions by saturation with ammonium sulphate, while true peptone is not. They have named the two products of digestion of elastin, proto-elastose (this corresponds to Horbaczewski's hemi-elastin) and deutero-elastose (this corresponds to Horbaczewski's elastin-peptone); the former is precipitable by saturation with sodium chloride, the latter is not. Both are precipitable by saturation with ammonium sulphate, and both give the xanthoproteic and biuret tests. The names just mentioned are analogous to the names of the albumoses, the first cleavage products in the digestion of albumin. These same substances can be obtained from elastin by the action of trypsin, or by the prolonged action of acidulated water at 100° C.

THE GROUND SUBSTANCE OF CONNECTIVE TISSUE

The ground substance of connective tissue, like the cement-substance of epithelium, has the power of forming a compound with silver salts, which becomes reduced in the light, and consequently brown or black from the deposit of metallic silver in it. This property is of great value to the histologist, as a means of demonstrating the spaces in the ground substance in which the cells lie. These spaces, which are connected to one another, form a branching network of irregular canals (Saft Kanälchen) in which lymph circulates.

The chief constituent of the ground substance is mucin. This is readily soluble in lime-water or other weak alkalis, and so the various tissue elements of fibrous tissue and other forms of connective tissue fall apart when they are treated for about twenty-four hours with limewater, owing to the solution of mucin. The other organic constituent of the ground substance is a proteid; this occurs in small quantities; it belongs to the class of proteids (which are insoluble in distilled water, soluble in dilute saline solutions, and insoluble in saturated saline solutions) called globulins. It is coagulated by heating its solution to 75° C.

Mucin is obtained in greater abundance from embryonic connective

1 Etzinger, Zeit. Biol. x. 84.

3 Morochowetz, Maly's Jahrsbericht, 1886, p. 271.

2 Loc. cit.

4 Loc. cit.

tissues than from those of the adult; in the fully formed connective tissues the ground substance is very largely replaced by fibrous (collagenous) material. In the vitreous humour and Whartonian jelly of the umbilical cord, in which the fibrous and cellular elements of connective tissues are reduced to a minimum, mucin can be obtained in abundance.

Mucin

Mucin is a substance which has a slimy consistency, and of which there are several varieties. It is found, not only in the ground substance of connective tissue and the cementing substance between epithelial cells, but, as we have already seen, in many epithelial structures (see Mucus, p. 444). We shall also have to consider it in a few secretions, such as the saliva. It must be carefully distinguished from certain nucleo-albumins which have similar physical characters, such as, for instance, the so-called mucin of the bile. Mucin, again, is an ingredient of the tissues of certain invertebrates, and the mucin obtained from the snail has been studied by Eichwald and Hammarsten.

Elementary analysis has shown that mucin from different sources differs in composition very much. Many of the minor reactions of the substance also vary, and it is now pretty generally granted that different mucins differ from one another in the nature of the proteid which is combined with a carbohydrate radicle to form the mucin molecule. The name given by Landwehr 2 to the carbohydrate which may be obtained from the various forms of mucin is animal gum. Hammarsten 3 gives the following three properties as characteristic of a mucin :

1. Its viscidity and stickiness.

2. Its solubility in dilute alkalis; it is precipitable from such solutions by acetic acid, being insoluble in excess of that reagent. 3. When heated with dilute sulphuric acid, it yields a reducing sugar.

Connective-Tissue Mucin

Preparation. The different methods that have been adopted for the preparation of mucin from connective tissue are all essentially the same, though they differ in detail (Rollett, Leebisch "). The tissue is finely minced, washed with water, and then extracted for twenty-four to

1 Gorup-Besanez (Physiol. Chem.) gives the following analysis of the vitreous humour by Lohmeyer: water in 1000 parts, 986'4; membranes 0'21; proteids and mucin 1:36; fats 0·016; extractives (urea &c.) 3'2; sodium chloride 7·76; other mineral matters 1:05. 2 Landwehr, Zeit. physiol. Chem. vols. vi. vii. viii. ix.

3 Chem. Centralbl. 1884, p. 814.

5 Læbisch, Zeit. physiol. Chem. x. 40.

4 Rollett, Stricker's Handbuch, i. 72.

forty-eight hours with a very large excess of lime-water, or baryta-water diluted to five times its bulk with distilled water. The extract is then

precipitated with excess of acetic acid, the precipitate is allowed to stand a few hours; in this time it collects into large flocculi or stringy masses if a large quantity of mucin is present, as when one is dealing with the vitreous humour. The substance is collected and may be purified by redissolving it in lime-water, filtering, and reprecipitating it from the filtrate by acetic acid.

Some recommend that the tissue should be first placed in alcohol for a week or two to coagulate the proteids that are present. This, however, is quite unnecessary; for if any proteid is dissolved by the lime-water it is precipitated, as alkali-albumin always is, by the acid, but is readily soluble in excess. The spirit has the disadvantage of rendering the pieces of tissue hard, and so they cannot be permeated easily by the lime-water; ultimately, also, it renders mucin insoluble.

Instead of using lime-water or baryta-water, other weak alkalis, like a 1 per cent. solution of sodium carbonate, may be employed; or even distilled water, no doubt in virtue of the alkaline salts in the tissue, will extract a considerable quantity of mucin.

Estimation of mucin.-The amount of tissue taken is weighed in the first instance; it is extracted with lime-water repeatedly till no more mucin goes into solution; and the mucin precipitated from this by acetic acid is collected on a weighed filter and washed with 2 per cent. acetic acid, distilled water, alcohol, and ether; it is finally dried, weighed, and incinerated, the amount of ash being deducted. The percentage of mucin can be thus calculated. In view of the alleged increase of mucin in a disease known as myxedema, it is important to have certain data concerning the amount present in normal tissues. Although the above method cannot claim to be absolutely accurate, it gives very good comparative results. On p. 478 is the result of a number of analyses made by Dr Stevenson and myself. In all the cases there enumerated the percentages refer to the organ as weighed in the fresh condition.

The following numbers represent the averages obtained in normal tissues. The details are given in the tables on the next page.

1 Clin. Soc. Transactions, vol. xxi. supplement, report of Myxedema Committee.