Chapter VII.), in which extract of pancreas is combined with ammonium

chloride, and some inert material.

The effect of puering on the fatty matter in the skin is well known. The fat and grease are partially emulsified, and set free, so that they can be removed by scudding. This action is a most important one, and one in which artificial bates have hitherto been wanting. The emulsification of the fats is brought about by means of an enzyme either identical with, or closely resembling, lipase.[105] This enzyme is found in the pancreatic juice, and in the seeds of many plants. It brings about the emulsification of the fat by saponifying a portion of it, i.e. the fat is split up into glycerin[106] and a fatty acid, according to the equation--

C_{3}H_{5}(C_{18}H_{35}O_{2})_{3} + 3H_{2}O Stearin.

= C_{3}H_{5}(OH)_{3} + 3(C_{18}H_{35}OHO) Glycerin. Stearic Acid.

[105] Also known as steapsin, or pyolin (Allen, p. 357, vol. iv., Comm. Org. Analysis).

[106] In the digestion of fat by dogs the glycerol produced is all absorbed before the ileum is reached, so that none exists in the excrement. (Levites, Chem. Soc. Abst. 1907, vol. iv. p. 891.)

Lipase was one of the first enzymes in which the reversibility of the reaction was shown, i.e. it is not only capable of hydrolysing a fat, but also of causing the formation of one by the combination of the fatty acid and glycerin.[107] This explains why the reaction of such an enzyme is never complete. An equilibrium is reached just as with ordinary reversible chemical reactions like the precipitation of magnesium hydroxide by ammonia.

[107] Another instance of the reversibility of enzyme action is the secretion of a peptolytic enzyme, by B. pyocyaneus; i.e. it has the power of synthesizing proteins, as well as of decomposing them. (Zak. Chem. Soc. Abst. 1907, p. 996.)

Lecithin, and possibly other fatty compounds, are known[108] to be important auxiliaries in the ferment-like actions produced by toxins; cholesterol has a similar effect, and as this body is a constituent of dung, it may play some part in puering. Here, again, is a further problem awaiting investigation.

[108] Chem. Soc. Annual Reports IV., 1907, p. 252.

Loevenhart has shown that the bile salts, sodium cholate, and sodium glycocholate, greatly increase the activity of lipase, and Magnus found that synthetic bile salts have the same effect; such bodies are known as co-enzymes. I have shown that bile itself is not favourable to the bating action, but the bile salts, by stimulating the action of the fat splitting enzymes, are probably essential to the full action of the dung bate.

Another enzyme which may be of importance in puering is erepsin, the enzyme of the intestinal juice, which is responsible for the completion of the digestive process. The pancreatic enzymes act upon the peptones produced by the pepsin of the stomach, splitting them up into simpler compounds, while the erepsin acts further upon these products. It dissociates albumoses and peptones into amino acids, taking as it were the last traces of nutriment from the food passing through the intestine. It acts best in alkaline solutions.

This ferment is very widely distributed in the animal kingdom, and occurs in other organs and tissues besides the intestines. The quantity of erepsin in the fresh fæces must be considerable, since a dog secretes from 400 to 500 c.c. of intestinal juice per day. It remains to be shown whether this retains its properties after excretion, and for how long.

A most important point in connexion with the activity of enzymes is the reaction of the medium, i.e. its acidity or alkalinity, or, more strictly speaking, its hydrogen ion concentration. A very slight increase or decrease of the acidity or alkalinity of the liquor will diminish the rate of action of the enzyme by a large amount, and in some cases cause the action to cease. In all cases enzymes have an optimum acidity or alkalinity; in other words, for every enzyme there is a particular hydrogen ion concentration at which its activity is at a maximum. The work of Soerensen, to which I have already referred, gives a very complete account of this aspect of enzyme action and also of the methods he employed to investigate it. His work should certainly be carefully studied by anyone wishing to take up this part of the subject.

The same remarks as to optimum conditions apply to temperature, although the effect of this is better known. Most tryptic enzymes act best at the body temperature, viz. 98° to 100° F. (37° to 40° C.), and hence puering should be conducted at this temperature. In the case of the hen or pigeon-dung bate the enzymes have not yet been studied, so far as I am aware, so that it is not possible to give an account of them. At the same time it seems highly probable that, as these bates are employed at comparatively low temperatures, the enzyme action is kept back, and therefore the chief action would be a chemical one.

Enzymes are retarded in their action in the first place by their own products, in a very similar manner to bacteria, in the second place the retarding or inhibiting action is brought about by the so-called _anti-bodies_. Of these the longest known and best studied are those which are produced by toxins, and which neutralize the action of the toxins upon the animal organism (anti-toxins).[109] Normal serum also inhibits the action of trypsin and many other enzymes. Another very important group of anti-bodies are the _precipitins_. If the serum of an animal be injected repeatedly into another animal of different species, a precipitin appears in the serum of the animal treated, which causes a precipitate when added to the serum of the first animal. The special importance of this fact is, that it can be utilized as a method of distinguishing between human blood and that of animals, which is often of importance in medical juris-prudence.[110]

[109] Hammarsten, p. 70.

[110] Gulland, Ency. Britt. iv., p. 83.

This fact has been made use of to distinguish dog dung from other matters, with which it has been adulterated.[111] A perfectly clear extract of dog dung filtered free from bacteria was injected into a rabbit. The serum obtained from the rabbit was found to contain a precipitin, and on addition of the serum to the dog dung extract a precipitate was produced. When the serum was added to the extract from the dung of another animal no precipitate was formed. When added to the extract of the adulterated dung a very much smaller precipitate was produced than with the extract from dog dung alone.

[111] Appelius, Technische Briefe, No. 23, April 1909.

The coli bacteria in the puer also produce an anti-body--_agglutinin_.[112] If a culture of B. coli be examined under the microscope, the bacteria are seen moving here and there in the liquid, evenly distributed. On the addition of a trace of the serum of an animal which has been previously injected with coli bacteria the bacteria on the slide cease their movements and collect together in clumps. They are said to be agglutinated. This property is used to diagnose bacteria in suspected cases of cholera and some other diseases.

[112] Harden, see Bibliography.

The wonderful discoveries that have been made in this direction constitute one of the most marvellous chapters in the history of science. Serum diagnosis and serum therapeutics are now firmly established as invaluable aids to the physician in his fight against disease and death.

The extent of our present knowledge of the action of enzymes in puering may be summed up as follows. Active enzymes are produced by bacteria growing in the infusion of dung, in addition to digestive enzymes which may be originally present in the dung; the bacterial enzymes are produced more rapidly in a dilute infusion, as employed in the puer wheel, than in the dung itself. The enzymes are of various kinds, proteolytic, peptolytic, lipolytic, etc., but the proteolytic and lipolytic are the most important. These have a solvent action on the fibres of the skin, but little or no action on the hyaline layer, at the concentration usually found in the puer liquor. The fatty matters and soaps in the skin are acted upon by the lipolytic enzymes, and the fats to some extent emulsified, so that they may be easily removed from the skin by scudding or pressing.

It must be clearly understood that enzyme action alone is not sufficient, as has been previously explained, but that the dung enzymes, acting in conjunction with the chemical compounds present, produce the specific puering effect.

There is still much work to be done before the action of the enzymes in dung is fully understood, but from the above short account it will be seen that the part they play in the bate is of great importance.