brown colour, often in star-shaped clusters, and with rounded angles (fig. 61), separate out.

In the case of an old blood stain, when one wishes to apply this test, it is necessary to add a small crystal of sodium chloride in addition to the glacial acetic acid. Fresh

blood contains sufficient sodium chloride in itself.

On a large scale, hæmin may be prepared in the following ways:1. A solution of hæmatin in alcohol acidified with sulphuric acid is heated with a solution of sodium chloride. 2. Defibrinated blood is mixed with a large excess of dilute sodium chloride (1.5 per cent.) solu

tion;

the

corpuscles when they have FIG. 61. - Hæmin crystals magnified (Preyer). subsided are extracted with ether; the ethereal extract is evaporated to dryness, and the residue heated with glacial acetic acid (Hoppe-Seyler). Hæmin=hæmatin+ 2HCl (Hoppe-Seyler). Hæmin is insoluble in water, ether, chloroform, alcohol, and in cold dilute acetic or hydrochloric acids. It is soluble in an alcoholic solution of potassium carbonate, in caustic alkalis, and in boiling acetic and hydrochloric acids. The crystals are decolourised by alcoholic ammonia (Shalfeeff). 2

Analogous compounds to hemin are formed with hydrobromic acid (HBr) and with hydriodic acid (HI). They may be called bromohæmatin and iodohæmatin respectively, while hamin may be termed chlorohæmatin. The crystalline form and colour of all three compounds are identical (Harris). It has been stated that the preparation of iodohæmatin crystals is a more delicate test for blood stains than that of chlorohæmatin crystals (Bufalini).

Cyan-hamatin. When potassium cyanide is added to an ammoniacal solution of pure hæmatin, or to a solution of oxyhæmoglobin, a broad band extending from D to E is seen on spectroscopic examination. On adding reducing agents, a spectrum with two well-marked absorption bands is obtained. These optical supposed to depend on the production of a compound which has

characters are

Gamgee, Physiol. Chem. p. 117.

Shalfeeff, Journ. Russ. Chem. Soc. 1885, p. 203.

3 V. D. Harris, Brit. Med. Journ. vol. ii. 1886, p. 103.

Centr. 1886, p. 499.

See also K. Bikfalvi, Chem.

Bufalini, Arch. Pharm. (3) xxiii. p. 682. The method consists in heating the aqueous extract of the blood stain with a drop of iodine tincture and a little acetic acid on a glass slide. Crystals form in 1-2 minutes. MacMunn obtained a crystalline compound of

hæmatin with sulphuric acid, but the chemical constitution of this substance still remains to be worked out (Journ. Physiol. vi. 24).

been designated cyan-hæmatin. We are, however, merely acquainted with the spectroscopic characters of the supposed compound."

Hæmatoporphyrin.-When hæmatin is heated with fuming hydrochloric acid to 160° C. the iron is removed from it, as a ferrous salt, and iron-free hæmatin or hæmatoporphyrin is formed. The same result is obtained when hæmatin is dissolved in concentrated sulphuric acid, the solution being of a purple-red colour. For spectroscopic purposes only, hæmatoporphyrin may be obtained by adding a small quantity of blood or oxyhæmoglobin solution to a large quantity of strong sulphuric acid.2 Hæmatoporphyrin may be precipitated from its acid solution by the addition of water. This precipitate is soluble in water, and also in caustic alkalis; the optical properties of the aqueous solution are the same as those of the acid solution, viz. a broad dark band a little to the right of D, and a narrow fainter band to the left of D (fig. 59, spectrum 10). When hæmatoporphyrin is dissolved in caustic alkalis, it appears to undergo some amount of decomposition (Gamgee); the solution has a reddish-brown tint, and the absorption spectrum shows four bands (fig 59, spectrum 11).

Hæmatoporphyrin is interesting as being not only an artificial product of hæmoglobin, but as also occurring in the integument of certain invertebrate animals, viz. starfishes, slugs, the common earth worm, and various sponges (MacMunn).3 Polyperythrin (Moseley), a pigment of various actiniæ and deep sea polypes, is also probably identical with hæmatoporphyrin. It is also found in the eggshells of some birds. Urohematoporphyrin is a kind of hæmatoporphyrin found in the urine in certain diseases; it will be fully described under Urine. Hamatoporphyroidin is a decomposition product of hæmatoporphyrin described by le Nobel.4

The formula given by Hoppe-Seyler to hæmatoporphyrin is C68H74N8O12; and its formation from hæmatin may be thus represented (see further p. 294):

C68H70N,Fe2O10+2H2SO4+O2=C68H74N8O12+ 2FeSO,

[hæmatin] [sulphuric acid]

[hæmatoporphyrin] [sulphate of iron]

1 Cyan-hæmatin will be found more fully described in Hoppe-Seyler's Med. Chem. Untersuchungen, Heft iv. The above short description I have taken from Gamgee's Physiol. Chem. p. 115.

2 Hoppe-Seyler has obtained hæmatoporphyrin by the action of nascent hydrogen, and MacMunn by the action of sodium amalgam on hæmatin.

3 MacMunn, Quart. Journ. of Mic. Science, 1877; Journal of Physiology, vols. vii. and viii. It may be added that in certain species of actinia a pigment very similar to hæmochromogen, and convertible into hæmatoporphyrin, is found (Phil. Trans. 1885). 4 Chem. Centralbl. 1887, p. 538.

5 Hoppe-Seyler has described another iron-free derivative of hæmatin, to which he gives the provisional name of hæmatolin (C6sH78NgO7). It differs from hæmatoporphyrin in being insoluble in sulphuric acid and caustic alkalis (Physiol. Chemie, p. 397).

Hæmatoidin.-In old blood clots, such as occur in the brain after cerebral hemorrhage, in the interior of aneurisms, and in the corpora lutea of the ovary, small rhombohedral crystals of a brick-red colour are often found, together with an amorphous deposit' of the same colour (see fig. 62). The name hæmatoidin was given to this substance by Virchow. It is insoluble in water, alcohol, ether, acetic acid, dilute mineral acids and alkalis; soluble in concentrated acids and caustic alkalis.

:

When treated with fuming nitric acid, the crystals give the same colour reaction as the bile pigment does 3 (Gmelin's reaction). Hæmatoidin is undoubtedly a derivative of hæmoglobin, and it is free from iron so also is bilirubin, the pigment of the bile; and Salkowski found hæmatoidin to be identical chemically with bilirubin. Berzelius found in the gall-bladder crystals of bilirubin exactly similar to those of hæmatoidin. Preyer, however, states that the two substances differ spectroscopically; solutions of bilirubin showing no bands, solutions solutions of hæmatoidin showing one band between b and F, and a weaker one between F and G. Holm obtained similar results. Thudichum 6 has pointed out that Preyer and Holm mistook the lipochrome (lutein) in the cow's ovary for hæmatoidin, and hence they concluded that it was not identical with bilirubin. Neither hæmatoidin nor bilirubin shows bands, but both possess a strong absorptive power for the violet end of the spectrum.

FIG. 62.--Hæmatoidin crystals.

Hemosiderin is the name given by Neumann to a pigment often occurring in extravasations and thrombi with hæmatoidin, but differing from it in containing iron.

So far, in describing the composition of hæmatin and its derivatives, I have followed Hoppe-Seyler pretty closely. More recently, however, the subject of hæmatin and its allies has been reinvestigated by Nencki and Sieber, and the

8

1 The amorphous variety was first described by Robin, Ann. Chem. Pharm. cxvi.

89. See also Städeler, Ibid. cxxxii. 328.

2 Virchow's Arch. d. pathol. Anat. u. Physiol. vol. i. (1847), p. 383.

3 Jaffe, Arch. f. path. Anat. xxiii. 192. Hoppe-Seyler, Ibid. xxiv. 10.

4 Salkowski, Hoppe-Seyler's Med. Chem. Unters. Heft iii. p. 436.

5 Holm, Journ. f. prakt. Chem. c. 142.

Thudichum, Proc. Roy. Soc. xvii. 255. Dr. MacMunn kindly furnished me with this and several of the foregoing references.

7 Virchow's Archiv, cxi. 25.

Nencki and Sieber, Berichte d. deutsch. chem. Gesellschaft, xvii. 2267, xviii. 392. Monatsh. Chem. ix. 115.

conclusions at which they have arrived are on many points different from those of Hoppe-Seyler': to certain of these, however, much importance cannot be attached; for instance, until we know a rational formula for hæmatin it matters but little whether in its empirical formula there are a few atoms more or less of any one element. In certain other matters, for instance, in the composition of hæmin, their views demand confirmation; I have, therefore, retained the description of this substance as usually given, and here add a résumé of Nencki and Sieber's work :

32

They ascribe to hæmatin the formula C,H,N,FeO,; and the word hæmin is applied, not to the hydrochloride of hæmatin, but to an anhydride of hæmatin, of which the formula is CH,N,FeO. What is usually called hæmin (chlorohæmatin) they call hæmin-hydrochloride, of which the formula is C2H30N,FeO,.HCl. This is a crystalline substance, and in its preparation these observers employ amyl alcohol, and a molecule of amyl alcohol of crystallisation is combined with the crystals, the full formula for which would therefore be CH,N,FeO.HCl.C,H12O. This molecule of amyl alcohol can be driven off by heating the crystals to 130° C.

30

Hæmatoporphyrin may be obtained by the action of concentrated sulphuric acid on either hæmatin (C„H„N‚FeO) or hæmin (С„H„N ̧FeO). Its formula is CHNO, and its formation may be represented by this equation:—

CHN,FeO, + H2SO,

[hæmatin] [sulphuric acid]

=

CHNO, + FeSO, + H2O [hæmatoporphyrin]

They subsequently found that when hæmin or hæmatin is heated with a saturated solution of hydric bromide in glacial acetic acid it is readily converted into hæmatoporphyrin, which shows differences from that obtained in the usual way. It has the formula C,,H,,N,O,. It is insoluble in water and dilute acetic acid, slightly soluble in ether, amyl alcohol, and chloroform, and readily soluble in alcohol, dilute mineral acids, and solutions of the alkalis. It is reddish-brown in colour, is amorphous, and turns brown and becomes more insoluble at 100° C. Its alkaline solution shows the four bands previously described. The hydrochloride (CHIN2O,.HCl) crystallises in tufts of needles, the sodium salt (CH,,NaNO3 + H2O) in microscopic prisms. This substance is considered to be pure hæmatoporphyrin, and the substance prepared by the action of sulphuric acid on hæmatin is probably its anhydride (CH„NO, = 2C,H12O,- 2H2O). Hæmatoporphyrin has the same empirical formula as bilirubin, which it resembles in many of its properties. The formation of bilirubin from hæmatin is represented by Nencki and Sieber by the equation:

17

16

16 18

When introduced into the living subject hæmatoporphyrin is partly expelled in the urine, but the greater portion is retained, and is probably utilised in the formation of hæmoglobin.

Taking a general survey of the subject, we are able, in spite of contradictory assertions on minor points, to draw a few conclu

1 Hoppe-Seyler has discussed the experiments of Nencki and Sieber, and reaffirmed his own views in the Berichte d. deutsch. chem. Gesellsch. xviii. 601.

Figured in Archiv f. exp. Path. u. Pharmakol. xxiv. plate iv.

sions. There seems to be a group of iron-free derivatives of hæmatin, and not a single one. Some of these can be produced artificially, such as hæmatoporphyrin with its anhydride, and hæmatolin; certain others occur in the organism of certain lower animals as such (hæmatoporphyrin), and certain others are formed during the normal or abnormal disintegration of hæmoglobin that occurs in the course of the manufacture of bile and urine pigments (bilirubin, urobilin and uro hæmatoporphyrin); and lastly one is formed in the disintegration of the blood pigment, that occurs in an old blood clot (hæmatoidin). In spite, however, of differences between these different forms of ironfree hæmatin (in solubilities, optical characters, &c.), one cannot help being more struck with the resemblances between them. It therefore appears possible that we may eventually find we are dealing with a number of isomeric or polymeric substances, for in three of them already (bilirubin, hæmatoporphyrin, and hæmatoidin) the same empirical formula has been described.

TESTS FOR BLOOD

In medico-legal cases it is often necessary to ascertain whether or not a red fluid or stain upon clothing is or is not blood.

The tests to be applied are microscopic, chemical, and spectroscopic. Microscopic tests.-The corpuscles of the blood should be sought for. If the blood is fairly fresh it is possible to distinguish the human red corpuscles from the red corpuscles of those animals in which they are nucleated, or differ from them greatly in size and shape. Exceedingly careful measurements have shown that there are small but very small variations in the diameter of the human red corpuscles and those of the common mammals, but practically it is not possible to discriminate between them.

Chemical tests. The old test with tincture of guaiacum and hydrogen peroxide, the blood causing the red tincture to become green, is very untrustworthy, as it is also given by many other organic substances, such as potatoes, certain forms of filtering paper, &c. &c. The only trustworthy chemical test is the formation of hæmin crystals; if one only has a piece of stained clothing to deal with, this is boiled with glacial acetic acid, and a small crystal of sodium chloride on a slide; on cooling the crystals form as already described.

Spectroscopic tests. If the blood is present in any quantity, the typical bands of oxyhemoglobin can be readily seen through the spectroscope; these give place to the single band of hæmoglobin on the addition of a reducing agent. One must be prepared, however, in the case of old stains for the presence of methæmoglobin, or of hæmatin.