THE RETENTION OF IRON I N THE ORGANS IN HBMOLYTIC ANBMIA.'

By ROBERT MUIR, M.D., F.R.S., and JOHN SHAW DUNN, M.A., M.D. From the Pdhlogical hborcllories of the Ufaiwraity and W e d m Injrmary, (flaagow.

(PLATE XXXIV.)

THE present rebearch arose out of certain observations on the change in the organs in hzemolytic anaemia (Muir and M"ee, 1910 l). It wae found that an enormous destruction of red corpuscles might occur in a short period of time, whilst the amount of haemoglobin passed in the urine might be comparatively small. The question then came to be as to the disposal of the haemoglobin and its derivatives. In all cases of the anzemia experimentally produced, there was a well-marked haemosiderin reaction in the liver, spleen, and kidneys, but it could not be said to what extent this represented the haemoglobin that had been lost from the blood. We accordingly determined to investigate the matter by making quantitative estimations of the iron in the organs of the anaemic animals as compared with normal animals. The results not only yield information as to the disposal of the iron, but also, owing to the comparatively short course of the anaemia, throw a strik- ing light on the rapidity with which the iron can be stored up in the organs.

The method of carrying out the experiments is similar to that in the previous series. Rabbits were rapidly rendered anaemic by the intravenous injection of an anti-serum obtained from the goat, which had been treated with injections of rabbit's corpuscles. The activity of the serum had fallen considerably, the haemolytic dose in vitro for 1 C.C. of a 5 per cent. suspension of the red corpuscles being now 0.04 C.C. instead of 0.01 0.C. A larger amount of the serum had thus to be injected. The progress of the anzmia was observed by examination of the blood from day to day, and when the anaemia was sufficiently severe, the animal was killed by bleeding under an anaesthetic, and the circulation was washed out by a solution of 1.5 per cent. sodium citrate and 0.5 per cent. sodium chloride. The blood volume was ascertained

[Received November 10, 1914.1 Towards the expenses of this research a grant WM

received from the Carnegie Trustees, for which we have pleasure in recording our indebted- D W .

2841. or PATE.-VOL. xu.

418 ROBERT MUIR AND JOHN SHA W BUNN.

at the same time by estimating the amount of hemoglobin in the blood and washings obtained by a colorimetric method. The organs thus rendered blood-free were removed, and dried to constant weight in a hot-air oven at 100" C. The iron waa then estimated by the wet-ash method of Neumann (1902 e), which we found well suited for the purpose. The iron derived from the haemoglobin lost from the blood may manifestly be disposed of in two ways. It may be retained in the organs, or it may be excreted in the urine and feces. Any excess in the urine can be readily detected, as the daily amount is relatively constant and does not exceed about 0.5 mgrm. per day. With regard to the fsces, however, we found that it was not practicable to obtain definite results. It is difficult to find how much iron is ingested, as the proportion in green vegetables varies considerably in different samples. We also found that the amount of feces, and also the percentage of iron in them, varied greatly from day to day. After some preliminary trials we accordingly abandoned the estimation of the iron passed through the intestine.

OF ANIMAL.

IRON IN NORMAL ANIMALS. In the first place, estimations were made of the iron in the organs

of normal rabbits, and the results obtained by one of u8 (J. S. D.) in the case of the liver, kidneys, and spleen, are given in Table I. In every case the organs ' were previously rendered blood-free by perfusion with salt solution.

- Liver.

c;

z !? 2

B

I.

11.

111.

IV.

V.

A v q __

Kidneys.

0.32

I_--

TABLE I. I I

Spleen.

0'26

~

Total. ~

5'78

Liver. __._

0'06 1680

1830

1590

1380

1260 --- 1548

5.20

4.29

3.43

2.70

2.10

3'54

0.65

0.41

0.36

0.35

0-49

I 0.25

0'20

0.13

0.14

0'19 ~~

3'20

2'60

4-15 - --

0.038

0.025

0'038

I 5'19 I 0'042

4'04 1 0-025

Kidneys. Spleen. I I

0.018 I 0'236

0'040 1 0'156l I

0'027l I 0.156

0.027 I 0'156

0.027 I 0.156

0.027 I 0'176 -__

1 Owing to t h e smdl amounta of iron to be estimated, the dried kidneys of animals 111.-V., and the dried spleens of animals 11.-V., were "pooled" and the total amounts of iron estimated. The figures in thew cases are thus averages.

-.__

It is interesting to compare theee results with those of Boycott and Price Jones (1913 s), obtained, also in the case of rabbits, by a

RETENTION OF IRON IN ORGANS IN A N A M I A . 419

colorimetric method. They found that, per 100 grms. of dried organ, the number of mgrms. of iron was, in the liver, 51 ; in the kidneys, 1 2 ; and in the spleen, 253-as compared with our results, namely, in the liver, 38 ; in the kidneys, 27 ; and in the spleen, 176. We can also take the average weight of the animals from the tables and calculate the average amount of iron in the organ per kilo of body weight. The results are, mgrms. of iron per kilo in Boycott and Price Jones' series-Liver, 3.3 : kidneys, 0.1 6 ; spleen, 0.1 3. In our series -Liver, 2.3 ; kidneys, 0.28 ; spleen, 0.12.

Our figures are higher than theirs in the case of the kidneys, whilst theirs are higher than ours in the caae of the liver.

We have also estimated the total amount of iron in the stomach and intestinks taken together, of three other rabbits of about 1500 grms. in weight ; the results are 3.49, 2.7, and 3.3 mgrms., the average being 3.16.

We may accordingly take it that the blood-free abdominal organs of a rabbit of about 1500 grms. contain about 7 mgrms. of iron.

In calculating the amount of iron lost it is essential to know the amount normally present per C.C. of blood. This of course depends on the percentage of haemoglobin, which varies considerably in rabbits. We have found, as an average of three estimations, that 1 C.C. of rabbit's blood, with a percentage of 85 of haemoglobin (on the human standard), contains 0.45 mgrm. of iron. As the fall of hzemoglobin has been observed throughout the experiments, and as the blood volume is known (in all cases except one it was found directly by washing out the circulation), the total amount of iron lost from the blood can be readily calculated. From the figures given above, with regard to the iron content of normal organs, the excess in the organa after haemolysis is easily estimated, and this can be compared with the .amount lost from the blood. We thus obtain knowledge aa to the proportion of iron which is stored in the organs and also as to the rapidity with which this can take place.

These sets of results are in pretty close agreement.

EXPERIMENTS.

Eayeriment I.-On 25th September the animal passed 200 C.C. of twine, .in which no haemoglobin could be detected, and on the 26th 110 cc., in which a doubtful trace of haemoglobin was found on spectroscopic examination. The animal w a ~ killed under an anathetic, and the circulation was washed out on 26th September. The blood volume obtained was 79.7 C.C. The liver, spleen, and kidneys gave a well-marked iron reaction, which, however, WBB deepest in the cortex of the kidneys.

Microscopic examination of the liver showed that there was a consider8ble .amount of hmmosiderin and remains of red corpueclee in the endothelial cells of the capillanea 'The hsmosiderin was in the form of fragments or granules of varying size and giving also a varying depth of ataining. When cold hydro- dhloric acid was used with potassium ferrocyanide, there was diffuse blue stain- .ing in the liver cells around the portal tract ; but when the acid was used at a

420

Date.

September 23 . . . . . . ,, 2 4 . . . . . .

ROBERT MUZR AND J O H . SHA W DUNhrr.

Red Corpuscles. Hemoglobin.

5,480,000 72' * 5,000,000 652

temperature. of 55" C., there were also seen fine blue granules of hsmosiderin within the cells (Plate XXXIV. Fig. 1). As is usually the case the iron within the liver cells faded off towards the centre of the lobule. The spleen showed a considerable but not excessive amount of hremosiderin within the phagocytes in the pulp and sinuses. Within these cells, just as in the case of the endothelial cells of the liver, red corpuscles in all stages of disintegration were present. The kidneys gave a diffuse blue reaction in the cells of the convoluted tubules, but this was comparatively slight. No granular hsmosiderin was found. In the intestines no hmmosiderin of any variety was detectable on microscopic examination.

Estimations of the amount of iron in the organs gave the following results, namely-Liver, 14.29 mgrms. ; kidneys, 1-54 mgrm. ; and spleen, 1-76 mgrm. The total amount in these three organs equalled 17.59 mgrms., and if we sub- tract the estimated amount for normal organs, namely, 5.6 mgrms., we get practically 12 mgrms. in excess over the normal. In this case the amount of iron in the stomach and intestines was not estimated. In the total amount of urine passed the amount of iron did not exceed 1 mgrm., even although there was a suspicion of hmmoglobin in the last sample. At no timp, however, was there distinct haemoglobinuria.

The Malpighian bodies were enlarged and free from pigment.

Elxperiment I.

. . . . 4,230,000 .. 2 6 . 2,840,000

,, 2 5 . 553

35

Sept. 23.--0.5 C.C. of I.S. (immune-serum) injected intravenously at 4 pm. * ,, ,,

24.-1 C.C. of 1.8. injected at 12 noon, and 1 C.C. of I.S. at 3 p.m. 25.-1 C.C. of I.S. injected at 12 noon.

I I

The estimated amount of iron lost from the blood amounted to 15-2 mgrms., and this exceeds the excess found in the three organs mentioned by 3.2 mgrms. It is thus seen that the iron lost, with the exception of a small proportion, has been stored up in the liver, kidneys, and spleen.

Experiment II.-The animal was killed under an anzesthetic on 29th September. The circulation waa washed out and the blood volume was found to be 100 C.C. Well-marked hmmoglobinsemia was present. The bladder con- tained 20 C.C. of urine, in which there was a considerable amount of h a m e globin. I n this w e there waa an extreme ansmia which, towards the close of the experiment, was of a rapidly advancing character.

* In every awe the injaction of Berum was given after the astimatiom of red corpuscles and hemoglobin were made. .

RETENTION OF ZRON IN ORGANS IN ANAMZA.

Experiment 11.

421

I RaBexT.-weight, 2140 grms.

Red Corpuscles. -_

5,820,000

5,380,000

4,200,000

850,000

Date.

September26 . . . . . . ,, 2 7 . . . . . . ,, 2 8 . . . . . . ,, 2 9 . . . . . .

_.

Hsemoglobin.

85'

759

6OS

10

Sept. 2 6 . 4 ' 8 C.C. of I.S. injected intravenously. ,, ,,

27.-0'9 C.C. of 1.8. injected at 10 a.m., and 1.1 C.C. of I.S. at 1 p.m. 28.-1'1 C.C. of I.S. injected at 12 noon, and 1-2 C.C. of 1.9. at 8'30p.m.

The urine passed was- Sept. 27.--50 C.C. ; no hsmoglobin. ,, ,,

28.-110 C.C. ; no hsmoglobin. 29.--13 c.c., which had a distinctly reddish colour owiiig to the presence of

hsmoglobin.

On applying the naked-eye test a well-marked iron reaction was given as before by the liver, kidneys, and spleen, the coIour being most intense in the cortex of the kidneys. Microscopic examination showed that when hot hydrochloric acid was used the cells in the periphery of the liver lobules gave a diffuse blue reaction, and also cou tained small granules of hilemosiderin, situated especially in the outer part of the cells. There was also much iron, the result of phagocytosis in the capillary endothelium. There was marked bluing in the convoluted tubules of the kidneys, and in Henle's tubules, but none in the Malpighian bodies. In the splenic pulp there was abundant hamosiderin within phagocytes, and there were also numerous red corpuscles in all stages of disintegration. In the intestines, especially in the large intestine, small groups of hEmo- siderin granules were found within the epithelial cells. These granules were very distinct, but, on the whole, not very numerous.

Analysis gave the following amobnts of iron:-In the liver, 34.58 mgrms.; in the kidneys, 346 mgrms. ; in the spleen, 6.02 mgrms. ; and in the stomach and intestines, 18.8 mgrms. The total quantity of urine contained 1.97 mgrm. The total amount recovered from the abdominal organs and the urine is thue 64-83 mgrms., and if we deduct 9-8 as the normal quantity, we get an excess over the normal of 55 mgrms. The calculated amount of iron lost from the blood was 33.75 mgrms., so that the amount found in excess exceeds the amount lost by 21.2 mgrma. This is largely accounted for by the high iron content of the intestines, which ia several times in excess of that found in any other case. The spleen also contained an abnormally large quantity of iron. It is quite likely, in view of

422 ROBERT MUIR AND JOHN SHA W DUNN

I Date. -

October20 . . . . . . ,) 21 . . . . . .

our other estimations, that both the spleen and intestines of this animal had previous to the experiment an abnormally high content of iron.

Experiment 111. -

I BABBIT-weight, 1540 grrns.

Red Corpuscles. Hremoglobin.

5,250,000 85l

5,000,000 76'

I I - I

Oct. 20.-0'8 C.C. of 1.5. injected at 1 p i . , and 0'5 C.C. of 1.5. at 3 p.m. * ,, 21.--1'25 C.C. of I.S. injected at 12 noon, and 1 c.c. at 5 p.m.

,, 22.- 2 C.C. of 1.5. injected at 12 noon. '

The amounts of urine passed were- Oct. 21.-65 C.C. ; no hemoglobin. ,) 22.4310 c.c., tho latter portion showing a small amount of hzemoglobin. ,, !&-lo7 c.c., containing a considerable amount of hemoglobin, the bands

being visible down to a 1:lO dilution.

. . . 3,780,000

1,880,000

, , 2 3 . . 553

25

The animal was killed under ether on 23rd October, and the blood volume was found to be 74 C.C. The liver, kidneys, and spleen gave an iron reaction aa in the previous experiments, and there was also a fairly distinct iron reaction in the large intestine, especially in the cscum. Microscopic examination showed almost identical appearances to those previously recorded, there being, with hot hydrochloric acid in the liver cells, distinct bluing, and also collections of hmmosiderin granules, whilst the capillary endothelium throughout the lobules contained hsmosiderin and breaking down corpuscles. The changes in the spleen and kidneys were similar to those already recorded.

The results of estimations of iron are, in the liver, 23.63 mgrms. ; in the kidneys, 3.06 mgrms. ; in the spleen, 1.57 mgrm. ; and in the stomach and intestines, 3.71 mgrms. The totalamount in the urine passed was 2-61 mgrms. The total amount of iron thus obtained reached 34-58 mgrms., and deducting 8.5 as the estimated normal amount, we get 26.08 aa the amount of iron in excess in the organs and passed in the urine. The calculated amount lost from the blood was 26-47 mgrms. This is slightly less than the amount found in excess, but the two figures correspond very closely.

Ezperinient 1V.-The animal died in the evening of 5th November, an8 the circulation was not washed out. The organs were, however, minced and washed free of blood, as far as possible, in water. The liver, kidneys, and spIeen gave a well-marked iron reaction as usual. The liver was considerably en- larged, its weight heing 126.5 grms., and it showed a distinct cirrhosis. The kidneys were much swollen and had a distinct brown colour. The cscum gave a dietinct iron reaction, whilst the rest of the intestines and the stomach gave a very slight reaction. Microscopic examination gave corresponding results to those already recorded, but the amount in the convoluted tubules of the kidneys did not seem specially great, whilst there was much haemorrhage with hsmoglobin casts in all the tubules.

Distinct hsmoglobinsmia was present.

RETENTION OF IRON I N ORGANS I . ANAMXA. 423

Red Corpuscles.

6,230,000

5,710,000

1,930,000

1,630,000

Experiment IV.

BABBIT-Weight, 2324 grms.

Hemoglobin.

90'

852

35

25

Date.

November3 . . . . . . : , 4 . . . . . .

9.30r.m. . . . . 5p.m. . . . .

NOT. 3.-1 O.C. of I.S. injected a t 5.30 p.m. ,,

NOV. 4.-150 C.C. ; no hemo lobin.

4.-1'8 C.C. of 1.8. injected at 11 a.m., and 1'5 C.C. injected a t 5 p.m. The amounts of urine passed were-

,, 5.-I30 c.c., in which t%ere wm marked haemoglobin.

The amounts of iron found were as follows :- I n the liver, 36.45 mgrms. ; in the kidneys, 8-49 mgrms. ; in the spleen,

1.3 mgrm.; in the stomach and intestines, 4-51 mgrms.; and in the urine, 5.29 mgrms. The total amounts to 56.03 mgrms., and if we deduct 12 mgrms. as the normal, the excess obtained is 44.03 mgrm. The estimated amount lost is 38.7 mgrms., so that the amount found in excess is somewhat greater. It is, however, to be noted that the last estimation w a ~ some time before the death of the animal, and that accordingly the iron lost is calculated from this last estimation. The actual amount lost would probably be somewhat in excess of this. There is also the possibility that there may have been some pre-existent excess of iron in the cirrhotic liver.

Experiment V.

I RABBIT-weight, 1904 grms.

I ____ I Red Corpuscles. !

Date.

I November 25 . . . . . . I 6,580,000 .. 2 6 . 1 5,200,000 .. . . . . 3,540,800 2 7 .

990,000 (?) .. 2 8 . . . . .

Hremoglobin.

90'

802

7OS

55

__

1 Nov. 25, 5.30 p.m.-1 C.C. of 1.S. injected intravenously.

* ,, 26, 11.30 a.m.-1 C.C. of I.S. injected. ,, 27, 11 a.m.-1 C.C. of I.S. injected, and a t 5 p.m. 0.75 C.C. of I.S. injected.

The amount of urine passed wns- NOV. 26.-60 C.C. ,, 27.-105 C.C.

28.-200 C.C. The)iast had a dark brownish-green colour, but gave no bands of hemoglobin on

spectroscopic examination.

424 ROBERT MUIR AND JOHN SUA W DUNN.

The animal was killed on 28th November, and the circulation washed out as usual. The blood volume was 80 c.c., and very slight haemoglobinzemia was present. There was a marked iron reaction in the cortex of the kidney and in the liver ; that in the spleen being less marked than in the other experiments. There was a fairly distinct reaction in the ctecum and a very faint reaction in the stomach and small intestine. On microscopic examination there was a considerable amount of iron in the capillary endothelium throughout the lobules of the liver; and there were small heemosiderin granules in the liver cells at the periphery, and also the usual diffuse staining in that situation. A diffuse iron reaction was distinct in the kidneys in the usual eites. and there were also a few minute granules of hzemosiderin, most marked in the outer part of the cells. A few small granules of hzemosiderin were present in the epithelium of the cecum, but none in the stomach and small intestine.

Analysis of the organs gave the following anounts of iron, namely:- I n the liver, 16.17 mgrms.; in the kidneys, 1.93 mgrm.; in the spleen, 1.81 mgrm. ; in the stomach and intestines, 2.83 mgrms. ; and in the urine, 1-41 mgrm. The total amount is thus found to be 24-15 mgrms. and the calculated excess over the normal about 15 mgrms. The total amount of iron lost from the blood was found by calculation to be about 25.42 mgrms. There is thus an amount of about 10 mgrms. unaccounted for either in the organs or in the urine.

The spleen showed the usual changes.

DISCUSSION OF RESULTS.

The five experiments detailed clearly demonstrate the great rapidity with which iron is stored up in the organs. They are closely comparable as regards duration, namely, a period of three days, and in all of them more than half of the blood had been destroyed; in fact, we possess in such a haemolytic serum an agent which leads to very rapid hmnolysis, with remarkably little toxic action on the other tissues of the body. In some experiments more than two-thirds of the blood had been destroyed, and as there is relatively little destruction on the first day, and most on the third day, it follows that the abdominal organs can store up about one-third of the whole iron of the blood in twenty-four hours. The great increase of the iron content of the organs is shown in Table IL, which may be compared with that on page 418.

Boycott and Price Jones found that in trypanosome anaemia the iron of the destroyed hamoglobin is deposited in the liver and spleen, but in their experiments the ansmia was less acute and extended over a longer period of time.

It is thus seen that in the ansemic animals the average percentage of iron in the liver and kidneye is about five times the normal, and in the spleen about three times the normal. The amount of iron in the stomach and intestines was as follows :-Experinlent I., not estimated ; Experiment II., 18.8 mgrms. ; Experiment III., 3.7 1 mgrms. ; Experiment IV., 4.51 mgrms. ; and Experiment V., 2.83 mgrms. Thus in only one experiment was any marked inorease over the normal observed.

RETENTION OF IRON IN ORGANS IN ANEMIA. 425

1'54

3'46

3-06

8'48

1'9s

TABLE 11.

1.76

8-02

1-57

1-30

1-81

$ I OF 2 j ANIMAL.

I 0'114

0'266

0'253

0'143

0'122

I. I 2100

11. 1 2140

111. 1 1540

IV. j 2324

I 0.077 I 0'343

0.158 I 0'602

0'198 0.680

0'168 I 0.650 I

0.078 , 0.340 1

Liver.

Ironlost from blood inmgrms.. . .

Iron in exceas in organsandurine .

Kidneys. Spleen. I 1

15'2 33.75 25'47

12.0 55'0 26.08

14.29

34-56

23-83

36-45

16-17

38.7

44'03

25.42

15.0

Urine.

? trace

1'97

2.61

5'29

1.41

Average1 2001 ! 25'02 1 3'69 1 2.49 1 2.25

PERCENTAGE OF IRON IN RELATION TO DRY WEIGHT.

Liver. Kidneys. Spleen. l i

The amount of iron in the urine is seen to be relatively small, the 'largest amount being in Experiment IV., but even there it is only about .one-seventh of the iron in excem in the organs. The increase in the urine seems to depend almost entirely upon the hemoglobinuria, and *our experience is that unless hamoglobin is present in the urine, .practically no increase of iron can be demonstrated by the method used. One interesting point is that the amount of iron in the urine i6 roughly proportional to the amount in the kidneys, as will be seen tfrom the table. In other words, the excess of iron in the kidney would appear to depend mainly upon the amount of hsmoglobin free in the blood.

If we compare the calculated amounts of iron lost from the blood .with the surplus over the normal found in the various organs, we find certain variations in the different experiments. I n two the amount found in excess in the organs was greater than that lost; in :two it was approximately equal, whilst in only one experiment was it distinctly less. This is brought out in Table 111.

TABLE 111.

Experiment Experiment 1v: 1 v. j

When the amount of iron in excess in the organs exceeds the .amount lost from the blood, the discrepancy must of course be due to

426 ROBERT MUIR AND JOHN SB.4 W DUNN;

a preceding excess of iron in the organs over the normal. In one of the two cms the excess was chiefly present in the stomach and intestines, the amount present being 18.5 mgrms., which is several times that observed in any other experiment ; whilst in the other the liver was enlarged and cirrhotic, and might have previously contained an excess of iron, In only one case was the amount found in the organ distinctly less than the amount lost, namely, in Experiment V. This may have been due to retention in other tissues, or, what is more likely, to excretion by the bowel. Variations in the amount. excreted by the bowel probably occur, but the results of analysis taken as a whole show that as a rnle only a small amount of iron is lost in this way.

CHARACTERS OF HAZMOSIDERIN.

When the deposits of iron are examined by the microscope, some interesting facts are obtained. In a previous paper the importance of the distinction between intracellular and extracellular was insisted on, and this is borne out in the present research. Phagocytosis and destruction of red corpuscles is the outstanding feature in the spleen, though some of the iron is probably derived from free hzinoglobin in the plasma. In the kidneys, on the other hand, there is no evidence of phagocytosis, and the iron in the cells of the convoluted tubules i s manifestly derived from free haemoglobin in the circulating blood. This is taken up by the cells of the kidneys and broken up by them, but if the amount exceeds a certain degree, the hsmoglobin is in part excreted as such, and haemoglobinuria is the result. It is to be noted that the cells of the kidneys give a diffuse Prussian blue reaction, and there are few granules of haemosiderin (Plate XXXIV. Fig. 2). In the experiments haemoglobinamia could usually be directly demonstrated, but even when the amount of free hcmoglobin is too small to be shown by ordinary methods, it may, if sufficiently long continued, lead to deposits of haemosiderin in the kidneys. W e

. may, in fact, recognise three degrees of haemoglobinaiiiia, all of which lead to accumulations of iron in the kidneys :-(a) A degree too. slight to be detected directly (this is probably the case in perniciou? ansmia); (6) a degree sufficient to enable the free haemoglobin to be recognised in the plasma ; and (c) a degree sufficient to produce also hzmoglobinuria. Corresponding to these three degrees of hzmo- globinaemia there will be increasing amounts of hsmosiderin in the kidneys.

In the case of the liver there are deposits of iron, resulting from both types of blood destruction. Within the liver cells we find a deposit which appears first iu the cells in the peripheral zoue of the lobule and which gradually increases in amount and spreads inwards. This, a t first, gives a diffuse blue reaction like that in the kidneys, but.

RETENTION OF IRON IN ORGANS IN ANAMIA. 437

a little later minute granules of bmosiderin appear, these also occurring first a t the periphery. We consider that this form of deposit results from the absorption of free bmoglobin by the liver cells, that is, depends dn hamoglobinaemia. There is also, however, a second form of deposit, namely, that within the endothelial cells of the capillaries. In this form, however, the process of phagocytosis of red corpuscles can be readily followed, these undergoing disintegration and the fragments coming to give the iron reaction. From these cells part of the haemosiderin may be passed on to the liver cells, but this cannot be considered the main source of the hamosiderin in the latter. It is further to be noted that the distribution of the hamo- siderin is different in the two situations, that in the endothelial cells being not specially related to the periphery, but occurring all through the lobules. The chief points in the distribution of the hamo- siderin in the two situations are illustrated in Plate XXXIV. Fig. 1. One further point may be referred to. The amount of “phagocytic” iron in the capillary endothelium of the liver is greater in the present series of experiments than in the previous. We have mentioned that the haemolytic value of the serum had fallen considerably, and if there were no corresponding fall in the opsonic property, this might explain the difference. We have not the means, however, of testing this hypothesis.

CONCLUSIONS.

1. In acute haemolytic auamia, attended with destruction of more than half of the blood within three days, nearly all the iron from the destroyed haemoglobin is deposited in the liver, spleen, and kidneys ; a certain amount escapes in the urine when there is haemo- globinuria, and the amount deposited in the kidneys is roughly proportional to this.

2. A third of the total iron of the blood may be deposited in the organs mentioned within twenty-€our hours.

3. With regard to the deposits of hsmosiderin, a broad distinction can be made between that resulting from lysis and that resulting from phagocytosis of red corpuscles.

REFERENCES.

1. Mum AND MWEE . . . Joz~rm. Path. and Baeteriol., Cambridge,

2, NEUMANN . . . . . . Ztschr. f. PhysiOl. Chem., Strassburg, 1902, 1910, vol. xvi p. 410.

Bd. XxxviL S. 115. 3. BOYCOTT AND PRICE JONES. Jam. Path. and Bacterial., Cambridge, 1913,

vol. xvii. p. 347.

428 RETENTION OF IRON IN ORGANS IN AN&MIA.

DESCRIPTION OF PLATE XXXIV.

FIG. 1.-Section of liver, Experiment II., showing haemosiderin within the capillary endothelium throughout the lobule, and also hvernosiderin both in diffuse aud granular state in the liver cells at the periphery.

PIG. 2.-Section of kidney, showing the diffuse hacmosiderin reaction in the cells of the convoluted lobules.

Carmalum ; hydrochloric acid (hot) and ferrocyanide of potassium.

JOURNAL OF PATHOLOGY.-VOL, XIX. PLATE XXXIV.

FIQ. 1.

FIQ. 2.