THE ABSORPTION OF IRON PROM THE ORGANS AFTER HBM0LYSIS.l

By ROBERT MUIR, M.D., F.B.S., and JOHN SHAW DUNN, MA., M.D.

Fyom thc Pnthologicnl Laboratories of Glnsgow Uieiucrsity and Glasgow Western I@rmary.

IN a previous paper (1 9 1 5 I) we gave the resrdts of the estiniation of the amount of iron in the o r p i s of rabbits after rapid destruction of the blood by means of a hamolytic serum. These results showed that nearly all the iron derived from a destruction of more than half of the total blood within three days, was present in the liver, spleen, and kidneys, the process of storing up being thus a remarkably rapid one. Such being the case a t the height of hmnolytic anizmia, it appeared a que tion of interest to determine to what extent the storediron is used up in the regeneration of blood, which Muir and McNee (1 9 1 2 2, have shown to occur very rapidly after this form of anzemia. The plan of the experiments is a simple one. It is merely to produce an anzmia corresponding in degree with that in the previous experiments, to allow the condition of the blood to return practically to normal, aud then to estimate the amount of iron in the organs after these have been washed free of blood by perfusion with salt solution. The methods used are the same as those in the previous series, the only difference being that the iniinune body was obtained from the cat instead of the goat. The following is an account of the individual experiments.

Experiment I. As is shown in Table I., a rapid anzmia resulted after two injections of

serum, the percentage of hiemoglobin falling to a third of the original on the fifth day. Thereafter fairly rapid regeneration occurred, the percentage of hiemoglobin reaching 75 on the thirteenth day. The aninial was killed on the seventeenth day of the experiment, arid the circulation was washed out with salt solution. The following results were obtained on applying the naked-eye test for iron with ferrocyanide of potassium and hydrochloric acid. The most marked reactioii was found i n the cortex of the kidneys, the colour developing somewhat slowly, but ultimately becoming a distinct greenish-blue. The liver

[ReceiTed for publication July 10, 1915.1 Towards the expenses of this research a grant was obtained from the Carnegie Trustees for which we have pleasure in recording our thanks.

43 ROBERT MUIR AND JOHN SHA W DUNN.

gave a very slight reaction, a faint greenish colonr only resulting; whereas the spleen, stomach, and intestines gave almost no reaction. Microscopic exaiiiination showed that the liver cells gave no iron reaction, but this was still present, though not i n great amount, i n the endothelial cells of the capil- laries throughout the lobules. There was a distinct though not very marked

TABLE I.

iron reaction in the cells of the convoluted tubules of the kidneys, this being of a diftilse character and no granules being present. A certain amount of granular hamosiderin was present in the splenic pulp, chiefly within the phagpcytes.

kstiniation of the iron gave the following results, which are put in tabular form, the percentage given being in the relation to the dry weight of t h e organ :

The stoiiiach and intestines gave no iron reaction.

Liver 3 7 6 5 mgrms. = 0.034 per cent. Spleen 0.404 ,, =0.546 ,,

Stoiiitich . 0.404 ,, =0*014 ,,

Large intestine . 1.00 ,, =0'016 ,,

Kidneys . 1.87 ), =0.013 ,)

Small intestine . 0.64 ,, =O*OOY ,,

The total in these organs equals 8.079 mgrms., that is, only 1.5 mgrm. above the normal ; and it IS to be noted that the excess is almost accounted for by the excess in the kidneys. (According to our previous estimations the abdominal organs of a normal animal of 1500 grms. contain about 7 mgrms. of iron.)

The total amount of iron in the ha?moglobinuric urine was 3.57 m, nrms., that is, an amount corresponding to ratlier more than 8 C.C. of blood. More than 40 C.C. of blood had been lost by the blood destruction.

ABSORPTION OF IRON AFTER HAMOLYSIS. 43

Experiment 11.

This animal was less susceptible to the action of the serum, and several injections were given. The hsmoglobin fell on the sixth day to less than a third of the original amount (Table II.), and on the fourteenth day it had almost regained its former level. The animal was killed on the fifteenth day of the experiment and the organs were washed out with salt solution as before.

TABLE 11.

The iron reaction in the liver was very slight ; in the cortex of the kidneys it was marked, though not very deep, whilst in the spleen it was slight. The stomach and intestines gave no iron reaction. Microscopic examination gave, as regards the liver and kidneys, the same result as in the previous experiment. There was a fair amount of hwmosiderin in the spleen in the form of aggregates of coarse granules. Estimation of the amounts gave the following results :

Liver Spleen . 0.35 ,, =0,203 ,, Kidneys . 2.2 ,, =0'118 ,, Stomach . 0.389 ,, =0.014 ,, Small intestine . 0'808 ,, =0.017 ,, Large intestine . 1.29 ,, =0.021 ,,

6.2 mgrms. = 0.066 per cent.

The total amount of iron in the organs is thus 11.23 mgrms., that is, about 3.2 mgrnis. above the normal, this being the largest excess which was inet with in any of the experiments. This circumstance was due to a slight excess in the liver and to a relatively marked excess in the kidneys. The haemoglobin- uric urine which was passed on two days contained 1.14 mgrm. of iron.

44 ROBERT MUIR AND J O H N SHA W D UNN.

Experiment 111. The greatest amount of anzniia was on the fourth day of the experiment,

the percentage of hwmoglobin being then 35 instead of 75 as a t the beginning of the experinient (Table 111.). The aniinal ~ v m killed on the sixteenth day, micl a t this time both the percentage of lizenioglobin and the number of red corpuscles were higher t h a n a t the outset. In this experiment there was no liiemoglohinuria. The renal cortex a n d the spleen gave a di,itiiict reaction, whilst the stomach and intestines gave no reaction. Microscopic euamin,ttion showed that a very few of the

The liver gave a slight iron reaction.

TABLE 111.

! ;; 19 . ' ) ) 21 . ,) 22 . 1 ,) 26 .

' I

. !

5,000,000 4,900,000 2,800,000 1,700,000 1,550.000 1,900,000 2,610,000 3,400,000 3,620,000 4,400,000 6,300,000

7 5 1

70 60 35 55 40 42 G O 70 7 5 55

1 Aiay 11 . , . . 1.9 C.C. I.S. injected intrsvenously. '' ), 12, 11 a.m. . . 2'4 ,) ,

) ) ,, 4 p . m . . . 2 4 ,, ,> 2 ,, 13 . . . . 2.0 ) )

cntlothelial cells of the capillaries througliout the lobules of the liver g,zve s difhise bluish-green colonr. I n the spleen hainosiderin was scanty on the whole, and occurred chiefly in the form of sinall scattered granules. In the kidneys a faint difliise reaction was present i n some of the cells of t.he convol- uted tubules. KO iron conld be detected in the storrinch and intestines.

T h e estiniation of iron gave the following results :

Liver Spleen 0.34 ,, =0.158 ,, Kidneys . 0'86 ,, =0'051 ,,

2.6 nigrnis. = 0.017 per cent.

Stornnch . 0.47 ,) =0.012 ,) Sniall intestine . 0.73 ,, =@a1 ,, Large intestine . 0.SS ,, =0.014 ,,

I n this experiment t h e tot~al ttniount of iron in the organs was not above nornial, ncrerthrless there is :L distinct excess in the kidneys, and t h e unioiint in the sideen is also a little above nornial.

Experiment IV.

In this experiment, after four injections of the serum, the hwmoglobin fell on the fifth day to a half of the original ainonnt (Table IV.). The percentage of corpuscles arid of hzmoglobiii had been more than fully restored by the

ABSORPTION OF IRON AFTER H&MOLY.YIS. 45

, , 6 . . . . . . . , , 8 . . . . . . . , , 9 . . . . . . . ,, 11. . . . . . . ,) 1 3 . . . . . . . ), 1 5 . . . . . . .

TABLE ITT. - _ _

EXPERIMENT IV.-RABBIT (Weight, 1320 grms.).

Date. i Red Corpuscles. Ha3moglo'uin. I

~

5,230,000 4,570,000 3,870,000 2,700,000 2,100,000 1,750,000 2,060,000 2,570,000 3,000,000 3,880,000 4,150,000 4,290,000 4,440,000 5,800,000

I 80 ' i

j 55

7 0 2 60 50 40

I

1 2 I 60

70 70 80 I 80

1 85

May 29.-2'4 c.e. I.S. injected intravenously. ,, 30.-1.0 ,, ,, t , ,, 31.-1'0 ,,

*Jnne 1.-1.0 ,, ,,

eighteenth day, when the animal was killed. The liver gave a very slight iron reaction, whilst t h e cortex of the kidney and the spleen gave a marked reaction. There was also a distinct, though slight, reaction in the caecum and the large intestine. Microscopic examination gave the same result as i n the previous experiment, with the exception that the hamosiderin granules in the spleen were more abundant.

The aniounts obtained by estimation of the iron were as follows : Liver Spleen . 0.48 ,) =0'450 ,, Kidneys . 0.71 ), =0'043 ),

Small intestine . 1.37 ,, =0'023 ,,

4.05 mgrms. = 0.066 per cent,

Stomach . 0.44 ,) =0.012 ,, Large intestine . 1.22 ,, =0.022 ,,

In this case the total amount of iron in t h e abdominal organs wsa a little above the normal, 8-27 mgrms. instead of about 6 .5 mgrms. There is a slight excess in the liver, but a greater relative excess in the kidneys and spleen. There is also a slight excess in the intestines.

DISCUSSION OF RESULTS.

In all the experiments the percentage of hzmoglobin was reduced a t least to half t,he original amount, in one case to less than a third ; whilst the number of corpuscles per cubic millimetre fell to a still greater degree. The maximum reduction occurred on the fourth or fifth day after the first injection, and the animal was killed nine t o twelve days later, when regeneration had occurred. In two cases both the percentage of haenloglobin and the number of the red corpuscles

46 ROBERT MUIR AND J O H N SHAW DUNN;

90

75

had risen to a higher level than the original. I n Table V. are given for each experiment the original figures, the minimum recorded and the day of its occurrence after the first injection, and the final condition just before the animal was killed.

Fifth Day. 990,000

Fourth Day. 1,550,000

TABLE V. ~ ~ -~ ~

RED CORPUSCLES. EXPERIWLNT.

-~ - ~-

1 Initial Figures. I. . . . ' 7,140,000

11. . . . 6,250,000

111. . . . 5,000,000

IV. . . 5,230,000 ' I

Fourth Dar. 1,420,000

Fifth Day. 80 ~ 1,750,000

30

28

35

40

RED CORPUSCLES.

~ ~~

Sixteenth Day. 4,400,000

Fourteenth Daj. 5,000,000

Fifteenth Day. 6,200,000

Seventeenth Day 5,800,000

HB.

75

85

85

85

From the results of our former experiments we are justified in concluding that on the fourth or fifth day nearly all the iron resulting from the destruction of red corpuscles was deposited in the liver, spleen, and kidneys. W e have thus to consider in view of these results the amounts found in these organs after regeneration had occurred. W e may summarise by saying that in all four experiments the iron content of the organs was little above the normal amount; that is, the iron deposited as a result of the hzmolysis had been nearly all absorbed during the process of blood regeneration. It appears to us that we are also justified in concluding that it has been utilised for purposes of regeneration, though this cannot be said to be absolutely proved. The iron required for the newly-formed corpuscles must of course have been derived either from the stored iron or from the iron of the food, and i t is to be noted that in the ordinary diet of a rabbit there is sufficient iron to make up the blooa loss, provided it is entirely utilised. The generally accepted view is that, under normal conditions, an absorption of iron takes place in the upper part of the small intestine and a corresponding amount is excreted by the large in- testine ; there is t,hus, as it were, a norinal stream of iron. Accord- ingly, in the experiments during the process of regeneration, there would be iron in the blood absorbed from the organs, and also iron obtained by alimentation : but we have no means of distinguishing the iron from the two sources. I t might theoretically be supposed that the iron in the organs was simply excreted, whilst the iron of the food was utilised to a greater extent than normal, but such an occur- rence is extremely unlikely. It is an established fact that the iron from pathological as well as from normal hzmolyeis is saved by the organism as far as possible. It is also known that regeneration is

ABSORPTION O F IRON AFTER HAMOLYSIS . 47

Liver.

more rapid after hzmolysis, when iron is stored, than after bleeding, when of course the iron is lost. Moreover, the liver cells are the structures chiefly concerned in the saving and storing of the iron from normal haemolysis, and it is also the liver cells which most rapidly give up the stored iron resulting from abnormal destruction of blood. The conclusion thus seems inevitable that the iron resulting from hzemolysis is stored up in a purposive way, as it were, and when it is absorbed from the organs, as occurs rapidly, it is used for purposes of blood regeneration. The collected results of the analyses are given in Table VI.

Stoniach Spleen. Eidneys. 1 and

TABLE VI.

3-76 (0.034) 1 0'404 (0'546)

6'2 (0.066) ' 0'35 (0'203)

2'6 (0.017) 0'34 (0'158)

4.05 (0.066) 0'48 (0'457) -

I.

11.

111.

IV.

1 Intestines.

1'87 (0.12) 2.04 ~

2'2 (0.118) 2'47

0,86(0.051) 2.07

0'71 (0'043) 3.03

WEIGHT

INIMAL. OF

1344

1680

1800

1320

IRON IN ORGANS, IN MILLIGRAMYES.

Petal.

8.07

11'22

5.87

8.27 __

Approxi- mate

Excess.

1.5

3 '2

None

1 .7

Note.-The figures within brackets give the percentage of iron in relation to the dry weight of the organ.

As we have stated in our previous paper, the average amount of iron in the abdominal organs of a rabbit of 1500 grms. weight is about 7 mgrms. The greatest excess occurs in Experiment II., where there is a surplus of 3.2 mgrms. On the other hand, in Experiment 111. the total amount is rather below than above the normal.

We have now t o consider the iron content of the various organs. With regard to the liver, there is a slight excess in Experiments I., II., and IV., this being 0.5, 2.4, and 1 mgrm. respectively. In Table VII. we have given, for comparison, the amounts of iron in the livers of four normal animals, of four animals which had been rendered anmnic (these figures being taken from our former paper), and of the four animals which had practically recovered. We have also calcu- lated the amount in the liver per 1000 grms. of animal weight, and the results show that in the experimental animals after recovery the average amount is not much more than in the normal animals, being 2.7 mgrms. as compared with 2.4 mgrms. Microscopic examination shows that in all the animals which had recovered the hzmosiderin had disappeared from the liver cells, whilst some remained, chiefly in a diffuse form, within the endothelium of the capillary walls, these appearances certainly indicating that the haemosiderin is in the latter

48 ROBERT MUIR AND JOHN SUA W DUNiV.

situation less easily utilised than in the former. The contrast between the condition of the liver cells in the anmnic animals and in the animals that recovered, is thus a very striking feature, and we conclude that the hlemosiderin in the liver cells, whether in the diffuse or in the granular form, is very readily and rapidly utilised in the process of blood formation.

TABLE T'TL ~~ ~ _ _

I

I I R O N I N LIYEII, I.\ &1ILI,IGRAMMhS. I I

.- .-

~ Norins1 Animals. I

I .~ ..

1 5 " 2 ~ (1830)1 '29 ' (1590) 3 '43

! (1380) ' 2 . 7

Average per

I Experimental Animals. I

-

~nr t . i n i c . .~ -

(1904) 16'17

(2100) 14'29

(1540) 23'63

(2140) 34'58

11.5

~

I After Rrcovery. -

(1344) 3.76 I (1680) 6.2

(1800) 2'6 I

(1320) 4'05 1

1 - _ . I

2.7 I

1

The nmouut of iron in the spleen is still distinctly increased, being between two and three tiiiies the uornial. On the other hand, there is a distinct fall as coniparecl with the anmiic aninials, in which the aniount was froin tive to twenty times the normal. The total amount of iron concerned is, however, compar:itirely small. The results show that although the hzniosiderin in the spleen is utilised in process of regeneration of the blood, this does not occur SO quickly as in the case of the lwmosiderin in the liver cells, probably owing to the fact that in the spleen the 1i:vmosiderin is in the form of coarser and larger granules.

Both the naked-eye and the niicroscopic test for iron indicated that there was a distinct excess in the renal cortex, and this is borne out by the analyses, The largest excess was in Experiment 11.. where the amount was five times the normal, and this excess accounted for fully half of the total excess in all the abdominal organs, namely, 3.2 nigrms. It is to be noted that the figures are highest in Experinleiits 1. and TI., in which there was marked hznioglobinuria, whereas this was absent in Experiments 111. and IV. The absorption of iron from the cells of the convoluted tubules is distinctly slower than from the liver cells, and this cannot be due to the form of the hzniosiderin, as in the former site i t is in the diffuse form. Possibly it may be due to

The condition of the kidneys is of considerable interest.

ABSORPTION OF IRON AFTER HBMOLYSIS. 49

the fact that the kidney cells are not concerned in the normal metabolism of iron as the liver cells are, and accordingly, when iron is deposited in them as a result of hzemoglobinzmia, which is a dis- tinctly pathological condition, it is not so readily re- absorbed.

CONCLUSIONS.

1. After acute hzmolytic anzemia in rabbits the excess of iron which has been stored in the organs, has been nearly all absorbed by the time of complete regeneration of the blood, and has been pre- sumably utilised for purposes of blood formation.

2. The absorption is most complete from the liver cells ; it is less complete, a certain excess still remaining, in the endothelium of the liver capillaries, in the pulp of the spleen, and especially in the cortex of the kidneys.

REFERENCES.

1. Mum AND SHAW DUNN .

2. MUIR AND WNEE . . Ibid., 1912, vol. xvi. p. 410.

Journ. Path. and Bacte~iol., Cambridge, 1915, vol. xix. p. 417.

4-JL. OF PATII.--VOL. XX.