THE ROLE OF THE PORTAL CIRCULATION OF THE

LIVER IN BILE FORMATION AND JAUNDICE

C. VOEGTLIN AND B. M. BERNHEIM

Prom the Department of Pharmacology and the Hunterian Laboratory of Experi-

mental Medicine, Johns Hopkins University

Received for publication, April 21, 1911

* From the classical experiments of Joh. Muller,’ Kunde,2 Mole-

schott3 on frogs, of Minkowski and Naunyn on geese, we know
that bile pigments are almost exclusively formed within the liver.
Virchow,5 however, was able to demonstrate the formation of
an iron-free pigment in old blood extravasations and also at the
site of subcutaneous injections of hemoglobin. He named this
pigment hematoidin. Later researches furnished sufficient proof
for the assumption that hematoidin is identical with bilirubin.
Under normal conditions this second mode of formation of bile
pigment may be neglected, as by far the greatest quantity of
bile pigment results through the activity of the liver cells. For
the bile acids an extrahepatic origin has not been demonstrated.
Knowing that the typical bile constituents are exclusively derived
from the activity of the liver cells, the question arises, ‘What are
the factors influencing this function of the liver? Since the liver
is a gland supplied by nerves, one might think that possibly the
productiOn Of bile is under the direct control of the nervous sys-
tem As a matter of fact this does not appear to be the case, as
PflUger6 and many later investigators could demonstrate that

‘Joh. Muller: Lehrb. d. Physiol., 1844, p. 131.

2Kunde: De hepatis ranarum extirpatione. Berolini, 1850.
J. Moleschott: Vierordt’s Arch. f. physiol. Heilk., 1852, ii, 479.
‘Minkowski and Naunyn: Arch. f. exp. Path. u. Pharm., 1886, xxi, 1.
Virchow: Arch. f. Path. Anat., 1857, xii, 48.
‘Pfluger: Arch. f. d. ges. Physiol., 1868, ii, 192.
455
456 C. VOEGTLIN AND B. M. BERNHEIM

section of the nerves leading to the liver did not abolish the forma-
tion of bile. It cannot be denied however, that the excretion and
formation of bile is dependent at least to some extent upon nervous
influences.
As far as the excretion of bile is concerned, the studies of Heiden-
ham and his pupils, and more recently of Doyon,7 have conclu-
sively demonstrated that stimulation of the splanchnic nerves
is followed by a constriction of the gall bladder, the bile ducts,
and the sphincter muscle of the common duct in the duodenum.
In this manner the flow of bile into the duodenum is temporarily
increased. Doyon furthermore pointed out that certain drugs
which are known to stimulate the nerve terminals contract the
gall bladder (pilocarpin) or cause a relaxation of the bile passage
(atropin).
That the nervous system, on the other hand, controls through
a vasomotor mechanism the bile formation, had been shown by
Heidenhain8 and Munk,9 and was later confirmed by many others.
As experimental proof for this assumption these investigators
called attention to the fact that continuous stimulation of the
splanchnic nerves sometimes caused a decreased flow of bile from
a cannula inserted into the gall bladder or common duct. This
observation was explained by a vasoconstriction of the liver capil-
laries, which lead to a diminished perfusion of this organ.
Afanassiew’#{176} pointed out, that section of the splanchnic nerves
causes a dilatation of the liver capillaries associated with hyper-
aemia resulting in a better perfusion of the liver and causing bile
pigments to appear in the urine and feces. This author explains
the excretion of bile pigments in this condition by the dilatation
of the liver capillaries causing a compression of the biliary capil-
laries, bringing about a stasis of bile, and consequently the bile
constituents diffuse into the lymphatics and blood vessels and are
finally excreted by the kidney.

Doyon: Arch. de physiol., 1893, p. 678 and 710; ibid, 1894, p. 30.
8 Heidenhain: Studien physiol. Inst. Breslau, 1868, iv, 226.
‘Munk: Arch. f. d. ges. Physiol., 1874, viii, 151.
10 Afanassiew: Arch. f. d. ges. Physiol., 1883, xxx, 385.
 BILE FORMATION AND JAUNDICE 457

It is a common belief, that red blood corpuscles break down

and lose their hemoglobin in the general circulation. The free
hemoglobin is brought to the liver with the blood and gives rise
to the formation of bile pigments. Stimulated by this assump-
tion, a number of physiologists became interested in the ques-
tion, To what extent are the hepatic artery and the portal vein respec-
tively responsible for the formation of bile? The first experimental
attempts in this direction were made by Ore,” who experiment-
ing on dogs succeeded (as he thought) in eliminating the portal
circulation by a gradual obliteration of the portal vein at the hilus
of the liver. As iii this case there was still some bile formed, he
concluded that the blood of the hepatic artery was the source of
bile formation.
Schiff 12 made extensive studies on the same subject and came to
the following conclusions:
1. Ligation of the hepatic artery in cats does not prevent bile
formation.
2. Ligation of the portal vein stops the bile secretion imme-
diately and the animal dies within one or two hours.
3. Gradual obliteration of the portal vein (Ore) is not followed
by a cessation of bile formation. A collateral circulation between
the portal vein* and the liver is established, and therefore this
method does not yield conclusive results.
That the experiments of Ore and Schiff were not free from criti-
cism was shown by Schiff’ himself in a fundamental research.
He was able to demonstrate that the bile which is secreted into
the duodenum is reabsorbed and carried by the portal circulation
to the liver, to be re#{235}xcreted into the intestines. Hence there
exists a so-called circulation of the bile. In dogs with a biliary
fistula Wertheimer’ more recently ligated the hepatic artery and
found that the secretion of bile was not interrupted by such a
procedure.

11 Ore: Jour. de l’Anat et de la Physiol., 1864, i, 565; Compt. rend, 1856, ii, no. 9.
12 M. Schiff: Schweiz. Zeitsch. f. Heilkunde, 1862.
“M. Schiff: Giorn. di Scienze naturali ed Econ., Palermo, 1868, iv; Arch. f.d.
ges. Physiol., 1870, iii, 598.
14 Wertheimer: Arch. de Physiol., 1892, iv. 577.
458 C. VOEGTLIN AND B. M. BERNHEIM

With these facts in mind we began a series of experiments the

object of which was to study the influence of the portal circulation
on the formation of bile. This work was made possible by the
introduction of a method for establishing a permanent anastomo-
sis between the portal vein and the vena cava inferior, thus lead-
ing the portal blood to the heart and preventing its passage through
the liver. This method was first devised by Eck. In a previous
communication’5 we described a modification of this operation
which simplifies the tecnic and decreases the mortality from 40
per cent to almost 0.
It was found that an Eck fistula combined with ligation of the
portal vein at the hilus did not prevent the formation of bile.
On the contrary,. dogs thus operated upon remained healthy for
many months, the operation obviously having no ill effects at
all. In all these cases the gall bladder contained apparently
normal bile, as shown by the usual tests for the various bile con-
stituents. The stools did not have a fatty appearance, so that
sufficient bile seemed to be excreted into the duodenum to bring
about a complete absorption of fats. Evidently in the absence
of the portal circulation, the blood of the hepatic artery alone is suffi-
cient for bile formation.
The question then arose, What proportion of total bile forma-
tion is due to the portal circulation?, This problem must of course
be studied from a quantitative standpoint
Two methods are at our disposal:
I. In animals with a biliary fistula the amount of bile formed
is determined for a given time, after which an Eck fistula is made
and the portal blood is led directly to the vena cava. Any influ-
ence of the portal circulation should now become evident by an
estimation of the flow of bile for the same period of time as used
before the establishment of the Eck fistula.
II. Ligation of the common duct in dogs always leads to a
severe and fatal jaundice. On the assumption that the portal
blood passing through the liver gives up some hemoglobin for the
formation of bile pigments, this obstructive jaundice might possi-
sibly be modified in intensity by an Eck fistula.

“Jour. Pharmacol. and Exp. Ther., 1910, i, 463.

 I

BILE FORMATION AND JAUNDICE 459

We have chosen the second method and submit the results

obtained in the following protocols. We hope to report in the
near future further studies using the first method here mentioned.
It is obvious that there are three possible ways of proving our point
by means of the second method: The Eck fistula can be made
(1) before, (2) simultaneously with, and (3) after the ligation of
the common bile duct.
Before proceeding we may briefly refer to the symptoms occur-
ring in dogs after the ligation of the common bile duct. To pro-
duce a permanent complete obstructive jaundice the common duct
is dissected free near the duodenum and two strong ligatures are
placed around it about 1 cm. apart, after which the part included
between the two ligatures is resected in order to prevent the
regeneration of the duct. Rosenbergl6 has pointed out that one
must take into consideration the possiblilty of an accessory duct
which in case of obstruction of the main duct may still take up
the function of the latter.
The succession of the symptoms is as follows: A few days (2
to 4) after the ligation of the bile duct, bile-pigments appear in
the urine. A short time afterwards a jaundice is noticed in the
mucous membranes and conjunctiva. The animal loses weight
very rapidly and takes very little nourishment. The stools are
clay colored. No urobiin is found in the urine. An anaemia
develops and the number of red cells drops to one-half of the nor-
mal. Death occurs within three to four weeks. On autopsy it
is noticed that the tissues are markedly janndiced. Histological
changes occur constantly in the liver. Small areas of necroses
are found with regeneration of the biliary capillaries.’ 18

As a basis for our conclusions we have experimented upon six

animals and submit the detailed reports of three of these experi-
ments representing the three different types of procedure.

3810. Medium-sized female collie.

February 9. Dog had been starved for 24 hours.

‘ Rosenberg: Arch. f. Anat. u. Physiol. (Physiol. Abt.), 1896, p. 191.

‘ D. Gerhardt: Arch. exp. Path. u. Pharm., 1892, xxx, 1.
18 Joannovicz: Zeit. f. Kinderh., 1904, xxv, 25, (Pathol. Abt.).
460 C. VOEGTLIN AND B. M. BERNHEIM

Operation. Ether anaesthesia. Portal vein and vena cava sewn

together side by side, following the same steps as in making an Eck fistula
with the exception that no communication is made between the two
vessels. Common bile duct cut between two ligatures. Wound closed.
Dog recovers well.
February 11. Dog shows severe jaundice. Mucous membranes
and skin yellow. Urine gives marked reaction for bile pigments.
Ether anaesthesia. Eck fistula completed and portal vein ligated
near liver. Gall bladder much distended. Wound closed.
February 15. Dog takes some milk. Mucous membranes yellow.
Dog shows marked improvement from this tijne on. Abdominal wound
which had broken down healed and jaundice disappeared slowly.
March 1. Dog in fairly good condition. No jaundice. Is on a meat
diet.
March 10. Dog lost some weight. No jaundice.
March 12. Eats very little. No jaundice. No bile pigments in
urine.
March 14. Doesnot take food. Condition is getting worse.
March 15. Dog is very weak. Refuses food. No jaundice. No
bile pigments nor bile acids in urine.
March 16. Found dead.
Autopsy: Dog is very emaciated. No subcutaneous fat left. Heart
normal, contains some firm Llood clots. Right lung normal. Left
lung shows some areas of consolidation. Spleen normal. Adhesions
between liver and diaphragm. Liver of dark brown color. Gall blad-
der contains a flui#{231}l
of pale yellow color with a flocculent precipitate.
Mucous membranes of stomach pale. Intestines normal. Pancreas
pale. Right lobe of liver contains an abcess of considerable size
involving the whole lobe and forming a cavity containing yellow pus
(300-350 cc.). The wall of this cavity is 0.5 cm. thick and covered with
yellow mucous.
Bile duct completely
obstructed and separated from duodenum. Ad-
renals normal. Left kidney normal in size, capsule strips easily, on
section pale. Right kidney shows a compression on superior pole,
due to pressure from abscess. Capsule strips easily. Portal vein com-
pletely obstructed near liver hilus. No collateral circulation. Lymph
glands in neighborhood of abcess enlarged. Peritoneum normal with
exception of portions near abcess, where yellow patches are seen. No
trace of jaundice in any tissue. Causes of death: Liver abcess, origin-
ating from upper end of infected wound. Microscopical examination
 -, .

BILE FORMATION AND JAUNDICE 461

of section.” Liver-Some acute congestion. Bile ducts appear per-

fectly normal and not distended. Liver cells in places contain small
amount of bile pigment. No increase in connective tissue. Liver cells
starning well. Practically no fat. Very little iron (Berlin blue stain).
3010. Small fox terrier. Weight 5 kg.
October 26. Starved.
October 28. 3 p.m. Ether in linea alba. Two
anaesthesia. Incision
ligatures are placed around close to duodenum,
common part of
bile duct
duct included is resected. Gall bladder emptied. Strong silk ligature
placed around portal vein near hilus but not tied. Eck fistula is made,
after which ligature around portal vein is tied. Wound closed. Dog
lost very little blood during operation. Comes out of ether very rapidly.
Put into metabolism cage and is given water but no food.
October 29. Urine 400 cc. Test for bile pigments negative. Dog
in fairly good condition but somewhat depressed. Pulse 90- 100.
October 31. Dog seems perfectly normal. Passed 800 cc. of dark
brown urine. Gmelin positive. Hammersten strongly positive. Liquid,
clay colored stools. Pulse 120, regular, strong. No trace of jaundice.
November 1. Is put on meat and bone diet. Urine highly pigmented.
Does not contain sugar nor albumin.
November 2. Slight yellowish tinge in conjunctiva. Urine contains
much bile pigments.
November 5. Weight 6 kg. Skin wound broken down. Stools clay
colored, no bilirubin nor urobilin. Jaundice conpletely cleared up.
Animal bright and active.
November 8. Animal is quite well. Is still on meat diet. No trace
of jaundice. Stools bulky, clay colored. Urine gives faint test for bile
pigments.
November 14. No jaundice. Appetite poor, but animal is apparently
in good condition.
November 15. Weight 5.25 kg. No bile pigments in urine.
November 23. Dog is emaciated. No trace of jaundice. Refuses
meat, takes some bread.
November 24. Dog is in poor condition.
November 25. Found dead.
Autopsy. Is very emaciated. No adipose tissue left. No trace of
jaundice. Heart and lungs normal. On upper end of wound, which

“'We are indebted to Dr. John H. King for the histological examination of the
sections.
462 C. VOEGTLIN AND B. M. BERNHEIM

is badly infected, an abcess had developed which extends into perito-

neal cavity in the region of the liver. Liver dark brown. Spleen, pan-
creas and kidney normal. No general peritonitis. Common bile duct
well cut off from duodenum. No bile flows out of duct when pressure is
put upon gall bladder. Bile ducts much distended. Ligature around
portal vein secure. Eck fistula perfect, 8 mm. long. No collateral cir-
culation. Sections taken from liver, adrenals, kidney and thyroid.
Microscopical examinations-Liver: Some bile pigments in liver
cells and caniculi, otherwise normal. Kidney: practically normal.
Small amount of parenchymatous degeneration of convoluted tubules.
3210. Male dog, weight 9 kg.
November 2. Dog had been starved for two days.
Operation: Ether anaesthesia. Eck fistula made and portal vein
tied off near liver hilus. No hemorrhage. Dog recovers well, but
refuses food for two days after operation.
November 4. Eats boiled beef. Looks perfectly well.
November 7. Wound healing.
November 8. Dog quite well. Turned out into yard and fed on boiled
meat and bones.
November 14. Dog in very good condition. Wound healed.
December 3. Weight 9.35 kg. In excellent condition.
December 8. Hemoglobin 12.76 per cent. Red cells, 6,390,000.
Leucocytes, 15,000.
December 16. Weight 8.7 kg. Dog had been starved for 24 hours.
Operation: Ether anaesthesia. Right rectus incision. ,A few adhe-
sions loosened. Bile duct cut near duodenum between two ligatures.
Wound closed. Dog recovers rapidly.
December 17. Takes some milk. Urine does not contain bile
pigments.
December 18 to December 20. Faint trace of bile pigments in urine.
Meat diet.
December 21. Weight 8.2 kg. Dog in excellent condition. Wound
healed. No bile pigments in urine. No trace of jaundice.
February 6. Dog has been well up to this date. Has lost some weight,
probably on account of poor care. No jaundice. No bile pigments in
urine.
February 22. No jaundice. Emaciated.
February 23. Eats a cgnsiderable quantity of meat in the morning.
Dies in the course of the afternoon. Cause of death: Ammonia intoxi-
cation.
 BILE FORMATION AND JAUNDICE 463

Autopsy: Heart and lungs normal. 6 mm. Eck fistula

Few adhe-
sions between liver and stomach andbut no collateral
intestines,
blood supply. Liver dark brown in color, congested. Bile duct not
regenerated. Kidney normal. No trace of jaundice in tissues. Micro-
scopical examination. Liver: Moderate amount of acute congestion,
localized in lobules about the hepatic vein, in others about the portal
vein. Occasional trace of bile pigment. Bile ducts appear normal,
with slight increase in connective tissue around them. Liver cells con-
tain a good deal of blood pigment.

NO. ANI)4AL HEOOLOBIN RED CELLS LEUCOCYTES REMARKS

per cent
3110 12.46 4,250,000 11,800 Eckfistula.
3210 12.76 6,390,000 15,000 Eck fistula + Obstruc-
tion of common bile
duct.
3410 11.48 5,820,000 16,400 1
3610 14.04 5,880,000 8,600 Eck fistula.
4410 10.20 4,860,000 33,400
Male 17.2 7,800,000 20,000
Female 13.4 5,150,000 16,400 Normal.
Normal Average. 12 to 14 6,500,000 10,000

Methods used: Hemoglobin, Hemoglobinometer of Miescher-Fleischl: Cell

count-Zeiss-Thoma.

This table illustrates the fact that there exists no marked

variation in the hemoglobin content and the number of erythro-
cytes of the blood of animals with an Eck fistula and an obstruc-
tion of the common bile duct as compared with normal controls.
The figures given in the above table are taken at random as typi-
cal from a large number of estimations.
The feces of these animals were tested a number of times for
bile pigment and urobiin, but always with a negative result.
This rules out the possibility of a compensatory excretion of bile
pigment by the intestines.
From our experiments we are justified in drawing the following
conclusions: In the absence of the portal circulation, ligation
of the common bile, duct in dogs did not produce jaundice. Should
a mild jaundice develop during the first few days following the
operation, it is probably due to a hemolytic action of the anaes-
464 C. VOEGTLIN AND B. M. BERNHEIM

thetic as a result of which more free hemoglobin is circulating with

the blood and hence more bile pigments are formed. This over-
production of bile pigments is probably sufficient to explain this
temporary jaundice.
In the six experiments five animals died of pneumonia, other
infections (liver abscess) or meat intoxication; in no case, however,
was jaundice present at death. One animal has survived and
seems to be perfectly normal at the present time. As to the ex-
planation of these facts, it seems obvious that the portal blood
passing through the liver yields the material for the greater part
of the total bile pigment formation; in other words, if the portal
blood does not flow through the livr less bile is formed. How can
this diminution in bile formation be explained? One might imag-
ine that the portal blood contains a specific substance acting
as a stimulant to the liver cells. This explanation is not very
plausible since it has been shown that the blood from the hepatic
artery alone is sufficient for the formation of bile.
Another explanation based on the following facts seems to us
more rational. The liver is a very vascular and voluminous
organ. Might it not be possible that red cells arebroken down in
this extensive network of blood capifiaries? Before citing any
evidence in favor of this new theory we shall sum up in short the
generally accepted theory of bile formation, according to which
red blood corpuscles disintegrate constantly in the general cir-
culation. The liberated hemoglobin is removed by the liver and
gives rise to bile pigments, the iron being deposited within the
liver cells. Our own experiments are obviously not in accord-
ance with this theory for the following reason: Removal of the
portal blood, which is by far the greater amount of blood supplied
to the liver in comparison with the arterial blood flowing to this
organ, would lead to an accumulation of hemoglobin in the circu-
lation, unless some other organ is able to take care of this free
hemoglobin. This last possibility is not ruled ou( as such a func-
tion is often ascribed to the spleen. Joannovicz for instance
claims that the spleen is the organ which takes up injured red
cells. We have never been able to demonstrate any free hemo-
globin in the serum of Eck fistula dogs.
 BILE FORMATION AND JAUNDICE 465

The evidence in favor of our view that the liver is normally

a blood destroying organ may now be considered. In this con-
nection a series of investigations by Browicz2o are of particular
interest. This author found that the livers of normal dogs, which
are killed during the process of digestion, re’eal a remarkable
histological picture. Within the liver cells are found complete
red corpuscles and also hemoglobin crystals. Sometimes clumps
of red cells are seen lying in well defined vacuoles. Furthermore,
if a dog has received an intravenous injection of hemoglobin this
substance is found in the cyto- and karyoplasm of the liver cells.
According to Browicz such hemoglobin crystals are also observed
in human livers from cases of chronic venous congestion and new-
born children. Browicz claims to have demonstrated the pres-
ence of an intracellular network of capillaries which is in com-
munication with the intercellular blood capillaries. The liver
cells obtain their nourishment by means of these intracellular chan-
nels. Among other substances hemoglobin enters the nucleus,
as is evident from the presence of hemoglobin crystals.
Herring and Simpson21 have been able to confirm these findings
of Browicz. These authors injected the livers of various animals
from the portal vein with carmin gelatine. Microscopical exam-
ination of such livers showed well defined intracellular capillaries.
Red blood corpuscles in a process of disintegration and also hemo-
globin crystals were frequently met with.
Herring 22 in another publication gives a detailed description
of the hemoglobin crystals found in the liver of dogs killed by chlo-
roform. As the nuclear membrane in such cases is found intact,
Herring believes that the crystals which he considers to be oxy-
hemoglobin are formed intra vitam, because a sudden crystalliza-
tion after the hardening of the cells is completed would lead to a
rupture of the nuclear membrane. The importance of these
histologic studies is evident in connection with the experimental
work reported in this paper, as they furnish additional evidence

20 Browicz: Arch. path. Anat. u. Allg. Path., 1902, clxviii, p. 1.

21 Herring and Simpson: Jour. of Physiol., 1906, xxxiv, Proc.
“Herring: Jour. of Physiol., 1906, xxxiv, Proc.
466 C. VOEGTLIN AND B. M. BERNHEIM

for the following considerations: The liver in extrauterine life is

actively engaged in the destruction of red cells. The erythro-
cytes are brought to the liver, enter the liver cells, disintegrate,
the hemoglobin is changed into globulin, bile pigment and iron.
The iron derived from the hemoglobin is deposited within the
liver, whereas the bile pigments leave the cell by way of the intra-
cellular bile capillaries, which are in communication with the
intercellular system of bile ducts.
Kupffer2 and Heinz24 do not accept the existence of an intra-
cellular capillary system, but claim that the endothelium of the
liver capillaries has a phagocytic property for erythrocytes,
allowing these cells to enter the liver cells. On this assumption
our results can be explained just as well as with the view held by
Browicz.
The liver has been shown by many investigators to form red
cells during embryonic life (Howell2s). In recent years a number
of have
investigators discovered erythroblasts in the liver in cer-
tain diseases (pernicious anaemia), and are therefore inclined to
adopt the view that this organ can assume a hematopoetic func-
tion after birth, if there is a demand for such an activity on the
part of the body.2627
The liver of an adult mammalian animal (dog) is apparently
therefore an organ which is actively engaged in the destruction
and under certain conditions the reproduction of red cells.
Numerous authors have suggested that the spleen is concerned
in the destruction of red blood corpuscles. The evidence in favor
of this view is based chiefly on the findings of fragments of red cells
within the cells of the spleen and on the comparatively high iron
content of this organ. It is obvious that such proof is very defi-
cient. On the othr hand Joannovicz23 has shown that after
the introduction of hemolytic substances into animals (dog) the

“ Kupifer: Arch. mikr. Anat., 1899, liv. 252.

24 Heinz: ibid, 1901, lviii,576.
“'Howell: Jour. of orph., 1891, iv, 58.
“ Lobenhoffer: Beitr. path. Anat. u. allg. Path., 1908, xliii, 124.
27 Meyer Erich: Munch. med. Woch. 1908, lv, 1161.
“ Loc. cit.
 ,. ..,..,....

BILE FORMATION AND JAUNDICE . 467

spleen takes up the injured blood cells and destroys them com-
pletely. The liberated hemoglobin is carried to the liver and.
formsbie pigments in such amounts that jaundice is produced.
Removal of the spleen in obstructive jaundice has no influence
on the course of the disease. It is therefore hardly possible that
the spleen, if it takes any part at all in the etiology of jaundice,
will occupy the same importance as the liver and its portal cir-
culation.

CONCLUSIONS

1. The amount of bile formed by the liver depends on the blood

flow through this organ.
2. The jaundice and fatal toxemia resulting from occlusion of
the common bile duct can be avoided by an Eck fistula made at
the time of the ligation of the duct.
3. In cases in which, owing to occlusion of the duct, jaundice
has already developed, an Eck fistula causes the symptoms to
disappear.
4. It is shown that the liver has a hemolytic function.