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Studies of the mechanism of elimination of egg albumen and its effect on the elimination of iron by the rabbit's kidney.

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8TUI)IKS OF THE MECHANISRI OE’ E~LThlINATIONO F
F X G ALBUMEN AND TTS E F F E C T ON THE
B~TJMTNATIONO F IRON BY THE
RABBIT’S K I D N E Y
In a consideration of the mechanism of the elimination of
egg albumen several questions prcsent themselves. First, iii
what part of the nephron is the elimination localized? Second,
is the eliminatioii of this albumeii a process of filtration o r
secretion? Third, does the passage of the albumen affect tlic
cells through which it passes?
Ifany investigators (notably bdami, Schmidt, Miller,
Fukuda, Oliver, Whipple and Gersh) have sought to answer
thew questions f o r the elimiiiatioii of hemoglobin. Variously
they coriclude that hemoglobin may be eliminated through tlic
glomei*ulus or through the proximal tubule or through both.
ofsfiltration, by others
By some it is considered to be a p r o c ~ ~
ti process of secretion, by others a combination of both, aiicl
(:ersli ( ’36) postulates a process of ‘lcaking ’ in addition.
Among the few who consider at all the effect of the hemoglobin on the cells through which it passes, some find no effect
and others conceive changes in the permeability of the capillary walls.
Bayliss, Kerridge and RusseII (’33) in a series of experimciits with various proteiiis injected into aiiaesthetized cats
This is a. biief report of a prcliiniiiary series of observations on thc eliminatioll
of egg albuiiicii and iron. The author wislies t o express her gratitude t o
Dr. R. 1T. Jiciislc~yfor her suggestions and (witiniirrl intc.1 est i l l the work.
233
234
CIIARLOTTE G . BABCOCR
a i d rabbits and with perfusion of dog's kidney concluded that
tlic glomerular membrane is normally permeable to some proteins, specifically those having a molecular weight of less than
70,000. Histological examination showed no evidence of renal
damage and protein was seen in the capsular space in two
expcrimcrits in which egg albumen (with a molecular weight
between 33,000 and 35,000) was excreted in high conccntration.
Hemoglobin, with a molecular weight of 68,000, was found to
be excreted only when its plasma eoncentration exceeded a
certain level, and its concentration by the kidney was small.
I n considering this group of experiments the question
arises : Are tlie criteria for tlic exclusively glomerular elimination of these substances and for the absence of changes in the
iwial epithelium adequate? At present there a r e 110 histological methods by which foreign proteins may be demonstrated in cells (except for hcmoglobin, in which case tlie coloi*
i n the section is distinctive), so that the possibility of elimination of protein (except hcmoglobin) by the proximal tubulc
cclls cannot be determined in sections alone. The treatment
of the tissue in order to preserve and dcmoiistrate the protein
at the sitc of its elimination is not always adequate for prcserving cytological detail and for the demonstration of minute
cliaiiges in sti*acturcs which a r e a s delicate and intirriatcly
i-cllated as the various components of the glomei.ular memk)rane and the cytological constituents of tlic tubule cells.
it numbcr of investigators (notably Sticglitz, '21, and
Holton and Bcrislcy, '31) have shown that normally iron injccted in the form of ferric ammonium citrate is eliminated
by the pi-oximal tubule cclls arid the glomerular spaces arc
uniformly free of iron. It had been noted by Bcnsley (personal communication) that in the rare instances when minute
amounts of iron occurred in the glomerular space, precipitated
protein was also present therc. If it-on injected in moderate
amounts cannot go through the glomei-ular membrane but
does in combination with albumen, in concentrations great
enough to be detected microchemically, this would suggest a
change in the permeability of the glomerular membrane.
ELIMINATION O F EGG ALBUMEN A N D IRON
235
Gcrsh (’36) indicates tlirtt this is so. I n discussing the
mechanism of the elimination of hemoglobin he refers, in
passing, to one experiment “in which iron ammonium citrate
WRS introduced intravenously in a rabbit some time after an
injection of hemoglobin. Iron was definitely found in the
glomerular space although it has never been demonstrated
there in normal rabbits injected with iron alone.”
One such experiment, however, cannot indicate whether the
qlomcrular membrane was made permeable to iron by the
presence of hernoglobin, or by the combination of hemoglobin
and iron, for i t gives us no data showing whether the glonicrular passage of liemoglobin causes or follows changes in the
permeability of the glomerular membrane.
The following experiments which were a p a r t of a study of
csperimental albumenuria in rabbits may indicate a method
of approach for the solution of this complex (and confused)
problem.
The experiments were made on five groups of animals:
1. Twenty-one male rabbits injected intravenously with 5
cc. each of a 50% solution of egg white in water, filtered
th1,ough filter paper.
2. Eleven male rabbits injected with 5 cc. each of 50% aqueous egg white a second time, 2 to 8 days after the first injection.
3. Nine male rabbits injected with 10 cc. each of a lo:%
aqueous solution of ferric ammonium citrate (green scales).
4. Eight male rabbits each injected intravenously with
a ) 5 cc. of 50% aqueous egg white, immediately followed bj7
I,) 0.2 gm. per kilogram body weight of ferric ammoniuni
citrate in 10 cc. of water.
5. Eight male rahbits injected with egg white and iron salt
in the same manner a s those in group 4 and sacrificed at
appropriate intervals for histological studies.
The rabbits were kept in metabolism cages for several days
preceding the experiments. The urine was collected and a m lyzed to determine a s far a6 possible that it was normal. From
catheter specimens of urine, collected after the intravenous
injection, elimiiiatioii c u i ~ e swei’e plot tecl. Tlie acetic acid
t r s t with heat was. used f o r thc detection a n d estimation of
a1lmmeii7tlit. v a l w s , chcclied agaiiist staiidarcl solutions, being
rtvordcd a s trace, I+, 2 4 , 3+ and 4+.
The F ‘ i x b h i a i i blue
reaction clevelopetl with 1 ‘/. H(’1 atid <554 sodium fei~rocyanide,
fi*eslily prepared, was used f o r the estimation of iron, tlw
values, checlied against slariclard solulioiis, I)ciiig rworded i t s
1, 2, 3 aiicl 4 plus. Because of iiwgalarity in the collection of
saml)les, volumes were corrected by tlie factor :
Vo I unw i ri ciil)iF cen t i n i ct
~ n m G or f miiiutrs siiicc las
wliiclli ~vouldhe a corrected rate, read in tcntlis of a cubic
cciitimrter per minute.
Pieces of kidney from the animals iii gronp 5 wi’i’e fixed
1) hy absolute alcohol chilled t o - (i2”C., 2) by boiling f o r
1 minute tint1 transferring tliem to 83% alcoliol, 3) by 9574
alcohol at rooni tempera tiire, and 4) by modified formaliiiZeriker, i.r., 1 part neutral formalin to 9 parts Kensley ‘i
chrome-sublima te solution.
The chilled alcohol provcci to he tlir best fixation f o r localizinq the iron, \\hie11 was visualized hy tlie Piwssian blue reaction (Sticglitz, ’21). There was lcss fading after fixatioii
in 957; ;~lcohol,howerer. The study of iron plimiiiation was
made on 20 p sections. Sectioiis of tisiuc fixed in formaliiiZenker, 6 p in thickness, staintit1 with R1allor.y’s coiiricctivr
lissuc stain p i - o ~ e dto be the most valunble for the detection
of albumcn a i d of cellular changes.
Before describing the results of tliese experiments tlicl possible sources of errors, which must be takcn into wccount iii
iiiterprrtiiig arid evaluating the results, sliciulcl hc meiitioiiril.
I\lthough the rabbits appeared to he iiormal before expel+
mentatioii sorue of the animals d i d o r developecl diai*i.lica
during the course of esyerimcntation. Tliis source of error
IWS eliminated in the lrtter expciirncnts by o1)taiikig a pui*c*
sti*ain o f healthy Rwift’s muffle-free rabhits.
The catheter specimens coulcl often be co1lectt.d only a t very
irregular periods. Although these were corrected by a factor
for comparison of rate, they cannot thereby express what was
occurring at the time when tlie sample was collected. Obviously if a Iong interval elapses between specimens with a considerable change in the concentration of the injccted substance, no evidence can be obtained a s to when during that
period the progress of change occurred.
At the time of these experiments only lferck’s iron ammonium citrate (green scales) was available. This product, as
Stieglitz ( ’21) points out, is toxic when injcctcd intravenously
and therefore the iron injection experiments had to be acute.
Stieglitz found, however, no apparent changes in any tissues
after the injection of as small quantities as were used in these
experiments.
It is obvious that the tcsts used for determining the amounts
of albumen or iron in the w i n e a r e only grossly quantitative.
Doubtless the maximum plateau eshibitcd for iron elimination
should be resolved into a rise and fall. R u t the intensity of
the bhw color attained at the 4+ reading is so great that no
further increase can be detected a t this concentration. No
attempt was made to determine whether the albumen excreted
was piirel~7egg albumen or a mixture of it will1 proteins derived from blood or tissue destruction.
Since the elimination c ~ r r e swere made primarily for the
purpose of determining the time intervals a t which to sacrifice
the animals in group 5 , complete histological studies were not
made on animals injected mitli albumen alone.
,4 study of the eliinination curves (of which those in figures
1 and 2 a r e fairly representative) reveals the f o l l o ~ ~ i i ireg
sults, which a r e summarized for convenience :
1. Following the first injection of albumen, this substance
appeared in the urine within the first 20 minutes only three
times, even though samples were colle d within that interval
in seventeen animals. I n five animals the urine was negative
f o r albumen f o r from 25 to 63 minutes.
238
CHABLOTTE G. BABCOCK
2. Following the second injection of albumen, it did not
appear in the w i n e in any case within the first 20 minutes,
even though samples were collected within that interval in
five animals. In five animals the urine was negative for alhumen f o r from 30 to 91 minutes.
b
Group l
JI
239
ELIMINATIOS O F EGG ALBCMEN AND I RO N
3. In both groups, when frequent samples could he collected,
albumen elimination usually reached the maximum within 2 to
6 minutes after its first appearance or following a negative
specimen.
11/4/36
albumen i n j e c t e d introvenous1Lj
followed
Te NH
4
i
in 6 rnin.
by
C-C.
inlOcC H O injected introvenoudq
in 7 m i n u t e s
Time intervals a r e given far specimens in r e s p e c t to t h e beginning of
9 minutes for the iron
a l b u m e n injection a n d therefore should be c o t - r e c t e d
elimination
curve.
Figure 2
4. Out of nine animals injected with iron as a single injection only one animal failed to show iron in the urine when
samples could be collected, in the first 20 minutes, but in only
three animals 1v:as the maximum plateau attained in that time.
240
CHA4RLOTTE G. BABCOCK
5. Out of seven animals injected with albumen arid iron:
1) Albumen appeared in the urinp twice within the first 20
minutes but no samples collected after 20 minutes were negative. 2 ) The maximum level of albumen elimination was
somewhat sustained. 3 ) The albumen elimination curve was
somewhat shortened. 4) Iron was present in all samples collected within 20 minutes and with onc exception bad reaclied
the maximum plateau within that time. 5) The plateau of
maximum elimination of iron mas considerably lengthened.
Studies of the fixed tissue from the animals in group 5 which
were sacrificed at 5, 8, 10, 13, 15, 20, 25 and 31 minutes after
the beginning of the double injection, reveal that :
1. I n the animals sacrificed at 5 to 20 minutes, the glomerular epithelium was continuous and swollen.
2. From 10 t o 20 minutes the glomerular capillaries were
dilated.
3. Coagulum appeared in the loops of He&, distal tubules
and collecting ducts within 5 minutes and from 10 minutes on
was mixed q-ith desquamated cells, cell debris and red blood
cells.
4. Progressive degenerative changes in all the segments of
the nephron occurred from 5 to 25 minutes. After this the
various segments of some tubules appeared more normal.
5. I n the 10-minute animal the capillaries in the medulla
were ruptured and erythrocytes were estravasated into partially denuded caollecting (hids and loops of Henle.
6. In the 13-minute animal there x-as considerable interstitial hemorrhage, the tissue spaces were enlarged aid filled
with coagulum; and coagulum, cell debris and red blood cellr
were found in the calyx.
7. Iron appeared in the proximal cells and in the glomerular
capillaries in 3 minutes and increased in amount subsequently.
8. In 4 minutes iron was found in the lumen of all segments
of the tubule.
9. In 10 minutes iron appeared in the swollen glomerular
epithelium.
ELIRTISATION 03’ EGG A L B I 3 I E N A N D I K O N
241
10. I n 13 minutes iron and coagulum occurred in some
glomerular spaces. I n some the coagulum was mixed with
fragmenting erythrocytes and cell debris.
11. From 15 to 20 minutes the iron and coagulum mixed
with cell debris reached a maximum in the glomerular space.
12. After 20 minutes the capillaries were not as distended,
the glomerular epithelium was not as swollen, and the content
of iron and coagulum in the glomerular space was reduced.
13. At the height of degenerative changes in the various
segments of the nephron (10 to 20 minutes) the coagulum and
iron was less concentrated in tlie loops of Henle, the distals
and in the collecting ducts.
SUMMSRY
From the elimination curves it would appear that 1) egg
alhumen is not simply filtered through the glomerular membrane ; 2) one injection of egg albumen delays the elimination
of albumen following a second injection; 3) elimination oE
albumen reaches its maximum shortly after it is initiated;
4) when albumen and iron are injected together there is a
greater elimination of both than when either is injected singly.
The histological studies of the elimination of albumen and
iron when injected together indicate that albumen and iron
are eliminated by the glomerular membrane coincident with
progressive degenerative changes of it and of the tixbular
epithelium, and suggest that both albumen and iron arc elimiiiated more freely through the damaged tubules, with a coincident loss of cencentration of these substances by the tubules.
CONCLUSIONS
1. The elimination of egg albumen injected intravenously
into rabbits is not a simple process of filtration through the
glomerular membrane.
2. Iron, injected intravenously in the form of ferric ammonium citrate in combination with egg albumen, appears in the
glomerular space following progressive degenerative changes
in the glomerular membrane.
242
CHARLOTTE G . BABCOCK
LITERATURE CITED
BAYIJSS,L. E., P. M. T. KERRIWEAXD D. S. RUSSJGU 1933
protein by the mammalian kidncy. J. Physiol., vol.
GERSH, I. 1936 The site of renal elimination of hemoglobin in
Ree., vol. 65, p. 371.
HOLTON,
S. G., AND R. R. BFANSLFY
1931 The functions of
The exeretion of
77, p. 386.
the rabbit. Anat.
the dlfferentiated
parts of the uriniferous tubule in the mammal. Am. J. Anat., col. 47,
p. 241.
Ssrrimmz, E. J. 1921 Histochemical studies on the mechanism of renal eecretion. Am. J. Anat., vol. 29, p. 33.
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