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AN EXPERIMENTAL STUDY OP THE EFFECTS OF STEROIDS ON THE OSMOTIC FRAGILITY OF RED BLOOD CELLS
A Thesis Presented to the Faculty of the Department of Physiology School of Medicine University of Southern California
In Partial Fulfillment of the Requirements for the Degree Master of Science
by Joseph Abrahamson September 1950
UMI Number: EP63578
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T his thesis, w ritten by
JOSEPH ABRAHAMSON under the guidance of h . . ^ . . F a c u lty C o m m ittee, and app ro ved by a l l its members, has been presented to and accepted by the C o u n cil on G raduate S tudy and Research in p a r t ia l f u l f i l l ment of the requirements f o r the degree of
MASTER OF SCIENCE .....
rw - September 1950__
C . V Chc^rhtan ...
TABLE OP CONTENTS CHAPTER I.
PAGE THE P R O B L E M ........................... Statement of the p r o b l e m .................. ..
Importance of the study
. . . . ...........
Preview of organization of thesis II.
REVIEW OF THE LITERATURE
The state of the problem when this project was b e g u n ................................ III.
EXPERIMENTAL PROCEDURE . . . .
Method used for this investigation ........
D I S C U S S I O N .................. P r o c e d u r e .......................
The red blood cell membranes in general and this problem in p a r t i c u l a r ...........
EVALUATION OF RESULTS AND C O M M E N T S ..........
SUMMARY AND C O N C L U S I O N S .....................
CHEMICAL STRUCTURE OF STEROIDS ........
GRAPHS AND T A B L E S .....................
LIST OP TABLES TABLE
NaCl S o l u t i o n s ................................
Cortin Sample of Unknown O r i g i n .............
Wilson C o r t i n ................................
Cholic A c i d ........................
Desoxycholic A c i d ............................
Dehydrocholic A c i d ............................
Dehydrodesoxycholic A c i d ......................
Commercial Cortins ............................
James Wilson1s C o r t i n s ........................
James Wilson1s Cortin with Ringerfs
Desoxycholic Acid in R i n g e r ' s ...............
James Wilson's Cortin— Dilution Experiment . .
X. XI. XII.
LIST OF FIGURES FIGURE
Preliminary E x p e r i m e n t ........................
Desoxycholic Acid and N a C l ...................
Dehydrocholic Acid and N a C l ....................
Dehydrodesoxycholic Acid and NaCl . . . . . . .
Concentration Ringerfs Solution ...........
Desoxycholic Acid in Ringer’s .................
CHAPTER I THE PROBLEM Statement of the problem.
To investigate the effects
of steroid compounds, as represented by certain bile salts and water soluble extracts of adrenal cortical tissue, on the osmotic behavior of red blood cells. Importance of the study.
In view of the beneficial
effects which have been achieved with cortical extracts on various human ills, it becomes important to know the ways in which these compounds influence living tissue.
in membrane permeability are one important effect they are thought to exert.
If effects could be found with
steroid concentrations at low physiological levels, the actions of cortical extracts and bile salts on clinical anemias might be postulated*— since it has long been known that red blood cells have diminished resistance to hemoly sis in acholuric jaundice and other hemolytic anemias, and an increased resistance in other types of anemias and jaundice (Creed)(Dacie, Lond, and Vaughan). Preview of organization of thesis. taken up in the following order:
Topics will be
(1) a brief review of
the most important papers on the subject to indicate where
2 the problem stands today;
(2) presentation of the methods
used in this investigation; the reasons for their use, and the shortcomings and experimental error of such systems; the results of the experiment;
(3) discussion and theore
tical considerations; evaluation of results and comments; and (4) conclusions and summary.
CHAPTER IX REVIEW OP THE LITERATURE The state of the problem when this project was begun.
There are several papers relating directly to the
topic under discussion. In 1939 & German investigator, Raushwalbe, reported on tests he made with swine red blood cells and cortin1 (,!cortidynn ), in hypotonic saline and hypotonic glucose solutions.
His NaCl solutions ran from .4 - .Q%, with
cortin concentrations of 1:1000.
He found that hemolysis
in the controls (i.e. without cortin) started at .7$ NaCl, and that cortin prevented any hemolysis at this concentra tion.
All of the effects he mentions were gross enough
to be visible to the naked eye, although he states that he checked his results on a colorimeter.
At .6$ NaCl he
noticed marked hemolysis in the controls, but very little in the cortin test solutions. Interestingly enough, he found that when hypotonic glucose was used in place of NaCl,
just the reverse hap
pened— cortin promoted rather than inhibited hemolysis. In his summary, this worker reverses himself, and
1 An extract of the adrenal cortex containing the active steroids of the tissue.
4 says that cortin is hemolytic with salt and protective with hypotonic glucose.
It is to be assumed that that
statement made in the body of his paper, along with the supporting data, represents the facts a3 he found them. Dr. Chester Hyman of the University of Southern California ran some similar experiments in 1947.
were preliminary studies only, and are not intended to be the basis for any conclusion on this problem.
rabbit blood cells and various concentrations of cortin (1:2000 - 1:1000,000) in NaCl solutions which, without cortin, gave approximately 50$ hemolysis, he found the cortin inhibited hemolysis up to as much as 50$ of the con trol value in some instances, thus tending to confirm the experiments of Raushwalbe.
Hyman determined the relative
hemolysis from the color of the supernatant fluids as read on a Klett photocolorimeter. In 1938, Berliner and Schoenheimer studied the hemolytic and anti-hemolytic properties of bile acids and sterols, and attempted to relate these effects to their chemical structure.
(See Appendix I for brief review of
steroid and sterol structural formulae.)
differed from those described above in that the controls were isoplethecontic, and hence were non-hemolytic. test solutions were made up of this isoplethecontic
5 mixture to which lysins were added to promote hemolysis, and the effect of the steroids on this ”lysin induced” hemolysis was tested.
(This lysin induced hemolysis is
thus different from the osmotic hemolysis used by Raushwalbe and in the investigation to be described herein.) The isotonic system employed involved use of veronal buf fers, alcohol, and NaCl.
Lithocholic acid was used as the
lysin for most of their experiments.
They found all
sterols protective against this hemolysis, and all the monohydroxy bile acids protective, provided they were of cis configuration at carbon 3 in relation to the methyl group at carbon 10, and in which there was either a double bond at 5, 6 or in which rings A & B were fused in a trans decalin.
Those bile acids of reverse structure were found
to be hemolytic.
Additional substituents on a bile acid
(i. e., hydroxyl or keto group) modified but did not reverse its protective or lytic property. Of additional interest, they were able to find no evidence to show a chemical combination between the lytic lithochotic acid (or any of the other lysins used) and those sterols and bile salts which, when added to the system, prevented hemolysis. In 19^1* Grodins, Berman and Ivy tested the hemo lytic effects of some bile acids on human red blood cells. They complain of difficulties in obtaining cell suspensions
6 with uniform properties.
The results of their work are
presented below. Weakly hemolytic: dehydrocholic acid Moderately hemolytic: synthetic glycoehollc acid synthetic taurocholic acid cholic acid dehydrodesoxy cholic acid Strongly hemolytic: apocholic acid desoxycholic acid natural glycholic acid natural taurocholic acid
CHAPTER III EXPERIMENTAL PROCEDURE Method used for this investigation. Hopps and Shedelen.
NaCl solutions were mixed as shown on
Table I (all tables and figures will be found in Appendix II).
These solutions were so made up that 1.3 nil. of a
given solution plus 0,3 ml. of blood (or a red blood cell \
suspension in isotonic salt) would leave the final solu tion with the NaCl percentage shown on Table I.
be seen, in no experiments carried out by this investigator were the final test solutions mixed exactly this way; hence, NaCl concentrations were never exactly those stated. This is unimportant, however, as the salt concentrations of the control and test solutions were identical; and, hence, had the same degree of hypotonicity.
out this paper, when reference is made to a ".65$ NaCl solution,11 it will refer to a solution which will have been made up with a salt solution which, only when mixed with plasma, or .9$ NaCl in a 13:3 ratio, would give a true .65$ NaCl concentration.
However, in a series of test tubes
containing NaCl solutions from ".2$ to .86$,11 while the percentages will not be exactly as stated, the intervals between the various percentages in the series will be the same as if they were.
8 The red cells used throughout were fresh rabbit red cells, obtained from ,rV rabbits” normal.
which were otherwise
Heparin was used as an anticoagulant.
were thrice washed in .9$ saline (which, according to Saslow  does not affect their osmotic properties) and then suspended in .9$ NaCl. The procedure for the preliminary experiments was as follows: two series of tubes were made up containing 2 ml. of NaCl solutions from .2 to .8^ in steps of .05$, with the final tube of the series containing .86^ NaCl. This is isotonic according to Hopps and Shedelen.
control series was added 0.5 ml. distilled HgO and 0.5 ml. of red blood cells suspended in .9# NaCl.
The test series
was the same except that 0.5 ml. of a 1:1000 solution of cortical extract was added in place of the 0.5 ml. of dis tilled water.
These solutions were allowed to stand for
two hours at a constant temperature of l8 °C. mixed by inversion every 15 minutes.
At the end of this
two hour period the unhemolysed red blood cells and ghosts were centrifuged down for 10 minutes at approximately J? r.p.m.
^ 11V Rabbit” is the term used in the Physiology Department of the University of Southern California to des ignate a rabbit whose vena cava has been partially tied off to enlarge the mammary veins and, hence, facilitates the extraction of blood samples.
9 One ml. of supernatant fluid was pipetted into a Klett cuvette to which 4.5 ml. of distilled water had been added from a burette and this was subsequently mixed by inversion.
(This was done to lower the concentration to
that range where the Klett is most accurate.)
photoelectric colorimeter was used with a #52 (green) fil ter to determine the relative amount of hemoglobin in the sample. One hundred per cent hemolysis of each suspension was determined by adding 0.5 ml. of the red blood cell suspension used to 2 ml. of distilled water at the begin ning of each experiment.
This tube was then treated like
the other tubes of the series, and its hemoglobin deter mined with a Klett using the same 9:1 dilution.
ing was then used as the basis of comparison for all expexi mental readings in the series. A check was made in order to determine whether or not two hours was sufficient time to allow the system to come to equilibrium.
It was found that at l8°C at .65$
NaCl there was a certain constant degree of hemolysis immediately (or, more exactly, within the first five min utes until a measurement could be taken) upon addition of the red blood cells, and that the percentage of hemolysis increased slowly with a direct relation to time from this point to the point of maximum hemolysis,
just about two
10 hours later.
Determination of hemolysis after this two
hour period, up to as much as four and one-half hours later showed no change. Figure 1 and Table II are typical of the results of all the preliminary experiments.
The test and control
series were almost identical; which, in addition to demon strating the typical shape of the curve and showing that the cortin sample was inactive, also showed that experi ments of this type were reproducible.
series were observed, all giving approximately the same results. This inactive cortin extract (origin not known), and some commercial cortin prepared by Wilson and Company at p concentrations of 1:1000 which also proved inactive, was abandoned and tests were begun on four different bile salts.
These steroids were brought into aqueous solution
by adding equivalent amounts of dilute NaOH solution to weighed quantities of the bile salts, and dissolving them
In one experiment the final cortin concentration was made up to 1:300 by volume. In this test (using Wilson and Company’s cortin) an attempt was made to get an effect with high concentration. Buffers were used in this test, as will be described later in the text. As shown by the data in Table III, a slight protective effect was achieved. But calculation indicated that the effect could be accounted for solely on the basis of. the additional osmotic pressure resulting from the high concentration of cortin.
11 as their Na salt.
A slight excess of NaOH was needed,
which afterward was neutralized with very dilute HC1. This left trace quantities of NaCl, but careful quantita tive determinations showed this amount to be so small as not to materially affect the results when these solutions were used.
All neutralizations were carried out with the
aid of a Beckman pH meter, and the final pH of these bile salt solutions was kept between 6.5 and 7 .2 . The first experiments with these bile salts gave widely divergent results.
The same compound on different
days was shown to be both hemolytic and protective.
the fragility of red blood cells in dilute suspension changes markedly with variations in hydrogen ion concen tration (Jacobs and Parpart, 1931), buffers were then used in an attempt to eliminate this variation.
A mixture of
NaHgPOjj. and Na-pHPO^ was made up so that it showed a pH of 7*0 on the Beckman pH meter.
All remaining experiments
were then conducted as follows: 2 ml. of this phosphate buffer; 2 ml. of NaCl at stated concentration; 0.5 ml. of distilled water for a control or 0.5 ml. of bile salt or cortin solution in distilled water as the test substance, and 0.5 ml. of washed red blood cells in .9$ NaCl.
100$ hemolysis determination was now found by adding 0.5 ml. of red blood cell suspension to 4.5 ml. distilled water.
12 Variations which were found in the earlier experiments before the buffer was used, were now almost completely eliminated. M/lQOO solutions of cholic acid, desoxycholic acid, dehydrocholic acid, and dehydrodesoxycholic acid of high purity (Van Camp Laboratories^) were made up in the manner already stated.
The included graphs (Figures 2, 3, 4;
Tables IV, V, VI, VII) represent the results of fragility tests made with these solutions.
With the exception of
cholic acid, several complete series using .4$ to .86$ NaCl, some involving two checks at each point, and several 2 or 3 point series (using .65$ and .7$ NaCl; or .65, .7 and .75$ NaCl) were observed for these reagents; all gave results which checked with each other.
No complete series
were made for cholic acid as six 2 and 3 point series failed to Indicate any activity. With the above information to show that certain steroids do affect red blood cell fragility, the investi gation returned to cortical extracts. Commercial extracts (Armour, Upjohn, and Parke Davis) were tried at concentrations (i.e., the final concentration in the test tubes when mixed with blood) of 1:6000.
3 Kindly supplied by Dr. Ernest Geiger.
13 effect could be found in carefully controlled experiments with single or duplicate tubes for each test and control. (See Table VIII.)
It will be noted that in each instance
the duplicates check very closely against one another.
test the possibility of subjective error in the Klett determinations, other persons were on two instances asked to check the readings of the investigator.
less than 1$. A graduate student at the University of Southern California, Mr.,James Wilson, had made a water soluble extract of bovine adrenals which he very kindly permitted the investigator to use.
In Table IX is shown the results
of one experiment with this extract.
The rabbit cells
here were treated exactly as those in previous experiments. A similar test was made with defibrinated bovine red blood cells 24 hours old.
Apparently these bovine cells were
more fragile than the rabbit cells, as phosphate-sodium chloride concentrations which preserved almost half of the rabbit cells hemolysed virtually all of the bovine cells. Far more significant, however, is the fact that this cortical extract acted consistently as a lysin to the rabbit cells. A slightly different type of experiment was made to check the role of inorganic salts.
The same 2 ml. phosphate
buffer was used; but in place of the NaCl solutions, a 1%
14 mammalian Ringer's solution was added along with distilled water to this series of tubes so that a series was made up of from .4$ - .85$ Ringer's salt mixture. shown in Figure 5 and Table X.
The results are
The same lytic activity
of this cortin was again demonstrated. Just as a cross check, this Ringer's series was tested with desoxycholic acid. Figure 6 and Table XI.
The results are shown in
Apparently NaCl and Ringer's act
very similarly as hemolytic agents in hypotonic concentra tions.
Tests were then made using Mr. Wilson's cortin
extract and buffered NaCl to see if there was a change in effect by decreasing the concentration of cortin. XII shows a typical result.
It will be seen that concen
trations of 1 :6,000 and 1 :60,000 give the same results, but that at higher dilutions the effect falls off. The major possible sources of error have been men tioned (the large number of times pipette and burette readings had to be made).
This was minimized, however, as
all fluids were measured at the same constant temperature— l 8°C.
Perhaps the greatest error in these measurements
was made in the pipetting of the 0.5 ml. ol* red blood cell suspension, which is more viscous and more difficult to manipulate than the water or salt solutions, although a special blood pipette was used.
CHAPTER IV DISCUSSION Procedure.
The only part of the procedure discussed
in Chapter III which seems to call for justification is the fact that per cent hemolysis is being determined when it has been stated that the problem was one of the effect of steroids on membrane permeability.
This difficulty is
in part resolved by the work of Jacobs (1927), in which he demonstrated that hemolysis produced by hypotonic solutions is due largely to simple osmotic swelling.
The point of
hemolysis may be considered to represent the attainment of a certain degree of swelling— or, stated in other words, the entrance of a definite amount of water into the cell. Each individual erythrocyte has its own point of swelling at which it hemolyzes, but in a relatively homogeneous group of a great number of cells there is a relationship between hemolysis and the amount of water taken into the cell. The red blood cell membranes in general and this problem in particular.
As soon as an attempt is made to
give an accurate picture of the red blood cell, a serious problem arises.
In spite of the fact that a wealth of
material has been collected by innumerable workers on the
16 chemical and physical properties of the red cell, its real structure is still only guessed at.
Many theories of
“simplified 11 structure have been proposed, but each of these has had to be changed as new material has been made available. The chemical composition of the red blood cells of various animals has been studied in detail, and this in formation is available in Ponder's (19^3) excellent mono graph.
The difficulty is integrating these constituents
into their places in an intact erythrocyte.
theory is that of
Bidloo who, in 1685,
red blood cell is
a bag-like structure
with a solution of
hemoglobin and other salts. This was
opposed by other theories, notably that of Rollet (1862), who thought of the erythrocyte as a colorless stroma or matrix in which the hemoglobin was held (cited in Ponder, 19^3).
Actually, the ideas of a surface envelope and an
structure are not mutually exclusive. The exis
tence of an internal stroma has never been proved or dis proved by any method so far used to test it. That the red cell has a surface ultrastructure is demonstrated by many of the erythrocyte's characteristics, including its selective permeability to certain ions and the great electrical resistance of its surface components as compared to that of the plasma (Davson and Danielli,
That this ultrastructure acts as a preferential
barrier to diffusion is probably true to a large extent, but it must be remembered that the red cell has a measur able metabolic rate (Ramsey and Warren, 1932), and that this metabolism must also play a part in maintaining the different ion relationships within the cell (Davson and Danielli, 1933)(Dean, Noonan, Haege, and Linn, 1941) (Wilbrandt, 1937)*
When living cells die, metabolism
stops, and their membranes lose the ability to maintain internal ion concentrations different from their surround ing medium (Hober, 1945). Very briefly, this surface ultrastructure of the erythrocyte is thought to be approximately 200 fi thick (Waugh and Schmitt, 1940), and to be made up of a lipidprotein mosaic, with the lipid arranged radially and protein tangentially (Furgott and Ponder, 1941).
lipid layer is 50 - 100 2 thick (depending upon the method used to measure it); and electrical studies indicate that the surface is dominated by acid groups (Parpart and Dziemian, 1940), probably the phosphoric groups of cephalin and related compounds. face is not one of protein.
It is clear that the sur
(Surface here refers to that
part of the ultrastructure which possesses the charged groups responsible for electrokinetic phenomena— not necessarily to the region of outermost molecules.)
18 portion of the red cell envelope which is referred to as the 11membrane 11 and which is responsible for selective permeability of ions is probably a layer of oriented o molecules about 30 A thick, either at the surface of the envelope or included within its thickness (Fricke, 1923)• Now, the main problem of this discussion must be dealt with— namely, which of these characteristics of the erythrocyte determines its osmotic fragility; and, what is the probability that each or any of these are altered by steroids? Before discussion is made on those effects which are most probable, and which have been dealt with at length by the most capable workers in this field, this investiga tor would like to present a possible mechanism by which lysins, inhibitors and accelerators might produce their effect on the erythrocyte.
There is no direct laboratory
evidence to support this theory, but while reviewing the literature, several articles were found which, when examined together, indicated its possibility. It is here suggested that an increased or lowered resistance of the red cell to hemolysis might be brought about by altering the metabolism of the red cell; and that some substances increase or decrease the fragility of the erythrocyte by the effect they exert on its meta bolism.
19 Obviously, simply lowering the metabolic rate will not cause hemolysis, as red cells have been kept at 4°C for days without any apparent ill effect.
Glassman and Parpart (193&) report on some work which sug gests the possibility of the above theory.
erythrocytes and mixed them in a ratio of 1:500 with .077 M NaCl (approximately .45^), buffered to pH 7*3*
hypotonic salt concentration there was no hemolysis at 40°C, while upon lowering the temperature to 0°C, almost complete hemolysis was obtained.
Of course, it is most
probable that a change of 40°C is going to affect more components of the cell than its metabolism; but it seems doubtful if any other aspect of the cell would be so greatly disturbed. It has been shown (Tipton, 1933) that there is a temperature coefficient of between 1.55 and 2.75 for res piration of nucleated red cells.
It is not improbable to
suppose that similar values are true for mammalian ery throcytes. Other known facts would tend to support this theory, although they are by no means submitted as proof.
experiments have shown that the red cell is permeable to potassium (Harris, 194l)(Davson and Reiner, 1942).
experiments suggested that some sort of metabolic pump must exist to maintain the high potassium concentration
20 found in the red cell— which, if true, would mean that altering the metabolism of the cell woudl also alter the osmotic forces in and around it.
As a further indication
that this metabolic pump exists, and that it can be affected (in this instance by temperature), it has been shown that potassium lost while the red cell is kept at 4°C will re-enter the cell when this pump is "speeded up,” by raising the temperature to 37°C.
The rate of re-enter
ing is even more greatly accelerated if glucose ("fuel for the pump") is added to the system (Harris, 1941). Admittedly, the above theory is very highly specu lative.
But it must be remembered that nothing is known
for sure as to how lysins and sensitizers affect the cells upon which they act. How well this theory might be applied to the prob lem here discussed is not known, as no data are available as to whether or not steroids in low concentration affect red blood cell metabolism.
Some experimental evidence on
this is needed. The most -probable explanations which workers in the field have hypothesized for changes in red cell fragility concern changes in either the mechanical properties of the membrane— that is, changes in the membrane which would permit it to swell to greater size, or prevent it from reaching as great a size before breaking down to release
21 hemoglobin; or the other usual explanation for alteration of fragility has to do with membrane permeability effects --changes in the ability of the membrane to control the entry or exit of water and other substances to and from the cell.
Various authors, using different types of
hemolytic systems have come to different conclusions as to what in the cell has been affected.
All seem to agree
that the effect is on surface components of the cell, however. In one of his many papers, Ponder (1937) discusses the disk-sphere transformations of red cells in saline when in contact with surface (such as a cover glass), or in plasma upon addition of lecithin.
The cell will go
through the same series of changes in either case— normal disk to ffthorn apple 11 (crenate) to sphere and eventual hemolysis.
Ponder claims these changes and the hemolysis
are due to effects on the surface components of the cell. He is not more specific.
Again Ponder (l9^5a), in another
paper states that inhibitors and accelerators of lysin induced hemolysis work in approximately the same way. They affect some component of the surface membrane of the cell, rather than affecting the lysin.
He thinks it is
probably the same component of the membrane that the lysin is attacking.
Again he is not more specific, for here he
is at the edge of knowledge on the subject.
But it should
22 be noted that the main line of evidence for the above is a negative one only; namely, that there is no indication of the formation of complexes between lysin and inhibitor, or lysin and accelerator* In another paper, Gordon, Kleinberg and Ponder (1937), in discussing the decrease in red cell fragility six weeks after spleenectomy of the donor animal, state that this decrease is due to the ability of the red cell to reach a greater size before hemolyzing.
This means the
effect was one of ,5membrane mechanical effect,M i.e., the membrane was so changed that it could stretch more before breaking down to permit the hemoglobin to escape. Raushwalbe, in his paper which was referred to earlier, went further than Just testing the effect of cortical extract on red cell hemolysis.
He attempted to
see how these compounds affected the swelling of red blood cells in hypotonic salt.
Working with salt concentrations
which gave some hemolysis with cortin and a greater per centage of hemolysis without it, he found the per cent hemolysis by testing the supernatant fluid in a photocolorimeter for hemoglobin, as was described earlier.
he took other aliquots of the same samples and spun them down in hematocrit tubes.
He found the hematocrit on the
sample with cortin higher than on the sample without— but it was higher than it should have been on the basis of the
23 differences in degree of hemolysis as shown on the colori meter.
He therefore concluded that the reason there was
less hemolysis with cortin than without in hypotonic salt was due to the fact that the cells in the test solutions were able to swell to greater size before hemolyzing. Raushwalbe ran similar tests on swine blood and hypotonic glucose and, interestingly enough, found just the opposite effect--i.e., he claims that the reason red cells hemolize to a greater degree with cortin than without in hypotonic glucose is that the cells are not able to reach as large a critical volume. Upon analyzing the available .information, the only conclusion that can be drawn is that it is probably the erythrocyte envelope rather than any internal structure that is affected in any change of the hemolytic properties of red blood cells.
However, it is possible to speculate
on probable ways the steroids can affect the red blood cell envelope and its fragility. In the first place, these substances are very sur face active.
In M/1000 solutions of the sodium salt of
cholic acid and those of its derivatives mentioned earlier, foaming was obtained on shaking; proving that even at these low concentrations the steroids used decreased the surface tension of water.
No experiments were made on the
final test solutions of m /6000 steroid to determine
24 whether or not there was a drop in surface tension, but presumably there was.
Since the physical state of the
cell surface is believed to be essentially that of a liquid rather than a solid film (Chambers, 1938)(Chambers and Kopac, 1937)(Kopac and Chambers, 1937)* such differ ences in the surface tension of the surrounding media might be expected to make a difference in permeability phenomena. Another highly probable mechanism through which steroids could exert their effect on the surface ultra structure, would involve actual chemical combination with some surface components.
Good experimental proof of this
is lacking; but in an attempt to justify it, reference is made to another of Ponder1s papers (1943b) in which he demonstrates that lysins are rendered non-lytic by the stroma of the red blood cells during the hemolytic reac tion.
However, this is only an indication and not proof
of chemical combinations, because Ponder points out that while the lrdelytifyingM reaction may be due to combination with surface components, it may also be due to a definite inhibitory effect of the cell. demonstrate which was the case.
His experiments did not But, here again, even if
it could be definitely shown that there is a chemical combination between steroid and surface components, there is as yet no apparently obvious way of telling for sure
25 whether or not this combination is affecting the membrane permeability or its mechanical strength. The only method this investigator has been able to devise which would attempt to find out which of the above two phenomena is actually occurring, is based on that of Raushwalbe, which was mentioned earlier.
tates the hypothesis that if only the permeability of the cell is being affected, it will hemolyze at the same critical volume in hypotonic NaCl with or without steroid present.
The steroid will only affect the reaching (or
the nnot-reachingft) of this critical volume.
If, on the
other hand, the steroid is affecting the surface ultra structure's mechanical strength, then the red blood cells of the test solution should hemolize at a different criti cal volume than the controls.
This hypothesis unfortunate
ly would not demonstrate the third possibility— namely, that both phenomena occur at the same time, and steroids, in reacting with the surface components, affect both membrane permeability and mechanical strength. The theory behind this experiment is simple.
concentration of salt is chosen which gives partial but not complete hemolysis in both the controls and test solutions, the per cent hemolysis can accurately be deter mined by the previously stated method of determinating the hemoglobin concentration in the supernatant fluid in a
How, if the effect of the steroid is only on
membrane permeability, the average volume of all the remain ing cells which have not hemolized should be the same both in the controls and test solutions.
For example, let it
be supposed that two tubes, A and B, are set up.
contains the same quantity of a hypotonic NaCl solution such that there is partial but incomplete hemolysis both with steroid and without. the test.
Let A be the control and B be
Now, to these tubes is added the same quantity
of the same red blood cell suspension (washed!), and they are allowed to stand for two hours at about 20°C.
this, samples of each are taken and spun down in a hemato crit, while the rest of both samples are spun down so the supernatant fluids can be read colorimetrically.
example, let it be supposed that control A gave 20$ hemolysis, while test B showed 30$ hemolysis.
permeability of the red cell was the only characteristic affected, then the hematocrits of A and B should have the ratio of 8:7*
If they have any other ratio, then on the
basis of the above stated hypothesis, membrane mechanical effects are being demonstrated. While this investigator has never performed the above experiment, it appears to be a logically valid one. A more precise means of measuring the red cell volume may be wanted, and these are available.
27 or defractrometric methods may be used; or perhaps the T-182^ method of Hunter and Shohl could be adapted.
attempt will be made to explain these here, but reference to them and others‘.will be found in Ponder's monograph. (Ponder, 19^8).
CHAPTER V EVALUATION OP RESULTS AND COMMENTS Contrary to the conclusions presented by Raushwalbe, water soluble extracts of adrenal cortex could not be shown to exert marked effect on red blood cell fragility. Certainly, they do not at concentrations of 1:1,000,000 or less, which might be found in normal physiological systems. From the data here presented one has the choice of deciding either that cortin has no effect at all, and that perhaps there was some impurity in the student preparation, or that the results obtained with this last sample are cor rect and that the commercial samples were inactivated in some way.
This investigator would tend to agree with the
second theory, since the commercial preparations were not freshly prepared; and while they were refrigerated in stoppered bottles, their effect on fragility is apparently slight and any deterioration or oxidation might affect it. Desoxycholic acid was found to be quite hemolytic in salt solutions which were at normal physiological con centrations.
This phenomena has been reported previously
in literature which has been collected and presented in a text by Sobotka (1937)*
29 As is seen from the data presented herein, however, it is apparent that this substance is less effective as a hemolytic agent as the NaCl concentration becomes more hypotonic, until at a concentration of .65$ NaCl or less it has no effect at all. The only explanation that this investigator could offer would be to theorize that this steroid attacks only one type of cell of the many that are normally present in a sample of normal blood— perhaps the oldest cells which are most easily destroyed, or perhaps, the reticulocytes, although it is by no means certain that these are less resistant to hypotonic saline (Ponder, 1948).
At any rate,
it is obvious that desoxycholic acid hemolyzes those cells most readily hemolyzed by hypotonic saline; for those cells which withstand salt concentrations of .65$ or lower are apparently unaffected by this steroid. In experiments with desoxycholic acid and NaCl, the curve flattens between NaCl concentrations of .7$ and .75$ (see Figure 2).
Such a phenomenon is possibly an indica
tion of a bimodal red cell population.
In the one experi
ment conducted with desoxycholic acid and Ringer1s, there seems to be an increase in hemolysis from .7$ to .75$ salt (see Figure 6).
More work is necessary in order to
demonstrate whether or not this is an antifact.
30 From the data, it is seen that cholic acid gave no demonstrable effect.
Grodins, Berman and Ivy (l94l) (see
results of their work on page 6) found cholic acid to be a moderately hemolytic substance in an otherwise isoplethecontic solution.
But in Table XXII in Sobotka*s text
it is seen that most investigators found no effect with this steroid at concentrations less than one part in a few hundred. The results obtained with dehydrocholic acid and dehydrodesoxycholic acid are difficult to interpret.
the basis of the material in Table XXII in Sobotka and the other papers previously mentioned, one would certainly expect that they either show slight hemolizing powers or no effect at all.
There is one fact which might possibly
be used as the basis of an explanation of the phenomenon demonstrated.
Both Ponder in his monograph, and Davson
and Danielli in their text, upon examination of the work in the field to date, caution that what has been found to be true for the red blood cells of one species cannot, without experimental evidence, be inferred to be true for the erythrocytes of another.
Chemical compositions of
cells vary from one species to another, as do their other properties. Thus it might be possible that the results here
31 given for dehydrocholic and dehydrodesoxycholic acid differed from other similar experiments conducted with it in that blood from different species was used.
CHAPTER VI SUMMARY AND CONCLUSIONS The effects of five water soluble extracts of adrenal cortex tissue and four bile salts (cholic, desoxy cholic, dehydrocholic, and dehydrodesoxycholic acid) on the permeability of the erythrocyte membrane have been tested.
This has been done by determining the per cent
hemolysis of red cells in hypotonic NaCl and mammalian Ringer’s solution with and without the steroids present. It appears that desoxycholic acid is hemolytic in. salt solutions which are close to physiological concentra tions; while dehydrodesoxycholic acid and dehydrocholic acid seem to be slightly protective in hypotonic salt solutions.
Cholic acid showed no activity.
Commercial cortical extracts (Armour, Upjohn, ParkeDavis, Wilson) were inactive.
One student preparation
seemed to be slightly hemolytic.
B I B L I O G R A P H Y
BIBLIOGRAPHY Berliner, Frieda, and Rudolf Schoenheimer, f,Hemolytic and Anti-hemolytic Properties of Bile Acids and Sterols in Relation to Their Structure,11 J. Biol. Ohem. 124:525, 1938. Chambers, R., lfThe Physical State of Protoplasm with Special Reference to Its Surface,” Am. Naturalist, 72:141-59, 1938. _______ , and Kopac, M. J., "The Coalescence of Living Cells with Oil Drops: I Arbatia Eggs Immersed in Sea Water,H J.C.C.P., 9:331-43, 1937. Creed, F.f.f., ”The Estimation of the Fragility of Red Blood Corpuscles,” J. Path. & Bacti., 46:331-340, 1938. Dacie, J. V., M. B. Lond and Janet M. Vaughan, ”The Fragility of the Red Blood Cell: Its Measurement and Significance,” J. Path. & Bacti., 46:341-356, 1938. Davson, H., and J. F. Danielli, ”Studies on the Permea bility of the Erythrocyte: V Factors in Cation Permeability,” Biochem. J., 32:991, 1938. , The Permeability of Natural Membranes., New York: The Macmillan Co., 1943. 353 PP. Davson, J., and Reiner, J. M., ”lonic Permeability: An Enzyme-like Factor Concerned in the Migration of Sodium Through the Cat Erythrocyte Membrane,” J.C.C.P. 20:325, 1942. Dean, R., T. R. Noonan, L. Haege, and W. I. Linn, "Per meability of Erythrocytes to Radioactive Potassium,11 1- G e n - Physiol.. 24:353, 1941. Fricke, H., ”The Electrical Capacity of Suspensions with Special Reference to Blood,” J. Gen. Physiol., 9:137, 1925. Furgott, R. F., and E. Ponder. "Electrophoritic Studies on Human Red Blood Cells/1 J. Gen. Physiol., 24:447, 1941. Gordon, Albert S., Wm. Kleinberg, and Eric Ponder, "Decreased Red Cell Fragility After Spleenectomy,” A m . J. Physiol., 120#1: Sept., 1937.
34 Grodins, Fred S., A. L. Berman, and A. C. Ivy, "Observations on the Coxecities and Choleritic Activities of Certain Bile Salts,” J. Lab. & Clin. Med., 27:181. 1941-42.-------------------- -------- ----Harris, J. E., ”The Influence of the Metabolism of Human Erythrocytes on Their Potassium Content,” J. Biol. Chem. 141:579, 1941. Hober, Rudolf, Physical Chemistry of Cells and Tissues. Philadelphia; The Blakiston Co., 1945. £>35 PP* Hopps, 'Howard C., M.D., and A. M. Shedelen, ”A Quantitative Method for Determination of Fragility of Erythrocytes,” A m . J. Clin. Path., 17:418-421, May, 1947Jacobs, M. H., Harvey Lecture, 22:146-164, 1927. _______ , H. N. Glassman, and Arthur K. Parpart, ”Osmotic Properties of the Erythrocyte: VIII on the Nature of the Influence of Temperature on Osmotic Hemolysis,” J.C.C.P., 8: #4, p. 403, 1936. _______ , and A. K. Parpart, ”The Osmotic Properties of the Erythrocyte,” B iol. Bull., 60:95, 1931* Kopac, M. J., and R. Chambers, ”The Coalescence of Living Cells with Oil Drops: II Arbatia Eggs Immersed In Acid or Alkaline Calcium Solutions,” J.C.C.P. 9:345-61, 1937. Parpart, A. K., and A, J. Dziemian, ”The Chemical Composi tion of the Red Cells Membrane,” Cold Spring Harbor Symposium on Quantitative Biology, 8:17, 1940. Ponder, E., 1,The Physical Structure of the Red Cell Membrane, with Special Reference to its Shape,” Tr. Faraday Society, LXXIII, #193, part 8, August, 1937. _______ , ”The Mechanism of the Inhibition of Hemolysis: V Inhibitory Processes Occurring in*the Course of Simple Hemolytic Reactions,” J. Gen. Physiol., 28:203, 1945 (a). ~ _______ , ”The Mechanism of the Inhibition of Hemolysis: V Inhibitory Processes Occurring in the Course of Simple Hemolytic Reactions,” J. Gen. Physiol., 29:203. 1945 (b).
35 Ponder, E., Hemolysis and Related Phenomena, New York: Green and Stratron Company, 1946. 3^5 p p . Ramsey, R., and C. 0. Warren, nThe Rate of Respiration of Erythrocytes,11 Quant. J. E x p . Physiol., 22:49, 1932. Raushwalbe, Herbert, "Permeabilitats Studren mit Cortidyn,a Arch. f. E x p . Path. u. Pharm., 195:^25, 1940. Saslow, G., f,The Effect of Washing upon the Resistance of Erythrocytes to Hypotonic Solutions,ff J. Physiol., 74:262, 1932. Sobotka, Harry, Physiological Chemistry of the B ile. Baltimore: The Williams and Walkins Company, 1937* Tipton, S. R., ,!Pactors Affecting the Respiration of Vertebrate Red Blood Cells," J.C.C.P. 3:313, 1933* ' Waugh, D. F., and F. 0. Schmitt, HInvestigation of the Thickness and Ultrastructure of Cellular Membranes by the Analytical Leptoscope, ** Cold Spring Harbor Symposium on Quantitative Biology, 8:1940, 17Wilbrandt, W., f,The Properties of Membranes, Natural and A r t i f i c i a l , T r . Faraday Soc., 33:956, 1937.
A P P E N D I X
CHEMICAL STRUCTURE OF STEROIDS
3, 7, 12, trihydroxychol&nic acid*
Desoxycholic Acids .+/U \
3, 12, dihydroxyeholanic acid.
Dithocholie Acid: Cholanlc Acid
N c»(v- 14
* Concentrations, all 1:6000. Solutions buffered to pH - 7.0. One of two identical experiments.
TABLE IX JAMES WILSON fS CORTIN* Per cent NaCl
Colorimetric control reading
.4 A .6 .6 .8 .8
233, 244) 208 , 214) 110. 113'
Average of control reading 24l 211 111.5
Colorimetric test reading
250 246 221
228 122 125
Per cent of control average 104 102 104.5 108 109 111
* Concentration 1:6000. Solutions buffered to pH 7.0, Cortin diluted one-half hour before use. Only one experiment performed.
TABLE X JAMES WILSON*S CORTIN WITH RINGER*S* Number of m l . H 2O
1.2 1.0 .8 *7 .6 *5 -.4 *3
Number of ml. 1 per cent Ringer1s
0.8 1.0 1.2 1 -3 1.4 1*5 1.6 1.7
Final per cent salt CONTROL .4 *5 .6
.65 *7 *75 .8
236 218 159 • 121 66 48
Per cent hemolysis
82 65*5 60.5 44 34
TEST 1.2 1.0 .8
.7 .6 *5 .4 *3
0.8 1.0 1.2 1*3 1.4 1*5 1.6 1*7 100$ Hemolysis
.4 *5 .6
.65 *7 *75 .8
310 294 249 227 180
238 89 59
82 69 63 50 38 25
Reading : 360
* Concentration of cortin 1:6000. Solutions buffered to pH 7*0. Cortin diluted one-half hour before using. One of two identical experiments (see Figure 5)*
TABLE XI DESOXYCHOLIG ACID IN RINGERfS* Number ml. Ringer1s
Number m l . H 2O
0.8 1.0 1.2
1.2 1.0 .8 .7 .6 • *5 .4 .3
Final per cent salt CONTROL .4 .5 .6
1.3 1.4 3L*5
101 76 34 15 5
Per cent hemolysis
88.5 79.5 54 40
28 13 6
1.2 1.0 .8
0.8 1.0 1.2
.7 .6 .5 .4 .3
1.3 1.4 1.5 1.6 1.7 100$ Hemolysis
.6 .65 .7 .75
228 191 142 117 90
90 73 56 46 35 39 33 31.5
* Concentration m/6000. Solutions buffered to pH 7«0. Only one such experiment performed (see Figure 6 ).
TABLE XII JAMES WILSON!S CORTIN— DILUTION EXPERIMENT* Cortin concentration 1:6,000 1:6,000 1:60,000 1 :60,000 1 :600,000 1 :600,000 1:6,000,000 1:6,000,000
123 123 124 123 115
48 48 48 48 45 45 45 46
Standard solution (without cortin) readings: 113, 112 * All solutions contain 0.6 ml. H 2O, 1.4 ml. 1$ Ringer's solution, and 2 ml. phosphate buffer, pH 7*0. Cortin was diluted one-half hour before use. One of two identical experiments.
N o . 6201, U n iv e r s ity B o o k s to re, Los Angeles
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