University of Southern California Dissertations and Theses

The effect of steroids and lecithin on the resistance of the beef erythrocyte to osmotic stress

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The effect of steroids and lecithin on the resistance of the beef erythrocyte to osmotic stress

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Content THE EFFECT OF STEROIDS AND LECITHIN
ON THE RESISTANCE OF THE BEEF ERYTHROCYTE
. TO OSMOTIC STRESS
Presented to
the Faculty of the Department of Zoology
University of Southern California
In Partial Fulfillment
of the Requirements for the Degree
Master of Arts
By
James Wilson
August, 1950
UMI Number: EP67198
All rights reserved
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a note will indicate the deletion.
Disssrtaîiorii PüMisNmg
UMI EP67198
Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author.
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unauthorized copying under Title 17, United States Code
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2. 'fi
This thesis, written by
......................... Jam.e^...WXl.s.on..........................
under the guidance of hÂ^—Faculty Committee,
and approved by a ll its members, has been
presented to and accepted by the Council on
Graduate Study and Research in partial fu lfill
ment of the requirements fo r the degree of
........................M asjt.er.. p .f„ ArÆ .S........................
........ DeDartment..pf_.ZpQlpgy:........
Date
Faculty Committee
Chairman
^ .......
____
TABLE OF CONTENTS
PAGE
STATEMENT OF THE PROBLEM........................ 1
REVD^f OF T^IJTER^^mE
I in&ATIŒV(WPE&#%UnLITY]^STER&næ . . . . 3
II PERMEABILITY ............................... 6
III METHODS OF MEASURING PERMEABILITY .......... 9
ME]?H(%DE; jlND T3EC]8inC%UE%3
I GENERAL ............... 11
II PREPARATION AND SELECTION OF RED BLOOD CELL
SUSPENSIONS......................... 12
III PREPARATION OF ADIHUNO-CORTICAL EXTRACT .... 15
IV INlEa)ARA]?iC»% (IF CËKILIüSIICROL, IIEE%]Xin:KIRTIC()8T%SIK)N]S
ACETATE, AND LECITHIN SOLUTIONS ......... l8
V OSMOTIC PRESSURE EXPERIMENT ................ 20
VI HEAT EFFECTS ............................. 21
VII COLORIMETRY..................... 21
EXPERIMENTAL RESULTS ................................. 23
DISCUSSION OF EXPERIMENTAL RESULTS .................... 60
CONCLUSIONS......................... 66
SUMMARY................... * 67
BIBLIOGRAPHY ........................................ 68
The size of this type is satisfactory.
Frederick Fey Shelden
Committee Chairman
Zoology Department
University of Southern California
STATnEÜGDN]? CÜT T7HE fTR0BLB3W
It has been noted by many investigators that hypo-function of
the adrenal cortex, as in Addison’s syndrome, produces marked changes
in water and sodium metabolism, and in the fluid balance throughout
the entire body. Harrop, et al (1935), state that deranged water
metabolism and cell shrinkage are in evidence and Turner (1948) notes
that hemoconcentrâtion and tissue dehydration are apparent in hypo-
cortidism. Because of the derangement in fluid balance it is probable
that permeability of both cells as individual units and of tissues as
a v/hole are influenced by adrenal cortical hormones. Hyman and
Chambers (1943) have obtained evidence that adreno-cortical extracts
reduce the rate of edema formation in the perfused hind limbs of frogs.
Information more pertinent to this research comes from Rauchschwalbe
(1940) who states that in hypotonic NaCl solutions the rate of hemo
lysis of red blood cells is affected by Cortidyn, an adrenal cortex
extract.
It can readily be seen that the adrenal cortex plays an impor
tant part in the water metabolism of the organism through the steroid
hormones it elaborates. In considering the effects of these hormones
it would be interesting to note the effects of other important ster
oids, such as cholesterol or desoxycorticosterone acetate, on the
permeability of cells.
Cholesterol and desoxycorticosterone acetate have, as their
major component, the steroid nucleus, thus relating then closely to
the adreno-cortical hormones. Furthermore, according to Kleiner
2
(1948) and Abelin (1946), cholesterol is thought to be the precursor
of all steroid compounds in the body, particularly the adreno
cortical steroids. Desoxycorticosterone acetate, commonly referred
to as DOC A, .is a synthetic steroid v/hich has an action similar to
some of the fractions of the adrenal cortex extract. Lecithin, a
phospholipid, has no structural kinship to the steroids, but the
study of its effects on permeability was included in this research
because of its lipoidal nature, its prominence in all cells as
stated by Gortner (1929), and its antagonistic action to cholesterol
in some physiological processes as discussed by Heilbrunn (1948).
Since both cholesterol and lecithin are pre-eminent physio-chemical
compounds having an intricate interrelationship and prominence in the
structure of the plasm membrane, it was thought that to study them
in conjunction with DOGA, and other cortical steroids, would perhaps
throw some light on the physiological mechanics of permeability.
Cartland and Kuizenga (1936) warned against raising the extract,
prepared according to their method, above 45°C. The effect of heat
is to destroy all physiological activity. It was decided to stuay
the effects of heat on the steroids employed in this research, to
ascertain if, in heating the cortical extract, the factor which in
fluences permeability is also destroyed when physiological, or. life
sustaining activity is destroyed.
The problem resolves itself into the study of the influence of
whole water soluble adreno-cortical extracts, desoxycorticosterone
acetate, cholesterol and lecithin on the permeability of the plasma
membrane and the demonstration of the effects of heat on the activity
of the steroids regarding permeability.
REVnm OF THE LITERATURE
I RELATION OF PERMEABILITY TO STEROIDS
Turner (1948) sumnarizes the effect of adrenal insufficiency
in saying that there is a fluid derangement with a hemoconcentra
tion, the fluid balance being swxing to the tissues; and there the
fluids are apparently immobilized.
In 1933 Hartman found that in the adrenalectomized rat, water
content of the liver and the skin is increased considerably. On the
other hand, Harrop, et al (l935), found that in the dog there is a
derangement in water metabolism accompanied by cell shrinkage and
tissue dehydration. Using the rate of edema formation in the hind
limbs of a frog, Hyma.n and Chambers (1943) developed a method which
indicates the reduction of experimental edema rate when adrenal cor
tex extract is present,
Cameron (1947) also reviews that much contradictory evidence has
been uncovered concerning true permeability dysfunction, but most of
the prominent authorities maintain that hypo-cortidism leads to a
fluid shift accompanying a change in permeability, the exact nature of
this change being unknown. Hartman and Brownell (1949) throw some
light on the situation by stating that the apparent high loss of water
and hemoconcentration that accompanies adrenal insufficiency, could be
traced, in some cases, to membrane failure regarding permeability.
More specifically Hartman (1933) found an increase in water content of
liver and skin after adrenalectomy, suggesting that these facts support
the theory of permeability dysfunction.
4
As has been mentioned Hymen and Chambers (l943) found that cor
tex extract, present in the perfusate, decreased the rate of forma
tion of edema in frog’s hind limbs. This reduction in the rate is a
direct indication that fluid was hindered in its passage through the
tissues as a result of a decrease in permeability apparently caused
by the cortex extract. On the other hand, Palmer and Joseph (1946)
could not reproduce Hyman and Chamber’s effect, so that their con
clusions were not in accord v/ith the former experiment*
Rauchschwalbe (l940) states in the body of his paper that in hypo
tonic NaCl solutions the rate of hemolysis of red blood cells is in
creased by Cortidyn, an adrenal cortex extract; the reverse being true
in hypotonic glucose solutions. His summary states that Cortidyn in
creases hemolysis in both cases. Although this report is confusing,
these facts indicate that adreno-cortical extracts do exert an influ
ence on the permeability of the plasma membrane. Sphering of red blood
cells in isotonic NaCl was produced by Netzcky and Jacobs (1941) using
desoxycorticosterone derivatives. Sphering indicates a change in shape
with an increase in volume due to the entrance of some penetrating sub
stance at a rate higher than normal. The permeability to the penetra
ting substance must therefore be increased to allow this passage.
Desoxycorticosterone is much more pov/erful than corticosterone in its
effect on electrolyte balance according to Kleiner (l946).
There evidently is some profound effect on permeability exer
cised by the adrenal cortex secretions. This effect can be seen in
studies on entire organisms by Harrop, et al (1935), on whole tissues
by Hyman (1943) and on cells by Rauchschwalbe (1940). Kleiner (1948)
5
states that no single extract fraction is the equivalent of a good
whole adrenal cortical extract. If, therefore, any synergistic
effect is to be demonstrated, whole extract should be employed.
Some information on the role of cholesterol in the adrenal cor
tex may aid in answering the questions concerning the steroid nucleus
and its permeability effects. Dalton, et al (19^4), Selye (1937);
Sayers, et al (19^^) and Abelin (19^1-6) all agree that in an organism
under stress, the cholesterol content of the adrenals increases marked
ly and Abelin (19^6) states further that this increase is associated
with the augmented production of adrenal cortex steroids of which
cholesterol is the precursor.
Mason et al (1937) and Mason (1939) discovered that all physio
logical activity of steroid compounds is destroyed if the ethyl-
enic bond is destroyed, provided, of course, the compound possesses
this feature. Cartland and Kuizenga (193^) state emphatically, that
to raise the temperature of their adrenal cortex extract above .
will affect the activity of the whole extract. It is possible, then,
that the increase in temperature renders the compound ineffective,
or alters the activity of the adrenal cortex extract by destroying the
4-5 double bond.
Without a doubt, the steroids play an important part in the entire
permeability picture, either as non-adreno-cortical compounds, such as
cholesterol, the raw material for adreno-cortical synthesis, or in
fractions of the adreno-cortical synthesised compounds. It can be
readily seen, then, that the steroids are closely linked to the per
meability phenomena.
6
II PERÎ'ÎEABILITY
Since, according to Keilin and Hartree (1946), the rates at
which many of the physiological reactions of metabolism may take
place are often controlled by the permeability of cells to various
metabolites, activators, and inhibitors, the study of the mechanism
of cell permeability becomes of considerable theoretical and prac
tical importance.
Permeability, as defined by Barnes (1937) and Davson and
Danielli (1943), is essentially the property of a membrane which
allows substances to pass through it. The amount passed is expressed
in gram moles per square micron of surface area per unit difference
in concentration on either side of membrane expressed in moles per
liter per unit time. Permeability can be non-selective, obeying the
laws of simple osmotic diffusion as does parchment, or permeability
can be highly selective, allowing only certain substances to pass.
Selective permeability is associated with all life processes, and
any derangement of permeability, or dysfunction of the membrane,
produces dire results. Free diffusion spells instant death. Little
is known about the chemical nature of the cell membrane and its per
meability, but both Lillie (1918) and Chambers (1922) agree that
this permeability is a function of the surface membrane alone, and
that metabolism is directly controlled by the membrane and its selec
tive permeability. This is also the view of Heilbrunn (1948).
Osterhout (1923) and Adolph (1936) also conclude that permeability
is a characteristic of life and living activity. Naturally, the
structure of a membrane governs the passage of substances, so it is
7
logical to assume that permeability is a direct function of struc
ture. Many authorities have reviewed the theories of membrane struc
ture. Davson and Danielli (l943) take up all the prominent theories.
Heilbrunn (1948), Sharpe (1926) and Schmitt, Bear and Ponder (1936)
(1938), discuss the Lipo-Protein-Active-Patch Theory, which states
that the membrane is composed of areas of protein and areas of lipoid
and that substances pass through each "patch" depending on their
relative solubilities in protein or lipoid; all discussions and con
clusions in this research will be founded on this theory. The basis
for this assumption is Rideal’s work (l945), which correlates many of
the widely separated facts. He attempts a plausible explanation of
the Lipo-Protein theory. He states that proteinoids pass through the
protein portion, fats through the lipoid portion, and all other com
pounds through the junction of the lipbid-protein areas. Naturally,
no one theory, per se, can completely answer all questions or explain
all a.nomalies, but for the purpose of this research, Rideal's explan
ation will be adhered to since many of his conclusions are in keeping
with the results obtained in this research.
Interesting physical reactions of lipoids, possibly related to
permeability effects and membrane behavior, have been discovered.
The most pertinent is the work of J, B. Leathes (1923) regarding
cholesterol and lecithin and their effects on mono-molecular films
of fatty acid. Essentially, Leathes (1923) believes that the be
havior of cholesterol and lecithin is to contract and expand mono-
molecular layers of fatty acids, respectively. That this effect is,
no doubt, in agreement with their tendencies to retard, or to in-
8
crease hemolysis, respectively, is the unsupported contention of
Bayliss (1924). The effects of surface active compounds on the
plasma membrane which is the Lipo-Protein system under consideration
are of importance» Ponder (1946) states that the action of lysins, or
compounds that readily produce hemolysis by chemically attacking the
plasma membrane, is similar to the penetration and breakdown of mixed
protein-lipoid films and cholesterol can combine with these lysins
and render them ineffective. Rideal (1945) also expresses the view
that biological activity is a function of the amount of the substance
adsorbed at lipoid-aqueous or lipoid-air interfaces thus correlating,
to some degree, the relationship between physical and physiological
effects of surface active compounds, such as lysins, and the impor
tance of the membrane and its relationship to its immediate environ
ment .
Jacobs (1927) states that water is one of the most important
penetrating fluids since it comprises 60f o of the weight of the eryth
rocyte and water is constantly passing across the plasma membrane,
water balance throughout the entire organism is important since de
ranged water metabolism, as in Addison’s disease, causes, in the later
stages, tissue dehydration and cell shrinkage resulting in death
according to Harrop and his co-workers (1935)• Water occupies a promin
ent position in the physiology of the organism, tissue and particularly
the cell, so, to study the effects of adreno-cortical steroids on the
penetration of water into the red blood cell is particularly appro
priate .
9
III METHODS OF MEASURING PERMEABILITY
The hemolytic method is used for measuring permeability to water
in this research. Hemolysis is best measured by photoelectric means,
as described by Davson and Danielli (1943),
Regarding the erythrocyte and permeability studies. Fonder and
Jacobs seem to be the leaders although their results do conflict
occasionally. Fundamentally, erythrocytes are permeable to water
and to anions according to Jacobs (1927) and Barnes (1937).
Jacobs (l927) states that erythrocytes, as individual cases,
show varied resistances to hemolysis, but that hemolysis in a homo
geneous mixture indicates the entrance of a given amount of water.
Concerning hemolysis, Jacobs remarks (1938) that the hemoglobin dur
ing hemolysis remains constant, therefore, the change in volume,
which must precede hemolysis, is due solely to water uptake.
As to the surface change during swelling preceding hemolysis.
Ponder (1933) states that until there has been a 25^ increase in
volume, no change in surface due to sv/elling occurs, and Jacobs (1938)
states that the cell membrane does not change until immediately before
rupture and hemolysis occur. Jacobs also states (1938) that the final
stages of hemolysis occur rapidly and there is no pre-hemolysis leaking
of the cell contents.
Keilin and Hartree (1946) summarize the advantages in using the
red cell by reviewing that the vertebrate red cell forms the most con
venient material for the study of cell permeability, cell resistance,
and lysis for the following reasons: cells are easily accessible; even
in small volumes the cell population is large enough to minimize the
10
effects of biological variability on results, no other tissue being
so suited; all surfaces are exposed to the environment; cells are
easily washed and suspended in the test solutions; hemolysis provides
a clear-cut end point and an efficient indicator; and complete hemo
lysis can readily be detected.
It is logical to assume, then, that using water as a penetrating
fluid and erythrocytes in hypotonic suspensions, a well defined end
point can be discerned, namely hemolysis, and that the end point is
abrupt and depends solely on the amount of water that has penetrated
the erythrocyte.
11
METHODS AND TECHNIQUES
I GENERAL
The basic plan of the practical program of this research was to
allow an indicator, Y/ith sharply demarcsted extremities, to corao in
contact with a suitable effector and to study this indicator in the
presence of this effector with and without an external influence
being present.
The indicator chosen was the beef erythrocyte since it could be
procured in limitless quantities sufficient for this work at a nominal
fee, and could be maintained with little or no laboratory equipment.
The suitable effector in this case was distilled v/ater. The red
cell can only hold so much v/ater; then it bursts, or heraolyzes.
The external influences were a water soluble extract of the
adrenal cortex, an aqueous solution of cholesterol, an aqueous solu
tion of desoxycorticosterone acetate, and an aqeous solution of leci
thin, Two adrenal extracts were used; one, an experimental extract
prepared by the Upjohn Laboratory (l-A SCL 17) and the other prepared
in conjunction v/ith this v/ork.
The most complicated phase of the problem was the bringing
together of the erythrocytes, water, and various test reagents, so that
optimum conditions v/ould prevail.
The method employed in reading the results was photo-electric
colorimetry and the Klett-Summerson colorimeter gave satisfactory
12
results.
It was decided to confine the work to fragility studies since
the Klett-Summerson colorimeter is unsuited for rate studies. The
pointer dial is uncalibrated therefore no indication of exact rate
can be ascertained, and there is else appreciable lag between con
ditions in the specimen being tested and the indication on the dial.
II PREPARATION AND SELECTION OF RED BLOOD CELL SUSPENSIONS
A series of standards was devised but only approximate values
and range values for these standards can be appreciated since the
character of the erythrocyte varies from one day to the next as will
be shown in the experimental data, but consistent values for any one
experimental period were obtainable, and proved to be within the
range of experimental error reliable for all practical purposes.
Preliminary studies involved the preparation of suitable concen
trations of erythrocytes in keeping with the effective range of the
Klett instrument. In all studies beef erythrocytes, from the Hereford
breed of cattle, were used. The whole blood was collected immediately
after the animal was slaughtered, defibrinated as quickly as possible
by whipping, and refrigerated at 4°C. within a period of tv;o hours.
Thus, the blood was stored until ready for use. It was found that the
blood remained in a fairly stable condition for a period approaching
two weeks, the individual erythrocyte maintaining experimental inte
grity for that period. Periods of storage exceeding two weeks pro
duced erratic and unpredictable responses in the erythrocytes, so it
was considered necessary to renew the material after two weeks.
It was determined by storage experiments of twenty-four hour
13
duration, that the erythrocj’ te in a Ringer's solution consisting of
9.0 grn NaCl, 0.24 gm CaGl^, 0.48 gm KOI, 0.30 gm NaHCOg and 0.18 gm
Glucose diluted to 1000 c.c. with distilled water maintained a con
stant resistance to osmotic stress for a period of two v/eeks. This
was considered as a 1 Normal Ringer's solution in all these experi
ments.
In the preliminary sensitivity tests, a 1.0^ suspension was pre
pared. The red cells v/ere v;ashed five times in 1 Normal Ringer’s
solution, the separations being performed by centrifugation. The
final period of centrifugation was standardized to thirty minutes, to
insure close and uniform packing before volume of washed cells was
determined. A five cubic centimeter portion of the 1.0^ suspension
was taken and to it v/as added prepared Ringer solutions of varying
normalities in five cubic centimeter portions. The fundamental cal
culations were the if 5 c.c. of a 1,0% suspension of red cells in
1 Normal Ringer’s solution were diluted with 5 c.c. of a 1.0 Normal,
0.8 Normal, 0.6 Normal, 0.4 Normal, 0.2 Normal Ringer’s solution and
finally distilled water the result would be six suspensions of 0.5^
red cells in 1.0 Normal, 0.9 Normal, 0.8 Njormal, 0.7 Normal, 0.6
Normal, and 0.5 Norraa.l Ringer’s solution, respectively. These 10 c.c.
portions were then suitable for preliminary work involving optimum
red cell concentrations, and also, the behavior of the red cells in
dilute solutions v/ith respect to the optimum colorimeter range.
Variations of the above method were employed involving all ranges
of red cell concentration from 1.0^^ to 0.01^ and the optimum red cell
concentration was found to be 0.2^. Higher concentrations gave
14
readings too high in the range of the colorimeter to be read with great
accuracy, and lower concentrations were too susceptable to even minute
changes, due to experimental error, to be reliable.
A 0,6% suspension of red cells was made in the aforementioned
manner in 1 Norme1 Ringer’s. To 5 c*Cè of this standard 0,6% suspen
sion 10 c.c. of 1 Normal Ringer’s were added giving a 0.2^, red cell
suspension in 1 Normal Ringer’s solution. This was the upper extremity
of colorimeter range, being 0.2^ red cell solution, 100^ non-hemolysed.
The lower colorimeter extremity was determined in like manner substi
tuting 10 c.c. Of aistilled water in place of the 1 Normal Ringer’s.
Thus a 100^ hemolysed 0.2^ red cell solution was obtained.
All subsequent readings fell between these two, and the optimum
range was determined for extract experimental effects to be in the
0.6 Normal to 0.3 Normal Ringer’s, a state of 100^ hemolysis existing
at 0.3 Normal Ringer’s, 0,2% erythrocytes.
The technique employed in arriving at intermediate normalities
between 1.0 and 0.3 Nornml Ringer’s is as follows.
To 5 c.c. portions of the 0.6% red cell suspension in 1 Normal
Ringer’s solution 10 c.c. of the following Ringer’s dilutions were
added; 0.85; 0.7; 0.5; 0.4; 0.25; 0.1. The resulting mixture was a
0.2% red cell solution in 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 Normal Ringer’s
solution, respectively. This had the range from 0.3 Normal Ringer’s
giving total hemolysis to 1.0 Normal Ringer’s and non-hemolysed cells
with 0.1 Normal subdivisions. The elaboration of this system to
cover the test reagents will also be discussed, the above method being
used as the standard.
15
III PREPARATION OF ADRENO-CORTICAL EXTRACT
Regarding the reagent studies, adreno-cortical extract prepara
tions v/ere used. The first, an experimental product of the Upjohn
Laboratories (Beef, 14 gCL 17, Upjohn), Adrenal Cortical Extract, and
another made in conjunction with this research, the preparation of
which is discussed below.
Jacobs (1930), Hartman and Brownell and Hartman (1930), Grollman
and Firor (1933), Kendall and McKenzie (1933), Kendall, et al (1934)
and Cartland and Kuizenga (1936) have all put forth a variety of
methods for preparing adreno-cortical extracts. Cartland and Kuizenga
(1936) in a long program, testing mainly solvents, discarded the weak
acid or base solvent as being too sensitive and liable to destroy much
of the activity, and also discarded the alcohol solvent method, since
subsequent purification procedure resulted in the loss of much of the
active extract. Most of the methods were not suitable for laboratory
facilities on the small scale.
Following is an outline, for the preparation of an active extract
from the adrenal cortex compatible with the procedures and facilities
of this research adapted from Cartland and Kuizenga (1936).
Whole, fresh, and unfrozen beef adrenals were secured locally at
an abattoir operated by the Swift Meat Packing Company and placed imme
diately into acetone as they were cut from the freshly slaughtered
animal. The glands were finely divided in a Waring Blender at the
laboratory and transferred to acetone as the initial solvent for a
period of not less than twenty-four hours, under refrigeration at 4°C.
One liter of acetone per kilo of gland was found to be optimum. The
16
acetone containing the extract was separated from the tissue residue
by suction filtration.
The acetone extract was next concentrated by evaporation in vacuo
below 4-5^C. to remove the acetone. Here the volume was reduced to
approximately 100 c.c. This resulting aqueous extract was next ex
tracted v/ith first, two 200 c.c. portions and then one 100 c.c. portion
of petroleum ether. This removed large quantities of inert lipoid
material. The aqueous extract was next submitted to extraction with
250 c.c. of ethylene dichloride thus separating the cortical and
medullary products, the cortical products going into the ethylene di
chloride and epinephrine remaining in the aqueous solution. The
ethylene dichloride was next concentrated in vacuo below 45^0., and the
residue was dissolved in 200 c.c. of absolute ethyl alcohol. To this
was added 200 c.c. of petroleum ether. The alcohol was progressively
reduced from 100% to 90% to 80% and finally 70%, by adding appropriate
quantities of distilled v/ater. Petroleum ether, being only miscible
with absolute alcohol, separates as the portions of water are adaed.
This petroleum ether is drawn off as it separates. The 70% alcohol
extract solution was concentrated by evaporation in vacuo below 45°C.
until an aqueous colloidal solution of the extract remained. Here one
normal beef Ringer's solution v/as added. A 50 c.c. portion was added
first. An inactive tarry substance precipitated and was removed and
then the extract was diluted so that 1 c.c. of solution in Ringer's
solution represented approximately 25 Dog Units according to figures
given by Cartland ana Kuizenga (1936).
Cartland and Kuizenga (1936) using the same method, arrived at an
17
assay of tiieir extract by dog survival test, of 2500 Dog Units per
kilo of gland extracted, with no appreciable amounts of epinephrine.
This extract is suitable for clinical study.
The Upjohn proauct will subsequently be referred to as extract
"Upjohn," ana the other as extract ’ Y/."
The cortical extracts were prepared for use in the following
manner. A concentration of 0.^ milliliters of extract "Upjohn" per
liter was considered sufficient, therefore a solution of 1 Normal
Ringer’s solution plus 0.4 milliliters was made so that one liter of
1 Norml Ringer’s solution contained the desired extract concentra
tion. The solution was then diluted to the following normalities;
0.85; 0.7; 0.55; 0.4; 0.25; 0.1. Now diluting a 0.6% suspension of
red cells in the following manner: 5 c.c. of 0.6% suspension of red
cells in 1.0 Normal Ringer's plus 10 c.c. of the listed prepared nor
malities, would give solutions ranging in normality; 0.9; 0.8; 0.7;
0.6; 0.5; and 0.4, and containing the extract.
Since extract "W" was unassayed the exact concentration was un
known, but could be approximated at twenty-five Dog Units per c.c.
To insure some activity being present, 500 Dog Units per liter was the
concentration strength, amounting to 20 c.c. of extract "W" per liter
of solution. The mechanics of preparation for use were the same as
for extract "Upjohn." Fresh dilutions had to be made daily in both
cases since all activity was apparently lost due to some indiscernable
factor after storage for a period of twelve hours.
In studying permeability effects of extracts, a method in which
the test reagent concentration would be constant in each tube, with
18
only the Ringer normality variable, was required; a three portion
method was devised for the study of the DOGA, cholesterol and lecithin.
Five cubic centimeters of a 0.6% suspension of beef erythrocytes in
isotonic Ringer’s plus five cubic centimeters of the aqueous test solu
tion plus five cubic centimeters of the variable normality of Ringor’s
were mixed, adding the erythrocytes last. Thus, at all times the con
centration of the test reagent in the individual test group was kept
constant, the normality of Ringer’s solution only being varied at 0.8,
0.5, and 0.2 Normal giving 0.6, 0.5 and 0.4 Normal solutions, respec
tively. This method proved satisfactory in keeping the test reagent
concentration constant in all tubes, yet varying the normality of the
Ringer’s easily.
IV PREPARATION OF CHOLESTEROL, DESOXYCORTICOSTERONE ACETATE,
AND LECITHIN SOLUTIONS
Cholesterol and desoxycorticosterone acetate (DOCA) are slightly
soluble in distilled wat er. Obtaining an aqueous solution of these
compounds was accomplished by first finely dividing the compounds with
mortar and pestle and mixing thoroughly for a period of thirty minutes
on each of two days in a Waring Blendor. This agitation produced a
satisfactory solution.
It was decided to keep all solutions as close to equi-molarity as
possible, so a stand8.rd quantity of cholesterol amounting to 0.0022
grams was dissolved in one liter of distilled v/ater in the following
manner. Powdered reagent cholesterol amounting to 0.027 grams was
placed in a Waring Blendor v/ith 500 c.c. of distilled v/ater and mixed
for thirty minutes. The 500 c.c. with the cholesterol was placed in
19
a 1 liter flask and the blendor we shed with 500 c.c. distilled water.
This was added to the original 500 c.c. For a period of two hours,
the flask was agitated by hand every thirty minutes for one minute.
The second day the flask was emptied into the blendor and contents
agitated again for thirty minutes. The 1000 c.c. were returned to the
original flask and the blendor rinsed with 500 c.c. distilled water.
A Gooch crucible had been cleaned, half filled with glass wool, dried
and weighed until constant. The contents of the flask were then fil
tered by suction through the crucible and flask washed with 500 c.c.
distilled water adding v/ash through crucible. The crucible was heat
dried until it weighed constant and the crucible weight subtracted.
This gave the amount of cholesterol not in solution. The amount of
cholesterol in solution was then 0.0044 grams in two liters, or 0.0022
grams per liter. From this solution, referred to as CHOL I, subsequent
dilutions were made. As a result, the four other solutions and their
concentrations were numbered as follows: CHOL II, 2.2 x lO”"^; CHOL III,
2.2 X 10~^; CHOL IV, 2.2 x 10“* ^ ; and CHOL V, 2.2 x 10~^. All concen
trations are expressed in grams per liter.
The chemical purity of the lecithin available being in question, a
series of tests and purification procedures were instituted to deter
mine if; first, were any steroids present; secondly, were any other
fat soluble compounds present; and last, the water content. By per
forming the Iodine-Sulphuric acid test, and employing the Salkowski,
Lieberman-Burchard and Rosenheim TriChlorAcetic Acid reactions as
outlined in Koch (1934), no color reaction due to the presence of
sterols was discernible. From the results of these tests, it was con-
20
eluded that no sterols were present in the sample of lecithin used in
this research. Further purification by dialysis through a fine rubber
membrane, as outlined by Gortner (1929), insured a maximum of purity,
the sample being free particularly from sterols which would interfere
with the series of experiments involving lecithin and its comparison
to cholesterol and DOCA* Also, a sample of lecithin was dried in an
oven until the weight appeared constant. It was assumed that weight
loss during heating was due to water loss and, subsequently, correc
tions were made, so that the lecithin solutions closely approached
the same molarity as the others.
A table of the concentrations of cholesterol, DOCA and lecithin,
in grams per liter and moles per liter, has been included in the Exper
imental Results.
V OSMOTIC PRESSURE EXPERIMENT
The possibility of observed results being due to osmotic effects,
was considered and the possibility tested as follows; a solution of
glucose which would have little or no specific effect on permeability
in proportion to its osmotic effect was tested in the same manner as
the other substances, and in the same molar concentration range.
Since osmotic activity is due solely to .particle concentration in the
solution, an equi-molar glucose solution serves to indicate whether or
not the effect of steroids is merely due to the Py they exert or a
specific physiological action of the compounds tested. Thus, any osmo
tic activity on the part of the compounds could be ascertained and com
pared to a suitable standard.
21
VI HEàT EFFECTS
Since Cartland and Kuizenga (1936) cautioned against raising the
temperature of the water soluble adrenal cortex extract above 45°G. in
their method of preparation, and since it is the contention of Mason
(1939) and this research that said heating ma.y destroy all activity of
those compounds having the 4-5 double bond in the steroid nucleus, it
was decided to see what effect heat would have on the water soluble
extract and also on cholesterol and DOGA, and if the heating, when it
destroys the physiological or life sustaining activity, also destroys
the factor which affects permeability. The water soluble extract,
cholesterol solution and the DOGA solution were placed in tightly
stoppered containers, heated to a temperature of 60°C. for one hour
and tested in the usual manner after cooling.
VII COLORIMETRY
In order to eliminate as many sources of error as possible, a
plan of standardization covering the use of the Klett-Summerson
colorimeter was adopted. The same instrument was used in all work,
since preliminary tests proved individual differences existed that
confused the results when more than one instrument was used. The
instrument was moved as little as possible, since movement and jarring
also affected results. All test tubes used were chosen to give snug
fit and identical readings when empty. The colorimeter was read in the
following manner. A control tube was placed in the holder and the dial
so adjusted to bring the galvanometer to zero deflection. The figure
read from the dial was considered as the control value. The first
experimental tube was placed in the holder and the galvanometer pointer
22
was again brought to zero position. The dial reading for this setting
was the first experimental value. The next experimental tube was
placed in the holder and if the galvanometer needle showed no deflection
the reading of the second was taken to be the same as the first, but if
the pointer was deflected, then the dial was reset to zero deflection
and that reading noted. The dial was only moved when the pointer v/as
deflected except for an occasional intentional deflection of the galvan
ometer pointer produced by moving the dial to any setting which effec
tively deflects the galvanometer pointer. This was done to see if the
experimental value could be reproduced. All subsequent tests were
made and recorded in the above manner.
The experimental procedure is as follows: A series of three test
tubes into which were placed 5 c.c. of distilled water, 5 c.c. of the
three variable normalities of Ringer’s solution and lastly, 5 c.c. of
a 0.6^ suspension of red cells, mixed in that order, were placed in
the instrument after thirty minutes from the mixing time, and the
readings of each tube taken. This \ms the control figure. In the
experimental tests an aqueous solution of the reagents tested was sub
stituted for the distilled water, and the readings taken. Each ex
perimental group consisted of the three controls in 0.6, 0.5, 0.4
Normal Ringer’s solution and as many other groups of three tubes as
there were dilutions of reagent.
23
I G X J P E R J l Æ B O N T j A l i f ü G E n J I V T :
I, Table of Abbreviations and Concentrations of Compounds Tested.
Abbreviation Concentration
Used
Concentration
Cholesterol CHOL
CHOL I 6 X 10-6
2.2 X 10-3
CHOL II 6 X 10-% 2.2 X 10-4
CHOL III 6 X
10-8
2.2 X 10-6
CHOL IV 6 X
10-9^
2.2 X 10-6
CHOL V 6 X 10-1° 2.2 X 10-7
Desoxycortico-
sterone Acetate DOCA
DOGA I 5.7 X 10-6 2.1 X 10-3
DOCA II 5.7 X lo-T 2.1 X
10-4
DOCA III 5.7 X 10-8 2.1 X 10-6
DOCA IV 5.7 X 10-9 2.1 X 10-6
DOCA V 5.7 X 10-1° 2.1 X 10-7
Lecithin LEG
LEG I 6.1 X 10-7 4.9 X 10-3
LEG II 6.1 X
10-8 4.9 X
10-4
LEG III 6.1 X 10-9 4.9 X 10-5
Glucose GLUC
GLUC I 6 X 10-6 1.08 X 10-3
GLUC II 6 X 10-7 1.08 X
10-4
GLUC III 6 X
10-8 1.08 X
10-5
24
II. Following is a table giving the range of readings on the KLETT-
SUIÆ'ÎERSON colorimeter at given percentages of hemolysis.
0
-
145
145 - 146
146
-
190
190
-
265
265 - 310
'310 -
315
315 -
325
325
-
340
340 -
345
345
-
375
Above 375
100^ Hemolysis
90^
80^
60^
50^
40^
30^
20^
10^
A lower value indicates a greater degree of hemolysis.
III. Tables of Standardization.
A. Blood Suspensions: To determine the optimum concentration of
red cells for use in the Klett-Summerson colorimeter.
Working standards of 1.0^ suspensions of red cells in 1
Normal Ringer’s solution diluted with distilled water to give
a range of dilution from 0.5^ to 0.1^ R.B.C., 100^ hemolysed
and with 1 Normal Ringer’s to give a range from 0.5^ to 0.1%
R.B.C., non-hemolysed.
Dilution of
Red Cells
Non-Hemolysed Hemolysed Reading
Below 0.10% No visible reaction on instrument
0.10% 93 22
0.20% 232 100
0.30% 390 173
0.40% 520 260
0.50% 750 390
Above 0.50% No visible reaction on instrument
25
III# A# (Continued)
Dilution of
Red Cells
Non-Hemolysed Hemolysed Reading
Below 0.10% No visible reaction on instrument
0.10% 92 20
0.20% 230 102
0.30% 390 173
0.40% 520 260
0.50% 750 390
Above 0.50% No visible reaction on instrument
Below 0.10% No visible reaction on instrument
0.10% 92 22
0.20% 232 100
0.30% 390 173
0.40% 520 260
0.50% 750 390
Above 0.50% No visible reaction on instrument
0.10% 133 34
0.20% 296 130
0.30% 450 216
0.40% 580 300
Above 0.40% Over upper limit of colorimeter
OilO% 135 32
0.20% 296 132
0.30% 448 216
0.40% 580 300
Above 0.40% Over upper limit of colorimeter
0.10% 133 34
0.20% 296 130
0.30% 450 216
0.40% 580 300
Above 0.40% Over upper limit of colorimeter
A 0,2% suspension of red cells put the readings of the colorimeter
in the most sensitive and most finely calibrated section of the
dial, so a 0*2% suspension will be employed in experimental work.
26
III. (Continued)
B. Normality: Using a 0.6% standard diluted to 0.2% concentra
tion of red cells, the normality of the Ringer’s is varied
from 1.0 Normal to 0.3 Normal. Thus, the most sensitive
range on the colorimeter dial can be determined with rela
tion to tonicity of suspending fluid.
Reading 1 Reading 2
Normality of Taken Immediately Taken 30 Minutes
1.0 N 360
_ -— z z a——
370
0.9 360 370
0.8 360 360
0.7 335 335
0.6 320 320
0.5 274 274
0.4 176 175
0.3 174 174
1.0 N 370 370
0.9 365 365
0.8 360 360
0.7 335 335
0.6 320 320
0.5 274 274
0.4 176 176
0.3 175 174
1.0 N 370 370
0.9 360 360
0.8 360 360
0.7 335 335
0.6 320 320
0.5 274 ' 274
0.4 176 176
0.3 175 175
27
III. B. (Continued)
Normality of
Ringer’s Solution
Reading 1
Taken Immediately
After Mixing
Reading 2
Taken 30 Minutes
After Reading 1
1.0 N 365 365
0.9 360 360
0.8 355 355
0.7 340 340
0.6 320 320
0.5 253 254
0.4 176 174
0.3 168 167
1.0 N 365 365
0.9 360 360
0.8 355 355
0.7 340 340
0.6 320 320
0.5 253 253
0.4 176 174
0.3 168 167
0.6 N 320 320
0.5 253 253
0.4 176 174
0.6 N 300 300
0.5 252 252
0.4 177 177
0.6 N 300 300
0.5 252 252
0.4 177 177
0.6 N 305 305
0.5 252 252
0.4 175 175
28
III. B. (Continued)
Normality of
Reading 1
Taken Immediately
Reading 2
Taken 30 Minutes
0.6 N 305 305
0.5 252 252
0.4 175 175
0.6 N 286 289
0.5 254 254
0.4 169 169
Using a normality range of from 0.6 to 0.4 Normal Ringer’s
solution as the suspending fluid, the values fall into the most
highly calibrated section of the colorimeter dial, giving
greatest accuracy. This norimlity range will be employed.
IV. MaTHEMATIGAL TREATMENT OF THE DATA.
The data were treated in the following manner; The experimental
value was subtracted from the control value, algebraically, so
that a negative result v/ould indicate a decrease in the degree of
hemolysis and a positive result would indicate an increase in the
degree of hemolysis. This value is the hemolysis change factor.
From all these factors in a given reagent concentration and nor
mality of Ringer’s solution, the standard deviation of the mean
difference between the control and experimental was determined
using the following formula,
~ T ^
29
V. Extract Studies: Behavior of a homogeneous suspension of beef
erythrocytes to variations in tonicity of the suspending fluid
in the presence of a water soluble adreno-cortical extract.
A. Preliminary Studies on the Effect of Extract "Upjohn." -
Reading 1 was made immediately after mixing. Reading 2 was
made 30 minutes after reading 1.
Reading 1 Reading 2 Control
Normality of Without With Without \7ith ■ .Minus
0.6 N 286 286 288 288 0
0.5 250 185 249 181 + 68
0.4 168 160 167 159 + 8
0.6 N 286 286 288 288 0
0.5 250 185 249 181 + 68
0.4 168 160 167 159 + 8
0.6 N 288 288 288 288 0
0.5 250 185 249 181 + 68
0.4 168 160 167 159 4 - 8
0.6 N 288 288 288 288 0
0.5 250 185 249 181 + 68
0.4 168 160 167 159 + 8
0.6 N 288 288 288 288 0
0.5 250 185 249 181 +68
0.4 168 160 167 159 + 8
Reading 2 is the equilibrium reading and will be considered as
the experimental value in the statistical treatment of the values.
30
V, (Continued)
B. Preliminary Studies on the Effect of Extract "W."
Normality of
Reading 1
Y/ithout With
Reading 2 Control
Y/ithout With Minus
Extract Extract
0.6 N 204 284 284 284 0
0.5 244 210 244 210 + 34
0.4 168 148 163 148 + 15
0.6 N 284 284 284 284 0
0.5 244 210 244 210 + 34
0.4 168 148 163 148 + 15
c<► Comparison of Effects of Extract ’ % ¥ ' ' * and Extract "Upjohn."
Control Control
Experimental Minus Experimental Minus
Normality Control ’ ‘ Upjohn " Experimental Experimental
0.6 N 284 284 0 284 0
0.5 244 188 ■+ 56 210 + 34
0.4 163 156 + 7 148 + 15
0.6 N 284 284 0 284 0
0.5 244 188 + 56 220 + 24
0.4 163 156 + 7 145 + 18
0.6 N 284 284 0 284 0
0.5 244 210 + 34 220 + 24
0.4 163 156 + 7 157 + 6
0.6 N 284 284 0 284 0
0.5 244 210 + 34 230 + 14
0.4 163 155 + 8 157 + 6
0.6 N 284 284 0 284 0
0.5 244 220 + 24 215 + 29
0.4 163 156 + 7 145 + 18
31
V# C. (Continued)
Normality Control
Experimental
.."Upj ohn"
Control
Minus
Experimental
Experimental
Control
Minus
Experimental
0.6 N
0.5
0.4
284
244
163
284
188
160
0
-+56
+ 3
284
205
150
0
+ 39
+ 13
0.6 N 284 280 + 4 275 + 9
0.5 244 210 +34 200 + 44
0.4 163 145 -+18 145 + 18
0.6 N 305 300 + 5 300 + 5
0.5 228 191 + 37 218 + 10
0.4 158 158 0 160 -2
0.6 N 305 282 f 23 294 + 11
0.5 228 208 + 20 228 0
0.4 158 153 + 5 158 0
0.6 N 305 294 + 11 298 + 7
0.5 228 218 + 10 228 0
0.4 158 155 + 3 145 +13
0.6 N 305 296 + 9 300 + 5
0.5 228 210 + 18 218 + 10
0.4 158 150 + 8 145 + 13
0.6 305 300 + 5 298 + 7
0.5 228 220 + 8 200 + 28
0.4 158 156 + 2 145 +13
0.6 305 294 +11 300 + 5
0.5 228 188 + 40 220 + 8
0.4 158 145 +13 150 + 8
0.6 305 300 + 5 305 0
0.5 228 205 + 23 220 + 8
0.4 158 145 +13 145 +13
32
V. (Continued)
D. Mathermtical Table Covering Results of Experiments on Water
Soluble Adreno-cortical Extracts "Upjohn" and "¥/•"
Test
Reagent
Solutions
Normality of
Ringèr's
Mean Difference
Between
Control & Experimental (T
Upj ohn 0.6 + 3.2 + 4.8
0.5 +40.9 + 27.3
0.4 + 8.1 ± ^'6
"Vv" 0.6 + 3.5 + 3.8
0.5 +21.4 +14.1
0.4 +11.4 + 5.3
VI# Cholesterol Tests#
Am Effects of CHOL I on a 0.2% Suspension of Red Blood Cells
in a 1.0 Normal Ringer's Solution: This experiment was per
formed to see if cholesterol affected the colorimeter value
by absorbing light, thus reducing the deflection of the
galvanometer pointer.
Normality of Ringer* s Control Reading Experimental Reading
1.0 305 305
1.0 305 305
1.0 310 310
1.0 300 300
1.0 300 300
1.0 305 305
1.0 305 305
1.0 300 300
1.0 300 300
Since the controls minus the expérimentais of this test are zero, it
can be concluded that the reduction in the amount of light transmitted
is not due to light absorbtion by the cholesterol.
33
VI. (Continued)
B. Behavior of a Homogeneous Suspension of Beef Erythrocytes
to Variations in Tonicity of the Suspending Fluid in the
Presence of an Aqueous Solution of Cholesterol.
1. Using 1 Solution of Cholesterol: Reading 1 was made
immediately after mixing. Reading 2 was made 30
minutes after reading 1.
Normality
Control Reading CHOL I Heading
1 2 1 2
Control
Minus
0.6 415 415 425 425 -10
0.5 335 335 346 350 -15
0.4 228 228 232 230 — 2
0.6 415 420 425 430 -10
0.5 334 335 355 360 -25
0.4 228 230 240 236 — 6
0.6 415 415 427 430 -15
0.5 335 335 350 355 —20
0.4 230 228 236 236 - B
0.6 420 420 425 430 -10
0.5 332 333 350 350 -17
0.4 228 230 230 230 0
0.6 415 420 430 430 -10
0.5 336 335 355 345 • -10
0.4 228 228 240 238 -10
0.6 535 535 540 550 -15
0.5 480 480 490 485 - 5
0.4 339 330 342 350 -20
34
VI. B. 1. (Continued)
Control
Control Reading CHOL I Reading Minus
Morrcelity 1 2 1 2 Experimental
0.6 538 535 545 545 -10
0.5 475 480 490 490 -10
0.4 330 330 350 345 -15
0.6 540 53,5 545 545 -10
0.5 475 480 490 490 -10
0.4 335 330 350 345 -15
0.6 545 538 550 548 -10
0.5 476 475 485 495 -20
0.4 330 330 342 354 —24
0.6 545 540 550 555 -15
0.5 480 474 480 485 -11
0.4 335 330 340 340 -10
0.6 540 545 555 555 -10
0.5 473 482 495 500 -18
0.4 340 340 350 355 -15
0.6 535 540 545 547 - 7
0.5 470 470 480 485 -15
0.4 340 340 350 355 -15
0.6 . 535 535 545 - 545 -10
0.5 475 475 490 490 -15
0.4 340 340 350 350 -10
VI. B. (Continued)
35
2. Using Three Dilutions of Cholesterol
•
Normality Control CHOL I C-E CHOL III C-E CHOL V G-E
0.6 350 350 0 358 - 8 350 0
0.5 305 320 -15 310 - 5 305 0
0.4 212 218 — 6 220 - 8 212 0
0.6 350 350 0 358 - 8 350 0
0.5 305 320 -15 310 - 5 305 0
0.4 212 218 - 6 220 - 8 212 0
0.6 350 360 -10 365 -15 350 0
0.5
305 320 -15 310 - 5 305 0
0.4 214 218 - 4 218 - 4 212 2
0.6 350 360 -10 365 -15 350 0
0.5 305 - 320 -15 310 — 5 305 0
0.4 214 218 - 4 218 — 4 212 2
0.6 350 350 0 365 -15 350 0
0.5 305 320 -15 310 - 5 305 0
0.4 212 218 - 6 218 — 6 212 0
0.6 350 350 0 365 -15 350 0
0.5 305 320 -15 310 - 5 305 0
0.4 212 218 — 6 218 — 6 212 0
0.6 350 360 -10 365 -15 345 5
0.5 305 320 -15 310 - 5 305 0
0.4 212 216 - 4 214 - 2 214 — 2
0.6 350 360 -10 365 -15 345 5
0.5 305 320 -15 310 - 5 305 0
0.4 212 216 - 4 214 — 2 214 - 2
C-E means control minus experimental.
VI. B. 2. (Continued)
36
Norirality Control CHOL I C-E CHOL III C-E CHOL V C-E
0.6 350 365 -15 360 ' -10 345 ' 5
0.5 305 320 -15 310 - 5 310 - 5
0.4 212 214 - 2 212 0 212 0
0.6 350 365 -15 360 -10 345 . 5
0.5 305 320 -15 310 - 5 310 - 5
0.4 212 214 - 2 212 0 212 0
0,6 350 358 — 8 358 - 8 350 0
0.5 305 320 -15 310 - 5 305 0
0.4 212 218 - 6 216 - 4 212 0
0.6 350 358 - 8 358 - 8 350 0
0.5 305 320 -15 310 - 5 305 0
0.4 212 218 - 6 216 - 4 212 0
0.6 350 358 - 8 358 — 8 350 0
0.5 305 320 -15 310 - 5 305 G
0.4 212 218 - 6 215 - 3 212 0
0.6 350 358 - 8 358 - 8 350 0
0.5 305 320 -15 310 - 5 305 0
0.4 212 218 - 6 215 - 3 212 0
0.6 350 365 -15 360 -10 350 0
0.5 305 320 -15 310 - 5 305 0
0.4 212 218 . - 6 214 - 2 212 0
0.6 350 365 -15 360 -10 350 0
0.5 305 320 -15 310 - 5 305 0
• 0.4 212 218 - 6 214 — 2 212 0
0.6 350 365 -15 355 - 5 350 0
0.5 305 320 -15 315 -10 305 0
0.4 212 218 - 6 215 - 3 212 0
37
VI. B. (Continued)
3. Using Five Dilutions of Cholesterol.
Normalitv Control CHOLI G-E CHOL II C-E CHOL in G-E CHOL IV G-E CHOLV G-E
0.6 350 350 0 358 — 8 358 - 8 354 4 350 0
0.5 305 320 -15 318 -13 310 - 5 308 - 3 305 0
0.4 212 218 - 6 218 - 6 220 - 8 214 2 212 0
0.6 350 360 -10 360 -10 365 -15 352 2 350 0
0.5 305 320 -15 316 -11 310 - 5 307
-
2 305 0
0.4 212 218 - 6 216 - 4 218 - 6 214 2 212 0
0.6 350 350 0 357 - 7 358 - 8 355 5 355 - 5
0.5 305 320 -15 316 -11 310 - 5 305 0 305 0
0.4 212 216 - 4 216 — 4 215 - 3 214 2 214 - 2
0.6 350 360 -10 360 -10 365 -15 350 0 345 + 5
0.5 305 320 -15 315 -10 310 - 5 306 -
1 305 0
0.4 212 216 - 4 216 - 4 214 - 2 215 3 212 0
0.6 350 365 -15 362 -12 360 -10 354 4 345 +5
0.5 305 320 -15 315 -10 310 - 5 306 -
1 305 0
0.4 212 214 - 2 214 - 2 212 0 214 2 212 0
0.6 350 358 - 8 360 -10 358 - 8 352
_
2 350 0
0.5 305 320 -15 316 -11 316 -11 305 0 305 0
0.4 212 218 - 6 218 - 6 216 - 4 214 2 212 0
0.6 350 360 -10 358 - 8 358 — 8 354 4 350 0
0.5 305 320 -15 318 -13 310 - 5 308
- 3 305 0
0.4 212 220 + 8 216 - 4 215 - 3 214 — 2 212 0
0.6 350 365 -15 360 -10 360 -10 355 5 350 0
0.5 305 320 -15 314 - 9 310 - 5 308 - 3 305 0
0.4 212 218 - 6 218 - 6 214 — 2 214 2 212 0
0.6 350 360 -10 358 - 8 355 - 5 355 5 350 0
0.5 305 318 -13 314 - 9 312 - 7 308 - 3 305 0
0.4 212 218 - 6 218 - 6 216 - 4 214 2 212 0
38
VI# (Continued)
G# Mathematical Table Covering Results of Experiments on
Cholesterol#
Test Solutions Mean Difference
Normality of Between
(T Reagent Ringer* s Control & Experiment al
CHOL I 0.6 - 9.4 + 4.7
0.5 -14.3 + 3.0
0.4 - 7.3 +4.9 .
CHOL II 0.6 - 9.3 +1.5
0.5 —10.8 +1.4
0.4 - 4.7 +1.0
CHOL III 0.6 -10.4 +3.4
0.5 - 5.5 -n.5
0.4 - 3.5 +1.8
CHOL IV 0.6 - 3.4 +1.6
0.5 - 1.8 + 1.5
0.4 2;i +0.25
CHOL V 0.6 + 0.96 +3.0
0.5 — 0.4 +1.2
0.4 - 0.15 + 0.76
39
VII. DOCA Tests.
A. Effects of DOCA I on a 0.2% Suspension of Red Blood Cells
in a 1.0 Normal Ringer's Solution: This experiment was per
formed to see if DOCA affected the colorimeter value by
absorbing light, thus reducing the deflection of the galvan
ometer pointer.
Normality of Ringer's____ Control Reading____ Experimental Reading
1.0 288 288
1.0 288 288
1.0 288 288
1*0 288 288
1.0 S#8 288
1.0 288 288
1.0 288 288
1.0 288 288
1.0 288 288
Since the controls minus the expérimentais of this test are zero, it
can be concluded that the reduction in the amount of light transmitted
is not due to light absorbtion by DOCA.
B. Behavior of a Homogeneous Suspension of Beef Erythrocytes
to Variations in Tonicity of the Suspending Fluid in the
Presence of an Aqueous Solution of DOGA.
1. Using Three Solutions of DOCA.
Normality Control DOCA I C-E DOCA III C-E DOCA V G-E
0.6 350 372 -22 365 -15 350 0
0.5 305 330 -25 320 -15 310 - 5
0.4 212 230 -18 218 - 6 212 0
0. 6 350 368 -18 358 — 8 350 0
0.5 305 332 -27 320 -15 310' - 5
0.4
212 230 -18 218 — 6 212 0
VII. B. 1. (Continued)
40
Normality Control DOCA I C-E DOCA III C-E DOCA V C-E
0.6 350 370 -20 360 -10 350 0
0.5 305 328 -23 318 -13 305 0
0.4 212 228 -16 220 - 8 212 0
0.6 350 368 -18 360 -10 350 0
0.5 305 330 -25 320 -15 305 0
0.4 212 230 -18 220 - 8 210 +2
0.6 350 372 -22 358 - 8 350 0
0.5 305 332 -27 318 -13 310 - 5
0.4 212 224 -12 214 - 2 214 - 2
0.6 350 375 -25 360 -10 350 0
0.5 305 330 -25 320 -15 305 0
0.4 212 230 -18 224 rl2 212 0
0.6 350 372 -22 368 -18 350 0
0.5 305 328 —23 316 -11 305 0
0.4 212 228 -16 218 — 6 210 + 2
0.6 350 372 -22 364 -14 350 0
0.5 305 333 -28 315 -10 305 0
0.4 212 230 -18 218 — 6 212 0
0.6 350 372 —22 365 -15 350 0
0.5 305 330 -25 320 -15 310 - 5
0.4 212 230 -18 218 — 6 212 0
0.6 350 368 -18 358 - 8 350 0
0.5 305 332 -27 320 -15 310 - 5
0.4 212 230 -18 218 - 6 212 0
0.6 350 370 -20 360 -10 350 0
0.5 305 328 -23 . 318 -13 305 0
0.4 212 230 -18 220 - 8 212 0
41
VII# B. 1# (Continued)
Normality Control DOCA I C-E DOCA III C-E DOCA V C-E
0.6 350 368 -18 360 -10 350 0
0.5 305 330 -25 320 -15 305 0
0.4 212 230 -18 220 - 8 210 + 2
0.6 350 372 -22 358 - 8 350 0
0.5 305 332 -27 318 -13 310 - 5
0.4 212 224 -12 214 - 2 214 - 2
0.6 350 375 -25 360 -10 350 0
0.5 305 330 -25 320 -15 305 0
0.4 212 230 -18 224 -12 212 0
0.6 350 372 -22 368 -18 350 0
0.5 305 328 -23 316 -11 305 0
0.4 212 228 -16 218 - 6 210 + 2
0.6 350 372 —22 364 -14 350 0
0.5 305 333 -28 315 -10 305 0
0.4 212 230 —18 218 - 6 212 0
2. Using Five Solutions of DOCA.
Normality Control DOCA I C-E DOCA II C-E DOCA ni Q-E DOCA IV C-E DOCAV G-E
0.6 350 372 -22 368 -18 365 -15 355 - 5 350 0
0.5 305 330 -25 325 —20 320 -15 315 -10 310 - 5
0.4 212 230 -18 220 - 8 218 — 6 218 — 6 212 0
0.6 350 368 -18 366 -16 358 — 8 357 - 7 350 0
0.5 305 332 -27 327 -22 320 -15 315 —10 310 - 5
0.4 212 230 -18 226 -14 218 - 6 216 - 4 214 - 2
0.6 350 370 -20 368 -18 360 -10 354 - 4 350 0
0.5 305 328 -23 328 -23 318 -13 312 - 7 305 0
0.4 212 228 -16 224 -12 220 - 8 214 - 2 212 0
42
VII. B. 2. (Continued)
0.6 350 368 -18 367 -17 360 -10 355 - 5 350 0
0.5 305 330 -25 324 -19 320 -15 315 -10 305 0
0.4 212 230 -18 226 -14 220 — 8 214 - 2 210 + 2
0. 6 350 372 -22 ' 366 -16 358 - 8 356 - 6 350 0
0.5 305 332 -27 325 -20 318 -13 314 - 9 310 - 5
0.4 212 224 -12 224 —12 214 - 2 215 ■ - 3 214 — 2
0.6 350 375 -25 368 -18 360 -10 354 - 4 350 0
0.5 305 330 -25 322 -17 320 -15 312 - 7 305 0
0.4 212 230 -18 . 222 -10 224 -12 216 - 4 212 0
0.6 .350 372 -22 370 -20 368 -18 358 - 8 350 0
0.5 305 328 -23 325 -20 316 -11 315 -10 305 0
0.4 212 228 -16 222 -10 218 — 6 218 - 6 210 + 2
0.6 350 372 -22 368 -18 364 -14 355 — 5 350 0
0.5 305 333 -28 326 -21 315 -10 310 - 5 305 0
0.4 212 230 -18 220 — 8 218 — 6 215 - 3 212 0
0.6 350 370 -20 368 -18 358 - 8 355 - 5 350 0
0.5 305 330 -25 324 -19 316 -11 312 - 7 310 - 5
0.4 212 228 -16 220 - 8 218 - 6 216 — 4 214 - 2
0.6 350 368 -18 366 -16 360 -10 353 - 3 350 0
0.5 305 330 -25 325 -20 320 -15 315 -10 310 - 5
0.4 212 230 -18 222 -10 224 -12 214 - 2 212 0
43
VII* (Continued)
C. Mathematical Table. Covering Results of Experiments on DOCA.
Test Solutions Mean Difference
Normality of Between
6^ Reagent Ringer’s Control & Experimental
DOCA 1 0.6 -20.9 + 2.1
0.5 —23.3 + 3.0
0.4 -17.1 + 1.3
DOCA II 0.6 -17.5 + 1.1
0.5 -20.1 + 4.3
0*4 -10.6 + 2.2
DOCA III 0.6 -12.2 + 3.6
0.5 -13.4 + 1.9
0.4 - 6.9 ± 0*72
DOCA IV 0.6 - 5.2 + 1.4
0.5 - 8.5
± 1-7
0.4 — 3.6 + 1.4
DOCA V 0.6 0 0
0.5 - 2.1 + 2.5
0.4 0 + 1.2
44
VIII. Lecithin Tests.
A* Effects of LEG I on a , 0.2% Suspension of Red Blood Cells
in a 1.0 Normal Ringer's Solutions This experiment v/as per
formed to see if lecithin affected the colorimeter value by
absorbing light, thus reducing the deflection of the galvan
ometer pointer.
1.0 288 288
1.0 288 288
1.0 288 288
1.0 288 288
1.0 288 288
1.0 288 288
1.0 288 288
1.0 288 288
1.0 288 288
Since the controls minus the expérimentais of this test are zero, it
can be concluded that the reduction in the amount of light transmitted
is not due to light absorbtion by the lecithin.
B* Behavior of a Homogeneous Suspension of Beef Erythrocytes
to Variations in Tonicity of the Suspending Fluid in the
Presence of an Aqueous Solution of Lecithin.
1. Using Three Solutions of Lecithin.
Normality Control LEG I G-E LEG II G-E LEG III G-E
0.6 350 350 0 350 0 350 0
0.5 280 292 -12 290 -10 286 — 6
0.4 180 218 -38 206 -26 212 -32
0.6 350 350 0 350 0 350 0
0.5 280 292 -12 290 -10 286 - 6
0.4 180 221 -41 206 —26 214 -34
VIII. B. 1. (Continued)
45
Normality Control LEG I G-E LEG II C-E LEG III C-E
0. 6 350 350 0 350 0 350 0
0.5 280 298 -18 290 -10 288 - 8
0.4 180 224 -44 208 —28 212 —32
0.6 350 350
* 0
350 0 350 0
0.5 280 296 -16 290 -10 286 - 6
0.4 180 220 -40 206 -26 212 -32
0.6 350 350 0 350 0 350 0
0.5 280 298 -18 292 -12 284 - 4
0.4 180 222 -42 208 -28 214 -34
0.6 350 350 0 350 0 350 0
0.5 280 292 -12 288 — 8 284 - 4
0.4 180 224 —44 204 -24 214 -34
0.6 350 350 0 350 0 350 0
0.5 280 294 —14 290 -10 286 — 6
0.4 180 220 —40 206 -26 216 -36
0.6 350 350 0 350 0 350 0
0.5 280 292 -12 288 - 8 286 - 6
0.4 180 224 -44 204 -24 212 -32
0.6 350 350 0 350 0 350 0
0.5 280 294 -14 290 -10 282 - 2
0.4 180 224 -44 206 -26 212 -32
0.6 350 350 0 350 0 350 0
0.5 280 298 -18 . 288 - 8 288 - 8
0.4 180 222 -42 208 -28 214 -34
0.6 350 350 0 350 0 350 0
0.5 280 292 -12 290 -10 286 — 6
0.4 180 222 -42 206 —26 212 -32
VIII. B. 1. (Continued)
46
Normality Control LEC I C-E LEC II C-E LEC III C-E
0.6 350 350 0 350 .0 350 0
0.5 280 294 -14 288 - 8 282 - 2
0.4 180 224 -44 208 -28 212 —32
0.6 350 350 0 350 0 350 0
0.5 280 298 -18 290 -10 284 - 4
0.4 180 219 -39 206 -26 214 -34
0.6 350 350 0 350 0 350 0
0.5 280 296 -16 290 -10 280 0
0.4 180 218 -38 204 —24 212 -32
0.6 350 355 - 5 350 0 350 0
0.5 300 320 -20 315 -15 310 -10
0.4 240 260 -20 258 -18 256 -16
0,6 350 355 - 5 350 0 350 0
0.5 300 320 -20 315 -15 310 -10
0.4 240 260 -20 258 -18 256 -16
0.6 350 355 - 5 350 0 350 . 0
0.5 300 320 -20 315 -15 310 —10
0.4 240 260 -20 256 -16 254 -14
0.6 350 355 - 5 350 0 350 0
0.5 300 320 —20 315 -15 310 -10
0.4 240 260 -20 258 —18 256 -16
0.6 350 355 - 5 350 0 350 0
0.5 300 320 -20 315 -15 310 -10
0.4 240 260 -20 258 -18 244 - 4
0.6 350 355 - 5 350 0 350 0
0.5 - 300 320 -20 315 -15 310 -10
0.4 240 260 -20 258 -18 256 -16
VIII. B. 1. (Continued)
4V
Norimlity Control LEC I C-E LEC II C-E LEC III C-E
0.6 350 355 - 5 350 0 350 0
0.5 300 320 -20 315 -15 310 -10
0.4 240 260 —20 258 -18 254 -14
0.6 350 355 - 5 350 0 350 0
0.5 300 320 -20 315 -15 310 -10
0.4 240 260 -20 256 -16 254 -14
0.6 350 355 - 5 350 0 350 0
0.5 300 320 -20 315 -15 310 -10
0.4 240 260 -20 258 -18 256 -16
0.6 350 355 - 5 350 0 350 0
0.5 300 320 -20 315 -15 310 -10
0.4 .240 260 -20 258 -18 256 -16
0.6 350 355 - 5 350 0 350 0
0.5 300 320 -20 315 -15 310 -10
0.4 240 260 -20 258 -18 254 -14
0.6 350 355 - 5 350 0 350 0
0.5 300 320 -20 315 -15 310 -10
0.4 240 260 -20 258 -18 254 -14
0.6 350 355 - 5 350 0 350 0
0.5 300 320 -20 315 -15 310 -10
0.4 240 260 -20 256 -16 256 —16
0.6 350 355 5 350 0 350 0
0.5 300 320 -20 315 -15 310 —10
0.4 240 260 -20 258 -18 254 -14
VIII* (Continued)
C. Mathema.tical Table Covering Results of Experiments on
Lecithin*
48
Test
Reagent
Solutions
Normality of
Ringer* s
Mean Difference
Retween
Control & Exjperimental ( T
LEC I 0.6 - 2.5 + 2.5
0.5 -17.4 + 3.1
0.4 -30.8 + 9.1
LEC II 0.6 0 0
0.5 -12.3 + 2.8
0.4 -21.1 +4.5
LEC III 0.6 0 0
0.5 - 7.43 + 2.5
0.4 —23.6 +9.6
IX* Glucose Tests; Effects of a solution of glucose isosmotic T/ith
the other reagents tested*
A* Using Three Solutions of Glucose.
Normality Control GLUC I C-E GLUC II C-E GLUC III C-E
0.6 350 350 0 350 0 350 0
0.5 300 300 0 300 0 300 0
0.4 240 240 ■ 0 240 0 240 0
0.6 350 350 0 350 0 350 0
0.5 300 300 0 300 0 300 O'
0.4 240 240 0 240 0 240 0
IX. Â. (Continued)
49
Normality Control GLUC I C-E GLUC II C-E GLUC III C-E
U. 6 350 350 0 350 0 350 0
0.5 300 300 0 300 0 300 0
0.4 240 240 0 240 0 240 0
0.6 350 350 0 350 0 350 0
0.5 300 300 0 300 0 300 0
0.4 240 240 Ü 240 0 240 0
0.6 350 350 0 350 0 350 0
0.5 300 300 0 300 0 300 0
0.4 240 240 0 240 0 240 0
0.6 350 350 0 350 0 350 0
0.*5 300 300 0 300 0 300 0
0.4 240 240 0 240 0 240 0
0.6 350 350 0 350 0 350 0
0.5 300 300 0 300 0 300 0
0.4 240 240 0 240 0 240 0
0.6 350 350 0 350 0 350 0
0.5 300 300 0 300 0 300 0
0.4 240 240 0 240 0 240 0
0,6 350 350 0 350 0 350 0
0.5 300 300 0 300 0 300 0
0.4 240 240 0 240 0 240 0
0.6 350 350 0 350 0 350 0
0,5 300 300 0 300 0 300 0
0,4 240 240 0 240 0 240 0
IX. (Continued)
B. Mathematical Table Covering Results of Experiments on
Glucose.
50
Test
Reagent
Solutions
Normality of
Ringer's
Mean Difference
Between
Control & Experimental (T
GLUC I 0.6 0 0
0.5 0 0
0.4 0 0
GLUC II 0.6 0 0
0.5 0 0
0.4 0 0
GLUC III 0.6 0 0
0.5 0 0
0.4 0 0
Heat Tests: Effect of heat on a water soluble adreno-cortical
extract (ACE), an aqueous solution of cholesterol and an aqueou;
solution of DOCA.
A. Effects on ACE "W"; Concentration of ACE I is the same, as
page 17; ACE II, 0.1 of ACE I; ACE III, 0.01 of ACE I.
1. Before Heating.
Normality Control ACE : I C-E ACE II C-E AGE III C-E
0.6 375 345 +30 340 +35 335 +40
0.5 315 286 +29 290 +25 296 +19
0.4 220 183 +37 210 +10 206 +14
51
X. A. 1. (Continued)
C-E ACE III C-E
n o r m a i J L u y
0.6
0.5
0.4
VJUIiOi VJL
375
315
220
345
286
194
^ 30
29
+ 26
345
288
208
-+30
+- 27
f 12
340
294
190
+ 35
+ 21
+ 30
0.6 375 345 / 30 350 + 25 345 + 30
0.5 315 286 / 29 288 + 27 288 + 27
0.4 220 200 / 2 0 189 ^ 31 208 V 12
0.6 375 345 / 30 345 + 30 350 + 25
0.5 315 286 + 29 288 + 27 290 + 25
0.4 220 198 ^ 22 210 ^ 10 206 / 14
0.6 375 345 / 30 350 +-25 335 + 40
0.5 315 286 /29 288 + 27 292 + 23
0.4 220 190 + 30 210 + 10 208 + 12
0.6 375 345 ^ 30 340 + 35 340 + 35
0.5 315 288 +-27 290 + 25 296 + 19
0.4 220 200 ^.20 210 + 10 206 t 14
0.6 375 345 + - 30 350 + 25 346 + 29
0.5 315 284 + 31 286 + 29 290 ^ 25
0.4 220 194 + 26 210 + 10 208 / 12
0.6 375 345 + 30 350 + 25 335 + 40
0.5 315 286 + 29 296 + 19 290 ^ 25
0.4 220 188 + 32 210 + 10 206 + 14
0.6 375 345 + 30 350 + 25 350 + 25
0.5 315 286 +-29 288 + 27 296 +-19
0.4 220 200 +- 20 210 / 10 206 + 14
52
X* A. (Continued)
2. After Heating.
Norirality Control ACE I G-E ACE II C-E ACE III C-E
0.6 375 375 0 375 0 375 0
0.5 315 315 0 315 0 315 0
0.4 220 220 0 220 0 220 0
0.6 375 375 0 375 0 375 0
0.5 315 315 0 315 0 315 0
0.4 220 220 0 220 0 220 0
0.6 375 375 0 375 0 375 0
0.5 315 315 0 315 0 315 0
0.4 220 220 0 220 0 220 0
0.6 375 375 0 375 0 375 0
0.5 315 315 0 315 0 315 0
0.4 220 220 0 220 0 220 0
0.6 375 375 0 375 0 375 0
0.5 315 315 0 315 0 315 0
0.4 220 220 0 220 0 220 0
0.6 375 375 0 375 0 375 0
0.5 315 315 0 315 0 315 0
0.4 220 220 0 220 0 220 0
0.6 375 375 0 375 0 375 0
0.5 315 315 0 315 0 315 0
0.4 220 220 0 220 0 220 0
0.6 375 375 0 375 0 375 0
0.5 315 315 0 315 0 315 0
0.4 220 :220 0 220 0 220 0
0.6 375 375 0 375 0 375 0
0.5 315 315 0 315 0 315 0
0.4 220 220 0 220 0 220 0
53
X. (Continued)
B. Mathematical Table Covering Results of Experiments of Heat
Effects on Adreno-Cortical Extract
Test Solutions Mean Difference
Normality of Between
( T Reagent Ringer's Control & Experimental
Before Heating
ACE "W" I 0.6 + 30.0 0
0.5 + 28.6 + 0.83
0.4 + 25.9 ± 5.8
ACE "W" II 0.6 + 28.3
+ 4.1
0.5 + 25.9 ± 2.7
0.4 + 12.6 + 6.7
AGE "W" III 0.6 + 33.2 + 5.9
0.5 + 22.6 + 2.9
0.4 + 15.1
+ 5.3
After Heating
AGE "W" I 0.6 0 0
0.5 0 0
0.4 0 0
ACE "W" II 0.6 0 0
0.5 0 0
0.4 0 0
ACE "W" III 0.6 0 0
0.5 0 0
0.4 0 0
54
X. (Continued)
G. Effects on Cholesterol,
1* Before Heating
Normality Control CHOL I C-E CHOL III G-E
0.6 325 335 -10 330 - 5
0.5 264 274 -10 270 — 6
0.4 187 200 -13 190 - 3
0.6 325 ' 335 -10 330 - 5
0.5 264 274 -10 270 - 6
0.4 187 200 -13 190 -rs
0.6 325 335 -10 330 - 5
0.5 264 274 -10 270 — 6
0.4 187 200 -13 190 - 3
0.6 325 335 - —10 330 - 5
0.5 264 276 —12 272 - 8
0.4 187 202 -15 188 - 1
0.6 325 340 -15 330 - 5
0.5 264 272 — 8 270 - 6
0.4 187 202 -15 190 - 3
0.6 325 330 - 5 330 - 5
0.5 264 270 - 6 268 — 4
0.4 187 202 -15 190 - 3
0.6 325 340 -15 330 - 5
0.5 264 268 — 4 266 - 2
0.4 187 200 -13 188 - 1
0.6 325 335 -10 330 - 5
0.5 264 274 -10 270 — 6
0.4 187 198 -11 190 — 3
X. c. 1. (Continued)
55
Normality Control CHOL I C-E CHOL III C-E
0.6 325 335 -10 330 - 5
0.5 264 268 — 4
272 - 8
0.4 187 202 -15 190 - 3
2. After Heating.
Normality Control CHOL I G-E CHOL III C-E
0.6 325 335 -10 330 - 5
0.5 264 274 -10 270 - 6
0.4 187 200 -13 190 - 3
0.6 325 340 -15 330 - 5
0.5 264 274 -10 270 - 6
0.4 187 200 -13 192 - 5
0.6 325 340 -15 335 —10
0.5 264 274 -10 270 — 6
0.4 187 198 -11 188 - 1
ÛC
0.6 325 330 - 5 330 - 5
0.5 264 276 -12 270 — 6
0.4 187 202 -15 190 - 3
0.6 325 335 -10 330 - 5
0.5 264 274 -10 268 — 4
0.4 187 200 -13 190 - 3
0.6 325 335 -10 330 - 5
0.5 264 274 -10 276 -12
0.4 187 200 -13 192 - 5
0.6 325 335 -10 330 - 5
0.5 264 274 -10 270 - 6
0.4 187 202 -15 188 - 1
X. c. 2. (Continued)
56
Normality Control CHOL I C-E CHOL III C-E
0.6 325 335 -10 330 - 5
0.5 264 274 -10 270 - 6
0.4 187 198 -11 190 - 3
0.6 325 335 -10 330 - 5
0.5 264 274 -10 268 — 4
0.4 187 200 -13 188 . - 1
D. Mathematical Table Covering Results of Experiments of Head
Effects on Cholesterol.
Test Solutions Mean Difference
Normality of Bet we en
Reagent Ringer's Control & Experimental 0
Before Heating
CHOL I 0.6 —10.6 -+ 2.9
0.5 — 8.2
t
2.7
0.4 -13.7
±
1.3
CHOL III 0.6 - 5.0 0
0.5 - 5.8 + 1.7
0.4 — 2.6
t
0.84
After Heating
CHOL I 0.6 -10.6 2.9
0.5 -10.2
+
0.63
0.4 -13.0 1.3
CHOL III 0.6 - 5.6
±
1.5
0.5 — 6.2
t
2.2
0.4 — 2.8 1.5
57
X* (Continued)
E. Effects on DOCA.
1. Before Heating
Normality Control DOCA I G-E DOGA III G-E
0.6 325 340 -15. 330 - 5
0.5 264 270 - 6 268 - 4
0.4 187 210 -23 194 - 7
0.6 325 350 -25 335 -10
0.5 264 270 - 6 260 + 4
0.4 187 210 -23 194 - 7
0.6 325 335 -10 330 - 5
0.5 264 270 - 6 268 - 4
0.4 187 210 -23 194 - 7
0.6 325 345 -20 330 - 5
0.5 264 268 — 4 268 — 4
0.4 187 208 -21 196 - 9
0.6 325 350 -25 335 -10
0.5 264 270 — 6 268 - 4
0.4 187 206 -19 195 - 8
0.6 325 345 -20 330 - 5
0.5 264 272 — 8 266 - 2
0.4 187 208 -21 192 - 5
0.6 325 340 -15 330 - 5
0.5 264 268 - 4 268 — 4
' 0.4 187 210 -23 194 - 7
0.6 325 340 -15 330 - 5
0.5 264 270 - 6 268 - 4
0.4 187 208 -21 194 - 7
X. E. 1. (Continued)
58
Normality Control DOCA I C-E DOCA III C-E
0.6 325 345 —20 330 - 5
0.5 264 270 — 6 268 - 4
0.4 . 187 206 -19 194 - 7
2. After Heating.
Normality Control DOCA I C-E DOCA III C-E
0.6 325 340 -15 330 - 5
0.5 264 270 - 6 268 - 4
0.4 187 210 -23 194 - 7
0.6 325 345 -20 335 -10
0.5 264 270 - 6 268 - 4
0.4 187 210 -23 194 - 7
0.6 325 335 -10 330 - 5
0.5 264 272 - 8‘ 268 - 4
0.4 187 210 -23 194 - 7
0.6 325 346 -21 330 - 5
0.5 264 270 — 6 266 — 2
0.4 187 208 -21 198 -11
0.6 325 345 -20 335 -10
0.5 264 270 - 6 268 - 4
0.4 187 210 -23 198 -11
0.6 325 345 -20 330 - 5
0.5 264 270 - 6 268 - 4
0.4 187 208 -21 196 - 9
0.6 325 340 -15 335 -10
0.5 264 268 - 4 266 — 2
0.4 187 210 -23 190 - 3
59
. 2. (Continued)
Normality Control DOGA I C-E DOCA III C-E
0.6 325 340 -15 330 - 5
0.5 264 270 - 6 268 — 4
0.4 187 210 -23 194 - 7
0.6 325 345 -20 330 - 5
0.5 264 270 - 6 - 268 - 4
0.4 187 210 -23 194 - 7
F. Mathematical Table Covering Results of Experiments of
Heat Effects on DOCA.
Test Solutions
Normality of
Mean Difference
Between
Reagent Ringer's Control & Experimental
Before Heating
DOCA I 0.6 -18.3 + 4.7
0.5 - 5.8 + 1.1
0.4 -21.6
± 1.6
DOCA III 0. 6 — 6.1 + 2.1
0.5 — 3.8 + 0.52
0.4 - 7.1
± 1.03
After Heating
DOCA I 0.6 -17.3 ± 3.5
0.5 - 6.0 - t 0.95
0.4 -22.6 ± 0.84
DOCA III 0.6 — 6.7 ± 2.4
0.5 - 3.6
± 2.5
0.4 - 7.7
d L 2.3
60
DISCUSSION OF EXPERIÎ/IENTÂL RESULTS
The experimental results of this research clearly indicate that
steroids influence the osmotic fragility of the beef erythrocyte.
There are several theories which could poosibly explain the mode of
operation of the reagents tested. In the case of an increase in
osmotic fragility the reagents which produce this increase could
affect the membrane in the following ways. The reagents could com
bine with the membrane in such a way as to actually alter the chem
ical bonds in the membrane resulting in a weaker structure which
breaks down under osmotic stress more readily than it ordinarily
would. Also, the reagent could chemically dissolve portions of the
membrane, thus leaving a v/eaker structure which collapses under
osmotic stress more readily than under normal conditions. Another
possible action is that the reagent actually changes the permeability
of the membrane either to vmter, to the solutes of the cell, or to
both. The implications of an increase in water permeability can
readily be seen; that is, more water can penetrate before solutes
diffuse out, thus producing greater degrees of hemolysis, swelling,
or both. In the case of the cellular solutes if the permeability of
the membrane to these compounds is decreased then in hypotonic solu
tions their passage out is hindered, and water flows into the cell
reducing the concentration gradient. This increase of water in the
cell produces swelling and possibly hemolysis to a degree greater
than if the reagent is not present. If, on the other hand, the
61
permeability to the cellular solutes is increased less swelling or
hemolysis of the erythrocyte will occur in hypotonic solutions.
A decrease in fragility could be caused by reagents which be
have in the following manner; A direct physical adsorption on the
surface of the membrane which, by virtue of the amount of material
added to the membrane, increase the tensile strength with a subse
quent increase in resistance to osmotic stress; also, these com
pounds could combine chemically with the constituents of the plasma
membrane thus binding it together more firmly, resulting in a struc
ture more resitant to osmotic stress. A change in the permeability
of the plasma membrane to either water or the cellular solutes is
possible. This change in permeability could be of such nature as to
inhibit the penetration of water, or facilitate the escape of the
cellular solutes and thus reduce the effects of placing the blood
cell in hypotonic solutions.
The method employed in reading the results, photo-electric color
imetry, depends upon the transmission of light. In this research
there are two possible methods by which the light transmission may be
affected. The cells may either hemolyze or swell. There is also the
possibility of both occurring simultaneously. Hemolysis allows more
light to be transmitted, and swelling, according to Davson and
Danielli (1943), cannot be detected as separate from hemolysis in the
same suspension. This means that as some cells may only have swollen
while others have hemolysed, so that the entire picture appears to be
that, at equilibrium the net effect of swelling and hemolysis is to ■
increase the amount of light transmitted by the w/hole suspension. In
62
the interpretation of the experimental results it will be assumed
that the change in the transmission of light is due to both hemolysis
and swelling since the suspension was mixed before reading. Tests
are now in progress to determine the exact nature of the change in
amount of light transmitted, that is the relative contributions of
hemolysis and swelling.
1, Effects of the water soluble adreno-cortical extract. Results
using both extract ’ ’ Upjohn** and extract ’ * \ Y ’ * were well defined and
showed that water soluble adreno-cortical extracts appreciably in
crease the osmotic fragility of the red blood cell, as is evidenced
by the decrease in the Klett reading when the extract is present in
the suspending fluid. The possible mode of action of compounds
which produce this effect has been discussed.
It is Interesting to note tho.t since the action of the extract
is so pronounced, and since both extracts exhibited similar actions,
perhaps some method of assay for water soluble adreno-cortical ex
tracts could be devised using the osmotic fragility method as out
lined in this research. The work of Rauchschwalbe (1940) indicates
that adrenal cortex products cause certain changes in the osmotic
fragility picture. The concensus of opinion is that cortical steroids
tend to increase the penetration of water into red cells. The results
of the extract experiments in this research are in keeping with the
results presented in the literature.
2. Effects of cholesterol. The experimental results clearly indi
cate that cholesterol reduces the amount of light transmitted,
probably due to a depressing action of cholesterol on hemolysis and
63
swelling, even in dilute solutions, as is shown in the results of
tests with CHOL III and CHOL IV# Possible modes of action of com
pounds producing these results have been discussed*
The results found using cholesterol agree fairly well v/ith the
information,available in the literature.
3* Effects of DOCA* The results of the DOCA experiments show
clearly that DOCA behaves in a manner similar to that of cholesterol,
but its mode of operation may be quite different. Although DOCA,
being lipoidal in nature, could conceivably be adsorbed to the sur
face of the cell, increasing the tensile strength, it is interesting
to note that the effect of DOCA in the adrenalectomized animal, as
discussed by Turner (1948), is to increase sodium retention. This
fact indicates an action attributable to DOCA which is more than the
mere physical adsorbtion which may occur in the case of cholesterol.
If cholesterol is given to the adrenalectomized animal it has no
effect on sodium retention. Although the results using DOCA and
cholesterol in red blood cell suspensions are similar, their mode of
action, because of this difference in sodium retention activity, may
not be the same. It is possible that the effect of DOCA on the red
cell is to increase the permeability of the membrane to sodium, the
constituent of the Ringer’s solution having the highest osmolar con
centration, so that in hypotonic solutions the sodium .readily escapes,
reducing the concentrât ion gradient for v/ater, thus reducing the flow
of water into the cell and the subsequent swelling, or hemolysis.
4. Effects of lecithin. Since lecithin is related to cholesterol in
that it is a lipoidal surface active compound, it is not surprising
64
to find that it ha.s an action on the red blood cell similar to that
of cholesterol. Lecithin and cholesterol exhibit an antagonistic
action on fatty acid films as described by Loathes (1943). Also,
V/illiams (l94l), in discussing fat metabolism, states that cholesterol
and lecithin are involved in oomo obscure way in fat transportation,
phosphoylation and utilization. Free cholesterol and lecithin also
appear to be antagonistic to each other in these reactions and are
present in a given tissue at a fixed ratio. Ponder (1945) states
that cholesterol has the power to combine directly with the components
of the cell membrane. Since, according to Sharpe (1926), Gortner
(1929) and Heilbrunn (1948), lecithin is an important constituent of
all cells, it is quite possible that lecithin also may be chemically
attached to the membrane thus reducing the effects of osmotic stress
by simply increasing the tensile strength of the membrane. Also, due
to the actual increase of materials at the surface of the blood cell,
the permeability may be altered, thus producing the apparent decrease
in hemolysis when lecithin is present.
5. Osmotic experiment with glucose. This phase of the research
was included to ascertain if the action of cholesterol, DOCA and
lecithin in reducing the amount of light transmitted, with a possible
decrease in hemolysis, Y/as merely an osmotic one, in that the concen
trations of the tested compounds were such that the osmotic pressure
of the entire system was affected, thus changing the effective amount
of water available for penetration.
The results clearly show that this was not the case, and that
some other activity was involved, rather than an osmotic one.
65
6. Heat effects, a; Adreno-cortical extract. Heating the adreno
cortical extract to 60^C. for one hour destroyed its effect on the
red cell. This probably indicates that the factor in the extract
which influences the blood cell is the same as the factor which is
responsible for life sustaining action since Cartland and Kuisenga
(1936) state that raising the temperature of the extract during its
preparation seriously impairs its life sustaining activity. Mason^
(1937) states that reduction of the 4-5 double bond destroys all
physiological activity of steroids possessing that configuration;
heat could possibly reduce the 4-5 double bond, thus destroying the
activity of the compound.
b: Cholesterol and DOGA. Heating these two compounds in a manner
similar to the-method employed with the adreno-cortical extract, did
not affect their respective effects on the red cell. One possible
interpretation of this fact is that the steroid nucleus is not
connected with the life sustaining activity and the effects of the
adreno-cortical extract in decreasing the resistance of the red cell
to osmotic stress as measured by light transmission.
66
CONCLUSIONS
Two Y7hole adreno-cortical extracts tested increase the osmotic
fragility of the erythrocyte as measured by light transmission methods.
The extracts, when present in the suspending fluids of the erythrocyte
suspensions, cause an increase in the amount of light transmission
which indicates either an increase in hemolysis, swelling or both.
Cholesterol, desoxycorticosterone acetate and lecithin have the
opposite effect on the red cells; decreasing the amount of light trans
mitted by their action on the red cell suspension. This decrease in
light transmitted indicates either a decrease in hemolysis, sv/elling,
or both.
Heating to 60^0. for one hour destroys all effect the whole adreno
cortical extract exerts on the erythrocytes. Heating the cholesterol
and DOCA has no effect on their respective effects on the erythrocytes.
67
SUMÆARY
Ty; o wa,ter soluble whole extracts of the adrenal cortex, one an
experimental preparation of the Upjohn Company, the other prepared in
conjunction with this research ucing the method outlined by Cartland
and Kuizenga (1936), were tested. The extracts were introduced into
the suspending fluid of red cell suspensions. Appropriate controls
were maintained and the results were read using light transmission
methods.
Aqueous solutions of cholesterol, DOCA and lecithin were also
tested in the above manner.
The results indicated that the whole adreno-cortical extracts
increased the amount of light transmitted by the erythrocyte suspen
sions. This was interpreted as meaning that the degree of hemolysis,
swelling or both was increased due to the presence of the adreno
cortical extract. The results of the cholesterol, DOCA and lecithin
tests indicate that these compounds decreased the light transmission
of red blood cell suspensions, probably by decreasing the degree of
hemolysis, swelling or both. These effects of cholesterol, DOCA and
lecithin were not osmotic phenomena since comparison v/ith an isosmotic
glucose solution tested in the same manner had no effect on the red
cell suspension.
Heating to 60®C. for one hour destroyed Sill activity of the adreno
cortical extracts on the red cell suspension, but had no effect on the
activity of cholestérol and DOCA solutions. All containers were stop
pered during the heating process to insure the retention of volatiles.
68
BIBLIOGRAPHY
A. BOOKS
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Onlwrsrty of Southern CaHforn,. u . r . „

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Creator Wilson, James (author)

Core Title The effect of steroids and lecithin on the resistance of the beef erythrocyte to osmotic stress

Contributor Digitized by ProQuest (provenance)

Degree Master of Arts

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Biological Sciences,OAI-PMH Harvest

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Permanent Link (DOI) https://doi.org/10.25549/usctheses-c39-277120

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