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

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

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2. ‘ f t

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T h is thesis, w ritte n by

............Jam.e^..WTi2.s.on, under the guidance of hi=§—F a c u lty C o m m ittee, and a p p ro ved by a l l its members, has been presented to and accepted by the C o u n cil on G raduate Study 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

Mast.er.. o.f..Arts, P.apartment„pf...Zpplogy. D ate..

Faculty Committee

Chairman

gtzM.,..

TABLE OF CONTENTS

PAGE

STATEMENT OF THE P R O B L E M ..............................

1

REVIEW OF THE LITERATURE I II III

RELATION OF PERMEABILITY TO STEROIDS

....

3

PERMEABILITY .................................

6

METHODS OF MEASURING PERMEABILITY

9

...........

METHODS AND TECHNIQUES I II

GENERAL

................

11

PREPARATION AND SELECTION OF RED BLOOD

CELL

S U S P E N S I O N S ....................... III IV

PREPARATION OF ADRENO-CORTICAL EXTRACT

VI VII

....

15

PREPARATION OF CHOLESTEROL, DESOXYCORTICOSTERONE ACETATE, AND LECITHIN SOLUTIONS

V

12

..........

18

.................

20

...............................

21

OSMOTIC PRESSURE EXPERIMENT HEAT EFFECTS

-COLORIMETRY......................

21

EXPERIMENTAL RESULTS ..................................

23

\

DISCUSSION OF EXPERIMENTAL RESULTS ....................

60

C O N C L U S I O N S ..........................

66

S U M M A R Y .................... BIBLIOGRAPHY ..........................................

*

67 68

The size of this type is satisfactory.

Frederick Fey Shelden Committee Chairman Zoology Department University of Southern California

STATEMENT OF THE PROBLEM

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 hemoconcentration and tissue dehydration are apparent in hypocortidism.

Because of the derangement in fluid balance it is probable

that permeability of both cells as individual units and of tissues as a whole 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 Rauehschwalbe (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 DOGA,.is a synthetic steroid which 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 plasma 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. is to destroy all physiological activity.

The effect of heat

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.

3

REVIEW OF THE LITERATURE

I

RELATION OF PERMEABILITY TO STEROIDS

Turner (1948) sunmarizes the effect of adrenal insufficiency in saying that there is a fluid derangement with a hemoconcentra­ tion, the fluid balance being swung 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 (1935), 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, Hyman 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 Hyman and Chambers (1943) 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 with the former experiment* Rauchschwalbe (1940) 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. creases hemolysis in both cases.

His summary states that Cortidyn in­ 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 powerful than corticosterone in its effect on electrolyte balance according to Kleiner (1946). 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 v/hole 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. Sayers, et al ( 1 9 ^ )

Dalton, et al (19^-), Selye (1937);

and Abel in (19^6) 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 phys i o ­

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 ( 1 9 3 0

state emphatically, that

to raise the temperature of their adrenal cortex extract above 4 5 ° C . 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 A-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 p e r ­ meability phenomena.

6 II

PERMEABILITY

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

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 (1943) take up all the prominent theories.

Heilbrunn (1948), Sharpe (1926) and Schmitt, Bear and Ponder (1936) (1938), discuss the Lipo-Protein-Active-Fatch 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 (1945), which correlates many of the widely separated facts. the Lipo-Protein theory.

He attempts a plausible explanation of

He states that proteinoids pass through the

protein portion, fats through the lipoid portion, and all other com­ pounds through the junction of the lipoid-protein areas.

Naturally,

no one theory, per se, can completely answer all questions or explain all anomalies, 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 monomolecular 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, Bayliss (192^).

is the unsupported contention of

The effects of surface active compounds on the

plasma membrane which is the Lipo-Protein system under consideration are of importance*

Ponder (19^6)

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 (19^5) 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

60'$

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 d e ­ ranged water metabolism, as in A d d i s o n ’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 pro m i n ­

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 I.II

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, Ponder 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 (1927) 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 swelling 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 h e m o ­ lysis can It is

readily be detected. 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, with sharply demarcRtad extremities, to come 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 The cell can The

could be maintained with little or no laboratory equipment. suitableeffector in this case was distilled water.

The

red

only hold so much water; then it bursts, or hemolyzes. 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 with this work. The most complicated phase of the problem was the bringing together of the erythrocytes, water, and various test reagents, so that optimum conditions would 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-Suramerson 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 v/hipping, and refrigerated at 4°C. within a period of two 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 erythrocyte in a Ringer's solution consisting of 9*0 gm NaCl, 0.24 gm CaCl^, 0.48 gm KG1, 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 weeks.

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 were washed 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 was 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 Normal 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 with 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/o 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, clue to experimental error, to be reliable. A 0.6% suspension of red cells was made in the aforementioned manner in 1 Normal Ringer’s.

To 5 c*e. 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 uistilled 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 Normal 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 were used.

The first, an experimental product of the Upjohn

Laboratories (Beef, 14 SCL 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 Heat 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 Blendor 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. approximately 100 c.c.

Here the volume was reduced to

This resulting aqueous extract was next ex­

tracted with first, two 200 c.c. portions and then one 100 c.c. portion of petroleum ether. material.

This removed large quantities of inert lipoid

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 4-5°G., and the residue was dissolved in 200 c.c. of absolute ethyl alcohol. was added 200 c.c. of petroleum ether.

To this

The alcohol was progressively

reduced from 100$ to 90$ to 80$ and finally 70$, by adding appropriate quantities of distilled water.

Petroleum ether, being only miscible

with absoluxe alcohol, separates as the portions of water are adaed. This petroleum ether is dravrn off as it separates.

The 70$ alcohol

extract solution was concentrated by evaporation in vacuo below 45°G. until an aqueous colloidal solution of the extract remained. normal beef Ringer's solution was added. first.

Here one

A 50 c.c. portion was added

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 (ly36) using the same method, arrived at an

17 assay of tneir 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 product will subsequently be referred to as extract "Upjohn,” ana the other as extract "W." The cortical extracts were prepared for use in the following manner.

A concentration of 0.3r 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 Normal 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

" 17"

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 v/as 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 DOCA, cholesterol and lecithin. Five cubic centimeters of a 0,6% suspension of beef erythrocytes in isotonic Ringer's plus five cubic centimeters o’ f the aqueous test solu­ tion plus five cubic centimeters of the variable normality of Ringer’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 water.

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 standard quantity of cholesterol amounting to 0.0022 grams v/as dissolved in one liter of distilled water in the following manner.

Powdered reagent cholesterol amounting to 0.027 grams was

placed in a Waring Blendor with 500 c.c. of distilled water and mixed for thirty minutes.

The 500 c.c. with the cholesterol was placed in

a 1 liter flask and the blendor washed with 500 c.c, distilled water. This v/as 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 v/as emptied into the blendor and contents agitated again for thirty minutes,

Tho 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 wash 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 CH0L I, subsequent

dilutions were made.

As a result, the four other solutions and their

concentrations were numbered as follows:

CH0L II, 2.2 x lCT4 ; CH0L III,

2.2 x 1CT6; CH0L IV, 2.2 x 10~7 ; and CH0L V, 2.2 x 10~9.

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 TriChIorAcetic 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. loss during heating m s

It v/as assumed that weight

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, v/as considered and the possibility tested as follows:

a solution of

glucose v/hich would have little or no specific effect on permeability in proportion to its osmotic effect v/as 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

HEAT EFFECTS

Since Cartland and Kuizenga (1936) cautioned against raising the temperature of the water soluble adrenal cortex extract above 45°C, in their method of preparation, and since it is the contention of Mason

(1 939) and this research that said heating may destroy all activity of those compounds having the 4-5 double bond in the steroid nucleus, it was decided to see v/hat effect heat would have on the water soluble extract and also on cholesterol and DOCA, 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. following manner,

The colorimeter was read in the

A control tube was placed in the holder and the dial

so adjusted to bring the galvanometer to zero deflection. read from the dial was considered as the control value.

The figure The first

experimental tube was placed in the holder and the galvanometer pointer

22 was again brought to zero position. was the first experimental value.

The dial reading for this sett'ing 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 was

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. experimental value could be reproduced.

This was done to see if the All subsequent tests were

made and recorded in the above manner. The experimental procedure is as followss

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 »6f> 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 was 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

EXPERIMENTAL RESULTS

I,

Table of Abbreviations and Concentrations of Compounds Tested*

Compound Tested Cholesterol

Desoxycorticosterone Acetate

Lecithin

Glucose

Abbreviation Used

Concentration Moles per Liter

Concentration Grams per Liter

CHOL CHOL CHOL CHOL CHOL CHOL

I II III IV V

6 6 6 6 6

x x x x x

DOCA DOCA DOCA DOCA DOCA DOCA

I II III IV V

5.7 5.7 5.7 5.7 5.7

X

LEC LEC LEC LEC

I II III

6.1 x 10~7 6.1 x 10“8 6.1 x 10~9

4.9 x 10~3 4.9 x 10~4 4.9 x 10“5

GLUC GLUC I GLUC II GLUC III

6 x 10 ~6 6 x 10~7 6 x 10 ~8

1.08 x 10~3 1.08 x 10“4 1.08 x IQ -5

X X X X

10 -6 10 -7 10 -8 10 -9 10 -10

10“® 10~7 10”® 10~9 10"10

2.2 2.2 2.2 2.2 2.2

2.1 2.1 2.1 2.1 2.1

x x x x x

10 -3 10 - 4 10 -5 10 -6 10 -7

x 10"® x 10"’ x 10"® x 10"® x 10"7

24 II,

Following is a table giving the range of readings on the KLETTSUMMERSON colorimeter at given percentages of hemolysis.

0 145 146 190 265 '310 315 325 340' 345 Above

145 146 190 265 310 315 325 340 345 375 375

100^ Hemolysis 90% 80%

10% 60% 50% 4:0%, 30% 20 %

10% 0%

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 Below 0.10%,

0.10% 0 .20% 0.30% 0.40% 0.50% Above 0.50%,

Non-Hemolysed R e a d i n g __

Hemolysed Reading 100%,

No visible reaction on instrument 22 93 100 232 173 390 260 520 390 750 No visible reaction on instrument

25 III. A. (Continued) Dilution of Red Cells Belov/ 0.10%

Non-Hemolysed Reading

Hemolysed Reading 100$ __

No visible reaction on instrument

0.10?C 0 20% 0.30/C 0 40 0.50$ Above 0.50$

No visible reaction on instrument

Belov/ 0.10/C

No visible reaction on instrument

. .^

0.10$ 0*20%

0.30$ 0.40$ 0.50% Above 0.50$ 0.10$ 0*20% 0*30% 0*4:0% Above 0.40/C

0.10$ 0.20$ 0*30%

0.40/C Above 0.40/C 0.10$ 0.20$ 0.30$ 0.40/C Above 0.40$

92 230 390 520 750

92 232 390 520 750

20 102 173 260 390

22 100 173 260 390

No visible reaction on instrument

133 296 450 580

34 130 216 300

Over upper limit of colorimeter

135 296 448 580

32 132 216 300

Over upper limit of colorimeter

133 296 450 580

34 130 216 300

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/C standard diluted to 0.2/C 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 Taken Immediately After Mixing

Reading 2 Taken 30 Minutes After Reading 1

1.0 N 0.9 0.8 0.7 0.6 0.5 0.4 0.3

360 360 360 335 320 274 176 174

370 370 360 335 320 274 175 174

1.0 N 0.9 0.8 0.7 0.6 0.5 0.4 0.3

370 365 360 335 320 274 176 175

370 365 360 335 320 274 176 174

1.0 N 0.9 0.8 0.7 0.6 0.5 0.4

370 360 360 335 320 274 176

370 360 360 335 320 274 176

Normality of Ringer's Solution

27 III# B. (Continued)

Normality of Ringer’s Solution 1.0 N 0*9

Reading 1 Taken Immediately After Mixing

Reading 2 Taken 30 Minutes After Reading 1

0. 3.

365 360 355 340 320 253 176 168

365 360 355 340 320 254 174 167

1.0 N 0.9 0.8 0.7 0.6 0.5 0.4 0.3

365 360 355 340 320 253 176 168

365 360 355 340 320 253 174 167-

0.6 N 0.5 0.4

320 253 176

320 253 174

0.6 N 0.5 0.4

300 252 177

300 252 177

0.6 N 0.5 0.4

300 252 177

300 252 177

0.6 N 0.5 0.4

305 252 175

305 252 175

0.8 0.7

0.6 0.5 0.4

28 III. B. (Continued) Reading 1 Normality of Taken Immediately Ringer* s Solution_______After Mixing___

Reading 2 Taken 30 Minutes After Reading 1

0 #4

305 252 175

305 252 175

0.6 N 0.5 0.4

286 254 169

289 254 169

0.6 N 0.5

Using a normality range of from 0.6 to 0.4 Normal Ringer’s solution 0.s the suspending fluid, the values fall into the most highly calibrated section of the colorimeter dial, giving greatest accuracy.

iv.

This normality range will be employed.

mathematical 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 would 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 v/as determined using the following formula,

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. Am

Preliminary Studies on the Effect of Extract "Upjohn." Reading 1 was made immediately after mixing.

Reading 2 was

made 30 minutes after reading 1.

Normality of Ringer's Solution

Reading 1 Without With Extract Extract

Reading 2 Without With ■ Extract' Extract

Control .Minus Experimental

0.6 N 0.5 0.4

286 250 168

286 185 160

288 249 167

288 181 159

0 + 68 + 8

0.6 N 0.5 0.4

286 250 168

286 185 160

288 249 167

288 181 159

0 + 68 + 8

0.6 N 0.5 0.4

288 250 168

288 185 160

288 249 167

288 181 159

0 + 68 +• 8

0.6 N 0.5 0.4

288 250 168

288 185 160

288 249 167

288 181 159

0 + 68 + 8

0.6 N 0.5 0.4

288 250 168

288 185 160

288 249 167

288 181 159

0 +68 + 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 Ringer*s Solution 0.6 N 0.5 0.4

204 244 168

0.6 N 0.5 0.4

C.

Normality

Reading 1 Y/ithout With Extract Extract

284 244 168

Reading 2 Without With Extract Extract

284 210 148

284 210 148

Control Minus Experimental

284 244 163

284 210 148

0 +34 +15

284 244 163

284 210 148

0 + 34 +15

Comparison of Effects of Extract "WM and Extract "Upjohn.” Control Control Minus Experimental Minus Experimental Experimental "¥" Experimental Control "Upjohn"

0.6 N 0.5 0.4

284 244 163

284 188 156

0 + 56 + 7

284 210 148

0 + 34 + 15

0.6 N 0.5 0.4

284 244 163

284 188 156

0 + 56 + 7

284 220 145

0 + 24 + 18

0.6 N 0.5 0.4

284 244 163

284 210 156

0 + 34 + 7

284 220 157

0 + 24 + 6

0.6 N 0.5 0.4

284 244 163

284 210 155

0 + 34 + 8

284 230 157

0 + 14 + 6

0.6 N 0.5 0.4

284 244 163

284 220 156

0 +24 + 7

284 215 145

0 +29 +18

31 V. C. (Continued)

Normality

Control Control Experimental Experimental Minus Minus "Up.iohn” Experimental Control **W*» 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 0.5 0.4

284 244 163

280 210 145

+ 4 +34 +18

275 200 145

+ 9 + 44 + 18

0.6 N 0.5 0.4

305 228 158

300 191 158

+ 5 4 37 0

300 218 160

+ 5 + 10 -2

0.6 N 0.5 0.4

305 228 158

282 208 153

f 23 4 20 + .5

294 228 158

+ 11 0 0

0.6 N 0.5 0.4

305 228 158

294 218 155

+ 11 4 10 + 3

298 228 145

+ 7 0 +13

0.6 N 0.5 0.4

305 228 158

296 210 150

+ 9 + 18 + 8

300 218 145

+ 5 + 10 + 13

0.6 0.5 0.4

305 228 158

300 220 156

+ 5 +8 +2

298 200 145

+ 7 + 28 +13

0.6 0.5 0.4

305 228 158

294 188 145

+11 + 40 +13

300 220 150

+ 5 +8 +8

0.6 0.5 0.4

305 228 158

300 205 145

+ 5 + 23 +13

305 220 145

0 + 8 +13

32 V. (Continued) D.

Mathematical Table Covering Results of Experiments on Water Soluble Adreno-cortical Extracts "Upjohn" and "W."

Test Solutions Normality of Ringer's Reagent

VI.

Mean Difference Between Control & Experimental