LIPOID SOLUBILITY, PERMEABILITY AND HEMOLYTIC ACTION OF THE SATURATED FATTY ACIDS.*

BY MEYER BODANSKY.

(From the Laboratory of Biological Chemistry, School of Medicine, University o-f Texas, Galveston.)

(Received for publication, June 25, 1928.)

Hemolysis by fatty acids is closely related to the ability of these substances to penetrate the red blood cell membrane. Jacobs (1) has studied hemolysis produced by ammonium salts of the fatty acids and found that these owe their hemolytic effect to the permeability of the corpuscle to ammonia and the fatty acids, both entering the cell with relative ease even in the undissociated form. The accumulation of ammonium salts of the fatty acids inside the corpuscle leads to the osmotic effects which ultimately result in the disintegration of the cell.

The penetration of fatty acids into living tissues has been the subject of many excellent investigations (Loeb, Harvey, Crozier, Taylor, and others), but with the exception of the work of Jacobs with the ammonium salts and the essentiahy qualitative study of Fiihner and Neubauer (2), the permeability of the erythrocyte to fatty acids and the hemolytic action of these substances have received little attention.

Loeb (3) studied the effect of different acids in the formation of artificial fertilization membranes in sea urchin eggs and found the order of effectiveness to be as follows: formic <acetic < propi- onic < butyric < caprylic < nonylic.

Harvey (4) investigated the penetrat,ion rates of different acids into the testis of the “prickly fish,” which contains a purple water- soluble pigment sensitive to acids, and found that valeric acid penetrated much more rapidly than any of its lower homologues. Croder (5) measured the penetration rates of various acids into

* A brief report of these observations was presented at the Ann Arbor meeting of the American Society of Biological Chemists, April, 1928.

241

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242 Hemolytic Action of Fatty Acids

the mantle fold of the mollusc, Chromodoris zebra and noted the following order of effectiveness: Valerie > butyric > propionic > acetic. The effectiveness of formic acid was intermediate between valeric and butyric acids, but in view of the fact that Crozier compared the action of solutions that were 0.01 N in strength, this result is not surprising since the dissociation of formic acid is considerably greater than of its homologues. Crozier has also studied sensory activation by acids (6), using for this work the common earthworm, Allobophora. The effectiveness of the various fatty acids was in the order: acetic <propionic < butyric < valeric <formic < caproic < caprylic. A summary of related investigations is to be found in Jacobs’ review in Cowdry’s “Gen- eral Cytology” (7).

More recently Taylor (8) determined the threshold concentra- tions necessary to produce a sour taste and found that for the lower members of the saturated fatty acid series higher concentra- tions are needed than for the higher members. He found that for solutions of equal pH a high degree of sourness is associated with a high penetration velocity of the undissociated acid or of the anion. It is to be seen therefore that in all the phenomena that have been studied in which primary penetration of the acid is a factor, a given fatty acid is invariably more active than its lower homologues and less active than its higher homologues.

In the present investigation a comparison has been made of the hemolytic action of the members of the saturated series of fatty acids, from formic to capric, including isobutyric, isovaleric, and isocaproic acids. A sufficient number of concentrations in isotonic saline were employed in order to establish for each acid the re- lationship between hydrogen ion concentration and the rate of hemolysis. In this work, parallel studies were made with dog and human corpuscles. The methods employed have been described in an earlier paper (9).

Even a condensed tabulation of the results that have been accumulated would require a considerable amount of space. The data for the dog corpuscle are therefore represented by means of the curves in Chart I. The data for human corpuscles will be considered separately. These curves bring out for the various acids their relative hemolytic power, and for the individual acids the relation between dilution and the rate of hemolysis. The

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M. Bodansky 243

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Hemolytic Action of Fatty Acids

close parallelism of the results upon which these curves are based and the results of those who have studied the permeability of various tissues to acids makes it difficult to escape the conclusion that the permeability of the fatty acids is intimately related to their hemolytic effect. Formic acid resembles in its action more nearly the inorganic acids than the remaining members of its series. The character of its dilution curve sets it apart from the other fatty acids and the fact that moderate increases in the osmotic pressure of the outside fluid produce relatively little effect on the rate of hemolysis leads one to suspect that primary penetration of the acid is not the main factor involved in the hemolysis by formic acid, but that this acid resembles the inorganic acids in injuring the corpuscles in some other way (9).

Two curves are given for acetic acid. The broken line is based on the results obtained with solutions prepared in isotonic salt solution. The hemolytic action of acetic acid is low. It requires a normal solution to hemolyze a standard cell suspension in about 0.3 minute. Now, a normal solution of acetic acid in water is approximately 4 X isotonic, so that solutions of this acid, more concentrated than 0.25 N, even though prepared in water, are still hypertonic with respect to the cells. In view of the limited range of effectiveness of the acid, the concentrations above 0.25 N could not be excluded from the series, and the fact that these solutions were not isotonic is therefore to be borne in mind in interpreting the solid line for acetic acid, which is based on the results obtained with isotonic solutions of acetic acid of concentra- tions lower than 0.25 N and with solutions above this concentration that were unavoidably hypertonic. This applies also in the case of propionic acid, but for all the higher acids it was possible to adjust all the necessary concentrations to isotonicity. Two curves are given for propionic acid, the broken line being based on the data for the solutions prepared in saline and the solid line for the isotonic solutions. Two sets of curves are likewise given for butyric, isobutyric, valeric, and isovaleric acids in order to bring out the effect on the rate of hemoIysis of small differences in the osmotic pressure of the outside fluid. The remaining curves are based on the results obtained with solutions prepared by dissolving the acid in 0.85 per cent sodium chloride. In the dilutions in which these acids were used, the addition of the acid to the physi-

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M. Bodansky

ological salt solution was insufficient to produce any appreciable change in tonicity. A more detailed study of the relation of osmotic pressure to the rate of hemolysis is contemplated.

It will be observed that for a given pH, isovaleric acid is less effective than n-valeric acid and isocaproic acid is less effective than n-caproic acid in hemolyzing the corpuscles of the dog.

The higher fatty acids are very active hemolytic agents. These acids have a low solubility in water; the statements given in various handbooks that they are insoluble are, however, mis- leading. For example, a ~/1500 solution of pelargonic acid is easily prepared. Pelargonic acid and its lower homologues are liquid at room temperature, and all the measurements with these acids were made at 25”. Capric acid, on the other hand, melts at 31”. The determinations with this acid were therefore made at 35”.

Under approximately uniform physiological conditions the properties of the red blood corpuscle of a given species are ap- parently such that experimental results of the type considered in this paper reproduce themselves from day to day with a uniformity that is very remarkable. Ponder (10) has observed this sort of consistency in his studies on saponin hemolysis. Corpuscles obtained from different normal individuals have approximately the same average resistance to saponin and the results which Ponder obtained with his own blood from time to time showed little variation. The present author has verified Ponder’s as- sertions using his own blood, the data obtained being practically identical with those given by Ponder. Acids behave similarly in that over a long period of time there is little variation in the resistance of the corpuscles of a given animal towards a given acid. This does not mean, however, that the same mechanism is involved in the hemolysis by these two groups of substances; in fact the mechanisms are probably different.

The constancy in the behavior of the washed corpuscles to a given acid need not lead to the assumption that the permeability of the corpuscle is not subject to variation. The composition of the erythrocyte is far from being constant either physiologically or in a variety of pathological conditions. It is well known, for example, that the lipoids of the corpuscle are altered both quali- tatively and quantitatively during fat absorption. Other changes

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TABL

E I.

Data

Sh

owing

Re

lative

Re

sista

nce

of

Dog

and

Hum

an

Corp

uscle

s lo

Va

ryin

g Co

ncen

tratio

ns

of

Fatty

Ac

ids.

*

Tim

e fo

r he

mol

ysis

in

m

inut

es.

Acid.

Norm

ality

t..

pH$.

.

Dog.

. .

M

an.

. . .

.

Acid.

Norm

ality

.

pH

. .

. .

Dog.

.

Man

. . .

.

Form

ic.

Acet

ic.

Prop

ionic.

0.05

0.

02

0.01

1.

0 0.

5 0.

25

0.1

0.5

0.25

0.

1 ~-

_-__

--

2.52

2.

73

2.93

2.

32

2.47

2.

63

2.85

2.

58

2.70

2.

92

~__-

____

---__

-

0.62

1.

25

2.30

0.

38

0.63

1.17

2.

750.

951.

25

2.7C

0.

44

1.08

2.

33

0.33

0.

421.

00

>4.0

01.2

23.4

2 >6

.OC

I I

L

Isova

leric.

I

capr

oio.

I Iso

capr

oio.

Buty

ric.

Isobu

tyric.

Va

le&.

0.5

0.25

0.

1 0.

5 0.

25

0.1

0.05

0.

02

-_

-___

_

2.49

2.

67

2.86

2.

52

2.68

2.

89

3.06

3.

27

___

-__

--__

:0.0

5 0.

33

1.80

<0

.050

.500

.070

.85

5.75

:0

.055

.75

>12.

00

<0.0

57.0

00.1

12.0

8 >1

4 1

I

I He

ptyli

c.

I.1

0.05

0.

02

0.1

0.05

0.

02

0 01

-_

_ -_

--

1.86

3.

03

3 22

2.

92

3.05

3.

24

3.42

-_

_ ---

~

.23

1.83

7.

75

<0.0

20.0

50.3

3 3.

1;

.94>

6.00

>1

3.0

0.02

0.

062.

00

17.0

0.1

0.05

0.

02

0.01

0.

02

0.01

0

005

0.002

--

__--

2.89

3.

03

3.22

3.

42

3.36

3.

43

3.61

3.

78

___

~-~-

InsD

antly

. 0.

05 0

.75

5.60

0.09

0.21

1.37

17

0.

02

0.07

3.15

>2

2.0

0.12

0.28

3.50

>3

5

Capr

ylic.

I I

-__

4.33

4.

46

4.53

-I-/-

1.16

4.

00

1 i

7.45

2.

58 1

2.5

>20

* Th

e so

lutio

ns

used

in t

hese

de

term

inat

ions

we

re

prep

ared

by

diss

olvin

g th

e ac

id i

n ph

ysio

logi

cal

salt

solu

tion.

t

The

norm

ality

va

lues

ar

e clo

se

appr

oxim

atio

ns

and

repr

esen

t th

e co

ncen

tratio

ns

of a

cid

befo

re

the

addi

tion

of t

he

stan

dard

ce

ll su

spen

sions

. $

The

pH

valu

es

repr

esen

t th

e ac

tual

hy

drog

en

ion

conc

entra

tions

at

th

e be

ginnin

g of

hem

olys

is

and

were

ob

tain

ed

in s

epar

ate

dete

rmin

atio

ns

by d

ilutin

g th

e ac

id

solu

tions

wi

th

salin

e in

th

e pr

opor

tion

of 1

cc.

of

salin

e to

4

cc.

of

acid

. In

de

term

inin

g th

e tim

e re

quire

d fo

r co

mpl

ete

hem

olys

is,

0.4

cc.

of t

he

stan

dard

co

rpus

cle

susp

ensio

n is

ad

ded

to

1.6

cc. o

f th

e ac

id

solu

tion.

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M. Bodansky 247

occur in experimental anemia. It is conceivable that changes such as these in the composition of the cell are likely to be as- sociated with variations in the rate of penetration of various substances into the corpuscle.

Variations in permeability of acids would alter somewhat the position and shape of the dilution curves of the type given in Chart I, but despite such variations, the order of effectiveness of the different members of the series as represented by these curves will probably be found to hold under a large variety of conditions for all mammalian corpuscles.

A comparison of hemolysis of human and dog corpuscles by fatty acids has brought out certain distinctions. It will be recalled that human corpuscles are somewhat less resistant to inorganic acids than dog corpuscles. This is also true for formic and acetic acids. The resistance to propionic acid, on the other hand, is somewhat greater in the case of human cells, and for all the higher fatty acids the human corpuscles show a distinctly greater resistance to hemolysis than dog corpuscles. This is brought out by the data outlined in Table I.

The greater resistance of the human corpuscles appears to be related to their higher content of buffer substances. In measuring the shift in potential of the acid solutions during hemolysis, the washed human corpuscles invariably neutralized a greater amount of acid than dog corpuscles. In hemolysis with the lower fatty acids this effect is bound to be of relatively little importance because of the high concentration of acid needed to bring about hemolysis and of the relatively small proportion of this acid neutralized by the constituents of the cells. Yet, even in a 0.1 N solution of acetic acid the human corpuscles exhibit greater resistance than dog corpuscles. In the case of the higher fatty acids where lower concentrations suffice to bring about hemolysis, the difference in the neutralizing capacity of the con- stituents of the two types of cells may be sufficient to reduce appreciably the amount of available acid and lead to a reduction in the rate of hemolysis. It is to be seen from the data in Table I, as well as from the shape of the curves in Chart I, that a small decrease in the concentration of acid may result in a very pro- nounced retardation of hemolysis. For example, in the case of caproic acid, at pH 3.4, hemolysis of the standard cell suspension

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248 Hemolytic Action of Fatty Acids

occurred in about 2 minutes, whereas when the initial pH was 3.45, complete hemolysis did not occur until after an interval of 8 minutes.

The data in Table II have been selected at random from the results obtained in the measurement of hemolysis by means of the potentiometer. The initial pH values of the acid solutions are given, as well as the values at the end of the reaction between the acid and cell constituents. While the differences in the pH shift between the human and dog corpuscles for the various acids is not great, nevertheless differences of like magnitude have a

TABLE II.

Shift in pH Due to Neutralization of Various Acids by Constit,uents of Dog and Human Corpuscles.*

Acid. . . . . . . . . . . . . . . 1 Acetic. Pro- Butyric. Valerio. ISO- pionic. valerio. Caprylic.

______ -~ Approximate normality.. 0.5 0.5 0.1 0.1 0.02 0.001

--

Dog. Initial PH.. . . . 2.40 2.50 2.77 2.80 3.12 4.30 Final PH.. . . 2.58 2.73 3.12 3.11 3.62 5.02

Man. Initial PH.. , . . . 2.40 2.50 2.77 2.80 3.12 4.28 Final PH.. . 2.63 2.80 3.18 3.17 3.73 5.20

* To 16 cc. of acid, 4 cc. of the standard red blood cell suspension were added.

considerable effect on the rate of hemolysis, especially for the higher fatty acids, as may be seen by projecting these values on certain portions of the curves given in Chart I.

Mechanism of Penetration of Fatty Acids into Red Blood Corpuscle.

In his study of cell permeability, Harvey (4) did not obtain clear cut evidence either to support or contradict Overton’s well known lipoid solubility theory. Harvey states, however, that lipoid solubility, or capillary activity, for the two run more or less parallel, constitutes one of the factors upon which the penetra- tion of acids depends.

Taylor’s work on taste (8) has led him to conclude that sourness, is not purely a function of the stoichiometric acid concentration, nor of the hydrogen ion concentration. In attempting to fmd an

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M. Bodansky 249

explanation for his results he has assumed that the production of a given degree of sourness is due to the establishment within the cells of the taste bud of a definite hydrogen ion concentration and from his experimental results he has calculated the con- centration gradients of equally sour acids. He recalculated similarly the taste data of Paul and Bohnen (see Paul (II)), and from the data of Crozier (5, 6) computed the concentration gradients of acids penetrating Chromodoris tissue, as tie11 as the

TABLE III.

Concentration Gradients of Acids Penetrating the Red Blood Corpuscle and Producing Hemolysis of a Standard Cell Suspension in 1 Minute.

Acid.

Formic. ............. Acetic ............... Propionic. ........... Isobutyric. .......... Butyric. ............ Isovaleric ........... Valerie .............. Isocaproic ........... Caproic ............. Heptylic. ............ Caprylic. ............ Pelargonic ...........

PH

2.67 2.64 2.86 2.87 2.87 3.03 3.14 3.28 3.35 3.58 4.33 4.67

: IO-n, x 10-3&l X 10-8,

23.5 2.14 21.4 86 2.3 184 41 1.4 40 22 1.35 .21 24 1.35 .23 51.9 0.93 51 34.1 0.73 33.4 19.2 0.53 18.7 14.4 0.45 13.9

4.9 0.26 4.64 0.2 0.047 0.153 o.ot 0.021 0.04

1.00 1.4 11.5 4.7 1.00 15.2 9.2 0.372 14.3 8.46 0.342 14.5 8.48 0.344 12.6 4.05 0.164 13.4 2.50 0.101 14.3 1.31 0.053 14.7 0.95 0.038 14.7 0.315 0.0128 14.9 0.01030.0004 19.4 0.00210.000084

concentration gradients causing retraction in the earthworm. The resulting values, not only showed agreement with each other, but resembled Freundlich’s data (12) on the relative concentrations of undissociated fatty acids (formic, acetic, propionic, and butyric) which are necessary in order that charcoal may adsorb the same quantity of acid in each case. Accordingly, Taylor suggests that the fatty acids are taken into tissues by an adsorption process.

It is probably unnecessary to postulate an adsorption mech- anism, for the results of acid penetration into tissues can be more

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250 Hemolytic Action of Fatty Acids

fully correlated on the basis of solubility d&a which are outlined in this paper. In order to compare our results on hemolysis with the data that have been obtained by others in a variety of biologi- cal phenomena involving the penetration of acids into tissues, the relative concentration gradients of undissociated acids across the red blood cell membrane, causing hemolysis (a) in 1 minute, and (6) in 5 minutes, have been determined by the methods sug- gested in’ Taylor’s paper. The results are outlined in Tables III and IV. The relative concentrations of undissociated acid

TABLE IV.

Concentration Gradients of Acids Penetrating the Red Blood Corpuscle and Producing Hemolysis of Standard Cell Suspension in 5 Minutes,

Acid. PH

Butyric............. Isobutyric Isovaleric . Valerie Isocaproic. . Caproic. Heptylic. . Caprylic Pelargonic

3.12 3.12 3.23 3.30 3.42 3.44 3.75 4.58 4.78

-

x IO-~Y x lo-~& x 1 o-339

39.9 0.76 39.1 39.3 0.76 38.5 21.1 0.59 20.5 16.2 0.50 15.7 10.0 0.38 9.6

9.3 0.36 8.9 2.3 0.175 2.1 0.07: 0.026 0.04; 0.04: 0.017 0.02t

14.5 2.70 1.00 14.3 2.70 1.00 12.6 1.63 0.60 13.4 1.17 0.43 14 3 0.67 0.25 14.7 0.60 0.22 14.7 0.143 0.053 14.9 0.0032 0.0012 19.4 0.0013 0.0005

-

inside the cell are based on the dissociation constants of the fatty acids, as given in the “Landolt-Bornstein Physikalisch-chemische Tabellen.” The present writer has been unable to find data for the dissociation constant of capric acid; the results for this acid are therefore omitted from the tables.

The ratios given in the last column of Tables III and IV may be taken to represent the relative gradients or driving forces which are necessary to cause the various fatty acids to penetrate into the corpuscles of the dog to a comparabie degree. When al- lowance is made for the higher concentrations of acid necessary to

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M. Bodansky 251

bring about hemolysis than are required to produce a sour taste or to cause sensory activation as in Crozier’s experiments (6), the relations indicated by the data in Tables III and IV neverthe- less bear a close resemblance to the relations that have been shown to exist in other biological phenomena which depend upon the primary penetration of the acids into tissues. That the primary penetration of the fatty acids into the erythrocyte depends on their solubility in the lipoids of the cell will now be considered.

It is generally known that the lipoid solubility of the members of the saturated series of fatty acids increases according to the size of the molecule. However, adequate data bearing on the distribution of the fatty acids between water and fatty substances seem to be lacking. In his paper on the permeability of cells to acids, Harvey (4) includes certain data obtained from Waste- neys on the partition coefficients between water and olive oil of several of the fatty acids in 0.01 N concentrati0n.l In view of the possible relationship between the penetrability of the fatty acids into the corpuscle and lipoid solubility and because of the lack of solubility data, it seemed desirable to determine the partition coefficients of the fatty acids between water and olive oil. (The same results were obtained with aqueous solutions as with solutions of the fatty acids in saline.)

The solubility of the fatty acids was determined at 23”. 100 cc. portions of the various acids of known concentration (N, 0.1 N, 0.01 N, 0.001 N) were transferred to flasks. After the removal of 25 cc. for the preliminary titration with alkali, 5 cc. of olive oil (Squibb) were added to the remainder; the flasks were tightly stoppered and the contents shaken in a mechanical shaking device for 13 hours. As a rule the distribution was found to reach equilibrium within 30 minutes. After the oil was allowed to separate, a second 25 cc. portion of the solution was titrated. The oil was then transferred to narrow test-tubes and set aside for 24 hours or longer to insure complete separation of the oily layer. Accurately measured portions of the oil were then dissolved in neutral alcohol and titrated with standard alkali. Suitable control analyses were made for the acid content of the olive oil. Excellent agreement was obtained in nearly all of the experiments

l Except for Harvey’s citation, this work has not been published, accord- ing to a personal communication from Dr. H. Wasteneys.

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TABLE V.

Solubility of Patty Acids in Olive Oil.

Acid.

Acid lost from 75 cc. Acid in Acid in water and 1 cc. water 1 cc. oil at Oil: water Water: oil

recovered at equilib- equilib- distribu- distribu-

in5 t-c. Cum. rium. tion ratio. tion ratio.

oil.

Cl CZ c*:c* cc cz

1.0 N.

cc. cc. cc. Formic

acid = 1.

Formic. . . . . . . . . . . . 0.11 0.9985 0.022 0.022 1.00 Acetic................... 0.15 0.998 0.03 0.030 0.73 Propionic................ 1.05 0.986 0.21 0.213 0.104 Butyric . . . . . . . . . . 7.5 0.900 1.5 1.67 0.013

0.1 N.

Formic .................. 0.025 Acetic ................... 0.15 Propionic. ............... 0.7 Isobutyric. .............. 3.1 Butyric .................. 3.1 Isovaleric ............... 11 .O Valerio .................. 17.2

Propionic ................ Isobutyric. .............. Butyric. ..... : .......... Isovaleric ............... Valerie .................. Isocaproic ............... Caproic ................. Heptylic ................

-

-

0.9997 0.998 0.991 0.959 0.959 0.853 0.77

-

- 0.01 N.

0.75 0.99 0.15 2.15 0.972 0.43 2.15 0.972 0.43 8.5 0.887 1.7

11.5 0.847 2.2 26.0 0.653 5.2 29.0 0.613 5.8 57.5 0.233 11.5

0.005 0.005 0.03 0.03 0.14 0.141 0.62 0.645 0.62 0.645 2.2 2.58 3.45 4.48

0.151: 0.443 0.443 1.92 2.60 7.96 9.47

49.3

-

-

-

-

-

0.001 N.

Propionic. ............... 0 1.00 0 Isobutyric ............... 1.5 0.98 0.3 Butyric ................ 1.5 0.98 0.3 Isovaleric ............... 7.5 0.90 1.5 Valerie .................. 9.0 0.88 1.8 Isocaproic ............... 16.5 0.78 3.3 Caproic ................. 22.5 0.70 4.5 Heptylic. ............... 40 .O 0.47 8.0 Caprylic ................. 51.0 0.32 10.2

252

iz i$ 2 .

1.00 0.342 0.342 0.079 0.058 0.019 0.016 0.003

-

-

i

-

-

-

0.306 0.306 1.67 2.05 4.23 6.43

17.0 31.9

Formic mid = 1.

1.00

0.165 0.035 0.0077 0.0077 0.0019 0.0011

Butyric beid = 1.

1.00 1.00 0.184 0.150 0.072 0.048 0.018 0.0095

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M. Bodansky 253

between the amount of fatty acid lost from the aqueous layer and the amount of fatty acid recovered in the oil. The results, which are based on several series of determinations, are outlined in Table V.

Owing to the low solubility of pelargonic acid, concentrations lower than 0.001 N were employed. With such dilutions, the procedure of titrating the acid solutions uefore and after shaking with oil was subject to considerable error. The more accurate

TABLE VI.

Solubility of Fatty Acids in Olive Oil.

Acid.

Isocaproic. .

Heptylic. . . . .

Caprylic. . . . .

Pelargonic . .

-

I

-

.4 --

.4

.4 --

.4 -

.15 4.2: --

.17 4.3! --

.144.6, --

.245.0:

-

3

5 --

4 __

2 -

Total acid concentration.

i a p .a p *. X 5”

2 24 .t: i

:x 4

-

-- --

0.4070.291 --

2 --

0.3750.184 --

3 --

0.4430.060 6 -- --

0.3630.009: 147 -

] Cl 1 cz

CC. 0.000407 N.

1.5 1 0.7131 4.30

CC. 0.000375 N.

8.2 IO.49 1 7.64

CC. 0.000443 N.

4.85 1 0.135112.97

Cc. 0.000363 N.

3.12510.025 114.62,

_

_-

-.

-.

5, -

15.6 1.064

96.1

585

I.014

1.0017

1.166

procedure was therefore adopted of measuring the change in hydrogen ion concentration. Determinations were made with solutions of isocaproic, heptylic, caprylic, and pelargonic acids of approximately the same concentration. The results are given in Table VI.

An examination of the distribution ratios given in Tables V and VI and comparison of these data with the relative concentra- tion gradients given in Tables III and IV, as well as with the other

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Hemolytic Action of Fatty Acids

data contained in this paper, lead to the conviction that lipoid solubility is an important factor in determining the rate of penetra- tion of the fatty acids into the erythrocyte and the order of effectiveness of the various members of the series in producing hemolysis.

Since the data outlined in this paper are not difficult to correlate, a detailed discussion seems unnecessary, but for the purpose of illustrating how well the results agree, the following example will be considered. It is to be noted that the distribution ratios for butyric and isobutyric acid are identical for the concentrations included in Table V. The hemolysis curves (Chart I) are likewise identical, and accordingly the relative concentration gradients for these acids are the same (Table III and IV). On the other hand, the position occupied by isovaleric acid in the order of effectiveness as a hemolytic agent is intermediate between butyric and n-valeric, and isocaproic acid occupies a position intermediate between valeric and n-caproic acids. These are precisely the positions occupied by the oil : water (and water : oil) distribution ratios of isovaleric and isocaproic acids.

Incidentally, it is to be observed that the oil : water distribution cz ratios - for the members of the series above propionic acid in- C*

crease with increasing concentration of the acids. This indicates that the degree of molecular association of the fatty acids is greater in olive oil than in water. It is likely that this is also true for formic, acetic, and propionic acids, but in view of the low solubility of these in oil, the methods employed in this work were inadequate to bring this out with certainty.

SUMMARY.

The order of effectiveness of the fatty acids in penetrating the red blood corpuscle and producing hemolysis is: acetic < propionic < butyric = isobutyric < isovaleric < valeric < iso- caproic < caproic < heptylic < caprylic < pelargonic < capric.

The relations between pH and hemolytic action have been determined for each of these fatty acids and the results represented by means of curves.

Increasing the osmotic concentration of the outside fluid retards

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M. Bodansky

hemolysis by fatty acids. This shows that osmotic effects are involved in the process.

Formic acid resembles the inorganic acids in its hemolytic action.

Human corpuscles are more resistant to hemolysis by fatty acids than dog corpuscles. This is apparently due to the greater content of buffer substances in washed human corpuscles than in washed dog corpuscles and the neutralization of a greater amount of acid.

Determinations have been made of the relative concentration gradients of the acids, penetrating the corpuscle and producing hemolysis. The relations which are shown to exist in hemolysis resemble closely the relations that have been observed by other workers in a variety of phenomena in plant and animal tissues involving the primary penetration of the acids.

Determinations have been made of the distribution of the fatty acids between water and olive oil. A close parallelism exists between lipoid solubility and the effectiveness of the fatty acids in producing hemolysis.

The writer wishes to express his appreciation to Dr. B. M. Hendrix of this laboratory for a number of valuable suggestions.

BIBLIOGRAPHY.

1. Jacobs, M. H., The Harvey Lectures, 1926-27, xxii, 146. 2. Fiihner, H., and Neubauer, E., Arch. exp. Path. u. Pharmakol., 19Om7,

lvi, 333. 3. Loeb, J., Artificial parthenogenesis and fertilization, Chicago, 1913,

134. 4. Harvey, E. N., Science, 1914, xxxix, 947; Internat. 2. physik.-them.

Biol., 1914, i, 463. 5. Crozier, W. J., J. Biol. Chem., 1916, xxiv, 255; 1916, xxvi, 225. 6. Crazier, W. J., Am. J. Physiol., 1917-18, XIV, 323. 7. Jacobs, M. H., Permeability of the cell, in Cowdry, E. V., General

cyt,ology, Chicago, 1924, 125. 8. Taylor, N. W., J. Gen. Physiol., 1928, xi, 207. 9. Bodansky, M., J. Biol. Chem., 1928, Ixxix, 229.

10. Ponder, E., Biochem. J., 1926, 507. xx, 11. Paul, T., 2. Electrochem., 1922, xxviii, 435. 12. Freundlich, H., Kapillarchemie, 3rd edition, Leipsic, 1923, 265.

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Meyer BodanskySATURATED FATTY ACIDS

AND HEMOLYTIC ACTION OF THE LIPOID SOLUBILITY, PERMEABILITY

1928, 79:241-255.J. Biol. Chem. 

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