Electrolyte Excretion in Bile

By HENRY 0. WHEELER, M.D., OSWALDo L. RAMOs, M.D., AND

ROBERT T. WHITLOCK, M.D.
The high concentration of certain test substances in both urine and bile suggests that
similarities exist between the biliary and renal tubular functions. Evidence from dogs
with permanent duodenal fistulas indicates that the rate of elaboration of bile depends
primarily on the rate of secretion of bile salts. The variations in flow and composition
of hepatic bile seem to result from the addition of a fluid whieh is similar in some
respects to pancreatic juice.

IN COMMON with other extrarenal secre- major end-product of cholesterol metabo-

tory structures, the biliary tract appar-
ently depends upon energy-consuming cellu-
lar processes rather than upon a hydrostatic
filtration system for the production of its
characteristic effluent. Nevertheless, there ap-
pear to be certain rather fundamental similar-
ities between biliary and renal tubular func-
tion. These similarities become apparent when
one considers the number of compounds which
are secreted in high concentrations in both
urine and bile. Examples of such compounds
are phenolsulfonphthalein,l fluorescein,2 p-
aminohippurate,3 penieillin,3 and phlorhizin.4
Downloaded from http://ahajournals.org by on August 25, 2020
lism.6' 7 In the dog and in other carnivores
they are conjugated with taurine. Because of
the free sulfonic acid group of taurine, the
resulting conjugates are completely dissoci-
ated and highly soluble in the physiologic pH
range, having a pK of about 1.5. These 2 bile
salts, which can be measured accurately by the
method of Mosbach et al.,8 will be referred to
hereafter simply as " taurocholate."
In the intact animal, the bile salts undergo
extensive enterohepatic recirculation so that,
as shown in figure 1, approximately 85 to 90
per cent of the bile salt in the bile at any

It is noteworthy that among the substances
which are most actively secreted into the bile
are included the most potent known choler-
eties. The correlation between active secretion
and choleretic potency may well have im-
portant implications with regard to the gen-
eral nature of bile formation. Outstanding
choleretic substances are the natural bile
salts and their synthetic derivatives, eincho-
phen5 and, to a lesser extent, the phthalein
dyes such as Bromsulphalein, phenol red and
bromeresol green.1 All of these, it should be
noted, are organic acids. Of these choleretic
compounds, the natural bile salts are, of
course, normally present in abundance and
are therefore of the greatest physiologic in-
terest.
The 2 major bile acids in the dog are the di-
and tri-hydroxy cholanic acids, deoxycholic
and cholic acid; of the 2, the latter is the
more abundant. These bile acids represent the
From the Department of Medicine, College of
Physicians and Surgeons, Columbia University, NeNi
York, N. Y.
988
given time represents material previously ex-
creted and reabsorbed from the bowel.9 The
remainder represents new bile salt synthesized
by the liver. The phenomenon of recirculation
was described as early as 1870 by Schiff, and
in the course of his experiments he also ob-
served that bile itself, when introduced into
the duodenum, was the most potent available
stimulant for increased bile production.10 It
will be obvious, then, that interruption of this
enterohepatic cycle must have major physio-
logic consequences which have to be consid-
ered in the planning and interpretation of any
study in which bile is removed from the ex-
perimental subject and not replaced.
Time does not permit a review of the many
ingenious technies which have been developed
for repeated collections of bile from un-
anesthetized animals. The particular device we
have employed is a duodenal cannula develop-
ed by Thomas."' This apparatus, which is held
in the duodenum by a hard rubber flange,
permits the creation of a permanent duodenal
fistula opening directly over the ampulla of
Circulation, Volume XXI, May 1960
 ELECTROLYTE EXCRETION IN BILE 989

Vater. Between studies it is kept stoppered,

but during an experiment it is opened, and a
small ureteral catheter is inserted directly
through the ampulla of Vater and advanced
well into the common duct. Except for the
fact that the animals are cholecystectomized,
this technic leaves the biliary tract in normal
condition between studies and free of per-
manent foreign bodies. All of the studies
which will be considered in this paper were
conducted on 4 such dogs. In each study, the
dog was held in the upright position by means
of a sling. Bile was collected either by gravity
or with the assistance of gentle suction pro-
vided by a tuberculin syringe.
When bile is collected continuously over a
period of hours, there is progressive diminu-
tion in bile flow because of the interruption of
enterohepatic circulation of bile salts. Figure
2 illustrates tLis phenomenon and also certain
general features of bile composition with
which we shall be concerned. At the top is
shown bile flow in ml./min., in the middle is
the pH, relative to a line drawn at 7.4, at the
bottom is shown the electrolyte composition of
Downloaded from http://ahajournals.org by on August 25, 2020

individual bile specimens. In each block dia-

gram the major cations, sodium and potas-
sium, are on the left and the anions, tauro-
cholate, bicarbonate and chloride, are on the
right. The specimen of bile labeled CD is
typical of bile removed from the common duct
at the moinent of catheterization. We shall
shortly examine this type of bile in more de-
tail. It should be noted here, however, that,
in contrast to subsequent samples of flowing
hepatic bile, the concentration of taurocholate
in "common duct bile" is very high, the total
ionic concentration is high, the pH is low,
and there is very little bicarbonate or chloride.
Over the course of 2 to 3 hours there is pro-
gressive diminution in bile flow and also in
taurocholate concentration. Thus, in this type
of experiment the output of all solutes, but
particularly the output of bile salt, diminishes
as expected. Not only is the electrolyte com-
position of bile difficult to interpret under
these circumstances, but actually it is often
difficult even to obtain bile in sufficient quan-
tity for analysis. It was found that this "un-
steady" state can be averted by the constant
Circulation, Volume XXI, May 1960
Excretion
1 5 °/a
Figure 1
Enterohepatic circulation of bile salts. About 85
to 90 per cent of the bile salt excreted into the
duodenmum is reabsorbed and returned to the liver
by way of the portal vein.
intravenous replacement of bile salt. Before
proceeding to the results which were obtained
by using this technic, let us examine more
closely the composition of " common duct
bile. "
The specimen on the left of figure 3 is typi-
cal of many specimens of "common duct bile"
obtained upon catheterization of fasting dogs.
Presumably it represents bile which was
formed over a period of several hours prior to
catheterization and held in the duct system
by the normal action of the sphincter of Oddi.
In the center is shown the composition of
canine gallbladder bile, and on the right, for
contrast, that of canine plasma. There is ob-
viously a striking similarity between " com-
 990

cc/min

0.06-8r
mEq/L
280
240
200
160 -
120
80 "
0.1 _
0008 _______- -_____-

-
6

.
.
t..
h

PLASMA
pH

"*
CrG
-u

BILE FL OW

12 Na+
m K +

.~~~~~~:
...:.:..-.....
........ ........
WHEELER, RAMOS, WHITLOCK

rD Taurocholate
E HC03
fS CI-

,,~~~~~~~~~~~~~~~~: ,.1.- .114 mOsm

..
C2

40
DOG Da ...x~: j I(ILI -A
1
MINUTES 0 80 160
Figure 2
Bile flow and composition during continuous collectio7z. Bile withdraawn from the common
duct immediately after catheterization (CD) has a high concentration of taurocholate
and low concentrations of chloride and bicarbonate. During continuous collection, with-
out bile salt replacement, the flow of bile and the concentration of taurocholate diminish
progressively. Composition of plasma is showvn at left for comparison.
Downloaded from http://ahajournals.org by on August 25, 2020

mEq/L pH 6.2 pH 6.5 El Na+ Taurocholate

-A , _ * K<+ m Hr.n z
mI

~-7.
~ ~ ~ ~ ~
250

200
150 :- :: : ::
::300m Osny <gI

100
500

"COMMON GALL PLASMA

DUCT BLADDER
BILE B ILE

Figure 3

Comnparison of typical fa ting canine "'commnon duct" and gallbladder bile. Bile retained
in the bile ducets by the sphincter of Oddi ga.llbladder bile in that it has a
is similar to

high concentration of taurocholate, lowl concenztrations of chloride and bicarbonate and

a low p11. Note that the total ionic conlcentration is highJ, although osmnolality is equal
to that of pla,sma (at right). (Republished by permission of the Journal of Clinical

Investigation^.'6)
Circulation, Volume XXI, May 1960
 ELECTROLYTE EXCRETION IN BILE 991
TAUROCHOL ATE* - w~ ~ ~ ~P
c-/i 13AM/min /V
0.20
BILE FLOW
0.10 _

8r
7
!

pH
Na+
mEq/L ED No+ Tau rochol ate
250 6L- - K + Ea HC03-
C I-,. .

200
I50
100
50
r
I
F
Dog Norma.4 4 __zz
_zA

0 tOo 200 300

MINUTES
Figure 4
Bile composition and flow during constant intravenous jifusion of taurocholate. The rate
of bile salt excretion is constant, but there are spontaneous variations in flow and electro-
lyte composition. Highest flows are accompanied principally by increased pH and con-
centration of bicarbonate.
TAUROCHOLA TE
1/3,iM/min IV -
Downloaded from http://ahajournals.org by on August 25, 2020

cc /min
0.30 BILE Scretn
e

0.20 K FLOWIOuI
0.10
0
8 _

pH EZ2 Na+ rJ Taurocholate

250 - K+ EE HC03
E CI-
200
50 7
100
50
Dog Co
0 100 250 2 00 250
MINUTES
Figure 5
Effect of secretin on the flow and the composition of bile. Intra!renous administration of
secretin causes a marked choleresis and a very high pH and bicarbonate concentration.
The excretion of bile salt is unaffected. (Republished by permission of the Journal
of Clinical Investigation.16)
Circulation, Volume XXI, May 1960
 992 WHEELER, RAMOS. WHTIjTOCK
rA UROCHOL A rE bo
/3,vM/min /V
.1- - - -

cc / min Introduodenal HCI

30 r F 0.05N 7.7cc/min+
BILE FLOW
20
10
0

7
mEq/L pH r;;;l Noa+ Tourocholate
250 K + EM HC03
= CI-
200
150 1-. .
100
50
Dog Co 1b, 0--i
100 150 200 2 50 30C
MINUTES
Figure 6
Effect of intraduodenal infusion of hydrochloric acid. The choleresis and changes in
electrolyte composition are similar to those produced by exogenous secretin and are
probably attributable to release of endogenous secretin.
Downloaded from http://ahajournals.org by on August 25, 2020

mon duct bile"' and gallbladder bile in that,
so far as anions are concerned, each bile is
practically a pure solution of bile salt. It
seems quite probable that, at least in chole-
cystectomized dogs, the common duct and its
major branches serve in effect as a gallbladder
in concentrating the bile salts by removal of
other solutes and water.
The osmolality of bile, according to all re-
ports and under all the circumstances we shall
discuss, is very close to that of plasma (that
is, about 300 mOsm./Kg.). In all bile speci-
mens, however, and particularly in those of
the type illustrated in figure 3, the total ionic
concentration (sum of anions plus cations)
is much greater than 300 mEq./L. This
marked discrepancy between ionic concentra-
tion and osmolality can be attributed to the
fact that tauroeholate, like many other sur-
face-active substances, forms large multipolar
aggregates, or micelles, when its concentratioin
exceeds a critical value known as the "micelle
point." The "micelle point" of pure tauro-
cholate, as determined by Pethica and Schul-
man,12 is about 0.007 M, which is much lower
than the concentration of taurocholate in bile.
Thus, the taurocholate ion itself is virtually
inactive osmotically. Its osmotic significance
is, in effect, attributable solely to the cation
which must accompany it to preserve electro-
neutrality. Consistent with this view is the
finding that, regardless of taurocholate con-
centration, virtually all the osmotic activity
of any bile specimen may be accounted for as
the sum of sodium, potassium, chloride and
bicarbonate.
To return to the composition of flowing
hepatic bile, figure 4 illustrates a typical study
in which sodium taurocholate was infused in-
travenously at a constant rate of 14.5 ,LM/
min. In this and similar studies, such a con-
stant infusion resulted in stabilization of
taurocholate excretion at a constant rate ap-
proximately equal to the rate of infusion.
There was no progressive diminution in bile
flow. Nevertheless, as shown in figure 4, sig-
Circulation, Volume XXI, May 1960
 ELECTROLYTE EXCRETION IN BILE 993

nificant fluctuations in bile flow often oc- 90v

curred. There was a reciprocal relationship 80-

between bile flow and taurocholate concentra- 70- A

tion, consistent with the constant output of 60 H
A~~~~~
this constituenit. There were also eharacteris- 0~~~~ 0~~~~~~~
50 4 0
tic changes in other electrolytes. As flow in-
Dog Norma
creased, the chloride concentration increased z 40 0 6uiR OCHOLATE /45,uM/min IV

slightly, but there was a more striking in- E 30 - CH-LORIDE . Sponloneous flow
A /nfroduodenol HCI
crease in bicarbonate concentration and a cor- 20- * Introvenous secrelln
respondilng rise in pH. V Intrcvenous ocefozo/omlde

When 100 units of the intestinal hormone, 0-

secretin, was adminiistered intravenously (fig. O0 60
ICA RBONATr
5), a nmarked increase in bile flow occurred, 50
exceeding the highest spontaneous flows ob- 40 W
served in the same animal. The excretion rate
of taurocholate was unchanged, but the secre- E 30
tin choleresis was accompanied by a very 20 _

high concentration of bicarbonate (reaching lo!r r

values as great as 60 mEq./L.) and pH (to as 0 l
high as 7.8). 775F pH U
The intraduodenal infusion of hydrochloric 7.50_ A

acid is known to stimulate the release of en- 7.25_/ 0

dogenous secretin.'3 As shown in figure 6, this 7 000 Y

maneuver resulted in a change similar to that

produced by exogenous secretin. There was 0 0 05 0 10 0.15 0 20 0 25 0.30
Downloaded from http://ahajournals.org by on August 25, 2020

an impressive choleresis accompanied, once
again, by a high bicarbonate concentratiorL
and pH. As with exogenous secretin, these
changes occurred in the face of a constant rate
of taurocholate secretion.
In each animal there was a reproducible
relationship between bile composition and bile
flow as shown in figure 7. Whenever bile flow
inereased, whether spontaneously or under
the influence of secretin (on the extreme
right), there was a marked increase in pH and
concentration of bicarbonate. The concentra-
tion of chloride also increased, but this change
was small compared to the increment in bicar-
bonate. In contrast to these changes, the en-
tirely different effect of intravenous acetazole-
amide is also shown in figure 7. This agent, in
a dose of 60 mg./Kg., resulted in a choleresis
which was characterized by a relatively high
chloride concentration and a comparatively
low bicarbonate concentration and pH.
All of the studies to which we have alluded
were conducted with the rate of secretion of
taurocholate arbitrarily fixed at about 15
Circulation, Volume XXI, May 1960
BILE FLOW-ml/min
rigure 7
Relationship between bile flow and composition
during constant infusion of taurocholate. At higher
rates of flow, the concentrations of bicarbonate
and chloride and the p11 were increased. The
increment in bicarbonate was greater than that
in chloride. Highest flows were observed after
intravenous administration of secretin or intra-
duodenal administration of hydrochloric acid.
Acetazoleamide produced a choleresis in which
chloride was the predominating anion. (Repub-
lished by permission of the Journal of Clinical
Investigation.16)
,uM/min. It should be mentioned, however,
that we have observed a very similar qualita-
tive relationship between bile composition
and flow at a taurocholate secretion rate of
about 40 ptM/min.-although, of course, the
absolute values of bile flow were much higher
than those shown here. It is also worth noting
that the arbitrary rate of taurocholate secre-
tion employed in the present studies repre-
sents only about one-tenth of the maximal rate
at which the liver is apparently capable of
secreting this bile salt. The transport maxi-
 994

-j

d
E
300

250

200

50

100

5 c
.*
...9
TAUROCHOLATE
FRACT ION
...
*::::
.... ......
..
.......
. . . .

.......
..

'. '.'''.'
..

:-:-:-:-:-:-:
......
*:-:-:-:-:-:-
.

. . ....

................
......
............
.............
.F...........
............
.........
............
............
........
... .
.:....,..
0 05ml/min U.
BILE

15m
CG-

/min
9WHEELER, RAMOS, WHITLOCK

ELECTROLYTE
FRACTION

0.1OmI/mi n
Figure 8
Hypothetic fractions of bile. The concentra,tions and flows are based on the assumption
that each solution is isosmotic with respect to plasma. When the output of taurocholate
is constant, all variations in bile flow and composition can be attributed to changes in
flow and composition of the "electrolyte fraction." (Republished by permission of the
Downloaded from http://ahajournals.org by on August 25, 2020

Journal of Clinical Investigation.16)

mum for taurocholate is well over 100 [M/
min. in dogs of this size.
One way of explaining the observed varia-
tions in bile flow and composition would be
to postulate that bile is formed by the admix-
ture of a number of solutions which differ
from one another in comnposition and mode of
production. With this thought in mind, we
have arbitrarily elected to regard each bile
specimen as a mixture of 2 hypothetic isosmot-
ic solutions as shown in figure 8. On the left
is a pure solution of taurocholate which-be-
cause of the associative properties of the
taurocholate ion-would be isosmotic at a con-
centration of about 300 muM/L. On the right
is a solution of chloride and bicarbonate
which we shall call the "electrolyte fraction ";
the sum of these ions would be equal to about
150 mEq./L. for an isosmotic solution. Thus,
on the basis of the osmotic behavior of all
these constituents, it is possible to calculate,
for each bile specimen, the flow and composi-
tion of these 2 hypothetic constituents. Under
the conditions we have emnployed, the output
of the "tauroeholate fraction" is maintained
at a constant rate. Hence, the changes in bile
flow and composition must be attributed to
changes in the output and composition of the
"electrolyte fraction," and we shall therefore
examine these changes.
When one examines the relationship be-
tween composition and output in the "elec-
trolyte fraction" (fig. 9), it is apparent that
increasing output is accompanied by recipro-
cal changes in chloride and bicarbonate con-
centration. At the very highest rates of flow
-after secretin administration-the concentra-
tion of bicarbonate achieves its highest value
of about 75 mEq./L. and chloride concentra-
tion reaches a minimum at about the same
level. This figure bears a striking resemblance
to illustrations of the behavior of pancreatic
secretion14 although, of course, much higher
concentrations of bicarbonate and lower con-
Circulation, Volume XXI, May 1960
 ELECTROLYTE EXCRETION IN BILE 995

centrations of chloride have been observed in 120

pancreatic juice under maximal secretin stim-

ulation. Note again the fact that acetazoleam-
10 K CHLORIDE
Dog Norma
TAUROCHOLATE
145A,M/mm in V

ide administration results in high concentra-

tions of chloride and low concentrations of E
90 4%.
bicarbonate. a
0
R
The results of these studies are consistent x- 80

with the view that at least 2 processes are in- 70

volved in the elaboration and modification of
bile. First, it is obvious that the rate of bile 60

production is profoundly affected by the rate B8CARBONATE

of secretion of a number of substanees of 020
°0
which the bile salts are of the greatest physi-
z
ologic importance. In spite of the variations .
noted there is, in fact, a rough proportionality 0

between total bile flow and rate of bile salt Ge Spontaneous f/ow
,a /ntraduodenol HGC
secretion. Also, as noted earlier, bile flow be- l0 I 03 InarGvenous secretin
VT tntravenous acetazolamide
comes almost vanishingly small in the animal lIl

which is acutely deprived of recirculating bile D 0005 0.10 0 15 0 20 0.25

salt unless the bile salt is replaced by another
route. It would seem entirely reasonable to
postulate that the priluary event in bile for-
mation is the active secretion of bile salts and,
to a lesser extent, of certain other substanees.
The addition of water and many diffusible
Downloaded from http://ahajournals.org by on August 25, 2020
OUTPUT OF ELECTROLYTE FRACTION-ml/min
Figure 9
Composition of "electrolyte fraction" during
chazges in its output. A reciprocal change in
bicarbonate and chloride, similar to that observed
in pancreatic juice, is observed as output increases.
After the administration of acetazoleamide, the

constituents could then occur passively along
the resulting osmotic and electrochemical gra-
dients. This viewpoint has been enunciated in
a recent review by Sperber'5 with which our
data are wholly in accord.
The second process involves the modifica-
tion of the final composition of bile by the
addition-at an unknown site in the biliary
tract-of a solution which is similar in many
respects to panereatic juice. This bicarbonate-
rich fluid appears to be responsible for the
spontaneous variations in bile flow which oc-
cur in spite of the constant rate of secretion
of bile salts, and its output is maximal fol-
lowing stimulation by exogenous or endoge-
nous secretin.
Finally, the possibility of reabsorptive mech-
anisms in the bile ducts must not be over-
looked, although at present the only evidence
of the existence of such mechanisms is that
which can be inferred from the similar com-
position of gallbladder bile and bile resting
in the common duct.
Circulation, Volume XXI, May 1960
concentration of chloride is high and the coceen-
tration of bicarbonate is low. (Republished by
permission of the Journal of Clinical Investiga-
tion.'6)
References
1. SPERBER, I.: Biliary excretion and choleretic ef-
fect of somiie phenolsulfonephthaleins. Acta
physiol. scandinav. 42: Suppl. 145, 129, 1957.
2. HANZON, V.: Liver cell secretion under normal
and pathologic conditions studied by fluores-
cence microscopy on living rats. Acta physiol.
scandinav. 28: Suppl. 101, 1, 1952.
3. CooK, D. L., LAWLER, C. A., CALVIN, L. D., AND
GREEN, D. M.: MIechanisnis of bile formation.
Am. J. Physiol. 171: 62, 1952.
4. JENNER, F. A., AND SAIYTH, D. H.: Excretion of
phlorrhizin by the liver. J. Physiol. 137: 18P,
1957.
5. BERMAN, A. L., SNAPP, E. P., ATKINSON, A. J.,
AND IVY, A. C.: Effect of cinchophen on bile
formation. J. Lab. & Clin. Med. 28: 682, 1943.
6. BLOCH, K., BERG, B. N., AND RITTENBERG, D.:
Biological conversion of cholesterol to cholic
acid. J. Biol. Cheai. 149: 511, 1943.
7. SIPERSTEIN 2M. D., AND MURRAY, A. W.: Choles-
terol metabolism in man. J. Clin. Invest. 34:
1449, 1955.
 996 WHEELER, RAMOS, WHITLOCK
8. MOSBACH, E. H., KALINSKY, H. J., HALPERN, 13. THOMAS, J. E., AND CRIDER, J.
0.: A quantitative
E., AND KENDALL, F. E.: Determination of study of acid in the intestine as a stimulus
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9. SCHMIDT, C. R., BEAZELL, J. M., BERMAN, A. L.,
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the secretion of bile. Am. J. Physiol. 126: aiid chloride of pancreatic juice secreted inl
120, 1939. response to various stimuli.Gastroenterology
10. SCHIFF, M.: Gallenbildung, abhaingig von der 4:409, 1945.
Aufsauguing der Gallenstoffe. Pfluigers Arch. 15. SPERBER, I.: Secretion of organic anions in the
ges. Physiol. 3: 598, 1870. formation of urine and bile. Pharmacol. Rev.
11. THOMAS, J. E.: An improved cannula for gastric 11: 109, 1959.
and intestinal fistulas. Proe. Soc. Exper. Biol. 16. WHEELER, H. O., AND RAMOS, 0. L.: Determi-
& Med. 46: 260, 1941. nants of the flow and composition of bile in
12. PETHICA, B. A., AND SCHULMAN, J. H.: Haemoly- the unanesthetized dog during constant infu-
tic and surface activity of sodium taurocho- sions of sodium taurocholate. J. Clin. Invest.
late. Nature 170: 117, 1952. 39: 161, 1960.

The Origin of Life

Downloaded from http://ahajournals.org by on August 25, 2020

At first there were the simple solutions of organic substances, whose behavior was
governed by the properties of their component atoms and the arrangement of those
atoms in the molecular structure. But gradually as a result of growth and increased
complexity of the new molecules new properties have come into being and a new colloid-
chemical order was imposed upon the more simple organic chemical relations. These
newer properties were determined by the spatial arrangement and mutual relationship
of the molecules. Even this configuration of organic matter was still insufficient to give
rise to primary living things. For this, the colloidal systems in the process of their
evolution had to acquire properties of a still higher order, which would permit the attain-
ment of the next and more advanced phase in the organization of matter. In this process
biological orderliness already comes into prominence. Competitive speed of growth,
struggle for existence and, finally, natural selection determined such a form of material
organization which is characteristic of living things of the present time.-A. I. Oparin.
The Origin of Life. Translated with annotations by S. Morgulis. Ed. 2. New York, Dover
Publications, 1953, pp. 250-251.

Circulation, Volume XXI, May 1960