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This is to certify that the

thesis entitled

THE COMPARATIVE DISTRIBUTION OF ISOTOPIC
ELECTROLYTES IN GUINEA PIGS
DURING HISTAMINE SHOCK

presented by
MARVIN MURRAY

has been accepted towards fulfillment
of the requirements for

Master Wegree in_Eh¥.aJ.Olagy

”MW

){ajor professor/

Date—Maw—

0-169

 

THE CCMPARATIVE DISTRIBUTION (F ISOI'OPIC EIECTROLITES IN
GUINEA PICS DURING HISTAMINE SHCXJK

by
MARVIN 33mm}:

ATEBIS

Submitted to the School of Graduate Studies at Michian
State College of Agriculture end Applied Science
in partial Miflment of the mquiremente
for the degree of

MASTER (F SCIENCE
Department at Pimeiolog and Phumcolog

1950

THEQ:

(D

 

ACKNWIEWN‘I‘S

The author wishes to express his gratitude to
Professor 1:. D. Collings of the Department of Plysiol-
ogy and Pharmacolog, for the valuable advice and assist-
ance rendered both during the experimental work, and the
preparation of this manuscript; also to Professor B. A.
Alfredscn for the use of the facilities of the department.
A special debt of gratitude is due to Professor L. r.
Wolterink for his tmtirmg assistance and inspiration in
the field of radiobiolog.

5339290

;.

TABLE OF CONTENTS

INTRODUCTION ....................................
METHODS AND MATERIALS ............................

CALCULATIONS ..............................
RESULTS .........................................
DISCUSSION ......................................
SUMMARY AND CONCLUSIONS .........................
BIBLIOGRAPHY ....................................

APPENDH OOOOOOOOOOOOOOOOOO...OOOOOOOCCOOOOOOOOOO

10
26
29
36
38

Recent progress in chemical and physical methods
has given the physiologist a better opportunity to continue
investigations or the functions of the organism at the cellular
level. Current advances in atonic physics have provided both
materials and interest in isotopic tracer investigations. Gon—
sequently a tool has been provided for the stub out elemental
dynamics in the organism.

Since the early days of protein chemistry, it has
been known that protein reactions pg; 22 depend upon minute
quantities of cations and anions, which moderate and direct pro—
tein actions and interactions. This has been found to be true
in natural reactions as well as those which occur outside the
organism.

' In the field or manolog, it is known that antigen-
antibody reactions depend upon the presence of salts. The exact
nature of the relationship between the cations and anions to n-
actions of proteins has never actually been established, although
sane investigators have tried to define such a relationship (Sunt-
0701'811, 1949).

The past half century has sham varied interest in
anaplwlazis, histamine and psptone shock and related phsnmsna. A
few authors have attempted to show sans relationship between miner—
alsandtheabovephenansna. Chieflybecaueeorthslackotothsr

I9

 

'—

 

 

Il"‘
..

4 a.
I u

 

methods, investigators were confined to studying mineral concen-
trations in the blood or tissues. for the most part these studies
were indecisive.

The principal cations which react with protein in
the organism are sodiun, potassium, calcium and mains. The
ions are conteinedinandact physiologicallyinone ornore of
the organism's phases. It is just as important to establish the
mineral dynamics in physiological states as well in pathological
conditions such as anaplvlsxie and related phenasena, where the
barriers which contain the normal phases are altered.

The present work was therefore undertaken to invest-
igate the dynamics of calcium redistribution in the various pint
siologicsl fluid canpartnents during a phenomenon such as histamine
shock. Themajor premise atthe wtsetwasthatsuchananaphyw-
lactoid reaction, which is probably accompanied by a distinct in-
crease in membrane pemsability, should show an ionic redistribu-
tionofsane sort. Iodine spacewasdeterminedasagrossexper-
inental control. This ion was chosen because of its availability,
and wealth of previous investigation of its distribution in the
various body oompartnents. It is the ccmensus of opinion that
thebulkofiodineintheorganismexistsintheertracellular
fluid with the exception of the intracellular thyroid pool . Thus,
a comparison of calcium and iodine distributions should be worth-
while.

Perhaps the first difficulty the investigator en-

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counters in reviewing pertinent literature is in the division of
topics. Can one group anaplvlsxis, histamine shock, peptone
shock, trypsin shock, allergy, tapersensitivity, and other libs
reactions in one general category, or should one specifically
separate them and treat them individually? Apparently it seem
to be a matter of convenience , and there is a general tolerance
of each viewpoint. In view of the general scarcity of literature
available with reference to this specific topic and lack of proof
that these reactions are not similar mechanisms, it would seem
expedient to discuss the group as highly similar phenomena.

The very nature of the relationship between calciua
and the aforementioned phenomena has been a highly debatable sub-
Ject. Various investigators align themelves into three groups:
those who claim an increase of blood calcius during the crisis. of
the phenanena, those who hold the opposite, and those who say there
is no change at all.

Clinically, Brown and Hunter (1925) showed that in a
plurality of cases of asthma, hayfevsr, etc., a definite caloius
deficiency existed. Furthermore, they stated that increased dietary
oalcimn, administration of calcium lactate, and thyroid and para-
tmroid hormone therapy alleviated severe clinical syndrmes.
Kallos and Kalloe-Deffner (1938) showed that injections of calcium
to allergic guinea pigs inhibited the respmses of isolated uteri
and bronchial mecles to specific antigens. Also, these investiga-
tors showed that calcium and atropine prevented asthntic attacks

in humans.

Kaetle, Healy and Buckner (1943) demonstrated that
excess calcium lactate intraperitoneallv did not protect guinea
pigs fran anapmrlsxis, whereas small amounts did. Arloing, Lang-
erson, and Mounier-kuhn (1925) showed that injections of calcitm
chloride at the time of sensitization in no way influenced ana-
plvlaxis. However, if calcium chloride was injected at the time
of the shock dose of antigen, shock was attenuated.

A rarely cited work by Sohittenhelm, Erhardt, and
Warnat (1928) contains a complete account of calcium and potassium
levels in blood and tissues of dogs and rabbits during sensitiza- ‘
tion and anaptvlsxis. These data showed that during shock there
was a small but definite decrease in blood and serum calcim. More-
over, they found that calciun levels in liver increased, whereas
in lung they decreased during shock.

. mischnaryew (1930) confirmed the data of Arloing et
a1. (1926) and in addition showed that anaplvlsxis was augeented
by the inaction of potassium chloride. By chemical analyses,
Kuschinsky (1929) found that histamine shock produced a slight

' rise in command plasma calciutn, however total blood calciunwas
lowered.

Conflicting with the previous reports, use not
(1922) stated that results of his investigations of calcium blood
levels in guinea pigs in anaphylactic shock were so varied that no
conclusions were possible. also with guinea pigs, Brown and Ram-
dell (1929) reported that total blood calcitn did not change during

anaplwlsxis, but diffusible calcium increased during shock.
Similarly, Averianow (1926) found no deviations in blood calcium
of dogs during anaphylaxis.

Still more puzzling are the following reports which
indicate that blood calcium increases during anaplrvlefln. inan
31:11 (1927) reported raised blood levels in guinea pigs and rabb-
its during anaphylaxis, histamine and peptone shock. Similarly
m‘ilhon, Cloque, Galup, Debedcur (1934) reported that in asthna
in hlmans there was a general increase in blood calcium. In 1939
Yosito Sidara, taking issue with Anan SinJi, reported decreased
serum calcium in rabbits during peptone shock. However, he con-
firmed the previous observations that histamine shock increased
blood calcium levels .

MB‘I‘HOIB AND MATERIALS

All work was done with male and female guinea pigs
varying in weight between 550 and 1100 grams. Four groups of an-
imsls were established. Groups I and II were designated "Iodine -
131 control" and "Iodine - 131 histamine" respectively. Groups
III and IV were designated "Calcium - 45 control" and "Calcium - 45
histamine" respectively.

The iodine 131 isotope was used in the form of potass-
ium iodine 131 with potassium iodide carrier. The concentration
was made by adding the isotopic potassium iodide to physiological
saline to form a dilution per 25 ml. which would read 14,750 counts
per minute at zero distance from a thin end window Geiger-Muller
tube with aluminium filter. The dosage was then defined as 0.5 ml.
per one hundred grams of body weight.

The calcium 45 isotope was in the form of calcium 45
carbonate with calcium carbonate carrier. The calcium 45 carbonate
was treated with 4 normal lvdrochloric acid in distilled water.

The pH was adjusted to 6 with the addition of 6 normal sodium hydrost-
ide, and the solution was diluted to 100 ml. The dosage was then
established as 0.5 ml. per 100 grams of bochr weight.

Histamine acid phosphate was used in concentration
of 2.75 mg. per ml. The dosage to produce shock was 1 ml. per 1000
grams of body weight.

Iodine Qontrol group I.
The animals were injected subcutaneously at zero time

with the iodine 131 solution. At two, four, and six hours re-
spectively, 1 ml. of blood was withdrawn by means of heart punc-
ture. The blood was placed in previously tared porcelain cruc-
ibles which had been weighed at constant weight. Two sizes of
crucibles were used: 00 and 0000. At six hours the animals
were sacrificed by asphyxiation with dimethyl ether. The animals
were innnediately autopsied and samples of liver, lung, bladder,
thyroid, uterus, and pectoralis major muscle were taken. These
were weighed on a torsion balance accurate to the third decimal.

All of the samples were dried to constant weight in
a dry air oven at 102 degrees centigrade. They were placed in a
desiccator and weighed on an analytical balance (which had pre-
viously been used to weigh all of the crucibles) which was accur-
ate to plus or minus 0.00005 grams. The weights were recorded.

The samples were then measured for radioactivity
with a thin end window type Geiger-Muller tube which recorded by
means of an electruic register. The samples were shielded in lead
counting chambers. Geometry was fixed for small and large cruc-
ibles, the air path for the large crucibles was 5 cm. and for the
small ones 2 cm. No attempt was made to correct for the obvious
discrepancy in geanetries, since no direct cross correlations
were made between counts. Since all counts were below 1500 per
minute, no corrections for coincidental counting were made. The
ranges of weights of materials counted indicated that self-ab—
sorption was within 2 percent (Lee, 1950).

The samples were then allowed to decay until the

amount of radio-activity was practically nil. Afterward they were
ashed in an electric oven at a temperature of 1500 degrees Fahren-
heit. The crucibles containing the ash were weighed at constant
weight.

1am: as mm 9.2”! .

The procedure followed for this groupwas motlythe
sane as in Group I. However, at six hours histamine acid phosphate
was injected intothe heart to produce shock. Shortly before death,

oneml. otbloodwaswithdrawnfraneachsnimlbyheartpuncture.
I The animals were autopsied inmediately, and the en-
suing procednre followed that of Group I.
gem gamma Won

TheprocedurewasthesameasinGroupriththe
following exceptions: The animals were injected with isotopic cal-
cium at zero time. All crucibles used in the calcium experiments
were «mama-an... Afterdryingtheussuesinthedryair
oven, they were ashed in the electric muffle furnace at 1500 degrees
centig'ade. Then, after weighing at constant weight, the samples
were measured for radioactivity by the same type Geiger-Muller tube
and counter as before.
seen n____stan1ne mm

ThemethodfollowedwasthesameasinGrmpIII,
with the exception that death was induced at six hours by mans of
injection with histamine acid phosphate.

In both calcium experiments, no correction was rude
for coincidental counting as none of the tissues emanated over

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1500 particles per second. The geometry was fixed for all samples
and the air path was 2 cm. Self absorption was within 2 percent
with reference to curves determined by Atens (1950) and Murray and
Refson (1950).

Previous investigators have shown that there is no
appreciable variation in calcium content of the erythrocytes dur-
ing anaphylaxis (Schittenhelm, 1928). However, due to the nature
of the experiment it was thought desirable to check the calcium
distribution in the red blood cells. Three guinea pigs weighing
1280, 1140, and 1080 grams respectively were injected with calchmm
45 chloride subcutaneously at zero time. At six hours, the animals
were injected with histamine acid phosphate by heart puncture.
During the ensuing spasms, seven m1. of blood were removed from
the heart of each animal. The blood was heparinamized and centri-
fuged. The hematocrit was determined. The supernatant plasma
was decanted, and the cells were washed in saline four times. The
cells were then dried and ashed and subsequently counted for part-

icle emanation.

Average hematocrits were determined for all four

experimental groups.

Standards were prepared of each injection solution
of isotopic substance by micrOpipetting 0.01 and 0.02 ml. in each
size crucible and assigning standards for each experimental group.

These were then dried or ashed depending on the procedure in the

experimental. group, and subsequently counted in the same manner
as the samples of tissue.

W
W
Una-mime or. no. iaei—iis main means
a“?
Na Noe N lumber ofparticles emanating
after 1 half life
_ N Number of particles emanating
ing a Att- ° at zero time
o
t? Time of half life
- 2 - ..
1n 1% " A Specific activity constant
W i 257
.At
A a ice A Activity at time of measmement

1 Activity at zero time

t Time
Specific activity content

 

Activity of 1 35 standard x injection fluid volume = diluting
activityoflm1.ofplasma voluse m1.)

Equilibrim of the absorbed materials was arbitrarily
seemed to have taken place by two hours following injectim, and

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the value calculated was equated to unity of extracellular
volume. Succeeding volumes were then plotted as percentages
of unity.

W
Mannery and Huge, 1941

Activity of l a, tissue x .95 x .93 x 100 = 1120.1 gin/100 gm.
Activity of 1 ml. plasma fresh
tissue
1120:: is expressed as the water mineral equilibrium in gun.
per hundred grams fresh tissue. 0.95 is the Gibbs» Donnan ratio
and 0.93 is theoorrection for the water content of plasma.
W
The average hematocrit was fmmd to be 1.5 percent.
Therefore, counts per ml. of plasma were calculated as follows:

c ts r m1 whole bl 3 counts per m1. plasma
.55

22.1.3.1

THE DISTRIBUTION CF WATER AND ASH IN THE IODINE

CONTROL AND IODINE HISTAMINE EXPERDIENTS

 

Tissue

Animal nmnbers

Parcentage

of
Water

Percentage

of
Ash

 

 

Control Histamine Control Histamine Control Histamine
fiver

#3 74.9 71.9 5.6 5.9
#2 #4. 76.0 78.0 6.8 5.5
#6 #3 69.5 71.8 7 .1. 5.2
#8 #10 68.9 72.0 5.1 6.6
X * I 72.3 73.4 6.2 508
i? #3 80.7 77.9 8.3 7.7
#2 #1. 78.7 79.2 7.0 12.7
i6 #8 71.1. 72.8 10.2 9.5
#7 #10 33.7 77.8 7.2 15.9
Sledge;
I2 #4 84.1. 84.6 15.3 10.6
#6 #8 ”e0 8404 1207 907
’7 #10 82.8 80.8 7e6 15e2
I I . 82.6 83.2 12.0 11.1
W7 77 4 78 0 8 2 9 1

3 e e O 9
#2 #4 77.4 See 7e 5
#6 #3 70.0 76.1 11.0 7.5
#7 #10 70.0 88.3 3.8 10.3
x x 72. 5 80.0 8.0 8.6
#6 #3 67.6 70.7 5.8 8.3
f7 #10 67.1 69.4 8.9 9.7
x x 69.1 71.2 6.7 9.1
item

83.7 3.8 14.2

’7 8°. 5 a .9 6. 1 130 5
I 83.3 5.0 13.9

 

* X .- Average

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w CWNTS HER SECOND PER ML. CF WHOLE BLOOD IN IODINE
HBTAMI‘E AND IODINE CONTROL mm
*0 #1 C #2 C #6 C #7
H #3 H #4 H #8 H #10
2 hr. Control 19.90 7.37 10.69 9.89
2 hr. Histamine 21.23 13.42 12.29
4 hr. Control 3.96 3.85 11.29 8.75
4 hr. Histamine 7.15 5.50 6.42 10.22
6 hr. Control 2.62 .01. 10.25 12.56
6 hr. Histamine 2.42 2.86 8.37 6.85

 

* C #1 a Control Animal 1, etc.

a #3 . Histamine Shockedinimal Number 3, etc.

.0».
a t
v
.
C .‘n

w. THE DISTRIBUTION a? 1131 IN TISSUE ASH mm IN muss
TISSUE 101nm: CONTROL AND 101mm HISTAMINE EXPERI-
Minus

 

Tissue Counts/an. Counts/mg.
Animal numbers Fresh Tissue Ash Per
Per Second Second

Control Histamine Control Histamine Control Histamine

 

 

295mg Mole

C 1 H 3 .30 .00 .02 .00
2 4 .13 .00 .01 .00
6 8 2.04 1.13 0% 006
7 10 1.64 5.75 .14 .28

C 1 H 3 .97 .23 .07 .01
2 4 .96 .63 .06 .05
6 8 3.23 14.10 .14 .96
7 10 .95 3.81 .06 .21

C l H 3 .98 .99 .06 .06
2 4 1.08 .92 .07 .03
6 8 8.38 7.91 .29 .30
7 10 4.19 1.1.36 .27 .32

masses-

0 1 H 3 1.32 .49 .07 .03
2 4 11.69 .00 .49 .00
6 8 5.56 10.67 .21 .07
7 10 5.28 6.94 .40 .24

Thyroid

C l H 3 23.23 52.41 1.47 1.63
2 4 15.10 37.75 .76 1.97
6 8 264.22 267.85 14.15 10.89
7 ‘ 10 154.34 199e91 5.28 6070

yterus

C 7 H 10 7.15 6.60 .56 .29

. .0

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-15...

 

 

 

TABLE I! coma. VALUES FOR TISSUE SPACE IN IODINE

CONTROL - GROUP I
Ln—ihlal Number - 1 2 6 7 w I*
Tim 1. hr. 6 hr. 6 hr. 6 hr. 6 hr.
Iissue .
Factor-11s 1.6 5.7 9.7 9.9 6.7
Muscle
Liver * 11.1. 18.1 15.3 5.7 12.6
Lung 13.6 1800 1.0.0) 2503 2402
Bleddcr 185.0 25.0 2706 32e0 6704
Uterus 4300

All values given in m./100 gm. fresh tissue

_—K

m EXPERIMENTAL VALUES FOR TISSUE SPACE IN IODINE
HISTAMINE - camp II

 

 

 

Animal Number 3 4 s m X *
T1130 6 hr. 6 hr. 6 hr. 6 hr. 6 hr.
Pectcrallis 0.00 00.0 6.6 4.1 2.7
Muscle

Liver 4.7 10.7 81.0 27.0 30.9
Lung 20.0 15.7 46.0 81.0 40.7
Bladder 9.9 0.0 60.0 . 1.9.5 29.9
Thyroid 292.0 640.0 1525.0 1940.0 1099.0

Uterus . 41.0 4608 43.9

 

All values given in gnu/100 gm. fresh tissue

* X 3 Average

-16..

M THE DISTRIBUTION (F WATER AND MNERAIS IN THE CALCIUM
CONTROL AND CALCIUM HISTAMINE EXPERDENIB

 

 

 

Tissue Percentage Porcentege
Animal Numbers of Of
Water Ash

Control Histamine Control Histamine Control Histamine
Ecfifls Ec%e ‘
#1 3 72.6 77.2 5.8 5.8
#2 #5 73.0 80.5 5.3 5.9
I * I 74.4 78.7 6.8 5.7

ver

1 #3 80.1 76.3 7.5 6.3
#2 #5 78.6 27.2 7.3 5.6
#7 #6 69.0 73.7 7.0 5.3
I I 75.9 75.0 7.3 5.7
51 #3 78.7 ‘ 80.0 7.3 7.1
#2 #5 79.1 83.0 6.6 7.0
#7 #6 59.8 82.3 6.5 6.9
X X 72.5 81.8 6.8 7.0
laid—
#1 #3 83.3 92.4 5.3 7.2
#2 #5 82.7 86.8 6.3 1.9
67 #6 82.4 86.7 1.2 8.0
x 82.8 88.0 5.8 7.6

torus
£1 #3 83.2 7.6
#2 #5 81.6 6.2 '
#7 6‘6 79.5 83.0 7.7 6.5
X 81.1. 7.2

 

* X = Average

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-17..

mm VI COUNTS PER SECOND PER ML. OF WHOLE BLOOD IN

CALCIUM CONTROL AND CALCIUM HBTAMINE EX-

 

 

PERDEMS
*c #1 C #2 c #7
H #3 H #5 H #6
2 Hr. Control 10.69 7.05 20.34
2 Hr. Histamine 10.69 7.80 13.61
4 Hr. Control 15.85 6.70 11.41
1. Hr. Histamine 14.85 8.77 15.50
6 Hr. Control 19.52 6.97
6 Hr. Histamine 11.51 4.15 12.23

 

*C #1 = Control animal number 1, etc.

H#3-Histamineshoclnedenine1nmher3, etc.

~18-

 

W THE DISTRIBUTION a? 0.45 IN TISSUE ASH AND TRISH
TISSUE CALCIUM CONTROL AND CALCIUM HISTAMINE Ix-
PERDIENTS -
Tissue Counts/gm. Counts/mg.
Animl number Fresh Tissue 18h Per '
Per Second Second

 

Control Histamine Control Histamine

Control Histamine

 

#2 #5
#7

Liver

#1 #3
#2 #5
#7 #6
#1 #3
#2 #5
#7 #6
11549.91

#1 #3
#2 #5
#7 #6
m

#1 #3
#2 #5
#7 #6

7.47
6.14

10.60
7.28
30.9

50.08
8.79
54.89

31.79
7.61

18.25
7. 56
41.54

15.87
18.07
67.47

11.31
15.86
29.84

18.30
10.04
17.11

5.74
12.66
35.57

18.63

.44

.67
.51
1.42
3.38

1.85

3.63
.70

1.43
2.63

1.18
1.57
5.73

.76
2.24
1.29

.85
1.39

1.05
4.92
2.90

1.70

 

-19..

 

 

 

W *CONTROL VALUES FOR TISSUE SPACE IN CALCIUM
CONTROL- GRUJP III

Animal Number 1 v 2 7

Time 6 hr.. 6 hr. 6 hr.

11193.2

Pectoralis Muscle 20.9 41.8

Liver 29.7 50.5 130.0

Lung 140.0 61.0 233.0

Bladder 89.4 53.0

Uterus 51.0 52.7 176.0

‘All Values Given in gm./100 gm. of Fresh Tissue

 

 

 

12111211113 *EXPERDIENTAL VALUES FOR TISSUE SPACE IN
CAICIUM HISTAMINE - GRWP IV

Animal Number 38w 5 6

1‘1an 6 hr. 6 hr. 6 hr.

119.222

Pastoralis Muscle 67.0 238.0 268.0

Liver 47.6 210.0 109.0

Lung 77.0 108.0 68.3

Bladder 24.2 148.0 142.0

Uterus 74.0

*111 Values Given in gl./100 an. at Fresh Tissue

 

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Iodine ggpgriment.
Fran the data on blood iodine radioactivity in

Table II, the diluting fluid volumes were calculated (according
to the equations on page 10). They indicate that the injected
iodine was absorbed and diluted for a period of about four hours.
After this the diluting volume became smaller progressively. How-
ever, during histamine shock, the iodine was further diluted five
to six times above control values as shown in Figure 2.

The ash and water content of the tissues differed
in control and histamine shocked animals, but all tissues did not
vary in the same direction (Table I, Figure 1). Specifically the
ash content of liver decreased, whereas the water content increased.
A similar condition was noted for the bladder. The lung tissues
demonstrated a contrary effect wherein the percentage ash increased,
and the percentage water decreased. Pectoral muscle, tlvroid gland,
and uterus showed an increase in ash and water content.

The data in Table III showing the distribution of
iodine radioactivity in the various tissues, both test and control,
were used to calculate the fluid space in which the iodine was dis-
tributed within the tissues. As indicated in Table IV and Figure
3, experimental and control iodine space determinations increased
as much as 200% in the case of liver. However, lung, thyroid and
uterus showed a smaller increase, and bladder and pectoral muscle

-27-

actually'underwent considerable reduction in the iodine space.
Calcium Experiment.

From.the data in Table IV, the diluting volumes
were calculated as for iodine (vide supra). These indicate
apparently that absorption from the subcutaneous pocket at the
injection site was slow. Thus control values show a decreasing
diluting volume, although experimental values followed the
controls closely. Upon injection of histamine and conSequent
shock, the diluting volume increased to about twice the control
value (Figure 4).

The SXperimental and control percentages of water
content varied similar to those in the iodine experiment (Table
V, Figure 5). However, percentage ash varied in the Opposite
direction excepting lung and bladder which increased in.both
'water and ash content. Pectoral muscle and uterus decreased in
percentage ash, but water content increased. Liver,on the other
hand, decreased both in ash and water content.

The calcium spaces (Table VIII) were calculated from
the data in Table VII in the same manner as for iodine spaces.
These were in no way related to the corresponding iodine spaces.
However, as in the iodine experiment, the calcium spaces varied
perceptibly among different tissues (Figure 6). Calcium space
increased about four times in pectoral muscle and to a lesser
degree in liver and uterus. In lung and bladder, the calcium
spaces were markedly decreased.

In a single experiment as described in the section

-23-

above on methods, it was found that Ca45 and 1131 failed to
enter the erythrocytes either in control animals or in those
subjected to histamine shock.

-29..

new

The phase determination experiments described above
Show that the injected isotopes of calcium and iodine do not enter
the erythrocytes. Therefore, the intracellular fluid of the red
cells plays no role in the dilution Of these isotopes as discussed
below.

The calculated diluting volumes of the radioactive

iodine indicate rapid dilution or uptake of iodine from.the extra-
cellular phase, as shown in Figure 2. It should be noted that the
slaps of the plot of the diluting volume versus time (Figure 2)
depended largely upon the rate of absorption of injected iodine from
the subcutaneous injection site. Under the experimental conditions,
however, it is further possible that there was a rapid dilution of
the iodine by means of a shift of intracellular fluid to the extra-
cellular phase. If this were true, a highly vascular organ.which
becomes engorged during shock, should show a great increase in iod-
ine content during shock. This is apparently true since the iodine
concentration in liver increased threefold (Table III). Another
factor in determination of iodine concentration in extracellular
fluid is its uptake by one or more tissues and dilution by intracell-
ular water. Possibly, this occurred during shock in the case of thy-
roid, lung, liver and uterus (Table IV).

The plots of the diluting volumes of calcium.versus
time (Figure 4) indicate a slow but progressive concentration of
calcium in the extracellular fluid Of the organism. Under con-

trol conditions, this is prObahly due to the slow rate of absorp-

tion of the calcium at the site of the injection. Under experi-
mental conditions the dilution increased as in the iodine experi-
ment. Similar reasoning indicates that the dilution of the cale

s.

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cellular fluid) and an uptake of calcium.by specific tissues.

The question then arises: what is the role of calr
cium as compared to iodine during the shock phenomenon, and what
is then their relationship to the distribution Of water in the
organism?

Definite shifts of water are indicated by the data
in Tables I and V. These data are summarized in Table IX which
contains the average values for total water content of the tiss-
ues. They indicate that under conditions of histamine shock,
there is a general increase of total water content of tissues,

with but two exceptions, namely lung and liver.

 

 

TEL}? IX. JA'IER SHIFTS IN VARIOUS TISSUES UNDER THE
CONDITION OF HISTAMINE SHOCK
Iodine Calcium
Tissue Histamine Control Histamine Control
Liver 73.4% 72.3% 75.0% 75.9%
Lung 76.9 79.9 81.8 72.5
Bladder 83.2 82.6 88.0 82.8
Pectoral 80.0 72.5 78.7 74.4
muscle
Therid 71.2 6901
Uterus 83.3 80.5 83.0 81.4
AV. H20 7800 76.15 81.3 7704

 

-31..

It is therefore established that the dilutions of
iodine and calcium shown in Figures 2 and 4 are real as the re-
sult of a shift of water into the tissues. Since it is well known
that movement of water into and out of the cells is accompanied by'
ionic shifts, some movement of calcium and iodine and possibly
other ions might be expected to have occurred in these experiments.
This may be indicated by the rate of exit of the isotopic ions
from the blood (extracellular volume). The average rate of radia-
tion from whole blood shows that calcium and iodine leave the
blood at the same rate under shock conditions. Data showing this

are summarised in Table X and accompanying ratios.

TABLE I IMEAN RADIATION COUNTS IN“WHOLE BLOODIFOR
CALCIUM AND IODINE

 

 

 

 

odine- l Calciumgéfi
Ayerage Blood Counts Average Blood Counts
Time Histamine Control Histamine Control
2 Hours 15.65 * 11.96 10.70 12.69
4 Hours 7.32 6.96 13.04 11.32
6 Hours 5.13 6.37 9.30 13.25
.ku.2, and 10.89 9.46 11.87 12.01

4 Hours
* Counts per md./hec.

 

From.Table X the following ratios show the compar-

- 32 -

able rates of diffusion of iodine and calcium under control and
experimental conditions:

2,%% 3 0.47 for iodine and histamine 6,27 = 0.67 for iodine
1 . 9. control

2%? . 0.78 for calcium and histamine 12,35 : 1.lO
11. 12.01

Therefore ‘61 a 1.426 for the iodine experiment
.47

Therefore‘l,lg : 1.410 for the calcium.experiment
.7

The last two ratios above show that both iodine and
calcium leave the blood (extracellular fluid) at about the same
rate.

In view of the fact that water appears to enter the
tissues and that calcium and iodine seem to leave the extracellue
lar fluid, the tissue content of these isotopes during shock be-
comes of interest.

From.the data in Tables III and VII, the general
trend in redistribution of calcium and iodine can be seen. Table
II summarizes the mean changes in concentrations of isotopes in

the various fresh tissues.

 

TAQLE XI SHIFT OF ESOTOPES DURING HISTAMINE SHOCK
Iodine Calcium
Tissue Histamine Control Histamine Control

Counts per second per gm. tissue

 

L179? 14.69 1.53 19.00 16.23
Bladder 4.35 5.96 17.99 19.70

_3_3-

 

TAB XI SHIFT 0F BOTOPES DURING HISTAMINE SHOCK
(Continued)
Iodine Calcium
Histamine Control Histamine Control

Counts per second per gm. tissue

 

Pectoral 1.72 1.03 33.80 6.81
muscle

Thyroid 139.43 114.22

Uterus 6.60 7. 15

 

Figures 3 and 6 depict graphically a resume of the
data on iodine and water, and calcium and water, respectively.
These show that apparently in shock, with the notable exceptionef
pectoral muscle, water and calcium move in Opposite directions.
That is to say, wherever calcium enters a tissue the total water
content decreases and vice versa. No such conclusion can be drawn
fran the iodine data.

Considering the total ash content of the tissue, it
appears as if there was sane sort of change in this material as
well as tissue water in the shock experiments. The extent of this
shift can be seen in Table XII which sulmnarizes data in Tables II

 

 

and “Is
T LE X REDISTRIBUTION 0F ASH DURING HISTMNE SHCXJK IN
IODINE AND CALCIUM EXPERD’IENTS
Iodine 2 Ash Calcium % Ash
T133119 Histamine Control Histamine Control
Liver 5.8 6.2 507 703

Lung 11.5 8.2 7.0 6.8

(I

 

- 34 _

 

 

TABLE XII REDISTRIBUTION OF ASH DURING HISTAMINE SHOCK IN
Wd) IODINE AND CALCIUM EXPERDIEN‘I‘S

Iodine 5 Ash Calcium sh
Tissue Histamine Control Histamine Control
Bladder 11.1 12.0 7.6 5.8
Pectoral 8.6 8.0 5.7 6.8
muscle
Thyroid 9.1 6.7
Uterus 13.9 5.0 6.5 7.2
Av. 10.0 7.7 6.5 6.8

 

From Tables XI and XII it can be seen that calcium
and ash.move in the Opposite directions. Therefore, it can also
be said that water and ash accompany each other under shock con-
ditions. In the case of iodine, however, the figures indicate
with few exceptions iodine, ash and water travel in the same dir-
ections.

In general the tissue spaces as calculated on page
11 (Figures 3 and 6) represent a dynamic picture of the previous
discussion. However, from the standpoint of actual space, it is
to be noted that the standard method of calculation of space as

given by Mannery and Haege (1941) cannot apply in its entirety

-35..

in this problem. It is Obvious that in any biological phen-
omenon where permeability is altered, the Gibbs-Donnan ratio
as given in the calculations will not apply. Similarly, in
any investigation where water shifts are as extensive as those
noted above, no standard fraction of water content can be given
for plasma. Nevertheless, even though these aforesaid con-
stants do have errors, if they are constant, the calculated
tissue spaces will be relatively correct.

The evidence presented indicates that there is a
shift of calcium from lung and bladder, whereas calcium enters
pectoral muscle. If these tissues can be considered represent-
ative of smooth and striated muscle respectively, it is prob-
ably quite sipificant that calcium should leave a tissue such
as 11mg which is so prominently involved in the cause of death
in histamine shock in the guinea pig. Moreover, there is some
indication that mineral ash may increase in smooth muscle dur-
ing shock of this type.

-36-

SUI-MAR! AND CONCLUSIOIIB

1. Two experimental groups of histamine-shocked
(2.75 mg. histamine acid phosphate per kilogram body weight),
which were previously injected with radioactive calcium 45 and
iodine 131 respectively, were compared to two control groups of
unshocked guinea pigs with respect to total extracellular volume.

For measurements of water, and isotope content 1 ml.
blood samples were secured by heart puncture at 2, 4 and 6 hours
following isotope injection. Experimental blood samples were
taken shortly after injection of histamine at 6 hours. All an-
imals were then sacrificed and samples of liver, lung, pectoralis
muscle, bladder, uterus, and in some instances, tlwroid tissue
were obtained for isotope, water and ash determinations.

2. The data indicate that there was a tendency
for body water to shift into the tissues during histamine shock.

3. Some evidence is presented to indicate that in
shock tissue calcium and ash content varied in opposite direct-
ions. 4

4. Ionic iodine, not having been previously im-
plicated in this type of shock, was used for comparison with
calcium. Contrary to calcium, tissue iodine was found to vary
in the same direction as tissue ash content.

5. It was shown that during shock both calcium
and iodine leave the blood at the same rate.

-37..

6. It is considered significant that calcium
space decreased in smooth muscle - containing tissues, i.e.
lung and bladder, whereas calcium space increased in striated
since severe smooth muscle spasm is so characteristic in guinea
pig histamine shock, muscle, i.e. pectoral muscle. Concanit-
antly ash content of striated muscle appeared to decrease where-
as ash in smooth muscle increased.

7. The data presented do not justify a firm con-
clusion based upon a comparison of iodine and calcium tissue
space. However, if any trend is indicated it is that iodine
space increases in lung and decreases in pectoral muscle contrary
to the calcium data.

- 33 -

1W

Arloing, P., Langerson, L., and Mounier-Kuhn, P. L. 1926.
Action of Calcium Chloride on Sensitization and

Shock in the Guinea Pig. 92223,. Bend. Egg. £1.21-
95: 845. ,

Aten, Jr., A. H. N. 1950. Corrections for Beta Particle Self
Absorption. W. Vol. VI. 1:68.

Averianov, P. P. 1926. Blood Calcium in Anaphylactic Shock.
Ions. Had. 21.1. 2(6)=90.

Azzo Azzi. 1923. Content of Na, K, Ca, and Mg of the Blood of
Guinea Pigs in Anaphylactic Shock. Arch. _S_<;j.. Med.
45:356.

Brown, C. 1., Hunter, 0. B. 1925. Calcium Deficiency in Asthma,
Hay Fever, and Allied Conditions. Ann. £1.13. lied.
4:299.

Brown, H. and Ramsdell, S. G. 1929. Blood Calcium in Anaphylaxis
in the Guinea Pig. £9112. @3525. Med. 49:705.

Drilhon, A., Cloque, R., Galup, J., and Debedour, T. 1934.
Calcium and Potassium in Asthma. m Med. 42:816.

Kallos, P., Kallos-Deffner, L. 1938. Calcium Therapy in Allergy.
Arch. Iniezn- Eharsesssznanisa. 59:253.

Kastle, J. H., Healy, D. J., and Buckner, G. D. 1913. The Relation
of Calcium to Anaphylaxis. m. Infect. Dis, 12:127.

Xuschinsky, G. 1929. Changes in Blood Potassium and Calcium in
Dogs During Histamine Shock. _Z_. figs. 23523}. M. 64:
563.

Xuschnaryew, M. A. 1930. Potassium and Chloride in Anaphylaxis.
Z. lssusiiais. 67:9.

Mannery, J. P., and Haege, L. P. 1941. The Extent to Which Radio-
active Chloride Penetrates Tissues, and Its Significance.

A_m. ,1. £225.12].- 134883.

- 39 _

Murray, H., and Refson C. 1950. Beta Particle Self Absorption
(Calcium 45) Studies in Bone Ash. (Unpublished data)
Michigan State College.

Schittenhelm, A. , Erhardt, H., and Warnat, K. 1928. Anaphylaxis

Studies in Man and Animals. VII 1- 933;. £523.. lied.
58:662.

Sinji, Anan. Nagasaki Igaku. 1927. Uber die Schwankung des Kalk
und Kalium spiegels im Blute bei der Serum und anaphy-
laxie sowei bei den Histamine und Pepton shock. KER;
Sggghi. 5:751.

Szent—Gyorgyi. 1947. The Chemistry of Muscular Contraction.
Academic Press Inc., Publishers, New York, New York.

Yosito Sidara. 1939. Inorganic Substances in the Blood of Rabbits.
Eras- hman 22.2. Fmen ° Med. 23:9.

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