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TRACER EXPERIMENTS iN CAMNE SKELHAL MUSCLE

Thesis for the Degree of M. S‘ ‘
MICHIGAN STATE UNIVERSITY
MlCHAEL L. GOODNIGHT

1978

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ABSTRACT
TRACER EXPERIMENTS
IN CANINE SKELETAL
MUSCLE
By

Michael L. Goodnight

In order to understand more fully the function of the
blood vessels in nutritive supply and waste removal, it was
desired to investigate experimentally the question of whether
blood flow through skeletal muscle occurs in both nutritive
and non-nutritive paths.

This study concentrated on precise development of the
apparatus and techniques by which to study this problem. In
addition, obvious signs of separate parallel blood flow

1&
paths were sought for in the resulting data. C sucrose and

H3 dextran were injected into the canine gracilis muscle
blood supply. Blood samples were collected and the concen-
tration of tracer in the venous outflow measured according
to the developed techniques. Although no definite signs of
parallel paths were seen, the experiments yielded smooth,
consistent data and demonstrated their utility in future ex-
periments of this kind. Further experiments on vascular beds
which are known to have shunting pathways, eg. the skin of

the canine paw, would be recommended.

TRACER EXPERIMENTS
IN CANINE SKELETAL
MUSCLE

By
Michael L. Goodnight

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Chemical Engineering

1978

For my parents, Lynn and JoAnn

and my wife, Judith

ii

ACKNOWLEDGMENTS

The author gratefully acknowledges the assistance of

Dr. Donald K. Anderson, Dr. J.B. Scott, the Michigan Heart

Association, and his co-worker, Mark Holmes.

iii

TABLE OF CONTENTS

LIST OF TABLES . . . . . .
LIST OF FIGURES. . . . . .
INTRODUCTION. . . . . .
EXPERIMENTAL METHODS
The Choice of Tracers.
Experimental Layout . . . .
The Sampling Valve. . . . . .
Electronic Controls .
Experimental Preparation.
Experimental Procedure
Analysis of Samples . .
RESULTS
SUMMARY AND CONCLUSIONS . . . .
LIST 0? REFERENCES. . . . . . .

iv

LIST OF TABLES

Table Page

1. Tracer Recoveries . . . . . . . . . . 16

Figure

0\

O\Om\)

11a.
11b.
12a.
12b.
120.
13a.
13b.
130.
14a.
14b.
140.

LIST

Experimental Setup.

Valve Configuration

Controlling Signals

Experiment
Experiment
Experiment
Experiment
Experiment
Experiment
Experiment
Experiment
Experiment
Experiment
Experiment
Experiment
Experiment
Experiment
Experiment
Experiment
Experiment

Experiment

1 0

OF FIGURES

vi

Page

10
18
19
20
21
22
23
24
25
26
27
28
29
3o
31
32
33
34
35

INTRODUCTION

At present, there is a limited amount of knowledge about
transport of solutes in vascular beds. For example, there is
a controversy brewing over the mechanism by which vasocon-
striction or vasodilation influences transcapillary exchange.
There are two major hypotheses proposed to describe the mech—
anism. First, Renkin (1) has suggested permeability limited
transport i.e. permeability of the capillary wall limits
the rate of exchange between blood and surrounding tissue.
Therefore, as the blood passes through the capillary, equi-
librium is not achieved between the blood and the extravas-
cular fluid. Thus, the amount of solute transported to or
from the tissue is at least partially dependent on the time
available for exchange.

Friedman (2) has advanced the second hypothesis that, as
blood perfuses a vascular bed, its flow is in effect split
into two parallel streams- nutritive and non-nutritive flow.
The nutritive flow stream passes through the capillary bed
and achieves equilibrium with the tissue. The non-nutritive
flow stream bypasses the capillary bed. Therefore, changes
in the amount of solute exchanged with the tissue result
from shifts in the flow distribution between both pathways.

To experimentally test their hypotheses on the canine

gracilis muscle, both Renkin and Friedman used radioisotopes

42

of highly exchangeable ions. The former used K and the

/
latter Rbao

. However, the question is still unresolved
since both investigators have interpreted their results to
support their own hypotheses.

In our attempt to study this problem, we have used tech-
niques developed for the analysis of flow distribution in
chemical reactors. Levenspiel (3) has shown that pulse in-
jection studies (similar to the studies done by Renkin and
Friedman) can, if done properly, yield information on the
flow patterns in the catalyst bed of a chemical reactor.

For instance, it is possible to detect the presence of par-
allel streams with different residence times (channelling)
or dead space in the bed.

We have attempted to apply these same methods to flow in
a vascular bed. The method consists of the following pro-
cedures:

1) A non-diffusible tracer, H3 labeled dextran, is in-
jected as a bolus into the blood stream perfusing a vascular
bed. Serial samples of the venous outflow are taken during
the time required for all of the tracer to exit the bed.
Using radiotracer counting techniques, the concentration of
tracer in each sample is determined.

2) A diffusible tracer is also injected in the same bolus
in order to test for nutritive versus non-nutritive flow. If
the tracer in one or both of the parallel streams (providin

they exist) can exchange with surrounding tissue, we would

expect a delayed washout of tracer from the muscle. One
2

stream would show this delay while the second parallel stream
would not if Friedman‘s hypothesis is correct.

3) The resulting concentration versus time data are plot-
ted and inspected for obvious signs of parallel paths which
would show as two peaks in the curve (3).

4) Electrical stimulation of the muscle was conducted in
the experiments to determine the effects, if any, of exer-
cise on the vascular flow and the resulting tracer curves.

In addition, several experiments also involved a hista-
mine infusion prior to tracer injection to view its effect
on tracer recovery.

C1“ tagged sucrose was used as this will pass through the
capillary wall (though not as easily as Rb86) but will not
enter the cells. This permits back—diffusion of extracted
tracer into the blood and allows recovery of a larger frac-
tion of the injected tracer.

A diffusible and non-diffusible tracer were used in these
experiments in anticipation of a need for data concerning
diffusible and non-diffusible tracer washout curves such as
would prove useful in an analysis conducted along the lines I

developed by Borghi (A).

EXPERIMENTAL METHODS
The Choice of Tracers

In accordance with the diffusible models (4), a tracer
was needed which would cross capillary membranes and diffuse
into the interstitial fluids, but would not be taken up and
retained by the tissue cells. It was important that this
tracer not be taken up by the cells to enhance the process
of diffusion back into the capillary flow stream and insure
detection.

The tracer selected for this purpose was Cl“ labeled
sucrose. Sucrose readily crosses capillary membranes, dis-
tributing itself in the extracellular space of the tissue,
but is not taken up by the cells. t is for this reason, in
fact, that sucrose is commonly used for measuring the extra-
cellular fluid volume of the body (5,6).

In accordance with the non-diffusible models (4), a tra-
cer was required which would not cross the capillary mem-
branes (referred to as the non-diffusible tracer).

H3 labeled dextran was selected for this purpose. Dextran
is a polysaccharide of molecular weight of 60,000-90,000.
This was chosen over I131 labeled albumin, which was origi-
nally suggested, because tritium has a much longer half life

131

than I131 (t% of I is 8.03 days) and could be considered

to have a constant activity. Tritium labeled dextran may be

differentiated from 014

labeled sucrose in liquid scintilla-
tion counting (7), thus both the diffusible and the non-dif-

fusible tracers may be injected at the same time. As for the

4

permeability of the capillary membranes to 60,000-90,000 MW
dextran, studies have shown that it is less permeable than
blood proteins (8). The major excretory pathways of dextran
in the body are the liver, kidneys, and the gastrointestinal
tract (9).

C14 sucrose alone was used,

For those experiments in which
the tracer solution consisted of 3.0 izci/cc in 0.85% normal
saline. For the dual tracer experiments, the solution was

1.25 }ACi/cc of 014 sucrose and 23.33 i1Ci/cc of H3

dextran,
also in 0.85% normal saline. Injection volumes were 0.25 cc

with ClL‘L sucrose alone or 0.5 cc with the dual tracer.

Experimental Layout

Figure 1 shows a schematic of the experimental setup
used. The vascular bed was pump perfused by blood from the
femoral artery. Venous blood passed through the sampling
valve and the portion not collected was discarded when the
experiment was in progress. Washout solution was supplied by
a constant flow pump.

A solenoid valve (No. 2) directed the flow of the washout
solution either back to the solution reservoir or to the
sampling valve to flush the sample loop into collection
vials which were serially positioned by an automatic fraction
collector.

A three way solenoid valve was used to direct a supply of
high pressure air to the sampling valve for activation in
order to shift the internal flow pattern of the samplin

valve.

 

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Washout solution consists of; 1200 ml

of 0.85% saline

solution and 4 ml of sodium heparin (5000 USP units/ml)

................ Control connections

Figure 1. Experimental Setup

._—#-_—_—-——’-*-——_-————-I-——o——-__—-_—¢————

Finally, the controller is shown with its control path-
ways shown as dashed lines. More about the controller will

be given in the section marked Electronic Controls.

The Sampling Valve

 

The heart of the experimental apparatus was the sampling
valve used to take intermittent blood samples from the ve—
nous side of the vascular bed being perfused. A valve was
needed which would collect blood samples of a constant size
for scintillation counting, while not interfering with the
normal flow of blood through the vascular system. The Altex
Scientific Inc. Model no. 201-63 stream sampling valve cho-
sen had the advantage of having little or no dead space and
provided samples of reproducible volume. The flow pathways
of this valve were enlarged to 1/16 inch to reduce the flow
resistance through the valve. The flow pattern through the
valve (refer to Figure 2) was determined by the position of
an internal slide which was pneumatically activated and
spring returned.

In the filling position, the blood from the dog flowed
into the valve, through the collection loop, out of the
valve, and was either returned to the dog or discarded.
Meanwhile, the flow path from the washout solution was con-
nected to a return path to the dog (however, there was no
flow of washout fluid at that time). The path to the collec-
tion vials was connected to a dead end during this filling

period.

To Dog/Waste

 

 

Not Activated
(Filling Position)

 

 

 

 

 

 

 

 

 

 

 

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Figure 2.

Valve Configuration

Upon pneumatic activation by a high pressure air source
directed by a three-way valve, the internal flow pattern was
changed to the sampling position (see Figure 2). In this
position, the sample loop was washed out with solution and
collected in a sample vial. During this period, the blood
from the dog went either back to the dog or to waste, depen-
ding upon whether samples were being taken at that time or
not.

The volume of the sample loop was determined by passing a
solution of hydrochloric acid of a known concentration through
the loop, washing out the 100p with distilled water into a
sample vial, and then titrating several such samples with a
standard sodium hydroxide solution. By this method, the loop
volume was determined to be 0.2464 i 0.00079 ml (95% level,

n=25).

Electronic Controls

To obtain samples at regular time intervals, an electronic
controller was constructed. This control unit is responsible
for activation of the sampling valve from the filling to the
collecting position, for activation of the solenoid to direct
washout fluid through the sampling valve, and for advancement
of the fraction collector to position a new vial beneath the
sampling valve.

Figure 3 shows the various signals generated in the con-
trol unit. Signal A is a variable time base which is used to

set the overall time span between samples. As this signal

 

 

 

 

 

 

 

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u...“—

 

 

 

 

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- Time base

- Sampling valve activation

- Washout valve activation

- Delay

- Fraction collector activation

- Delay between valve activations

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Figure 3. Controlling Signals

10

 

 

falls off, the pulse B is generated activating the sampling
valve from the filling to the collecting position.

The fall of the time base signal A also activates a delay
relay which, after a short time to allow complete positioning
of the sampling valve, activates signal C to start the flow
of washout fluid through the sampling valve.

Upon the cessation of signals B and C, a delay signal (D)
is activated to insure that any drop of liquid remaining on
the tubing above the sample vial has a chance to drip into
that vial before a new one is moved into position.

The fall in D produces signal E causing the fraction col-
lector to advance.

The duration of each of these signals was adjustable with

the time base having a range up to 30 seconds in length.

Experimental Preparation

Mongrel dogs (16-20 kg.) were anesthetized with 18 to 20
cc of a sodium pentobarbital (Nembutal) solution (50 mg/cc)
and respirated mechanically (Harvard Apparatus dual phase
control respirator, 15 rpm, 250 cc/stroke, output ratio=45).

With the gracilis muscle exposed, all blood vessels com—
municating with the muscle except the major artery and vein
and those entering at the origin and insertion were ligated.
The gracilis artery was connected to a pump to be perfused
at constant flow (using blood from the femoral artery) and
the venous outflow directed to the sampling valve. Blood

from the normally open path of the valve was returned to the

11

femoral vein. In addition, all nerves to the muscle were cut
and the obturator nerve leading to the gracilis muscle was
attached to an electrical stimulator.

The washout solution consisted of a 0.85% saline solution
with 4 ml of 5000 USP units/ml sodium heparin solution added
per 1200 ml of saline solution. This was circulated by the
washout pump through the directional valve (see Figure 1)
and back to the supply reservoir.

0.2 ml of a 60 wt % perchloric acid solution was placed
in each sample vial. This solution, along with a 30 wt %
hydrogen peroxide solution added later, was used to bleach
and solubilize the blood to reduce interference during liquid
scintillation counting. Perchloric acid-tracer standard so-
lution (0.2 ml) was added to determine counting efficiencies
of the H3 dextran and C14 sucrose tracers used. This is dis—
cussed in more detail in the analysis of samples section
later on.

Statham pressure recording transducers (Model no. P23Gb)
were used to monitor and record onto a strip chart the arter-
ial pressure, perfusion pressure, and venous pressure of the
dog. An indexing signal (actually, the sampling valve pulse)
from the controller was also recorded to provide a record of
the time interval between samples.

In the later experiments, when both C14 sucrose and H3
dextran were injected, the blood was discarded during sam-
pling to prevent recirculated tracer from being collected.

Dextran (6% w/v) in 0.9% saline aws used to replace this

12

lost volume during the experiment.
The flow rate to the muscle was adjusted to give a per-

fusion pressure approximately equal to systemic pressure.

Experimental Procedure

Several blanks and standard samples were obtained before
introduction of the tracer. Then, the tracer was injected at
a point close to the muscle, while the time was noted on the
strip chart for future reference. At that time, the blood was
diverted to a waste container and the (nonradioactive)
dextran infusion was begun.

Between 50 and 60 samples (plus standards and blanks)
were collected for each experiment. For the "exercise" ex-
periments, electrical stimulation was used on the obturator
nerve (6Hz @ 6 volt, 1.6 msec duration) for 30 seconds imme-
diately prior to the tracer injection.

In some cases a third run was made during infusion of a
histamine solution (10 g/cc @ 1.23 cc/min). The infusion was
started approximately 5 minutes prior to the injection.

After each run, the blood was directed back to the dog to
prevent excessive loss, the dextran infusion stopped, and the

muscle allowed to recuperate from the exercise.

Analysis of Samples

The determination of the amount of tracer(s) present in
each sample was accomplished by means of liquid scintillation
counting. Detailed explanation of the general counting prin-

ciples will not be given here due to the amount of time

13

needed to cover the material adequately. There are many
excellent sources on the subject readily available (7,10,11).

To reduce the amount of quenching due to the highly
colored blood samples, a bleaching procedure was employed
as outlined in a Beckman Instruments Co. publication (11).
Briefly, this involved the addition of 60% HClOu (0.4 ml/1.0
ml blood) and 30% H202 (0.8 ml/1.0 ml blood) as bleaching
and solubilizing agents. After the samples were bleached for
3-4 hours, 3-4 drops of 15% fresh ascorbic acid solution
(per 1.0 ml blood) were added to act as oxygen scavengers
in order to alleviate chemiluminescence. Then, 10 ml of
Beckman ReadySolve HP scintillation cocktail were added and
the samples shaken. The samples were ready to count after
the solid matter was allowed to settle.

Standardized samples were used to determine the counting
efficiency for each set of samples. The standards consisted
of preparations of known concentrations of tracers mixed
separately in a perchloric acid solution (one of the bleach-
ing agents). This solution was then used in place of the nor-
mal HClOu solution for 10 samples. The counting efficiency
was the same for the standard and the regular samples since
the standard solution mixed with the unlabeled blood before
the injection in the same way that the pure H0104 solution
mixed with the labeled blood. Thus, the amount of tracer in
the labeled blood samples was calculated from the net CPM
(counts per minute) of a given sample divided by the average

net CPM per ml tracer in the standard samples. This was done

14

14 and H3

for both C14 and HB/Clu injections because the C
counts could be separated onto different channels and com-
pared to either set of standards.

All samples were counted on a Nuclear Chicago liquid
scintillation machine (Model no. 6860 Mark I) for 20 minutes
each. For that length of time per sample, the percent error
in the counting is only 4.4% for CPM as low as 100 (95% level
of confidence).

After the ml of tracer in each sample was determined, a
value E was calculated for each. This value E was defined
by:

E - £LE%—X (sec-1)

where: f(t) fraction of the total injected tracer which

appeared in the sample collected at time t.

ml tracer in sample collected at time t
ml tracer injected

or f(t)

v blood flow in ml/sec
V = volume of blood sample = sample loop volume.

This value E is the same as the E discussed in Levenspiel
(3). Levenspiel defines the fraction of the exit stream of
age between t and t+dt as Edt. If t is defined as the time
when the first bit of our sample enters the sample loop and
t+dt as the time when the sample is collected, then Edt is
equivalent to the term f(t) the fraction of the total tracer
which is in the sample. And dt, the time from when the sam-
ple first starts to enter the sample loop to when the sample

is collected (which is a small time interval), may be written

as V/v, where V is the sample loop volume and v is the

15

volumetric flow rate through the sample loop. Then

E V E : f(t) v

T: 1(t) or V

as stated before.

Edt =
CO
Levenspiel also states that Of Edt should equal 1. Table
1 shows the tracer "recoveries" for the various experiments.
were calculated from a numerical inte—

These "recoveries"

gration of the E vs. time data over the length of the ex—
periment. These values give an idea as to how much of the

tracers exited the vascular bed during the experiment.

 

Table 1. Tracer Recoveries
Figure 014 H3
4 0 0.7035395 -
5 0 0.7148130 -
6 0 0.9496238 —
6 0 0.9216082 -
7 0 0.8251231 -
7 0 0.6812981 -
8 0 0.7952803 -
8 0 0.8097591 -
9 0 0.9460958 -
9 0 0.9714919 -
10 0 0.9270879 -
10 0 0.9852182 -
11a 0.8547825 0.9855676
11b 0.8469506 0.9636616
12a 0.9098015 0.8880417
12b 0.7162707 0.6958571
12c 0.8354927 0.5238641
13a 0.9054458 0.8619089
13b 0.7465478 0.4401488
13c 0.6588887 0.4171850
14a 0.8087099 0.7340553
14b 0.8376521 0.5925857
14c 0.7493408 0.4821045

16

RESULTS

The following graphs, Figures 4 through 14c, show the
results of the experiments employing the equipment and the
methods outlined.

Figures 4 and 5 show the E vs. time curves following in-
jection of C14 sucrose and no stimulation of the muscle.
Each figure represents an experiment performed on a parti-
cular muscle specimen.

Figures 6 - 10 show the E vs. time curves following in—
jection of Gig sucrose both before and after electrical sti-
mulation of the muscle (see page 13). Each figure represents
a set ofexperiments performed on a particular muscle spe-
cimen.

Figures 11 a.b show the E vs. time curves for H3 dextran
and C14 sucrose after both were injected simultaneously.

Note that for each sample an E value was calculated for C14

3

sucrose as well as for H dextran and both plotted versus
time. Figure 11a shows the results prior to stimulation
while 11b shows the results after electrical stimulation.
Figures 12a,b,c, 13a,b,c, and 14a,b,c show the E vs. time
curves for H3 dextran and 014 sucrose after simultaneous in-
jection. The figures marked 'a' show the results before sti-
mulation; those marked °b' show the results after stimulation;
and those marked '0' show the results after histamine infu-

sion (see page 13). Each set of figures a.b,c, represents a

set of experiments performed on a particular muscle specimen.

17

0.1

 

 

 

 

 

 

 

 

 

 

— I I I I I I
I—— .—_I
: Experiment 1 j
__ U C14 sucrose control ._
"" Blood flow 5.00 ml/min -‘
-- Muscle weight 68.9 gms. —
—- ‘—I
0.01 Mb ._::
I. 0 Z
_. U .—
__ U Q: _

D
1 I-— Ch ——I
E(sec- ) ... IE .—
‘15
O
0.001 D .._.._I
: D I
: aunt: :
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D D 0

0.0001 __ ._::.
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I I I I I I I I I I

0'000010 100 200 300 400 500
Figure 4. Expt. 1 t(sec)

0.1

 

 

 

 

 

 

 

 

" I I ‘ I I I —
'" Experiment 2 :
L- U C sucrose control _
'— Blood flow 6.00 ml/min '-
-- Muscle weight 67.6 gms. -—I
..._ D _..I
D
D
0.01 hi1
: D j
I—— —-l
a
—- —4
- — D ‘
E(sec ) -— D “
c1
-— D —
D
0
0.001 ,_.. ‘32 _,
: '81 I:
__ D :
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($30,953 0 __
Do
._ ‘58: D ._
0.0001 ——- ———-
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. 001 I I I l l
O 00 0 100 200 300 400 500
Figure 5. Expt. 2 t(sec)

0.1

 

 

 

 

: I I I I I I I I I I
"" Experiment 3 Z
_. D C1 sucrose control _...
~— 0 C sucrose after -‘
r— electrical stimulation —
00 Blood flow 7.80 ml/min
5 Muscle weight 65.6 gms.
D D
O
0.01 —— <9 _.
I-— .—
Z 0 I
-— a —+
__ O .1
(sec 1.) ._o (g __
—o 032 -—
Q:
00
0.001 ._ ‘3)qu _
I— 03 ——
I- ""I
_ ._I
_. O _
-- b69900 -—
000001 '—"_ ——.
--- —-I
D
I I I I I I I I I I I
0.000010 100 200 300 400 500
Figure 6. Expt. 3 t(sec)

20

0.1

 

 

 

 

 

E I I I I I .I .I tlu I I j
4- prffiimen _q
_. 0 C14 sucrose control __
—- O C sucrose after -+
- electrical stimulation -—

Blood flow 5.53 ml/min

Muscle weight 67.34 gms.
a:
0.01 —— 5’3 _,
2 95:90 :
I—— (1% .-
__ O _.
.— 8. —
__ I9 __
a -1 c9
1:.(sec ) ,... E9 __
@O
__ Do __
00
DO
U .
0'0“ :t9 c9830 —:
_ Ci; ._
..... W .—
_. o __
0
0.0001 ——-:’
— —I
0.00001 I I I I I I I VI I I I
0 100 200 300 400 500
Figure 7. Expt. 4 t(sec)

21

0.1

 

 

 

-—1

I f I I

 

 

 

7 I

I—-— —-I
E: Experiment 5 -
14 . -4
_. 0 C1“ sucrose control .1
—- O C sucrose after -
electrical stimulation -—

Blood flow 11.45 ml/min

_4E§ Muscle weight 100.0 gms.

O
0.01 ——— .___
:0 ‘8 I
_. cp __
_ c9 —.
_ c8) _.
E(sec-1) ...D Q) .L
(m
—— § .—
0.001

—- —4
0.0001 ~—— —_—_-I»

.0 001 I I I I I I I I I I

O O 0 100 200 300 400 500

 

Figure 8. Expt.5

t(sec)

22

0.1

 

 

 

 

 

-— I I I I I I I I I I
I—
“ Experiment 6 '—
_. D C sucrose control .L
r- O 01LL sucrose after -
—- electrical stimulation -
Blood flow 6.70 ml/min
_— ~ Muscle weight 93.88 gms.
6
0. I———D ___I
01 __ 8 __
I—— % —-I
E a --I
- 0 Q) .1
-1 _- U .—
E(sec ) __ 9] _..
0.001 ——— ___.
:O l
L— -4
—- -a
a
0.0001 ———

- -I
"" "1

.0 o 1 I I I I I I I I I I

O O O O 100 200 300 400 500

 

Figure 9. Expt.6

t(sec)

23

0.1

 

 

 

 

 

: I I I I I I I I I I-
_- Ex eriment 7 _‘
I: p4 ‘I
D C 4 sucrose control ‘4
"-D O C1 sucrose after ‘7

'2? electrical stimulation
__@1 Blood flow 14.5 ml/min _q

Muscle weight 78.45 gms.

D
0.01__I5I _.
I—- --I
:: I: _L
:: fig .4
- _ 8 ‘-
E(SeC 1) —— E? .—
__ a; _,
O
O
0.001 ——— .__,
:3 Z
—— —4
0

0,0001... —:-
u. .1
__ .I

I I I I I I I I I

0.000010 100 200 300 400 500
Figure 10. Expt. 7 t(sec)

24

0.1

 

 

 

 

 

I— I I I I I I I I
I— I I I , -—
'" Experiment 8 :
._ A H3 dextran control _.
‘I
-A D 0‘4 sucrose control '—
—% Blood flow 9.6 ml/min —-
DA Muscle weight 74.80 gms.
_ D
.A
0.01 -——0A __:.
I: U I:
|—— A -
r— C] ~—
E(sec'1) e
1 £1 _.
C] A
I—— D —l
43
0.001 __ ASE. _.
: 43%: _I
I. 4, _
81%
A
0.0001 %40 —_:
b ma 3
-— —-I
_ 4583A __
m __
A _.
D D
I I I I I I I I I I I
O'OOOOlO 100 200 300 400 500
Figure 11a. Expt. 8 t(sec)

25

0.1

 

 

h— I I
I——
:0
O
_- O
O
_.0<9
O
O
0.01
E(sec-1) 1.

“E?
O
0.001 ———

I

0.0001I

 

3? I:

'o 80 -

_ 089 ’1

CL _.
‘0

:1 ‘6 °o _
00

o 00001‘D I I I I I I<><> I I I I
O 0 100 200 300 400 500

I I I
Experiment 9
O H3 dextran after

electrical stimulation

0 C14 sucrose after

electrical stimulation

Blood flow 9.6 ml/min

Muscle weight 74.80 gms.

 

I I II

I

I LIIIIIII

| I II IIII

 

 

Figure 11b. Expt. 9

0.1

 

 

 

 

 

_.. I I I I I I I I I I I
I..—
I: Experiment 10 j
3% A H3 dextran control A
I-A D C11+ sucrose control "‘
I._.CI Blood flow 16.8 ml/min -1
Muscle weight 78.98 gms.
_ —1
A0
A
0.01 ._ —'
: a ::
.._. c3 '—
_ A .—
_. U _
-1
E(sec ) _ [p .4
m
_ m _"I
0
AC!
0
A
0.001 I-——— [g ._._.,
_ % d
: 2
0.0001 ______ % ___._.
— -I
4- "I
— —I
-— '_'I
I I I I L I I I I I I
0.000010 100 200 300 “00 500
Figure 12a. Expt.10 t(sec)

27

0.1

 

 

 

 

 

 

 

 

I:- I I I I I I I I .4
"' Experiment 11 :
_ o H3 dextran after _
*8 elictrical stimulation ‘-
— O C1 sucrose after —-—
£8 electrical stimulation
Blood flow 16.8 ml/min
Muscle weight 78.98 gms.
0.01 ._8 .___.
:0 I
._ <2) __
__ (p ._
E(sec-1) _ g _
O
I-I—I % —-l
O
2.3,
O
0.... :_ .03!» _____,
: ”it _.
_. 0 .—
00
__ 06% W
O
(3%) <9 —
O
{2% °
0.0001 __ (596% ——_:.
—— —-I
— -_I
O
I I I I I I I I I
0‘000010 100 200 300 7400 500
Figure 12b. Expt. 11 t(sec)

28

0.1

 

:: I I I
_.
I;
_’
.0

0.01 _§
LI

E(sec-1)
0,001.

—

 

 

 

 

 

I I I
Experiment 12
‘0 H3 dextran after
histamine infusion

‘

 

 

 

0 014 sucrose after
histamine infusion

Blood flow 16.8 ml/min

Muscle weight 78.98 gms.

 

 

I I III

I

I I I IIIII

I

| I II LIII

I

 

 

0”
"is”

0.0001 ’ ___.

I:

—-- ——I

I I I I I I
0.000010 100 200 300 400 500
Figure 12c. Expt. 12 t(sec)

29

0.1

 

 

 

 

 

 

: I I I I I I I I I I I __
: Experiment 13 L:
—- A H3 dextran control —
"' D Cw sucrose control '—
—- Blood flow 6.25 ml/min -—
Muscle weight 65.2 gms.
" 4}
fl
0 01 —Q%
: [B ::
— 6 —-I
_ Q __
E(sec-1) _. g _
ACE:
0.001 I...— A0 _...
: ‘0 :
._ ‘31 .1
_ Q % ”WEED __
LII-*2:
_. A __
. ‘ 4%
0.0001 _
E I;
A
I I I I I I I I I I
0'000010 100 200 300 400 500
Figure 13a. Expt. 13 t(sec)

30

 

0.1

 

 

 

 

 

: I I I I I I * I I I
: Experiment 13 :
-- A H3 dextran control —-
I" D C14 sucrose control ‘—
"' Blood flow 6.25 ml/min —
Muscle weight 65.2 gms. __
’— 0
E:
O 01 9%
O — qI
: I} ::
_ g .1
- Q .4
E(sec’l) __ g __
0%
0.001 I——— AD _,
C: % Z
: ’3 W10 :
U “30:50
_ _I
_ A __
‘ 1%
0.0001 _ .....:.
A
0, 0001 I I I I I I I I I
O 0 100 200 300 400 500
Figure 13a. Expt. 13 t(SEC)

30

0.1

 

 

 

 

 

 

 

:: I I I I I I I I
" I Experiment 14 -s
_. .0 H3 dextran after ._
__ electrical stimulation -
-— O O C14 sucrose after -—

electrical stimulation
_.0 . __

95 Blood flow 6.25 ml/min
0 Muscle weight 65.2 gms.
0 . 01 I—
—O —.I
E(sec-1) _. _l
0.001 _.._I
0.0001 I—e- %) _.
_ o _
I 8&0 Z
_ <9 __
Q90

_. 69 _.
—— _—I

0.00001 I I I L, I I I I I I

0 I00 200 300 400 500
Figure 130. Expt. l4 t(sec)

0.1

 

 

 

 

 

 

-— I I I I I I f I I I
I...
7"" Experiment 15 :
I... O H dextran after _.
— histamine infusion —
— 0 C14 sucrose after --
histamine infusion
— 0 Blood flow 6.25 ml/min
~ Muscle weight 65.2 gms.
0 01 -—1. ....__.
:: o I
-— -I
._ o .—
_ .g .—
..... ’0 _
E(sec-1) _ 0‘. ——
’ °.
I-——. .0 .. —
o
’ ’o 'o
0.001 I...— 0 0.
:: t 0, z
I- O ‘ "I
_ . -
s ‘ -
__ 't \5 __
’z.
__ .. ~ ..
\ a
0
0.0001 :— ‘. _
L... ‘ ._
3 " "
’o __
F— .\
_. O ..
O
I I I I I I I I I I I
0'000010 100 200 300 400 500
Figure 13c. Expt. 15 t(sec)

32

 

0.1

 

 

 

 

 

 

I

 

 

: I I I I '--I
" Experiment 16 -
:: A.H3 dextran control .4
r— U C14 sucrose control _.
-J3 Blood flow 10.4 ml/min ——
Muscle weight 48.15 gms.
-— ——I
B
E
0.01 ——° _:
III— ——I
L— -a
B
—- -I
E(sec-1) .. 8 .1
D
— A d
O
0.001 .1:
C: ..
7’ "I
"— A90°°009o
_ m 0
2955A ac? ‘1
_. m9 @951: ..
a%%¢ [Eadbfifi
, 45
0.0001 - m. —-
Q .—
: @195 ::
._ m ..
.. 4%» ..
_. ‘&§§% ._
I I I I I I I
0.000010 100 200 300 400 500
Figure 14a. Expt. 16 t(sec)

33

0.1

E(sec-1)

0.001

0.0001

0.00001

 

 

 

 

 

 

Figure 14b. Expt.17

:: I I I I I .I I I I _J
— Experiment 17 :
—- .0 H3 dextran after ..
'43 eIectrical stimulation -
—J9 O C sucrose after CI

0 electrical stimulation
—<> Blood flow 10.4 ml/min

0 Muscle weight 48.15 gms.

o nuns—I
I: o I:
P— A
:0 Q: __
49 ..

<9
__ O _.
O
__ 0 ..
0o
00
"'__."" 0° —:
I—- 00 _I
_. o ..
_. O OO .1
h’ ch> ‘1
—o 0 00
__ O ..
0 <2) Q5
06b
O o
Io C’00
85 06;) "12'
: <> <30; _.
_. *0 d%? ..
% Q23
9' 0%69 "'I
.. 40 ..
($839
_. 00 _
‘00209
I I I I I I f0 I I I
0 100 200 300 400 500
q t(sec)

0.1

 

 

 

 

 

 

 

 

 

.. I I
I_ I I I I .1
”‘ Experiment 18 ‘t
_. ‘9 H3 dextran after .4
F- histamine infusion -
Li’ 0 C sucrose after -
histamine infusion
Blood flow 10.4 ml/min
. Muscle weight 48.15 gms.
0.01 _I
—O "—I
I— . ——I
I «- I
._ O ..
E(sec-1) ._. . ..
O
O'.
0.001 ——-- .____I
I: so ._
.. 0‘ I]
._ O. ..
._ 0'. -
_ ‘0 -
O
__ Q _.
. O
._ O ..
O il~
V.
I...
0.0001 :— "i- . -
_ $ ..
o“. '
O
I.— —I
'5’.
I I " I I
0.00001 1 J L I I
0 100 300 400 500
Figure 14c. Expt. 18 t(sec)

SUMMARY AND CONCLUSIONS

Figures 4 through 14c show the E vs. time data obtained
from the experiments. The obvious sign of parallel paths
which might be seen on such a graph would be a distinct sec-
ond peak in the data. In most of the graphs, no secondary
peak could be seen, only a random fluctuation of one or two
points at a time.

In Figure 5, however, there seems to be a distinct, well
defined secondary peak. Whether or not this is due to a par-
allel path flow pattern is uncertain as this is the only
graph which shows such a definite peak. Figure 12c shows
what may be a second peak near the end of the experiment,
but it is not as well defined as that in Figure 5.

It is suggested that many more experiments be performed
with these same techniques and studied to see if any more of
them yield a second peak in the data.

Table 1 shows the recoveries of the tracers based on a
trapezoidal numerical integration of the E vs. time curves.
Very little pattern can be seen in these values. In the dex-
tran recoveries, the control values are highest followed by
the values after electrical stimulation. The lowest values
were those obtained after histamine infusion. The only con-
sistent pattern in the sucrose recoveries is that the values
obtained after the histamine infusion were the lowest. No
consistent patterns are seen when comparing the sucrose and
dextran recoveries. The fact that, in both the dextran and

sucrose, the values obtained after the histamine infusion

36

are the lowest may be due to the effect of histamine on the
capillary membrane. Histamine causes the membrane to become
much more permeable (12); thus allowing a high rate of dif-
fusion into the extracellular space where it may not have
returned to the capillary system readily to have been de—
tected.

One possible reason why there is no clearcut pattern to
the tracer recoveries is seen when one looks at the factors
determining the accuracy of those numbers. The accuracy of
the individual E values depends on three items: the accuracy
of the scintillation counting, the reproducibility of the
volumes sampled, and a small loop volume i.e. small dt (see
page 15). These criteria were fairly well met and the re-
sulting data graphs were for the most part fairly smooth,
much more so than those obtained by previous methods (4).

However, the accuracy of the tracer recoveries was affect-
ed by not only the accuracy of the individual E values, but
also by having a sufficient number of points to insure that
the entire graph was well defined. For example, if a sample
was taken just before the concentration peaked and the next
point was taken after that peak, there would be an area which
was not counted in the recovery, one which may result in a
significant error since it is in the region having the high-
est E value. If, in another experiment, a sample was taken
right at the peak, a good representation of that area could
be calculated and added to the tracer recovery. This value

could not really be compared to the recovery calculated when

37

the peak E was missed. In addition, if any sharp secondary
peaks exist and are missed by the sampling techniques, then
additional error will follow.

If the scintillation counting sensitivity or the tracer
activity could be increased, the sample loop volume could
be decreased. This would have two results. It would lower
the value dt and give greater accuracy to the individual E
values and it would allow more samples to be taken since the
time between samples could be decreased. These two factors
combined would result in the calculation of more accurate
recoveries.

Further experiments using these same techniques are sug—
gested with possible refinements in the scintillation count-
ing using perhaps a more sensitive machine or a higher tra-
cer activity. In addition, experiments performed on vascular
beds known to have shunting pathways, such as the skin of
the canine paw (13), are highly recommended to see if any
obvious signs of channeling are seen in the resulting E vs.

time curves.

38

LIST OF REFERENCES

LIST OF REFERENCES

1. Renkin,E.M., "Transport of potassium-42 from blood to
tissue in isolated mammalian skeletal muscle," Am. J.

Physiol. 121, 1205 (1959).

2. Friedman,J.J., "Single passage extraction of Rb86 from
the circulation of skeletal muscle," Am. J. Physiol.

216(3), 1205 (1959).

3. Levenspiel,0., Chemical Reaction Engineering Wiley, New
York (1972), Chapter 9. PP.253-315.

4. Borghi,M.R., "Mathematical simulations of isotope
extractions in nutritional and non-nutritional flow
channels in vascular beds," Thesis (M.S.), Michigan
State University, Dept. of Chem. Eng., 1977.

5. Lassiter,W.E.,Gottschalk,C.W., "Volume and composition
of the body fluids," Medical Physiology 13th edition,
Mountcastle,V.B.(Ed.) Volume II, C.V.Mosby Co., St. Louis

(1974). p. 1052.

6. Starling,E.H., Principles of Human Physiology 14th
edition, Davson,H.,Eggleton,M.G.IEds.) Lea & Febiger,
Philadelphia (1968), pp.373-374.

7. Newman,F.M., "Introduction to Liquid Scintillation
Counting," Biomedical Technical Report TR-567, Beckman
Instruments Inc., Fullerton,Ca. (1973).

8. Gronwall,A., Dextran and its use in colloidal infusion
solutions, Academic Press, New York (1957).

9. Segal,A., The Clinical Use of Dextran Solutions, Grave
and Stratton, New York and London (1964), pp.5-13

10. Horrocks,D.L., "Liquid Scintillation Counting of Quenched
Samples - Application and Advantages of Automatic Quench
Compensation (AQC)," Biomedical Technical Report TR_560,
Beckman Instruments Inc., Irvine,Ca. (1973).

11. Long,E.C., "Selective Aspects of Sample Handling in
Liquid Scintillation Counting," Biomedical Technical
Report TR-600, Beckman Instruments Inc., Fullerton,Ca.

(1976)-
39

12. Milnor,W.R., "Autonomic and peripheral control mecha—
nisms," Medical Physiology 13th edition, Mountcastle,
V.B.(Ed.) Volume II, C.V.Mosby Co., St. Louis (1974),

P-956-

13. Hertzman,A.B., "Vasomotor regulation of cutaneous cir-
culation," Physiol. Rev. 39, 280 (1959).

40

IIIII IIIII III IIII III IIIIII IIII IIII III IIII II IIII III IIII IIIII

 

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