CAPILLARY PERMEABILITY TO NARROW-RANGE

MACROMOLECULAR DEXTRANS AT NORMAL

AND HYPOBARIC PRESSURES

APPROVED:

Major Professor

Minor Professor

Comnwttee Mepvper

Director of the Department of Biology

Dean (of the Graduate School

Norris, John Anthony, Capillary Permeability to

Narrow-Range Macromolecular Dextrans at Normal and Hypobaric

Pressures. Master of Science (Biology), December, 1972,

130 pp., 3 tables, 15 illustrations, bibliography, 90 titles.

In view of its varied concepts and interpretations, and

because of the discrepancies produced by the previous

utilization of polydispersed dextrans, a study using

extremely narrow-range molecular weight dextran fractions

was initiated to reevaluate and consolidate some of the

aspects of capillary permeability. A portion of the study

was performed under decreased barometric pressure in order

to clarify further some of the mechanisms involved in

particulate transfer across the capillary endothelial

membranes. Gel filtration procedures augmented the study

as an assessment of the polydispersity effects of the

dextrans employed.

Mongrel dogs were chosen as the experimental animal.

Their capillary permeability to dextrans was facilitated

by monitoring dextran concentrations in plasma, thoracic

lymph, and leg lymph. Control and simulated altitude

groups of animals were examined in four molecular weight

fractions of dextran.

The concentrations of macromolecular, narrow-range

dextran fractions increase in the thoracic lymph during

exposure to a simulated altitude of 28,000 feet (247 mm Hg).

The increases are suggested to originate from a combination

of changes in the capillary endothelial membranes, increased

central blood volume, and changes in the gel reticulum and

pressure balances of the interstitial compartment.

The lymphatic network of the popliteal fossa is found

unsatisfactory for use in studying capillary permeability

at decreased barometric pressures. A possible alternate

method to monitor leg lymph would be the lymphatics of

the inguinal fossa.

Gel filtration procedures reveal substantial poly-

dispersity differences between the acquired, narrow-range

dextran fractions and commercially prepared dextran.

However, no significant differences are evident in their

capillary permeability.

It is suggested that further investigations should be

made to determine the pooling of dextran and the maximum

molecular weight limits of dextran for capillary transport.

An increase in both the control and treatment animals

would also enhance statistical evaluation.

CAPILLARY PERMEABILITY TO NARROW-RANGE

MACROMOLECULAR DEXTRANS AT NORMAL

AND HYPOBARIC PRESSURES

THESIS

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfillment of the Requirements

For the Degree of

MASTER OF SCIENCE

By '

John Anthony Norris, B. A.

Denton, Texas

December, 1972

TABLE OF CONTENTS

Page

LIST OF TABLES iv

LIST OF ILLUSTRATIONS v

Chapter

I. INTRODUCTION . . . . 1 Capillary Permeability Altitude Studies Gel Filtration Statement of the Problem

II. METHODS AND MATERIALS . . . 37

Experimental Design Preparation of the Animals Decompression Chamber Sampling Procedure Dextran Analysis Molecular Weight Determination by

Gel Filtration

III. RESULTS . . . . . . . . . . . . 54

Capillary Permeability Physiological Parameters Molecular Weight Determination by

Gel Filtration

IV. DISCUSSION 93

Capillary Permeability Physiological Parameters Molecular Weight Determination by

Gel Filtration Summary

BIBLIOGRAPHY . . . . . . . . . . . . . . 123

111

LIST OF TABLES

Table Page

I. Physical Properties of Dextran Fractions as Determined by Granath 38

II. Intercomparison of Plasma vs. Plasma and Thoracic Lymph vs. Thoracic Lymph at Normal and Hypobaric Pressures 79

III. Column Characteristics of Dextran Fractions . . 87

IV

LIST OF ILLUSTRATIONS

Figure p a g e

1. Summation of Physical Forces Governing New Fluid Movement in the Capillaries . . . 10

2. Circulatory and Lymphatic Drainage of the Dog's Leg 42

3. Dextran Concentration Means in Plasma and Thoracic Lymph of Combined Control Groups (A, B, C, and D) 57

4. Dextran Concentration Means in Plasma and Thoracic Lymph of Combined Altitude Groups (A, B, and D) 59

5. Dextran Concentration Means in Plasma and Thoracic Lymph of Group A Ground Level Control Animals 62

6. Dextran Concentration Means in Plasma and Thoracic Lymph of Group A Altitude Animals 64

7. Dextran Concentration Means in Plasma and Thoracic Lymph of Group B Ground Level Control Animals 66

8. Dextran Concentration Means in Plasma and Thoracic Lymph of Group B Altitude Animals 68

9. Dextran Concentration Means in Plasma and Thoracic Lymph of Group C Ground Level Control Animals 71

10. Dextran Concentration Means in Plasma and Thoracic Lymph of Group D Ground Level Control Animals 74

11. Dextran Concentration Means in Plasma and Thoracic Lymph of Group D Altitude Animals 76

VI

Figure Page

12. Comparison of the Effects of Sodium Azide on the Mean Pulse Pressure 84

13. Mean Hematocrit Values of All Experimental Animal s 86

14. Comparison of Elution Patterns of Dextran Fractions on Sepharose 6B 89

15. Calibration Curve of Sepharose 6B Column . . . 91

CHAPTER I

INTRODUCTION

The capillary wall forms the barrier between the

closed mammalian circulatory system and the tissues which

it serves. This wall has the distinct characteristic of

having a selectively restrictive permeability. With this

unique characteristic the wall functions to retain the

fluid volume of the blood, yet still allow the numerous

components of the circulating plasma to pass across in

amounts necessary to the physiological function of the

system. This special permeability of the capillary barrier

and the mechanisms by which materials are transported

across the barrier have long been the subject of physio-

logical investigation. The present work is submitted as

an extension of that investigation.

Capillary Permeability

Theory

Rudiments of the study of capillary permeability and

the related lymphatic system have been reported as early as

the seventeenth century. The most significant developments,

however, were brought forth in the middle of the nineteenth

century. With the theory of a closed lymphatic system

gaining impetus, related questions concerning the mechanism

of capillary permeability and lymph formation arose. Two

schools of thought developed to answer these questions (20).

The first group, those following the Heidenhain line of

thought, claimed that lymph was formed by active secretion

on the part of the lymphatic endothelial membrane. A

second group, led by Carl Ludwig, adhered to the idea that

lymph was formed by filtration. The latter line of

reasoning gained experimental prominence, and in 1873 one

of Ludwig's students, H. P. Bowditch (7), presented a paper

describing some of this experimental evidence. In this

paper the connections between the lymphatic vessels and what

is now called interstitial spaces in connective tissue were

established. It still remained, however, for Ernest Starling

to set forth the underlying principles that regulated the

dynamic process of capillary permeability.

Prior to Starling's basic pronouncement in 1896, experi-

mentation had been primarily involved with injecting foreign

substances into the previously described interstitial space

and observing their absorption through natural diffusion

into the blood vessels (52). With this in mind, Starling (53)

set out in a series of experiments to explain the absorption

effects of fluids having the same tonicity and same approx-

imate constitution as the circulating plasma. From the

results of these experiments he recognized the distinction

between total osmotic pressure and colloid osmotic pressure

as the force regulating fluid balance across the capillary

membrane. Thus he was able to establish the simplest

description of capillary membrane selectivity: free

permeability to crystalloids and water, and relative

impermeability to colloids. With a few minor additions,

the basic thrust of the Starling hypothesis has remained

valid through present investigations.

For several years after Starling submitted his

hypothesis to the scientific community, it remained

untested. It was not until 1926, when Landis (32) intro-

duced his micro-injection technique, that anyone had the

capability to test the theory. His method permitted

measurement of mean pressure flucuations in successive

periods without disturbance of blood flow in individual

capillaries. Through the use of this technique, he was able

to study and report several physiological parameters

involving the capillary dynamics in frog mesentery (33, 34).

In 1930 Landis (35) applied his technique to mammalian

mesenteric capillaries, and his observations indicated a

balance between the average capillary blood pressure and

the osmotic pressure of the plasma proteins. He reported

that the average arteriolar capillary blood pressure was

above the colloid osmotic pressure, and the average venous

capillary pressure was below this value. These findings

provided evidence in favor of Starling's hypothesis for

fluid interchange in mammals.

At the same time that Landis was setting forth the

results of his experiments, another group of investigators

was discovering some facts that did not seem to coincide

with some of Landis's findings. Churchill, Nakazawa, and

Drinker (10) measured colloid osmotic pressure of blood

serum and lymph from subcutaneous lymph spaces in frogs.

With this procedure they were able to show physical and

chemical forces acting to produce a normal movement of

blood plasma, including a fraction of its protein, from the

blood vessels into the lymphatic system. In a more

elaborate set of experiments, Conklin (11) demonstrated

the presence of serum proteins in lymph. She also

discovered that an almost total depletion of protein from

plasma and lymph occurred with injection of large amounts

of Ringer's solution into the frog's circulatory system.

In addition to this, foreign proteins, when injected into

the circulatory system, were found to pass through the

capillaries and to be recovered in the lymph. This latter

phase of experimentation was thought to be somewhat

dependent on molecular weight.

With this evidence of serum proteins in amphibian

lymph, Drinker and Field (13) published a paper citing

references of lymph content from various regions of

different mammals. In almost all instances, the lymph

contained serum proteins, usually in concentrations above

one per cent. This led Field and Drinker (14) to develop

a series of experiments involving the quantitative detection

of horse and dog serum in lymph. By ligating various

lymphatic branches and by injecting one of the above sera

into strategic locations, they were able to show a

generalized though graded permeability to protein on the

part of blood capillaries in different parts of the dog.

This and previous findings modified but did not invalidate

Starling's hypothesis. It was determined that the

difference between the osmotic pressures of the intra- and

extravascular fluids was the important factor. This meant

that the variations between the capillary pressure and this

"effective" osmotic pressure determined filtration and

absorption by the capillary endothelial wall.

This theory was further refined in 1948 by Pappenheimer

and Soto-Rivera (41). They developed a technique for

perfusion of dog and cat hindlegs so that the protein

osmotic pressure of the plasma could be independently

adjusted to desired constant values. Included in this

perfusion method was the delicate, individual control of

capillary and venous pressures. The resulting rate of

filtration of fluid from blood to tissues or the absorption

6

of fluid from tissues to blood was determined respectively

as a gain or loss in weight of the leg. By manipulating

each of the parameters individually or in pairs, these

investigators determined that with any given protein osmotic

pressure, there were an infinite number of pairs of values

of arterial and venous pressures at which no net transfer of

fluid occurs. Two or more pairs of such values were thus

defined as the mean hydrostatic pressure in the capillaries.

Using this method the two colleagues also established that

the principal osmotic factor regulating the fluid exchange

normally involves changes in the plasma protein concentration

rather than changes in the composition of tissue fluid. As

a result of these findings, a clarification and a delicate j

method of measurement were attained for capillary pressure

and effe tive osmotic pressure described by Field and

Drinker (14).

The study of the kinetic aspects of this effective

osmotic pressure was greatly enhanced by the use of radio-

isotopic tracers. In experiments using trace amounts of 131

albumin and globulin labeled with radioactive iodine (I ),

Wasserman and Mayerson (57) were able to follow the pathway «

of these serum proteins as they traveled from the plasma to

the lymph and back to the plasma. They discovered that

both proteins passed through the capillary wall, albumin

passing approximately 1.6 times faster than globulin.

After mixing with the pericapillary interstitial fluid,

about two-thirds of these "leaked" proteins were returned

to the circulatory system via the thoracic lymph duct.

Newly metabolized proteins were not detected in the thoracic

duct lymph.

In a later study using the same techniques, Wasserman,

Joseph, and Mayerson (60) established that the extravascular

albumin mass exists as a separate entity. Net movements

from this mass occurred when the equilibrium between the

vascular and extravascular masses was disturbed. With this

last information, the importance of "effective" osmotic

pressure, its role and its maintainance in capillary

permeability, was essentially determined.

This meant that the only aspect of the Starling hypo-

thesis left to be investigated was the effect of interstitial

pressure on the net transcapillary pressure. Early attempts

at actual measurement of this pressure centered around the

needle or micropuncture technique. It involved inserting a

very small needle into a tissue and then determining the

minimum pressure required to make fluid flow from the needle

into the tissue. A good example of this technique was given

by Swann and co-workers (56) in 1950. A major criticism of

the method, however, was the fact that at best the inserted

needle was still some 300 times as large as the widths of

the tissue spaces. As a result, distortion of these spaces

and false measurements were thought to occur.

8

In order to bypass this criticism, Guyton (22)

developed a technique using perforated capsules implanted

in the tissue spaces. Two to three weeks after implantation,

his histological examination of the capsules revealed that

they were filled with interstitial fluid, tissue and blood

vessels had begun to fill the capsule, and the intra- and

extracapsular fluids communicated freely. In essence, he

had created a comparatively large interstitial space into

which he could insert a small needle and measure the

pressure without distortion of the surrounding tissues.

Using this method the workers in Guyton's laboratory

set out a series of experiments in 1963 to investigate the

importance of interstitial fluid pressure on capillary

permeability. Guyton (22) initially established that this

pressure was negative and that it only became positive in

cases of tissue edema. He also demonstrated that the

pressure within the capsule was affected by the classic

permeability mediators of arterial pressure, venous pressure,

and effective osmotic pressure. A later study (23) depicted

these effects in the form of pressure volume curves.

In a series of experiments that climaxed their studies

in 1966, Guyton and several of his co-workers (24) used the

implanted capsules to compare the effects of changes in

interstitial fluid pressure with the effects of changes in

venous and arterial pressures. Used in this manner the

implanted capsules were described as internal plethysmo-

graphs, and the net movement of fluid through the capillary

membranes Wc(S measured. As a result of this modification,

they were able to demonstrate that a decrease in inter-

stitial fluid pressure of 1 mm Hg increased the filtration

out of the capillaries 1.20 times as much as did a 1 mm Hg

increase in venous pressure. In addition, a filtration

coefficient for fluid movement through the capillary wall

per unit change in interstitial fluid pressure was

calculated, and this value was compatible with the coeffic-

ient calculated by Pappenheimer and Soto-Rivera (41)

following changes that they induced in capillary and

osmotic pressures.

The primary significance of the experimentation cited

to this point was that it unquestionably confirmed the

principles of Starling's basic hypothesis. The net transfer

of fluids through capillary endothelial membranes was

governed by a delicate balance and interplay of two factors:

net transcapillary pressure, which was determined by

arterial pressure, venous pressure, and interstitial fluid

pressure; and (2) effective colloid osmotic pressure, which

was determined by plasma colloid osmotic pressure and

interstitial fluid colloid osmotic pressure. A pictorial

summation of these factors can be seen in Figure 1.

10

BLOOD FLOW

ARTERY

C.R 25

RC.O.P. 28

VEIN

C.R » RC.O.R * 28

- 7 I.F.P.

4.5 I.C.O.P.

- 7

I.F.P. 4.5

I.C.O.P.

Net Outward Force

8.5 mm Hg Net Inward Force =

7.5 mm Hg

Fig. 1. Summation of physical forces governing net fluid movement in capillaries. Heavy arrows indicate main force affecting fluid movement and direction of fluid movement; C.P. « Capillary Pressure; I.C.O.P. = Interstitial Colloid Osmotic Pressure; I.F.P. = Interstitial Fluid Pressure; P.C.O.P. = Plasma Colloid Osmotic Pressure.

Mechanism

Although the basic principles for capillary permeability

were established, the exact mechanisms and criteria for the

endothelial membrane selectivity have been and still are

a highly debatable subject. Numerous theories have been pro-

11

posed as an explanation, but as yet none have emerged as

a completely acceptable answer.

The process of diffusion has been regarded by most as

an integral part of the transfer mechanism, especially that

of low molecular weight molecules. As early as 1926, Arnold

and Mendel (3) studied the composition of total solids,

chlorides, calcium, phosphorus, sugar, non-proteins, and

proteins in serum and lymph of dogs. Applying some abnormal

physiological concentrations, they concluded that inter-

changes occurred in response to alterations in concentration

gradients. They also observed that diffusable substances

passed easily and rapidly at all times between the blood,

lymph and tissues. Renkin (48) extended this observation

to include carbon dioxide and lipid-soluble molecules.

Several other investigators have independently substantiated

these findings.

In a comprehensive review of the capillaries presented

in 1947, Chambers and Zweifach (9) cited some endothelial

properties which were proposed to account for the diffusion

observations. Using accepted histological staining tech-

niques and the light microscope, they distinguished three

types of cellular membranes on the basis of structural

factors regulating the passage of materials. The first two

types involved active secretory work by the endothelial cell

constituents. This involved either total transport by

secretion, as in the endothelial cells of renal proximal

12

tubules; or partial cell secretory action aided by passive

transport through intercellular material, as in intestinal

mucosa. The third type that they identified limited

permeability and transport to the intercellular cement

substance. In this instance the role of the cells was

relegated to being building blocks and maintaining the

intercellular cement. Thus the relatively undiscriminating

nature of permeability in the blood capillary could be

explained by this type of endothelium.

This apparent indiscrimination to some molecules on the

part of the capillary led Pappenheimer, Renkin, and Borrero

(42) to a direct, experimental comparison of them with

artificial, porous membranes. The similarities were so

evident that they proposed a system of endothelial pores to

explain the capillary diffusion. In a 1953 review,

Pappenheimer (43) attempted to clarify and summate the

existing evidence for capillary endothelial pores. He

proposed that uniform, cylindrical pores of radius 30-45 A

and a population density of 1-2 x 10^ per cm^ of capillary

wall would account for the observed rates of passage of

water and lipid insoluble molecules of various sizes.

Using these "effective" dimensions, a total cross-sectional

area of the pores would comprise less than 0.2 per cent of

the histological surface of the capillaries. This fact

indicated that the pores were limited to areas between endo-

thelial cells as proposed by Chambers and Zweifach (9).

13

Pappenheimer made the additional distinction that most or

all of the capillary endothelial surface was available for

the passage of oxygen, carbon dioxide, and other lipid

soluble molecules. These substances were not dependent on

pores for transcapillary exchanges.

The transport of larger molecules proved to be a weak

point in Pappenheimer's (43) reasoning. Previous investi-

gators had reported decreased diffusion rates for the serum

proteins and other large molecules. To explain these

decreases, Pappenheimer developed the concept of restricted

diffusion or molecular sieving. According to this theory

the effective pore size in the capillary wall was

sufficiently great to allow even the plasma protein molecules

to penetrate the pores. The degree of molecular sieving

of any given solute depended upon the ratio of its

restricted diffusion coefficient to the filtration rate

through the capillary wall. Thus the degree of transport

for large molecules was dependent upon their restricted

filtration rates through the pores. This explanation left

many investigators in doubt, and as a result, experimentation

and hypotheses became abundant in this area.

One group of investigators headed by H. S. Mayerson

initiated some experimentation in 1955 that dominated the

thought in the area of-capillary permeability for the next

few years. Prior to this time Wasserman and Mayerson (58)

had studied the effects of the polysaccharide dextran on

14

plasma expansion and transfusion. Some of their results

suggested that a biological fractionation occurred when

the dextran was infused as a heterogenous mixture of

molecular sizes;. This led them into a concentrated study

of dextran and capillary permeability.

Wasserman, Loeb, and Mayerson (59) obtained eight

"well-characterized" fractions of dextran and infused

then as mixtures and as discrete fractions. Their results

showed that all sizes of dextran fractions, including that

with an average molecular weight of 412,000, penetrated the

capillaries and entered the thoracic duct lymph. The

possibility of large and small pores with unequal distri-

bution in capillary beds was suggested to account for this

observation. These studies also suggested the possibility

that pore sizes stretched with increased blood volume. This

possibility was later substantiated by Shirley and

co-workers (50), and the concept of capillary permeability

was modified to include a labile capillary pore size,

subject to change with variations in plasma volume as well

as other factors.

Wasserman and Mayerson's (59) explanation that pores

might exist in different sizes and in different proportions

in various capillary beds, led Grotte (20) into an attempt

to characterize capillary membranes in the dog in terms of

their permeability to dextran molecules. This character-

ization was accomplished by studying dextran in steady state

15

lymph/plasma ratios and by monitoring the rate of disap-

pearance of dextran from the plasma. Using these proce-

dures, he studied capillary permeability in three regions:

the leg, the cervical region, and the liver.

In the leg region, Grotte calculated an approximate

pore radius of 35-45 A. The studies further revealed that

larger molecules passed from plasma to lymph in bulk flow,

indicating the presence of capillary leaks of at least 120 A

radius. Results for the cervical and liver capillaries were

similar except that the number of "leaks" greatly increased

with each region. In a subsequent study, Grotte, Juhlin,

and Sandberg (21) used solid spherical particles of methyl-

methacrylate (radius 300-700 A ) to further characterize the

capillary "leaks". The radii of the "leaks" were thought to

be in the range of 120-300 A in the leg, heart, and bron-

chial capillaries and above 700 A in the liver.

At about this point in time, the morphologists and

electron microscope began to have some bearing on the

physiologists viewpoint of capillary permeability. Palade

(40) had first reported the presence of vesicles in the

endothelial cells of capillaries in 1953. He proposed then

that they were the mechanism for capillary transport. His

reports were largely ignored, however, because physiologists

regarded his vesicles as artifacts in tissue preparation

for the electron microscope. This remained the circumstance

16

even with increasing reports of vesicular presence in

capillary endothelium.

In 1959, Bennet, Luft, and Hampton (6) presented the

morphological features of blood capillaries from various

vertebrate forms of organs. They attempted a simple classi-

fication using structural features as criteria. The most

significant part of their work, however, was the fact that

they could find no perforations or pores as described by

Pappenheimer (43) in capillary endothelial cells. They

termed this failure very significant and suggested that a

reconsideration and reinvestigation of the concepts of

capillary physiology was in order.

This reevaluation was begun by Mayerson and his

colleagues (39). They used the same procedures as in a

previous study (59), but the dextran fractions were moni-

tored from three regions (cervical, intestinal, and hepatic)

rather than from one as before. The data they obtained were

somewhat similar to Grotte's (20) regional study. It indi-

cated the presence of two discrete sets of "pores": (1) a

set of "small pores" allowing passage of molecules not

greater than 250,000 MW; (2) a set of "large pores" permit-

ting passage of molecules of at least 412,000 MW. In view

of the lack of favorable electron microscopic evidence,

however, Mayerson and his co-workers proposed an alternate

theory of transport for the molecules greater than

250,000 MW. The proposal was essentially vesicular

17

transport, but they called it "cytopempsisThis descrip-

tion was employed to convey the idea that substances were

being transported through the cytoplasm rather than being

engulfed for cell use as in "pinocytosis

As the physiologists began to reconcile their position,

the morphologists also had to change their views. With

improved techniques in electron microscopy, Luft (37)

reported some of the same observations as Chambers and

Zweifach (9) had seen with a light microscope.. He used

ruthenium red with the electron microscope and confirmed

for the first time the presence of the "endocapillary layer"

and its continuation between the cells as the "intercellular

cement". Luft further observed that the tight junction of

the intercellular cement at the region of endothelial

contact could still remain porous. In.this case, Starling's

hypothesis had a plausible foundation in structure without

the need to invoke vesicular transport of fluid across the

capillary wall. This fact did not account for the transport

of large molecules, however.

A survey of the current literature does not resolve

this question any more satisfactorily than earlier reports.

Numerous vesicular transport models based on electron micro-

scope studies are available. Shea and Karnovsky (49) seem

to have the most prominent model. It employs the use of

horseradish peroxidase, and it is based on Brownian movement

of vesicles through the endothelial cells. The work of

18

Casley-Smith (8) in measuring, numbering, and calculating

the rate of transport of these vesicles supports the model

fairly well.

Garlick and Renkin (15) on the other hand, have

attacked the question from an experimental aspect. Using

classical infusion and monitoring techniques of dextran,

they study the permeability characteristics of capillaries

in the dog's paw. With the addition of mathematical cal-

culations to their experimentation, they favor a system of

small pores of 40 X radius for transport of small molecules,

In order to carry the larger molecules, Garlick and Renkin

propose either a few large pores of approximately 800 X

radius or pinocytotic vesicles, of approximately 250 A

radius. It is obvious that much knowledge has been gained

since Starling set forth his hypothesis in 1896, but it is

also evident from the lack of decisive agreement of these

and other investigators that much more knowledge still

remains to be attained.

Altitude Studies

The effects of high altitude were probably first

noticed by the early explorers as they scaled the mountains

in search of various goals. Indeed, their reports of some

of these effects stimulated some of the initial scientific

expeditions. Douglas and his associates (12) reported one

of these classical scientific adventures in 1913. They

19

traveled to the top of Pike's Peak and described how hypoxia

or oxygen deficiency progressively stimulated their venti-

lation. As a result, their alveolar CC>2 tension fell as

they ascended to the summit. Such reports relating to

hypoxia and its associated effects have dominated the

literature in the area of altitude physiology.

Alberto Hurtado (29), who with his research group in

Lima, Peru, has been the most active investigator in this

area, related the rationale behind the predominance of

hypoxia-related reports. At reduced barometric pressure,

he explained that the partial pressure of oxygen in the

inspired air was low. As a result, the hemoglobin of the

blood circulating through the lungs became less saturated

with this gas. This fact, together with the decreased

tension of the fraction physically dissolved in the plasma,

made its diffusion and utilization at a tissue level more

difficult. A variety of coordinated mechanisms compen-

sated in an attempt to relieve the stress placed on the

tissues.

Reports of these compensatory mechanisms, both long

and short term, have covered a myriad of physiological

phenomena. Shifts in oxygen-hemoglobin dissociation curves,

changes in pH of body fluids, increases in hemopoeitic

activity, and various aspects of pulmonary edema are a few

of the areas in which altitude research has flourished. The

primary concern of the present study, however, involves

20

cardiovascular responses to short-term, simulated altitude

with an attempt to eliminate the effects of hypoxia.

To eliminate the effects of hypoxia, the most natural

answer was to administer a supply of oxygen to the experi-

mental subject during the test period. Whitehorn, Edelman,

and Hitchcock (62) examined this solution in 1946. Allowing

their subjects to breathe 100 per cent oxygen at normal

barometric pressure, they determined resulting cardiac out-

puts from ballistocardiograms. Their results indicated a

maintainance of the blood pressure level in spite of reduced

cardiac output. This was interpreted as evidence of an

increase in the general peripheral vascular resistance. The

investigators suggested that the effects be remembered in

conditions of hyperoxygenation of the blood, such as denitro-

genation, and that their presence may play a part in the

physiological responses to changes in barometric pressure.

A direct application of these suggestions was presented

by Girling and Maheux (16) in 1952. They studied the

peripheral circulation of rabbits at 10,000, 20,000, and

30,000 feet simulated altitude. Animals taken above this

level demonstrated fatal signs of anoxia, but animals

supplied with oxygen could safely withstand the additional

decreased pressure. The peripheral resistance exhibited

the same response with or without the supplemental oxygen;

small change in resistance to 20,000 feet, but an appreci-

able increase in resistance from ground to 30,000 feet. In

21

order to eliminate the possibility of nitrogen bubbles

forming in the blood and increasing peripheral resistance,

a subsequent study (17) by the same investigators employed

preoxygenation periods of up to four hours. The results

remained the same, however, and they concluded that the

observed vasomotor response was due to reduced barometric

pressure causing decreased critical closing pressures of

peripheral blood vessels.

In an attempt to observe the effects of simulated

altitudes of 60;,000 and 80,000 feet, Sullivan and De Gennaro

(54) studied the peripheral circulation in the web of a

frog's foot. Their observations suggested that the extent

of decrease in blood flow was dependent on the altitude,

the length of time the animal was subjected to this altitude,

and whether or not air was supplied to the animal during

decompression. These results were thought to be primarily

due to the hypoxia accompanying decompression, but the

investigators also suggested that the initial, reduced

venous flow was due to stasis following increased capillary

endothelial permeability under the influence of hypoxia.

At any rate, the effects could be alleviated by reducing

hypoxia.

In 1966 Marotta and Boon (38) extended the studies of

oxygenation at simulated altitudes to dogs. They monitored

femoral arterial blood flow and related cardiopulmonary

parameters during exposure to altitude (9,000, 17,000, and

22

25,000 feet simulated altitude) while breathing 100 per cent

oxygen or a mixture of oxygen and nitrogen. One series of

experimental animals were subjected to per-altitude denitro-

genation with 100 per cent oxygen at ground level. A decrease

in femoral arterial blood flow was observed in all animals

except those denitrogenated prior to ascent. Initial and

final blood flows in the latter animals, however, were all

less than in the other animals. To this extent, denitro-

genation exhibited some slowing effect in all of the animals

examined. It thus appeared that increased peripheral

resistance to blood flow was an unavoidable legacy in the

attempt to overcome the more severe effects of hypoxia.

Experimentation has thus proceeded with and without the aid

of supplemental oxygen depending on the nature of the

experiment involved.

Another cardiovascular response that interested

investigators in recent years has been the fluid shifts and

the blood component imbalances that developed with the

encounter of low barometric pressure. A general plasma

volume decrease had long been accepted as a part of the

altitude acclimatization process, but it had been thought

this loss was due to body dehydration. In 1969 Hannon,

Chinn, and Shields (25) used a new, more reliable method

for determining total body water. As a result they reported

a decreased plasma volume with a static or slightly raised

total body water. Further investigation (26) of the extra-

23

vascular compartments showed decreases in interstitial and

extracellular spaces and an increase in intracellular space.

This was thought to be caused by osmotic effects associated

with a transfer of electrolytes, principally, sodium,

chloride, and bicarbonate. Krzywicki and associates (31)

recently reported, however, that their experimentation

indicated that the loss in body weight was still due to loss

in total body water. They maintained that this hypo-

hydration was an adaptive mechanism to altitude. At this

point in experimentation, the purpose and destination of

fluid shifts in subjects maintained at altitude remains

unresolved.

Another baffling question of recent years has been the

shift in components of the blood with the advent of altitude,

Surks (55) reported an initial albumin shift from intra-

vascular to extravascular compartments with an increased

degredation of albumin during initial stages of altitude.

After six days at altitude, however, the shift was reversed

from the extravascular to the intravascular compartments.

This latter shift was accompanied by a decrease in the rate

of albumin synthesis and net transfer. Westergaard and his

associates (61) also observed the initial shift in albumin

from intravascular to extravascular compartments. They

suggested that this might be due to increased capillary

permeability, but a direct measurement was not performed.

Independent attempts were recently made by Parker (44) and

24

Reaves (47) to directly measure the capillary permeability

of dogs at altitude. Their results both indicated an

increase in capillary permeability possibly due to stretched

pores. As in the case of fluid shifts, however, the exact

purpose and mechanism of blood component shifts at high

altitude levels still remains to be determined.

Molecular Weight Determination by Gel Filtration

The class of polysaccharides called dextrans consists

of a great variety of a-polyglucosans produced by the coccus

Leuconostoc mesenteroides and closely related bacteria under

suitable environmental conditions. In their native state,

the dextran chains are composed of about 200,000 glucose

units, corresponding to a molecular weight of about 40 mil-

lion. Partial hydrolysis of these chains yields smaller

molecular weight molecules that exhibit desirable character-

istics for experimental use. They appear to be electrically

neutral, metabolically inert (no mammalian tissue extract is

known to break them down), apparently non-toxic in high

concentration, and can be assayed accurately and easily (51).

Their one drawback in most experimental endeavors is that

they are very difficult to obtain in narrow-range molecular

weight fractions. Thus, experimentors employing their use

must in some way lessen the effects of the molecular weight

dispersity, or they must acknowledge the limitations placed

25

on the validity of their results by the use of the wide

range of molecular weight molecules.

One of the methods by which the molecular weight range

of dextrans can be narrowed, or at least estimated, is gel

filtration. In this process, a mixture of molecules is

passed through a column of porous gel granules. The

molecules appear in the effluent in order of decreasing

size. Fractionation is believed to occur when diffusion of

the molecules into the pores is restricted but not prevented

because of their size. As a result of this restriction, the

molecules pass through the column at rates that are related

inversely to the fluid volume accessible to them within the

column. This molecular sieving is the basis by which

different and somewhat narrow-range dextran fractions can

be obtained. An estimation of their molecular weight may

aldo be determined as a result of this basic principle.

An early-application of gel filtration to large

molecules was attempted by Lathe and Ruthven (36) in 1956.

Using columns of starch grains, they were able to achieve

some separation of polypeptides and proteins. They proposed

that their method be used as a means of molecular weight

determination, and they reported some interesting results

in this connection. Their starch columns, however, did not

effect enough separation between proteins for further

development of the idea with this particular gel.

2 6

Numerous other types of porous gels were introduced,

however, and the idea of molecular weight determination was

applied to them. One of the more successful gels was a

cross-linked dextran gel introduced by Porath (46) in 1960.

He reported that highly cross-linked dextran gels separated

amino acids from proteins and large peptides. Gels

possessing a low degree of cross-linking even retained

proteins for fractionation. This dextran gel was later used

by Granath and Flodin (18) to fractionate low molecular

weight dextrans. These two investigators were able to

establish the limits of the molecular weight sizes of the

gels involved. With further use and development a series of

cross-linked dextran gels was marketed under the trade name

of Sephadex.

By using G-75 and G-100 of the Sephadex series, Andrews

(l) was able to' correlate the elution volumes, Ve, and

molecular, weights of proteins of known molecular weight.

Within the limits of certain ranges, the gels gave optimum

separation of proteins according to molecular weight, but

their useful working ranges extended only to about 70,000

and 150,000 respectively. The complete exclusion limits

for proteins was estimated somewhat higher, however. As a

final test, Andrews used his columns to estimate the molec-

ular weights of various enzymes, and he found that these

values were in agreement with those reported in the litera-

ture. He had essentially calibrated Sephadex columns within

27

prescribed limits for the determination of molecular weights

of proteins.

In 1967 Granath and Kvist (19) were able to apply the

same basic procedures to calibration of dextrans. This was

more difficult considering the polydispersity characteristics

of dextrans, and certain physical and mathematical modifi-

cations had to jbe employed. The results were comparable to

those obtained for proteins. Thus, the calibration of

porous gel columns for molecular weight determination of

proteins and dextrans had been accomplished. They were only

useful, however, for molecular weights up to approximately

800,000 for compact, globular molecules like proteins, or

approximately 200,000 for randomly coiled chain molecules

like dextran (30). The fractionation and molecular weight

determination of larger molecules and particles required a

gel filtration medium of much higher porosity.

In 1961, Poison (45) had described a method in which

granulated agar was used for separating protein compounds

of different molecular weights. The method demonstrated

that molecules ranging in molecular weights from 13,000 to

several millions could be distinguished. Hence, Poison

proposed that his gel be used in conjunction with the linked

dextran gels for a powerful fractionation of substances of

very low to very high molecular weights. The use of granu-

lated agar, however, was severely limited due to variable

gelling and adsorption properties encountered. These

28

variations were due to the poorly defined composition of

commercially available agar which varied from batch to

batch.

Araki (2) reported that the unsatisfactory character-

istics of granulated agar as a medium for gel filtration

was due to the fact that it consisted of two different

polysaccharides, agaropectin and agarose. It was determined

that agaropectin was a typical pectic substance containing

a high percentage of sulfate and carboxyl groups. These

constituents seemed to impart the undesirable adsorption

effects seen in gel filtration. Agarose, on the other hand,

was classified as an unchanged polysaccharide that consisted

of alternating D-galactose and 3,6-anhydro-L-galactose units,

It had the same good gelling properties as agar and was thus

a superior medium for gel filtration.

Even with this discovery, the use of agarose in gel

filtration was not widespread until Hjerten (27) developed

a simple technique to remove the agaropectin from agar

solutions by precipitation with cetylpyridinium chloride. %

A further development by Hjerten (28) and Bengtsson and

Philipson (5) established it as a bonafide gel filtration

medium. These investigators independently developed

procedures for bead-gelling of agarose solutions. Photo-

micrographs of these beads showed a granular structure, but

the nature of the granules was not readily explainable (30).

The beaded agarose gels have been marketed in series,

29

similar to the Sephadex series, under the trade name of

Sepharose. Their fractionation characteristics are similar

to those of Sephadex, but their molecular weight exclusion

limits extend into the millions.

In a recent article, Arturson and Granath (4) used

Sepharose 4B as the gel medium for molecular weight cali-

bration of narrow-range, high molecular weight dextrans.

Their method was similar to that used by Granath and Kvist

(19), who used Sephadex as the gel medium. The procedure

thus yielded the first gel filtration columns capable of

fractionating and detecting extremely high molecular weight

dextrans. Its use was demonstrated in the study of biolog-

ical membranes, and it was evident that its future use in

this area could aid in the explanations of capillary

dynamics and permeability.

Statement of the Problem

In view of its varied concepts and interpretations,

and because of the discrepancies produced by the previous

utilization of polydispersed dextrans, a study using

extremely narrow-range molecular weight dextran fractions

was initiated to reevaluate and consolidate some of the

aspects of capillary permeability. A portion of the study

was performed under decreased barometric pressure in order

to clarify further some of the mechanisms involved in

particulate transfer across the capillary endothelial

30

membranes. Gel filtration procedures augmented the study

as an assessment of the polydispersity effects of the

dextrans employed.

CHAPTER BIBLIOGRAPHY

1. Andrews, P, 1964. Estimation of the molecular weights of proteins by sephadex gel-filtration. Biochem. J. 91: 222-233.

2. Araki, C. 1956. Structure of the agarose constituent of agar-agar. Bull. Chem. Soc. Japan. 29: 543-544.

3. Arnold, R. M. and L. B. Mendel. 1926. Interrelation-ships between the chemical composition of the blood and the lymph of the dog. J. Biol. Chern. 72: 189-211.

4. Arturson, G. and K. Granath. 1972. Dextrans as test molecules in studies of the functional ultra-structure of biological membranes. Clin. Chim. Acta. 37: 309-322.

5. Bengtsson, S. and L. Philipson. 1964, Chromatography of animal viruses on pearl-condensed agar. Biochim. Biophys. Acta. 79: 399-406.

6. Bennett, H. S., J. H. Luft? and J. C. Hampton. 1959. Morphological classifications of vertebrate blood capillaries. Am. J. Physiol. 196: 381-390.

7. Bowditch, H. P. 1873. The lymph spaces in fasciae. Proc. Am. Acad. Sci. 8: 508-510.

8. Casley-Smith, J. R. 1969. The dimensions and numbers of small vesicles in cells, endothelial and meso-thelial and the significance of these for endo-thelial permeability. J. Microscopy 90: 251-268.

9. Chambers, R. and B. W. Zweifach. 1947. Intercellular cement and capillary permeability. Physiol. Rev. 27: 436-463.

10. Churchill, E. D., F. Nakazawa, and C. K. Drinker. 1927. The circulation of body fluids in the frog, J. Physiol. (London). 63: 304-308.

11. Conklin, R. E. 1930. The formation and circulation of lymph in the frog. III. The permeability of the capillaries to protein. Am, J. Physiol. 95: 98-110,

31

32

12. Douglas, C. G., J. S. Haldane, Y. Henderson, and E. C. Schneider. 1913. VI. Physiological obser-vations made on Pike's Peak, Colorado, with special reference to adaptation to low barometric pressure. Phil. Trans. Hoy. Soc. B 203: 185-318.

13. Drinker, C. K. and M. E. Field. 1931. The protein content of mammalian lymph and the relation of lymph to tissue fluid. Am. J. Physiol. 97: 32-39.

14. Field, M. E. and C. K. Drinker. 1931. The permeability of the capillaries of the dog to protein. Am. J. Physiol. 97: 40-51.

15. Garlick, D. G. and E. M. Renkin. 1970. Transport of large molecules from plasma to interstitial fluid and lymph in dogs. Am. J. Physiol. 219: 1595-1605.

16. Girling, F. and C. Maheux. 1952. Peripheral circu-lation and simulated altitude. J. Aviation Med. 23: 216-217, 270.

17. Girling, F. and C. Maheux. 1953. Peripheral circu-lation and simulated altitude. Part II. J. Aviation Med. 24: 446-448.

18. Granath, K. A. and P. Flodin. 1961. Fractionation of dextran by the gel filtration method. Makromol. Chem. 48: 160-171.

19. Granath, K. A. and B. E. Kvist. 1967. Molecular weight distribution analysis by gel chromatography on sephadex. J. Chromatogr. 28: 69-81.

20. Grotte, G. 1956. Passage of dextran molecules across the blood-lymph barrier. Acta Chir. Scand. Suppl. 211: 1-84.

21. Grotte, G., L. Juhlin, and N. Sandber. I960. Passage of solid spherical particles across the blood-lymph barrier. Acta Physiol. Scand. 50: 287-293.

22. Guyton, A. C. 1963. A concept of negative interstitual pressure based on pressures in implanted perforated capsules. Circulation Res. 12: 399-414.

23. Guyton, A. C. 1965. Interstitial fluid pressure. II. Pressure-volume curves of interstitial space. Circulation Res. 16: 452-460.

33

24. Guyton, A. C., J. Prather, K. Scheel, and J. McGehee. 1966. Interstitial fluid pressure. IV. Its effect on fluid movement through the capillary wall. Circulation Res. 19: 1022-1030.

25. Hannon, J. R., K. S. K. Chinn, and J. L. Shields. 1969. Effects of acute high-altitude exposure on body fluids. Fed. Proc. 28: 1178-1184.

26. Hannon, J. P., K. S. K. Chinn, and J. L. Shields. 1971. Alterations in serum and extracellular electrolytes during high-altitude exposure. J. Appl. Physiol. 31: 266-273.

27. Hjerten, S. 1962. A new method for preparation of agarose for gel electrophoresis. Biochim. Biophys. Acta. 62: 445-449.

28. Hjerten, S. 1964. The preparation of agarose spheres for chromatography of molecules and particles. Biochim. Biophys. Acta. 79: 393-398.

29. Hurtado, A. 1971. The influence of high altitude on physiology, p. 3-13. In Porter and Knight, High Altitude Physiology: Uardiac and respiratory aspects. Churchill Livingstone, Edinburgh and London.

30. Joustra, M. K. 1969. Gel filtration on agarose gels, p. 183-198. In J. Gerritsen, Modern separation methods of macromolecules and particles. Wiley-Inter science, New York.

31. Krzywicki, H. J., C. F. Consolazio, H. L. Johnson, W. C. Nielsen, Jr., and R. A. Barnhart. 1971. Water metabolism in humans during acute high-altitude exposure (4,300 m). J. Appl. Physiol. 30: 806-809.

32. Landis, E. M. 1926. The capillary pressure in frog mesentery as determined by micro-injection methods. Am. J. Physiol. 75: 548-570.

33. Landis, E. M. 1927. Micro-injection studies of capillary permeability. II. The relation between capillary pressure and the rate at which fluid passes through the walls of single capillaries. Am. J. Physiol. 82: 217-238.

34

34. Landis, E. M. 1928. Micro-injection studies of capillary permeability. III. The effect of lack of oxygen on the permeability of the capillary wall to fluid and to the plasma proteins. Am. J. Physiol. 83: 528-542.

35. Landis, E. M. 1930. The capillary blood pressure in mammalian mesentery as determined by the micro-injection method. Am. J. Physiol. 93: 353-362.

36. Lathe, G. H. and C. R. J. Ruthven. 1956. The separation of substances and estimation of their relative molecular sizes by the use of columns of starch in water. Biochem. J. 62: 665-674.

37. Luft, J. H. 1966. Fine structure of capillary and endocapillary layer as revealed by ruthenium red. Fed. Proc. 25: 1773-1783.

38. Marotta, S. F. and D. J. Boon. 1966. Femoral arterial circulation in nonhypoxic dogs at reduced baro-metric pressures. Am. J. Physiol. 210: 953-956.

39. Mayerson, H. S., C. G. Wolfrom, H. H. Shirley, Jr., and K. Wasserman. 1960. Regional differences in capillary permeability. Am. J. Physiol. 198: 155-160.

40. Palade, G. E. 1953. Fine structure of blood capillar-ies. J. Appl. Phys. 24: 1424.

41. Pappenheimer, J. R. and A. Soto-Rivera. 1948. Effective osmotic pressure of the plasma proteins and other quantities associated with the capillary circulation in the hindlimbs of cats and dogs. Am. J. Physiol. 152: 471-491.

42. Pappenheimer, J. R., E. M. Renkin, and L. M. Barrero. 1951. Filtration, diffusion, and molecular sieving through peripheral capillary membranes. A contribution to the pore theory of capillary permeability. Am. J. Physiol. 167: 13-46.

43. Pappenheimer, J. R. 1953. Passage of molecules through capillary walls. Physiol. Rev. 33: 387-423.

44. Parker, P. E. 1969. Capillary permeability to macro-molecules at normal and hypobaric pressure. M. S. Thesis, North Texas State Univ., Denton, Texas. 45 p.

35

45. Poison, A. 1961. Fractionation of protein mixtures on columns of granulated agar. Biochim. Biophys. Acta. 50: 565-567.

46. Porath, J. 1960. Gel filtration of proteins, peptides, and amino acids. Biochim. Biophys. Acta. 39: 193-207.

47. Reaves, T. A., Jr. 1971. Effects of high altitude exposure on capillary permeability. M. S. Thesis, North Texas State Univ., Denton, Texas. 62 p.

48. Renkin, E. M. 1952. Capillary permeability to lipid-soluble molecules. Am. J. Physiol. 168: 538-545.

49. Shea, S., M. and M. J. Karnovsky. 1969. Vesicular transport across endothelium: simulation of a diffusion model. J. Theoret. Biol. 24: 30-42.

50. Shirley, H. H., Jr.., C. G. Wolfram, K. Wasserman, and H. S. Mayerson. 1957. Capillary permeability to macro-molecules: stretched pore phenomenon. Am. J. Physiol. 190: 189-193.

51. Squire, J. R. 1951. Background to biological studies with dextran. Proc. Roy. Soc. Med. 44: 557-558. (Abstr.)

52. Starling, E. H. and A. H. Tubby, 1894. On absorption from and secretion into the serous cavities. J. Physiol. (London). 16: 140-155.

53. Starling, E. H. 1896. On the absorption of fluids from the connective tissue spaces. J. Physiol, (london). 19: 312-326.

54. Sullivan, B. J. and L. D. DeGennaro. 1953. Micro-scopical observations of peripheral circulation at simulated high altitudes. J. Aviation Med. 24: 131-137.

55. Surks, M. I. 1966. Metabolism of human serum albumin in man during acute exposure to high altitude (14,100 feet). J. Clin. Invest. 45: 1442-1451.

56. Swann, H. G.j A. V. Montgomery, J. C. Davis, Jr., and E. R. Mickle. 1950. A method for rapid measure-ment of intrarenal and other tissue pressures. J. Exp. Med. 92: 625-636.

36

57. Wassexman, K. and H. S. Mayerson. 1952. Dynamics of lymph and plasma protein exchange. Cardiologica. 21: 296-307.

58. Wasserman, K. and H. S. Mayerson.* 1954. Relative importance of dextran molecular size in plasma volume expansion. Am. J. Physiol. 176: 104-112.

59. Wasserman, K., L. Loeb, and H. S. Mayerson. 1955. Capillary permeability to macromolecules. Circulation Res. 3: 594-603.

60. Wasserman, K., J. D. Joseph, and H. S. Mayerson. 1956. Kinetics of vascular and extravascular protein exchange in unbled and bled dogs. Am, J. Physiol. 184: 175-182.

61. Westergaard, H., S. Jarnum, R. Preisig, K. Rams0e, J. Tauber, and N. Tugstrup. 1970. Degredation of albumin and IgG at high altitude. J. Appl. Physiol. 28: 728-732.

62. Whitehorri, W. V., A. Edelmann, and F. A. Hitchcock. 1946. The cardiovascular responses to the breathing of 100 per cent oxygen at normal barometric pressure. Am. J. Physiol. 146: 61-65.

CHAPTER II

METHODS AND MATERIALS

Experimental Design

Dextran was chosen as the experimental molecule in

the study of capillary permeability because of its use

in previous studies (l, 2, 8), and because of its

desirable physical and chemical properties (7). Through

the courtesy of Dr. Kirsti Granath, Uppsala, Sweden, five

extremely narrow-range molecular weight fractions of

dextran were obtained. These fractions were much

narrower than those used in previous studies, and

therefore, the false indications of permeability due

to the trailing smaller molecular weight molecules

would be decreased significantly. Some lower molecular

weight contamination could still bias the results, but

the realization of this fact would eliminate the

tendency to make erroneous permeability conclusions.

With these limitations established, experimentation

was initiated using these narrow-range fractions as

test molecules. Their physical characteristics as

determined by Dr. Granath can be seen in Table I.

37

38

TABLE I

PHYSICAL PROPERTIES OF DEXTRAN FRACTIONS AS DETERMINED BY GRANATH

Fraction No. Mw* /V V^n**

5 720,000 0.622 Between 6 581,000 0.590 1.22-1.25 7 467,000 0.565 for all 8 409,000 0.530 fractions. 9 312,000 0.496

* Determined by light scattering

** Analyzed by gel chromatography on Sepharose 4B

A total of twenty mongrel dogs, with an average weight

of 10 kg, were utilized to study the transport of selected

fractions of these macromolecular dextrans across the capil-

lary endothelial barrier. On these experimental animals,

seven involved the use of Fraction 9 (312,000 mol wt); five

involved the use of Fraction 8 (409,000 mol wt); and two

involved the use of Fraction 5 (720,000 mol wt). The six

remaining animals were examined using Dextran 500 which had

a Myy of approximately 500,000 (Lot no. 4376 obtained from

Pharmacia Fine Chemicals, Uppsala, Sweden). With these

molecular weight divisions as identifying characteristics,

the animals were classified respectively as Groups A, B, C,

and D.

Two dogs were designated as ground level control

animals within each experimental group. Those animals

remaining in the groups were subjected to 28,000 feet

39

(247 mm Hg) simulated altitude. The period at altitude

lasted 2 hr, and it was preceded by a 2 >-hr equilibration

period. A 1-hr, post-altitude stabilization period

concluded the experimental run. The rate of ascent and

descent during the run was 4,000 ft/min. The animals

were denitrogenated by breathing 100% oxygen 30 min prior

to altitude, and their respiration was continually sup-

ported by this oxygen supply until 30 min after the period

at altitude. Ground level control animals were studied

under the same experimental parameters, except they

remained at ground level during the period designated for

simulated altitude. These altitude studies were deleted

in Group C.

Under these criteria established for experimental

study, each individual animal was examined under identical

surgical and analytical procedures. Every attempt was

made to reduce the occurance of procedural variations.

This experimental format was thus designed to test

capillary permeability under the individual and collective

influences of dextran molecular weight polydispersity and

decreased barometric pressure.

Preparation of the Animals

It was necessary to monitor both the circulatory

system and the lymphatic system in order to accurately

observe the permeability of the capillary walls. Since

it has been established that the permeability of

40

capillaries may vary in different regions of the body (3)

it was also desirable to monitor more than one lymphatic

drainage region. The following surgery and cannulation

procedures were developed to satisfy these monitoring

requirements. These procedures involve the thoracic

lymph duct, which collects lymph primarily from the liver,

intestines, and spleen; the popliteal lymph "network,"

which collects lymph from the subcutaneous and super-

ficial muscles of the lower leg; and the femoral artery,

which was used as the common monitor of the circulatory

system as a whole.

Surgery Procedures

Approximately 30 min before administration of anes-

thesia, the dog was fed 50 ml of condensed milk. The

absorption of this fatty substance into the intestinal

lymph subsequently rendered the' thoracic lymph a milky

white. As a result, the thoracic lymph duct could be

easily identified in later dissection. The animal was

the anesthetized by an intravenous injection of sodium

pentobarbital, 30 mg/kg body weight, in the cephalic vein.

A glass endotracheal cannula was inserted in the trachea,

and the proposed surgical areas were shaved.

A 3-inch incision, beginning at the base of the neck,

was made directly above the left external jugular vein.

The external jugular, subclavian, and transverse scapular

veins were exposed by blunt dissection. The entrance of

41

the thoracic lymph duct into the venous system was generally

located in an area under the external jugular and around

the junction of this vein with the subclavian vein.

Anatomical variations were observed due to developmental

anomalies and the use of mongrel dogs. Once it was

identified, however, approximately 2 inches of the duct

were dissected free of adipose and connective tissue for

cannulation. A small portion of the transverse scapular

vein was also cleared for subsequent bypass cannulation.

At this point the incision was packed with moist, saline

pads, and the remaining surgery was performed.

After performing a left femoral cut down to expose the

femoral artery, the animal was turned on its ventral side

and arranged for maximum exposure of the right popliteal

fossa. The popliteal lymph nodes were located in the fossa

by palpation, and a lJ£-inch incision was made immediately

distal to the lymph nodes and medial to the clearly visible

external saphenous vein. Extraneous bleeding from small

skin vessels was eliminated by cauterization. Two larger

lymphatics could be found by blunt dissection on either

side of and adjacent to the external saphenous vein. The

lymphatic usually chosen for cannulation, however, was on

the medial side of the vein. The remaining lymphatic

branches, which were sometimes very numerous, were ligated.

A schematic diagram of the lymphatic drainage of the dog

leg can be seen in Figure 2,

42

J B W V W

<4H O

- P C

•H O

03 cd •»-p

_ 0) »H P h 3 rH

o a c o

• a) a . CT*rC II a) a II

rH co • co a

w • •—I Q>

cn co o c -

Tf N CD

CD - P - P X X a j -p a>

a £

0) rH CD *11 D > 0 II 05 • C »H

O •H

Mh O

•H • • CO <0 «*

H rH (0 T5 CD 3

JQ o O CO c

•H 1—5 CD - P x : CO a

r C • (0 a x * (0 £ CD £>h-P rH

rH CO CO O c

" d - H H C T3 CD CO C - P

•H C •H

JH <0 H O »H II - P •

CO c rH O •

D *H •H O - P H (0 • *

•H rH C O D •H

CD > a c • CO

CM O rH • CO CD

• a H TJ o v h o o

•H - P £ C CO CD

CL it CL a II £ pN « >H

H C H H

43

Cannulation Procedures

When all dissection was completed, the dog was

heparinized by injecting sodium heparin, 500 USP units/kg

body weight, into the left external saphenous vein. All

cannulae were also filled with heparinized saline. The leg

lymphatic selected for cannulation was then ligated and

allowed to swell from lymph pressure increased by squeezing

the paw. Using iris scissors, a small, diagonal slit was

cut in the side of the duct. This created an open flap

through which Clay Adams PE 10 Intramedic polyethylene

tubing could be inserted. Since numerous valves were

present in the duct, the tip of the cannula had to be

positioned between the valves to insure continuous lymph

flow. The cannula was then secured with two ligatures, and

the incision was closed with three stay sutures.

After completion of the leg lymphatic cannulation, the

animal was returned to his dorsal side, and the left femoral

artery was cannulated proximally and distally using PE 280

polyethylene tubing. This created an arterial bypass for

blood sampling and further injections.

The thoracic lymph duct was then cannulated by a

procedure similar to that used for the leg. The natural

thoracic lymph pressure, however, was sufficient to greatly

enlarge the duct after initial ligation without additional

external pressure. The diagonal cut was made, and PE 190

or PE 90 polyethylene tubing was inserted a distance of

44

2-3 cm. One ligature was made to secure the cannula in

place. A second cannula of PE 205 polyethylene tubing was

inserted proximally and secured in the transverse scapular

vein. The distal end of this cannula was then joined to

the thoracic duct cannula. A lymph-external jugular bypass

was thus formed that only slightly modified the natural

route of the lymph. This completed the surgery and

cannulation procedures, and the dog was moved to the

decompression chamber.

Decompression Chamber

The decompression chamber utilized in this study was

designed and built by Dallas Radionics, Inc., Dallas, Texas.

It consisted of a large steel cylinder with large side

windows and end port of plexiglass allowing continual

observation of the experimental animal. The chamber was

equipped with a Welch Scientific Company vacuum pump, and

the pump was connected to a Wallace and Tiernan model

FA-193100 absolute pressure contractor. Through these

connections, the barometric pressure could be regulated

over a very narrow range. The rate of decompression was

monitored by a Pioneer Company model 1636-6AB1 rate of

climb control mechanism also connected to the vacuum pump.

The absolute pressure was continuously measured by a

Wallace and Tiernan model FA 129 absolute pressure gauge.

These gauges and controls allowed the intricate manipulation

and observation of the pressure within the chamber.

45

The dog was placed in this- decompression chamber for the

experimental period. The animal's right foot, having been

previously wrapped in a constant heat pad, was fitted in a

sling attached to a rotary device. The toe of the dog's

foot was then anchored so that a passive muscular action of

the leg was achieved when the rotary device was set in

motion (20-30 rpm). This setup was designed to produce a

milking action on the lymphatics and hence, a constant flow

of lymph. The temperature of the dog's foot was monitored

at 38lt20 C by a Yellow Springs Instrument Company, Inc.,

Tele-thermometer, model 43 TD. It was equipped with a

thermistor probe, model 402, and the temperature was

controlled manually through a Powerstat, Superior Electric

Company type 116, connected to the constant heat pad. Both

the rotary device and the Powerstat were joined to an

external power source in one wall of the chamber, and the

power source could be externally controlled during

decompression.

Additional apparatus was employed to obtain a complete

monitoring picture of the dog's physiological parameters at

altitude. The arterial and thoracic lymph bypasses were

exteriorized through one side of the chamber and connected

to three-way luer stopcocks to allow sampling. Lymph

samples from the leg were collected continuously in a tube

inside the chamber. A Statham differential pressure strain

gauge, model PR 23-40-300, located inside the chamber was

46

used to monitor the femoral arterial blood pressure.

Additional electrical outlets on the side of the chamber

connected the strain gauge to a Brush Universal amplifier,

model RD-5612-00, and ink writing oscillograph, model

RD-2321-00. The 100% oxygen that was supplied to the dog

was vented through a valve located on the floor of the

chamber. Tygon tubing was used to connect the valve to

a T-tube on the endotracheal cannula. The open arm of the

T-tube had a flutter valve to aid in regulating the oxygen

flow and to assure that the animal did not inhale chamber

air. Experimentation was ready to proceed when these

procedures were completed.

Sampling Procedures

Initial samples of 4 ml of blood, 1% ml of thoracic

lymph, and approximately 0.5 ml of leg lymph were obtained.

An initial blood pressure reading was also observed. After

these preliminary observations were made, a 12% dextran

solution containing a measured dosage of 20 mg dextran/kg

body weight was injected into the femoral artery bypass.

This injection also contained minute portions (ca. 0.2 mg/ml)

of sodium azide for the fractions other than Dextran 500.

From this point until termination, blood pressure, readings,

blood samples, and thoracic lymph samples were taken at

15-miri intervals. The volume was maintained at a constant

level by immediate replacement of sample volume with

47

isotonic saline. The leg lymph samples were collected as

flow would allow, preferably at 30- or 45-min intervals.

After the samples were acquired, several preparatory

steps occurred before analysis could proceed. Hematocrits

were obtained from each fresh blood sample using an Adams

Autocrit centrifuge, model CT 2905. The remainder of each

blood sample and the lymph samp.les were then centrifuged

in an International Equipment Company clinical centrifuge,

model CL 46417M-6, for 10 min at 3,000 rpm. The plasma

and cell-free lymph thus obtained were pipetted to sterile

plastic refrigerator tubes^ The samples were then stored

at 4° C until analysis procedures could be performed.

Dextran Analysis

The detection and quantitation of dextran in plasma

and lymph was accomplished using the combined, modified

procedures of Semple (5) and Scott and Melvin (6). A 1-ml

portion of each experimental sample was deproteinized with

5 ml of 5% trichloroacetic acid (J, T. Baker Chemical Co.).

Each was mixed on a Vortex shaker and allowed to stand for

10 min. They were then centrifuged for 20 min at 3,000 rpm

in the IEC centrifuge. Volumetric portions of 4 ml of the

resulting deproteinized supernatants were placed in 3/4-inch

dialysis bags. The bags had been previously soaked in

distilled water for 12 hr, washed thoroughly, tied at one

end, and fitted with a short piece of tygon tubing which

was held tightly in place by a rubber band. The open end

48

of the tygon tubing was securely plugged with a glass hook,

and the whole assemblage was tied to a metal dialysis rack.

When all of the deproteinized plasma ox lymph samples from

one dog were tied on the rack, they were dialyzed overnight

against distilled, deionized water to remove the blood

glucose. Dialysis was carried out for an initial minimum

period of 2 hr at 40° C in a 17-liter water bath equipped

with a Precision Scientific Company heater and circulator,

model 66590. During this period, an additional 21 liters

of distilled, deionized water was passed through the bath

by continuous flow. The samples were transfered to 5 ml

volumetric flasks when dialysis was completed, and the flasks

were filled to mark with distilled, deionized water.

Volumetric samples of 2 ml were removed from the flasks

and placed in test tubes in an ice water bath. Anthrone

reagent (200 mg anthrone/100 ml of concentrated Reagent

ACS sulfuric acid) was added to each tube in 4-ml quantities.

A truncated 4-ml pipette was used for fast, uniform reagent

addition. The tubes were then mixed by the Vortex shaker,

topped with a marble, and placed in an Electric Hotpack

Company water bath, model 106. The samples were thus

incubated at 90 — 2° C for 16 min to develop the color.

Immediately after removal from the hot water bath, the

samples were returned to the ice water bath. They were

removed from the ice water bath after a short period of

time and allowed to return to room temperature.

49

Each sample was then transferred to a quartz cuvette

and read in a Perkin-Elmer double beam spectrophotometer,

Coleman model 124. The reference cell contained a distilled

water-anthrone blank, and the wavelength was set at 625 nm.

The optical density readings for the samples were converted

to dextran concentration in mg/100 ml according to the

following formula:

OD..-OD. Cy = x C s x 6f x k,

0DS

where Cu = concentration of dextran in unknown sample

mg/100 ml; 0DU and 0DC = the optical densities of extracts

from unknown and control (initial) samples respectively;

ODs = optical density of the standard; C s = concentration

of the standard (always 5 mg/100 ml); 6f = the dilution

factor resulting from deproteinization and dilution to 5 ml

(f = 1.25); and k = a hydrolysis constant (l.O for dextran

standards). Calculated in this manner, the converted

concentrations were then presented as data for evaluation

and statistical analysis.

Molecular Weight Determination by Gel Filtration

The gel filtration procedures employed in this study

were used to compare the polydispersity of the individual

dextran fractions. The column used for the study was an

50

Ace Glass, Inc. stacked column, composed of two 600 x 25 mm

columns joined by a column connector. An 18-inch flow

adaptor was placed on the lower end of the column, and the

column was packed with Sepharose 6B (Sigma Chemical Company)

to a height of 93 cm. A double-vented plug was inserted

into the top of the column to allow sample addition and

fluid flow from the reservoir. The reservoir was filled with

0.85% sodium chloride for the liquid phase. In addition,

0.02% sodium azide was added as a bacteriostatic agent. A

bubble trap was also incorporated between the reservoir and

the top of the column. The column was allowed to run by

descending flow for a few days until the gel bed had

stabilized at a height of 91.5 cm. At that time, a 2.5 cm

diameter filter paper disc was placed on top of the gel

bed, and the void volume was determined.

Determination of void volume (VQ) was accomplished

using Blue Dextran 2000 (10 mg/ml in 0.2 M NaCl) was layered

on the filter paper disc. The vents were closed, and the

column was run at a hydrostatic pressure of 10-20 cm H2O.

The samples were collected by an Instrumentation Specialties

Company fraction collector, model 327. Equipped with an

ISCO drop counter, model 600, the fraction collector was

programed to allow 150 drops/collection tube. The volume

was checked by graduated cylinder measurement. As the Blue

Dextran began to appear in the collection tubes, the samples

were transferred to cuvettes and read in the double beam

51

spectrophotometer at 623 nm. A plot of the optical density

vs. volume yielded the void volume as the extrapolated high

point of the curve.

The dextran fractions described in Table I were

individually applied to the column. The procedure employed

was identical to that used for determining V0, except ml

were applied to the column instead of 3 ml. After a volume

equal to V0 had been collected, the collections were saved

and diluted 1:4 with distilled water. A 2-ml sample of

each dilution was analyzed using the anthrone reaction

described previously. The optical density vs. volume curve

was plotted for each fraction, and the extrapolated high

point was determined to be the elution volume (Ve) for that

particular fraction.

The total volume (V .) of the column was determined in

the same manner as VQ and Ve# L-Tryptophan (lJ£ ml at 1 mg/ml

in 0.2 M NaCl) was the molecule used to determine V^, and

the optical density was read at 280 nm. The extrapolated

high point of the optical density vs. volume curve was used

as the determining point of V ., After this value was

established the void volume was rerun to see if any shifts

had occurred in the gel bed.

Calibration of the Sepharose 6B column was completed

by calculating the partition coefficient (Kav) between the

liquid phase and the gel phase for each Dextran fraction used.

This value was obtained by using the following formula:

52

Kav = Ve - V0

V-t - V,

The Kav for each fraction was then plotted against its

logarithmic molecular weight. The line resulting from this

plot represented the calibration curve for the column (4).

The molecular weight of an unknown dextran fraction was

then examined on the calibrated Sepharose 6B column. The

unknown fraction was composed of a combined collection of

highly concentrated aliquots obtained from gel filtration of

Dextran 500. The column used for this additional gel filtra-

tion study was a Pharmacia K 15/90 (I.D. = 1.5 cm, length »

90 cm) packed with Sephadex G-200 (Sigma Chemical Company).

Operation of the filtration procedure was similar to that of

the Sepharose 6B column, except that 2* ml of sample (5 mg/ml

in 0.2 M NaCl) was layered on the bed surface. The aliquots

containing the most dextran were determined by the anthrone

reaction. These samples were then pooled, dialyzed by proced-

ures previously described, and lyophilized. The resulting

dextran flakes were dissolved in 0.2 M NaCl at a concentration

of 10 mg/ml. A sample of 2)£ ml was layered on the Sepharose

6B column, and the Ve was determined. Kav for the unknown

dextran was then calculated and compared to the calibration

curve previously plotted. In this manner, an estimate of

the degree of polydispersity comparison of Dextran 500 was made

with the narrow-range dextran fractions described in Table I.

CHAPTER BIBLIOGRAPHY

1. Garlick, D. G. and E. M. Rankin. 1970. Transport of large molecules from plasma to interstitial fluid and lymph in dogs. Am. J. Physiol. 219: 1595-1605,

2. Grotte, G. 1956. Passage of dextran molecules across the blood-lymph barrier. Acta Chir. Scand. Suppl. 211: 1-84.

3. Mayerson, H. S., C. G. Wolfram, H. H. Shirley, Jr., and K. Wasserman. 1960. Regional differences in capillary permeability. Am. J. Physiol. 198: 155-160.

4. Pharmacia Fine Chemicals AB. 1970. Sephadex. Gel filtration in theory and practice. Pharmacia Fine Chemicals, Inc., Uppsala, Sweden. 63 p.

5. Semple, R. E. 1957. An accurate method for the estimation of low concentrations of dextran in plasma. Can. J. Biochem. Physiol. 35: 383-390.

6. Scott, T. A., Jr. and E. H. Melvin. 1953. Determina-tion of dextran with anthrone. Anal. Chem. 25: 1656-1661.

7. Squire, J. R. 1951. Background to biological studies with dextran. Proc. Roy. Soc. Med. 44: 557-558. (Abstr.)

8. Wasserman, K., L. Loeb, and H. S. Mayerson. 1955, Capillary permeability to macromolecules. Circulation Res. 3: 594-603.

53

CHAPTER III

RESULTS

Capillary Permeability

The movement of macromolecular dextran fractions across

the capillary endothelial membrane was monitored by periodic

concentration determinations of dextran in plasma and lymph.

These determinations were organized and averaged according

to their specific molecular weight group and barometric

pressure treatment. The dextran concentrations in thoracic

and leg lymph were separately compared to the dextran

concentration in plasma.

Plasma vs. Thoracic Lymph Concentrations

A constant decrease in plasma dextran was observed in

all animals. This decline was somewhat enhanced by decreased

barometric pressure in some groups, but no definite dextran

loss was observed with the change in pressure. The dextran

levels gradually approached zero as the experiments proceeded

to termination.

Small concentrations of dextran generally appeared in

the thoracic lymph within 15 min. This concentration grad-

ually rose to a constant plateau after 1 hr, and then it

began to decline slowly until termination of the experiments.

Administration of decreased barometric pressure halted this

54

55

decline and significantly increased the dextran concentra-

tions in thoracic lymph. These concentrations decreased to

values that approached zero when the barometric pressure was

returned to normal.

The general trends cited above for dextran concentration

in plasma and thoracic lymph can be seen in the combined

group means. These graphs are presented in Figures 3 and 4.

Group A (321,000 mol. wt.).—The general decline in plasma

concentration of dextran was observed in both the ground

level control and the altitude experimental runs. A rather

erratic pattern was exhibited by the plasma means of the

control animals. This was contrasted by the relatively

smooth decline of the altitude mean plasma concentrations.

The control plasma values reached zero after 4^ hr, and the

altitude values were leveled off at 10 mg/100 ml at the end

of experimentation.

Only a small amount of dextran was identified in the

thoracic lymph of the control animals. A slight rise in

concentration was observed later in those experimental

animals, but the dextran concentration was essentially zero

after 3 3/4 hr. On the other hand, the mean thoracic lymph

concentrations of the altitude runs demonstrated the general,

characteristic pattern described for them. The concen-

tration rise with decreased barometric pressure was not as

dramatic as observed in some of the other groups, but it was

56

w O *H •H O -P

C •H O OuJQ

(0 H O JC2 -P

XI«—I Tf O (0 £ 03 ro m

o •H -P H 0) >

m S CO# 05 Q r~l p-t a/dx* c c 05 -o •h a>

*4J if) O 03 C O 03 «**H d) CQ T5 S C

•**rl c <

•H »H -P if) 03 a s " 3U4

O CO N

-P a a> a> o c. o u

if) •H CO e

£ 03

O H •P

C C 03 O H O •p X TJ +» 0 (DXl Q C O )

•H *H Xt CD • £

CO O^H o o

cn^ •H O P-i

x: a S a> >^x; H -P

c 03 0) 6

57

Hrtfci

»n xf o CN ( % Bui) NOI1VU1N3DNOD

58

CO o H >H CO CO O . H X I CO H - P r H O C CO

H O O - H a - p

N

- p

T f c x ; CO o

CO C0PL3 £ t o CO •

rH«"—N a n

a> >

jQ

TS Q)

- P CO a rH

c * a • H C

CO * 0 CO c C * * « H c o m a> co £ * # H <

o • H - P CO H

- P C 0 ) a a> c T J o 3 a - p

• H C - P CO H

• P X T * 0 ) Q)

Q C • H <4-1 £* O • £ ^ o

o

U ) CO CO a p o • N CO CT>rH

CO

• H C CO

a> >

H

I - P

CO i

c n H - i • H O PU

<D crs CO H a> >

CO j C a 6 <l> p n x i

r H - P

59

(%6u i ) NOI1VHJLN3DNOD

60

still significant. The mean concentration values fox control

and altitude runs can be seen in Figures 5 and 6.

Group B (409,000 mol. wt.).--Mean plasma dextran concen-

trations for both the control and altitude groups were similar.

Each of them exhibited the same slow decline, and they both

had a final dextran concentration around 2.5 mg/100 ml. The

average dextran concentrations of the altitude animals,

however, were initially much higher than the averages of the

control animals. This resulted in a more rapid rate of

decline during the first few hours for the altitude runs.

Administration of oxygen and decompriession created an erratic

pattern in thses animals that eventually developed into

dignificant decreases in mean plasma dextran concentrations.

The dextran concentrations in thoracic lymph demon-

strated exaggerated rises in the initial dextran concen-

trations of both the ground level control and the altitude

runs. These values decreased, however, to constant levels

which, in the case of the control concentration means,

stayed around 5 mg/100 ml. The dextran concentrations of

thoracic lymph were increased when the animals were subjected

to decompression. To a limited degree, these increased con-

centrations were mirrored by the decreases in the plasma

dextran concentrations. The thoracic lymph dextran decreased

to zero after the barometric pressure was returned to normal.

Graphs of these results can be seen in Figures 7 and 8.

61

0) x:

o -P •H O CO fl5 *rl H O -P • x: c (o •P*H H

O 03 "0 a-O c CO XI «HI

O CO 03 ctJ O e w -H 0) -p CO N HI • <D a co >

r—I c co >. •H 6 rQ

•rl « CTJ C CO <1> (0 +> <D H (0 e o o

H -H C 4-> T3 O C C •H O »H -t-> O (0 0) H i—I *rl +» <1) c a) <D yj OHt/3 C O TJ O C •

3 V) C O t-1 CO fH CO H cn S 4-> .H X »C <D < CO Q

a o 3 5

• o +> m H

a m o

a>M-t •h o a> (jli cn

jC co a n S a> > >

r-4 (0

62

o ' o ' <Sr o » o r

«© «0 CO CN ( % 6 U I ) N O I 1 V U 1 N 3 D N O D

63

o o •H O <D co cn H CO O H

JC 0 -P >

CO TJ C CP CO jC -P

CO 6 to <O.H • CO CO H + J H O.C CO

•H XI C O •H 0*1—I

CO CO JC O C O.H CO CO -P CD U4 H S CD

c • ^ O CO > •H «-H J3 •P (0 (0 S T5 H n H CD -P C -P C CO CO 0) O O CD «H C T * T * O 3 C O - P t 4

•rl C -P <0 (OrH .H H CO -P S CD < CO P

a •

• o CO \0 N H

CD CO • S

• H O C tt* CO

,c a <d S > >h«H f-l H-i

64

(%6u i ) N0I1VJI1N3DN03

65

CD x:

o -P •H U if) <0 *H H O -P • x: c co HP *H H

O CO -o ajQ c (OXJ rH

O <0 fO CO o S W d (0 -P CO H rH • 0) a w >

1 - 4 C fO > •H £ JQ

•H W C T 5 G CO <D

<dh nj g o o

H »H C -P T5 O C C •H O *H -P O CO <0 HtH *H •p a) c > S ( D O W O H CO c o"d o c: •

p (0 C O H <o H co H CJ> S -P »H X - C a> CQ co Q

a o D 5

• O -P r- N o

o o*H •HOC) Pu cn

x: <o a h £ o ^ > rH C0

66

{% 6ui) NOI1VU1N3DNOD

67

<+4 o o

•H O <1) co cn H f0 O N

x : a) - p > CO

T* C <D 05 j C

-P CO 5 <0 • 0 ) . H 0) CO H

rH -P CO a c x i

•H C O rH

•H 0 . <0 O

C O -P CO CO H <u PJ a> 6 > £ • > . O (OX*

•H rH -P CO TJ CO g <D H - H -P

• p a co C (0 o 0) *H u o n C t 3 C O O -P

•H (0 C -P *H CO rH H COS •P W X *CO O PQ Q

a • 2 <0

• O rH 00 N CO

O 6 • *H

C •H O CO PL,

jC 0) a < D 6 H

H *P

68

7

( % 6ui) NOI1VU1N33NOD

69

Group C (720,000 moi. wt.).—The ground level control

animals were the only animals examined in this molecular

weight group. Although the initial plasma dextran concen-

tration did not reach that of the other groups, it did show

the same general decline. After 3i hr the mean plasma

value was zero. Dextran of this molecular weight did appear

in the lymph, but it was present in only small quantities.

The mean concentration of dextran in thoracic lymph was

rarely above 2 mg/100 ml after 1% hr of experimentation.

Graphic analysis of these results can be seen in Figure 9.

Group D (Dextran 500).—The results obtained in this

group were similar to the general trend already set forth.

Plasma dextran concentrations in both control and altitude

groups demonstrated a slow decline. The values reached zero

in the control means and 6 mg/100 ml in the altitude means.

No noticeable change in altitude concentrations was present

in the plasma.

Dextran mean values presented in the thoracic lymph also

appeared similair to those expressed in the general trend.

The main exception, however, comes in the last samples of

the control thoracic lymph values. They show a definite

increase over both the previous thoracic lymph values and

the compared values for plasma dextran. The differences in

the concentrations of dextran in plasma and thoracic lymph

at this point seem to be significant. Another less obvious

exception to the general trend was the failure of the thoracic

70

o •H O (T5

O jC 4->

0) JC -p

•H

-P £

O

CO H CO

ax> T3 c rOXlH

O 03 CO CO e w CO co

a <o

o •H •p

N <D >

c •H

(0 C CO CD 6 a o •H -P CO fH -P C Q)

CO >* 6 X> •H C "0> CO CD

-P r-H CO o o H *H •P "0 C C 0«H O

40 H *H Q) o §

O rH CO c C O o c •

P CO O H H CO cr> g •H

* c CO

C CO H -P X <D O Q

ON

cr><+4 •H O [X,

a o 3 5 O *P H O

O

<D cr>

jc co a h S 0) > > H CO

71

(%6ui) NOI iVa iN3DNOD

72

dextran means to fall drastically twoard zero after the

period at decreased barometric pressure. Otherwise the

results were similar to those already expressed. Graphs

of these results can be seen in Figures 10 and 11.

Plasma vs. Leg Lymph Concentrations

Efforts to obtain leg lymph in usable quantities

encountered several physiological and mechanical difficulties,

The rate of flow was normally very slow at the beginning of

the experiment, and this flow was decreased with time to a

complete stoppage in most animals. If the lymph was flowing

at the time decompression was started, it stopped after a

slight increase in rate. Problems with lymph duct consis-

tency in size and number also hindered the attainment of

significant lymph flow rates. Because of these problems,

leg lymph samples were obtained from relatively few animals.

Most of the samples that were taken suffered from lack of

sufficient volume for valid analysis. *

A total of nine dogs yielded leg lymph samples for

dextran analysis. Representatives of each molecular weight

group and barometric pressure treatment were contained with-

in these animals. Using analysis procedures modified by

dilution techniques, only two of the animals were judged to

have concentrations of dextran in their leg lymph samples.

A Group D (Dextran 500), ground level control animal

exhibited slight increases in dextran concentration that

reached 3 mg/100 ml at the termination of the experiment.

73

a> ox:

•H - p o CO (0

o x : - P • • p c

•H N T J O CO c a x * (0

r C «--4 CO O 03 g <o u CO P4 *H CO - P

r—t H Q* * <U

CO > C •—l

•H CO >* 6 x>

CO C C T5 CO CO 0 0) - P g r-4 CO o a

C N - H O - P "D

•H C £ - P 0 » H CO O H 0)

• P H *ri C <P CD > S o a> w c to o o - d

c • C 3 « (0 O I-H H H <0

• p D i e X "H <D - G

O Q (0

Cu O , O S

O O - P H H

O O

crm-t •H O <D VLi CT>

x : co a h 6 <u > >

r - i 10

74

"SI o» o» cT

? % 6 m ) ^ N O I l V H i N B D N O D

75

OQ-I •H O O CO H O •P

T3 C CO

CD D> CO N 0) > <0

0) X

CO -P S CO <0 CO «H

A-P c c •H

CO H (0

CO

CO O <D CO G W

H JQ O O-RH

CO O •H -P N 0 > C

O •H CO •PHXI CO CO N -P«H 0) C G +•> CD CO CO O O C 0) OTF-0 O P G -P «H

C-H <0 -P N < -P CO X s OJI *JJ4 Q Q CO

a • 3 •

•HOC/) H N H

O CO

CO •rl

S •H C CO

•H O P-I .C a N E ^

R-I <+*

76

(%6ui) NOI1VU1N3DNOD

77

Also, a Group A (321,000 mol. wt.), altitude animal showed

inconsistent increases in dextran concentration, expecially

in the last sample collected after decreased barometric

pressure. The remaining animals revealed no measurable

amounts of dextran in leg lymph. Because of this lack, no

comparison could be made between leg lymph concentrations

and plasma concentrations of dextran.

Statistical Significance

Statistical analysis was applied to the plasma vs.

thoracic lymph results to determine their significance.

Standard errors of the mean (SEM) were calculated for each

sample of every experimental run. The results of these

calculations were presented as vertical bars on the graphs

in Figures 3-11. Loss of significant differences between

plasma and thoracic lymph concentration means were readily

observed by the overlap of the SEM vertical bars. This

overlap occurred in the combined means after 4 hr for the

control animals and after 3% hr for th'e altitude animals.

A further statistical evaluation was necessary to

establish the significance of lymph changes and plasma

changes occurring between ground level control and altitude

animals. A calculated Student's t was obtained for the thor-

acic lymph vs. thoracic lymph and the plasma vs. plasma

values of each barometric pressure treatment. These calcu-

lations were then compared to a table of t values (tQ 0 5 ) to

determine probability (P). The combined control runs were

78

compared to individual group altitude runs for this analysis.

In addition, combined control runs were compared to combined

altitude runs. Table II gives the results of these compared

calculations with their levels of significance.

The calculated t values obtained for the combined

control and altitude groups gave a general picture of the

overall significance of the experimental runs when they were

compared to the tabled t values. The thoracic lymph compar-

isons that were determined during the decompression period

revealed significant defferences in dextran concentrations.

The 2:45- through 3:45-hr samples were significant at the

0.01 percent level, and the 4:00- through 4:30-hr samples

were significant at the 0.05 percent level. These values

and two other initial values in the plasma were the only

comparisons that indicated any significant differences in

the combined control vs. altitude groups.

Group A (321,000 mol. wt.).—In both the ground level

controls and the altitude runs, the dextran concentration

differences between plasma and thoracic lymph became insig-

nificant after 3% hr. The intercomparison of lymph and

plasma values under different barometric pressure treatments

yielded values of a 0.05 percent level of significance for

all comparisons involving thoracic lymph at altitude. This

significance level was extended to one sample of thoracic

lymph in the post-altitude stabilization period. Other

79

H

U4 $

o

CO OJ 3 O N O

x: QJ B > »-4

T5 <1> C •H XI S o o

x: oj 6 4

Q Oj 3 o M CD

0Q Ol o H CD

a o H CD

XI 04 6 kJ

0-

6 N •H X?

iooo^T'Hr-or~-oooin^t^Hi>ir)cocx)'Hco'^oo H'HOlO'^h-vOiOh-COO'HOOO'crvOHOOD-CNO coo-HooN'srioor-oot^cMoo(Ni>ON<N<M'Hvor--o ^nCDOHOJCMHvOOOOhvOCOth-OOOvOh-H HHHHOOOOOHlNCOOCOWOJCSlOlHOOO I I 5j< 5§£ OOOOOlCMOJvO tOCO'HvOlO'HvOOO rOOCOinvOvO'H OCMtOOOOOOiOCO fOOOONO-HiOOCM-HONvOCOO mcOlfiCOvDvOO^vOh-Hin^OOvOH'-I CO vO 00 CM ^voocMvDf-r-co^oiOvoovocof-oiOvOiot^in • • • • • • • • • • • • • • • • • • • • • • CM '—1 CM CM r"H 1—I'—f •—1 ""H CM '—i 'H •—i •—i •—1 1—I CM 1—I •—I 1—! •—! *—I

% sjt 5 5 * * * ^•or-cMmcMOvor-oooincM-sriooo^roinoj-ioo ooMnocot^-ooJ-Hh-voiovoh-cocoio^roJoooo vOOOOr-h-HvOOHhODh-Oh-OJ^OCOO^iOCO HCOinOiniO-HCMOvOOOOr-vOOCMO t OOCOvO -HOOOOOOOO-HCOOJOOmCMHCMOJOO-HO

I I I I *{< >|c >fc

o^coocM^inr-t^-ooHinooocMmcoooocO'H r--t^cD\rioco—i-H^-^-cocMcM^ivOh-coiniooN-Ho lOOJ-HCOOOOO^OOHr-'HLnOOOr-OJh-O'HCM'HOO h-' OIXNininoOCMvOOOOCMCO 'H'HCSlOCM-H'H • • • • • • • • • • • • • • • • • • • • a *

HHOJHHHHOHHOOHHHHCMCJOJCJOlOl

COf-HOlCM^OOOOiOCOOt -OCO^OOOCOOOOOO CMOinvOHONOCMH-HunCM^COh-iOCOiOvOOOt O ^COHOlOCMCOOhONvO^ODvOOOOvOHOOH OOHOO^h-OOI^^OHHCMCOhOOCOOlCOHvOH HCJCOOJOOdOOOHinhCOfHHOlHOOH I I I I I I

CMCMin^COvOiOOOiOvOCOCOiDCOOOOOvOvOI ' Or*-CM\OOOinOOOCMOOCMiOLO\QONCMOOifiCM<tf VOOVO OVCM ONOOO fOOCMM-OJOiO^-OO iOvOO r-^rin'st-ooiOior-'HOh-voaDr-ooco'^fCM^^rvo • • • • • • • " • • • • • • • • • • • • • • • ( M H H H O O O O O H O H H H H l N H H H H W H

COOOCOOHt-vOt vOr OCOOOHiOCOCOCMvOONOO Ot-CMCMOOOOONr CMOvQl HvOf-vOf-OOvQCXW ^CO^vDHH^vOCOOOiDl^HONOvOHvOCMC^iOH coHr-uosrior-^cx)voir)^rcM^-oocovoc>vOin^io HHHHOOOOOOOIOJOI CM'CM CM CM CM CM O O O

OOvOOCMOCWvO vOCOO -OOCMCM- vOCO-H^OH csicO'Hoot^--Hoor--'3-i>csiooot^cMoor~-—tvoino—i coiOh-cocM^f-iO'HincD^voor-oor^^^T-r-vo cocM^oco^vocMcocor-r-HoooHininKi-vo^r HHHCMHHHHHHHHOlCJHOiCMHHHHH

o i o o i n o m o i o o m o i o o i n o i o o i o o i o o i o o OHCO^OHCO^OHCO^OHCO^rOHCO^OHCO

m m m mm mm mm ££ skm mm mm mm mm mm m* mm mm mm mm ** mm mm mm

• • • • • • • • • • • • • •

OOOOHHHHCMCMOKNCOCOCOCO^^r^^iOiOin

•H o o • o V a* %

•H o <1 o V a*

in o • o V a. *

80

comparisons in this group were not significant at the

prescribed level.

Group B (409,000 mol. wt.).—Insignificant differences

between plasma and thoracic lymph concentrations of dextran

became evident after 4 hr in the control animals and after

3'4 hr in the altitude animals. The plasma differences and

thoracic lymph differences demonstrated a varied pattern of

significance. The thoracic lymph samples taken at altitude

showed significant levels of from 0.001 to no significance.

Additional levels of significance (0.05) were found in the

thoracic lymph at the 0:45-hr and 1:00-hr samples. A

significant value was also found in the plasma at the 0:15-

hr sample. Other comparisons did not give any significant

values at this level.

Group C (700,000 mol. wt.).—In this molecular weight

group, differences between plasma and lymph were not

significant after 2/4 hr. No plasma and thoracic lymph

intercomparisons could be made since there were no altitude

runs.

Group D (Dextran 500).—The concentration means of

dextran in plasma and thoracic .lymph showed some patterns

that varied from the general trend, especially toward the end

of the control animals. Insignificant differences between

the two, however, could be established after 4 hr in the

control animals and after 2 3/4 hr in the altitude animals.

81 J

In the comparisons between the thoracic lymph values of con-

trol and altitude animals, all but one of the samples invol-

ving altitude exhibited significant levels of 0.05 percent

or better. The 3:30-hr sample was significant at a 0.001

percent level. Plasma comparisons demonstrated significance

in the 4:30-hr, 4:45-hr, and 5:00-hr samples. None of the

other calculations was significant at the prescribed level.

Physiological Parameters

Pulse Pressure

The pulse pressure of the experimental animals in

Groups A, B, and C were affected by the presence of minute

quantities of sodium azide (ca. 0.02 percent NaN3) present

in the injected dextran solution. These animals experienced

a mean pulse pressure drop of 30 mm Hg immediately after

injection. The pulse pressure then slowly rose until it was

approximately normal after 3 hrs. Animals in Group D did

not have sodium azide in their dextran injections, and they

did not demonstrate the drastic drop in pulse pressure. A

0.02$ sodium azide concentration was added to the dextran

injection of one animal in Group D. The pulse pressure of

this animal responded exactly like those pulse pressures of

animals in Group A, B, and C. Animals not having sodium

azide in the dextran injection showed a slight decrease in

pulse pressure throughout the experimental run. The use of

oxygen and decreased barometric pressure did not have drastic

82

effects on the pulse pressures. Comparisons of the pulse

pressures of azide and non-azide animals can be seen in

Figure 12.

Hematocrit

Average hematocrit values of the experimental animals

changed very little during the course of experimentation.

A maximum range of from 41 to 45 percent was observed, and

the tendency was for the hematocrit to rise slightly as the

experimentation period progressed. Some individual animals

showed small decreases after injection of dextran, and other

individual animals showed slight increases with the intro-

duction of decompression. No definite trends could be

established from these animals, however. The hematocrit

means for all animals can be seen in Figure 13.

Molecular Weight Determination by Gel Filtration

Column Calibration

Calibration of the Sepharose 6B column was accomplished

by determining the elution volumes (Ve) for each molecular

weight fraction and the void volume (VQ) and internal volume

(V^) of the column. Using these values, the partition coef-

ficient (Kav) was calculated. The void volume and the

internal volume of the column were 158.5 ml and 518.3 ml

respectively. Elution patterns of the dextran fractions

were symmetrical, and they allowed easy estimation of

83

c • CO (0 0) CD

<u £ D -C •—l +-> 0) CO

tJ > C »H O N C CO CO cd a> "O <u g •H x: N-p J) CO TJ

14 «H S o n 3 <+H CO •H I TJ TS C o a> o w cn c

CO 'H R 0) o a> sx

> 4-> tO CO -P H o a> o <u Mm •+-< a) <H g TS <1) <D •

w cnw OH (0 N Xi CO H CO +> £ <1> .Q

•H > 'H C (OH O ro CO

<D O C C H-H O <1) <D -P « H 5 H •H <D H W > to • cn a o o >• £ HT3XJ O P O M UK

to p aj <D o +>

• H Mh co cm a o H TS *H

<U CO • 0) CO C

D>rH -H •H p " (i< aw <o

a) »H CD CO H S CD CO UJ £ > W

84

1 * 1

( 6 H m m ) 3 u n S S 3 M d I S i n d

a> u

JJ 'jj "D ° N O O C

II

85

CO i—! CO S

•H C CO

CO -p

c 0) £

•H N 0) a X <D •

CO r-4 r—< co re e

•H 4H C O fO

<0 C <tf Q) D <D r-i +J ro x : > CD

•rl +> <u •H H a o o +> a> CO D> S <0 <D H x ; a> > £ CO <0 a) Q) S X -p

• <o CO -H H

4-> • C

cn-H •rl O ^ a

XI a CO (XI

86

JLIHDOJ.VW3H

87

elution volumes. The elution patterns of the respective

molecules can be seen in Figure 14, and their individual

elution volumes and partition coefficients can be seen in

Table III. Semilogarithmic plots of partition coefficient

vs. molecular weight established the calibration curve of

the column and can be seen in Figure 15.

TABLE III

Column Characteristics of Dextran Fractions

Molecular Weiqht

Ve (ml)

Kav

720,000 199.9 0.115 581,000 208.2 0.138 467,000 215.0 0.157 409,000 219.7 0.170 321,000 229.1 0.196 Unknown 227.9 0.193

Examination of Unknown Molecular Weight

The combination and lyophilization of selected samples,

fractionated on Sephadex G-200, yielded 34.0 mg of dextran.

The appropriate solution of this dextran was applied to the

Sepharose 6B column. A rather broad distribution was exhib-

ited with the elution of the dextran down the column. This

elution pattern did have an exaggerated peak at the point of

elution volume determination, but it was otherwise very flat

and widespread. The partition coefficient was calculated to

be 0.193, and this value corresponded to a molecular weight

88

CO C o •H -P O Ctf M <+•<

C (0 H •P X TJ m o CO c N <l> -P CO a c o •H -P 3

0

O c o CO •H H 03 a 6 o o

m vD 0)

H CO O

• H CP <0 •H ,C mu a <D tn c o

89

o o o O c ooo o £ o o o o o

K o o C O O O CN C 1 0 ^ ^ CO 3

uni t

% Z> mmJ

o >

( U I U 5 J 9 * ° ) A 1 I S N 3 Q I V D I i d O

90

Fig. 15. Calibration curve for Sepharose 6B column.

91

MOLECULAR WEIGHT

92

of 330,000 on the calibration curve (Figure 15). The

elution pattern of this dextran fraction can be compared

to the elution patterns of the previous dextran fractions

in Figure 14. The column characteristics of the unknown

can also be compared in Table III.

CHAPTER IV

DISCUSSION

Capillary Permeability

The Starling hypothesis depicts the direction and rate

of fluid transfer between plasma and tissue fluids as being

determined by three factors: (a) the hydrostatic pressures

on each side of the capillary membranes, (b) the colloid

osmotic pressures of plasma and tissue fluids acting across

the capillary membranes, and (c) the physical, chemical,

and histological properties of the capillary membranes them-

selves (8, 37, 42). If an inert, lipid-insoluble molecular

species is suddenly added to arterial blood, a disturbance

is created in the osmotic balance that initiates two related

processes to reestablish the Starling equilibrium (36).

The first process involves the diffusion of the added

molecule from the plasma across the capillary membrane to

the interstitial fluid, and the second process involves the

withdrawal of fluid from the interstitial compartment into

the capillary blood due to the concentration gradient formed

by the added molecules. Both processes continue until an

equilibrium is attained according to the new osmotic

pressures. The rate of these two processes and the time

required for them to reach equilibrium are dependent on the

93

94

hindrance to diffusion at the endothelial wall. This

membrane hinderance is primarily related to molecular size,

and through mathematical applications of the progressive

plasma and lymph concentrations, the larger molecular

species can be used to observe and describe some of the

characteristics unique to the capillary endothelial wall

(13, 35, 38). Likewise, changes in the hydrostatic

pressures can induce shifts in the transcapillary fluid

flow and/or in the osmotically active molecules to return

the system to the equilibrium portrayed by the Starling

hypothesis. Monitoring the shifts of the large, membrane-

selected molecular species can reflect additional properties c

of the capillary endothelial wall and the surrounding inter-

stitial compartment (25, 26). In essence, all three of the

factors experssed in the Starling hypothesis must be

considered in discussing the properties of capillary perme-

ability.

The macromolecular dextran fractions utilized in this

study were chosen because of their similarities with the

above hypothetical molecular species, both in physiological

inertness (41) and in biological fractionation according to

molecular size (21, 45, 46). The capillary permeability

characteristics of the fractions were studied both during

the initial equilibrium adjustments and during barometric

pressure changes after equilibrium had been reached. These

characteristics studies were facilitated by monitoring the

95

concentrations of dextran disappearing from the plasma and

appearing in the lymph obtained from.two different lymphatic

drainage areas. This was in response to the reports of

varied histological (3) and permeability (31) characteristics

of capillary endothelial membranes in different anatomical

regions. Within the limits of certain qualifications, the

results obtained from the two different regions substan-

tiated the reported variations in capillary permeability.

Plasma vs. Thoracic Lymph Concentrations

Molecular Weight Effects—The size of the molecular

species, as reflected by molecular weight, has been pointed

out as one of the primary considerations in capillary

membrane selectivity. This fact was especially evident in

the studies evaluating dextran as a plasma expander (4, 41,

45). In those studies, increased efficiency in rapidity

and duration of plasma expansion was noted with increased

molecular weights of dextran. Larger molecular weight

dextrans were simply retained longer by the restrictive

endothelial barrier. The increasing molecular weights in

the present study, however, did not appreciably alter the

permeability characteristics as they were monitored in the

thoracic duct drainage regions. With two exceptions that

will be explained later, there were no significant deviations

in the progressive dextran concentration patterns in either

96

the plasma or the thoracic lymph. This observation can

be explained by several factors.

The first explanatory factor involved, and probably

the most important one, is the region from which the

thoracic lymph is primarily derived, the spleen, intestine,

and liver. Several investigators (13, 21, 31, 46) have

shown the presence of dextran in thoracic lymph, and their

reported molecular weight average is within the range of

Groups A, B, and D. These workers have suggested the

presence of large "leaks", especially in the liver, to

account for this presence of macromolecular dextran. The

estimated size of these "leaks" is 250 to 300 A, and

electron microscope studies by Bennett and co-workers (3)

have suggested their existence. Other workers (34, 39) have

indicated that macromolecular transport may be by vesicles,

but no evidence has been offered to support the vesicular

transport of non-proteinaceous, lipid-insoluble molecules

like dextran. At this point, the endothelial "leaks"

attributed to the liver appear to be the most acceptable

theory of macromolecular transfer of dextran to the thoracic

lymph. The "leaks" must be larger than the effective

molecular size of dextrans in Groups A, B, and D, because

there are no selective differences between them in their

thoracic lymph concentrations.

A second factor that must be considered is related to

the dextran molecule itself. It has already been pointed

97

out that its molecular weight polydispersity has been a

major drawback in its use in capillary permeability studies.

Even though the dextran fractions used in this study were

much less polydispersed than those used in previous studies,

some lower molecular weight contamination could still be

expected. An example of contamination can be seen where the

thoracic lymph concentrations of dextran become greater than

the plasma concentrations in the latter samples of Group D.

This group is much more polydispersed than the other three

groups. In addition to the polydispersity effects, the

dextran molecule has been described as a flexible, linear

coil that can be subject to shear deformation in smaller

passageways (2, 22). These two molecular characteristics

of dextran indicate that some limited transfer could occur

within the proposed small pore system (13, 31, 35) or at

least through smaller "leaks". This indication is especially

significant when the comparatively small concentration

dosages are considered. The effects of the increasing

molecular weight of dextran may have been partially hidden

by the false permeability characteristics of the dextran

molecules.

Either one or combinations of both of the above factors

can explain the lack of observed concentration changes with

the use of increased molecular weight dextran fractions of

Groups A, B, and D. Molecular weight averages within these

groups present no drastic change. The molecular weight

98

average of Group C, however, is much greated than the

averages of dextrans used in previous studies. Although

only two ground level control animals are examined in

Group C, the results obtained indicate that the dextran

transfer is restricted. The average plasma concentrations

approach zero much sooner than the other groups, and very

small amounts of dextran appear in the thoracic lymph.

Conclusions based on two experimental runs are questionable,

but the tendency of the endothelial membranes in the thoracic

duct drainage regions to restrict dextrans of much higher

molecular weight averages seems to be present. Renkin (38)

has said that it would take much higher molecular weight

dextrans to establish the maximum permeability limits of the

capillary endothelial membranes, and this may be true if

the above tendency proves to be correct with further

experimentation.

Since the ground level control runs were somewhat

similar, their dextran concentrations were combined and

averaged for comparison with the altitude runs. This is

justified in the light of the above explanations for their

similarities. The statistical significance of the later

comparisons is also increased by doing this.

Loss of Dextran—One of the more puzzling aspects of

this study centers around the constant decline of dextran

concentration in both the plasma and the thoracic lymph of

all groups. At first glance, elimination by the kidneys

99

might be suspected for the decrease, but this is difficult

to support after further examination. Wasserman and his

associates (46) have shown that dextran molecular weights

greater than 40,000 are generally retained by the kidney

and returned to the circulation. This limit is far below

any of the average molecular weights used in this study,

and all of the fractions except Group D were sufficiently

narrow to believe that few if any molecules of this size

were present. Even Group D showed no change in concen-

tration patterns that would lead one to suspect significant

kidney elimination of dextran.

Metabolism could also be suspected for the decline of

dextran concentrations. Gray (19) reported that dextran

was broken down by the animal, incorporated into the carbon

pool, and distributed throughout the body in the form of

normal biochemical constituents. In this instance, the

experimental animals were mich, and the metabolic half-life

of the carbon in dextran was approximately 6.1 days. This

is contrasted to a metabolic half-life on dogs of 27 hr as

reported by Wasserman and Mayerson (44). The latter inves-

tigators also calculated the slope of exponential disap-

pearance to be -0.00043. When the 5J£-hr period of experi-

mentation for the present studies is considered, it is

unlikely that metabolism alone could have caused the dextran

decreases that were observed.

100

The factors involved in establishing an osmotic equi-

librium with the low concentration dosage is one of the more

probable causes for the decline in dextran concentration.

Previous reports (40, 43, 44, 46) give conflicting periods

of time (ranging 1-13 hr) that are required to establish an

osmotic balance, and the time periods involved depend some-

what on whether a true equilibrium or a steady state condi-

tion is acquired. All of these studies, however, used

dextran concentrations that were many times greated than the

concentrations used in the present investigation, and some

of the studies (40, 43) used the additional somotic effector

of radioactive iodinated albumen. This means that the small

quantities of dextran fractions used in this investigation

should have reached a steady state condition much sooner

than these previous studies. When the lymph to plasma con-

centration ratios are examined, it can be seen that they do

indeed become somewhat static after one hour of experimen-

tation, and even with the constant decline in respective

concentrations, this ratio remains almost constant until the

values begin to approach zero and become invalid indicators.

These facts suggest that the plasma and lymph concentrations

of the dextran fractions are basically in a steady state.

However, there is either an additional steady state equili-

brium that must be satisfied for constant concentration

values, or, as hinted previously, the metabolic activity is

accentuated by the low concentration values present.

101

The accentuated metabolic activity with small dextran

concentrations is a distinct possibility, because dextran

disappearance would be more noticeable in smaller total

quantities. In light of the reappearance of dextran in the

thoracic lymph with the advent of decreased barometric

pressure, however, metabolism seems highly unlikely. The

anabolic processes required under these circumstances are

physically impossible. Thus, metabolism must again be

discounted as a major cause for the dextran decline, and

other dextran-consuming processes or secondary equilibria

must be examined.

There are three mechanisms by which dextran could be

consumed or in which a secondary equilibrium might be

established. The first of these involves cirtain organs

that perform specific functions for the animal's circu-

latory system. In this instance, the organs are the liver

and the spleen. Both of these organs have storage and

phagocytic properties that could account for the disap-

pearance of dextran. Reports (16, 20) concerning these

properties do not indicate that dextran concentrations

would be altered from those seen in the plasma and thoracic

lymph, but no real evaluations are presented. The sinu-

soidal spaces and phagocytic characteristics of one or

both organs could cause the disappearance of dextran that

was observed.

102

The second mechanism deals with a peculiar erythrocyte

reaction in the presence of dextran. Bull and his collab-

orators (6) observed formation of rouleaux in smears of

blood previously infused with dextran. Formation was

inhanced by concentration and molecular weight increases.

Although the concentrations used in the present study were

below those reported, the molecular weights were much higher.

There is no way to know now if significant quantities of

rouleaux were formed or not, but if they were, they too might

be an explanation for the dextran decline, especially in

conjunction with the phagocytic properties suggested in the

first mechanism.

The third mechanism encompasses the theories of the

tissue spaces within the interstitial compartment. Histolo-

gists, biochemists, and physiologists (12, 14, 32) have all

pointed out that almost all normal tissue spaces are filled

with a gel or gel-like substance in addition to free inter-

stitial fluid. The gel is composed primarily of mucopoly-

saccharides, and in most tissues, the most important one is

hyaluronic acid. This substance is present in the form of

long coiled filaments, and it exists in concentrations

usually between 0.3 and 1.2 percent (33). Since the coiled

filaments are oriented in all directions, the resulting

network has been described by Ogston and Sherman (33) as a

"brush pile" that, with its entrapment effects, impedes

bulk movements of fluid and solutes within the fluid.

103

Guyton and co-workers (25) have shown that the degree of this

impedence by. this gel reticulum is inversely proportional to

the quantity of free fluid that is also in the interstitial

spaces. Under these conditions, it is conceivable that

dextran could be in a quasi-equilibrium between the plasma

and free fluid while still undergoing diffusion or entrap-

ment within the gel reticulum. This mechanism could easily

explain the disappearing pattern of the dextran fractions.

Any one of the three mechanisms cited above could have

had a part in the decline in dextran concentrations. Inter-

action between the mechanisms could also have had some part.

In view of the later results showing a reappearance of

dextran in the thoracic lymph, the latter mechanism seems

to be the most favorable. The first two mechanisms involve

a somewhat irretrievable loss in dextran that would not

allow for the observed increases in thoracic lymph dextran

at altitude. These increases can be rationalized by gel-

free fluid features present in the third mechanism. It must

be pointed out, nonetheless, that no definite answer can be

realized with the present data. Unitl further experimen-

tation involving radioisotopically tagged dextran is

performed, no conclusive statements about the disappearance

of dextran can be made.

Effects of Pressure Changes—The changes observed in

the thoracic lymph concentration of dextran at reduced

barometric pressures were the most significant results of

104

the entire study. The thoracic concentrations of the ground

level control animals continued their slow decline toward

zero throughout the experimentation period, but the thoracic

concentrations of the altitude animals demonstrated a *

dramatic increase in dextran concentration for the period

of reduced barometric pressure. In both instances, however,

the plasma dextran concentration sustained its slow decrease

without significant change during the periods at altitude.

These results could be due to one or combinations of all of

the following physiological explanations: 1) an actual

increase in the permeability characteristics of the capil-

lary endothelial membrane changes, 2) an increase in central

blood pressure that alters the dynamics of the Starling

equilibrium, and/or 3) a change in the gel consistency and

pressures in the interstitial compartment that shifts the

steady state dynamics and increases the thoracic lymph

dextran concentrations.

The stretched pore phenomenon elaborated by Shirley and

co-workers (40) is an example of a change that could take

place in the endothelial membrane itself. In their illus-

tration of the phenomenon, pore distortion was created by

enormous volumes of normal saline infused into the experi-

mental animals after a large quantity of dextran had reached

steady state conditions. The drastically increased plasma

volume supposedly stretched the small pore system to

stimulate transport of increased dextran molecular weight

105

sizes. Variations of this theory come from Grotte's system

of large pore "leaks" (21) and from Arturson and Granath's

"safety valve" system of increased large pore radii (l).

The present study, however, did not allow for the possibility

of increased pore size or leaks because of plasma volume

expansion. On the contrary, dextran concentration dosages

were intentionally kept low and no more than sample-replace-

ment volumes of normal saline were added to avoid signifi-

cant plasma expansion in the animals. Blood pressure was

also decreased substantially by the addition of minute quan-

tities of sodium azide. In this manner, any membrane chan-

ges that occurred, such as the stretched pores or "leaks",

had to be related to the decreased barometric pressure.

Under these experimental conditions, membrane changes

like those cited above, are difficult to support for the

capillary endothelial system as a whole. Decreased baro-

metric pressure, however, does have some profound effects

on the hemodynamics of experimental animals, and they may

be related to the present observations. Investigators

have identified increases in peripheral tonicity and

resistance (10, 15) and decreases in venous compliance (47)

with exposure to high altitude. The general conclusion

derived from these observations is that the central blood

volume is increased and the extreme peripheral circulation

receives a limited blood flow (10, 47). With these facts

in mind the question in the present study involves the

106

degree to which the central blood volume and peripheral

resistance is increased. If these increases are sufficient

to raise the capillary hydrostatic pressures significantly,

then stretched pores or "leaks" may indeed have caused the

the rise in thoracic lymph concentration of dextran. The

liver and spleen would again play an important part in this

supposition. Because of the large amount of supposition

imposed at this point, the stretched pore hypothesis must

remain plausible but not fully substantiated by the present

facts of this study.

The hydrostatic forces that purportedly allow the

stretched pore phenomena to operate come from inside the

capillaries. Since the conditions established in the

experimental design would affect the forces surrounding

the capillaries, the interstitial compartment and its

related forces must be examined. Guyton (25) has described

the interstitial compartment as being composed of the fol-

lowing three constituents: 1) the interstitial fluid within»

the interstitial spaces, 2) the cells and fibers of the

stroma, and 3) the mucopolysaccharide gel. The first con-

stituent forms the interstitial fluid pressure, and the last

two make up the solid tissue pressure. Accordingly, the

total tissue pressure is determined by the algebraic averages

of the interstitial fluid and solid tissue pressures that

occur on the surfaces of the capillaries.

107

Interstitial fluid pressure is commonly reported as

a negative pressure (23) that pulls the surface of the skin

toward the cell surfaces, as well as pulling the adjacent

cell surfaces together. In turn, the solid fibers in the

spaces are holding the surfaces apart. Hence, the solid

tissue pressures exerted by the solid fibers would counter-

act the pressures exerted by the negative interstitial fluid

pressure, and the total tissue pressure would be zero or

slightly positive (25). This reasoning involves a simple

application of physics that says that "at any single plane

in space in a nonaccelerating system, the forces acting in

one direction must be exactly opposed by forces acting in

the opposite direction." Within the limits of fiber strength,

skin turgor, and additional structural forces, this law

should apply both to increasing and decreasing interstitial

fluid pressures (25).

Assuming that the skin would be flaccid enough to

transmit pressures faithfully, the total tissue pressure on

the under surface of the skin is equal to zero with respect

to atmospheric pressure. A decreased atmospheric pressure,

like that presented in the present study, would cause a

decreased pressure under the skin. This same amount of

pressure is transmitted through the fluid and solid struc-

tural to the surface of the cells and capillaries because

of the "equal but opposite" physical principal stated

above. The total pressure in this instance would

108

still be around zero with respect to the reduced atmospheric

pressure, but the fluid pressure in the space between the

capillary and the surrounding tissues could be very negative

(23, 24). Thus, bulk movement and diffusion of fluid and

solute molecules would be increased in an outward direction

through the capillary pores which, in effect, is the

stretched pore phenomena in reverse. The solid forces

involved in this mechanism would reach a point of compaction

in the peripheral and cutaneous regions, and the blood ves-

sels supplying these regions would be closed or severely

restricted (7). This is in accord with the increased peri-

pheral resistance and decreased venous compliance obser-

vations noted earlier (10, 15, 47). In deeper and more

centrally located tissues, the compaction tendency would

not be as great, and the forces causing bulk fluid and

solute movement would predominate.

The influx of fluid into the interstitial spaces

increases the lymphatic flow (29) which in turn would

remove the dextran and other osmotic effectors that were

present in the tissue spaces. The dextrans would be returned

to the circulatory system in an attempt to reestablish the

balance of the Starling equilibrium, and this fact could

explain the lack of noticiable decline in plasma dextran

concentration with concomitant increases in thoracic lymph

dextran. The mechanism just explained is very similar to

the "safety valve" system proposed by Arturson and Granath

109

(1) fox the large pore system. Although their system was

set forth to explain the mechanism involved in the relief

of increased venous pressure, it also fits very well into

the explanation for the relief of unbalanced forces coming

from the interstitial compartment and decreased barometric

pressure.

In light of the above discussion, it appears that all

of the explanations initially presented combine to give a

whole picture of the mechanism involved. This could have

been expected because of the number of physiological vari-

ables that are inherent to capillary dynamics and capillary

permeability. Certainly additional work needs to be done

to establish the trends and reasoning presented in this

study.

Plasma vs. Leg Lymph Concentrations

The lack of sufficient data severely hinders the eval-

uation of capillary permeability as monitored in the leg.

The surgical procedures involving the leg lymphatics were

aborted only once due to impossible morphological formation

of the ducts, and in the remaining experimental runs,

successful cannulation was performed with lymph flow observed

at the onset of experimentation. However, only a few of

these leg cannulations continued to flow for a significant

period of time. Those cannulations which did continue to

flow for the entire experimental period were handicapped by

110

volumes insufficient for accurate analysis, especially near

the end of experimentation. Any discussion of the obser-

vations and general tendencies must be made with the real-

ization that much additional work must be done to substan-

tiate and explain them.

Initial responses of the leg lymph flow coincided with

those reported by Irisawa and Rushmer (29). The steady

state pressures appeared to be relatively low, but they

could be raised to extremely high levels by squeezing the

paw or otherwise manipulating the region distal to the

lymphatic cannulation. Although these initial results were

favorable to the success of the procedure, the subsequent

lymph flow exhibited a general decrease in rate as the

experiment progressed. The flow completely stopped in some

instances shortly after the experimentation period had

begun. These decreases and stoppages occurred in spite of

warming the paw and passively rotating the leg to stimulate

lymph flow. Similar difficulties have been indicated by

other investigators (11, 13, 48), and they are thought to

be primarily due to the overall inactivity of the anesthe-

tized animal. In addition, fibrin clots were formed in the

collecting tubes of some of the experimental animals in the

present study, and this may have been a factor in the flow

stoppage of some animals, even though thorough precautions

were taken to eliminate clotting problems. Other additional

physiological phenomena that could have slowed or stopped

1X1

the flow rate were as follows: 1) washout of the inter-

stitial osmotic effectors causing increased fluid reabsorp-

tion (11, 25), and 2) compaction of the cutaneous region

and occlusion of peripheral blood and lymph vessels with

altitude (7, 15, 25). All of these factors and probably

some that were undetected could have stopped or contrib-

uted to the slowing of the leg lymph flow.

It was additionally difficult to analyze and evaluate

the results of the few experimental animals that did yield

samples for the entire period. The dilution procedures

necessary to analyze the samples demanded that only qual-

itative results could possibly be considered valid. In

this light, only two dogs revealed the presence of dextran

in the leg lymph, and one of these seemed dubious. A ground

level control animal (Group D) showed the definite presence

of dextran and an altitude animal (Group A) showed sporadic

appearances of the test molecule. Previous reports (13, 21)

indicate that dextran molecules of this size have passed

through the capillary endothelial membranes, but the

present study cannot lend any evidence to support these

reports.

The results obtained from this portion of the study

indicate that it may be of little value to continue to use

leg lymphatics located in the popliteal fossa. It is very

difficult to obtain significant flow rates from this area,

especially with decreased barometric pressure studies. The

112

lymph obtained from these ducts is primarily derived from

subcutaneous areas (9, 30) that are dubject to compaction

and closing pressures of vessels (7, 25). As an alternate

position for the monitoring of leg lymph, the lymphatics in

the inguinal fossa should yield more satisfactory results.

Their lymph is derived from the deep muscles of the leg, and

as a result most of the problems involving consistent flow

rate and decreased barometric pressure should be diminished

(9, 30).

Statistical Significance

The standard error of the mean (SEM) was calculated

for the average of each sample of both barometric pressure

treatments within each molecular weight group. These

calculations presented rather wide standard errors of the

mean, especially in the ground level control averages, that

could have been reduced somewhat by an increased number of

runs. Additional variation from the mean could have come

from the fact that the experimental animals utilized were

mongrel dogs. Bulgin and associates (5) attributed vari-

ations in protein concentrations in purebred beagle dogs

to age differences, nutritional deficiencies, and/or disease.

Although an attempt was made to uniformly select the animals,

there was no real way of judging the above conditions in

mongrels. Consequently the variation had to be increased

by these factors.

113

Calculated Student's t values demonstrated significant

variations primarily in the thoracic lymph comparisons during

the altitude period. The significance of the thoracic lymph

increases in dextran is more consistently expressed in the

combined group comparisons. This is because more experimental

animals were evaluated, and a true expression of the sta-

tistical tool could be expressed. Individual group compar-

isons in the thoracic lymph showed essentially the same

period of significance, but the level of significance vari-

ation within the individual groups could be diminished by

increased numbers of animals. A general trend toward

decreasing levels of significance during the latter samples

of the altitude period could possibly be interpreted as the

beginning stages of a steady state equilibrium at the new

barometric pressure. No explanation other than the mongrel

variations and the small number of experimental animals can

be given for the spot appearances of significant t values

in initial samples. The obvious need is for more experiments

to be run, but the important trends can be seen from the

experimental runs presented in this study.

Physiological Parameters

Pulse Pressure

The pulse pressure drop observed in the experimental

animals of Groups A, B, and C were due to the presence of

minute quantities of sodium azide (ca. 0.02 percent Na^).

114

Graham (17) has described this chemical as a neutral salt

of hydrazoic acid that acts peripherally in small concentra-

tion dosages to relax the smooth muscle of blood vessels and

bronchi while increasing the force of cardiac contraction.

As was seen in Figure 12, the result of this action was a

drastic drop in systemic blood pressure. Graham also

reported other physiological reactions, including death,

could occur with further increases in azide concentration.

These reactions, however, required much higher concentra-

tions than were employed in this experiment.

Other than the hemodynamic effects discussed above,

there appeared to be no significant differences in the

results of the animals with or without the sodium azide.

No apparent difference was present in the dextran concen-

trations monitored. The general trend in animals not

injected with dodium azide was to show a slight decrease

in pulse pressure throughout the experiment, and the

treatment of oxygen and decreased barometric pressure had

no drastic effects on the pulse pressure. Including the

effects of sodium azide, the pulse pressure averages

demonstrated no phenomena that would suggest physiological

abnormalities in the capillary permeability studies.

Hematocrit

Average hematocrit values demonstrated no significant

changes during the pcsriod of experimentation. The individual

115

variations could have been responses to fluid shifts between

the vascular and extravascular compartments with the intro-

duction of dextran and decreased barometric pressure. These

changes were not uniform, however, and were therefore not

significant. Because of the relatively short experimental

period involved, no attempts were made to thoroughly examine

fluid shifts and hemopoietic activity reported in longer

stays under the influence of reduced atmospheric pressure

(27, 28). The hematocrit, as used in 'this study, was

simply employed as an indicator of the physiological status

of the experimental animals, and this status remained

constant throughout the period of experimentation.

Molecular Weight Determination by Gel Filtration

The method employed to calibrate the Sepharose 6B

column in this study was sufficient to reveal the molecular

weight characteristics pertinent to the involved dextran

fractions. The semilogarithmic plot revealed a fairly

straight line for molecular weight comparisons. If more

exact molecular weight determinations had been desired,

however, the polydispersity effects of the dextran calibra-

tion fractions would have had to be considered using

additional methods of calibration (1, 18).

In examining the unknown molecular weight, the most

significant result has to be the comparison of elution

patterns seen in Figure 14. The patterns of the calibrating

dextran fractions are symmetrical and somewhat concentrated

116

over a comparatively narrow volume range. The unknown

dextran demonstrated a broad, flat elution pattern. Compar-

ison of these two patterns suggests a much higher degree of

polydispersity for the unknown dextran fraction, even though

it had been previously fractionated on Sephadex columns.

Since the steps to consider the polydispersity of the

calibrating fractions were not taken, the molecular weight

determined for the unknown dextran cannot be viewed with a

great deal of exactness. It does appear, however, that the

average molecular weight for Dextran 500 is less than

500,000 and that it is extremely polydispersed in this

commercial form.

Summary

The outstanding aspects and conclusions derived from

this study can be summarized as follows:

1. The concentrations of macromolecular, narrow-range

dextran fractions were increased during exposure to a

simulated altitude of 28,000 feet. The increases were

suggested to originate from a combination of chanqes in the

capillary endothelial membranes, increased central blood

volume, and changes in the gel reticulum and pressure

balances of the interstitial compartment.

2. Further investigations should be made to determine

the pooling places of dextran and the maximum molecular

weight limits of dextran for capillary transport.

117

3. The lymphatic network of the popliteal fossa is

unsatisfactory for use in studying capillary permeability

at decreased barometric pressure. Future studies should

examine the possibility of using the lymphatics in the

inguinal fossa for monitoring leg lymph.

4. Gel filtration procedures reveal substantial

polydispersity differences between the acquired, narrow-range

dextran fractions and commercially prepared dextran. However,

no significant differences are evident in their capillary

permeability.

5. Increased significance can be achieved by

increasing the number of experimental animals in both the

control and treatment areas.

CHAPTER BIBLIOGRAPHY

1. Arturson, G. and K. Granath. 1972. Dextran as test molecules in studies of the functional ultra-structure of biological membranes. Clin. Chim. Acta. 37: 309-322.

2. Baker, R. W. and H. Strathmann. 1970. Ultra-filtration of macromolecular solutions with high-flux membranes. J. Appl. Polym. Sci. 14: 1197-1214.

3. Bennett, H. S., J. H. Luft? and J. C. Hampton. 1959. Morphological classifications of vertebrate blood capillaries. Am. J. Physiol. 196: 381-390.

4. Brewer, D. B. 1951. Renal clearances of dextrans of varying molecular weights. Proc. Roy. Soc. Med. 44: 561-563.

5. Bulgin, M. S., M. Shifrine, and S. J. Galligan. 1971. Electrophoretic analyses of normal beagle serum proteins. Lab. Anim. Sci. 21: 275-279.

6. Bull, J. P., C. Ricketts, J. R. Squire, W. d'A. Maycock, S. J. L. Spooner, P. L. Mollsion, and J. C. S. Paterson. 1949. Dextrin as a plasma substitute. Lancet 256 (l): 134-143.

7. Burton, A. C., J. T. Nichol, F. Girling, and W. Jerrard. 1950. A new view of relation between pressure and flow in a vascular bed, and validity of "peripheral resistance". Fed. Proc. 9: 20.

8. Chambers, R. and B. W. Zweifach. 1947. Intercellular cement and capillary permeability. Physiol. Rev. 27: 436-463.

9. Drinker, C. K., M. E. Field, and J. Homans. 1934. The experimental production of edema and elephantiasis as a result of lymphatic obstruction. Am. J. Physiol. 108: 509-520.

118

119

10. Field, M. E. and C. K. Drinker. 1931. The rapidity of interchanges between the blood and lymph in the dog. Am. J. Physiol. 98: 378-386.

11. Durand, J., J. Verpillat, M. Pradel, and J. Martineaud. 1969. Influence of altitude on the cutaneous circulation of residents and newcomers. Fed. Proc. 28: 1124-1128.

12. Fund, Y. C., B. W. Zweifach, and M. Intaglietta. 1966. Elastic environment of the capillary bed. Circulation Res. 19: 441-461.

13. Garlick, D. G. and E. M. Renkin. 1970.# Transport of large molecules from plasma to interstitial fluid and lymph in dogs. Am. J. Physiol. 219: 1595-1605.

14. Gersh, I. and H. R. Catchpole. 1960.# The nature of ground substance of connective tissue. Perspectives Biol. Med. 3: 282-319.

15. Girling, F. and C. Maheux. 1952. Peripheral circu-lation and simulated altitude. J. Aviat. Med. 23: 216-217, 270.

16. Godart, S. J. and W. F. Hamilton. 1963. Lymphatic drainage of the spleen. Am. J. Physiol. 204: 1107-1114.

17. Graham, J. D. P. 1949. Actions of sodium azide. Brit. J. Pharmacol. 4: 1-6.

18. Granath, K. A. and B. E. Krist. 1967. Molecular weight distribution analysis by gel chroma-tography on sephadex. J. Chromatog. 28: 69-81.

19. Gray, I. 1953. .Metabolism of plasma expanders studied with carbon -labeled dextran. Am. J. Physiol. 174: 462-466.

20. Greenway, C. V. and R. D. Stark. 1971. Hepatic vascular bed. Physiol. Rev. 51: 23-65.

21. Grotte, G. 1956. Passage of dextran molecules across the blood-lymph barrier. Acta Chir. Scand. Suppl. 211: 1-84.

22. Grotte, G., L. Juhlin, and N. Sandberg. J-^O. Passage of solid spherical particles across the blood-lymph barrier. Acta Physiol. Scand. 50: 287-293.

120

23. Guyton, A. C. 1963. A concept of negative inter-stitial pressure based on pressures in implanted perforated capsules. Circulation Res. 12: 399-414.

24. Guyton, A. C. 1965. Interstitial fluid pressure: II. Pressure-volume curves of interstitial space. Circulation Res. 16: 452-460.

25. Guyton, A. C., H. J. Granger, and A. E. Taylor. 1971. Interstitial fluid pressure. Physiol. Rev. 51: 527-563.

26. Guyton, A. C., J. Prather, K. Scheel, and J. McGehee. 1966. Interstitial fluid pressure. IV. Its effect on fluid movement through the capillary wall. Circulation Res. 19: 1022-1030.

27. Hannon, J. P., K. S. K. Chinn, and J. L. Shields. 1971. Alterations in serum and extracellular electrolytes during high altitude exposure. J. Appl. Physiol. 31: 266-273.

28. Hurtado, A., C. Merion, and E. Dalgado. ,1945. Influence of anoxemia on the hemopoietic activity. Arch. Intern. Med. 75: 284-323.

29. Irisawa, A. and R. F. Rushmer. 1959. Relationship between lymphatic and venous pressure in leg of dog. Am. J. Physiol. 196: 495-498.

30. Jacobsson, S. and I. Kjellmer.^ 1964. Flow and protein content of lymph in resting and exercising skeletal muscle. Acta Physiol. Scand. 60: 278-285.

31. Mayerson, H. S., C. G. Wolfrom, H. H. Shirley, Jr., and K. Wasserman. 1960. Regional differences in capillary permeability. Am. J. Physiol. 198: 155-160.

32. Meyer, K., E. Davidson, A. Lindker, and P. Hoffman.^ 1956. The acid mucopolysaccharides of connective tissue. Biochim. Biophys. Acta. 21: 506-518.

33. Ogston, A. G. and T. F. Sherman. 1961. Effects of hyaluronic acid upon diffusion of solutes and flow of solvent. J. Physiol. (London). 156: 67-74.

121

34. Palade, G. E. 1953. Fine structure of blood capil-laries. J. Appl. Phys. 24: 1424.

35. Pappenheimer, J. R. 1953. Passage of molecules through capillary walls. Physiol. Rev. 33: 387-423.

36. Pappenheimer, J. R., E. M. Renkin, and L. M. Borrero. 1951. Filtration, diffusion, and molecular sieving through peripheral capillary membranes. A contribution to the pore theory of capillary permeability. Am. J. Physiol. 167: 13-46.

37. Pappenheimer, J. R. and A. Soto-Rivera. 1948. Effective osmotic pressure of the plasma proteins and other quantities associated with the capillary circulation in the hindlimbs of cats and dogs. Am. J. Physiol. 152; 471-491.

38. Renkin, E. M. 1964. Eighth Bowditch Lecture. Transport of large moleaies across capillary walls. Physiologist. 7: 13-28.

39. Shea, S. M. and M. J. Karnovsky. 1969. Vesicular transport across endothelium: simulation of a diffusion model. J. Theoret. Biol. 24: 30-42.

40. Shirley, H. H., Jr., C. G. Wolfrom, K. Wasserman, and H. S. Mayerson. 1957. Capillary permeability to macromolecules; stretched pore phenomenon. Am. J. Physiol. 190: 189-193.

41. Squire, J. R. 1951. Background to biological studies with dextran. Proc. Roy. Soc. Med. 44: 557-558 (Abstr.)

42. Starling, E. H. 1896. On the absorption of fluids from the connective tissue spaces. J. Physiol. (London). 19: 312-326.

43. Wasserman, K. and H. S. Mayerson. 1951. Exchange of albumen between plasma and lymph. Am. J. Physiol. 165: 15-26.

44. Wasserman, K. and H. S. Mayerson. 1952. Plasma, lymph, and urine studies after dextran infusions. Am. J. Physiol. 171: 218-232.

45. Wasserman, K. and H. S. Mayerson. 1954. Relative importance of dextran molecular size in plasma volume expansion. Am. J. Physiol. 176: 104-112.

122

46. Wasserman, K., L. Loeb, and H. S. Mayerson. 1955. Capillary permeability to macromolecules. Circulation Res. 3: 594-603.

47. Weil, J. V., E. Byrne-Quinn, D. J. Battock, R. F. Grover, and C. A. Chidsey. 1971. Forearm circulation in man at high altitude. Clin, Sci. 40: 325-246.

48. White, J. C., M. E. Field, and C. K. Drinker. 1933. On the protein content and normal flowof lymph from the foot of the dog. Am. J. Physiol. 103: 34-44.

BIBLIOGRAPHY

Andrews, P. 1964. Estimation of the molecular weights of proteins by sephadex gel-filtration. Biochem. J. 91: 222-233.

Araki, C. 1956. Structure of the agarose constituent of agar-agar. Bull. Chem. Soc. Japan. 29: 543-544.

Arnold, R. M. and L. B. Mendel. 1926. Interrelationships between the chemical composition of the blood and the lymph of the dog. J. Biol. Chem. 72: 189-211.

Arturson, G. and K. Granath. 1972. Dextrans as test molecules in studies of the functional ultra-structure of biological membranes. Clin. Chim. Acta. 37: 309-322.

Baker, R. W. and H. Strathmann. 1970. Ultrafiltration of macromolecular solutions with high-flux membranes. J. Appl. Polym. Sci. 14: 1197-1214.

Bengtsson, S. and L. Philipson. 1964. Chromatography of animal viruses on pearl-condensed agar. Biochim. Biophys. Acta. 79: 399-406.

Bennett, H. S., J. H. Luft? and J. C. Hampton. 1959. Morphological classifications of vertebrate blood capillaries. Am. J. Physiol. 196: 381-390.

Bowditch, H. P. 1873. The lymph spaces in fasciae. Proc. Am Acad. Sci. 8: 508-510.

Brewer, D. B. 1951. Renal clearances of dextrans of varying molecular weights. Proc. Roy. Soc. Med. 44: 561-563.

Bulgin, M. S., M. Shifrine, and S. J. Galligan. 1971. Electrophoretic analysis of normal beagle serum proteins. Lab. Anim. Sci. 21: 275-279.

Bull, J. P., C. Ricketts, J. R. Squire, W. d'A. Maycock, S. J. L. Spooner, P. L. Mollsion, and J. C. S. Paterson. 1949. Dextran as a plasma substitute. Lancet 256 (1): 134-143.

123

124

Burton, A. C., J. T. Michol, F. Girling, and W. Jerrard. 1950. A new view of relation between pressure and flow in a vascular bed, and validity of "peripheral resistance". Fed. Proc. 9: 20.

Casley-Smith, J. R. 1969. The dimensions and numbers of small vesicles in cells, endothelial and mesothelial and the significance of these for endothelial perm-eability. J. Microscopy 90: 251-268.

Chambers, R. and B. W. Zweifach. 1947. Intercellular cement and capillary permeability. Physiol. Rev. 27: 436-463.

Churchill, E. D., F. Nakazawa, and C. K. Drinker. 1927. The circulation of body fluids in the frog. J. Physiol. (London). 63: 304-308.

Conklin, R. E. 1930. The formation and circulation of lymph in the frog. III. The permeability of the capillaries to protein. Am. J. Physiol. 95: 98-110.

Douglas, C. G., J. S, Haldane, Y. Henderson, and E. C. Sch-neider. 1913. VI. Physiological observations made on Pike's Peak, Colorado, with special reference to adaptation to low barometric pressure. Phil. Trans. Roy. Soc. B 203: 185-318.

Drinker, C. K. and M. E. Field. 1931. The protein content of mammalian lymph and the relation of lymph to tissue fluid. Am. J. Physiol. 97: 32-39.

Drinker, C. K., M. E. Field, and J. Homans. 1934. The experimental production of edema and elephantiasis as a result of lymphatic obstruction. Am. J. Physiol. 108: 509-520.

Durand, J., JT. Verpillat, M. Pradel, and J. Martineaud. 1969. Influence of altitude on the cutaneous circu-lation of residents and newcomers. Fed. Proc. 28: 1124-1128.

Field, M. E. and C. K. Drinker. 1931. The permeability of the capillaries of the dog to protein. Am. J. Physiol. 97: 40-51.

Field, M. E., and C. K. Drinker. 1931. The rapidity of interchanges between the blood and lymph in the dog. Am. J. Physiol. 98: 378-386.

125

Fund, Y. C., B. W. Zweifach, and M. Intaglietta. 1966. Elastic environment of the capillary bed. Circulation Res. 19: 441-461.

Garlick, D. G. and E. M. Renkin. 1970. Transport of large molecules from plasma to interstitial fluid and lymph in dogs. Am, J. Physiol. 219; 1595-1605.

Gersh, I. and H. R. Catchpole. 1960. The nature of ground substance of connective tissue. Perspectives Biol, Med. 3: 282-319.

Girling, F. and C. Maheux. 1952. Peripheral circulation and simulated altitude. J. Aviation Med. 23: 216-217, 270.

Girling, F. and C. Maheux. 1953. Peripheral circulation and simulated altitude. Part II. J. Aviation Med. 24: 446-448.

Godart, S. J. and W. F. Hamilton. 1963. Lymphatic drainage of the spleen. Am, J. Physiol. 204: 1107-1114.

Graham, J. D. P. 1949. Actions of sodium azide. Brit. J. Pharmacol. 4: 1-6.

Granath, K. A. and P. Flodin. 1961. Fractionation of dextran by the gel filtration method. Makromol. Chem. 48: 160-171.

Granath, K. A. and B. E. Kvist. 1967. Molecular weight distribution analysis by gel chromatography on spehadex. J. Chromatogr. 28: 69-81.

Gray, I. 1953. Metabolism of plasma expanders studied with carbon14-labeled dextran. Am. J. Physiol. 174: 462-466.

Greenway, C. V. and R. P. Stark. 1971. Hepatic vascular bed. Physiol. Rev. 51: 23-65. .

Grotte, G. 1956. Passage of dextran molecules across the blood-lymph barrier. Acta Chir. Scand. Suppl. 211: 1-84.

Grotte, G., L. Juhlin, and N. Sandberg. 1960. Passage of solid spherical particles across the blood-lymph barrier. Acta Physiol. Scand. 50: 287-293.

126

Guyton, A. C. 1963. A concept of negative interstitial pressure based on pressures in implanted perforated capsules. Circulation Res. 12: 399-414.

Guyton, A. C. 1965. Interstitial fluid pressure: II. Pressure-volume curves of interstitial space. Circulation Res. 16: 452-460. ,

Guyton, A. C., H. J. Granger, and A. E. Taylor. 27 "* Interstitial fluid pressure. Physiol. Rev. 51: 527-563.

Guyton, A. C., J. Prather, K. Scheel, and J. McGehee. 1966. Interstitial fluid pressure. IV. Its effect on fluid movement through the capillary wall. Circulation Res. 19: 1022-1030.

Hannon, J. R., K. S. K. Chinn, and J. L. Shields. 1969. Effects of acute high-altitude exposure on body fluids. Fed. Proc. 28: 1178-1184.

Hannon, J. P., K. S. K. Chinn, and J. L. Shields. ^ 7 1 . Alterations in serum and extracellular electrolytes during high-altitude exposure. J. Appl. Physiol. 31:. 266-273.

Hjerten,- S. 1962. A new method for perparation of agarose for qel electrophoresis. Biochim. Biophys. Acta. 62: 445-449.

Hjerten,. S. 1964. The preparation of agarose spheres for chromatography of molecules and particles. Biochim. Biophys, Acta. 79: 393-398.

Hurtado, A. 1971. The influence of high altitude on physiology, p. 3-13. In Porter and Knight, High Altitude Physiology: Uardiac and respiratory aspects. Churchill Livingstone, Edinburgh and London.

Hurtado, A., C. Merino, and E. Delgado.^ 1945. Influence of anoxemia on the hemopoietic activity. Arch. Intern. Med. 75: 284-323.

Irisawa, A. and R. F. Rushmer. 1959. Relationship between lymphatic and venous pressure in leg of dog. Am. J. Physiol. 196: 495-498.

Jacobsson, S. and I. Kjellmer. 1964. Flow and protein content of lymph in resting and exercising skeletal muscle. Acta Physiol. Scand. 60: 278-285.

127

Joustra, M. K. ;1969. Gel filtration on agarose gels, p. 183-198. In J. Gerritsen, Modern separation methods of macromolecules and particles. Wiley-Inter science, New York.

Krzywicki, H. J., C. F. Consolazio, H. L, Johnson, W. C. Nielsen, Jr., and R. A. Barnhart. 1971. Water metabolism in humans during acute high-altitude exposure (4,300 m). J. Appl. Physiol. 30: 806-809.

Landis, E. M. 1926. The capillary pressure in frog mesentery as determined by micro-injection methods. Am. J. Physiol. 75: 548-570.

Landis, E. M'. 1927. Micro-injection studies of capillary permeability. II. The relation between capillary pressure and the rate at which fluid passes through the walls of single capillaries. Am. J. Physiol. 82: 217-238.

Landis, E. M. 1928. Micro-injection studies of capillary permeability. III. The effectoof lack of oxygen on the permeability of the capillary wall to fluid and to the plasma proteins. Am. J. Physiol. 83: 528-542.

Landis, E. M. 1930. The capillary blood pressure in mammalian mesentery as determined by the micro-injection method. Am. J. Physiol 93: 353-362.

Lathe, G. H. and C. R. J. Ruthven. 1956. The separation of substances and estimation of their relative molecular sizes by the use of columns of starch in water. Biochem. J. 62: 665-674.

Luft, J. H. 1966. Fine structure of capillary and endo-capillary layers as revealed by ruthenium red. Fed. Proc. 25: 1773-1783.

Marotta, S. F. and D. J. Boon. 1966. Femoral arterial^ circulation in nonhypoxic dogs at reduced barometric pressures. Am. J. Physiol. 210: 953-956.

Mayerson, H. S., C. G. Wolfram, H. H. Shirley, Jr., and K. Wasserman. 1960. Regional differences in capillary permeability. Am. J. Physiol. 198: 155-160.

Meyer, K., E. Davidson, A. Lindker, and P. Hoffman. 1956. The acid mucopolysaccharides of connective tissue. Biochim. Biophys. Acta. 21: 506-518.

128

Ogston, A. G. and T. F. Sherman. 1961. Effects of hyaluronic acid upon diffusion of solutes and flow of solvent. J. Physiol (London). 156: 67-74.

Palade, G. E. 1953. Fine structure of blood capillaries. J. Appl. Phys. 24: 1424.

Pappenheimer, J. R. 1953. Passage of molecules through capillary walls. Physiol. Rev. 33: 387-423.

Pappenheimer, J. R., B. M. Renkin,and L. M. Borrero. 1951. Filtration, diffusion, and molecular sieving through peripheral capillary membranes. A contribution to the pore theory of capillary permeability. Am. J. Physiol. 167: 13-46.

Pappenheimer, J. R. and A. Soto-Rivera. 1948. Effective osmotic pressure of the plasma proteins and other quantities associated with the capillary circulation in the hindlimbs of cats and dogs. Am. J. Physiol. 152: 471-491.

Parker, P, E. 1969. Capillary permeability to macro-molecules at normal and hypobaric pressure. M. S. Thesis, North Texas State Univ., Denton, Texas. 45p.

Pharmacia Fine Chemicals AB. 1970. Sephadex. Gel filtration in theory and practice. Pharmacia Fine Chemicals, Inc., Uppsala, Sweden. 63 p.

Poison, A. 1961. Fractionation of protein mixtures on columns of granulated agar. Biochem. Biophys. Acta. 50: 565-567.

Porath, J. I960. Gel filtration of proteins, peptides, and amino acids. Biochim. Biophys. Acta. 39: 193-207.

Reaves, T. A., Jr. 1971. Effects of high altitude exposure on capillary permeability. M. S. Thesis, North Texas State Univ., Denton, Texas. 62 p.

Renkin, E. M. 1952. "Capillary permeability to lipid-soluble molecules. Am, J. Physiol. 168: 538-545.

Renkin, E. M. 1964. Eighth Bowditch Lecture. Transport of large molecules across capillary walls. Physiologist, 7: 13-28.

Semple, R. E. 1957. An accurate method for the estimation of low concentrations of dextran in plasma. Can. J. Biochem. Physiol. 35: 383-390.

129

Scott, T. A., Jr. and E. H. Melvin. 1953. Determination of dextran with anthrone. Anal. Chem. 25: 1656-1661.

Shea, S. M. and M. J. Karnovsky. 1969. Vesicular transport across endothelium: simulation of a diffusion model. J. Theoret. Biol. 24: 30-42.

Shirley, H. H., Jr., C. G. Wolfram, K. Wasserman, and H. S. Mayerson. 1957. Capillary permeability to macromolecules.: stretched pore phenomenon. Am. J. Physiol. 190: 189-193.

Squire, J. R. 1951. Background to biological studies with dextran. Proc. Roy. Soc. Med. 44: 557-558.

Starling, E. H. and A. H. Tubby. 1894. On absorption from and secretion into the serous cavities. J. Physiol. (London). 16: 140-155.

Starling, E. H. 1896. On the absorption of fluids from the connective tissue spaces. J. Physiol. (London). 19: 312-326.

Sullivan, B. J. and L. D. DeGennaro. 1953. Microscopical observations of peripheral circulation at simulated high altitudes. J. Aviation Med. 24: 131-137.

Surks, M. I. 1966. Metabolism of human serum albumin in man during acute exposure to high altitude (14,100 feet). J. Clin. Invest. 45: 1442-1451.

Swann, H. G., A. V. Montgomery, J. C. Davis? Jr., and E. R. Mickle. 1950. A method for rapid measurement of intrarenal and other tissue pressures. J. Exp. Med. 92: 625-636.

Wasserman, K. and H. S. Mayerson. 1951. Exchange of albumen between plasma and lymph. Am. J. Physiol. 165: 15-26.

Wasserman, K. and H. S. Mayerson. 1952. Dynamics of lymph and plasma protein exchange. Cardiologica. 21: 296-307.

Wasserman, K. and H. S. Mayerson. 1952. Plasma, lymph, and urine studies after dextran infusions. Am. J, Physiol. 171: 218-232.

130

Wasserman, K. and H. S. Mayerson. 1954. Relative importance of dextran molecular size in plasma volume expansion. Am. J. Physiol. 176: 104-112.

Wasserman, K., J. D. Joseph, and H. S. Mayerson. 1956. Kinetics of vascular and extravascular protein exchange in unbled and bled dogs. Am. J. Physiol. 184: 175-182.

Wasserman, K., L. Loeb, and H. S. Mayerson. 1955. Capillary permeability to macromolecules. Circulation Res. 3: 594-603.

Weil, J. V., E. Byrne-Quinn, D. J. Battock, R. F. Grover, and C. A. Chidsey. 1971. Forearm circulation in man at high altitude. Clin. Sci. 40: 235-246.

Westergaard, H., S. Jarnum, R. Preisig, K. Ramstfe, J. Tauber, and N. Tugstrup. 1970. Degredation of albumin and IgG at high altitude. J. Appl. Physiol. 28: 728-732.

White, J. C., M. E. Field, and C. K. Drinker. 1933. On the protein content and normal flow of lymph from the foot of the dog. Am. J. Physiol. 103: 34-44.

Whitehorn, W. V., A. Edelmann, and F. A. Hitchcock. 1946. The cardiovascular responses to the breathing of 100 per cent oxygen at normal barometric pressure. Am. J. Physiol. 146: 61-65.