•ar/67531/metadc131162/... · garding the permeability of capillaries to macromolecules. in...

CAPILLARY PERMEABILITY TO MACROMOLECULES

AT NORMAL AND HYPOBARIC PRESSURE

APPROVED:

C3ULAJUCSL

Major Professor

Minor Professor

•Ar

Director of the Department of Biology

Dean of the Graduate School

CAPILLARY PERMEABILITY TO MACROMOLECULES

AT NORMAL AND HYPOBARIC PRESSURE

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

Paul E. Parker, B. S.

Denton, Texas

August, 1969

TABLE OP CONTENTS

Page

LIST OF TABLES iv

LIST OF ILLUSTRATIONS V

Chapter

I. INTRODUCTION 1

Capillary Permeability Altitude Studies Sephadex Chromatography of Dextrans Dextran Analysis Statement of the Problem

II. MATERIALS AND METHODS . 13

Capillary Permeability Standard Curve Sephadex Chromatography of Dextrans

III. RESULTS 22

Capillary Permeability Standard Curve Sephadex Chromatography

IV. DISCUSSION 36

Capillary Permeability Sephadex Chromatography Summary

BIBLIOGRAPHY 43

iii

LIST OF TABLES

Table Page

I. Analysis of Variance of the L/P Ratios of Four Dextran Molecular Weight Fractions at Normal and Hypobaric Pressure .31

iv

LIST OF ILLUSTRATIONS

Figure Page

1. Concentration of the 70,000 MW Dextran Fraction in the Plasma and Lymph at Ambient and Hypobaric Pressure 23

2. Concentration of the 150,000 MW Dextran Fraction in the Plasma and Lymph at Ambient and Hypobaric Pressure . . . . 24

3. Concentration of the 250,000 MW Dextran Fraction in the Plasma and Lymph at Ambient and Hypobaric Pressure . . . . . 25

4. Concentration of the 500,000 MW Dextran Fraction in the Plasma and Lymph at Ambient and Hypobaric Pressure 26

5. Rate of Appearanoe of the Dextran Fractions in the Lymph at Ambient and Hypobaric Pressure 28

6. Total Lymph-Plasma Concentrations of the Dextran Fractions at Ambient and Hypobaric Pressure 30

7. Lymph to Plasma Concentration Ratios of the Dextran Fractions at Ambient and Hypobaric Pressure . . 32

8. Molecular Weight Distribution of Dextran Fractions • • • * . . . . . 34

CHAPTER I

INTRODUCTION

Capillary Permeability

In 1896 Starling (17) formulated a hypothesis which

proposed that the rate of net fluid movement through the

capillary walls was proportional to the difference between

the hydrostatic and osmotic forces acting across the walls

of the capillaries,, In work with the intravenous injection

of hypertonic salt solutions, he attributed the resultant

decrease in hematocrit to the osmotic removal of fluid from

the interstitial spaces to the circulation during the

diffusion of the salt molecules through the capillary walls.

Landis' (9,10) studies with capillary pressures and

permeability in isolated mesenteric capillaries of the frog

offered supporting evidence that the net rate of fluid flow

through the capillary wall is proportional to the difference

between the capillary hydrostatic pressure and the protein

osmotic pressure of the plasma. Landis found that when the

capillary hydrostatic pressure exceeded the protein osmotic

pressure, filtration from the capillary occurred. Conversely,

when the protein osmotic pressure exceeded the capillary

hydrostatic pressure, absorption into the capillary occurred.

Similar findings were obtained by Pappenheimer and Soto-

Rivera (13) in studies of the net fluid movement through

capillary walls;, by the use of various plasma protein per-

fusions in the hindlimbs of dogs and cats. The net filtration

or absorption was determined by the rate of gain or loss of

weight of the limb, which was proportional to the difference

between the capillary hydrostatic and colloidal osmotic

pressure.

Wasserman and Mayerson (20) investigated the dynamics

of lymph flow and the exchange of protein between the plasma

and lymph. The observations made on the fluid shifts between

the plasma and interstitial fluid provided information re-

garding the permeability of capillaries to macromolecules.

In addition to the use of proteins as test molecules in the

study of capillary permeability, the possibility of the use

of dextrans as a research tool became apparent following

studies on the use of dextran infusions as plasma volume

expanders. Dextrans were found to be useful because they

leaked from the plasma into the interstitial fluid more slowly

than did proteins. In this study it was also shown that

capillary permeability was increased in dogs from which

relatively large volumes of blood had been removed in order

to lower the blood pressure.

Wasserman, Loeb, and Mayerson (21) employed dextran

fractions having average molecular weights from 10,600 to

412,000 to obtain plasma-to-lymph concentration ratios.

These ratios were found to be directly proportional to the

molecular weight of the dextran fraction. The plasma-to-

lymph concentration gradient was found to decrease, however,

as the volume of the dextran infusion was increased, due to

a stretching of the capillary pores, resulting in less resist-

ance to the passage of the dextran molecules through the

capillary walls. The phenomenon of the stretched capillary

pore was studied in greater depth by Shirley, Wolfram,

Wasserman, and Mayerson (16), using dextran fractions of

average molecular weights of 51,000 to 255,000. The con-

centration of the dextran fractions was followed in the plasma,

thoracic duct lymph, and right duct lymph. An increase in

the lymph-t0-plasma concentration ratios was noted with all

dextran fractions when the plasma volume was expanded. These

findings called for a modification of the capillary pore

concept to include a labile capillary pore size due to varia-r

tions in the plasma volume.

In 1960 Mayerson et al. (12) employed the dextran

fractions to determine regional differences in capillary

permeability. Dextran fractions with average molecular

weights ranging from 10,600 to 412,000 were infused simul-

taneously and their appearance in the cervical, intestinal,

and hepatic lymph was determined as lymph-to-plasma concen-

tration ratios. Based on their results, they proposed the

presence of two distinct pore sizesi one size allowing passage I

of molecules not greater than 250,000 molecular weight, and a

second size allowing passage of molecules of at least 412,000

molecular weight. The proposal was made that the passage of

molecules attributed to the second pore size may be due to

cytopemphis.

Chien, Sinclair, Chang, Peric,and Dellenback (3) employed

a dextran molecular-weight fraction as a test molecule in

their simultaneous study of capillary permeability to several

macromolecules. The preparations used were similar to those

employed by Mayerson et al. Since an increase in arterial

pressure and a decrease in lymph flow were observed with time,

there was a tendency for capillary permeability to decrease

with time. Chien et al. recommended that this type of pre-

paration be employed for study conditions in which capillary

permeability is increased* ,

L/P. permeability coefficient = F ( 1-L/P )

The permeability coefficient (ml/min) is the rate of clearance

of the macroraolecule. P is the flow rate of the lymph (ml/min)

and L/P is the lymph-to-plasma concentration ratio. Utilizing

the capillary permeability coefficient formulated by Renkin,

Johnson (8) devised a model for indicating the influence of

lymph flow on the transient and steady-state extracellular

space distribution of a solute. He found the degree of this

influence to be dependent on the relative magnitude of the

lymph flow as compared to the capillary permeability coeffi-

cient-capillary area product.

Wiederhielm (23) simulated the fluid balance at the

capillary level with an analogue computer program based on

experimental data in the areas of hydrostatic pressures,

surface areas, and capillary permeability. Physiological

experiments and clinical disease states produced data that

were in agreement with the data obtained by computer analysis.

Vogel, Ulbrich, and Gartner (19) studied the exchange

of plasma protein between the extra- and intravascular space

in the kidney. On the basis of their data, they presented

the hypothesis that the interstitial space contains compartments

which permit the diffusion of macromolecules at different

rates.

Altitude Studies

Sullivan and De Gennaro (18) observed a decrease in the

rate of arterial and venous blood flow in the web of the frog

at simulated altitudes of 60,000 and 80,000 feet. A change

in blood flow was first observed in the distal capillaries

of the web. The extent of the decrease in blood flow was

found to be dependent on the altitude, the length of time

the animal was subjected to the altitude, and whether or not

air was supplied to the animal during decompression. They

proposed that hypoxia accompanying a reduction in barometric

pressure was the primary cause of the reduction in blood

flow, since changes in the rate of flow were delayed when

air was supplied to the animal during decompression. The

changes in peripheral circulation due to simulated altitudes

were also examined by Girling and Maheux (5). In a series of

experiments, two groups of rabbits were subjected to simulated

altitudes of 20,000 and 30,000 feet, with group one breathing

100 % oxygen and group two breathing ambient air. Their

results indicated that the resistance to blood flow was

increased by a reduction in barometric pressure. The change

in resistance was not significant up to 20,000 feet, but at

30,000 feet the resistance to flow increased by more than 100

%. This increase in blood flow was not due to anoxia,

since resistance was increased to the same extent both in

animals breathing 100 % oxygen and those not breathing 100

% oxygen.

In a later study, Girling and Maheux (6) examined the

use of 100 % oxygen to determine if preoxygenation altered

the response of the peripheral resistance to lowered

barometric pressure. Rabbits breathing ambient air were

subjected to thirty to sixty minutes of breathing 100 %

oxygen. The results of this study indicated that the breathing

of 100 % oxygen had no demonstrable effect on the peripheral

blood flow at ground level. Whitehorn et al. (22) investigated

the breathing of 100 % oxygen at normal barometric pressures

in man. This was found to effect a reduction in both heart

rate and stroke volume. In spite of the reduction in cardiac

outputi the systolic blood pressure remained unchanged and

only a slight increase in diastolic pressure was observed,

Sephadex Chromatography of Dextrans

Fractionation of a mixture of molecules may be carried

out by gel filtration chromatography with a dextran gel such

as the Sephadex G-series. Andrews (1) employed Sephadex gel-

filtration chromatography to estimate the molecular weights

of proteins. The protein molecules fractionated ranged in

molecular weight from 3,500 to 670,000. A relationship between

the molecular weights of proteins and their elution volumes

was observed. This relationship was similar to that found

8

between the molecular weights of dextrans and their elution

volumes as noted by Granath and Flodin (7).

The chromatographic fractionation of dextrans on Sephadex

G-200 was carried out by Laurent and Granath (11). Dextran

samples were separated into fractions with molecular weights

ranging from 11,000 to 63,000. Molecular weights for the

dextran fractions derived from the diffusion and sedimentation

constants were in close agreement with the molecular weights

as determined by conventional methods.

Dextran Analysis

The quantitative determination of dextran in the blood

is complicated by the presence of blood glucose in the sample.

Most practical methods of dextran analysis involve a total

carbohydrate analysis employing the anthrone-carbohydrate

reaction. A method for quantitatively determining the dextran

concentration without interference from blood glucose was

proposed by Bloom and Wilcox (2). This method was based on

the fact that dextrans, unlike simple sugars, are resistant

to digestion with hot alkali and can be precipitated with

ethanol.

An alternate method for dextran analysis was proposed

by Semple (15). The method offered an accurate means of

estimating low concentrations of dextran in the plasma.

Semple's method was a modification of an earlier blood

carbohydrate analysis (4), but was improved to allow removal

of blood glucose by dialysis prior to total carbohydrate analy-

sis. The method also provided for treatment of the colori-

metric samples in order to reduce the rate of degradation of

the color intensity. i

Statement of the Problem

Since there are many reports in the literature concerning

circulatory shifts, the purpose of this investigation was to

study the effects of decreased barometric pressure on the

transcapillary movement of molecules# by monitoring the

macromolecular capillary permeability with lymph derived

primarily from the hepatic and gastrointestinal regions of

the dog.

CHAPTER BIBLIOGRAPHY

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

.2. Bloom, W.L. and M.L. Wilcox. 1951. Determination of dextran in blood and urine. Proc. Soc. Exper. Biol. Med. 76:3 —4•

3. Chien, S., D.G. Sinclair, C. Chang, B. Peric and R.J. Dellenback. 1964. Simultaneous study of capillary permeability to several macromolecules. Am. J. Physiol. 207:513-517.

4. Durham, W.F., W.L. Bloom, G.T. Lewis and E.E. Mandel. 1950. Rapid measurement of carbohydrate in blood. Public Health Rep. 65:670-673.

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

6. • 1953. Peripheral circulation and simulated altitude. Part II. J. Aviat. Med. 24:446-448.

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

8. Johnson, J.A. 1966. Capillary permeability, extracell-ular space estimation, and lymph flow. Am. J. Physiol. 211:1261-1263.

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

10* . 1928. Micro-injection studies of capillary permeability. 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.

10

11

11. Laurent, T.C. and K.A. Granath. 1967. Fractionation of dextran and ficoll by chromatography on Sephadex G-200. Biochem. Biophys. Acta. 136:191-198.

12. 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.

13. Pappenheimer, J.R. and A. Soto-Rivera. 1948. Effective osmotic pressure of the plasma proteins and other quantities associated with the capillary circula-tion in the hindlimbs of cats and dogs. Am. J. Physiol. 152x471-491.

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

15. Semple, R.E„ 1957. An accurate method for the estimation of low concentrations of dextran in plasma. Can. J. Biochem. Physiol. 35:383-389.

16. 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.

17. Starling, E.H. 1896. On the absorption of fluids from the connective tissue spaces. J. Physiol. 19x312-326.

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

19. Vogel, G., M. Ulbrich and K. Gartner. 1969. The exchange of plasma-albumin (I-^l-albumin) between extra- and intra-vascular space of the kidney, the lymphatic flow of macromolecules (polyvinylpyrro-lidone) in the kidney, under conditions of normal and furosemide inhibited tubular reabsorption. [in German, English summary] Pfluegers Arch. 305:104-112.

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

12

21. , L. Loeb and H.S. Mayerson. 1955. Capil-lary permeability to macromolecules. Circul. Res. 3:594-603.

22. 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.

23. Wiederhlelm, C.A. 1968. Dynamics of transcapillary fluid change. J. Gen. Physiol. 52*29-63.

CHAPTER IX

MATERIALS AND METHODS


Twelve adult mongrel dogs ranging in weight from 10.0

to 17.3 kg were employed in this study. Thirty minutes

prior to surgery, the animals were fed approximately 3

ounces of condensed milk to facilitate indentification of

the thoracic duct. The animals were anesthetized with an

intravenous injection of sodium pentobarbital at a concentra-

tion of 33 mg/kg of body weight. Injections were given in

i

the cephalic vein. The trachea was cannulated with a glass

cannula fitted with a "Y" connector. One arm of the "Y"

connector was attached to a 100 % oxygen source. The other

arm was fitted with a flutter valve.

After the animal's neck was shaved, the area of the

anastomosis of the external jugular and subclavian veins was

exposed by an incision directly over the external jugular

vein at the base of the neck. The thoracic duct was located

and prepared for cannulation. An incision was made over the

left femoral triangle, and the left femoral artery and vein

were exposed. An injection of sodium heparin at a concentra-

13

14

tion of 5 rag/kg of body weight was given in the femoral vein.

The left femoral artery was by-pass cannulated with Clay-

Adams Intramedic PE 190 polyethylene tubing. The thoracic

duct was cannulated with polyethylene tubing (PE 60). Lymph

from the thoracic duct was returned to the venous circulation

by a by-pass cannula (PE 100) into the transverse scapular

vein.

After the cannulations had been completed, control

samples of blood and lymph were taken. A 12 % solution of

a specific dextran molecular weight fraction was injected

in the left femoral vein at a dosage of 1 mg/kg of body

weight. Pour dextran molecular weight fractions, obtained

from Pharmacia Fine Chemicals, were used in this study. The

fractions had average molecular weights of 40,000, 150,000,

250,000, and 500,000. The time of injection of the dextran

fraction was designated as time 0.

The animal was placed in a decompression chamber and

the thoracic duct and left femoral artery by-pass cannulas

were connected to extensions which passed through the chamber

wall and through three-way luer stopcocks located outside

the chamber. This arrangement permitted sampling during

altitude studies.

The altitude chamber employed in this study was a steel

cylinder 58 inches in length and 30 inches in diameter, with

15

observation windows located along the sides. The pressure

was reduced in the chamber by means of a Welch Scientific

Company vacuum pump which was equipped with controls capable

of maintaining the chamber at a specific pressure. The cham-

ber control panel contained a Wallace and Tiernan absolute

pressure gauge and a rate-of-climb indicator. One hundred

% oxygen was supplied to the chamber through a series of

regulator valves located outside of the chamber. Blood

pressure readings were taken from the femoral artery with

a Statham PR 23-40-300 differential strain gauge and a

Brush amplifier with an ink-writing oscillograph.

Blood pressure recordings, hematocrits, blood, and

lymph samples were taken at fifteen-minute intervals following

the dextran injection. The blood volume was maintained by

replacing the fluid taken as blood and lymph samples with i

isotonic saline. Ground-level sampling continued for 120

minutes to allow time for equilibration of the injected

dextran (1). Denitrogenation was begun 30 minutes prior to

decompression by administering 100 % oxygen through the

tracheal cannula. After 120 minutes of equilibration the

animals were decompressed to 18,000 feet (380 mm Hg) at a

rate of 4000 feet per minute. The animal was maintained at

this simulated altitude for 60 minutes.

16

The blood and lymph samples obtained during the ground

level and altitude study were treated in the following

manner: the samples were centrifuged at 3000 rpm for 10

minutes to separate the fluid and formed elements. The

centrifugation was carried out on an Internation Equipment

Company model UV centrifuge with a head radius of 17 centi-

meters. A 1-ml aliquot of the plasma and lymph was depro-

teinized with 5 ml of cold 5% trichloroacetic acid. The

precipitated protein was removed by centrifugation at 3000

rpm for 20 minutes in the IEC centrifuge. The deproteinized

samples were dialyzed against distilled water to remove the

blood glucose. Dialysis was carried out for 150 minutes at

40° C in a 17-liter thermostatic water bath equipped with

a Sargent heater and circulator. During dialysis, an addi-

tional 21 liters of distilled water was passed through the

bath by continuous flow.

A 1-ml aliquot of the dialyzed samples was diluted

with 9 ml of distilled water to obtain a colorimetric sample

of sufficient color intensity to be read on the colorimeter.

A 2-ml aliquot of the diluted sample was taken and 4 ml of

anthrone reagent were added (200 mg anthrone per 100 ml

95% sulfuric acid)• The contents of the tubes were mixed by

swirling and placed in an Electric Hotpack Company model

17

106 water bath at 80° C for 6 minutes to develop the color.

Immediately after removal from the water bath, the samples

were placed in a freezer at -22° C for 10 minutes and stored

at 3° C to reduce the rate of color degradation (2).

The samples were transferred to cuvettes and read in a

Bausch and Lomb Spectronic 20 colorimeter against a distilled

water-anthrone blank at a wavelength of 625 mjx. The absor-

bance readings for the samples were converted to micrograms

per milliliter from the standard curve and corrected by

subtracting the control values and multiplying by 60 to

correct for a sixty-fold dilution of the original sample.

The corrected values were converted to milligrams percent

for presentation as data.

Standard Curve

A standard curve was plotted for determining the con-

centration of dextran in a sample from the absorbance reading

it produced on the colorimeter. The dextran fractions used

in determining the curve were from three molecular weight

rangesi 60,000 to 90,000, 100,000 to 200,000, and 200,000 to

300,000. The dextran fractions were obtained from the

Nutritional Biochemicals Corporation. Five serial dilutions

of a 100 f*g/ral solution of each dextran fraction were made

to produce six samples containing 100.0 /*g/ml, 50.0 ^g/ml.

18

25.0 yug/ml, 12.5 yag/ml, 6.25 yjig/ml, and 3*13 jxg/ml for each

fraction. Twenty sets of the serial dilutions of known

concentration were analyzed for carbohydrate content by the

anthrone-carbohydrate method previously described. The

absorbance values were averaged and plotted against the

corresponding concentration values on standard graph paper.


The three dextran fractions employed in this study were

those used in determining the standard curve. The dextrans

had molecular weight ranges from 60,000 to 90,000; 100,000

to 200,000 and 200,000 to 300,000. Fractionation of the

dextrans was carried out by descending gel filtration chroma-

tography on a Sephadex K 15/90 column packed with Sephadex

G-200. The Sephadex K 15/90 column has a diameter of 1.5

cm, a length of 90 cm and a bed volume of 154 ml. The column

was packed with 5 g of Sephadex G-200, which was allowed to

swell in a 0.2-M NaCl solution for 24 hours in an 80°-C water

bath. The gel suspension was allowed to settle partially and

the liquid layer removed by suction to eliminate fine particles %

of Sephadexo

The gel was resuspended in 0.2 M NaCl and connected to

a vacuum line for one hour to remove air trapped in the gel

suspension. The gel suspension was poured into the Sephadex

19

column, filling it entirely. Suspended gel particles were

allowed to sediment until a layer a few centimeters in thick-

ness had formed. The column elution outlet was opened and

the packing of the gel proceeded as the eluant was eluted.

Additional gel suspension was added at intervals until the

gel bed filled the column. The eluant reservoir was connected

to the column and the column was washed for 12 hours to stabi-

lize the gel bed. A filter paper disc was placed on the

surface of the gel bed to protect it during application of

the samples to be chromatographed. One-cubic-centimeter

samples were applied evenly over the surface of the bed with

a 3-cc syringe having a 10-cm polyethylene tubing extension.

Following application of the sample, the eluant reservoir

was opened to the column and the collection of eluted fractions

was begun. The rate of flow of the eluant was regulated by

a Mariotte flask.

Individual and combined chromatographic studies of the

dextran molecular weight fractions were made at various

concentrations to attempt to identify the elution products

of the column. The following dextran molecular weight fractions

were chromatographed individually: the 60,000 to 90,000 at

concentrations of 1.25 mg/ml and 5.0 mg/mlj the 100,000 to

200,000 at a concentration of 2.5 mg/ml and the 200,000 to

300,000 at a concentration of 1.25 mg/ml. A glucose sample

20

was chromatographed for reference at a concentration of 1.0

mg/ml. Three mixtures of the dextran molecular weight fractions

and glucose reference were chromatographed! (1) a mixture

of 60,000 to 90i000# 100,000 to 200,000, 200,000 to 300,000,

and the glucose reference at concentrations of 1.25 mg/ml,

2,5 mg/ml, 1.25 mg/ml, and 0.25 mg/ml, respectively; (2) a

mixture of 60,000 to 90,000, 100,000 to 200,000, and 200,000

to 300,000 at concentrations of 1.25 mg/ml, 2.5 mg/ml and

1.25 mg/ml, respectively; and (3) a mixture of 60,000 to

90,000 and the glucose reference at concentrations of 2.5

mg/ml and 0.5 mg/ml, respectively. Sixty fractions were

collected at ten-minute intervals for each chromatographic

study to insure the complete elution of the dextrans from the

column. Fractions were continuously collected by either a

Microchemical Specialties Company model 6550 or a Research

Specialties Company model E-3 automatic fraction collector.

The eluted fractions were analyzed for carbohydrate content

by the anthrone-carbohydrate method described above.


1, Perlc, B. 1967. Simultaneous study of capillary-permeability to different macromolecules. Bibl. Anat. 9s494.

2. Semple, R.E. 1957. An accurate method for the estimation of low concentration of dextran in plasma. Can. J, Biochem. Physiol. 35*383-389.

21

CHAPTER III

RESULTS


The transcapillary movement of molecules at decreased

barometric pressure was investigated by monitoring the

movement of dextran molecular weight fractions from the

plasma to the lymph. The concentrations of the dextran

fractions in the plasma and lymph are illustrated in figures

1 through 4» Measurable amounts of the molecular weight fractions

were observed in the lymph within 15 minutes. The plasma

dextran concentrations fell and the lymph dextran concentrations

rose as the macromolecules passed out of the capillaries and

into the interstitial spaces. The plasma and lymph concen-

trations of the fractions, with the exception of the 70,000MW

fraction, continued to approach each other with time until

a state of equilibrium was reached at approximately 120

minutes. The concentration of the 70,000MW fraction did not

reach equilibrium but continued to decrease in concentration

in the plasma and to a slight degree in the lymph until 165

minutes. The 150,000MW fraction attained equilibrium but

showed a maximal decrease in plasma dextran concentration of

22

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11.2% and an increase of 4.3% in lymph dextran concentration

after decompression* The 250,000MW fraction showed a decrease

of 0.6% from the equilibrated dextran concentration in the

plasma and an increase of 4.2% in the lymph dextran concentra-

tion after decompression. A decrease in the plasma dextran ,

concentration was observed prior to decompression in the 500,000

MW fraction. The concentration curve maintained a downward

trend until a 6.7% decrease from the equilibrated concentra-

tion had been reached. The plasma dextran concentration then

increased until a value 2.3% below the equilibrium concentra-

tion had been reached. A corresponding increase of 2.5% in '

dextran concentration, followed by a decrease of 6.3% from

the equilibrium concentration, was observed in the lymph.

The hematocrit values for the four molecular weight ranges

remained consistent throughout the ground and altitude study.

The rate of appearance of the four dextran molecular

weight fractions in the lymph is illustrated in figure 5.

The 70,000MW fraction appeared most rapidly in the lymph.

This fraction had an initial dextran concentration of 29

mg/100 ml, and it gradually disappeared from the lymph after

a maximal concentration of 82 mg/100 ml was attained at 75

minutes. The 150,000 and 250,000MSf fractions differed only

slightly in their rates of appearance and concentrations in

28

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29

the lymph. The maximal concentrations reached in the lymph

were approximately 95 mg/100 ml, considerably greater than the

maximal concentrations of the other molecular weight fractions.

The 500|000MW fraction had the least rapid rate of appearance

in the lymph, with an initial concentration of 2 mg/100 ml.

From a peak concentration of 81 mg/100 ml, dextran concentration

decreased 8.6% during simulated altitude.

Since the dextran molecular weight fractions pass from

the capillaries through the interstitial space and into the

lymphatics, the concentration of the dextran retained in the

interstitial space can be estimated by monitoring the dextran

concentrations in the plasma and lymph. The sum of the

lymph and plasma dextran concentrations of the four molecular

weight fractions is illustrated in figure 6. The 70,000MW

fraction showed a significant decrease in concentration that

the other molecular weight fractions did not exhibit during

the study• Decreases in the dextran concentration of the

150,000 and 500,000MW fractions were noted after decompression,

while the concentration of the 250,000MW fraction increased

slightly.

The lymph-to-plasma concentration ratios of the dextran

molecular weight fractions provide an estimate of the capillary

permeability to those macromolecules. Figure 7 illustrates

30

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31

the L/P ratios for the four dextran molecular weight fractions,

An analysis of the variance by rneans of a split-piot design#

table I, was employed for this data.

TABLE I

ANALYSIS OF VARIANCE OP THE L/P RATIOS OF FOUR DEXTRAN MOLECULAR WEIGHT FRACTIONS AT

NORMAL AND HYPOBARIC PRESSURE

Source of Degrees Sum of Mean Variation of Freedom Squares Square F

Blocks 2 .0739 .0370

MW (A) 3 .1476 .0492 1.6966

Error (a) 6 .1742 .0290

Treatment (B) 1 .0042 .0042 0.8077

Interaction (AB) 3 .0077 .0026 0.5000

Error (b) 8 .0418 .0052

Total 23 .4494

i

The analysis was calculated with the L/P ratios from the

ground level equilibration sample at 120 minutes, and the

final altitude sample at 180 minutes. The analysis found

no significant difference between L/P ratios of the four

dextran molecular weight fractions. A comparison of the L/P

H* tQ

-J f I

f rt

o

v 0J W 3

L'P Ratio

CM O 3 O a- 3'

•< >d o tr a# H H-O

O ft) 3 rt *1 a* ft H* o 3 »1 0* ft h** o w

3

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01 3 Hi *1 & o ft H* o 3 03 0# ft

f* 3 a* fl>

ft

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ift U1 fO O t/i in o o o

s 5» I (M

zz

33

ratios at normal and hypobaric pressure showed no signi-

ficant difference. A comparison of the L/i? ratios for the

four molecular weight fractions and the normal and hypobaric

pressure showed no significant interaction between the two

factors.

The molecular weight distribution of 70,000, 150,000 and

500,000MW fractions, as determined by analytical gel filtration

chromatography by Pharmacia, is illustrated in figure 8. The

dextran fractions consisted of a broad range of molecular

weights, with the designated molecular weight as the major

portion of the molecular-weight range. Dextrans with molecular

weights of 70,000 ± 10,000 constituted 20% of the 70,000MW

fraction. Dextrans with molecular weights of 150,000 ±

10,000 constituted 33% of the 150,000MW fraction. Dextrans

with molecular weights of 500,000 - 10,000 constituted 1% of

the 500,000MW fraction.

Standard Curve

The standard curve was plotted for determining the dextran

concentration in the colorimetric samples. The absorbance

values produced a straight-line graph when plotted on standard

graph paper. The linear relation between the absorbance and

the dextran concentration of the samples was identical for all

of the molecular weight fractions at concentrations between

34

I

%

%

1 \

o o o o o o o o o % * % o o o h in o

ih in 0 ( g

SH

V %

%

•̂GCrv

^Br

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00 o v£>

o

V"

o <N

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.o <N

Ô

uoTôâd uejE^xaa j o ûao aaa

CO c 0 4-> 0 <0 *W c u -p X a •a *w 0 o

rH c 0 X •ri 4J

JSC 2 S •Q •H U «P 0) *H

* tJ 4J rC tP •H 0) £ *-» (d |H p 0 <t) ,„ i n 0 a * i oo *

tr> •H &4

35

1 and 150 /j.g.

Sephadex Chromatography

The attempt to separate a mixture of three dextran

molecular weight fractions by Sephadex gel filtration chroma-

tography failed to be practical because of the inability to

determine a distinct fractionation pattern for the individual

dextran molecular-weight ranges. Chromatography of the

individual dextran fractions, with molecular-weight ranges

from 60,000 to 90,000, 100,000 to 200,000, and 200,000 to

300,000, produced identical results when the chromatographic

fractions were analyzed. The glucose reference was chromato-

graphically separated and identified from a mixture of glucose

and dextran fractions.

CHAPTER IV

DISCUSSION


The lymph-to-plasma concentration ratios of several

dextran macromolecular fractions were determined to provide

an estimate of the capillary permeability. The passage of

the dextran fractions from the plasma to the lymph is depen-

dent on the size of the proposed pores in the endothelial

wall of the capillary (1). An estimate of the relative capil-

lary pore size can be deduced by monitoring the L/P dextran

concentration ratios* If the population of capillary pores

consisted primarily of pores which were large, in relation to

the dextran molecules, there would be little resistance to

the passage of the macromolecules from the plasma to the lymph.

The resultant L/P ratio, for pores of this size, would approach

unity. Howevesr, if the major population of the capillary

pores consisted of pores that were small, in relation to the

dextran molecules, the passage of the macromolecules would be

restricted and the resulting L/P ratio would approach 0.

A major difficulty encountered in this study was the

inability to obtain narrow molecular-weight dextran fractions.

36

37

This must be considered when discussing the results, since the

capillary permeabilities were determined by the use of broad

range dextran fractions.

The 70#000MW fraction failed to reach an equilibrium

between the lyxnph and plasma (figure 1) „ Its consistent

decline in total lymph-plasma concentration (figure 6) suggests

its removal from the circulation by glomerular filtration.

Although dextrans with a molecular weight of 70,000 would be

classified as nonrenal-excretable (3), a portion of the mole-

cular weight range of the dextran fraction is within the renal-

excretable range of molecular weights (figure 8). The leveling

off of the lymph and plasma concentration curves at 165

minutes, in figure 1# suggests that the major portion of the

smaller molecules of the 70,000 fraction had been excreted

and that an equilibrium between the lymph and plasma dextran

concentrations was being established. The L/P ratio curve

(figure 7) for'the 70,000MW fraction appears uneffected by

the removal of the dextran fraction from the circulation.

This is apparently caused by the concurrent loss of dextran

from the plasma and lymph. A comparison of the L/P ratios at

ground level and simulated altitude for the effects of

hypobaric pressure cannot be made because an equilibrium

between the lymph and plasma dextran concentrations was not

reached until 165 minutes.

38

The 150,000 and 250,000MW dextran fractions underwent

similar alterations in lymph and plasma concentrations until,

a ground level concentration equilibrium was established

between the plasma and lymph (figure 2 and 3)• This suggests

the existence of a population of pore sizes greater than the

size of the 250,000MW dextran molecule, since there was no

significant difference between the 250,000 and 150,000MW

L/P ratios (figure 7). The significant decrease in the

plasma dextran concentration cannot be explained with the

present data, and more studies employing additional molecular

weight fractions should be carried out to investigate this

phenomenon.

The decreases in the plasma dextran concentration, lymph-

plasma, and L/P ratio curves (figures 4, 6 & 7) of the 500,000

MW fraction observed upon decompression suggest the movement

of the 500,000MW fraction into the interstitial spaces. The

smaller molecules of the fraction travel through the inter-

stitial spaces more rapidly than do the larger molecules (2).

Vogel et al. hypothesized the presence of compartments within

the interstitial spaces that allow the diffusion of macromole—

cules at different rates. The time required for the passage

of the large molecules through the interstitial space could

account for the disappearance of the dextran from the plasma

39

without a corresponding increase in the lymph concentration.

Assuming the above hypothesis, it is concluded that there was

an increase in the capillary permeability to macromolecules

and a probable increase in pore size during decompression*

Further investigation of the capillary permeability to

macromolecules is being carried out by use of a wider series

of dextran fractions with narrow molecular weight ranges.

The overlapping of the molecular weight ranges of the dextran

fractions used in this investigation masked the effects of

specific fractions and made the results difficult to interpret.

Variations in the results due to the use of mongrel experi-

mental animals also produced difficulties in interpreting

the data from this study. A more advantageous experimental

design would include a greater number of replicates, the

use of dextran fractions with narrow molecular-height ranges,

and the use of a homogeneous strain of animals.


Gel filtration chromatography with Sephadex G-200 was

employed to attempt the separation of a mixture of several

dextran fractions ranging in molecular weight from 90r000

to 300,000. Sephadex G—200 separates substances with molecular

weights smaller than about 200,000 by acting as a molecular

sieve. Molecules with molecular weights greater than 200,000

40

cannot penetrate the porous gel and are eluted first.

Molecules with molecular weights less than 200,000 penetrate

the gel and the rate of their elution is dependent on the degree

of penetration. The difficulties encountered with the dextran

fractions in this study are similar to those encountered with

the broad-range fractions used in the previous study. The

molecular weight ranges of the dextran fractions were so

broad that analysis of the chromatographic fractions showed

identical elution patterns for all of the dextran fractions.

Further chromatographic studies employing narrow-range dextran

fractions would possibly improve the elution patterns so that

individual dextran fractions could be identified.

Summary

The conclusions of this investigation may be summarized

as follows t

1. The capillary permeability to macromolecules was

increased by a hypobaric pressure of 380 mm Hg.

2. Further investigation of the capillary permeability

to macromolecules should be carried out, use being

made of dextran fractions having narrow molecular-

weight ranges.

3. The statistical validity of the study was reduced

by variation in the results, caused by the use of

41

mongrel animals.

4. An attempt to separate dextran fractions by Sephadex

gel filtration chromatography failed to produce spe-

cific fraction elution patterns because dextran

fractions with broad molecular weight ranges were

used.


1. Areskog, N.H. G. Arthurson, G. Grotte and G. Wallenius. 1964. Studies on heart lymph II. Capillary per-meability of the dog's heart using dextran as a test substance. Acta. Physiol. Scand. 62:218-223.

2. Vogel, G., M. Ulbrich and K. Gartner. 1969. The exchange of plasma-albumin (I^l_cilbumin) between extra- and intra-vascular space of the kidney, the lymphatic flow of macromolecules (polyvinylpyrrolidone) in the kidney, under conditions of normal and furosemide inhibited tubular reabsorption. fjLn German, English summaryj Pfluegers Arch. 305:104-112.

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

AO

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Bloom, W.L. and M.L. Wilcox. 1951. Determination of dextran in blood and urine. Proc. Soc. Exper. Biol. Med. 76:3-4.

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Durham, W.F., W.L. Bloom, G.T. Lewis and E.E. Mandel. 1950. Rapid measurement of carbohydrate in blood. Public Health Rep. 65:670-673.

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Johnson, J.A. 1966. Capillary permeability, extracellular space estimation, and lymph flow. Am. J. Physiol. 211: 1261-1263.

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

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44

. 1928. Micro-injection studies of capillary permeability. 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.

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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.

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<

Vogel, G., M. Ulbrich and K. Gartner. 1969. The exchange of plasma-albumin (I ^^-albumin) between extra- and intra-vascular space of the kidney, the lymphatic flow of mac-romolecules (polyvinylpyrrolidone) in the kidney, under

45

conditions of normal and furosemide inhibited tubular reabsorption. [in German, English summary! Pfluegers Arch. 305:104-112.

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

L. Loeb and H.S. Mayerson. 1955. Capillary permeability to macromolecules. Circul. Res. 3:594-603.

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.

Wiederhielm, C.A. 1968. Dynamics of transcaplllary fluid change. J. Gen. Physiol. 52:29-63.

•ar/67531/metadc131162/... · garding the permeability of capillaries to macromolecules. in...

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