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Maximal Femoral Artery Blood Flow During Cycle Ergometry Maximal Femoral Artery Blood Flow During Cycle Ergometry

Tucker W. Squires University of Montana, Missoula

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MAXIMAL FEMORAL ARTERY BLOOD FLOW DURING CYCLE ERGOMETRY

By

Tucker William Squires

B.S. Health and Human Performance, University of Montana Missoula, Montana 2013

Thesis

Presented in partial fulfillment of the requirements of the degree of Master of Science, Health and Human Performance

University of Montana

Missoula, MT

December 2015

Approval determined by

Dr. Sandy Ross, Dean of The Graduate School

Dr. Matt Bundle Health and Human Performance

Dr. Charles Dumke

Health and Human Performance

Dr. Reed Humphrey Dean, College of Health Professions & Biomedical Sciences

X

X

X

X

Signatures

Dr. Sandy Ross, Dean of The Graduate School

Dr. Matt Bundle (Committee Chair) Health and Human Performance

Dr. Charles Dumke Health and Human Performance

Dr. Reed Humphrey Dean, College of Health Professions & Biomedical Sciences

Dedication

I would like to dedicate this work to my family and friends who have stood by my side during my

education. Without their encouragement I would not be where I am at today.

Not a day goes by that I don’t think about the great gift of education afforded to me by the hard

work and forethought of my parents, William A. and Sherry L. Squires and my grandfather, Albert

Squires. Your kind words of encouragement will never be forgotten and your input always valued.

To my sister Ashton Ann Squires, I cannot begin to measure and describe how immensely

helpful your perspective and humor about the completion of my graduate education was. I hope one

day that I am able to encourage you to pursue your educational and career goals in the same way that

you encouraged me.

To Mariah R. Williams, I am especially grateful for your support when things seemed

insurmountable. You have always pushed me to complete the seemingly impossible and to pursue a

career path that truly makes me happy.

Thank you all for everything you have done for me. It means a great deal to me to be able to

count on you for support and encouragement.

Acknowledgements

I would like to thank my thesis committee, Dr. Matthew W. Bundle (Chair), Dr. Charles L. Dumke,

Dr. Reed Humphrey, and Dr. J.B. Alexander “Sandy” Ross, for their work on getting me to the point of

the completion of my Master of Science degree.

I would like to thank Tyler Gallo for his technical expertise with working with the Doppler

ultrasound, his steady hand and expertise made this study possible.

To Alex and Nathan, your support and alternative perspective on my writing are greatly

appreciated. Both of you pushed me to do my best during the most difficult times during my education.

Your support throughout my graduate work will never be forgotten.

To my friends and colleagues in the Undergraduate Advising Center, Office for Student Success,

and across campus at the University of Montana, I am greatly appreciative for your perspective on my

education, for allowing me the flexibility to work while completing my degree, and always being willing

to help me in any way that you could. I hope that I am able to contribute to your education and lives like

you contributed to mine.

Squires, Tucker W., M.S., Autumn 2015 Health and Human Performance MAXIMAL FEMORAL ARTERY BLOOD FLOW DURING CYCLE ERGOMETRY

Chairperson: Matthew Bundle Ph.D.

The delivery of oxygen to working muscle in order to support aerobic respiration has long been

recognized as one of the critical elements conferring endurance performance. Oxygen is transported

from the environment to the active cells by the cardiovascular system, which is classically understood to

deliver progressively greater amounts of gas to support increased levels of aerobic metabolism.

However, the vast majority of existing studies have used modes of exercise that activate relatively small

volumes of muscle and consequently have relatively small demands for oxygen delivery. We measured

blood velocity using pulsed Doppler ultrasound and simultaneous measures of femoral artery diameter

using sonography while highly fit subjects (n=5; mass: 65.25 ± 3.25 kg; VO2 max: 71.3 ± 3.4 mL O2 ·kg-

1·min-1) exercised across a range of cycling power outputs (130W - 410W) supported by aerobic

metabolism. Artery diameter was unchanged between rest (9.9 ± 1.7 mm) and exercise at all intensities

(9.9 ± 1.1mm). Blood velocity increased up to 15.9 ± 5.4 cm s-1 at the greatest common power output

completed by the subjects (370 W). Peak single artery flows were 11.4 l min-1 at 250W, at greater power

outputs (up to 370W) the measured values of flow were largely unchanged or even decreased slightly

(mean = 9.7 ± 0.6 l min-1). Our results are 12.3% greater than the previously highest measures known to

science and indicate the importance of the structural and functional factors within the respiratory

system necessary to supply sufficient oxygen in order to sustain strenuous exercise.

Table of Contents

CHAPTER ONE: INTRODUCTION 1

INTRODUCTION 1 PROBLEM 6 SIGNIFICANCE 6 GOALS AND RESEARCH QUESTIONS 7 LIMITATIONS 7 DELIMITATIONS 8 DEFINITION OF TERMS 8

CHAPTER TWO: REVIEW OF LITERATURE 9

CHAPTER THREE: METHODOLOGY 13

PARTICIPANTS 13 AEROBIC METABOLISM 13 CYCLE ERGOMETERY 13 FEMORAL ARTERY BLOOD VELOCITY 14 ARTERIAL DIAMETER 15 ARTERIAL BLOOD FLOW 16 ANALYSIS 17

CHAPTER FOUR: RESULTS 18

AEROBIC METABOLISM 18 CYCLE ERGOMETER 18 FEMORAL ARTERY BLOOD VELOCITY 19 ARTERIAL DIAMETER 20 ARTERIAL BLOOD FLOW 21

CHAPTER FIVE: DISCUSSION 21

VASCULATURE REQUIREMENTS FOR HIGH RATES OF AEROBIC METABOLISM 23 ARTERIAL DIAMETER 25 ARTERIAL BLOOD FLOW 25 LIMITATIONS AND OTHER CONSIDERATIONS 26

REFERENCES 28

1

Chapter One: Introduction

Introduction

The delivery of oxygen to working muscle to support an aerobic respiration has long been

recognized as one of the critical elements conferring endurance performance (Hill & Lupton 1923).

Oxygen is delivered from the environment to muscle through a respiratory cascade that begins at the

air-body interface of the lung and descends to the mitochondria. The flux rates throughout the cascade

are dependent upon pulmonary ventilation, cardiac output, blood carrying capacity, and mitochondrial

density volume within the exercising tissue (Taylor 1996 Weibel 1984). The process of supplying

sufficient oxygen for aerobic metabolism across each of these steps is accomplished by the movement

and transportation of oxygen across the membranes of the individual structures and steps in the oxygen

cascade. Thus, greater rates of oxygen delivery depend on the size and quantities of the diffusive

pressure differences and membrane thickness present in the anatomical differences found between

individuals (Liguzinski & Korzeniewski 2007 Saltin 1985 Taylor 1996 Weibel 1981, 1992).

For oxygen to descend to the mitochondria it must diffuse across progressively thinner

membranes while being transported to the working muscle, such that larger surface areas generally

provide for higher conductance while thicker tissue membranes lessen conductance (Maina & West

2005). The thickest membranes are found in the lungs and are approximately 1 µm thick while the

mitochondria have the thinnest membranes (Weibel 1984 Brooks et al., 2005 Maina & West 2005 Roy &

Secomb 2014). Rates of oxygen transport depend on diffusion from the alveolar tissues in the lungs to

the blood, transport by the blood to the cells, and diffusion from the blood to the mitochondria, leading

to the creation of a driving force for oxygen transportation through tissues according to the

concentration gradient due to the partial pressure of oxygen being lower within the body than the

2

environment (Weibel 1984 Brooks et al., 2005). Fick (1870) demonstrated the importance of oxygen

delivery and VO2 in the calculation and measurement of cardiac output as shown by Equation 1, where

CO is cardiac output, VO2 is oxygen uptake, and Ca-Cv is the difference in content for arterial and venous

blood (Fick 1870).

Equation 1:

Thus, whether considered at the mouth or the muscle level, Fick’s equation identifies the circulatory

influence on aerobic metabolism (Fick 1870 Taylor 1996 Wagner 1999 Wagner 1996).

The concept of symmorphosis proposes that the maximal fluxes across the different parts of the

oxygen cascade are matched to accommodate similar oxygen flux across the respective steps of the

oxygen cascade. This implies that no single step limits aerobic performance but a combination of all of

the previously mentioned factors, with each one contributing equally to the total VO2, supporting the

theory of having matching oxygen carrying capacity at the different levels of the oxygen cascade (Taylor

1996 Weibel et al., 1981, 1991 Weibel 1981, 1992). In contrast, cardiac output in humans compared to

animals has been hypothesized to be one of the limitations to aerobic performance due to its structural

and physiological limitations such as stroke volume and maximal heart rate in its ability to meet the

oxygen demand of the working muscle during high intensity exercise (Hoppeler & Weibel 2000). In

addition to cardiac output, there are other factors that affect maximal aerobic performance that include

pulmonary function, decreased transit time of blood in the capillaries due to increased blood velocity

during intense exercise, and desaturated blood (Liguzinski & Korzeniewski 2007 Taylor et al., 1996 Roach

et al., 1999 Hoppeler & Weibel 2000).

3

Due to the inherent limitations of the cardiovascular system, exact measures of blood flow and

delivery to working tissues at higher oxygen consumptions such as cycling are unclear. The available

literature indicates great variability in expected flows during similar activities and a general lack of

consensus in the measurements of blood flow even at similar mechanical power outputs (Table 1). Prior

to 2003, many investigators used the thermodilution technique, a highly invasive measurement, to

measure blood flow in different working limbs. Knee extension exercises were generally used in order to

isolate the amount of working muscle (Saltin 1985). More recently, many investigators have begun to

use less invasive measurement techniques such as Doppler ultrasound. Some studies using Doppler

ultrasound and thermodilution to measure femoral artery blood flows during cycling have shown

femoral artery blood flows of 18.7 L/min and 20.3 L/min (Harms 1997 Harms 1999). These measures are

similar to what could be predicted based on the measured VO2 and the distribution of cardiac output

during exercise of 85% of cardiac output being distributed to the working limbs (Astrad et al., 1963

Hermansen et al., 1970 Norton et al., 1990).

Investigations focused on cardiac output have demonstrated that athletes can have a maximal

cardiac output of nearly 27.7 L/min during cycling while other studies have indicated that some Olympic

athletes can have a cardiac output in excess of 30 L/min (Blomqvist & Saltin 1983 Calbet et al,. 2007

Hermansen et al., 1970). Other research has developed prediction equations for cardiac output that

predict maximal cardiac outputs based on absolute oxygen consumption of over 28 L/min in subjects

with an absolute VO2 max greater than 5 L·O2 /min which is near the current ceiling of human oxygen

uptake in the world’s most elite runners (Astrad et al., 1963 Lucia et al., 2006). However, outside the

scientific literature there are undocumented maximal oxygen consumptions of 97.5 ml O2 ·kg-1·min-1 by

Oskar Svenden, a cyclist, which was measured at University College of Lillehammer by Joar Hansen in

2012. Other maximal oxygen consumption tests done on Bjorn Daehlie, a Nordic skier, have measured

oxygen consumptions of 96 ml O2 ·kg-1·min-1. Another undocumented maximal oxygen consumption test

4

put Espen Harald Bjerke, another Nordic skier, at a maximal oxygen consumption of 96 ml O2 ·kg-1·min-

1 and an absolute VO2 of 7.3 L/min leading to a predicted cardiac output in excess of 45L/min (Astrad et

al., 1963). With over 85% of cardiac output being put towards lower limb blood flow during exercise it is

possible to have femoral artery blood flows of nearly 15 L/min in highly fit athletes and potentially

closer to 23 L/min in some of the world’s most fit individuals (Norton et al., 1990). Currently there are

only a few studies that demonstrate femoral artery blood flows similar to what can be predicted, see

Table 1.

In order to address this uncertainty and the large degree of variability in the current body of

literature it is our goal to measure both the oxygen consumption and blood flow simultaneously that is

necessary to support high intensity cycling. Our method allows for a more precise calculation of blood

flow than has previously been possible. Finally, the subjects that were recruited for this study are able to

achieve higher oxygen consumptions compared to other exercise modalities, such as knee extension,

that typically activate much less than half of the body’s aerobic capacity. Using highly fit subjects will

also allow for measurements of flow at the high oxygen uptakes that they can attain and maintain. We

anticipate being able to measure some of the highest femoral artery blood flows with the highest

degree of accuracy possible.

5

Author YearMeasurement

TechniqueExercise Mode

Anatomical Location

VO2 MaxPeak Mechanical

Power Output

Saltin 1988 Thermodilution Knee Extension Femoral 6.8 L·min-1 -

Bangsbo 1992 Thermodilution Knee Extension Femoral 5.16 L·min-1 -

Radegran 1997Doppler &

ThermodilutionKnee Extension Femoral 2.85 L·min-1 50 W

Radegran and Saltin 1998 Doppler Knee extension Femoral 8.53 L·min-1 70 WSaltin et. al ,. 1998 Thermodilution Knee Extension Femoral 10 L·min-1 -Saltin et. al,. 1999 Doppler Knee Extension Femoral 10 L·min-1 -

Bangsbo 2000 Thermodilution Knee Extension Femoral 5.39 L·min-1 65.3 W

Radegran and Saltin 2000 Thermodilution Knee Extension Femoral 6.06 L·min-1 5.03 LO2·min-1 68 W

Hoelting et. al,. 2001 Doppler Knee Extension Femoral 2.16 L·min-1 3.43 LO2·min-1 16.2 W

Krustrup et.al, 2001 Thermodilution Knee Extension Femoral 4.32 L·min-1 65 WOsada and Radegran 2009 Doppler Knee Extension Femoral 6.4 L·min-1 40 W

Ridout et. al,. 2010 Doppler Knee Extension Femoral 2.1 L·min-1 3.19 LO2·min-1 40.4 W

Christensen 2013 Thermodilution Knee Extension Femoral 5 L·min-1 60 W

Garten et. al,. 2014 Doppler Knee Extension Femoral 4 L·min-1 3.65 LO2·min-1 25 W

Voliantis and Secher 2002 Thermodilution Arm Cycling Brachial 3 L·min-1 4.04 LO2·min-1 153 W

Gonzales et. al, 2007 DopplerHand Grip Ergometry

Brachial 0.1 L·min-1 4.2 W

Harms et. al,. 1997 Thermodilution Cycle Ergometer Femoral 18.7 L·min-1 4.4 LO2·min-1 150 W

Harms 1999 Doppler Cycle Ergometer Femoral 20.3 L·min-1 4.4 LO2·min-1 -

Wetter 1999 Thermodilution Cycle Ergometer Femoral 16.87 L·min-1 4.52 LO2·min-1 170 W

Proctor 2001 Thermodilution Cycle Ergometer Femoral 9.4 L·min-1 2.06 LO2·min-1 140 W

Alonso & Calbet 2003 Doppler Cycle Ergometer Femoral 2.6 L·min-1 4.7 LO2·min-1 -

Proctor et. al,. 2003 Doppler Cycle Ergometer Femoral 5.9 L·min-1 100 W

Murias et. al,. 2013 Near Infared Cycle Ergometer Femoral 14.6 L·min-1 4.2 LO2·min-1 -

Spectroscopy

Table 1 demonstrates previous research and different techniques of blood flow measurement across a number of intensities and modalities of exercise from hand grip exercises, knee extension exercises, along with high intensity cycling.

* indicates VO2 Max measured during experimental exercise

Peak Blood Flow

-

-

----

--

-

-

-

-

6

Problem

Currently, the body of knowledge of blood flow to the lower extremities is limited during

dynamic whole body exercise, such as cycling. Due to the previous limitations of the thermodilution

technique it has been impractical to continuously measure blood flow for an extended period of time.

Due to the limited body of knowledge along with the limitations previously encountered, it is necessary

to explore and quantify blood flow during high intensity exercise that recruits a larger muscle mass than

previous studies. It is also currently unknown what the interactions between the mechanical aspects of

cycling are with the limitations of the cardiovascular system.

This experiment could also demonstrate whether the mechanical interactions between the

body and the cycle ergometer or the limitations of the cardiovascular system are a greater contributor

to the changes in blood flow across a wide array of intensities. These data could allow for further

discovery leading to new knowledge of the limitations of blood flow and oxygen delivery on cycling

performance to resolve issues not yet reported in the scientific literature.

Significance

Findings from this study would contribute to the body of knowledge to allow future research on

blood flow during whole body high intensity exercise and potentially lead to new estimations of blood

flow based on workload during cycling. This study could also demonstrate the highest femoral artery

blood flows know in the currently available scientific literature that are closer to what could be

theoretically predicted based on previous research. In addition, this study allows for the evaluation of

some of the existing hypotheses that are used to describe the mechanisms associated with exercise

induced hyperemia.

7

Goals and Research Questions

1) What are the highest flows through the femoral artery during high intensity whole body

exercise?

2) Does the relationship of blood flow and work rate remain linear during higher intensity

exercise as it does during lower intensity knee extension exercises?

3) To observe and quantify blood flow through the femoral artery at varying intensities up to

the aerobic limit while simultaneously measuring oxygen uptake and the forces applied to

the external environment in order to determine possible mechanisms of fatigue, such as

blood occlusion.

Limitations

1) Convenience sampling was used to recruit subjects from a previously known pool of

highly trained cyclists in western Montana.

2) Synchronization of data from multiple sources and sampled at different frequencies

could lead to inconsistencies due to human error. All synchronization was done by one

person who was trained to ensure consistency.

3) Subjects’ lifestyles were not controlled for including training volume, fatigue, or amount

of exercise the day prior to testing.

4) Subjects were allowed exogenous carbohydrate intake along with water ad libidum

throughout their exercise bouts. Some subjects elected to ingest carbohydrate and

water while others did not.

8

Delimitations

1) All subjects that participated were highly trained competitive male endurance athletes.

Definition of Terms

• Thermodilution: A technique that uses a catheter to inject an iced saline solution into the

femoral artery in order to achieve a 1 degree Celsius drop in temperature to measure blood

flow. Previous studies have used differing amounts of the saline solution to achieve the needed

temperature drop. Generally between 90-180 ml/minute were injected for up to 1.5 seconds for

a one time measurement at the end of a trial (Andersen & Saltin 1984).

• Inert Gas Clearance Method (133 Xe): The 133Xe clearance technique involves injecting Xe133

that was dissolved in saline into the belly of the active muscle and determining the rate of

clearance using a scintillation crystal detector (Grimby et al., 1966).

• Doppler Effect/Doppler Shift: Using phase shift in the sound waves that are returned as an

object moves and provides data that can be converted to fluid velocity (Gill 1979, 1979 Herr et

al., 2010).

9

Chapter Two: Review of Literature

At the onset of exercise, the cardiovascular system increases cardiac output almost immediately

while simultaneously constricting vessels to non-contracting tissues such as organs and organ systems

like the gastrointestinal system (Radegran 1997, Saltin et al., 1985 Andersen & Saltin 1984). The

increase in blood flow to the exercising muscles, also known as exercise induced hyperaemia, begins

within 2-10 seconds after the onset of exercise (Saltin et al., 1998). Highly fit subjects have

demonstrated an increased ability to dilate their blood vessels in order to meet the demands of the

working muscles (Calbet et al., 2006 Walther et al., 2008). Some Olympic level athletes can have a

maximal cardiac output of over 30 L/min that can be predicted by examining the increases in absolute

VO2 while exercising; for every 1 L/minute increase in VO2 there is an expected increase of 5 L/minute in

cardiac output. Olympic athletes exercising at an average VO2 of 5.38 LO2 /min lead to a cardiac output

of 30.4 L/minute (Blomqvist & Saltin 1983 Saltin 1988). During cycling, cardiac output increases linearly

along with total oxygen delivery until 80% of peak power. After 80% of peak power there is a plateau in

cardiac output due to decreases in stroke volume due to the Frank-Starling mechanism. The demand for

blood is met by increases in heart rate to maintain cardiac output. Despite the changes in cardiac

output, both heart rate and oxygen consumption increase linearly until exercise can no longer be

maintained. Maximal oxygen delivery and blood flow occurred between 73-88% of maximal power

output during cycling (Brooks et al., 2005 Mortensen et al., 2005).

Previous research has been done that suggests that when a subject approaches their maximal

work capacity, blood flow is limited to the exercising muscles due to the demands of the chest muscles

and other muscles used in breathing. Oxygen demand of the breathing muscles can be around 10% of

total oxygen demand but up to 15% of total oxygen demand in highly fit athletes at a given work

intensity. When the work of breathing was either decreased or increased there were resulting changes

10

in the blood flow to the exercising limb. A 50% change in the work of breathing (Wb) lead to a change of

2 L/min of blood flow which is approximately 11% of the normal value. A drop in work of breathing also

allowed for a subject to work at a lower VO2 by 9.3% while performing the same task. These decreases

in the work of breathing also improved performance in time to exhaustion experiments done on a cycle

ergometer (Harms 1997 Harms 2000 Wetter 1999).

Previous studies have measured femoral artery blood flow of up to 9.1 L/min using knee

extension exercises up to 100 watts with VO2 measurements of up to 1.42 L O2 /min using a

thermodilution technique ( Andersen & Saltin 1984 Rowell et al., 1986 Richardson et al., 1993 Radegran

1999). Previous studies using knee extension exercises were able to achieve an absolute VO2 between .8

L/min and 1.42 L/min (Radegran 1999). Since VO2 and blood flow appear to be related it is important to

explore femoral artery blood flow at oxygen uptakes greater than 2 L/min. This increase in blood flow is

demonstrated by a 33 fold increase in absolute flow from approximately .3L/min at rest to up to 10

L/min to the working limb at the femoral artery at VO2 peak for knee extensor exercises. However, with

these large flows during knee extension exercises it is possible that if these results were generalized to

other modalities of exercise such as cycling, the demand for blood flow would exceed the heart’s ability

to produce the required cardiac output since cardiac output is relatively fixed (Richardson et al., 1995

Savard et al., 1989). This rise in blood flow is directly proportional with power output during knee

extension exercise up to maximal flows of 6-10 L/min at about 80% of the subject’s maximal oxygen

uptake (Rowell 1988 Saltin et al., 1998). Previous studies using a knee extension ergometer and

thermodilution culminated in a workload of 99 Watts and yielded flows through the femoral artery of up

to 10 L/min (Radegran 1999). These near instant adaptations to exercise work to meet the oxygen

demand of the exercise bout that the body has undertaken.

11

Historically, there have been many different attempts and techniques used to measure blood

flow in mammals and humans. These techniques all have inherent limitations that affect the accuracy of

their measurements. Some early attempts at measuring blood flows in mammals involved letting blood

from the subject. One of these early attempts was in 1887 in the levator labii superioris muscles of the

lip of a horse. While the horse was chewing oats, researchers collected blood without anesthesia

directly from the levator labii superioris muscles to measure blood flow (Chauveau & Kaufmann 1887).

In exercising humans, knee extension exercise has been the mode of choice for the study of blood flow

due to the ability to restrict movement in the leg and to isolate the amount of muscle mass that is active

during exercise. Knee extension exercise was coupled with the thermodilution technique to measure

blood flow at different work rates during exercise. This technique uses a catheter to inject an iced saline

solution into the femoral artery in order to achieve a 1 degree Celsius drop in temperature to measure

blood flow. Previous studies have used differing amounts of the saline solution to achieve the needed

temperature drop. Generally between 90-180 ml/minute, were injected for up to 1.5 seconds for a one

time measurement at the end of a trial (Andersen & Saltin 1984).

The thermodilution technique is limited in whole body exercise, such as cycling, due to subject

comfort with the catheter that must be placed into the subjects’ blood vessel. This technique is also

limited by it not being able to collect continuous date owing to the need to inject cooled saline into the

circulatory system. In order for this technique to continuously measure flow during exercise such a large

volume of saline would need to be introduced into the circulatory system that there is the potential for

oxygen carrying capacity of the blood to be changed and the potential for hypovolemia. Other

techniques have measured the clearance rates of inert isotopes of 133 Xenon (133Xe) from the belly of

working muscles. This technique has been shown to underestimate the blood flow when compared to

the thermodilution technique (Radegran 1999). The 133Xe method was potentially limited by the rate of

diffusion being lower than expected at higher intensity exercise out of the muscle belly leading to its

12

underestimation of blood flow (Bonde-Petersen 1975). Due to the limitations of the thermodilution and

Xe133 techniques, using ultrasound to measure blood flow was suggested in the late 1970s as a less

invasive alternative (Gill 1979, 1979 Grimby et al., 1966).

Using the Doppler Effect, calculation of blood velocity is possible so long as the angle of

approach of the Doppler probe is known or can be estimated (Gill 1985). Early studies using Doppler

ultrasound had RMS error rates of 14% and maximal random errors of 32% due to under and

overestimating the angle of insonation and artery diameter (Gill 1979, 1985). Flow is calculated by

determining the diameter of the artery based on the ultrasound videos collected from each trial along

with the blood flow velocities from each trial. Collection of blood velocity data along with acquiring

images of the artery has proven difficult over long periods when multiple ultrasound machines were

needed for both imagining and non-imaging data. Using a device to convert Doppler signal to an analog

signal via Fast Fourier transform (FFT) it is possible to collect data on any number of data acquisition

platforms. Having this technique available allows for real time data collection as opposed to using

multiple probes, multiple modes, or even multiple machines to collect data on blood flow during

exercise. This device was validated using a cornstarch solution that resembled blood cells on the

ultrasound machine. This solution was run through a loop of Tygon tubing and the known flow from the

pump was measured using the signal converter and Doppler ultrasound (Herr et al,. 2010).

13

Chapter Three: Methodology

Participants

Five male subjects (mean ± SD; Age: 33.2 ± 2.7 years; Mass: 65.25 ± 3.25 kg; VO2 max: 71.3 ± 3.4

mL O2 ·kg-1·min-1) who were currently training actively and competing as endurance athletes

volunteered and provided written informed consent in accordance with guidelines set by the

Institutional Review Board at the University of Montana.

Aerobic Metabolism

Rates of oxygen uptake (ml O2 ·kg-1·min-1) were determined by indirect calorimetry, using the

fractional and volumetric analysis of the subjects’ expired gases. The expired gas was collected and an

aliquot of expired gas was analyzed by a computerized metabolic system (TrueOne 2400, ParvoMedics,

Sandy Utah). Reported means from expired gases were determined from the measures obtained in the

final 2 minutes of each trial. Data are from the final 2 minutes of a 5 minute bout. Maximal rates of

oxygen uptake were determined from the greatest 30 second average obtained during the testing

procedures.

Cycle Ergometery

Subjects undertook a progressive discontinuous test to VO2 max test on a cycle ergometer

(Velotron, Racermate, Seattle, Washington). The subjects performed all testing at a constant cadence of

80 revolutions per minute, using pedal cranks of 0.165 m and completed 5 minute bouts of exercise until

they were unable to complete the bout despite putting forth a maximal effort. The test was initiated at a

power output of 130 watts and increased by an increment of 40 watts until the point of failure. Visual

feedback for cadence was provided to the subjects with a computer monitor connected to and mounted

within their field of view to ensure adherence to the prescribed cadence. Subjects were allowed as

much rest between trials as they needed to feel fully recovered and generally took 5 minutes between

14

trails at lesser power outputs and 15 minutes or more between higher power output trials. The

measurements of power output and cadence were obtained at 10 Hz using the Velotron package

software. Average pedal forces applied were estimated using the method described by Bundle &

Weyand 2011.

Equation 2:

ω•=

rPowerFpedal

Where Fpedal is the average force applied throughout a revolution, r is the ergometer crank arm and ω is

the angular velocity in units of radians per second.

Femoral Artery Blood Velocity

Pulsed Doppler ultrasound was used to insonate the right common femoral artery between the

bifurcation of the deep femoral artery and the inguinal ligament with a linear array transducer due to its

accuracy when measuring blood velocity in medium depth arteries (Acuson 8L5 Probe, 8.0 MHz,

Washington D.C. USA & Siemens Acuson Sequoia 512). The Doppler frequency spectrum data was

converted to a mean arterial velocity using the Fast Fourier transform (FFT) and a previously established

calibration relationship (Gallo 2014). The FFT and an analog transformation were performed with a

custom designed electronic device (DAT) described by Herr et al, 2010. The analog signals from the DAT

were recorded to a computer using an A/D board (Digidata 1330, Molecular Devices, California, USA)

that sampled at 3012 Hz.

The video record of the arterial sonogram was used to provide real-time feedback to the

researcher performing the insonation. This permitted more rapid replacement of the probe during

periods when the movement of the leg caused the probe to be focused on the adjacent tissues. Each

video record was also captured to a computer (Canopus ADVC-55, Quebec Canada), and used to exclude

portions of the data wherein the probe was not properly aligned with the femoral artery.

15

Arterial Diameter

We analyzed the video record (30 Hz) of the sonograms obtained throughout each trial and performed

frame-by-frame (>300,000 frames) digitization (Tracker Open Source, National Science Foundation, USA)

of the arterial cross section at the location of the ultrasound probe’s focus. The video image (Figure 1)

includes a visual representation of the frequency spectrum, sonogram, and the specific instrument

parameters used to obtain the measures. Linear distances obtained by digitization were calibrated

based on the x and y-axis provided by the ultrasound device.

Figure 1: The sonogram from the Doppler ultrasound shows the real-time feedback showing the femoral artery proximal to the deep femoral bifurcation along with the linear array grid that was used to calibrate the diameter calculations in pixels to convert to artery diameter. Blood velocity can also be seen at the bottom of the sonogram on a heartbeat-by-beat basis.

16

We excluded from further analysis the regions of data where the video record indicated that the

ultrasound probe’s insonation focus was not within the lumen of the common femoral artery. In this

study the subjects completed a total of 176.5 minutes of cycling of which 37.36 minutes, or 21.2%, of

the video record was excluded from further analysis. We assessed the extent of exercise induced artery

dilation by comparing the artery diameters obtained at rest, prior to the initiation of the cycling bout, to

the mean value obtained throughout the effort. To evaluate the possibility of greater numbers of

excluded frames occurring during higher vs lower power output trials, we tallied the number of frames

excluded in the three least powerful vs three most powerful trials administered to the subjects. Of the

total number of frames 51% occurred in the lesser power trials whereas 49% in the trials with greater

power output.

Arterial Blood Flow

The artery diameter data obtained from the video record was synchronized to the blood velocity

data to obtain instantaneous measures of artery diameter and blood flow throughout each administered

bout of cycle ergometry. Single leg femoral artery blood flow was calculated as the product of artery

cross sectional area and the velocity of the blood traveling through this space in accordance with

Equation 3:

BVADFlowBlood •

=

2

2_ π

Where AD is the arterial diameter in centimeters and BV is blood velocity measured in cm·s-1. This

treatment considers the cross-sectional shape of the artery as a circle. Total blood flow to the legs was

considered to be twice the single leg value.

17

Analysis

The blood velocity, arterial diameter and blood flow data, were synchronized, conditioned and analyzed

on a revolution-by-revolution basis in a using a custom written software package (Igor Pro 6.37,

Wavemetrics, Oregon USA).

Data are presented as means with their respective standard deviations. Statistical comparisons (Paired

Samples T-Test) were performed with an a priori significance level of α =0.05.

18

Chapter Four: Results

Aerobic Metabolism

VO2 increased linearly with power output up to max for all subjects (slope: 0.7746 ± .429,

intercept: 0.13327 ± .63236, R2: 0.98 ± .02). VO2 max across subjects was 71.31 ± 1.1 mL O2 / kg·min-

1 and 4.64 ± .17 L O2 · min-1. Two subjects were unable to achieve the desired maximal oxygen uptake of

70 mL O2 / kg·min-1 (minimum VO2: 67.97 mL O2 / kg·min-1 and 4.53 L O2 · min-1). Respiratory Exchange

Ratios (RER) were 1.10 ± 0.01 (minimum: 1.053 maximum: 1.12) across subjects indicating reliable

measures of VO2 max in the discontinuous power incremented cycling protocol.

Cycle Ergometer

Participants undertook the incremental protocol with all of the subjects completing the following bouts:

130W, 170 W, 210W, 250W, 290W and 330W. Three of the subjects reached the point of failure during

the 370W trial and the remaining two were unable to complete the 410W bout. From the measured

maximal oxygen uptakes and the highly linear relationships of VO2 and mechanical power output we

interpolated the minimum wattage necessary to elicit each subject’s VO2 max (370 ± 21 W). The power

outputs, obtained during the constant cadence trials, were achieved with average forces applied against

the pedals of the cycle ergometer of 94 N, 123 N, 152 N, 181 N, 210 N, 239 N, 268 N, and 297 N,

respectively.

19

Femoral Artery Blood Velocity

The per trial means of arterial blood velocity generally increased in relation to trial power

output and the greater metabolic demand of the working tissue (Figure 2). Mean trial velocities were

the least at 170 W with a velocity of 11.5 ± 5.3 cm s-1 and reached 15.9 ± 5.4 cm s-1 at the greatest

common power output. The greatest mean velocity measure obtained in the study was obtained with

an intermediate power output of 250 W.

Figure 2: Single leg femoral artery blood velocity at varying power outputs for all subjects. Part A shows a single subjects’ blood velocity (130W, 170W, 210W, 250W, 290W, 330W, 370W, and 410W) while Part B shows group averages of blood velocity at all common power outputs (130W, 170W, 210W, 250W, 290W, 330W, and 370W).

20

Arterial Diameter

Artery diameter was measured continuously throughout the duration of each trial by

digitizing the sonogram indicating the location of pulsed Doppler insonation. Femoral artery

diameter at rest prior to the onset of the exercise trials was 9.9 ± 1.7 mm while the mean value

determined continuously, throughout all the administered bouts of exercise, was similar (p= 0.96)

and measured 9.9 ± 1.1 mm. Similarly, artery diameter was indistinguishable (p=0.56) between the

trials with the least and greatest common power outputs (i.e. 370 W).

Subject

Power Output (Watts)

1 2 3 4 5 Average SD

Rest 8.2 (1.4) 9.8 (1.8) 9.8 (0.5) 10.7 (2.3) 10.5 (0.6) 9.9 1.7

130 9.9 10.7 10.5 10.9 9.5 10.3 0.6

170 12.0 10.8 11.4 11.4 0.6

210 9.7 7.8 9.7 9.2 9.6 9.2 0.8

250 9.1 8.0 9.9 9.8 10.5 9.5 0.9

290 10.9 10.3 9.8 10.8 10.4 0.5

330 8.5 8.0 10.4 11.5 10.5 9.8 1.5

370 8.3 10.3 10.3 7.2 10.9 9.4 1.6

410 9.6 9.6 9.6 0.0

Exercise 9.4 (0.9) 9.5(1.6) 10.2 (0.3) 9.9 (1.3) 10.5 (0.7) 9.9 1.1

Table 2: Artery diameters for all subjects at rest and during exercise mean(SD). Blanks indicate missing or incomplete data.

21

Arterial Blood Flow

The per trial means of single leg arterial blood flow increased with trial power output up to the level

eliciting the maximum blood velocity, i.e. 250 W and were 11.4 l min-1(Fig. 3 & Fig. 4). At greater power

outputs the measured values of flow were largely unchanged or even decreased slightly. Specifically,

flow values averaged 9.7 ± 0.6 l min-1 across the three greatest common power outputs administered.

Thus the greater rates of oxygen uptake were achieved with relatively lesser volumes of femoral artery

blood flow during the most demanding trials that the subjects completed. The single leg values

presented indicate that up to 22.8 l min-1 was delivered to both legs during the cycle ergometry trials

administered.

Figure 3: Single leg femoral artery blood flow fluctuations across the length of a 5 minute bout of cycling for one subject.

22

Figure 4: Single Leg femoral artery blood flow at varying power outputs for all subjects. Part A show a single subjects’ blood flows through the femoral artery (130W, 170W, 210W, 250W, 290W, 330W, and 370W) while Part B shows group averages of blood flow at all common power outputs (130W, 170W, 210W, 250W, 290W, 330W, and 370W)

23

Chapter Five: Discussion

The goal of this study was to obtain femoral artery blood flow measures in highly fit subjects

during exercise intensities representing the upper limit of cardiovascular and respiratory function. Here

we were largely successful. We obtained continuous, simultaneous, and direct measures of the two

contributing components of blood flow, blood velocity and femoral artery diameter, during cycle

ergometry at varying power outputs from 130 to 410 Watts. The incremental protocol used permitted

the evaluation of whether external mechanical factors such as the intermuscular pressure generated to

apply force against the pedal during the phase involving muscular contraction altered the delivery of

blood to the active muscle beds (Figure 5). This study also allowed for the basic measurements of blood

flow across the range of possible aerobic performances, from relatively easy power outputs all the way

up to the aerobic limit of highly fit subjects. Our maximum measure of single leg blood flows of 11.4

l·min-1 (expected 2 legged flows of 22.8 l·min-1 ) , compare favorably and are slightly greater, 12%, than

those reported in the only other known study to obtain these measures during dynamic highly aerobic

exercise (Harms 1997,1999).

Figure 5 a Figure 5 b

Figure 5: Part A demonstrates the changes in blood velocity throughout the pedal stroke based on the mechanical interactions between the leg and the cycle ergometer and shows a spike in velocity after the start of the upstroke during a low wattage trial. Part B demonstrates the changes in blood velocity throughout the pedal stroke based on the mechanical interactions between the leg and the cycle ergometer and shows a spike in velocity after the start of the upstroke during a high wattage trial.

24

Vasculature Requirements for High Rates of Aerobic Metabolism:

For subjects to meet the high metabolic demands of strenuous aerobic exercise there are two

main options available to increase the delivery of oxygen to mitochondria, either increase the flow of

oxygen or increase the extraction of oxygen from the blood in the working tissue beds. The measured

oxygen consumption rates of 4.64 ± 0.17 LO 2 ·min-1 support a predicted cardiac output of greater than

30 l·min-1 , further suggesting blood flows up to 15 l min-1 occurring in the femoral artery of each leg

(Astrad et al., 1963 Hermansen et al., 1970). Our observation of similar levels of femoral artery blood

flow within the 3 greatest common power outputs, Figure 2, suggests that during the trials administered

the cardio-respiratory system of these subjects utilized both strategies, primarily relying on increased

delivery at low and modest intensity efforts and extracting greater fractions of oxygen during the trials

requiring the greatest metabolic rates. The measured velocity data further inform the circulatory flow

patterns used by the subjects during these trials. The generally increasing trial average blood velocities,

Figure 2, used to support higher power outputs produced similar arterial flow rates within the

contraction cycle. The most likely explanation for these results is the peak velocities necessary to

elevate the trial average velocity values occurred during lesser fractions of the contraction cycle during

greater vs lesser power outputs. These data suggest applied pedal forces of 181 N and greater are

sufficient to alter the periods within the cycling revolution when blood is able to reach the muscular

beds.

25

Arterial Diameter:

The data indicate that the femoral artery diameter changes little with the onset of exercise nor

does this artery appear to dilate during intense aerobic exercise. Here we measured arterial diameter

continuously throughout the bouts of exercise. These measures suggest the long held practice of many

laboratory groups of using a single arterial sonogram obtained at rest either prior or subsequent to

exercise provides a reliable estimate of lumen size for the common femoral artery (Hoelting et al., 2001

Osada & Radegran 2009). Using a representative femoral artery diameter at the onset of exercise would

help to eliminate a significant amount of data analysis and artery digitization due to the lack of change in

diameter during exercise. This is especially useful during dynamic tasks similar to the cycle ergometry

used here because of the generally slow refresh rate of the sonogram imagery which lessens the quality

of the video record and contributes to the inability to fully automate this analysis step. Previous

research indicates an average femoral artery diameter of 10. ± 1.0 mm in healthy and active males with

similar ages to the subjects in this study (Sandgren et al., 1999).

Arterial Blood Flow:

Following the onset of an exercise workload, arterial flow progressively increased for the first

35-40 s before reaching the steady state level maintained throughout the remainder of the effort. The

increases seen at the beginning of each trial followed a similar time course to the increases in oxygen

uptake occurring during this same period (Krustrup et al., 2009). Blood flow increased 5-fold during the

initial period and varied by as much as 6.6-fold between the onset of exercise and the peak flow

measured in the 370W trial illustrated in Figure 4. While our data represent the greatest blood flows yet

measured during exercise, we did not observe the same highly-linear trends as observed by Saltin and

colleagues and many others during knee extension exercises (Table 1) (Saltin et al., 1985). Moreover,

their observed blood flows of up to 9 l·min-1 obtained during single leg knee extension exercise, and

those of many studies presented in Table 1, are by comparison surprisingly large, given our greatest

26

measure was only 25.5% higher than these, but was elicited from an activity that uses roughly 2-fold

more muscle and rates of oxygen uptake that are similarly 2-fold greater (Sundberg & Bundle 2015).

Previous reports of femoral artery blood flow during cycling reported flows in excess of 20 l·min-1 for

two legged flows whereas our numbers are reported as single leg values as we only measured one leg

during cycling. While flows in both femoral arteries should be roughly equal during cycling we are

unable to directly verify this based on our methodology (Harms 1997 Harms 1999).

Limitations and other considerations:

Due to the methods used in this study our measures of blood flow are conservative because we

eliminated portions of the data set during which time the ultrasound probe was not focused in the

center of the femoral artery, we used a filtering routine to remove spurious short duration spikes in

velocity, and we reported the trial means as 20-second averages obtained during the final two minutes

of the trial to most appropriately compare these measures with the steady state rates of oxygen uptake

we also obtained from these trials. Additional limitations with the method used caused the Doppler

ultrasound probe to not be on the artery for the full trial due to the modality of exercise involving rapid

leg movement which resulted in the probe occasionally being on the femoral vein or other adjacent

tissue.

With our current methods there are also limitations in our ability to measure artery diameter

with the same precision as blood velocity. Using the Digidata and A/D board we were able to sample

data at 3012 Hz whereas the artery diameter was measured from 30Hz video. However, the refresh rate

of the sonogram image was essentially 10Hz, and occasionally several consecutive frames of data

needed to be omitted from the digitizing procedure because no clear image was visible in the sonogram,

despite accurate measures from the pulsed Doppler portion of the equipment. This discrepancy in

sampling frequency could lead to a lower precision in blood flow measurements owing to the need for

27

interpolation of data points needed in order to successfully analyze the data that was collected.

Additional work on analysis methods and further modification of equipment is required in order to have

a more similar refresh and sampling rate between the Doppler ultrasound sonogram and the blood

velocity data that is recorded digitally.

While this study provides significant insight into the changes in femoral artery blood flow across

a wide range of power outputs and blood flow fluctuations during exercise, there is still work that

remains to further the understanding of femoral artery blood flow during cycling. In these experiments

we observed the highest femoral artery blood flows known to science.

28

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