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Global REACH 2018: High blood viscosity and hemoglobin concentration contribute to
reduced flow-mediated dilation in high-altitude excessive erythrocytosis
Joshua C. Tremblay, MSc1; Ryan L. Hoiland, PhD2; Connor A. Howe, BHK2; Geoff B. Coombs,
MSc2; Gustavo A. Vizcardo-Galindo, BSc3; Rómulo J. Figueroa-Mujíca, BSc3, Daniela
Bermudez, BSc3; Travis D. Gibbons, MSc4; Benjamin S. Stacey, BSc5; Damian M. Bailey, PhD5;
Michael M. Tymko, MSc2; David B. MacLeod, MBBS6; Chris Gasho, MD7; Francisco C.
Villafuerte, PhD3, Kyra E. Pyke, PhD1; Philip N. Ainslie, PhD2
Affiliations:
1Cardiovascular Stress Response Laboratory, School of Kinesiology and Health Studies, Queen’s
University, Kingston, Ontario, Canada
2Centre for Heart, Lung & Vascular Health, Faculty of Health and Social Development,
University of British Columbia – Okanagan, Kelowna, British Columbia, Canada
3Laboratorio de Fisiología Comparada, Departamento de Ciencias Biológicas y Fisiológicas,
Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Perú
4School of Physical Education, Sport & Exercise Sciences, Division of Sciences, University of
Otago, Dunedin, New Zealand
5Neurovascular Research Laboratory, Faculty of Life Sciences and Education, University of
South Wales, Wales, UK
6Human Pharmacology and Physiology Laboratory, Department of Anesthesiology, Duke
University Medical Center, Durham, North Carolina, USA
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7Division of Pulmonary, Critical Care, Hyperbaric and Sleep Medicine, Loma Linda University
School of Medicine, Loma Linda, California, USA
Short Title: Flow-mediated dilation in high-altitude Andeans
Correspondence to:
Joshua C. Tremblay, Queen’s University, 28 Division Street, Kingston, Ontario, Canada, K7L
3N6; email: [email protected]; phone number: 250–807–8089; fax: 250-807-9865
Total word count: 5751
Abstract word count: 243
Number of figures: 3
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Abstract
Excessive erythrocytosis (EE; hemoglobin concentration [Hb] ≥21 g dl-1 in adult males) is
associated with increased cardiovascular risk in highlander Andeans. We sought to quantify
shear stress and assess endothelial function via flow-mediated dilation (FMD) in male Andeans
with and without EE. We hypothesized that FMD would be impaired in Andeans with EE after
accounting for shear stress and that FMD would improve after isovolemic hemodilution.
Brachial artery shear stress and FMD were assessed in 23 male Andeans without EE (age: 40±15
years [mean±SD], Hb <21g dl-1) and 19 male Andeans with EE (age: 43±14 years, Hb ≥21 g dl-1)
in Cerro de Pasco, Peru (4330 m). Shear stress was quantified from Duplex ultrasound measures
of shear rate and blood viscosity. In a subset of participants (n=8), FMD was performed before
and after isovolemic hemodilution with blood volume replaced by an equal volume of human
serum albumin. Blood viscosity and Hb were 48% and 23% higher (both P<0.001) and FMD was
28% lower after adjusting for the shear stress stimulus (P=0.013) in Andeans with EE compared
to those without. FMD was inversely correlated with blood viscosity (r2=0.303, P<0.001) and Hb
(r2=0.230, P=0.001). Isovolemic hemodilution decreased blood viscosity by 30±10% and Hb by
14±5% (both P<0.001) and improved shear stress stimulus-adjusted FMD from 2.7±1.9% to
4.3±1.9% (P=0.022). Hyperviscosity, high Hb, or both, actively contribute to acutely reversible
impairments in FMD in EE, suggesting that this plays a pathogenic role in the increased
cardiovascular risk.
Key words: endothelial shear stress; endothelial function; hypoxia; chronic mountain sickness;
hemodilution; polycythemia; high altitude native
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Abbreviations:
CMS = chronic mountain sickness; EE = excessive erythrocytosis; FMD = flow-mediated
dilation; Hb = hemoglobin concentration; NO = nitric oxide; PaCO2 = arterial partial pressure of
carbon dioxide; PaO2 = arterial partial pressure of oxygen; RSNO = S-nitrosothiols; SaO2 =
arterial oxyhemoglobin saturation; SpO2 = oxyhemoglobin saturation from pulse oximetry;
SSAUC60 = shear stress area under the curve 60 seconds post cuff deflation
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Introduction
Chronic mountain sickness (CMS) is a maladaptive high-altitude disease characterized by
excessive erythrocytosis (EE) and severe hypoxemia that is associated with increased
cardiovascular risk1. Approximately 5-10% of high-altitude residents exhibit EE, which is a
major public health problem primarily in the Andes, but also in Kyrgyzstan, Northern India, and
among migrants to high-altitude in Tibet and the United States2-5. One of the highest reported
prevalences is in the mining city of Cerro de Pasco, Peru, where 15% of men aged 30-39 years,
and 34% aged 60-69 years exhibit EE (hemoglobin concentration (Hb) ≥21 g dl-1)6,7. Despite its
prevalence and potential for cardiovascular complications, the impact of EE on the systemic
vasculature has received little attention in the literature. To date, only one group has investigated
systemic endothelial function in Andeans with EE and observed impaired flow-mediated dilation
(FMD) compared to Andeans without EE8-10, which may partially reflect the increased oxidative-
nitrosative stress in EE indicated by a free radical-mediated reduction in the systemic
bioavailability of nitric oxide (NO)8,11,12. However, understanding of the mechanisms and extent
of endothelial dysfunction in Andeans with EE is limited because the shear stress stimulus (the
product of blood viscosity and shear rate) was not assessed.
Given the high hematocrit in EE, blood viscosity is likely much higher compared to non-
EE Andeans13-15. Moderate elevations in hematocrit, and therefore blood viscosity, can be
beneficial to vascular health due to increased shear stress-associated NO release16,17. However, if
the resistance to flow incurred by blood viscosity exceeds the shear stress-associated
vasodilation, hyperviscosity impairs perfusion and can increase arterial pressure16,18,19.
Additionally, in EE, NO scavenging by Hb may limit shear stress-associated NO-induced
vasodilation16,20,21. Thus, documenting the interplay between Hb, viscosity, shear stress, and FMD
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in a population characterized by marked hyperviscosity and noted endothelial dysfunction8,10 may
provide insight into a potential mechanism that could be directly responsible for the increased
cardiovascular risk in high-altitude EE.
We sought to determine [1] endothelial function by identifying the shear stress stimulus
and FMD in Andeans with and without EE, and [2] whether acute reductions in Hb and blood
viscosity via isovolemic hemodilution (IVHD) improve FMD. We hypothesized that FMD
would be impaired in Andeans with EE even after accounting for shear stress and FMD would
improve after IVHD. Our findings indicate that hyperviscosity and high Hb contribute to reduced
FMD and likely mediate the increased cardiovascular risk in Andeans with EE.
Materials and Methods
The individual data that support the findings of this study are available from the corresponding
author upon reasonable request.
Ethical Approval
The Clinical Research Ethics Board of the University of British Columbia (H17-02687 and H18-
01404), the Queen’s University Health Sciences Research Ethics Board (#6023174), and the
Universidad Peruana Cayetano Heredia Comité de Ética (#101686) approved all experimental
procedures and protocols in adherence with the principles of the Declaration of Helsinki (except
registration in a database). All participants read an in-depth study information form translated
into Spanish, spoke with a Peruvian research assistant and provided written informed consent in
Spanish prior to participating.
Participants and experimental protocol
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Male Andean participants who were born and have been permanently living in Cerro de Pasco
(4330m above sea-level) or the Pasco region who were not taking any medications and did not
work in the mining industry were included in the study (detailed in Tymko et al., under review
and the online-only supplement, please see http://hyper.ahajournals.org). Participants were
classified as either with EE (n=19, Hb ≥21 g dl-1) or without EE (n=23, Hb <21 g dl-1). The
severity of CMS symptoms was determined using the Qinghai CMS score22. A score of 0
(absent) to 3 (severe) was assigned for the following signs and symptoms:
breathlessness/palpitations, sleep disturbance, cyanosis, venodilation, paresthesia, headache, and
tinnitus. The presence of EE (≥ 21 g dl-1) adds 3 to the score. The sum of the score for each
symptom and EE defines CMS severity as absent (0-5), mild (6-10), moderate (11-14), and
severe (≥ 15). One participant without and five participants with EE were smokers and refrained
from smoking the day of testing. Participants were tested a minimum of 6h post-prandial, 12h
post-caffeine/alcohol, and 24h post-exercise23. A venous blood sample was drawn to measure
blood viscosity, Hb, and hematocrit. After laying supine for a minimum of 20 minutes, baseline
blood pressure (automated blood pressure, measured in duplicate, Life Source, UA-767FAM,
Canada) and oxyhemoglobin saturation (pulse oximetry, SpO2; Nonin Medical, USA) were
obtained. Participants rested in the supine position for 30 minutes prior to assessment of FMD.
All participants with EE during the screening visit were invited to participate in an IVHD study
and eight volunteered. FMD was performed before and after IVHD on a separate laboratory visit
(n=8).
Doppler Ultrasound
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All measurements were performed by experienced sonographers with a 10 MHz multifrequency
linear array probe (15L4 Smart Mark, Teratech, USA) attached to a high-resolution ultrasound
machine (Terason usmart 3300, Teratech, USA). Diameter and blood velocity recordings of the
left brachial artery were simultaneously recorded using Duplex ultrasound (Terason usmart 3300,
Teratech, USA). The angle of insonation for the acquisition of velocity was 60°. Screen capture
of the ultrasound was saved as an audio video interleave file (Camtasia Studio, Techsmith Co,
Ltd, USA) for future analysis.
Flow-mediated dilation
FMD was performed in adherence with recommended guidelines23,24. One-minute of baseline
brachial artery diameter and blood velocity was acquired via Duplex ultrasound. Subsequently, a
pneumatic cuff placed distal to the epicondyles was inflated to 250 mmHg for five minutes. The
cuff was then rapidly deflated and ultrasound imaging persisted for three minutes post-deflation.
Screen capture of the ultrasound was saved as an audio-video interleave file (Camtasia Studio)
for future analysis using edge-detection software25.
Isovolemic hemodilution
Under local anesthesia (Lidocaine, 1.0 %) and ultrasound guidance, a 20-gauge arterial catheter
(Arrow, Markham, ON, Canada) was placed in the radial or brachial artery. Following arterial
catheterization, baseline measurements of FMD were performed in the contralateral arm and
arterial blood gases, pH, total bioactive plasma NO (see below), oxyhemoglobin saturation
(SaO2), blood viscosity, Hb, hematocrit, and intra-arterial pressure were recorded. Afterwards,
~20 % of blood volume was removed and simultaneously replaced with an equal volume of 37
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ºC, 5 % human serum albumin (result: 1063±320 ml; 17.2±6.6 % of total blood volume). The
hemodilution protocol took between 60-to-120 minutes. All measures were re-assessed after
IVHD.
Hematological and biochemical analyses
Hematological and biochemical analyses were performed on venous blood samples in the
Andeans with versus without EE comparison and arterial blood samples for the IVHD protocol
(detailed in online-only supplement, please see http://hyper.ahajournals.org). Whole blood
viscosity was measured in duplicate at a shear rate 225 s-1 at 37 ºC using a cone and plate
viscometer (DV2T Viscometer, Brookfield Amtek, USA) and a circulating water heating bath
(TC-150, Brookfield Amtek, USA)26,27. Arterial blood gases, Hb, hematocrit, and SaO2 were
obtained from a calibrated blood gas analyzer (ABL-90, Radiometer, Denmark). In a subset of
participants in the IVHD trial (n=6), ozone-based chemiluminescence (Sievers NOA™ 280i,
Analytix Ltd, Durham, UK) was employed to detect the combined concentration of plasma-borne
nitrite (NO) and S-nitrosothiols (RSNO) via chemical reagent cleavage as previously described28.
The intra- and inter-assay coefficient of variations were <10 %.
Data analysis
Observers were blinded to participant status for all analyses.
Shear stress
Shear stress was calculated as the product of shear rate (4 × peak envelope blood velocity /
arterial diameter) and whole blood viscosity at a shear rate of 225 s-1 26,28. Antegrade, retrograde,
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and mean shear stress were calculated from positive, negative, and mean blood velocities,
respectively. The oscillatory shear index was calculated as |retrograde shear stress| / (|antegrade
shear stress| + |retrograde shear stress|)29.
Flow-mediated dilation
Edge-detection software (FMD/BloodFlow Software Version 5.1, Reed C, Australia)25
automatically detected peak diameter using a moving window-smoothed median across time
post-cuff deflation. FMD was calculated as the relative and absolute difference between peak and
baseline diameter. Although shear rate is a suitable surrogate when comparing homogenous,
healthy groups at sea level30, blood viscosity changes influence interpretation in high-altitude
studies28,31. The FMD stimulus was therefore quantified as the shear stress area under the curve
(SSAUC60) 60-seconds post-cuff deflation32. Peak reactive hyperemia, vascular conductance, and
total reactive hyperemia were acquired as indices of resistance vessel function33-37. Blood flow
was calculated as peak envelope blood velocity / 2 × [π(0.5 × diameter)2]38,39 and vascular
conductance was calculated as blood flow / mean arterial pressure. Peak reactive hyperemia was
calculated as the greatest three-second post-occlusion blood flow28, and peak vascular
conductance was calculated as peak reactive hyperemia / mean arterial pressure37. Total reactive
hyperemia was calculated as the blood flow area under the curve three minutes post-cuff
deflation28.
Statistical Analysis
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All statistical analyses were performed using IBM SPSS 24 (International Business Machines
Corp, USA). Data are presented as mean ± standard deviation. Data were assessed for normality
using the Shapiro-Wilk Test. When data were not normally distributed, Mann-Whitney Rank
Sum Tests were performed. All normally distributed data were analyzed using a linear mixed
model unless otherwise noted with a compound symmetry co-variance structure with
significance set at P<0.05. Participant characteristics, baseline cardiovascular variables, and
FMD-associated parameters were compared between groups. The effect of IVHD on
hemodynamic, blood, and FMD parameters was assessed with time (pre versus post) as a
repeated factor. To account for potential effects of shear stress stimulus on FMD, analysis was
additionally performed with the SSAUC60 as a covariate40. The slope of the Hb and blood viscosity
relationship in Andeans with and without EE was compared using analysis of covariance. Linear
regression analyses were performed between FMD and Hb, blood viscosity, and SpO2.
Results
Participant characteristics (Table 1)
Peak oxygen uptake and work rate did not differ between groups as reported and described in
detail elsewhere (Tymko et al., under review). CMS scores were higher and SpO2 lower in
Andeans with (n=19) versus without EE (n=23). The relationship between Hb and whole blood
viscosity is displayed in Figure 1A. The slope of this relationship was steeper amongst Andeans
with compared to without EE (P=0.016). Hb and blood viscosity were 23 % and 48 % higher in
Andeans with versus without EE (P<0.001; Figure 1B, C). Four participants (all with EE) had
blood viscosities that exceeded the upper limit of the viscometer, thus 10.22 cP (the upper limit
of the viscometer) was used as an underestimation of their blood viscosity. Including smoking
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status as a covariate did not impact our findings and FMD in smokers and nonsmokers were not
different (smokers: 6.7 ± 3.1 %, nonsmokers: 6.8 ± 3.0 %; P=0.959).
FMD and microvascular function (Table 2)
The baseline brachial artery diameter was 14 % greater in Andeans with compared to without EE
(P=0.001) and related to blood viscosity (r2=0.311, P<0.001). Baseline shear stress parameters
were not different between groups. FMD was 31 % lower in Andeans with versus without EE
(P=0.008; Figure 1D). After covariate-adjustment for the shear stress stimulus (SSAUC60; P=0.415
between groups), FMD was 28% lower in Andeans with versus without EE (P=0.013; Figure
1E). Indices of microvascular function (i.e. reactive hyperemia) did not differ between groups.
FMD was inversely correlated with Hb (r2=0.230, P=0.001) and blood viscosity (r2=0.303,
P<0.001) and positively correlated with SpO2 (r2=0.256, P=0.001) (Figure 2).
IVHD (Table 3)
IVHD decreased Hb by 14 ± 5 % and blood viscosity by 30 ± 10 % (both P<0.001; Figure 3A
and B). FMD increased from 2.9 ± 2.1 % to 4.1 ± 2.0 % following hemodilution (P=0.036;
Figure 3C). There was a trend towards a lower SSAUC60 post-hemodilution (P=0.076) and indices
of microvascular function were not different. The improvement in FMD persisted after covariate-
adjustment for SSAUC60 (P=0.022; Figure 3D). Of note, the only participant to experience a
decrease in FMD post-hemodilution had the lowest pre-hemodilution Hb. In contrast, IVHD did
not alter the vascular bioavailability of (plasma) NO.
Discussion
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We have provided the first comprehensive assessment of shear stress-associated endothelial
function in high-altitude Andeans, a population with a high prevalence of EE. The primary
findings were that 1) FMD was lower in Andeans with EE than without when accounting for the
shear stress stimulus, 2) FMD was inversely related to both blood viscosity and Hb, and 3) FMD
improved following IVHD-induced reductions in Hb and whole blood viscosity without changes
in plasma bioactive NO. Collectively, these findings suggest that hyperviscosity and/or high Hb
in Andeans with EE contributes to the reduced FMD by mechanisms that appear to be
independent of basal plasma bioactive NO. This reduced functional response to increased shear
stress may be considered an important vascular property that predisposes Andeans with EE to
increased cardiovascular risk.
The findings of the present investigation are consistent with previous observations of
impaired FMD in Andeans with CMS compared to those without8-10. Importantly, these findings
confirm the evidence of reduced FMD by measuring and adjusting for the shear stress stimulus
for FMD40-42. The magnitude of FMD in this population was inversely related to whole blood
viscosity and Hb, suggesting that the EE-associated hyperviscosity and/or high Hb contributes, at
least in part, to the impaired endothelial function. Hemodilution in polycythemic patients has
previously been shown to improve FMD43 and is a common, although incompletely understood,
treatment option in CMS44-49 that may help to liberate bioavailable NO by reducing Hb-mediated
scavenging16,20,21. In the present investigation, IVHD improved FMD and decreased blood
viscosity and Hb, without changing SaO2 or basal plasma bioactive NO. The finding of no net
effect of IVHD on plasma bioactive NO, the “reactant” of oxidative-nitrosative stress8,50,
suggests that acute improvements in FMD following IVHD were not dependent on changes in
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oxidative-nitrosative stress (as indexed via plasma NO). This finding provides experimental
evidence that the reduced FMD is acutely mediated, in part, by high levels of blood viscosity
and/or Hb scavenging of shear stress-mediated NO production. Rimoldi, et al. 10 did not observe
changes in FMD following hemodilution in nine participants with CMS; however, our
hemodilution protocol differed in two ways. First, we replaced blood with human serum albumin
as opposed to saline to ensure the fluid remained in the intravascular space. Second, we observed
a threefold greater reduction in Hb (22.1 ± 1.7 g dl-1 to 19.1 ± 1.7 g dl-1 versus 21.4 ± 1.0 g dl-1 to
20.4 ± 1.3 g dl-1). Thus, the magnitude of hemodilution in the study by Rimoldi, et al. 10 may not
have been sufficient to improve FMD. By contrast, Rimoldi, et al. 10 demonstrated that oxygen
supplementation improved FMD in hypoxemic participants, perhaps because oxygenated red
blood cells scavenge NO at a slower rate than deoxygenated red blood cells20. In line with this
observation, we noted a relationship between SpO2 and FMD (r2=0.256, P=0.001). Taken
together, the complex profile of EE and chronic hypoxemia appears to impair FMD and likely
plays a critical role in the association of EE and cardiovascular risk1 in high-altitude Andeans.
Blood viscosity elicits opposing resistive and shear stress-associated vasodilatory effects
on hemodynamics51,52. Modest increases in blood viscosity have been shown to reduce blood
pressure via increased shear stress-associated NO formation but increasing viscosity >50%
increased blood pressure52. Moderate polycythemia may be associated with greater FMD in
hypoxemic patients53. However, the relationship between blood viscosity and Hb becomes
steeper in Andeans with EE (Figure 1). Although the mechanisms that determine this relationship
remain to be established (see below), endothelial dysfunction in instances of high levels of blood
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viscosity and Hb may render individuals especially susceptible to increased cardiovascular risk
as both whole blood viscosity54,55 and reduced FMD56,57 predict cardiovascular risk and events.
Enlarged brachial artery diameters have previously been reported in participants with
EE10, which appears to be a phenotypic characteristic of this maladaptive response to chronic
hypoxemia. The large diameter may be the result of structural adaptations in response to high
blood viscosity-associated shear stress, the so-called shear stress normalization hypothesis58,59.
Additionally, there may be a role for chronic hypoxemia in maintaining the conduit artery in a
vasodilated state, supported by the observation that oxygen supplementation decreases brachial
artery diameter in patients with CMS1040. Thus, enlarged conduit artery diameter is a fundamental
component of EE pathophysiology. In non-EE populations, an inverse relationship between
baseline diameter and FMD has been observed60. This has been interpreted to suggest that the
same endothelial function is reflected by a progressively smaller percent and absolute FMD
response as baseline artery dimensions increase60. However, FMD increased with hemodilution
while baseline artery diameter did not change, providing support that the larger baseline
diameters per se do not explain the impaired FMD in EE.
We have identified a role for high Hb and/or blood viscosity in contributing to the
reduced FMD in Andeans with EE. However, the mechanisms responsible for the low FMD in
Andeans with EE are likely multifaceted and cannot be completely elucidated from the present
study. For instance, increased systemic free radical formation has been reported in Andeans with
EE compared to Andeans without8,12, and this likely contributes to the lower FMD by chronically
inactivating NO (oxidative-nitrosative stress)8,50. Additionally, cell-free Hb is a 1000-fold more
potent NO scavenger61-64 than red blood cell Hb and can impair endothelium-dependent
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vasodilation61,65. Polycythemic patients at sea level have elevated levels of cell-free Hb compared
to healthy control participants, and polycythemic patients with hypertension have higher cell-free
Hb compared to normotensive polycythemic patients66. Thus, cell-free Hb may play a role in the
NO scavenging and subsequent reduced FMD and increased cardiovascular risk in Andeans with
EE1. Further, measures of erythropoietin to soluble erythropoietin receptor (an erythropoietin
antagonist) ratio may have provided additional mechanistic insight, as this ratio is increased in
CMS67,68 and erythropoietin has been shown to impair endothelial function in humans69.
Therefore, future investigations should investigate markers of oxidative stress, NO
bioavailability (in both plasma and red blood cell), cell-free Hb, and levels of erythropoietin to
better dissect the mechanisms linking EE with reduced FMD and related adverse vascular
outcomes.
Perspectives
Andean highlanders with EE had reduced FMD compared to those without EE and blood
viscosity and Hb correlated with FMD in this population. IVHD reduced Hb and blood viscosity
and improved FMD. These findings identify an Hb/hyperviscosity-associated reduction in FMD
as a potential mechanistic link between EE and increased cardiovascular risk.
Acknowledgements
We would like to thank the entire Global REACH 2018 team and collaborators in Cerro de Pasco
for their support in the weeks leading up to and during data collection in Peru.
Funding
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This work was funded by an American College of Sports Medicine Doctoral Student Research
Grant (JCT and RLH), the Natural Sciences and Engineering Research Council of Canada
(PNA), and a Canada Research Chair (PNA) with travel support provided by The Physiological
Society (JCT). This study received funding from the Wilderness Medical Society Research-in-
Training Grant, supported by the Academy of Wilderness Medicine®. DMB is supported by a
Royal Society Wolfson Research Fellowship (#WM 170007) and funding from the Higher
Education Funding Council of Wales for BS.
Conflict of Interest/Disclosure
The authors have no conflicts of interest to disclose.
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Novelty and Significance
What is New?
- We tested the hypothesis that hyperviscosity and/or high hemoglobin concentration
contribute to reduced flow-mediated dilation (FMD) in highlander Andeans with
excessive erythrocytosis (hemoglobin concentration ≥21 g dl-1) by 1) comparing shear
stress and FMD between Andeans with and without excessive erythrocytosis, and 2)
performing FMD before and after isovolemic hemodilution in a subset of Andeans with
excessive erythrocytosis.
What is Relevant?
- In cases of excessive erythrocytosis, the high levels of blood viscosity and/or hemoglobin
concentration appear to contribute to reduced FMD.
- Impaired FMD is likely an important mechanism contributing to the increased
cardiovascular risk in high-altitude excessive erythrocytosis.
Summary
- Andeans with excessive erythrocytosis presented impaired shear stress-associated
endothelial function compared to Andeans without excessive erythrocytosis. FMD was
inversely correlated with blood viscosity and hemoglobin concentration. Isovolemic
hemodilution reduced blood viscosity and hemoglobin concentration and improved FMD.
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Figure Legends
Figure 1. The relationship between whole blood viscosity and hemoglobin concentration (Hb;
panel A). The slope of viscosity and Hb in individuals with excessive erythrocytosis is steeper
than healthy individuals (0.7328 versus 0.4108; P=0.0159). Dashed lines indicate the 95%
confidence intervals. Hb (panel B), whole blood viscosity (panel C), flow-mediated dilation
(FMD; panel D), and shear stress stimulus (shear stress area under the curve 60 seconds post-cuff
deflation, SSAUC60) – adjusted FMD (panel E) in Andeans with versus Andeans without excessive
erythrocytosis (EE). *, P<0.05 versus Andeans without EE. Hb was compared using the Mann-
Whitney Rank Sum Test.
Figure 2. Relationships between hemoglobin concentration (Hb, panel A), whole blood viscosity
(panel B), and SpO2 (panel C) and flow-mediated dilation (FMD) in highlander Andeans.
Figure 3. Hemoglobin concentration (Hb; panel A), whole blood viscosity (panel B), flow-
mediated dilation (FMD; panel C), and shear stress stimulus (shear stress area under the curve 60
seconds post-cuff deflation, SSAUC60) – adjusted FMD (panel D) pre- and post-hemodilution. *,
P<0.05 pre- versus post-hemodilution.
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Table 1. Participant characteristics.Group Without EE With EE P-valuen 23 19Age (years) 40±15 43±14 0.526Height (m) 1.61±0.04 1.61±0.06 0.924Mass (kg) 68±12 67±11 0.929†
BMI (kg m-2) 26±5 26±3 0.727Systolic Blood Pressure (mmHg) 115±12 117±11 0.662Diastolic Blood Pressure (mmHg) 72±10 78±10 0.059Mean Arterial Pressure (mmHg) 86±11 91±10 0.168Heart Rate (bpm) 73±13 67±9 0.075Hematocrit (%) 57±6 70±4* <0.001†
SpO2 (%) 87±4 83±4* <0.001CMS Score 1±2 7±3* <0.001†
Data are presented as mean±SD. BMI, body mass index; CMS, chronic mountain sickness; EE, excessive erythrocytosis; SpO2, oxyhemoglobin saturation from pulse oximetry. *, P<0.05 versus Andeans without EE; †, Mann-Whitney Rank Sum Test performed.
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Table 2. Parameters for flow-mediated dilation (FMD) and microvascular function assessment.Group Without EE With EE P-valuen 23 19Baseline Diameter (mm) 3.86±0.45 4.42±0.59* 0.001Peak Diameter (mm) 4.15±0.43 4.64±0.57* 0.003Absolute FMD (mm) 0.29±0.11 0.22±0.08* 0.035SSAUC60 (103) 12.1±3.9 11.0±4.5 0.415Time to Peak Diameter (s) 53±17 63±39 0.820†
Baseline Mean Shear Stress (dyn cm-2) 5.47±3.35 5.61±4.74 0.596†
Baseline Antegrade Shear Stress (dyn cm-2) 6.24±2.99 6.88±4.21 1.000†
Baseline Retrograde Shear Stress (dyn cm-2) -0.76±0.79 -1.28±1.19 0.180†
Baseline Oscillatory Shear Index 0.13±0.13 0.18±0.14 0.206†
Baseline Blood Flow (ml min-1) 33±26 32±25 0.705†
Baseline Vascular Conductance (ml min-1 mmHg-1) 0.39±0.32 0.35±0.27 0.544†
Peak Reactive Hyperemia (ml) 233±87 220±55 0.590Peak Vascular Conductance (ml min-1 mmHg-1) 2.77±1.15 2.44±0.62 0.270Total Reactive Hyperemia (liters) 13.4±6.7 11.1±5.1 0.312†
Data are presented as mean±SD. SSAUC60, shear stress area under the curve 60 seconds post cuff deflation. *, P<0.05 versus healthy; †, Mann-Whitney Rank Sum Test performed.
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Table 3. Participant characteristics and flow-mediated dilation and microvascular function assessment parameters pre- and post-isovolemic hemodilution.Time Pre Post P-valuen 8Age (years) 43±19BMI (kg m-2) 26±3CMS Score 6±3Systolic Blood Pressure (mmHg) 151±29 127±26 0.052Diastolic Blood Pressure (mmHg) 76±7 72±12 0.384Mean Arterial Pressure (mmHg) 101±14 90±16 0.063Heart Rate (bpm) 69±11 77±15 0.088Hemoglobin concentration (g dl-1) 22.1±1.7 19.1±1.7* <0.001Hematocrit (%) 68±5 58±5* <0.001SaO2 (%) 75±6 72±6 0.225PaO2 (mmHg) (n=6) 42±5 41±5 0.393PaCO2 (mmHg) (n=6) 36±3 36±2 0.847Arterial pH (n=6) 7.41±0.01 7.39±0.01* 0.028Total bioactive NO (nM) (n=6) 53±23 48±11 0.738Baseline Diameter (mm) 4.51±0.38 4.49±0.47 0.852Peak Diameter (mm) 4.64±0.39 4.67±0.50 0.725FMD (mm) 0.13±0.10 0.18±0.10* 0.038SSAUC60 (103) 13.7±6.3 9.9±5.2 0.076Time to Peak Diameter (s) 62±16 85±17* 0.038Mean Shear Stress (dyn cm-2) 8.07±5.49 5.10±3.02 0.092Antegrade Shear Stress (dyn cm-2) 9.77±4.56 7.70±2.33 0.105Retrograde Shear Stress (dyn cm-2) -1.70±1.07 -2.61±1.46 0.184Oscillatory Shear Index 0.18±0.14 0.25±0.14 0.290Baseline Blood Flow (ml min-1) 56±42 36±25 0.102Baseline Vascular Conductance (ml min-1 mmHg-
1)0.56±0.43 0.42±0.33 0.118
Peak Reactive Hyperemia (ml min-1) 239±85 280±123 0.296Peak Vascular Conductance (ml min-1 mmHg-1) 2.40±0.96 3.23±1.51 0.083Total Reactive Hyperemia (liters) 17.1±9.1 16.9±11.3 0.941Data are presented as mean±SD. CMS, chronic mountain sickness; FMD, flow-mediated dilation; SaO 2, arterial oxyhemoglobin saturation; SSAUC60, shear stress area under the curve 60 seconds post cuff deflation. Total bioactive nitric oxide (NO) refers to the combined concentration of (plasma) nitrite and S-nitrosothiols. *, P<0.05 versus pre.
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