J Appl Physiol 109: 959–965, 2010.
First published July 29, 2010; doi:10.1152/japplphysiol.00532.2010.
Measuring FMD in the brachial artery: how important is QRS gating?
Tinoy J. Kizhakekuttu,1 David D. Gutterman,1,2 Shane A. Phillips,3 Jason W. Jurva,1,4
Emily I. L. Arthur,1 Emon Das,1 and Michael E. Widlansky1,2
1
Cardiovascular Medicine Division, Department of Medicine, 2Department of Pharmacology, Medical College of Wisconsin,
Cardiovascular Medicine Division, Department of Medicine, Zablocki Veterans Affairs Medical Center, Milwaukee,
Wisconsin; and 3Department of Physical Therapy, University of Illinois-Chicago, Chicago, Illinois
4
Submitted 14 May 2010; accepted in final form 22 July 2010
information but also identifies the efficacy of medical interventions that enhance endothelial function (23, 25).
The potential of brachial FMD to be used as a barometer of
cardiovascular risk at a population level has led to efforts to
standardize the technique with the hope of maximizing the
validity and reproducibility of the measurement (7). Recent
reviews suggest further refinements to these recommendations
(8, 18, 29). These publications recommend measurement of
brachial artery diameter at the same time in the cardiac cycle,
most commonly gated to onset of the QRS complex at end
diastole (7). This recommendation stems from concerns that
the arterial distension occurring during systole may vary on the
basis of non-endothelium-dependent factors affecting arterial
compliance, reducing the measurement’s validity as a measure
of dynamic endothelial function. However, QRS-gated image
capture is not available on all vascular ultrasound machines,
particularly more economical and portable models. The lack of
readily obtainable QRS-gated still images for analysis increases the complexity of postimaging processing and analysis
and remains a barrier to any potential clinical application of
FMD (32). Prior investigations into the necessity of QRS
gating has yielded conflicting results (6, 13, 13). We hypothesized that deriving FMD using diameters from entire R-R
intervals would be in strong agreement with traditional methods that select diameters at the onset of the QRS complex
across a wide range of brachial artery distensibilities.
endothelium; shear stress; flow-mediated dilation; method
MATERIALS AND METHODS
ENDOTHELIAL DYSFUNCTION
Subjects
Address for reprint requests and other correspondence: M. E. Widlansky,
Medical College of Wisconsin, 9200 W. Wisconsin Ave., FEC Suite E5100,
Milwaukee, WI 53226 (e-mail: [email protected]).
We randomly selected 1) healthy, physically active young adults
(ages 18 –35 yr), 2) healthy middle-aged adults (age 36 – 45 yr),
3) healthy older adults (age 46 –70 yr), and 4) adults (age 36 –70 yr)
with type 2 diabetes mellitus (DM) from whom baseline FMD
measurements were obtained for other study protocols for this analysis. This was done to ensure that our alternative method for FMD
measurement remained robust across a wide range of baseline cardiovascular risk. Participants were recruited via flyers and internet
advertisements. DM adults qualified for enrollment if they were
between 35 and 70 yr of age and met the American Diabetes
Association criteria for a diagnosis of type 2 DM (14). Potential
healthy control subjects were excluded if they had a history of
cardiovascular disease, showed evidence of active chronic liver, renal,
or neoplastic disease, were pregnant at the time of screening, or used
tobacco products within 1 yr of enrollment. Additionally, control
subjects were excluded from enrollment during screening if they were
found to have a fasting serum glucose ⱖ126 mg/dl, elevated blood
pressure (systolic blood pressure ⱖ140 mmHg, diastolic blood pressure ⱖ90 mmHg, or previous diagnosis of or current treatment for
hypertension), or elevated triglycerides (⬎200 mg/dl) or if they
qualified for the diagnosis of metabolic syndrome (10). Young adults
were considered to be physically active if they ran ⱖ15 miles/wk,
is characterized by a proinflammatory, prothrombotic, and vasoconstrictive phenotype (16).
Maintenance of nitric oxide (NO) bioavailability through activation of endothelial NO synthase is of central importance in
maintaining vascular homeostasis.
Substantial evidence supports the concept that detection of
endothelial dysfunction in the brachial artery is an indicator of
systemic endothelial dysfunction (32). Brachial artery reactivity [flow-mediated dilation (FMD)], as measured by highresolution vascular ultrasound prior to and following a hyperemic flow stimulus, is highly dependent on NO bioavailability
(9, 20). Furthermore, endothelial dysfunction as detected by
FMD predicts future cardiovascular risk in those with and
without diagnosed coronary artery disease (4, 5, 11, 15, 25, 26,
28, 30, 35). FMD’s ability to stratify individuals into low-,
intermediate-, and high-risk categories for future cardiovascular events (35, 36) not only yields potentially clinically relevant
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Kizhakekuttu TJ, Gutterman DD, Phillips SA, Jurva JW, Arthur
EI, Das E, Widlansky ME. Measuring FMD in the brachial artery: how
important is QRS gating? J Appl Physiol 109: 959 –965, 2010. First
published July 29, 2010; doi:10.1152/japplphysiol.00532.2010.—
Recommendations for the measurement of brachial flow-mediated
dilation (FMD) typically suggest images be obtained at identical times
in the cardiac cycle, usually end diastole (QRS complex onset). This
recommendation presumes that inter-individual differences in arterial
compliance are minimized. However, published evidence is conflicting. Furthermore, ECG gating is not available on many ultrasound
systems; it requires an expensive software upgrade or increased image
processing time. We tested whether analysis of images acquired with
QRS gating or with the more simplified method of image averaging
would yield similar results. We analyzed FMD and nitroglycerinmediated dilation (NMD) in 29 adults with type 2 diabetes mellitus
and in 31 older adults and 12 young adults without diabetes, yielding
a range of brachial artery distensibility. FMD and NMD were measured using recommended QRS-gated brachial artery diameter measurements and, alternatively, the average brachial diameters over the
entire R-R interval. We found strong agreement between both methods for FMD and NMD (intraclass correlation coefficients ⫽ 0.88 –
0.99). Measuring FMD and NMD using average diameter measurements significantly reduced post-image-processing time (658.9 ⫾
71.6 vs. 1,024.1 ⫾ 167.6 s for QRS-gated analysis, P ⬍ 0.001). FMD
and NMD measurements based on average diameter measurements
can be performed without reducing accuracy. This finding may allow
for simplification of FMD measurement and aid in the development of
FMD as a potentially useful clinical tool.
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QRS GATING AND FLOW-MEDIATED DILATION
lifted weights at least three times per week, or both, for ⱖ6 mo prior
to enrollment. Young adults not meeting any of these three criteria
were excluded. All study protocols were approved by the Institutional
Research Board of the Medical College of Wisconsin, and all subjects
gave their verbal and written informed consent prior to all study
procedures.
Study Protocol
Brachial Artery Measurements
Flow- and nitroglycerin-mediated dilation. Brachial artery diameters were measured throughout the cardiac cycle at rest (baseline) and
during reactive hyperemia produced by 5 min of forearm cuff inflation
above suprasystolic levels (50 mmHg above systolic pressure or 200
mmHg, whichever was greater). Images of the brachial artery in
longitudinal cross section were recorded continuously throughout the
entire study onto a VHS tape. From the recorded images, 100 frames
(10 frames/s for 10 s) were captured and digitized using edgedetection software (Brachial Analyzer version 4.2.2, Medical Imaging
Applications) at baseline and 1, 2, and 3 min after cuff deflation.
We determined the set of post-cuff-release values to be used for
measurements of brachial diameter for each subject as follows. The
10-s interval 1 min after cuff release (55– 65 s), 2 min after cuff
release (115–125 s), or 3 min after cuff release (175–185 s) with the
highest mean percent FMD over the complete set of R-R intervals was
selected for comparison with baseline. Postreactive hyperemia FMD
average measures (FMD%Ave and FMDmmAve) were calculated for
each subject by averaging the mean brachial diameter of each complete, individual R-R interval during the selected captured 10-s interval following cuff release and subtracting this value from the mean
diameters for each complete R-R interval captured during the 10-s
baseline brachial artery measurement. Images for QRS analyses were
selected by the sonographer but were analyzed using the automated
edge-detection software. FMD%Ave is this difference divided by the
calculated mean baseline brachial diameter. QRS-gated FMD
(FMD%QRS and FMDmmQRS) were calculated in an identical
manner by substitution of manually selected diameters at the onset of
the QRS complex for the average diameter across each R-R interval.
From 10 subjects randomly selected from studies in our laboratory,
intra- and interobserver correlation coefficients are as follows:
FMD%QRS ⫽ 0.96 and 0.85, FMD%Ave ⫽ 0.99 and 0.87. The mean
absolute differences measured between observers was as follows:
FMD%QRS ⫽ 1.2 ⫾ 0.8%, FMD%Ave ⫽ 1.0 ⫾ 0.8%. Similar
assessments of nitroglycerin-mediated dilation (NMD) measurements
were made following sublingual administration of 0.4 mg of nitroglycerin. The 10-s span after nitroglycerin dilation with the greatest
average brachial diameter (sampled at 2, 3, 4, and 5 min after
J Appl Physiol • VOL
Statistical Analysis
SigmaStat 3.1 and SPSS 12.0 were used for the statistical analyses.
Baseline characteristics for DM and non-DM adults were compared
using ANOVA, ␹2 tests, or Fisher’s exact tests as appropriate. Data
were logarithmically transformed or analyzed using appropriate nonparametric testing if found to have a skewed distribution. Post hoc
testing using Student-Newman-Keuls test or Dunn’s method was
performed as applicable. Intraclass correlation coefficients were used
to compare average-based and QRS-gated based measurements for
FMD and NMD determinations using all participants in the analysis.
The entire dataset was used in Bland-Altman plots that were generated
by comparing the average of two sets of values with the difference
between the same two sets of values for average FMD and NMD
measurement protocols vs. QRS-gated FMD and NMD measurement
protocols. P ⬍ 0.05 was considered to be significant.
RESULTS
Participant Demographics
A total of 31 DM, 17 middle-aged, 17 older, and 12 young,
physically active adults were initially chosen at random for this
study. Five subjects (2 DM and 3 older adults) were excluded
because of technically inadequate scans, leaving 29 DM and 14
older adults. The baseline profiles of each group and comparisons between participant groups are shown in Table 1. As
discussed in MATERIALS AND METHODS, selection of our older
adult population excluded participants with cardiovascular risk
factors, including hypertension and hyperlipidemia, resulting
in healthy middle-aged and older adult populations on few
prescription medications. As expected, the DM cohort had a
significantly larger waist circumference and higher serum triglyceride levels than both nondiabetic groups. The medications
taken by the subjects are shown in Table 2.
Comparisons of Measurements of FMD, Shear, and Vessel
Compliance
Tables 3 and 4 demonstrate the brachial diameters, absolute
FMD (FMDmm) and percent FMD (FMD%), and absolute
NMD (NMDmm) and percent NMD (NMD%) for each method
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Subjects arrived at the Medical College of Wisconsin’s Adult
Translational Research Unit after an overnight (8 –12 h) fast. Vital
signs (heart rate and blood pressure) were measured in triplicate, and
height, weight, and waist circumference at the umbilicus were measured and recorded. All studies were performed between 8 and 10 AM
to limit the influence of circadian variations on endothelial function.
Participants were then asked to lie supine in a quiet, dimly lit,
temperature-controlled (22–24°C) study room for 20 min prior to any
measurements of brachial artery reactivity. B-mode ultrasound images
(Logiq 500PRO, GE, Milwaukee, WI; or MicroMaxx, Sonosite,
Seattle, WA) of the brachial artery in the dominant arm were obtained
with the arm supinated and abducted ⬃80°. Images were obtained at
a standard depth of 4 cm. The linear-array ultrasound probe (11 MHz)
was positioned longitudinally at a site 1–3 cm proximal to the cubital
fossa. A three-lead ECG system was attached to the participant, and
its input was sent to the ultrasound machines for simultaneous recording of the ECG representation of the cardiac cycle with our brachial
artery images to facilitate determination of the timing of events in the
brachial artery relative to the cardiac cycle.
nitroglycerin administration) was the time span chosen for analysis for
each individual subject.
Flow velocity and shear stress in the brachial artery. Baseline
brachial flow velocity and peak hyperemic flow velocity (recorded
immediately after cuff release) were measured using pulse-wave
Doppler signals with the sample volume placed at the center of the
lumen of the brachial artery signal with a 60° correction for the angle
of insonation. We measured baseline and hyperemic brachial artery
shear stress (dyn/cm2) using the following formulas: 2.8 ⴱ baseline
flow velocity/baseline diameter and 2.8 ⴱ hyperemic flow velocity/
baseline diameter, respectively. These equations assume a constant
viscosity of 0.035 dyn·s·cm⫺2.
Brachial artery compliance. Brachial artery compliance, as estimated by cross-sectional distensibility (10⫺3/mmHg), was measured
using the following formula: (2 ⴱ ⌬D ⴱ Dmin ⫹ ⌬D2)/(⌬P ⴱ D2min)
(12), where ⌬D is the difference between the average minimum and
maximum baseline brachial artery diameter for each complete R-R
interval recorded at baseline, ⌬P is the pulse pressure averaged from
three baseline blood pressure measurements, and Dmin is the average
minimum baseline brachial artery diameter. From 10 subjects randomly selected from studies in our laboratory, the intra- and interobserver correlation coefficients for brachial artery distensibility were
0.98 and 0.85, respectively. The mean absolute difference measured
between observers was 0.4 ⫾ 0.4 10⫺3/mmHg.
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QRS GATING AND FLOW-MEDIATED DILATION
Table 1. Subject demographics
Young Adults (n ⫽ 12)
Middle-Aged Adults (n ⫽ 17)
Older Adults (n ⫽ 14)
Type 2 DM Adults (n ⫽ 29)
26 ⫾ 3
42
24 ⫾ 4
79 ⫾ 11
41 ⫾ 3
59
29 ⫾ 6
96 ⫾ 17
50 ⫾ 4
71
28 ⫾ 4
95 ⫾ 10
54 ⫾ 8
63
35 ⫹ 8
113 ⫹ 18
118 ⫾ 18
71 ⫾ 11
64 ⫾ 11
130 ⫾ 21
78 ⫾ 14
65 ⫾ 10
133 ⫾ 20
74 ⫾ 10
71 ⫾ 12
172 ⫾ 25
102 ⫾ 22
52 ⫾ 15
97 ⫾ 39
205 ⫾ 50
128 ⫾ 37
60 ⫾ 19
86 ⫾ 34
179 ⫾ 32
100 ⫾ 30
50 ⫾ 13
147 ⫾ 67
a
Age, yr
Sex, %female
Body mass index,b kg/m2
Waist circumference,c cm
Blood pressure, mmHg
Systolicd
Diastolice
Resting heart rate,f beats/min
Cholesterol, mg/dl
Totalg
LDLh
HDL
Triglycerides,i mg/dl
115 ⫾ 8
55 ⫾ 9
54 ⫾ 7
of measurement by cohort. Within subject groups, there were
no significant differences between QRS-gated and averaged
measurements for any of these parameters. Between groups,
brachial artery diameter was larger in the young adult group
than all other groups (P ⬍ 0.05), and FMD% and FMDmm
were significantly lower in the DM group than all other groups.
NMDmm was significantly greater in the young compared with
older and DM adults (P ⬍ 0.05) and was also greater in
middle-aged than DM adults (P ⬍ 0.05). The between-subjects
findings were identical, regardless of the diameter measurement method employed.
Brachial artery distensibility was significantly lower in the
DM group than all other groups (Fig. 1). Brachial distensibility
in young athletes trended lower than in older healthy control
groups, but these differences did not reach statistical significance. Baseline shear was significantly lower in young athletes
than older adults, but there was no significant difference in the
peak hyperemic shear response [baseline shear: 28 ⫾ 10, 33 ⫾
10, 45 ⫾ 17, and 38 ⫾ 14 dyn/cm2 (P ⫽ 0.008, overall P ⫽
0.007, young athletes vs. older adults); peak hyperemic shear:
59 ⫾ 14, 65 ⫾ 26, 79 ⫾ 32, and 68 ⫾ 28 dyn/cm2 for young,
middle-aged, older, and DM adults, respectively (P ⫽ 0.32
overall)].
Table 2. Medication profiles of participant populations
Medications
Young
Adults
(n ⫽ 12)
Middle-Aged
Adults
(n ⫽ 17)
Older
Adults
(n ⫽ 14)
DM
Adults
(n ⫽29)
␤-Blockers
Ca2⫹ channel blockers
ACE inhibitors
ANG II receptor blockers
HMG-CoA reductase inhibitors
Aspirin
Metformin
Sulfonylureas
Thiazoladinediones
Multivitamin
Hormone replacement therapy
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5
1
0
0
0
0
0
0
0
0
0
3
2
7
3
6
8
12
13
22
11
3
12
2
ACE, angiotensin-converting enzyme. HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA.
J Appl Physiol • VOL
To determine whether average and QRS-gated measurements yielded comparable results along a range of brachial
artery distensibilities, we combined the populations and calculated the intraclass correlation coefficients between the QRSgated and average measurements. Measurements based on
average diameters showed a very high degree of similarity to
QRS-gated FMD measurements (0.98, 0.88, 0.97, and 0.99
FMD%, FMDmm, NMD%, and NMDmm, respectively, P ⬍
0.001 for all comparisons).
Furthermore, we generated Bland-Altman plots to find evidence of significant measurement bias or measurement discrepancies between QRS-gated and average FMD and NMD measurements (Fig. 2). The average bias approached zero for
average FMD and NMD measurements compared with the
QRS-gated measurements in all cases. The percent agreement
of the average-based measurements within the 95% confidence
interval of the average bias relative to the QRS-gated FMD and
Table 3. Brachial artery diameter and FMD measurements
FMD
n
Young adults
QRS wave-gated
Average
Middle-aged adults
QRS wave-gated
Average
Older adults
QRS wave-gated
Average
DM adults
QRS wave-gated
Average
12
17
14
29
Baseline
Diameter, mm
mm
%
5.29 ⫾ 0.84
5.33 ⫾ 0.82
0.35 ⫾ 0.12
0.35 ⫾ 0.13
6.6 ⫾ 1.9
6.5 ⫾ 1.7
3.79 ⫾ 0.72
3.86 ⫾ 0.72
0.25 ⫾ 0.06
0.25 ⫾ 0.06
6.4 ⫾ 0.7
6.4 ⫾ 0.8
3.51 ⫾ 0.61
3.46 ⫾ 0.56
0.22 ⫾ 0.05
0.23 ⫾ 0.04
6.6 ⫾ 1.3
6.6 ⫾ 1.0
3.63 ⫾ 0.71
3.64 ⫾ 0.70
0.13 ⫾ 0.06
0.13 ⫾ 0.06
3.9 ⫾ 2.0
3.8 ⫾ 1.8
Values are means ⫾ SD. FMD, flow-mediated dilation. There were no
significant differences in baseline diameter, FMDmm, and FMD% between
measurement methods within each of the 3 study groups (P ⬎ 0.05 for all
comparisons). For between-group comparisons, baseline diameter (QRS-gated
and average): P ⬍ 0.05, young adults vs. all other groups. For FMDmm ECG
and average-gated: P ⬍ 0.05, all groups vs. DM adults. For FMD% ECG and
average-gated, P ⬍ 0.05, all groups vs. DM adults. Findings are identical for
FMD ECG and FMD%.
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Values are means ⫾ SD. Lipid profile data were not available for the young adult group. DM, diabetes mellitus. aP ⬍0.001 overall; P ⬍ 0.05, young and
middle-aged vs. older and type 2 DM adults. bP ⬍ 0.001 overall; P ⬍ 0.05, young vs. type 2 DM adults. cP ⬍ 0.001 overall; P ⬍ 0.05, young and middle-aged
vs. type 2 DM adults. dP ⫽ 0.006 overall; P ⬍ 0.05, young and middle-aged vs. type 2 DM adults. eP ⬍ 0.001 overall; P ⬍ 0.05, young adults vs. all other
groups. fP ⬍ 0.001 overall; P ⬍ 0.05, young vs. older and DM adults. gP ⫽ 0.034 overall; P ⬍ 0.05, middle-aged vs. older adults. hP ⫽ 0.02 overall; P ⬍ 0.05,
older vs. middle-aged and DM adults. iP ⫽ 0.001 overall; P ⬍ 0.05, older and middle-aged vs. DM adults.
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Table 4. Brachial artery diameter and NMD measurements
NMD
n
Young adults
QRS wave-gated
Average
Middle-aged adults
QRS-gated
Average
Older adults
QRS wave-gated
Average
DM adults
QRS wave-gated
Average
12
14
11
26
Baseline Diameter
mm
%
5.36 ⫾ 0.81
5.42 ⫾ 0.82
1.21 ⫾ 0.22
1.18 ⫾ 0.21
22.9 ⫾ 4.8
24.9 ⫾ 5.6
4.00 ⫾ 0.70
4.04 ⫾ 0.70
1.00 ⫾ 0.21
0.98 ⫾ 0.23
25.6 ⫾ 6.1
25.2 ⫾ 6.0
3.44 ⫾ 0.63
3.48 ⫾ 0.62
0.86 ⫾ 0.22
0.85 ⫾ 0.22
25.4 ⫾ 6.0
24.7 ⫾ 5.3
3.63 ⫾ 0.67
3.65 ⫾ 0.67
0.76 ⫾ 0.20
0.75 ⫾ 0.19
21.3 ⫾ 5.5
20.9 ⫾ 5.4
NMD were as follows: 94.4% (FMDmm), 94.4% (FMD%),
93.6% (NMDmm), and 98.4% (NMD%).
The time saved by using average FMD and NMD measurements, rather than QRS-gated methods, was calculated (Table 5).
In the hands of our technician with 3 years of experience
with this technique, diameter measurements using the average diameter, compared with QRS-gated diameters, saved
an average of 3.2 min for FMD measurement alone (P ⬍
0.001) and an average of 6.1 min (P ⬍ 0.001) for studies
including assessment of NMD.
DISCUSSION
This study demonstrates that measuring brachial artery diameter as an average of all diameter measurements taken
throughout the cardiac cycle yields calculated FMD and NMD
values that are in strong agreement with FMD and NMD values
derived from traditional brachial diameter measurements at the
onset of the QRS complex. This agreement is seen across the
spectrum of brachial artery compliance represented in our
population. These data suggest that FMD and NMD calculations using nongated protocols and automated edge-detection
software are valid approximations of manual QRS-gated measurements. These data assuage concerns that nongated measurements would yield invalid results due to integration of
structural characteristics of the vessel into FMD calculations
(6, 7). Furthermore, these data suggest a valid method to
simplify image capture requirements and reduce offline analysis time for brachial artery FMD studies, important steps in
moving toward wider application of this technique in cardiovascular risk stratification (32).
The 2002 recommendations for the ultrasonographic assessment of FMD in the brachial artery represent a thorough review
of proper techniques for the acquisition, analysis, and reporting
of brachial FMD, along with discussion of appropriate training and
quality assurance protocols (7). Since the writing of these guidelines, further refinements have been suggested to increase the
validity and reproducibility of the results, including more specific suggestions for cuff location, justification for the use of a
J Appl Physiol • VOL
Fig. 1. Brachial artery distensibility between groups. Brachial distensibility
was significantly lower in adults with type 2 diabetes mellitus (DM) than all
other groups: 3.5 ⫾ 1.2, 5.1 ⫾ 3.2, 4.5 ⫾ 1.1, and 2.7 ⫾ 1.4 10⫺3/mmHg for
young, middle-aged, older, and DM, respectively (P ⬍ 0.001 overall; P ⬍
0.001, DM vs. middle-aged and older controls; P ⫽ 0.025 vs. young athletes).
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Values are means ⫾ SD. NMD, nitroglycerin-mediated dilation. There were
no significant differences in NMDmm and NMD% between measurement
methods within each of the 3 study groups (P ⬎ 0.05 for all comparisons).
Between-group differences for QRS-gated and average measurements: P ⬍
0.001 overall, P ⬍ 0.05, young adults vs. all other groups (baseline diameter);
P ⬍ 0.001 overall, P ⬍ 0.05, young vs. older and DM adults; P ⬍ 0.05,
middle-aged vs. DM adults (NMDmm); no significant differences detected
(NMD%). No differences between methods were detected.
5-min occlusion interval, and analysis of the shear stimulus (9,
18, 29). With respect to the timing of diameter measurements,
there have been longstanding concerns that FMD measurements that incorporate non-end-diastolic diameters may introduce measurement errors due to fixed structural issues (7) and
a very recent FMD review advocated using the QRS-gated
approach (18). However, only three prior studies have investigated whether non-QRS-gated brachial measurements show
significant differences from FMD measurements using only
end-diastolic measurements. One study of 24 subjects at increased cardiovascular risk demonstrated wide variation in
FMD% when the cardiac cycle was ignored and FMD% was
calculated using the single largest and smallest brachial diameters measured at baseline and following reactive hyperemia
(6). Our method of measuring FMD and NMD differs significantly from that reported in this prior work, and we find much
greater agreement with QRS-gated FMD measurements.
Another prior report compared measurement of FMD using
a novel filter to remove higher-frequency oscillations in the
brachial diameter waveform that occur because of the influence
of the cardiac cycle (13). The resultant waveform contains
diameters theoretically under the influence of only intrinsic
vasodilatory and vasoconstrictive influences. Using this
method, the group found significant agreement between their
method of brachial artery measurement and ECG-gated measurements. While promising, our results demonstrate that the
added expense and technical complication posed by the addition of a filter may not be necessary to obtain valid measurements of FMD and NMD.
A recent observation by Padilla et al. (27) supports our
findings. Internal data from their laboratory showed that continuous assessment of diameter at 5 frames/s yields the same
FMD results as R-wave-gated images (n ⫽ 10, FMD ⫽ 7.58 ⫾
0.9 vs. 7.62 ⫾ 0.9%, P ⫽ 0.655, intraclass correlation ⫽
0.998). Our data confirm these findings in a larger dataset and
demonstrate, for the first time, that utilization of the proposed
approach is acceptable in populations with a wide range of
compliance.
Fixed structural factors influencing vessel compliance, particularly those related to brachial artery remodeling in the
setting of risk factors, have been reported to affect FMD
responses. Structural remodeling involves complex interac-
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QRS GATING AND FLOW-MEDIATED DILATION
Fig. 2. Bland-Altman plots for absolute and
percent flow-mediated dilation (FMDmm and
FMD%) and absolute and percent nitroglycerinmediated dilation (NMDmm and NMD%) comparing average (Ave) with QRS-gated method
of measurement. Overall, average biases were
⫺0.0003, 0.06%, 0.017, and 0.57% for FMDmmAve (A), FMD%Ave (B), NMDmmAve (C),
and NMD%Ave (D), respectively. At least 93%
of all mean differences fell within the 95%
confidence interval for the reported average biases.
surements were used than when manually selected images on
the QRS complex were used. For an individual or a small study
the size of this report, a 3- to 6-min difference in the time
required for data analysis has relatively little impact on resource utilization or costs. However, the importance of a small
reduction in analysis time for an individual study is realized
when FMD is projected for use as a potential population
screening tool to assess cardiovascular risk. For example, using
our measurement method to screen a population of 5,000
would save 250 –500 h of analysis time over a QRS-gated
protocol. Protocol simplification, reductions in resource utilization, and increased accessibility, all of which can be realized
by application of our measurement protocol, are three of the
many important factors to consider when FMD is contemplated
as a population screening tool to detect individuals at greatest
risk for adverse cardiovascular events (32). Software designs
that allow for selection of ECG-gated images without additional hardware for ECG detection will also aid in the promulgation of FMD measurements in cardiovascular screening (34).
While not the primary purpose of this study, the vascular
structure of our young adult athlete group merits consideration.
Our young adults had a significantly larger baseline diameter
than older and DM adults. This finding is consistent with prior
exercise training, which is known to lead to vascular remodeling that includes increased luminal size (17, 21). We found
no significant difference in FMD% in young athletic subjects
relative to the other two, older control groups, a finding also
Table 5. Time required for QRS-gated vs. average FMD and NMD measurements
FMD
NMD
Total
Ave (sec)
QRS (sec)
Ave (sec)
QRS (sec)
Ave (sec)
QRS (sec)
301 ⫾ 33
495 ⫾ 88*
358 ⫾ 45
530 ⫾ 94†
659 ⫾ 72
1,024 ⫾ 168‡
Values are means ⫾ SD; n ⫽10 for all measurements. *P ⬍ 0.001 vs. FMD-Ave. †P ⬍ 0.001 vs. NMD-Ave. ‡P ⬍ 0.001 vs. total Ave.
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tions between mechanical forces, metabolic factors, and paracrine influences on vascular wall composition, leading to
fibrotic changes, increased collagen cross-linking, smooth
muscle hyperplasia, and, ultimately, reductions in overall arterial compliance. While earlier studies suggest that local
reductions in brachial artery compliance are modest with aging
and hypertension (19, 22), other work suggests that brachial
compliance modifies the relationship between smoking, age,
and FMD measured using QRS-gated diameters (33). However, our data demonstrate that use of average brachial artery
diameters to measure FMD and NMD yields strong agreement
with QRS-gated FMD and NMD measurements across a spectrum of individual brachial compliances. The use of average
diameters may limit the effects of differences in brachial artery
compliance on FMD and NMD by “averaging out” the extremes of the brachial diameter spectrum during the measurement period. A deeper understanding of the relative contributions of dynamic production of endothelium-derived NO and
vascular stiffness to FMD measurements requires further
study.
Viable clinical screening tools must be easily accessible,
valid, reproducible, safe, and appropriately stratify risk. Furthermore, in order for a clinical screening tool to reach a stage
where it can be applied at a population level, it must also be
used in a rapid and cost-effective manner. We found that
post-image analysis time required to measure FMD and NMD
was significantly shorter when average brachial diameter mea-
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QRS GATING AND FLOW-MEDIATED DILATION
J Appl Physiol • VOL
the average brachial diameter over the cardiac cycles measured
at baseline and peak reduces the technical complexity of the
procedure and offline analysis time. To be sure, multiple
issues, including standardization of protocols and normative
values, as well as technical improvements to improve reproducibility and image quality and reduce the technical demands
of the procedure, need to be addressed before the measurement
of FMD is advocated as a valid clinical prediction tool. Nevertheless, our data support a methodology of FMD and NMD
assessment that could allow for wider application of this
methodology.
GRANTS
T. J. Kizhakekuttu is supported by National Heart, Lung, and Blood
Institute (NHLBI) Ruth L. Kirschstein Training Grant T32 HL-007792-15,
D. D. Gutterman is supported by NHLBI Grants HL-094971 and HL-080704,
S. A. Phillips is supported by NHLBI Grant K23 HL-085614, and M. E.
Widlansky is supported by NHLBI Grant K23 HL-089326 and Advancing a
Healthier Wisconsin Grant 5520119-9520094. This work was supported by the
Clinical Translational Research Institute at the Medical College of Wisconsin
and NHLBI Grant HL-081587.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
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