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Normalization of flow-mediated dilation to shear stress area under the curve eliminates the impact of variable hyperemic stimulus
© Padilla et al; licensee BioMed Central Ltd. 2008
Received: 22 August 2008
Accepted: 04 September 2008
Published: 04 September 2008
Normalization of brachial artery flow-mediated dilation (FMD) to individual shear stress area under the curve (peak FMD:SSAUC ratio) has recently been proposed as an approach to control for the large inter-subject variability in reactive hyperemia-induced shear stress; however, the adoption of this approach among researchers has been slow. The present study was designed to further examine the efficacy of FMD normalization to shear stress in reducing measurement variability.
Five different magnitudes of reactive hyperemia-induced shear stress were applied to 20 healthy, physically active young adults (25.3 ± 0. 6 yrs; 10 men, 10 women) by manipulating forearm cuff occlusion duration: 1, 2, 3, 4, and 5 min, in a randomized order. A venous blood draw was performed for determination of baseline whole blood viscosity and hematocrit. The magnitude of occlusion-induced forearm ischemia was quantified by dual-wavelength near-infrared spectrometry (NIRS). Brachial artery diameters and velocities were obtained via high-resolution ultrasound. The SSAUC was individually calculated for the duration of time-to-peak dilation.
One-way repeated measures ANOVA demonstrated distinct magnitudes of occlusion-induced ischemia (volume and peak), hyperemic shear stress, and peak FMD responses (all p < 0.0001) across forearm occlusion durations. Differences in peak FMD were abolished when normalizing FMD to SSAUC (p = 0.785).
Our data confirm that normalization of FMD to SSAUC eliminates the influences of variable shear stress and solidifies the utility of FMD:SSAUC ratio as an index of endothelial function.
As a "barometer" for cardiovascular health status, brachial artery flow-mediated dilation (FMD) provides a bioassay for in vivo endothelial function . The evidence supporting the dependency of FMD on the endothelium is based on the observation that, after removal of the endothelial lining, arteries lose their ability to dilate in response to an increase in flow . Furthermore, FMD is partially to totally abolished after intra-arterial administration of a NO synthase blocker (i.e. L-NMMA) [3, 4]. Nitric oxide is an anti-atherosclerotic molecule, and decreased NO bioavailability is a hallmark of pro-atherogenic states . The biological significance of FMD, the relative simplicity and the non-invasive nature of the technique has motivated the dramatic growth in FMD research over the past 15 years. The widespread adoption of this ultrasound technique has been supported in part by published data suggesting that FMD correlates with invasive measurements of endothelial function in the coronary arteries [6, 7] and may predict future cardiovascular events [8, 9]. However, data are also available indicating that reduced FMD has weak or no association with other cardiovascular risk factors [10, 11], is a questionable marker of the presence and severity of coronary artery disease , fails to show a prognostic value , and has poor diagnostic accuracy for identifying older adults with subclinical cardiovascular disease . These contradictory results may be due to substantial discrepancies in FMD protocols across labs, but also due to the inter-subject variability in hyperemic shear stress (the stimulus for FMD). Large variability in reactive hyperemia has been documented among individuals and populations . The unadjusted FMD outcome may reflect conduit artery endothelial function as well as the magnitude of the hyperemic stimulus. Current work is moving to adjust the measured vasodilation response for the applied stimulus; in other words, dividing the peak FMD by the magnitude of stimulus achieved with reactive hyperemia. In 2005, Pyke and Tschakovsky , the initiators of this approach, suggested the utilization of shear rate (or shear stress (SS)) area under the curve (SSAUC) (individual area until peak FMD) as a method to quantify the entire and "relevant" hyperemic stimulus; and thus to be used for FMD normalization purposes. Two years later , the same authors provided experimental evidence of the physiological appropriateness of using this method. They concluded that the SSAUC, but not the peak shear, was the critical determinant of the peak FMD response. Following publication of this paper, a radical change in clinical practice within the FMD community was expected; however, to our surprise, since the time of Pyke and Tschakovsky's publication (April 2007), only two [17, 18] out of 92 FMD studies (PubMed search) have incorporated normalization of FMD to SSAUC. Reasons for this slow adoption of the recently proposed method to express FMD data may include 1) insufficient lead time after publication of the article for dissemination and implementation; 2) inability to simultaneously capture arterial diameter and velocity; 3) lack of clinical evidence supporting the utility of this approach; and 4) complicated comparison with the large body of evidence collected using the traditional FMD approach.
A "proof of concept" study was conducted herein to further evaluate the efficacy of FMD normalization. In a group of healthy individuals, we elicited five different magnitudes of reactive hyperemia-induced shear stress to mimic real-life inter-population differences. Considering this analogy, we created the following experimental scenario: comparison of five hypothetical "populations" with different magnitudes of reactive hyperemia-induced shear stress stimuli. Because the five "populations" do in fact have identical endothelial function, the correct outcome should be no detectable differences. We hypothesized that peak FMD:SSAUC ratio (normalization approach), but not peak FMD (traditional approach), would be the same among the five "populations" with identical endothelial function. This observation would corroborate that normalization of FMD to SSAUC eliminates the influences of variable shear stress found among populations, and solidify the utility of FMD:SSAUC ratio as an index of endothelial function.
Twenty healthy, physically active young adults (10 men, 10 women) volunteered for this study. All subjects were free of recognized cardiovascular, pulmonary and metabolic diseases, non-hypertensive (resting blood pressure < 140/80 mm Hg), non-obese (body mass index < 30 kg/m2), non-smokers, and had no family history of heart diseases. No subjects were taking medications with vaso-active effects, including contraceptives. All procedures were approved by Indiana University Committee for the Protection of Human Subjects. Written informed consent was obtained from each subject prior to participation in the study.
Procedures of the study consisted of a screening session and a vascular testing session. The screening session included completion of a medical history/health habits questionnaire, measurements of height, weight, resting blood pressure, and a fasting venous blood draw to obtain total cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, triglycerides, blood glucose, and high sensitivity C-reactive protein. In addition, all subjects were familiarized with the equipment/testing room to be used during the vascular session.
Brachial Artery Diameter and Blood Velocity
Whole blood viscosity
With the use of a pipette, 1 mL of blood sample was removed from the vacutainer tube, the mass of the sample was determined, and the density calculated. An additional blood sample was removed and transferred to a glass capillary viscometer  (Cannon-Manning Semi-Micro Viscometer, Cannon Instruments, Philadelphia, PA) and placed in a constant-temperature water bath (37°C). The viscosity of the sample was determined by measuring the time required for the sample to pass between two fixed points on the viscometer. Kinematic viscosity (mm2 • s-1) was obtained by multiplying a viscometer constant (0.007927 mm2 • s-2) by the efflux time in seconds. To obtain viscosity in mPa • s, kinematic viscosity was multiplied by the density in grams per milliliter. Measures were performed in duplicate.
Brachial artery shear stress
Brachial artery shear stress (dynes • cm-2) was calculated using the following formula: (4ηVm) • D-1, where η is blood viscosity (mPa • s), Vm is mean blood velocity (cm • s-1), and D is mean arterial diameter (cm) . To describe the magnitude of reactive hyperemia-induced shear stress elicited with increased duration of occlusion, shear stress AUC was calculated for each occlusion condition. Briefly, the AUC was calculated by summing the areas of successive postocclusion trapezoids (each with a base of 3-sec) for 60 sec (SS60secAUC; a.u.). To quantify the "relevant" hyperemic stimulus responsible for the peak FMD response, the shear stress AUC above baseline was individually calculated for the duration of time-to-peak dilation (SSAUC; a.u.). Normalization of FMD to shear stress was expressed as the peak FMD:SSAUC ratio (a.u).
A 50–75 μL blood sample was removed from the vacutainer tube, transferred into a capillary tube and centrifuged for 5 min using a micro-hematocrit centrifuge (Clay-Adams, New York, NY). Hematocrit (%) was read using a micro-capillary reader (International Equipment Company, Needham Heights, MA). Measures were performed in duplicate.
Magnitude of ischemia
Forearm oxygen tissue saturation (StO2; %) was monitored at baseline (30 sec), throughout each occlusion period, and during the reperfusion phase. The time course of StO2 was determined using a 3-sec moving average. To quantify the volume of ischemia, the StO2 AUC (area below baseline) was calculated by summing the areas of successive trapezoids (each with a base of 3-sec) for the total duration of the occlusion period (StO2AUC; a.u.). Peak ischemia (StO2peak; %) was considered as the StO2 change from baseline to immediately before cuff release.
Descriptive statistics were used to summarize the subject demographic data. One-way repeated measures ANOVA (5 levels) were performed to determine the effect of forearm occlusion duration on volume of ischemia (StO2AUC; a.u.), peak ischemia (StO2peak; a.u.), magnitude of reactive hyperemia-induced shear stress (SS60secAUC; a.u.), peak FMD response (%), and FMD normalized to shear stress (FMD:SSAUC ratio; a.u.). Tukey's HSD procedure was used when a significant F-ratio was found. All data are presented as mean ± standard error of the mean (SEM). For all statistical tests, the alpha level was set at 0.05. Statistical analyses were performed with SPSS v.15.0. (SPSS, Inc. Chicago, IL, USA).
Demographic characteristics of the subjects.
25.3 ± 0. 6
175.0 ± 2.1
68.6 ± 2.5
Body mass index, Kg/m2
22.3 ± 0.4
Resting systolic blood pressure, mmHg
112.0 ± 2.6
Resting diastolic blood pressure, mmHg
71.4 ± 2.2
Total cholesterol, mg/dL
159.3 ± 7.2
High density lipoprotein cholesterol, mg/dL
57.2 ± 2.4
Low density lipoprotein cholesterol, mg/dL
89.1 ± 6.2
64.9 ± 5.4
Fasting glucose, mg/dL
90.3 ± 1.5
High sensitivity C-reactive protein, mg/L
0.50 ± 0.1
Whole blood viscosity, mPa·s
3.74 ± 0.1
42.9 ± 0.8
Reported physical activity1, days/week
4.3 ± 0.2
Baseline hemodynamic information among forearm occlusion durations.
Arterial diameter, cm
0.352 ± 0.01
0.349 ± 0.01
0.349 ± 0.01
0.350 ± 0.01
0.350 ± 0.01
Blood velocity, cm • s-1
8.48 ± 1.9
8.99 ± 2.0
8.18 ± 1.8
8.81 ± 2.0
9.00 ± 2.0
Shear stress, dynes • cm-2
367.0 ± 32
392.4 ± 42
359.9 ± 32
385.1 ± 42
393.7 ± 40
Heart rate, bpm
51.5 ± 2
51.1 ± 2
52.5 ± 2
52.6 ± 2
52.4 ± 2
Forearm oxygen tissue saturation, %
56.87 ± 2.4
58.16 ± 2.3
58.46 ± 2.5
57.98 ± 2.5
58.41 ± 2.5
Given that reactive hyperemia varies among individuals and populations , the FMD outcome is reflective of both conduit artery endothelial function and magnitude of the hyperemic stimulus. Normalization of FMD to SSAUC has recently been proposed  to control for the presence of the large inter-subject variability in reactive hyperemia-induced shear stress and solely reflect conduit artery endothelial function. The present study was designed to further examine the efficacy of FMD normalization. In a group of healthy individuals, via manipulation of duration of cuff occlusion, we evoked five different magnitudes of reactive hyperemia-induced shear stress (Figure 4A) to create an idealized experimental scenario: comparison of five hypothetical "populations" with known identical endothelial function but different magnitude of reactive hyperemia-induced shear stress stimuli. Our findings demonstrate that, when presenting the data using the traditional approach (peak FMD), differences in FMD are detected across the varying shear stimuli (Figure 4B), which would suggest the erroneous conclusion that discrepancies in endothelial function exist among these populations. However, when presenting the data using the normalization approach (FMD:SSAUC ratio), these differences are abolished (Figure 4C); thus confirming that normalization of FMD to SSAUC eliminates the influences of variable shear stress on measured conduit artery endothelial function.
Pyke and Tshakovsky  were the first to examine the effect of normalizing FMD to SSAUC. The purpose of their study was to investigate the independent contributions of the peak and continued reactive hyperemia-induced shear stress on FMD. This was elegantly conducted by maintaining the peak shear stimulus constant while manipulating the shear stimulus duration (re-inflation of the cuff) or by maintaining the shear stimulus constant while manipulating the magnitude of the peak shear stimulus (application of arterial pressure). From these experiments, the authors concluded that the SSAUC, but not the peak shear, was the critical determinant of the peak FMD response; and thus to be used for normalization purposes. Because their findings were convincing and physiologically sound, we did not consider further exploration of the relative contribution of peak vs continued shear. Instead, we opted to manipulate the duration of cuff occlusion because this is an effective method to globally modify the hyperemic stimulus (both peak and duration); thus closely mimicking real-life between-subject differences in shear.
The only two ultrasound studies manipulating the duration of cuff occlusion as strategy to create a range in hyperemic stimuli and assess the subsequent vasodilation response at the conduit artery (radial and brachial) were published in 1997 [23, 24]. Both groups concluded that the duration of hyperemic stimulus played an important role on the vasodilatory response, observations that prompted Pyke and Tshakovsky's study  10 years later. Our data are in agreement with Leeson et al.  and Joannides et al.  in that longer duration of cuff occlusion was associated with greater hyperemic and vasodilation responses; however, these groups did not test whether normalization of FMD to SSAUC removed differences among trials; this is not surprising as the concept of FMD normalization was not introduced at that time. A limitation to the studies of Leeson et al. and Joannides et al. is the utilization of blood flow, instead of shear stress, as representation of the vasodilatory stimulus. Unfortunately, blood flow (velocity • π • (diameter2/4) • 60) and shear stress (viscosity • 4 • velocity/diameter) are parallel only in conditions where arterial diameters are constant, a situation that does not occur during the hyperemic phase where arterial diameters are significantly and dynamically altered. Furthermore, in contrast to Leeson et al. and Joannides et al., our study incorporated the NIRS technology to quantify the magnitude (volume and peak) of forearm occlusion-induced ischemia. Given that the forearm ischemia is the driving force for reactive hyperemia, characterization of the principal stimulus for FMD should not be neglected.
Shear stress area under the curve calculated using measured viscosity vs. assumed viscosity.
Shear stress area under the curve (a.u.)
Measured viscosity used
Assumed viscosity (4 mPa·s) used
t-test p value
ICC coefficient (p value)
11.7 ± 1.3
12.7 ± 1.5
0.985 (< 0.0001)
20.0 ± 1.4
21.7 ± 1.7
0.950 (< 0.0001)
26.3 ± 1.6
28.7 ± 2.2
0.941 (< 0.0001)
35.4 ± 2.1
38.3 ± 2.5
0.930 (< 0.0001)
41.5 ± 2.2
45.0 ± 2.8
0.919 (< 0.0001)
The significance of the present findings is notable. Our results support the view of Pyke and Tschakovsky , the original advocates of FMD normalization to SSAUC approach. The obvious lack of compliance to the recent guidelines in the clinical literature is intriguing and perhaps problematic. From a theoretical and physiological standpoint, there is robust evidence that FMD should be corrected by its dilator stimulus; however, the following question remains to be resolved: Does FMD:SSAUC ratio predict cardiovascular risk and events more accurately than peak FMD (the traditional FMD approach)? Further research is warranted to confirm that the apparent physiological appropriateness of normalization is in fact clinically pertinent. Given that arterial diameter and velocity measurements are typically performed continuously and simultaneously during the hyperemic phase, we encourage investigators to, at the minimum, report FMD data using both the traditional (peak FMD response) and normalization (peak FMD:SSAUC ratio) approaches. In supporting this new approach, while we do not intend to invalidate the large body of evidence collected using the traditional FMD approach, we advise the reader to interpret the existing literature with caution. Special attention should be devoted to studies that did not report any form of hyperemic stimulus.
The authors thank all the subjects for their time, effort, and willingness to participate in the study. This research was supported in part by the IU Graduate Student Research Grant-in-Aid, the IU HPER Research Grant-in-Aid, and the IU AAU/Bell-Updyke-Willett Kinesiology Research Fund. JP is sponsored by a fellowship from the Ministerio de Educación y Cultura de España.
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