CLINICAL RESEARCH: ENDOTHELIAL FUNCTION
Non-Invasive Assessment of Endothelial Function
Which Technique?
Ann E. Donald, AVS*,3,*,
Marietta Charakida, MD*,3,
Tim J. Cole, ScD*,
Peter Friberg, MD, PhD*,
Phil J. Chowienczyk, FRCP ,1,
Sandrine C. Millasseau, PhD,2,
John E. Deanfield, MB, FRCP* and
Julian P. Halcox, MD, MRCP*
* Institute of Child Health, University College London, London, United Kingdom
King’s College London, Cardiovascular Division, Department of Clinical Pharmacology, St. Thomas’ Hospital, London, United Kingdom.
Manuscript received March 20, 2006;
revised manuscript received June 26, 2006,
accepted July 3, 2006.
* Reprint requests and correspondence:
Ms. Ann Donald, Vascular Physiology, Cardiac Unit, Institute of Child Health, 30 Guilford Street, London, WC1N 1EH United Kingdom. (Email: A.Donald{at}ich.ucl.ac.uk).
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Abstract
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OBJECTIVES: The purpose of this study was to compare 3 non-invasive techniques for assessment of endothelial function in adults and children and evaluate their utility in acute inflammation.
BACKGROUND: Endothelial dysfunction is a key early event in pre-clinical atherosclerosis. Flow-mediated dilation (FMD), although the established technique, is expensive and technically demanding. Measurements of vascular responses to inhaled salbutamol by pulse wave analysis (PWA) or pulse contour analysis (PCA) are potential alternatives.
METHODS: Sixteen adults (mean age 28 years, range 18 to 39) and 16 children (mean age 13 years, range 7 to 17) underwent concurrent vascular function testing on 2 occasions with ultrasound, PWA, and PCA. Eighteen men were also studied before and after typhoid vaccination.
RESULTS: Reproducibility of FMD was high in adults and children (coefficient of variation [CV] = 7.1 and 6.3, respectively). Salbutamol responses were more variable with PWA (adults CV = 11.5, children CV = 17.1) and PCA particularly in children (adults CV = 18.2, children CV = 36.3). Flow-mediated dilation (p < 0.001) and PWA with salbutamol (p = 0.03) responses fell after typhoid vaccination, and PCA (p = 0.7) was unchanged.
CONCLUSIONS: Vascular dysfunction during acute inflammation can be measured by FMD and by PWA with salbutamol. Flow-mediated dilation is less variable than PWA. Variability of PCA makes this technique currently unsuited to serial measures of endothelial function in children. Flow-mediated dilation remains the most reproducible method.
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Abbreviations and Acronyms
| | D = diameter | | DB
= baseline diameter | | DP
= peak diameter | | FMD = flow-mediated dilation | | GTN = glyceryl trinitrate | | NO = nitric oxide | | PAI = peripheral augmentation index |  PAIG
= maximal change in PAI after GTN administration | | PAIS
= maximal change in PAI after salbutamol administration | | PCA = pulse contour analysis | | PWA = pulse wave analysis | | RI = reflection index | | RIG
= maximal change in RI after GTN administration | | RIS
= maximal change in RI after salbutamol administration |
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The vascular endothelium is a key signal transducer in atherogenesis (1
). Study of preclinical vascular disease has been facilitated by use of non-invasive ultrasound techniques (2
). The vasodilator response to increased conduit arterial flow (flow-mediated dilation [FMD]) is dependent on local nitric oxide (NO) bioavailability, and measurement of this response has been widely used in clinical studies (3,4
).
Recently, alternative non-invasive techniques have been developed with beta2
adrenoceptor agonist-mediated endothelial NO release, using measurement of the response with radial artery applanation tonometry (pulse wave analysis [PWA]) or digital photoplethysmography (pulse contour analysis [PCA]) (5–7
). Although the equipment is small, portable, and easy to use, the comparative reproducibility of PWA and PCA and their ability to detect acute vascular changes have not been determined. We designed this study to assess the reproducibility of PWA, PCA, and FMD in children and adults and compared their ability to detect inflammation-induced changes in vascular function.
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Methods
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Study population.
We studied 16 children (11 boys, mean age 13 years, range 7 to 17) and 16 adults (9 men, mean age 28 years, range 18 to 39) to determine reproducibility and 18 men (mean age 26 years, range 18 to 39) to study vascular responses during acute inflammation. All were healthy and free from cardiovascular risk factors. All adults and parents gave written consent; children gave verbal assent. The study was approved by the local ethics committee.
Study protocol.
All investigations were performed by 2 experienced investigators. Subjects refrained from caffeine-containing drinks and food for 4 h before study and from vigorous exercise on the day of study. All studies were undertaken in a warm, temperature-controlled room by the same operator at the same time of day on both occasions.
Study 1—reproducibility: Adults
Adults attended on consecutive days (Fig. 1
A). Brachial FMD was assessed. The effect of a 25-µg sublingual dose of glyceryl trinitrate (GTN) was then assessed simultaneously by ultrasound, PWA, and PCA for  15 min after administration, until the brachial artery diameter (D) had returned to baseline. The PWA and PCA were assessed before and at 2.5-min intervals for 20 min after salbutamol inhalation via a spacer device (adults 400 µg, children 200 µg, Allen & Hanbury’s, GlaxoSmithKline, Uxbridge, United Kingdom). Blood pressure, D, peripheral augmentation index (PAI), and reflection index (RI) were measured at baseline and before each intervention.
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Figure 1 Study timeline for (A)
adults and (B)
children. D = diameter; FMD = flow-mediated dilation; GTN = glyceryl trinitrate; PAI = peripheral augmentation index; RI = reflection index.
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Study 1—reproducibility: Children
Children attended on 2 occasions at the same time of day, 1 week apart (to avoid disrupting school attendance). The GTN was omitted to shorten the study and improve recruitment and compliance (Fig. 1
B).
Study 2—response to an acute inflammation
Eighteen men were studied before and 8 h after a typhoid vaccination (TYPHIM Vi 0.5-ml injection, Aventis Pasteur, Merck Sharp & Dohme; Hoddesdon, Hertfordshire, United Kingdom), which transiently attenuates FMD (8
) (Fig. 1
A).
Vascular function assessment.
Brachial artery FMD
The right brachial artery was imaged, 5 to 10 cm above the antecubital fossa, with a high-resolution ultrasound probe (Acuson Aspen, Siemens, Malvern, Pennsylvania) held in a stereotactic clamp. Brachial artery FMD was induced by a 5-min inflation of a pneumatic cuff placed around the forearm immediately below the medial epicondyle (adults 300 mm Hg, children 200 mm Hg) followed by rapid deflation with an automatic air regulator (Logan Research; Rochester, Kent, United Kingdom). Brachial artery diameter (D) was measured with edge detection software (Brachial Tools, Iowa City, Iowa) from electrocardiogram-triggered images captured every 3 s throughout the 11-min recording protocol (9
). The FMD was expressed as maximal percentage change in vessel D from baseline (with the average of 20 baseline [DB
] and 3 peak [DP
] D readings: FMD% = [DP
– DB
]/DB
x 100). Blood flow was recorded continuously by pulsed-wave Doppler. Reactive hyperemia (RH%) was calculated from the maximal flow within the first 15 s after deflation of the pneumatic cuff, relative to the baseline flow. All ultrasound measures were performed offline by one experienced investigator (A.E.D.) masked to the order of the study visit.
PWA
The radial pressure-pulse waveform was acquired with a micromanometer (SPC-301, Millar Instruments, Houston, Texas) (10
). Data were recorded directly onto a computer running proprietary software (SphygmoCor version 7.0, Scanmed; Moreton-in-Marsh, Gloucestershire, United Kingdom). Peripheral and central augmentation index can be derived from the peripheral pressure pulse (11
). We used PAI, because changes in the central pulse waveform with salbutamol are more subtle and the transfer function used to derive the central pressure-pulse waveform has not been validated in children.
PCA
The RI was obtained with a photoplethysmograph (Micro Medical; Gillingham, Kent, United Kingdom), on the index finger of the left hand and measured as previously described (5
).
Pulse wave analysis and PCA were performed simultaneously; 3 baseline readings, 1 min apart, were averaged, and the maximal changes in PAI and RI after GTN and salbutamol were calculated.
Statistical analysis.
For PWA and PCA, results are expressed as the maximal change in PAI (PAIS
; PAIG
) and RI (RIS
; RIG
) after salbutamol and GTN administration, respectively. Values are expressed as mean ± SD unless otherwise stated. Within-method reproducibility is expressed as the technical error of the measurement (TEM) (12
) and as the percentage of the coefficient of variation [(SD of the paired differences/the overall mean)/
x 100.
Data are also presented with Bland-Altman plots. Relationships between vascular responses were examined with the Pearson correlation. Student paired t
tests were used to assess the effect of typhoid vaccination on vascular function. Statistical analyses were performed with SPSS v12 (SPSS, Chicago, Illinois).
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Results
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Study 1—reproducibility.
All adults and all but 2 children completed both assessments (Table 1
, Fig. 2
). One child did not return and one engaged in strenuous exercise immediately before visit 2. Reproducibility of the baseline measures before endothelial stimulation was highest for D and similar for PAI and RI both in adults and children. Baseline flow and reactive hyperemia were the same on both visits in adults and children.
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Table 1. Summary of the Baseline Data and Endothelial-Dependent and Endothelial-Independent Function for Three Methods in Adults and Children
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Figure 2 Bland Altman plots for flow-mediated dilation (FMD), pulse wave analysis (PWA), and pulse contour analysis (PCA) (after Salbutamol) in adults (left)
and children (right)
. The dotted lines
represent the 95% confidence intervals.
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Flow-mediated dilation was the most reproducible measure between visits, followed by PAIS
, then RIS
. PAIS
and particularly RIS
were considerably less reproducible in children. Between-visit differences versus mean values for the 3 measures in adults and children show no obvious trends in mean or variability. Individual estimates of endothelial function by the 3 methods were uncorrelated with each other (FMD/PAIS
r = –0.05, FMD/RIS
r = –0.08, PAIS
/RIS
r = 0.03). Reproducibility of GTN-mediated changes was less good for all 3 methods.
Study 2—response to acute inflammation.
Baseline diameter, PAI, and RI were unchanged after typhoid vaccination (Fig. 3
). Baseline flow (p = 0.53) and reactive hyperemia (p = 0.45) were unchanged before and after typhoid. Flow-mediated dilation and PAIS
fell by 24% (p = 0.0005) and 22% (p = 0.03), respectively 8 h after vaccination. RIS
was unchanged (p = 0.7). Responses to GTN with all modalities remained the same.
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Figure 3 The impact of typhoid vaccination on endothelial-dependent and endothelial-independent function. Data are expressed as mean values ± SE. *p < 0.05. **p < 0.001. GTN% = glyceryl trinitrate-mediated dilation; other abbreviations as in Figure 1
.
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Discussion
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This study shows that FMD is the most robust of these non-invasive measures of endothelial function in both children and adults. The PWA was less reproducible in children, and PCA performed acceptably only in adults. Flow-mediated dilation and PWA can detect changes in vascular function during acute inflammation.
Endothelial dysfunction is central to early atherogenesis (1,4
). The development of non-invasive FMD as a surrogate measure of arterial health has enabled the study of children and healthy populations, providing an opportunity for early detection and prevention. This technique is conceptually simple, NO dependent, and can measure the effect of interventions (2,4,13
). Although methodological advances have diminished operator dependence, it remains expensive and technically demanding (14
), prompting the search for simpler and cheaper validated techniques.
Salbutamol, a beta2
agonist, causes vascular endothelial NO release, enabling measurement of global rather than local NO-dependent vascular changes (5,6
). The drug was administered via a spacer, providing reproducible pulmonary delivery in healthy subjects (7
). Pulse wave and contour responses with salbutamol are reproducible in adults and impaired in diabetes, hypercholesterolemia, and coronary artery disease (5–7
). However, there are few data on their ability to detect changes with interventions and no data on their applicability in children. Similarly, these methods have not been assessed in smokers, asthmatic patients, and those with other pulmonary conditions, limiting their potential applicability in such subjects.
Typhoid vaccination has been shown to induce transient conduit and microvascular endothelial dysfunction in  12 subjects (8,15
). In contrast to the findings of Vlachopoulos et al. (16
), baseline augmentation and reflection indexes were unaffected by vaccination, but PAIS
fell by 22%, consistent with the extent of endothelial dysfunction seen with FMD. The lack of change in RIS
after typhoid might be due to the greater variability of this technique, resulting in a type 2 error. Alternatively, the vascular effects of vaccination might be less readily detected by RIS
as a result of differences between the volume and pressure-pulse waveforms.
We have been able to demonstrate lower reproducibility of PWA and particularly PCA compared with FMD, clearly distinguishing between the methods even within this relatively small study. This might have important consequences for clinical use of these techniques. Thus studies based on PWA and PCA must be larger than for FMD to achieve the same power to detect a given effect size. Taking account of the intrinsic variability demonstrated for each method, power calculations indicate that studies using PWA and PCA would need to be 2 and 6 times larger, respectively, than those using FMD.
The strengths and weaknesses of available methods for endothelial function measurement should be considered in relation to any proposed clinical application. Flow-mediated dilation is accurate and reproducible after appropriate training and applicable to large population studies (3,17
). Pulse wave analysis is cheap and portable but requires considerable expertise, remaining in part operator dependent, and current analysis software algorithms are limited in the presence of sinus arrhythmia, common in children and young adults. Pulse contour analysis requires little training and is operator independent; however, it is more variable, possibly owing to the greater influence of sympathetic tone on hand circulation physiology.
We were interested in noting the lack of correlation between the 3 techniques. Although the greater variability of the newer methods is a possible explanation, it is more likely that our observations reflect distinct differences in the pathways responsible for endothelial regulation of arterial tone in different vascular beds. These mechanisms and their pathophysiologic implications require further investigation.
Conclusions.
Flow-mediated dilation remains the non-invasive technique of choice for study of endothelial function in adults and children. In addition, acute endothelial dysfunction is detectable with PWA, supporting its potential use in mechanistic vascular studies. Pulse wave analysis and PCA are promising methods for assessment of vascular function, but their reproducibility needs to be improved, especially if contemplating use in children. Furthermore, these methods might provide different information on vascular pathophysiology. The larger subject numbers required to demonstrate clinically significant differences reduce the financial and practical advantages of these newer techniques.
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Footnotes
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Support was provided by the Coronary Artery Disease Research Association (to Ms. Donald), the Greek State Scholarship Foundation (to Dr. Charakida), the UK Medical Research Council (to Prof. Cole), the British Heart Foundation (to Prof. Deanfield and Dr. Halcox), and the Swedish Medical Research Council (to Prof. Friberg).
1 Dr. Chowienzyck was a director of Micromedical until March 2005. 
2 Dr. Millasseau is supported by Micromedical.
3 Ms. Donald and Dr. Charakida contributed equally to this study.
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References
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1. Ross R. Atherosclerosis—an inflammatory disease N Engl J Med 1999;340:115-126.[Free Full Text]2. Celermajer DS, Sorensen KE, Gooch VM, et al. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis Lancet 1992;340:1111-1115.[CrossRef][Web of Science][Medline] 3. Benjamin EJ, Larson MG, Keyes MJ, et al. Clinical correlates and heritability of flow-mediated dilation in the community: the Framingham Heart Study Circulation 2004;109:613-619.[Abstract/Free Full Text] 4. Clarkson P, Adams MR, Powe AJ, et al. Oral L-arginine improves endothelium-dependent dilation in hypercholesterolemic young adults J Clin Invest 1996;97:1989-1994.[Web of Science][Medline] 5. Chowienczyk PJ, Kelly RP, MacCallum H, et al. Photoplethysmographic assessment of pulse wave reflection: blunted response to endothelium-dependent beta2-adrenergic vasodilation in type II diabetes mellitus J Am Coll Cardiol 1999;34:2007-2014.[Abstract/Free Full Text] 6. Hayward CS, Kraidly M, Webb CM, Collins P. Assessment of endothelial function using peripheral waveform analysis: a clinical application J Am Coll Cardiol 2002;40:521-528.[Abstract/Free Full Text] 7. Wilkinson IB, Hall IR, MacCallum H, et al. Pulse-wave analysis: clinical evaluation of a noninvasive, widely applicable method for assessing endothelial function Arterioscler Thromb Vasc Biol 2002;22:147-152.[Abstract/Free Full Text] 8. Hingorani AD, Cross J, Kharbanda RK, et al. Acute systemic inflammation impairs endothelium-dependent dilatation in humans Circulation 2000;102:994-999.[Abstract/Free Full Text] 9. Charakida M, Donald AE, Terese M, et al. Endothelial dysfunction in childhood infection Circulation 2005;111:1660-1665.[Abstract/Free Full Text] 10. Kelly R, Hayward C, Avolio A, O’Rourke M. Noninvasive determination of age-related changes in the human arterial pulse Circulation 1989;80:1652-1659.[Abstract/Free Full Text] 11. Chen CH, Nevo E, Fetics B, et al. Estimation of central aortic pressure waveform by mathematical transformation of radial tonometry pressureValidation of generalized transfer function. Circulation 1997;95:1827-1836.[Abstract/Free Full Text] 12. Ulijaszek SJ, Kerr DA. Anthropometric measurement error and the assessment of nutritional status Br J Nutr 1999;82:165-177.[Web of Science][Medline] 13. Joannides R, Haefeli WE, Linder L, et al. Nitric oxide is responsible for flow-dependent dilatation of human peripheral conduit arteries in vivo Circulation 1995;91:1314-1319.[Abstract/Free Full Text] 14. Deanfield J, Donald A, Ferri C, et al. Endothelial function and dysfunctionPart I: methodological issues for assessment in the different vascular beds: a statement by the Working Group on Endothelin and Endothelial Factors of the European Society of Hypertension. J Hypertens 2005;23:7-17.[CrossRef][Web of Science][Medline] 15. Clapp BR, Hirschfield GM, Storry C, et al. Inflammation and endothelial function: direct vascular effects of human C-reactive protein on nitric oxide bioavailability Circulation 2005;111:1530-1536.[Abstract/Free Full Text] 16. Vlachopoulos C, Dima I, Aznaouridis K, et al. Acute systemic inflammation increases arterial stiffness and decreases wave reflections in healthy individuals Circulation 2005;112:2193-2200.[Abstract/Free Full Text] 17. Sorensen KE, Celermajer DS, Spiegelhalter DJ, et al. Non-invasive measurement of human endothelium dependent arterial responses: accuracy and reproducibility Br Heart J 1995;74:247-253.[Abstract/Free Full Text]
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Abstract
Methods