Variability of Peripheral Arterial Tonometry in the Measurement of Endothelial Function in Healthy Men

Transcription

1 Variability of Peripheral Arterial Tonometry in the Measurement of Endothelial Function in Healthy Men Jianmin Liu, MD; Jingzhu Wang, MS; Yan Jin, MS; Hans J. Roethig, MD, PhD, FCP, FFPM; Martin Unverdorben, MD, PhD Research Development and Engineering, Altria Client Services, Richmond, Virginia Address for correspondence: Jianmin Liu, MD Research Development and Engineering Altria Client Services 601 E. Jackson Street Richmond, VA Measurement of endothelial function using peripheral arterial tonometry (PAT) has been reported to be significantly correlated with coronary blood flow. Repetitive PAT measurements were performed in 22 healthy male subjects at test intervals of 1 hour (5 times within a day) and hours (7 times within a day) to evaluate the variability of the reactive hyperemia index (). A total of 10 subjects underwent additional repetitive PAT at 2 hour intervals (7 times within a day) for 3 consecutive days to evaluate the diurnal effects and day-to-day reproducibility. The from each test was computed automatically based on a 15 minute recording of pulse wave amplitude changes of the fingers in response to reactive hyperemia induced by a 5 minute occlusion of the brachial artery. Intrasubject variability of at different test intervals, defined as the coefficient of variation(cv) was 15.3% ± 5.3%, 16.1% ± 7.8%, and 22.6% ± 3.9% for the tests at hour, 1 hour, and 2 hour intervals, respectively. Reactive hyperemia indices measured at the same time points on each of the 3 days were not statistically significant. The interday reproducibility, presented as intraclass correlation coefficients (ICC) ranged from 7 to 0.47.We conclude thatrepetitivepat measurementshaveno carryovereffecton at 1 hour, and 2 hour intervals, and the measured at hour intervals is associated with a trend of increase. The interday reproducibility is relatively low and the intrasubject variability of is similar to those observed in studies of flow-mediated dilation using brachial artery ultrasound scanning. Introduction Endothelial dysfunction results in abnormal regulation of blood vessel tone, impaired release of vasoprotective hormones, and the loss of the atheroprotective properties of the normal endothelium. 1,2 Endothelial dysfunction occurs in the early stages of atherosclerosis throughout the vascular system and, hence, is associated with cardiovascular events. Endothelial function has been widely studied in the peripheral arteries mainly using noninvasive techniques. Among these, flow mediated dilation (FMD) assessed by brachial artery ultrasound scanning (BAUS) is the most frequently used method. Endothelial function of the brachial arteries has been reported to reflect the degree of endothelial dysfunction of the coronary arteries, which represents a risk factor for cardiovascular disease. 3 Ashortcoming of BAUS encompasses considerable intrasubject variability (10% 50% coefficient of variation [CV]) while measuring approximately a 10% change in a brachial artery of 5 mm in diameter. Moreover, BAUS requires highly trained operators to measure the differences manually, and it is therefore prone to operator bias and high variability. Reactive hyperemia peripheral arterial tonometry (RH- PAT) has recently been reported as an alternative method to identify patients with coronary endothelial dysfunction by measuring pulse volume changes at the fingertips. 4 The device used in RH-PAT measurement, the Endo PAT 2000 (Itamar Medical Ltd., Caesarea, Israel), has received Financial support provided by Philip Morris USA, Inc. the Food and Drug Administration (FDA) approval as a diagnostic tool in the detection of coronary artery endothelial dysfunction in patients with signs or symptoms of ischemic heart disease, but without angiographic evidence of obstructive coronary artery disease. 5 It has also been reported that the reactive hyperemia index () measured by PAT correlates significantly (r = 0.405, P < 01) with coronary blood flow. 4 The results of PAT measurements are claimed to be operator independent and have been reported to correlate significantly with FMD measured by BAUS (r = 5, P < 001). 6 However,informationon the variability of repeat measurements within a day or on different days is limited for both methods. 7 In particular, no data have been reported on the carryover effect of repetitive hyperemia on with varying time intervals between measurements over a certain period of time. Recently, repeat PAT measurements were used in the evaluation of therapeutic intervention on improvement of endothelial function. 7 We became interested in the reliability of repeat measurements using this technique, and the minimumtime window requiredfor tests performedsequentially without causing a carryover effect. Peripheral arterial tonometry uses the digital pulse tonometry technique to measure the changes in the digital pulse volume due to reactivehyperemia.it is possiblethat measuringprocedures repeated at a short interval may cause the endothelium to respond differently to the same stimulus. In addition, if PAT is to be tested on different days, the interday reproducibility of this technique is equally important in the assessment of endothelial function. This study was therefore designed to 700 Clin. Cardiol. 32, 12, (2009) Received: April 21, 2009 Accepted with revision: July 2, 2009

2 investigate whether measurement of endothelial function using PAT is affected by the test intervals and the time of day. The study was also designed to evaluate the interday reproducibility of this technique, and to quantify intrasubject variability of during repetitive measurements in healthy men. Methods and Materials Endo PAT 2000 The Endo PAT 2000 system (Itamar Medical Ltd, Caesarea, Israel) consists of a main control unit, finger probes with tubing, and a signal analysis software package (version.3). The Endo PAT 2000 main control unit is connected to independent, thimble-shaped, finger-mountable probes attached to the index fingers of both hands. Peripheral arterial tonometry signals, which reflect pressure fluctuations sensedat the probe s distalcompartmentinducedby volume changes of the pulsatingdigital arteries,are sent to the main control unit from both probes. The signals of the occluded arm are corrected for the spontaneous variations of the pulse pressure amplitude by the signals derived from the contralateral arm. Then, a laptop computer generates the using proprietarysoftwareby calculatingthe ratio of the pulse pressure amplitude during hyperemia and the pulse pressure amplitude before inflation of the blood pressure cuff. Subjects A total of 22 healthy male volunteers were enrolled in the study conducted in the clinical unit at MDS Pharma Services, Lincoln, Nebraska. All subjects were free of cardiovascular, endocrine, hepatic, renal, and chronic inflammatory diseases. No subjects were taking any vasoactive medications or using tobacco products. Two PAT tests were performed on each of the subjects at a prestudy screening period and only subjects with an of or above for both tests were enrolled in the study. All subjects signed informed consent prior to the screening procedures. The study was approved by an independent institutional review board. Study Conduct All subjects stayed in the clinic for the entire study. Following overnight fasting, the subjects were allowed to have a low-fat, low-sugar snack at 0630 on each of the study days. The PAT tests were performed in a dark, noise- and climate-controlled (22 C 24 C) room. Daily blood pressures, taken at 0730, were entered into the Endo PAT 2000 system as reference. The subjects relaxed in a supine position with both their arms resting on arm support pads to avoid muscular activities of the arms and fingers. A blood pressure cuff was placed on the nondominant upper arm and the PAT probes were mounted on both index fingers. Following a 10 minute resting period, the 5-minuteRH-PATbaselinesignals were recorded.the blood pressure cuff was then inflated rapidly to 300 mm Hg and remained at that level for 5 minutes (occlusion) before being quickly released. The post occlusion RH-PAT was recorded for 5 minutes following cuff deflation. A total of 10 subjects underwent a series of 7 PATs at 2-hour intervals each day from 0800 to 2000 for 3 consecutive days. A total of 22 subjects had a series of 5 PATs at 1- hour intervals starting at On the subsequent days, these subjects were tested at -hour intervals for a total of 6 tests. The subjects were allowed to consume lowfat, low-sugar snacks between the tests at 2-hour and 1-hour intervals only. Alcoholic and/or caffeine/xanthinecontainingfood or beverageswere prohibited48 hours prior to and during the study. The subjects were instructed to remain quiet and relaxed during the tests, and to rest in a separate room between the tests at 1-hour and 2- hour The subjects remained in the test laboratory for the entire study when PAT was performed at -hour RH-PAT Data The was calculated automatically from each of the tests as described elsewhere. 6 Statistical Analysis Comparisons of repetitive were made using a linear mixed model for analysis of variance of repeated measurements (SAS version 8.2; SAS Institute,Inc, Cary, NC) to test the effect of day and the time point of measurement on. The interday reproducibility of the was determined by intraclass correlation coefficients (ICC), which were calculated for s measured with 2-hour intervals at the same time point on the 3 consecutive days, using the single score ICC for a 2-way mixed effect model (ICC[A,1]) as described by McGraw and Wong. 8 This method calculates the absolute agreement of measurements made under the fixed levels of the column (day) factor. Individual subject s CV (%) of measured at 2-hour, 1-hour, and -hour intervals and the mean CV for all subjects at each test interval was calculated to determine the intrasubject variability. For all statistical analysis, type I error was controlled at the 5 level for 2-sided tests. Data are expressed as mean ± SD unless otherwise mentioned. Results All 22 subjects completed the study. The baseline characteristics of these subjects are presented in Table 1. The at 2-hour intervals did not change significantly throughout the day and this observation remained true for all 3 days (P > 5 for all 3 days; Table 2). No statistical significant differences in the were observed among the 3 days (P = ; Figure 1A). The s measured at 1-hour Clin. Cardiol. 32, 12, (2009) 701

3 Clinical Investigations continued Table 1. Baseline Characteristics of 22 Participants Age (yrs) 35.4 ± 12.9 Weight (kg) 80.2 ± 11.3 BMI (kg/m 2 ) 25.0 ± 2.4 Systolic blood pressure (mm Hg) ± 8.8 Diastolic blood pressure (mm Hg) 69.3 ± 9.4 Serum cholesterol (mg/dl) ± 23.9 Serum triglyceride (mg/dl) 72.7 ± 28.3 Abbreviations: BMI, body mass index. showed similar results as those measured at 2-hour intervals, that is, no statistically significant differences in were found for the tests at any time point (Figure 1B). When measured at -hour intervals, the s showed a significant (P = 174) increase from measurement 1 to measurement 6; while the pair-wise comparison did not show a statistically significant difference in s between any adjacent PATs (Figure 1C). The ICC for s measured at 0800, 1000, 1200, 1400, 1600, 1800, and 2000 among the 3 days were 3, 7, 6, 6, 0.20, 0.29, and 0.46 respectively. Intrasubjectvariability of presented as CV (%) for PAT measured at 2-hour intervals was 22.6% ± 3.9%, which was statisticallygreater as compared to the ones measured at 1-hour intervals (16.1% ± 7.8%, P = 100) and at -hour intervals (15.3% ± 5.3%, P = 039), respectively (Figure 2). Discussion The main findings of the current study are: reactive hyperemia indices measured from repetitive peripheral arterial tonometry either at 2-hour intervals for 12 hours, Table 2. Reactive Hyperemia Index () at 2-Hour Intervals (n = 10) Time Point Day 1 Day 2 Day ± ± ± ± 2.1 ± 0.6 ± ± 2.1 ± 2.2 ± ± 0.6 ± ± ± ± ± 1800 ± 2.1 ± 1.9 ± ± ± ± 0.7 P Value a a For comparisons of s within each day. or at 1-hour intervals for 4 hours did not differ significantly. Trend analysis showed a significant increase in the when the PAT was measured at -hour intervals for hours. The reactive hyperemia indices measured at different days were not statistically different but with a relatively poor interday reproducibility. The intrasubject variability of was statistically, significantly smaller, when the measurements were repeated at - hour and 1-hour intervals as compared to those at 2-hour Studies using PAT and coronary angiography in patients with normal and abnormal endothelial function have shown a good correlation between and coronary blood flow. 4 However, the fact that digital pulse volume is modulated and influenced by various local, systemic, and environmental factors such as blood pressure, sympatheticparasympathetic nerve activities, blood vessel compliance, and temperature has reasonably lead to questioning the repeatability of test results when multiple measurements over a short period of time are needed. Until now, little was known about the reproducibility and variability of PAT in the measurement of repetitive reactive hyperemia. Studies measuring FMD of brachial artery using ultrasound scanning techniques, another noninvasive method used in the assessmentof endothelialfunction,showeda wide range (CV: 10.3% to >50%) of variability Most investigations in these studies compared only 2 FMD measurements taken at different times, with intervals ranging from several hours to several days. Brachial artery ultrasound scanning measurements of FMD at the radial artery has been reported to have similar variabilityas that measured from the brachial artery. 13 A study by Harris, in which multiple reactive hyperemia sessions were conducted at -hour, 1-hour, and 2-hour intervalsusing BAUS, showed that repetitivereactive hyperemia had little or no effect on the FMD measurements over a 2-hour period in the morning. 14 The findings from our study, that the measurements of the repetitive hyperemia at 1-hour and 2-hour intervals have no significant effect on the are in line with those in which BAUS was used to assess flow-mediated dilation of the brachial artery. However, in the current study, the time periods, that is, hours for the tests at -hour intervals, 4 hours at 1-hour intervals, and 12 hours at 2-hour intervals ( 3 d), were longer, and the number of tests performed was larger compared to those in other studies and thus, extended our understanding on the profiles of in repetitive hyperemia status in healthy subjects.our data suggestthat a diurnal variationof reactive hyperemia is very unlikely and the interday variability of reactivehyperemiais of small scale, which is in contrastwith the results reported by Etsuda et al 15 and Otto et al 16 who used BAUS and found significant changes of FMD between morning and afternoon measurements.in the current study, the subjects were allowed to consume snacks identical in compositionbefore and betweenthe measurementswhile in both Etsuda s 15 and Otto s 16 studies,the first measurements 702 Clin. Cardiol. 32, 12, (2009)

4 Day1 Day2 Day3 (A) hr PAT measurements (2-h interval) (B) PAT measurements (1-h interval) 3.5 a (C) PAT measurement (-h interval) Figure 1. Mean measured at 2-hour intervals during the first 3 days (A); PHI measured at 1-hour intervals (B); and PHI measured at -hour (C) a P = 174 for trend. were taken with the subjects in fasting status in the morning and the subsequentmeasurementswere taken followingthe subjects having consumed meals. It is therefore possible that the variations of FMD between morning and afternoon observed in those studies were due to the differences in the subjects fasting status, that is, measurements taken in fasting status vs postprandial rather than the time of the day. It is noticeable that showed a significant increase for PAT performedat -hour intervals in the current study. A detailed analysis indicated that the trend was statistically significantonly when the sixth PAT ( hours from the first PAT) was included, indicating that the s are relatively stable for 2 hours even measured at a -hour interval repetitively. Low interday reproducibility of PAT measurements was observed in the current study. Our study design could not determine whether it is due to the daily variability of endothelialresponses,or due to other factorsthat may affect the peripheral vascular responses to ischemia. However, this finding is clinically important when interpreting an individual s PAT results obtained from different days. The intrasubject variability in the current study is similar to or lower than those reported with FMD measurements using the brachial artery ultrasound method, suggesting that the reactive hyperemia varies substantially and can be detected by either method.an interestingfinding of the currentstudy is that the intrasubject variability tended to be smaller when the tests were repeated at shorter time intervals compared to those with longer intervals, that is, hourly or every - hour vs every 2 hours. This may be due to the fact that the subject s status was more stable since they stayed in the room for all of the measurements at the -hour test Limitation The limitation of our study pertains to the uncertainty among the causes of the variability. Since there is no gold standard for the noninvasive measurement of endothelial function, the observed variability might be due to the Clin. Cardiol. 32, 12, (2009) 703

5 Clinical Investigations continued CV% M ± SD 2h 1h 0 Test intervals Figure 2. Mean (large dot) and individual (small circle) percent coefficient of variation (CV%) of measured at 2-hour, 1-hour, and -hour a P = 100 for s at 1-hour intervals vs 2-hour b P = 039 for s at -hour intervals vs 2-hour spontaneous fluctuation of endothelial function or related to the measuring device itself. Similarly, repetitive angiographic measurements have not been performed either, and even an invasive gold standard for endothelial function test does not exist. Conclusions This study indicates that the variability of repeat measurements of endothelial function using PAT at intervals of -hour, 1-hour, and 2 hours is within the range of other noninvasive methods, and that there is no carryover effect of the PAT measurement on the when the tests are repeated as frequently as every hour. Low interday reproducibility was observed and further investigations are warranted. Acknowledgments The authors want to thank Drs. K. Shefy and R. Torish from Itamar Medical, Ltd. for their great technical support and Dr. Q. Liang for assistance in statistical analysis. References 1. Celermajer D. Endothelial function: does it matter? Is it reversible? J Am Coll Cardiol. 1997;30: Vane J, Anggard E, Botting R. Regulatory function of the vascular endothelium. NEnglJMed. 1990;323: Kuvin JT, Patel AR, Sliney KA, et al. Peripheral vascular endothelial function testing as a noninvasive indicator of coronary artery disease. J Am Coll Cardiol. 2001;38: Bonetti PO, Pumper GM, Higano ST, Holmes DR Jr. Kuvin JT, Lerman A. Noninvasive identification of patients with early coronary atherosclerosis by assessment of digital reactive hyperemia. J Am Coll Cardiol. 2004;44: US Food and Drug Administration. 510(K) SUMMARY ITAMAR MEDICAL. 510(k) Number KO32519, November 12, Accessed December 12, Kuvin JT, Patel AR, Sliney KA, et al. Assessment of peripheral vascular endothelial function with finger arterial pulse wave amplitude. Am Heart J. 2003;146: Bonetti PO, Barsness GW, Keelan PC, et al. Enhanced external counterpulsation improves endothelial function in patients with symptomatic coronary artery disease. J Am Coll Cardiol. 2003;41: McGraw KO, Wong SP. Forming inferences about some intraclass correlation coefficients. Psychological Methods. 1996;1: Liang YL, Teede H, Kotsopoulos D, et al. Non-invasive measurements of arterial structure and function: repeatability, interrelationships and trial sample size. Clin Sci. 1998;95: Herrington DM, Fan L, Drum M, et al. Brachial flow-mediated vasodilator responses in population-based research: methods, reproducibility and effects of age, gender and baseline diameter. J Cardiovasc Risk : De Roos NM, Bots ML, Schouten EG, Katan MB. Within-subject variability of flow-mediated vasodilation of the brachial artery in healthy men and women: implications for experimental studies. Ultrasound Med Bio. 2003;29: Malik J, Wichterle D, Haas T, et al. Repeatability of noninvasive surrogates of endothelial function. Am J Cardiol. 2004;94: Brook R, Grau M, Kehrer C, et al. Intrasubject variability of radial artery flow-mediated dilatation in healthy subjects and implications for use in prospective clinical trials. Am J Cardiol. 2005;96: Harris RA, Padilla J, Rink LD, et al. Variability of flow-mediated dilation measurements with repetitive reactive hyperemia. Vasc Med. 2006;11: Etsuda H, Takase B, Uehata A, et al. Morning attenuation of endothelium-dependent, flow-mediated dilation in healthy young men: possible connection to morning peak of cardiac events? Clin Cardiol. 1999;22: Otto ME, Svatikova A, Barretto RB, et al. Early morning attenuation of endothelial function in healthy humans. Circulation. 2004;109: Clin. Cardiol. 32, 12, (2009)