Factors Associated With Blood Pressure Response to the Cold Pressor Test: The GenSalt Study

Original article Factors Associated With Blood Pressure Response to the Cold Pressor Test: The GenSalt Study Mingzhi Zhang, 1,2 Qi Zhao, 2 Katherine T. Mills, 2 Jichun Chen, 3 Jianxin Li, 3 Jie Cao, 3 Dongfeng Gu, 3 and Jiang He 2 background Blood pressure (BP) response to the cold pressor test (CPT) has been associated with increased risk of cardiovascular disease. We studied risk factors associated with BP response to CPT. methods We conducted the CPT among 2,682 individuals in rural north China. BP was measured using a standard mercury sphygmomanometer prior to and at 0, 1, 2, and 4 minutes after the participants immersed their right hand in ice water for 1 minute. results Sex, age, and baseline BP levels were significantly associated with BP response to the CPT. For example, maximum systolic BP response (mean ± SD) was greater in women than in men (15.5 ± 10.7 vs. 13.8 ± 10.0 mm Hg; P < 0.0001), correspondingly higher with age (12.4 ± 8.7, 13.8 ± 10.0, and 16.4 ± 11.2 mm Hg for those aged < 35, 35 44, and 45 years, respectively; P for trend < 0.0001), and greater with higher BP (13.5 ± 10.0, 14.9 ± 10.2, and 17.4 ± 11.5 mm Hg for those with baseline BP < 120/80, 120 139/80 89, and 140/90 mm Hg, respectively; P for trend < 0.0001). In multivariable analyses, we also observed that higher body mass index, physical inactivity, and alcohol consumption were significantly associated with greater BP response to the CPT. conclusions Our study indicates that females, older age, and elevated baseline BP levels are associated with greater BP response to the CPT. In addition, physical inactivity, higher weight, and alcohol consumption may also be related to BP hyperreactivity to stress. Keywords: blood pressure; hypertension; physiological; risk factors; stress. doi:10.1093/ajh/hpt075 Hypertension is a leading risk factor for cardiovascular disease and premature death globally. Cardiovascular hyperreactivity to stress has been hypothesized to be an important risk factor for the development of hypertension and cardiovascular disease. 1 3 The cold pressor test (CPT), which measures blood pressure (BP) response to the stimulus of external cold, has been commonly used for the evaluation of cardiovascular reactivity to stress in normotensive and hypertensive subjects. Increased BP response to CPT has been associated with greater risk of hypertension in previous studies. 4 6 The increased activity of the sympathetic nervous system during the CPT is considered one of the major mechanisms mediating the cardiovascular response to CPT. 7,8 Several studies have investigated risk factors for BP response to the CPT. To date, a few factors have been identified to be associated with the pressor effect of the cold stimulus, such as age, sex, baseline BP level, and physical activity. 9 18 Blacks exhibited greater BP response to cold stimulation than whites. 10,18,19 However, most of the studies had small sample sizes, and the associations between risk factors and BP response to the CPT are still controversial. For example, 2 studies reported that men had greater BP response to the CPT than women, while a more recent study showed women had greater BP responses to the CPT. 9 11 The determinants of BP response to the CPT are still not fully understood. We investigated the risk factors for BP response to the CPT using data from the Genetic Epidemiology Network of Salt Sensitivity (GenSalt) study. A total of 2,682 GenSalt study participants completed the CPT, which provides a large sample to examine multiple risk factors for hyper reactivity of BP to cold stress. METHODS Study participants The GenSalt study was conducted in rural areas in northern China. The study design and methods for the GenSalt Corresponding to: Qi Zhao (qizhao@tulane.edu). Initially submitted January 2, 2013; date of first revision April 29, 2013; accepted for publication May 4, 2013; online publication June 1, 2013. 1 Department of Epidemiology, Soochow University School of Public Health, Suzhou, China; 2 Department of Epidemiology, Tulane University School of Public Health and Tropical Medicine, New Orleans, LA; 3 Department of Epidemiology and Population Genetics, Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. American Journal of Hypertension, Ltd 2013. All rights reserved. For Permissions, please email: journals.permissions@oup.com 1132 American Journal of Hypertension 26(9) September 2013

Factors Associated with BP Response to CPT study have been published elsewhere. 20 The main objective of the GenSalt study was to identify genetic factors associated with BP response to dietary sodium and potassium interventions and CPT. A family-based study design was adopted by the GenSalt study to maximize study power because the families with hypertensives are most likely enriched in genetic variants for hypertension and tend to be more sensitive to dietary sodium and potassium interventions and the CPT. In brief, a community-based BP screening was conducted among persons aged 18 60 years in the study villages to identify potential probands with mean systolic BP (SBP) of 130 160 mm Hg and/or diastolic BP (DBP) between 85 and 100 mm Hg and no use of antihypertensive medications. The probands and their parents, siblings, offspring, and spouses were recruited for the GenSalt study. Individuals who were older than age 60 years; had stage 2 hypertension, secondary hypertension, or a history of clinical cardiovascular disease, diabetes, or chronic kidney disease; were using antihypertensive medication; or were pregnant were excluded from the CPT. A total of 2,682 study participants completed the CPT. Institutional review boards at all participating institutes approved the GenSalt study. Written informed consent was obtained from each participant. Data collection A standard questionnaire was administered by trained staff to obtain data on demographic characteristics, medical history, and lifestyle risk factors, including cigarette smoking, alcohol drinking, and physical activity. Current cigarette smoking was defined as having smoked 100 cigarettes during a lifetime and smoking at survey, while current alcohol drinking was defined as consumption of 12 or more drinks in the past 12 months. The physical activity information obtained from the questionnaire was converted to metabolic equivalent hours per day. Body weight, height, and waist circumference were measured according to a standard protocol. Three BP measurements were obtained every morning during the 3-day baseline observation by trained and certified BP technicians using a random-zero sphygmomanometer according to a standard protocol. BP was measured with the participant in a sitting position after a 5-minute rest. Additionally, participants were advised to avoid alcohol, coffee/tea, cigarette smoking, and exercise for at least 30 minutes before their BP measurements. The mean of the 9 BP measurements collected during the 3-day baseline observation was considered the baseline BP level. Cold pressor test The CPT was conducted using a standardized protocol by trained and certified BP technicians. After the participant had remained seated for 20 minutes, 3 pre-cpt BP measurements were obtained using a standard mercury sphygmomanometer on the right upper arm before the ice water immersion. Then, participants immersed their left hand in the ice water bath (3 5 C) to just above the wrist for 1 minute. BP measurements at 0, 1, 2, and 4 minutes were obtained using a standard mercury sphygmomanometer on the right upper arm after the left hand had been removed from the ice water bath. The CPT was well tolerated in all subjects, and no side effects were reported. Statistical analyses Means and percentages of study participant general characteristics were calculated overall and by sex. Differences between men and women were examined using t test for continuous variables and χ 2 test for categorical variables. The magnitude of BP response to the CPT was assessed with BP responses at 0, 1, 2, and 4 minutes; maximum BP response during the 4 time points; and the total area under curve (AUC) above baseline BP levels. Responses at 0, 1, 2, and 4 minutes were calculated as the difference between BP at these time points and pre-cpt BP levels, respectively; maximum response was defined as the largest BP difference between BP at any of the 4 CPT tested time points and pre-cpt BP; and the AUC of BP response above pre-cpt BP levels was defined as the difference between the area under the response curve and the area below pre-cpt BP levels (from the time point of immersing the hand in ice water to 4 minutes post-cpt). BP responses to the CPT were presented by sex, age, and baseline BP subgroups, and differences among subgroups were tested using a mixed linear model that accounted for nonindependence of family members. In addition, multiple mixed-effect regression analyses were conducted to explore factors associated with BP response to the CPT. Age, sex, education, alcohol drinking, cigarette smoking, physical activity, body mass index (BMI), hypertension (BP 140/90 mm Hg), and baseline pulse were included in these regression models. All P values were 2-tailed, and a significance level of 0.05 was used. Statistical analysis was conducted using SAS statistical software (version 9.2; SAS Institute Inc., Cary, NC). RESULTS The general characteristics of study participants are presented in Table 1. Women were less likely to have graduated from secondary school, smoke, or drink alcohol and engaged in less physical activity than men. Women had greater BMI and pulse but smaller waist circumference and lower SBP and DBP levels compared to men. The mean SBP and DBP responses to the CPT in men, women, and the overall sample are shown in Figure 1. During the CPT, most participants achieved maximum BP response at 0 minutes after ice water immersion (92.3% and 82.1% of participants for SBP and DBP responses, respectively). Thereafter, BP levels gradually decreased and returned to the baseline level in 4 minutes after cold stimulus. The distributions of SBP and DBP responses to the CPT at 0, 1, 2, and 4 minutes are presented in Figure 2. In general, BP responses at the 4 time points are continuously and nearly normally distributed among the study population. Table 2 shows mean SBP and DBP responses to the CPT in sex, age, and baseline BP subgroups. Women had greater SBP response at 0 minutes and a greater maximum SBP response than men (both P < 0.0001). Participants who were older or had higher baseline BP levels showed greater SBP responses at 0, 1, and 2 minutes, as well as maximum American Journal of Hypertension 26(9) September 2013 1133

Zhang et al. Table 1. General characteristics of 2,682 study participants Characteristic Overall (n = 2,682) Men (n = 1,429) Women (n = 1,253) P value a Age, years 42.2 (10.7) 42.6 (10.7) 41.8 (10.6) 0.06 Secondary school or higher,% 64.4 76.3 50.8 < 0.0001 Married, % 93.8 93.2 94.5 0.17 Current alcohol drinking, % 28.6 51.5 2.4 < 0.0001 Current cigarette smoking, % 31.0 57.5 0.7 < 0.0001 Physical activity, MET 23.1 (11.8) 25.0 (12.0) 21.0 (11.1) < 0.0001 BMI, kg/m 2 23.9 (3.4) 23.7 (3.4) 24.1 (3.5) 0.001 BMI 25kg/m 2, % 34.4 33.0 36.0 0.11 Waist circumference, cm 81.9 (10.5) 83.4 (10.3) 80.3 (10.4) < 0.0001 Systolic BP, mm Hg 119.6 (15.2) 121.0 (13.9) 117.9 (16.5) < 0.0001 Diastolic BP, mm Hg 75.6 (10.6) 77.3 (10.2) 73.6 (10.7) < 0.0001 Hypertension, % 14.3 15.2 13.2 0.15 Pulse, beat/min 71.5 (7.6) 70.8 (8.0) 72.3 (7.2) < 0.0001 Values are presented as mean (standard deviation) or percentage. Abbreviations: BMI, body mass index; BP, blood pressure; MET, metabolic equivalent. a P values for comparisons between men and women. Figure1. SBP and DBP levels during the cold pressor test. Abbreviations: DBP, diastolic blood pressure; SBP, systolic blood pressure. 1134 American Journal of Hypertension 26(9) September 2013

Factors Associated with BP Response to CPT Figure 2. Distribution of SBP (upper panels) and DBP (lower panels) responses to cold pressor test at 0, 1, 2, and 4 min (from left to right). Black bars show zero changes in BP. BP response to CPT at 0 min = BP at 0 min pretest BP; BP response at 1 min = BP at 1 min pretest BP; BP response at 2 min = BP at 2 min pretest BP; BP response at 4 min = BP at 4 min pretest BP. Abbreviations: BP, blood pressure; DBP, diastolic blood pressure; SBP, systolic blood pressure. response and total AUC for SBP. On the contrary, older subjects showed less DBP response at 0 minutes and maximum DBP response. Table 3 shows regression coefficients of maximum BP responses and total AUC associated with risk factors from the multiple mixed-effect models. In these multivariable regression models, sex, age, and baseline BP were significantly associated with BP responses to the CPT after adjusting for covariates. In addition, greater BMI was associated with higher maximum SBP response to the CPT (β = 0.132, P = 0.04). Physical activity was inversely associated with total AUC of SBP response (β = 0.101, P = 0.01), and alcohol drinking was positively associated with total AUC of DBP response (β = 1.810, P = 0.04). Discussion To the best of our knowledge, this is the largest study to investigate factors associated with BP response to the CPT in a Chinese population. The present study provided further evidence for the associations of sex, age, baseline BP, and physical activity with BP response to the CPT. In addition, this study indicates for the first time that higher BMI and alcohol drinking may be risk factors for higher BP response to the CPT. The CPT is known to cause global sympathetic activation and results in significant arteriolar vasoconstriction with a subsequent increase in BP. 21 23 Our recent analyses have indicated that BP response to the CPT is a long-term reproducible and stable characteristic among the GenSalt study population. For example, the correlation coefficients of SBP responses to CPT over a 4.5-year follow-up period were 0.42 and 0.39 for the maximum and total AUC, respectively (both P < 0.0001). 24 Greater BP response to the CPT has been associated with an increased risk of hypertension in previous studies. 4 6 For example, Kasagi et al. reported that systolic hyperreactors (defined as maximum SBP response to the CPT >15 mm Hg) had a 1.37-fold increase in risk for hypertension compared with systolic normal reactors based on a follow-up study of 28 years. 6 The factors influencing BP response to the CPT have not been thoroughly investigated, although a limited number of studies with small sample sizes were conducted. Previous studies have shown inconsistent results regarding the sex difference in BP response to the CPT. LeBlanc et al. reported that men (n = 9) had a greater SBP response to the CPT than women (n = 8). 9 Similar results were found in a study including 117 college students. 10 On the contrary, another larger study has recently concluded that women showed greater BP response to the CPT by comparing 57 women with 94 men. 11 This finding was further supported by our study results. We observed that the SBP response at 0 minutes and maximum SBP response were significantly higher in women than in men. In addition, 4 minutes after the cold stimulus, both SBP and DBP fell below baseline BP levels to a greater degree in women than in men. The mechanism of the sex difference in BP response to the CPT is still unclear. However, it has been suggested that the effects of sympathetic nerve activity on cardiac output and peripheral resistance, 2 major factors of BP regulation, are different between men and women. 25 It also has been suggested that the greater responsiveness American Journal of Hypertension 26(9) September 2013 1135

Zhang et al. Table 2. BP responses to cold pressor test by sex, age, and baseline BP subgroups 0 min 1 min 2 min 4 min Maximum Total AUC Variable ΔSBP ΔDBP ΔSBP ΔDBP ΔSBP ΔDBP ΔSBP ΔDBP ΔSBP ΔDBP SBP DBP Sex Men 13.4 (10.5) 6.8 (6.9) 4.2 (6.3) 2.6 (4.5) 0.7 (5.3) 1.0 (4.1) 0.8 (4.8) 0.2 (3.7) 13.8 (10.0) 7.7 (6.3) 17.8 (23.5) 10.8 (16.7) Women 15.2 (11.0) 7.2 (6.8) 4.5 (6.7) 2.6 (4.7) 0.5 (5.4) 0.7 (4.1) 1.4 (5.1) 0.6 (3.9) 15.5 (10.7) 7.8 (6.3) 19.0 (24.2) 10.2 (17.3) P for difference by sex < 0.0001 0.18 0.22 0.96 0.33 0.03 0.001 0.003 < 0.0001 0.44 0.20 0.36 Age, years < 35 11.9 (9.2) 7.7 (7.1) 3.1 (5.5) 2.9 (4.8) 0.1 (4.9) 0.9 (4.3) 1.5 (4.9) 0.3 (4.2) 12.4 (8.7) 8.5 (6.4) 13.7 (21.2) 11.7 (17.4) 35 44 13.5 (10.4) 6.8 (6.8) 4.1 (6.1) 2.4 (4.5) 0.6 (5.0) 0.9 (4.0) 0.9 (4.5) 0.3 (3.6) 13.8 (10.0) 7.5 (6.2) 17.5 (22.5) 10.1 (16.8) 45 16.1 (11.5) 6.8 (6.8) 5.1 (7.1) 2.6 (4.5) 0.9 (5.8) 0.8 (4.1) 1.0 (5.3) 0.4 (3.7) 16.4 (11.2) 7.5 (6.3) 21.5 (25.7) 10.3 (16.9) P for difference by age < 0.0001 0.02 < 0.0001 0.17 0.01 0.85 0.06 0.94 < 0.0001 0.002 < 0.0001 0.18 Baseline BP, mm Hg < 120/80 13.2 (10.4) 6.9 (6.7) 3.9 (6.0) 2.5 (4.4) 0.4 (4.8) 0.9 (4.0) 1.2 (4.7) 0.4 (3.8) 13.5 (10.0) 7.6 (6.1) 16.4 (22.3) 10.2 (16.5) 120 139/80 89 14.5 (10.6) 6.9 (6.9) 4.3 (6.8) 2.7 (4.7) 0.5 (5.7) 0.9 (4.1) 1.1 (5.2) 0.3 (3.8) 14.9 (10.2) 7.7 (6.1) 18.6 (24.6) 10.6 (17.2) 140/90 17.0 (11.9) 7.9 (7.5) 5.6 (7.0) 2.8 (4.9) 1.5 (6.0) 0.8 (4.4) 0.7 (5.1) 0.4 (3.6) 17.4 (11.5) 8.3 (7.3) 24.3 (25.8) 11.5 (18.1) P for difference by BP < 0.0001 0.03 < 0.0001 0.25 0.001 0.96 0.17 0.93 < 0.0001 0.13 < 0.0001 0.41 BP responses are presented as mean (standard deviation). Abbreviations: AUC, area under the curve; BP, blood pressure; DBP, diastolic blood pressure; SBP, systolic blood pressure. 1136 American Journal of Hypertension 26(9) September 2013

Factors Associated with BP Response to CPT Table 3. Regression coefficients in the multiple regression models of maximum BP responses and total AUC of responses Maximum BP responses to CPT Total AUC during CPT ΔSBP ΔDBP ΔSBP ΔDBP Variable β SE P value β SE P value β SE P value β SE P value Age, years 0.148 0.020 < 0.0001 0.042 0.013 0.002 0.299 0.051 <0.0001 0.048 0.036 0.18 Women 1.674 0.560 0.003 0.057 0.332 0.87 1.387 1.234 0.26 0.589 0.965 0.54 Secondary school or higher 0.370 0.474 0.43 0.080 0.305 0.79 0.927 1.093 0.40 0.161 0.803 0.84 Current alcohol drinking 0.179 0.486 0.71 0.072 0.334 0.83 0.338 1.138 0.77 1.810 0.867 0.04 Current cigarette smoking 0.136 0.535 0.80 0.025 0.329 0.94 0.444 1.212 0.71 0.829 0.942 0.38 Physical activity, MET 0.018 0.017 0.29 0.022 0.011 0.05 0.101 0.040 0.01 0.018 0.031 0.57 Body mass index, kg/m 2 0.132 0.065 0.04 0.066 0.042 0.12 0.256 0.148 0.08 0.136 0.110 0.22 Hypertension 2.023 0.663 0.002 0.610 0.404 0.13 3.588 1.476 0.02 0.390 0.999 0.70 Baseline pulse, beat/min 0.019 0.024 0.45 0.022 0.015 0.15 0.002 0.060 0.98 0.028 0.042 0.50 Abbreviations: AUC, area under the curve; BP, blood pressure; DBP, diastolic blood pressure; CPT, cold pressor test; MET, metabolic equivalent; SBP, systolic blood pressure; SE, standard error. American Journal of Hypertension 26(9) September 2013 1137

Zhang et al. observed in women may be partially attributed to increased pain sensitivity to cold. 11 A few studies have indicated that aging affects BP response to the CPT, with older subjects showing greater BP responses. 12 14 In our study, we found that SBP responses to the CPT were greater in older subjects, while DBP responses were greater in younger subjects. These data are consistent with those of Hess et al., who observed the same trend for DBP response with aging. 14 With increasing age, there is a shift from a DBP reactivity pattern to a more SBP reactivity pattern during the CPT. 13 It has been suggested that the mechanisms underlying the greater SBP, but not DBP, response to cold stress in older adults may be partially due to central arterial stiffness. Structural changes in the aorta with age would be expected to reduce the ability of the vasculature to buffer pressure fluctuations across the cardiac cycle, resulting in increased SBP and widening of pulse pressure at rest and, possibly, in response to stress. 14 Our study indicates that BP response to the CPT is greater among participants with higher baseline BP levels and less physical activity. These findings are consistent with those of previous studies. It was reported that BP response to the CPT was higher in a hypertensive group compared to a normotensive group of 40 Indian medical students. 16 In addition, aerobic exercise has been indicated to attenuate BP reactivity to the CPT among young adult black women. 15 Physical activity at a level that increases the maximal oxygen uptake was reported to decrease sympathetic neural activity at rest and during provocative stress. 26,27 This may be a mechanism explaining the observed attenuating effect of physical activity on BP responsiveness to the cold stimulus. Greater BMI and alcohol drinking are 2 newly identified risk factors for higher BP response to the CPT in our study. Several studies have shown a positive correlation between BMI and sympathetic nerve activity. 28 30 However, 2 previous studies failed to detect significant association between obesity/overweight and BP response to the CPT. 31,32 For example, Park et al. did not observe a significant difference in BP response between overweight and lean women, although SBP response appeared to be higher in the overweight group (18.5 ± 4.2 mm Hg vs. 15.9 ± 4.2 mm Hg, P = 0.67). 32 The relatively low statistical power due to small sample sizes (29 obese vs. 12 lean subjects and 8 overweight vs. 8 lean subjects, respectively) might result in their negative findings. In addition, our study is the first to report a positive association between alcohol drinking and BP response to the CPT. Acute increase in plasma alcohol has been indicated to increase heart rate and sympathetic nerve activity, with or without elevating BP. 33 35 However, the long-term effect of alcohol drinking on cardiovascular reactivity is still unclear. Further studies are warranted to validate the observed association and to clarify the mechanism mediating alcohol drinking and cardiovascular reactivity. In conclusion, the current study indicates that females, older age, and elevated baseline BP levels are associated with greater BP response to the CPT. In addition, physical inactivity, increased weight, and alcohol consumption may also be related to BP hyperreactivity to stress. Furthermore, our findings are consistent with the hypotheses that these risk factors for hypertension can elevate BP through enhanced sympathetic neural activity. Acknowledgments The GenSalt study is supported by research grants (U01HL072507, R01HL087263, and R01HL090682) from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD. DISCLOSURE The authors declared no conflict of interest. References 1. Krantz DS, Manuck SB. Acute psychophysiologic reactivity and risk of cardiovascular disease: a review and methodologic critique. Psychol Bull 1984;96:435 464. 2. Manuck SB. Cardiovascular reactivity in cardiovascular disease: once more unto the breach. Int J Behav Med 1994;1:4 31. 3. Treiber FA, Kamarck T, Schneiderman N, Sheffield D, Kapuku G, Taylor T. Cardiovascular reactivity and development of preclinical and clinical disease states. Psychosom Med 2003;65:46 62. 4. Wood DL, Sheps SG, Elveback LR, Schirger A. Cold pressor test as a predictor of hypertension. Hypertension 1984;6:301 306. 5. Menkes MS, Matthews KA, Krantz DS, Lundberg U, Mead LA, Qaqish B, Liang KY, Thomas CB, Pearson TA. 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