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Eicosanoid Production following One Bout of Exercise in Middle-Aged African American Pre- and Stage 1 Hypertensives

Eicosanoid Production following One Bout of Exercise in Middle-Aged African American Pre- and... SAGE-Hindawi Access to Research Journal of Aging Research Volume 2011, Article ID 302802, 7 pages doi:10.4061/2011/302802 Research Article Eicosanoid Production following One Bout of Exercise in Middle-Aged African American Pre- and Stage 1 Hypertensives 1 2 1, 3 2 Sheara Williamson, Deepti Varma, Michael Brown, and Susan Jansen Hypertension, Molecular, and Applied Physiology Laboratory, Department of Kinesiology, Temple University, 1800 North Broad Street, Philadelphia, PA 19122, USA Department of Chemistry, Temple University, Philadelphia, PA 19122, USA Department of Kinesiology, The University of Maryland, College Park, MD 20742, USA Correspondence should be addressed to Sheara Williamson, shearatoy@gmail.com Received 23 August 2010; Revised 31 January 2011; Accepted 10 March 2011 Academic Editor: Iris Reuter Copyright © 2011 Sheara Williamson et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Endothelial dysfunction and a sedentary lifestyle may be involved in the development of hypertension which is proliferative among middle-aged African Americans (AA). Signaling molecules derived from the oxidation of 20-carbon fatty acid molecules known as eicosanoids influence vascular tone. The relationship between aerobic fitness and eicosanoid formation following exercise in middle-aged African American hypertensives is unknown. Purpose. To determine the relationship between aerobic capacity and eicosanoid formation after a bout of moderate-intensity exercise in middle-aged AA hypertensives. Methods. Ten sedentary hypertensive AA underwent 50 min of aerobic exercise at 65% VO max. Urine was collected for 24 hr on two occasions, prior to testing and immediately following the bout of exercise. Urinary metabolites of prostacyclin (6-keto PGF ) and thromboxane (11- 1α dTXB ) were measured during the day and night periods by high-performance liquid chromatography (HPLC). Results.6-keto PGF levels significantly increased (P = .04) following the bout of exercise compared to the control day. There was a significant 1α relationship (r = .49, P< .05) between 6-keto PGF levels and VO max during the exercise day. Conclusion. Based on this 1α 2 preliminary study, there appears to be a relationship between aerobic capacity and exercise-induced 6-keto PGF production in 1α middle-aged hypertensive AAs. AAs with lower VO max had lower 6-keto PGF formation. 2 1α 1. Introduction prevalence of endothelial-impaired function disorders such as hypertension is disproportionately higher in the African Hypertension is a multifactorial disease that has high preva- American population in contrast to Caucasians [1, 2, 10]. lence in African Americans [1, 2]. National surveys show Eicosanoids are 20-carbon molecules derived from that the majority of middle-aged, urban African Americans arachidonic acid, a polyunsaturated fatty acid. Cyclooxyge- engage in little or no leisure-time physical activity [3]. This nase 1 or 2 (COX-1 or COX-2) and cell-specific synthases high prevalence of physical inactivity contributes to the convert free arachidonic acid to a biologically active com- disproportionate burden of obesity, hypertension, diabetes, pound. The biologically active eicosanoid binds to receptors and coronary heart disease in African Americans [3–5]. on the target cell plasma membrane to cause a wide range A number of important causal factors for hypertension of biologic effects including platelet aggregation, lymphocyte have been identified. Research has implicated endothelial aggregation and proliferation, bronchoconstriction, and dysfunction as a factor involved in the pathogenesis of vasodilation or vasoconstriction. Stable metabolites of the hypertension and is evident in the early stages of the eicosanoids can be measured in urine, plasma, or tissue and development of coronary atherosclerosis [1, 6–9]. Hyper- are thought to represent whole-body synthesis of eicosanoids tension is also more prevalent with advancing age. The [11, 12]. 2 Journal of Aging Research Table 1: Study timeline. Baseline Testing Screening 24 hr Diet logs 24 hr Casual BP Exercise urine returned urine Screening blood draw Bruce maximal TM test Diet Diet Diet Diet Diet Diet Diet Diet Diet Diet Diet Diet Modified GXT with gas log log log log log log analysis Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Thromboxane (TXA ), a vasoconstrictor and platelet sure (JNC 7) guidelines [2] having an average systolic BP of aggregator, and prostacyclin (PGI ), a vasodilator and 120–139 mmHg (pre-HTN), 140–159 mmHg (stage 1), and antiplatelet aggregator, are the most well-characterized a diastolic BP of 80–89 mmHg (pre-HTN), or 90–99 mmHg eicosanoids influencing vascular tone. TXA , a very unstable (stage 1). No participants were taking antihypertensive compound produced by platelets, has thrombogenic prop- medications. BP level was determined by the average of three erties and is cytotoxic. The stable metabolite of TXA is 11- casual BP readings from three separate days according to dehydro-thromboxane B (11-dTXB ). The stable metabolite the standards established by the JNC 7 guidelines. Blood 2 2 of PGI is 6-keto PGF . samples were drawn in the morning following a 12-hour 2 1α Acute exercise enhances the generation of reactive oxygen fast and sent to Quest Diagnostics for routine chemistries. species (ROS) which can lead to the oxidation of lipids, In addition, blood lipids, serum creatinine, and glucose proteins, and nucleic acids altering cellular function. The were also measured. Blood lipids were assessed to rule out increase in ROS after acute exercise promotes an acute phase hyperlipidemia; serum creatinine was measured to rule out of local inflammation that characteristically induces the renal dysfunction; fasting glucose was measured to rule release of inflammatory cytokines. These cytokines stimulate out diabetes. Individuals with abnormal blood chemistries the release of arachidonic acid and ultimately an increase were excluded from further participation in the study. Each in PGI production. Thus, the exercise-induced increase in participant then underwent a physical examination by the PGI is a normal consequence following exercise [13, 14]. A study physician. Exclusion criteria included smoking, a body recent study by Zoladz reported an attenuated PGI release mass index (BMI) >35, alcohol intake of more than 3 following exercise in coronary artery disease and hyperten- drinks per day, diabetes (fasting glucose level >126 mg/dL), sive patients when compared to healthy controls [15]. There total cholesterol >240 mg/dL, and evidence of renal or is contrasting evidence reported on exercise-induced TXA cardiovascular disease. production. Some research indicates an increase in TXA The screening GXT was a maximal test to screen for following exercise when others have reported no change [15– signs and symptoms of coronary artery disease. During the 19]. test, the participants walked on a treadmill while their blood There is little, if anything, known about the acute pressure, heart rate, and ECG response were monitored. The exercise-induced responses of PGI and TXA in middle- speed and/or degree of incline of the treadmill increased 2 2 aged African Americans. The purpose of the study was to every 3 minutes until the participants could no longer con- assess the changes, if any, in PGI and TXA production tinue or showed signs or symptoms of cardiovascular events. 2 2 following a single bout of aerobic exercise in middle-aged Participants having a negative screening graded exercise hypertensive African Americans. stress test (GXT) were excluded from further participation in the study. The study protocol was approved by the Institutional Review Board of the University of Maryland, 2. Protocol College Park. Informed consent was obtained from each participant. A timeline of the protocol is provided in Table 1. 2.1. Participants. Participants were recruited from the Balti- more, MD and Washington, DC area. Ten volunteers (5 male, 5female; 58 ± 2.3 yrs) completed the study. The participants 2.3. 24-Hour Urine Collection. Urine was collected for a 24- were sedentary (regular aerobic exercise ≤2 sessions/wk and hour period one week prior to the exercise session (Baseline). <20 min/session, sedentary occupation) African Americans Samples were collected in five time periods (00:00–08:00, who were categorized as having prehypertension (pre-HTN) 08:00–12:00, 12:00–16:00, 16:00–20:00, and 20:00–0:00). or stage 1 hypertension (systolic and diastolic blood pressure Urine for each time period was collected in a separate (BP) 143 ± 7/87 ± 7 mmHg). container. All urine collection containers were kept on ice in a cooler for the entire 24-hour period. Subjects began 2.2. Screening. Subjects had a physical examination, routine urine collection after their first void in the morning and fasting blood chemistries, and BP measured under standard- ended after their first void the following morning. Total urine ized conditions. Inclusion criteria required participants to volume was measured to the nearest .5 mL and an aliquot be pre- or stage 1 hypertensive according to the Seventh of the pooled 24 hour urine was sent to Quest Diagnostics Report of the Joint National Committee on Prevention, for determination of creatinine concentration. Creatinine is Detection, Evaluation, and Treatment of High Blood Pres- a breakdown product of creatine phosphate in muscle. In Journal of Aging Research 3 the absence of kidney dysfunction, creatinine clearance is acid, HPLC water, and acetonitrile were purchased from relatively constant and can be used as a means to compare Fisher Scientific (Waltman, MA, USA). other excreted metabolites. Aliquots from each time period were frozen at −80 C until analysis. A second 24-hour urine 2.7.2. Instrumentation. The Jasco HPLC system consisted collection was repeated immediately following the exercise of a Jasco pumps (PU-980), a Jasco UV-VIS detector session. (UV-975; Jasco Incorporated Easton, MD, USA), and a Rheodyne manual injector (Rheodyne LLC, Rohnert Park, 2.4. Diet. Three days prior to beginning baseline testing, CA, USA). Jasco-Borwin software (version 3.3.5) was used subjects were instructed to maintain their usual diets and for data collection. The analysis was done on a Symmetry complete a 6-day dietary log. Each subject was given their C 4.6 × 250 mm column with 5 μmparticlesize(Waters dietary information recorded during the baseline period and Corporation, Milford, MA, USA). instructed to repeat the 6-day diet leading to the exercise day. 2.7.3. HPLC Method. Acetonitrile was used as the organic 2.5. Graded Exercise Test to Measure Aerobic Capacity. A phase. Phosphoric acid solution (pH = 4) was used as second GXT was completed a week prior to the exercise the aqueous phase as it gave much better chromatographic session. All subjects completed a maximal treadmill exercise results than acetic acid solution. An acidic solution (pH of test to derive a valid prescription for the acute exercise 4) was necessary to maintain the eicosanoids in the neutral session. When oxygen consumption (VO )isadded to 2 state. Creatinine, at pH = 4 which is below its pKa value aGXT,itispossibletomeasure VO max, an index of 2 (4.83) will be in the protonated form. Creatinine in this form cardiovascular fitness. Participants began at 70% of the peak will be retained for a very short period of time on a nonpolar heart rate achieved on the subject’s screening exercise test and stationary phase compared to the neutral form of 8-iso PGF , the treadmill grade was increased 2% every 2 minutes. Blood a long-chain (C20) polyunsaturated fatty acid. The flow rate pressure, heart rate, and ECG were monitored during the and ratio of the aqueous and organic phase were selected test which was terminated when the subject could no longer to obtain a method with a practical run time and which continue. VO was measured continuously throughout this 2 resulted in good separation of all the compounds. All the test using a mass spectrometer (Marquette), mixing chamber chromatograms showed a negative drop at around 3.0 min. (Rayfield), turbine volume meter system (model VMM, This is due to the change in the wavelength from 254 nm to Interface Associates), and customized validated metabolic 196 nm. The HPLC method employed an isocratic elution of software. Standard criteria were used to determine if 17 mM phosphoric acid (solvent A) and acetonitrile (solvent VO max had been achieved. Subjective criteria included 2 B) in the ratio of 65 : 35. The analytes were separated at the subject’s rating of their perceived exertion and their ambient temperature with an injection volume of 100 μLand physical inability to continue exercise. The objective criteria using a flow rate of 1.3 mL/min. The run time for the method that were monitored were a plateau in rise in heart rate was 16.5 min. and a respiratory exchange ratio of greater than 1.15. The Urinary metabolites 6-keto PGF and 11-dTXB were 1α 2 respiratory exchange ratio is determined by dividing the dissolved in 1 mL methanol such that the stock mass volume of CO expired per minute by the volume of O 2 2 concentration for all was 1 mg/mL. 1000 mg of creatinine inspired per minute. was dissolved in 100 mL HPLC water to obtain a stock having concentration of 10 mg/mL. Working standards for the analytes were prepared by serial dilution using 17 mM 2.6. Acute Exercise Session. The goal of the exercise session phosphoric acid and acetonitrile in the ratio of 1 : 1 as the was to accumulate 50 minutes of exercise. The exercise solvent. The solutions were stored at −80 Cwhennot in use. session began with a 10-minute warm-up consisting of 75 μL of the filtered urine samples was diluted to 500 μLwith walking and stretching exercises. The participants then a 1 : 1 mixture of 17 mM phosphoric acid and acetonitrile walked on a treadmill for 30 minutes, followed by 5 minutes before injecting into the HPLC system. of rest, and then 20 additional minutes of treadmill walking or cycle ergometry for a total of 50 minutes of submaximal exercise. A heart rate monitor (Polar CIC, Inc.) was used to 2.7.4. Linearity, Accuracy, and Precision and Recovery. The ensure that each subject’s exercise heart rate corresponded to HPLC method was validated as per the FDA guidance for the 65% of their aerobic capacity that was measured during the industry: bioanalytical method validation [20]. The linear VO max GXT. After completing the acute exercise session, range for 6-keto PGF and 11-dTXB , the metabolites of 1α 2 participants began a 24-hour urine collection period in the arachidonic acid, was established by injecting in triplicate same manner as during the baseline collection. standard solutions at mass concentrations ranging from their respective LOQ to 1000 ng. Both the compounds showed a good linear response with R > 0.999 in the range. 2.7. Measurement of 6-Keto PGF and 11-dTXB 1α 2 Accuracy was determined by analyzing the compounds at 2.7.1. Chemicals and Materials. The prostaglandins (6-keto three different mass concentration levels. Percent accuracy PGF and 11-dTXB ) were purchased from Biomol (Ply- for the prostanoids was between 90.3–101.5%. Interassay 1α 2 mouth meeting, PA, USA). Creatinine was purchased from and intra-assay precision were determined by injecting in Sigma-Aldrich (St. Louis, MO, USA). Ortho Phosphoric triplicate. Intra-assay and interassay precision as indicated 4 Journal of Aging Research Table 2: Baseline demographic and clinical characteristics. Male/female 5/5 Age (years) 58 ± 2.3 BMI (kg/m)30.5 ± 1.1 VO (mL/kg/min) 22.6 ± 1.2 TC (mg/dL) 214.8 ± 9.2 LDL-C (mg/dL) 131.8 ± 10.1 VLDL-C (mg/dL) 27.9 ± 4.0 HDL-C (mg/dL) 55.1 ± 3.4 10 Casual Avg SBP (mmHg) 143 ± 7 0 AM (00:00–08:00) PM (08:00–16:00) Casual Avg DBP (mmHg) 87 ± 7 Values shown as mean ± SEM. Baseline BMI indicates body mass index; VO , aerobic capacity; TC, total cholesterol; Final LDL-C, low-density lipoprotein cholesterol; VLDL-C, very low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; SBP, Figure 1: Urinary 6-keto PGF levels increased from 33.5 ± 1α systolic blood pressure; DBP, diastolic blood pressure. 11 ng/ug to 77.8 ± 14.1 ng/ug during the AM period after the bout of exercise when compared to baseline. Levels returned to near baseline during the PM collection following the exercise bout, by the percent relative standard deviation (RSD) were P< .05. <10%. The percent recovery for the prostanoids at the three concentration levels was within 95–108%. Instrument precision was determined by injecting a The urinary metabolites were examined at two time peri- single mixture containing all the compounds six times and ods: 08:00–16:00 (AM) and 16:00–00:00 (PM) by ANOVA. calculating the RSD values. RSD values were found to be Baseline levels of 6-keto PGF and 11-dTXB were 33.5 ± 1α 2 less than 5% for all the analytes. The RSD values for all 10.9 ng/ug and 50.8 ± 20.4 ng/ug in the AM, respectively. 6- injections were <10% for intra-assay precision, and all urine keto PGF levels increased by more that twofold from 33.5± 1α metabolites were normalized to ug of creatinine. 10.9 ng/ug at baseline to 77.8± 14.1 ng/ug (P< .05; Figure 1) during the AM collection period after the acute exercise session. ANCOVA using VO max as the only covariate, 3. Statistics followed by Fisher’s PLSD, improved the effect of the acute For statistical analyses, we redefined the time periods to AM exercise bout (P = .03). Based on regression analysis, (08:00–16:00) and PM (16:00–00:00) because of insufficient there was a significant relationship (P = .04) between 6- data collected between 00:00–08:00. Descriptive statistics keto PGF levels and VO max during the AM collection 1α 2 were performed for subject characteristics and are presented period following exercise (Figure 2). Regression analysis also as mean ± SEM in Table 2. For each urinary metabolite (6- identified VO max as a significant predictor of 6-keto PGF 2 1α keto PGF and 11-dTXB ), a repeated measures ANOVA following the exercise bout. 1α 2 comparing groups at the two time periods; before and after Correlational analysis conducted using the baseline val- acute exercise bout. Repeated measures ANCOVA was used ues revealed a significant positive relationship (r = .97, P = to covary for age, BMI, and aerobic fitness. Separate simple .04 AM collection; r = .99, P = .01 PM collection) between linear regression analyses were performed with VO max as 2 LDL-C and 11-dTXB . This relationship was not observed the independent variable and each of the urinary metabolites between LDL-C and 6-keto PGF . Analysis also revealed a 1α as the dependent variables. Values are presented as mean ± significant negative correlation between 6-keto PGF levels 1α SEM. A value of P ≤ .05 was considered statistically sig- and age (r =−.92, P = .003). nificant. Statistical analysis was performed using STATVIEW The urinary 11-dTXB concentrations were decreased (SAS Institute, Cary, NC). at both collection times; 51% AM and 35% PM relative to baseline levels; however, this trend, though reproducible and consistent throughout the study, did not meet the test of 4. Results significance as defined by P ≤ .05 following the acute exercise session. Subject characteristics are shown in Table 2.Ten African American men and women (5 male, 5 female), age 58 ± 2.3 years, participated in the study. VO max determined 5. Discussion from the GXT ranged from 17.4–29.3 mL/kg/min (mean 22.6 ± 1.2 mL/kg/min), which would classify all participants Increases in vascular shear stress and mobilization and as sedentary. The average LDL-C for the participants was activation of cytokines and immune cells occur as a result 131.8 ± 10.1, which is borderline high according to the of exercise. This leads to arachidonic acid liberation from American Heart Association guidelines. the plasma membrane. COX-1 or COX-2 and cell-specific 6-keto PGF (ng/μg) 1α Journal of Aging Research 5 Laustiola et al. and Carter et al. observed that plasma levels of 6-keto PGF increased following a low-intensity 1α (30–50% VO max) bout of exercise, which suggests that exercise at lower intensities may stimulate a greater produc- tion of 6-keto PGF [16]. This trend was seen in the present 1α study population with a 1.5-fold to 2-fold increase in 6-keto PGF . Todd et al. showed that 6-keto PGF levels decreased 1α 1α incrementally as exercise intensity increased to near maximal levels [16]. In the present study, moderate intensity aerobic exercise increased urinary levels of 6-keto PGF following 1α exercise in the AM urine sample. This is consistent with findings by Okahara et al. that have documented an increase in 6-keto PGF production as a result of increased vascular 1α shear stress and cytokine release [25]. The 6-keto PGF 1α levels during the PM sample were not different than the 16 18 20 22 24 26 28 30 baseline day. VO (mL/kg/min) Tokunga et al. demonstrated an age-related decline in PGI synthesis which is consistent with our findings. Figure 2: 6-keto PGF measured in urine collected in the 2 1α AM following a moderate intensity exercise bout was found to Reduced PGI production, as a result of aging or selective significantly correlate with VO max in sedentary African American COX-2 inhibition, has been proposed as a mechanism pre- and stage 1 hypertensives (r = .67, P< .05). involved in the atherogenic process, myocardial infarctions, and stroke [26, 27]. Nicholson et al. reported that forearm vasodilation responses to PGI were significantly impaired in an older group when compared to a younger group matched for BMI and sex. They further suggested age- synthases convert the free arachidonic acid to a biologically active compound with cytoprotective properties. related differences in PGI -mediated vasodilation are likely Platelet activation increases during exercise. It has been attributed to a reduced contribution of the endothelium- derived relaxing factor, nitric oxide (NO) [27]. In a race suggested that metabolic activity in the arachidonic acid cascade may be increased equally at various exercise intensity comparison study, healthy African Americans demonstrated levels, but that the synthesis of PGI and TXA may be more blunted NO-mediated vasodilation when compared to a 2 2 heavily influenced by the exercise intensity level [16]. Alter- group of healthy Caucasians supporting evidence of race- natively, the conversion of arachidonic acid to eicosanoids related defects in endothelial function [10]. other than PGI and TXA may be affected by exercise, which The highly reactive oxygen radical, superoxide anion 2 2 may be responsible for the observed decrease in 11-dTXB (O )has ahighaffinity for NO, which increases the 2 2 following the acute exercise bout. The increase in PGI likelihood of the inactivation of NO and the formation of peroxynitrite (ONOO ), a potent oxidant [28, 29]. generation could be compensating for the prothrombotic environment induced by exercise. Prostacyclin production is Kalinowksi et al. reported racial differences in the steady- initiated by an influx of calcium into the cytoplasm resulting state NO/O /ONOO balance in endothelial cells from African Americans. Basal levels are reportedly closer to from the emptying of intracellular stores, which occurs only in the initial minutes of cellular activation [21]. This could the redox states characteristic for endothelium-impaired explain why the increase in the excretion of 6-keto PGF function disorders [1]. In the current study, LDL-C was 1α occurred in the morning following the exercise bout and not highly correlated with the urinary metabolite 11-dTXB at later in the day. baseline. LDL-C has been reported to increase the generation Rodrigo et al. reported that blood antioxidant activity of O , along with NO. Weisser et al. showed that LDL- was lower in hypertensives when compared to normotensive C increases thromboxane synthesis in a dose-dependent controls [22]. Acute exercise enhances the generation of manner [30]. Kuklinska et al. reported a significant negative correlation between LDL-C and PGI but we did not observe ROS, which can lead to the oxidation of lipids, proteins and, nucleic acids leading to altered cellular function. such a finding [6]. ROS increases the expression of antioxidant enzymes, but Decreases of 11-dTXB following the exercise session could be due to arachidonic acid being used as a precursor this is attenuated in hypertensives [23, 24]. It has been documented that, in a hypertensive population, plasma levels for prostacyclin to compensate for the prothrombotic state. of 6-keto PGF were significantly lower than those of TXA synthase may be at the same expression level, but 1α 2 healthy controls [21]. In the current study, individuals who competing PGI synthase may be in higher concentrations. were more aerobically fit (assessed by VO max) produced Todd et al. suggested that exercise intensities below 70% of VO max may not be sufficient to stimulate significant higher concentrations of urinary 6-keto PGF following 1α 2 an acute bout of exercise. This may suggest that there is alterations in TXA activity [16]. There has been some greater antioxidant activity in those individuals who are conflicting evidence in regards to how exercise influences TXA concentrations following exercise. 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Eicosanoid Production following One Bout of Exercise in Middle-Aged African American Pre- and Stage 1 Hypertensives

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Copyright © 2011 Sheara Williamson et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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SAGE-Hindawi Access to Research Journal of Aging Research Volume 2011, Article ID 302802, 7 pages doi:10.4061/2011/302802 Research Article Eicosanoid Production following One Bout of Exercise in Middle-Aged African American Pre- and Stage 1 Hypertensives 1 2 1, 3 2 Sheara Williamson, Deepti Varma, Michael Brown, and Susan Jansen Hypertension, Molecular, and Applied Physiology Laboratory, Department of Kinesiology, Temple University, 1800 North Broad Street, Philadelphia, PA 19122, USA Department of Chemistry, Temple University, Philadelphia, PA 19122, USA Department of Kinesiology, The University of Maryland, College Park, MD 20742, USA Correspondence should be addressed to Sheara Williamson, shearatoy@gmail.com Received 23 August 2010; Revised 31 January 2011; Accepted 10 March 2011 Academic Editor: Iris Reuter Copyright © 2011 Sheara Williamson et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Endothelial dysfunction and a sedentary lifestyle may be involved in the development of hypertension which is proliferative among middle-aged African Americans (AA). Signaling molecules derived from the oxidation of 20-carbon fatty acid molecules known as eicosanoids influence vascular tone. The relationship between aerobic fitness and eicosanoid formation following exercise in middle-aged African American hypertensives is unknown. Purpose. To determine the relationship between aerobic capacity and eicosanoid formation after a bout of moderate-intensity exercise in middle-aged AA hypertensives. Methods. Ten sedentary hypertensive AA underwent 50 min of aerobic exercise at 65% VO max. Urine was collected for 24 hr on two occasions, prior to testing and immediately following the bout of exercise. Urinary metabolites of prostacyclin (6-keto PGF ) and thromboxane (11- 1α dTXB ) were measured during the day and night periods by high-performance liquid chromatography (HPLC). Results.6-keto PGF levels significantly increased (P = .04) following the bout of exercise compared to the control day. There was a significant 1α relationship (r = .49, P< .05) between 6-keto PGF levels and VO max during the exercise day. Conclusion. Based on this 1α 2 preliminary study, there appears to be a relationship between aerobic capacity and exercise-induced 6-keto PGF production in 1α middle-aged hypertensive AAs. AAs with lower VO max had lower 6-keto PGF formation. 2 1α 1. Introduction prevalence of endothelial-impaired function disorders such as hypertension is disproportionately higher in the African Hypertension is a multifactorial disease that has high preva- American population in contrast to Caucasians [1, 2, 10]. lence in African Americans [1, 2]. National surveys show Eicosanoids are 20-carbon molecules derived from that the majority of middle-aged, urban African Americans arachidonic acid, a polyunsaturated fatty acid. Cyclooxyge- engage in little or no leisure-time physical activity [3]. This nase 1 or 2 (COX-1 or COX-2) and cell-specific synthases high prevalence of physical inactivity contributes to the convert free arachidonic acid to a biologically active com- disproportionate burden of obesity, hypertension, diabetes, pound. The biologically active eicosanoid binds to receptors and coronary heart disease in African Americans [3–5]. on the target cell plasma membrane to cause a wide range A number of important causal factors for hypertension of biologic effects including platelet aggregation, lymphocyte have been identified. Research has implicated endothelial aggregation and proliferation, bronchoconstriction, and dysfunction as a factor involved in the pathogenesis of vasodilation or vasoconstriction. Stable metabolites of the hypertension and is evident in the early stages of the eicosanoids can be measured in urine, plasma, or tissue and development of coronary atherosclerosis [1, 6–9]. Hyper- are thought to represent whole-body synthesis of eicosanoids tension is also more prevalent with advancing age. The [11, 12]. 2 Journal of Aging Research Table 1: Study timeline. Baseline Testing Screening 24 hr Diet logs 24 hr Casual BP Exercise urine returned urine Screening blood draw Bruce maximal TM test Diet Diet Diet Diet Diet Diet Diet Diet Diet Diet Diet Diet Modified GXT with gas log log log log log log analysis Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Thromboxane (TXA ), a vasoconstrictor and platelet sure (JNC 7) guidelines [2] having an average systolic BP of aggregator, and prostacyclin (PGI ), a vasodilator and 120–139 mmHg (pre-HTN), 140–159 mmHg (stage 1), and antiplatelet aggregator, are the most well-characterized a diastolic BP of 80–89 mmHg (pre-HTN), or 90–99 mmHg eicosanoids influencing vascular tone. TXA , a very unstable (stage 1). No participants were taking antihypertensive compound produced by platelets, has thrombogenic prop- medications. BP level was determined by the average of three erties and is cytotoxic. The stable metabolite of TXA is 11- casual BP readings from three separate days according to dehydro-thromboxane B (11-dTXB ). The stable metabolite the standards established by the JNC 7 guidelines. Blood 2 2 of PGI is 6-keto PGF . samples were drawn in the morning following a 12-hour 2 1α Acute exercise enhances the generation of reactive oxygen fast and sent to Quest Diagnostics for routine chemistries. species (ROS) which can lead to the oxidation of lipids, In addition, blood lipids, serum creatinine, and glucose proteins, and nucleic acids altering cellular function. The were also measured. Blood lipids were assessed to rule out increase in ROS after acute exercise promotes an acute phase hyperlipidemia; serum creatinine was measured to rule out of local inflammation that characteristically induces the renal dysfunction; fasting glucose was measured to rule release of inflammatory cytokines. These cytokines stimulate out diabetes. Individuals with abnormal blood chemistries the release of arachidonic acid and ultimately an increase were excluded from further participation in the study. Each in PGI production. Thus, the exercise-induced increase in participant then underwent a physical examination by the PGI is a normal consequence following exercise [13, 14]. A study physician. Exclusion criteria included smoking, a body recent study by Zoladz reported an attenuated PGI release mass index (BMI) >35, alcohol intake of more than 3 following exercise in coronary artery disease and hyperten- drinks per day, diabetes (fasting glucose level >126 mg/dL), sive patients when compared to healthy controls [15]. There total cholesterol >240 mg/dL, and evidence of renal or is contrasting evidence reported on exercise-induced TXA cardiovascular disease. production. Some research indicates an increase in TXA The screening GXT was a maximal test to screen for following exercise when others have reported no change [15– signs and symptoms of coronary artery disease. During the 19]. test, the participants walked on a treadmill while their blood There is little, if anything, known about the acute pressure, heart rate, and ECG response were monitored. The exercise-induced responses of PGI and TXA in middle- speed and/or degree of incline of the treadmill increased 2 2 aged African Americans. The purpose of the study was to every 3 minutes until the participants could no longer con- assess the changes, if any, in PGI and TXA production tinue or showed signs or symptoms of cardiovascular events. 2 2 following a single bout of aerobic exercise in middle-aged Participants having a negative screening graded exercise hypertensive African Americans. stress test (GXT) were excluded from further participation in the study. The study protocol was approved by the Institutional Review Board of the University of Maryland, 2. Protocol College Park. Informed consent was obtained from each participant. A timeline of the protocol is provided in Table 1. 2.1. Participants. Participants were recruited from the Balti- more, MD and Washington, DC area. Ten volunteers (5 male, 5female; 58 ± 2.3 yrs) completed the study. The participants 2.3. 24-Hour Urine Collection. Urine was collected for a 24- were sedentary (regular aerobic exercise ≤2 sessions/wk and hour period one week prior to the exercise session (Baseline). <20 min/session, sedentary occupation) African Americans Samples were collected in five time periods (00:00–08:00, who were categorized as having prehypertension (pre-HTN) 08:00–12:00, 12:00–16:00, 16:00–20:00, and 20:00–0:00). or stage 1 hypertension (systolic and diastolic blood pressure Urine for each time period was collected in a separate (BP) 143 ± 7/87 ± 7 mmHg). container. All urine collection containers were kept on ice in a cooler for the entire 24-hour period. Subjects began 2.2. Screening. Subjects had a physical examination, routine urine collection after their first void in the morning and fasting blood chemistries, and BP measured under standard- ended after their first void the following morning. Total urine ized conditions. Inclusion criteria required participants to volume was measured to the nearest .5 mL and an aliquot be pre- or stage 1 hypertensive according to the Seventh of the pooled 24 hour urine was sent to Quest Diagnostics Report of the Joint National Committee on Prevention, for determination of creatinine concentration. Creatinine is Detection, Evaluation, and Treatment of High Blood Pres- a breakdown product of creatine phosphate in muscle. In Journal of Aging Research 3 the absence of kidney dysfunction, creatinine clearance is acid, HPLC water, and acetonitrile were purchased from relatively constant and can be used as a means to compare Fisher Scientific (Waltman, MA, USA). other excreted metabolites. Aliquots from each time period were frozen at −80 C until analysis. A second 24-hour urine 2.7.2. Instrumentation. The Jasco HPLC system consisted collection was repeated immediately following the exercise of a Jasco pumps (PU-980), a Jasco UV-VIS detector session. (UV-975; Jasco Incorporated Easton, MD, USA), and a Rheodyne manual injector (Rheodyne LLC, Rohnert Park, 2.4. Diet. Three days prior to beginning baseline testing, CA, USA). Jasco-Borwin software (version 3.3.5) was used subjects were instructed to maintain their usual diets and for data collection. The analysis was done on a Symmetry complete a 6-day dietary log. Each subject was given their C 4.6 × 250 mm column with 5 μmparticlesize(Waters dietary information recorded during the baseline period and Corporation, Milford, MA, USA). instructed to repeat the 6-day diet leading to the exercise day. 2.7.3. HPLC Method. Acetonitrile was used as the organic 2.5. Graded Exercise Test to Measure Aerobic Capacity. A phase. Phosphoric acid solution (pH = 4) was used as second GXT was completed a week prior to the exercise the aqueous phase as it gave much better chromatographic session. All subjects completed a maximal treadmill exercise results than acetic acid solution. An acidic solution (pH of test to derive a valid prescription for the acute exercise 4) was necessary to maintain the eicosanoids in the neutral session. When oxygen consumption (VO )isadded to 2 state. Creatinine, at pH = 4 which is below its pKa value aGXT,itispossibletomeasure VO max, an index of 2 (4.83) will be in the protonated form. Creatinine in this form cardiovascular fitness. Participants began at 70% of the peak will be retained for a very short period of time on a nonpolar heart rate achieved on the subject’s screening exercise test and stationary phase compared to the neutral form of 8-iso PGF , the treadmill grade was increased 2% every 2 minutes. Blood a long-chain (C20) polyunsaturated fatty acid. The flow rate pressure, heart rate, and ECG were monitored during the and ratio of the aqueous and organic phase were selected test which was terminated when the subject could no longer to obtain a method with a practical run time and which continue. VO was measured continuously throughout this 2 resulted in good separation of all the compounds. All the test using a mass spectrometer (Marquette), mixing chamber chromatograms showed a negative drop at around 3.0 min. (Rayfield), turbine volume meter system (model VMM, This is due to the change in the wavelength from 254 nm to Interface Associates), and customized validated metabolic 196 nm. The HPLC method employed an isocratic elution of software. Standard criteria were used to determine if 17 mM phosphoric acid (solvent A) and acetonitrile (solvent VO max had been achieved. Subjective criteria included 2 B) in the ratio of 65 : 35. The analytes were separated at the subject’s rating of their perceived exertion and their ambient temperature with an injection volume of 100 μLand physical inability to continue exercise. The objective criteria using a flow rate of 1.3 mL/min. The run time for the method that were monitored were a plateau in rise in heart rate was 16.5 min. and a respiratory exchange ratio of greater than 1.15. The Urinary metabolites 6-keto PGF and 11-dTXB were 1α 2 respiratory exchange ratio is determined by dividing the dissolved in 1 mL methanol such that the stock mass volume of CO expired per minute by the volume of O 2 2 concentration for all was 1 mg/mL. 1000 mg of creatinine inspired per minute. was dissolved in 100 mL HPLC water to obtain a stock having concentration of 10 mg/mL. Working standards for the analytes were prepared by serial dilution using 17 mM 2.6. Acute Exercise Session. The goal of the exercise session phosphoric acid and acetonitrile in the ratio of 1 : 1 as the was to accumulate 50 minutes of exercise. The exercise solvent. The solutions were stored at −80 Cwhennot in use. session began with a 10-minute warm-up consisting of 75 μL of the filtered urine samples was diluted to 500 μLwith walking and stretching exercises. The participants then a 1 : 1 mixture of 17 mM phosphoric acid and acetonitrile walked on a treadmill for 30 minutes, followed by 5 minutes before injecting into the HPLC system. of rest, and then 20 additional minutes of treadmill walking or cycle ergometry for a total of 50 minutes of submaximal exercise. A heart rate monitor (Polar CIC, Inc.) was used to 2.7.4. Linearity, Accuracy, and Precision and Recovery. The ensure that each subject’s exercise heart rate corresponded to HPLC method was validated as per the FDA guidance for the 65% of their aerobic capacity that was measured during the industry: bioanalytical method validation [20]. The linear VO max GXT. After completing the acute exercise session, range for 6-keto PGF and 11-dTXB , the metabolites of 1α 2 participants began a 24-hour urine collection period in the arachidonic acid, was established by injecting in triplicate same manner as during the baseline collection. standard solutions at mass concentrations ranging from their respective LOQ to 1000 ng. Both the compounds showed a good linear response with R > 0.999 in the range. 2.7. Measurement of 6-Keto PGF and 11-dTXB 1α 2 Accuracy was determined by analyzing the compounds at 2.7.1. Chemicals and Materials. The prostaglandins (6-keto three different mass concentration levels. Percent accuracy PGF and 11-dTXB ) were purchased from Biomol (Ply- for the prostanoids was between 90.3–101.5%. Interassay 1α 2 mouth meeting, PA, USA). Creatinine was purchased from and intra-assay precision were determined by injecting in Sigma-Aldrich (St. Louis, MO, USA). Ortho Phosphoric triplicate. Intra-assay and interassay precision as indicated 4 Journal of Aging Research Table 2: Baseline demographic and clinical characteristics. Male/female 5/5 Age (years) 58 ± 2.3 BMI (kg/m)30.5 ± 1.1 VO (mL/kg/min) 22.6 ± 1.2 TC (mg/dL) 214.8 ± 9.2 LDL-C (mg/dL) 131.8 ± 10.1 VLDL-C (mg/dL) 27.9 ± 4.0 HDL-C (mg/dL) 55.1 ± 3.4 10 Casual Avg SBP (mmHg) 143 ± 7 0 AM (00:00–08:00) PM (08:00–16:00) Casual Avg DBP (mmHg) 87 ± 7 Values shown as mean ± SEM. Baseline BMI indicates body mass index; VO , aerobic capacity; TC, total cholesterol; Final LDL-C, low-density lipoprotein cholesterol; VLDL-C, very low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; SBP, Figure 1: Urinary 6-keto PGF levels increased from 33.5 ± 1α systolic blood pressure; DBP, diastolic blood pressure. 11 ng/ug to 77.8 ± 14.1 ng/ug during the AM period after the bout of exercise when compared to baseline. Levels returned to near baseline during the PM collection following the exercise bout, by the percent relative standard deviation (RSD) were P< .05. <10%. The percent recovery for the prostanoids at the three concentration levels was within 95–108%. Instrument precision was determined by injecting a The urinary metabolites were examined at two time peri- single mixture containing all the compounds six times and ods: 08:00–16:00 (AM) and 16:00–00:00 (PM) by ANOVA. calculating the RSD values. RSD values were found to be Baseline levels of 6-keto PGF and 11-dTXB were 33.5 ± 1α 2 less than 5% for all the analytes. The RSD values for all 10.9 ng/ug and 50.8 ± 20.4 ng/ug in the AM, respectively. 6- injections were <10% for intra-assay precision, and all urine keto PGF levels increased by more that twofold from 33.5± 1α metabolites were normalized to ug of creatinine. 10.9 ng/ug at baseline to 77.8± 14.1 ng/ug (P< .05; Figure 1) during the AM collection period after the acute exercise session. ANCOVA using VO max as the only covariate, 3. Statistics followed by Fisher’s PLSD, improved the effect of the acute For statistical analyses, we redefined the time periods to AM exercise bout (P = .03). Based on regression analysis, (08:00–16:00) and PM (16:00–00:00) because of insufficient there was a significant relationship (P = .04) between 6- data collected between 00:00–08:00. Descriptive statistics keto PGF levels and VO max during the AM collection 1α 2 were performed for subject characteristics and are presented period following exercise (Figure 2). Regression analysis also as mean ± SEM in Table 2. For each urinary metabolite (6- identified VO max as a significant predictor of 6-keto PGF 2 1α keto PGF and 11-dTXB ), a repeated measures ANOVA following the exercise bout. 1α 2 comparing groups at the two time periods; before and after Correlational analysis conducted using the baseline val- acute exercise bout. Repeated measures ANCOVA was used ues revealed a significant positive relationship (r = .97, P = to covary for age, BMI, and aerobic fitness. Separate simple .04 AM collection; r = .99, P = .01 PM collection) between linear regression analyses were performed with VO max as 2 LDL-C and 11-dTXB . This relationship was not observed the independent variable and each of the urinary metabolites between LDL-C and 6-keto PGF . Analysis also revealed a 1α as the dependent variables. Values are presented as mean ± significant negative correlation between 6-keto PGF levels 1α SEM. A value of P ≤ .05 was considered statistically sig- and age (r =−.92, P = .003). nificant. Statistical analysis was performed using STATVIEW The urinary 11-dTXB concentrations were decreased (SAS Institute, Cary, NC). at both collection times; 51% AM and 35% PM relative to baseline levels; however, this trend, though reproducible and consistent throughout the study, did not meet the test of 4. Results significance as defined by P ≤ .05 following the acute exercise session. Subject characteristics are shown in Table 2.Ten African American men and women (5 male, 5 female), age 58 ± 2.3 years, participated in the study. VO max determined 5. Discussion from the GXT ranged from 17.4–29.3 mL/kg/min (mean 22.6 ± 1.2 mL/kg/min), which would classify all participants Increases in vascular shear stress and mobilization and as sedentary. The average LDL-C for the participants was activation of cytokines and immune cells occur as a result 131.8 ± 10.1, which is borderline high according to the of exercise. This leads to arachidonic acid liberation from American Heart Association guidelines. the plasma membrane. COX-1 or COX-2 and cell-specific 6-keto PGF (ng/μg) 1α Journal of Aging Research 5 Laustiola et al. and Carter et al. observed that plasma levels of 6-keto PGF increased following a low-intensity 1α (30–50% VO max) bout of exercise, which suggests that exercise at lower intensities may stimulate a greater produc- tion of 6-keto PGF [16]. This trend was seen in the present 1α study population with a 1.5-fold to 2-fold increase in 6-keto PGF . Todd et al. showed that 6-keto PGF levels decreased 1α 1α incrementally as exercise intensity increased to near maximal levels [16]. In the present study, moderate intensity aerobic exercise increased urinary levels of 6-keto PGF following 1α exercise in the AM urine sample. This is consistent with findings by Okahara et al. that have documented an increase in 6-keto PGF production as a result of increased vascular 1α shear stress and cytokine release [25]. The 6-keto PGF 1α levels during the PM sample were not different than the 16 18 20 22 24 26 28 30 baseline day. VO (mL/kg/min) Tokunga et al. demonstrated an age-related decline in PGI synthesis which is consistent with our findings. Figure 2: 6-keto PGF measured in urine collected in the 2 1α AM following a moderate intensity exercise bout was found to Reduced PGI production, as a result of aging or selective significantly correlate with VO max in sedentary African American COX-2 inhibition, has been proposed as a mechanism pre- and stage 1 hypertensives (r = .67, P< .05). involved in the atherogenic process, myocardial infarctions, and stroke [26, 27]. Nicholson et al. reported that forearm vasodilation responses to PGI were significantly impaired in an older group when compared to a younger group matched for BMI and sex. They further suggested age- synthases convert the free arachidonic acid to a biologically active compound with cytoprotective properties. related differences in PGI -mediated vasodilation are likely Platelet activation increases during exercise. It has been attributed to a reduced contribution of the endothelium- derived relaxing factor, nitric oxide (NO) [27]. In a race suggested that metabolic activity in the arachidonic acid cascade may be increased equally at various exercise intensity comparison study, healthy African Americans demonstrated levels, but that the synthesis of PGI and TXA may be more blunted NO-mediated vasodilation when compared to a 2 2 heavily influenced by the exercise intensity level [16]. Alter- group of healthy Caucasians supporting evidence of race- natively, the conversion of arachidonic acid to eicosanoids related defects in endothelial function [10]. other than PGI and TXA may be affected by exercise, which The highly reactive oxygen radical, superoxide anion 2 2 may be responsible for the observed decrease in 11-dTXB (O )has ahighaffinity for NO, which increases the 2 2 following the acute exercise bout. The increase in PGI likelihood of the inactivation of NO and the formation of peroxynitrite (ONOO ), a potent oxidant [28, 29]. generation could be compensating for the prothrombotic environment induced by exercise. Prostacyclin production is Kalinowksi et al. reported racial differences in the steady- initiated by an influx of calcium into the cytoplasm resulting state NO/O /ONOO balance in endothelial cells from African Americans. Basal levels are reportedly closer to from the emptying of intracellular stores, which occurs only in the initial minutes of cellular activation [21]. This could the redox states characteristic for endothelium-impaired explain why the increase in the excretion of 6-keto PGF function disorders [1]. In the current study, LDL-C was 1α occurred in the morning following the exercise bout and not highly correlated with the urinary metabolite 11-dTXB at later in the day. baseline. LDL-C has been reported to increase the generation Rodrigo et al. reported that blood antioxidant activity of O , along with NO. Weisser et al. showed that LDL- was lower in hypertensives when compared to normotensive C increases thromboxane synthesis in a dose-dependent controls [22]. Acute exercise enhances the generation of manner [30]. Kuklinska et al. reported a significant negative correlation between LDL-C and PGI but we did not observe ROS, which can lead to the oxidation of lipids, proteins and, nucleic acids leading to altered cellular function. such a finding [6]. ROS increases the expression of antioxidant enzymes, but Decreases of 11-dTXB following the exercise session could be due to arachidonic acid being used as a precursor this is attenuated in hypertensives [23, 24]. It has been documented that, in a hypertensive population, plasma levels for prostacyclin to compensate for the prothrombotic state. of 6-keto PGF were significantly lower than those of TXA synthase may be at the same expression level, but 1α 2 healthy controls [21]. In the current study, individuals who competing PGI synthase may be in higher concentrations. were more aerobically fit (assessed by VO max) produced Todd et al. suggested that exercise intensities below 70% of VO max may not be sufficient to stimulate significant higher concentrations of urinary 6-keto PGF following 1α 2 an acute bout of exercise. This may suggest that there is alterations in TXA activity [16]. There has been some greater antioxidant activity in those individuals who are conflicting evidence in regards to how exercise influences TXA concentrations following exercise. Aerobically trained more aerobically fit than those who are less aerobically fit. individuals seem to have an enhanced antioxidant system 08:00–16:00 6-keto PGF (ng/μg) 1α 6 Journal of Aging Research [29, 31], which could explain the lack of significant alter- National Health and Nutrition Examination Survey, 1988– 1994,” American Journal of Preventive Medicine, vol. 18, no. 1, ations in TXA activity at the low exercise intensity. pp. 46–53, 2000. Increased risk and incidence of disease are associated [4] W.L.Haskell,I.M.Lee,R.R.Pateetal., “Physicalactivityand with low aerobic capacity [5]. In the present study, there public health: updated recommendation for adults from the was a relationship between aerobic capacity as assessed American College of Sports Medicine and the American Heart by VO max and exercise-induced PGI production in pre- 2 2 Association,” Medicine and Science in Sports and Exercise, vol. and stage 1 middle-aged hypertensive African Americans. 39, no. 8, pp. 1423–1434, 2007. African American subjects with lower VO max had lower [5] B. A. Murray, C. J. Brahler, J. Baer, and J. Marotta, “Correla- PGI formation. Impaired PGI production in response 2 2 tions between activity and blood pressure in African American to exercise may be suggestive of endothelial dysfunction. womenand girls,” Journal of Exercise Physiology Online, vol. 6, When racial comparisons are made, the endothelium of no. 3, pp. 38–44, 2003. African Americans is closer to the state characteristic for [6] A. M. Kuklinska, B. Mroczko, W. J. 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