- Email: [email protected]
Cardiovascular Effects of Hyperhomocysteinemia in Conscious Unrestrained Rats
AJH
2006; 19:94 –97
Cardiovascular Effects of Hyperhomocysteinemia in Conscious Unrestrained Rats Richard H. Kennedy, Russell B. Melchert, and Jacob Joseph Background: Experiments were designed to determine whether hyperhomocysteinemia (Hhe) affects cardiovascular function when monitored in conscious unrestrained animals. Methods: Adult, male Sprague-Dawley rats were fed a homocystine-supplemented diet for 6 months. Blood pressure (BP), heart rate, and pulse pressure were monitored continuously, 24 h a day, using biotelemetry techniques. Results: The resulting intermediate level of Hhe was not associated with significant changes in heart rate, diastolic BP, systolic BP, or the circadian variation in heart rate. In spite of the lack of significant changes in systolic
S
and diastolic BP, there was a slight but statistically significant increase in pulse pressure after 4 months of treatment that returned toward control levels after 6 months. Conclusions: Current results indicate that Hhe alone does not have significant effects on BP. Furthermore, they suggest that the previously reported Hhe-induced adverse cardiac remodeling and diastolic dysfunction in this animal model are not the result of pressure overload. Am J Hypertens 2006;19:94 –97 © 2006 American Journal of Hypertension, Ltd. Key Words: Blood pressure, chronic hyperhomocysteinemia, heart rate, male rats, pulse pressure.
everal studies in both humans and animal models have suggested that hyperhomocysteinemia (Hhe) is associated with hypertension. For example, survey data from the Third National Health and Nutrition Examination Survey indicated that elevations in plasma homocysteine are associated with increases in blood pressure (BP) and the risk of hypertension,1 whereas other investigators reported that increased homocysteine levels are linked to hypertension in adolescents2 and type I diabetic patients.3 Similarly, studies in normotensive and spontaneously hypertensive (SHR) rats4 as well as in minipigs5 showed that experimentally induced Hhe results in elevated BP. In contrast, other investigators have suggested that hypertension is not related to Hhe. A report from the Framingham Heart Study showed no relationship between plasma homocysteine levels and the incidence of hypertension when adjusted for other factors such as age and gender,6 and recent work in our laboratory7,8 and others9 using normotensive and SHR rats showed no difference in
BP when compared between control and Hhe animals after chronic treatment of 6 weeks or longer. The cause of this disparity, especially in animal models of Hhe, is unknown but could be related to differences in species or strain, level or duration of Hhe, age of the animal, method of inducing the hyperhomocysteinemic state, or techniques used to measure BP. Current experiments were conducted using telemetered rats to examine the effects of Hhe during a 6-month exposure on BP, heart rate, and pulse pressure in a conscious unrestrained animal model. The Hhe, which was in the intermediate range, was induced by addition of homocystine to the chow. Results showed no significant changes in BP or heart rate; however, there was a slight effect on pulse pressure.
Received January 29, 2005. First decision July 7, 2005. Accepted July 16, 2005. From the Departments of Pharmaceutical Sciences (RHK, RBM, JJ), Internal Medicine (JJ), and Pharmacology & Toxicology (RHK, RBM), University of Arkansas for Medical Sciences, Little Rock, Arkansas; Department of Physiology (RHK), Loyola University of Chicago Stritch School of Medicine, Maywood, Illinois; and Department of Internal Medicine (JJ), VA Boston Healthcare System
and Boston University School of Medicine, West Roxbury, Massachusetts. This work was supported in part by a Grant-in-Aid from the American Heart Association, Heartland Affiliate (JJ). Address correspondence and reprint requests to Dr. Richard H. Kennedy, Research Services 120-400, Loyola University of Chicago Stritch School of Medicine, 2160 S. First Avenue, Maywood, IL 60153; e-mail: [email protected]
0895-7061/06/$30.00 doi:10.1016/j.amjhyper.2005.07.008
Methods Animal Model All procedures in this study were approved by the Institutional Animal Care and Use Committee at the University
© 2006 by the American Journal of Hypertension, Ltd. Published by Elsevier Inc.
AJH–January 2006 –VOL. 19, NO. 1
of Arkansas for Medical Sciences. Four-month-old male Sprague-Dawley (SD) rats (375 – 400 g; n ⫽ 5) were purchased from Harlan Sprague Dawley (Indianapolis, IN) and maintained in our institutional Division of Laboratory Animal Medicine on a 12:12 light-to-dark cycle (lights on at 6 AM) with free access to water and a purified amino acid diet (Harlan Teklad, Indianapolis, IN). After 7 to 10 days of acclimatization to this facility, the animals were anesthetized, and telemetry transmitters were surgically implanted as described later. After surgical recovery, baseline cardiovascular parameters were recorded, and the animals were placed on a Hhe-inducing diet (Harlan Teklad) for 6 months. As described previously,8 the Hhe diet was designed by supplementing the amino-acid-defined control diet with 9 g/kg homocystine (the disulfide form of homocysteine) to produce intermediate (30 to 100 mol/L) Hhe in the rats. Homocysteine levels were monitored in plasma. Blood was drawn into heparinized syringes from a tail vein before the animals were placed on the Hhe-inducing diet and at the end of the study, and total homocysteine in the plasma samples was measured by the fluorescence polarization immunoassay technique, which is routinely used in the clinical laboratories at the Central Arkansas Veterans Healthcare System Facility. Briefly, homocysteine and bound homocysteine were reduced to free homocysteine with dithiothreitol. The free homocysteine was converted to S-adenosyl-L-homocysteine (SAH) with S-adenosyl-Lhydrolase, and the concentration of SAH was determined by competition with fluorescein-tagged SAH for binding to an SAH-specific mouse antibody. Binding was assessed with an Abbott IMX System (Abbott Laboratories, Abbott Park, IL). This assay is comparable to chromatographic techniques.10 Telemetry Techniques for Monitoring Cardiovascular Function in Conscious Animals Cardiovascular function was monitored in conscious unrestrained rats using biotelemetry techniques. Animals were anesthetized with sodium pentobarbital (50 mg/kg, intraperiteonally), and biotelemetry transmitters (PA-C40, Data Sciences International, St. Paul, MN) were implanted in the abdominal cavity with the pressure-sensing catheter of the transmitter being inserted into the descending aorta just below the renal artery. After recovering from anesthesia, the rats were transferred to plastic cages that were positioned above telemetry receivers. Values for heart rate, diastolic BP, systolic BP, and pulse pressure were recorded continuously at 10-min intervals. Hourly values were obtained by averaging the six 10-min values within each hour. All data measurements were obtained using the Dataquest IV program (Data Sciences International). Postoperative recovery, which routinely required 7 to 10 days, was monitored through stabilization of BP and heart rate, as well as by the return of normal circadian variation in
HYPERHOMOCYSTEINEMIA AND BP
95
heart rate. After recovery was complete, baseline (control) parameters were captured for 1 week before the animals were placed on the Hhe diet for 6 months. Reported values are averages of the 3 days surrounding each interval. The telemetry transmitters were calibrated against standard pressure transducers before and after the study to insure that there were no significant drifts in calibration. Statistical Evaluation Data were evaluated by repeated measures ANOVA with a Student-Newman-Keuls post-hoc test or by t test as appropriate using Sigmastat (SPSS Inc., Chicago, IL). The criterion for significance was a P value ⬍ .05. Data are reported as means ⫾ SEM.
Results Animal Model Hyperhomocysteinemia did not appear to affect body weight in these male SD rats. Body weight before placing the animals on the Hhe diet (approximately 5 months of age) was 452 ⫾ 11 g (n ⫽ 5). At the end of the 6-month treatment period body weight was 533 ⫾ 15 g. These values are similar to those reported by the vendor for naïve animals (www.harlan.com/us/index.htm). The Hhe diet elicited a Hhe that was in the intermediate range (30 to 100 mol/L). Plasma homocysteine levels before treatment with the Hhe diet were 6.0 ⫾ 1.0 mol/L (n ⫽ 5). Values at the end of the 6-month treatment period were 32.7 ⫾ 1.4 mol/L. Similar plasma levels were observed in previous studies.7,8 Blood Pressure As shown in Fig. 1, chronic Hhe had no effect on diastolic or systolic BP in male SD rats at any time of day. Twentyfour-hour daily average values for systolic BP were 128 ⫾ 4, 130 ⫾ 4, 129 ⫾ 4, 131 ⫾ 4, and 130 ⫾ 5 mm Hg before and after 2 weeks, 6 weeks, 4 months, and 6 months on the Hhe diet, respectively (n ⫽ 5). Corresponding values for diastolic BP were 95 ⫾ 3, 95 ⫾ 3, 93 ⫾ 4, 92 ⫾ 4, and 92 ⫾ 5 mm Hg. Heart Rate As with BP, Hhe had no significant effect on heart rate at any time of day (Fig. 2). Twenty-four-hour daily average values for heart rate were 340 ⫾ 12, 333 ⫾ 12, 336 ⫾ 11, 339 ⫾ 13, and 325 ⫾ 14 beats/min before and after 2 weeks, 6 weeks, 4 months, and 6 months on the Hhe diet, respectively (n ⫽ 5). Maximum daily values (observed at 7 to 10 PM) were 379 ⫾ 13, 365 ⫾ 16, 378 ⫾ 11, 375 ⫾ 16, and 361 ⫾ 18 beats/min before and after 2 weeks, 6 weeks, 4 months, and 6 months treatment, respectively, with corresponding minimum daily values (observed between 7 AM and noon) being 290 ⫾ 8, 295 ⫾ 9, 297 ⫾ 14, 300 ⫾ 14, and 291 ⫾ 12 beats/min.
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FIG. 1. Mean hourly values for diastolic (dotted lines) and systolic (solid lines) blood pressure in conscious unrestrained male Sprague-Dawley rats before (closed circles) and after 2 weeks (open circles), 6 weeks (closed squares), 4 months (open squares), and 6 months (closed triangles) treatment with the hyperhomocysteinemia diet (n ⫽ 5). Values for blood pressure were recorded continuously at 10-min intervals with hourly values for each animal being obtained by averaging the six 10-min values surrounding each hour. Time of day is presented as a 24-h clock. Vertical bars represent SEM.
Pulse Pressure In contrast to BP and heart rate, pulse pressure showed slight but statistically significant changes over time (Fig. 3). Twenty-four-hour daily average values for pulse pressure increased gradually from the control level of 33 ⫾ 2 mm Hg to 35 ⫾ 2, 36 ⫾ 2, and 39 ⫾ 1 mm Hg after 2 weeks, 6 weeks, and 4 months on the Hhe diet (P ⬍ .05 control v 4 months; n ⫽ 5). At 6 months, the 24-h daily average returned
FIG. 2. Mean hourly values for heart rate in conscious unrestrained male Sprague-Dawley rats before (closed circles) and after 2 weeks (open circles), 6 weeks (closed squares), 4 months (open squares), and 6 months (closed triangles) treatment with the hyperhomocysteinemia diet (n ⫽ 5). Values for heart rate were recorded continuously at 10-min intervals with hourly values for each animal being obtained by averaging the six 10-min values surrounding each hour. Time of day is presented as a 24-h clock. Vertical bars represent SEM.
FIG. 3. Mean hourly values for pulse pressure in conscious unrestrained male Sprague-Dawley rats before (closed circles) and after 2 weeks (open circles), 6 weeks (closed squares), 4 months (open squares), and 6 months (closed triangles) treatment with the hyperhomocysteinemia diet (n ⫽ 5). Values for pulse pressure were recorded continuously at 10-min intervals with hourly values for each animal being obtained by averaging the six 10-min values surrounding each hour. Time of day is presented as a 24-h clock. Vertical bars represent SEM.
to 37 ⫾ 1 mm Hg, which did not differ from either the control or 4-month values. As shown in Fig. 3, changes in the hourly values generally reflect those in the 24-h averages.
Discussion Previous studies in animal models examining the effects of Hhe on BP have resulted in conflicting results. For example, a correlation between Hhe and elevated BP was observed in anesthetized normotensive and SHR rats after 12 weeks of intermediate Hhe (⬃30 mol/L) induced by adding homocysteine to the drinking water,4 as well as in conscious minipigs as measured by tail plethysmographic techniques after 4 months of mild Hhe (⬃10 mol/L) induced by dietary methionine.5 In contrast, Hhe was found to have no effect on BP in anesthetized normotensive or SHR rats after 10 weeks of intermediate (30 to 45 mol/L) or severe (⬃200 mol/L) Hhe induced by dietary homocystine,7,8 and another study in SHRs made mildly hyperhomocysteinemic (⬃12 mol/L) by dietary methionine showed that tail cuff-measured values for BP were not increased after 6 weeks treatment.9 Current experiments were designed to monitor the cardiovascular effects of Hhe in conscious, unrestrained animals during a 6-month exposure period with the Hhe being induced by addition of homocystine (the disulfide form of homocysteine) to the chow. Previous studies in this model of Hhe have shown that it results in a significant diastolic cardiac dysfunction with adverse remodeling after as little as 10 weeks of exposure.7,8 Current results indicated that intermediate Hhe (⬃33 mol/L) is not associated with elevations in BP after 2 weeks to 6 months of exposure. This does not entirely clarify the cause of the disparity in previous
AJH–January 2006 –VOL. 19, NO. 1
studies, but it does provide an evaluation in an animal model that is not anesthetized or subjected to the stress of plethysmographic techniques during hemodynamic measurement. In addition, current data show that Hhe does not influence heart rate or the circadian variation in heart rate, as there were no differences in minimum daily values, maximum daily values, or the times of day at which these minimums and maximums were observed. The absence of a difference in heart rate conflicts with the study by Rolland et al5 in minipigs, which showed that Hhe is associated with an increase in rate when monitored by tail cuff techniques. The absence of an observed effect of Hhe on BP in the current study is somewhat surprising in light of the numerous studies showing that Hhe is associated with decreases in endothelium-mediated vasodilation.11–13 However, this diminished endothelial function may be offset by direct effects of Hhe on vascular smooth muscle. For example, homocysteine has been shown to diminish the sensitivity of isolated vascular preparations to the vasoconstrictor action of norepinephrine,14 although other investigators found that homocysteine enhances the intracellular calcium response to angiotensin II.15 It is interesting to note that pulse pressure was increased after 4 months of Hhe but then decreased to levels not significantly different from control after 6 months. This slight, but statistically significant, increment in pulse pressure (an increase of ⬃6 mm Hg between control values and those at 4 months) was the result of a combined 3 mm Hg increase in systolic BP and 3 mm Hg decrease in diastolic BP. Because available studies have suggested that pulse pressure, like BP, does not change in rats during the age range included in current experiments,16,17 the observed increase in pulse pressure may suggest that Hhe is affecting vascular compliance or cardiac systolic function. In fact, our previous research using echocardiographic techniques showed that Hhe in normotensive rats is associated with ventricular hypertrophy and an increase in fractional area change, which is suggestive of increased contractility.7 Guazzi et al18 reported that ventricular hypertrophy can be associated with supernormal contractile function. Future studies are obviously required to ascertain the cause of this increased pulse pressure. In summary, current results demonstrate that chronic intermediate Hhe alone does not increase BP in a rodent model when monitored under conscious unrestrained conditions during a 6-month period. The absence of changes in BP in this model support the concept that the adverse cardiac remodeling and diastolic dysfunction caused by Hhe is not mediated by pressure overload.
HYPERHOMOCYSTEINEMIA AND BP
References 1.
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12.
13.
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15.
16.
17.
Acknowledgments We thank Kerrey Roberto and Nathan Petty for their excellent technical assistance.
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18.
Lim U, Cassano PA: Homocysteine and blood-pressure in the Third National Health and Nutrition Examination Survey, 1988 –1994. Am J Epidemiol 2002;156:1105–1113. Kahleová R, Palyzová D, Zvára K, Zvárová J, Hrach K, Nováková I, Hyánek J, Bendlová B, Kožich V: Essential hypertension in adolescents: association with insulin resistance and with metabolism of homocysteine and vitamins. Am J Hypertens 2002;15:857– 864. Neugebauer S, Tarnow L, Stehouwer C, Teerlink T, Baba T, Watanabe T, Parving H-H: Total plasma homocysteine is associated with hypertension in Type I diabetic patients. Diabetologia 2002; 45:1315–1324. Miller A, Mujumdar V, Palmer L, Bower JD, Tyagi SC: Reversal of endocardial endothelial dysfunction by folic acid in homocysteinemic hypertensive rats. Am J Hypertens 2002;15:157–163. Rolland PH, Friggi A, Barlatier A., Piquet P, Latrille V, Faye MM, Guillou J, Charpiot P, Bodard H, Ghiringhelli O, Calaf R, Luccioni R, Garcon D: Hyperhomocysteinemia-induced vascular damage in the minipig. Circulation 1995;91:1161–1174. Sundstrom J, Sullivan L, D’Agostino RB, Jacques PF, Selhub J, Rosenberg IH, Wilson PWF, Levy D, Vasan RS: Plasma homocysteine, hypertension incidence, and blood pressure tracking: the Framingham Heart Study. Hypertension 2003;42:1100 –1105. Joseph J, Joseph L, Shekhawat NS, Devi S, Wang J, Melchert RB, Hauer-Jensen M, Kennedy RH: Hyperhomocysteinemia leads to pathologic ventricular hypertrophy in normotensive rats: Am J Physiol 2003;285:H679 –H686. Joseph J, Washington A, Joseph L, Koehler L, Fink LM, HauerJensen M, Kennedy RH: Hyperhomocysteinemia leads to adverse cardiac remodeling in hypertensive rats. Am J Physiol 2002;283: H2567–H2574. Yen C-H, Lau Y-T: Vascular responses in male and female hypertensive rats with hyperhomocysteinemia. Hypertension 2002;40: 322–328. Blanco-Vaca F, Arcelus R, Gonzalez-Sastre F, Ordonez-Llanos J, Queralto-Compano JM: Comparison of the Abbott IMX and a high-performance liquid chromatography method for measuring total plasma homocysteine. Clin Chem Lab Med 2000;38:327–329. Ungvari Z, Csiszar A, Bagi Z, Koller A: Impaired nitric oxidemediated flow-induced coronary dilation in hyperhomocysteinemia: morphological and functional evidence for increased peroxynitrite formation. Am J Pathol 2002;161:145–153. Symons JD, Mullick AE, Ensunsa JL, Ma AA, Rutledge JC: Hyperhomocysteinemia evoked by folate depletion: effects on coronary and carotid arterial function. Arterioscler Thromb Vasc Biol 2002; 22:772–780. Dayal S, Brown KL, Weydert CJ, Oberley LW, Arning E, Bottiglieri T, Faraci FM, Lentz SR: Deficiency of glutathione peroxidase-1 sensitizes hyperhomocysteinemic mice to endothelial dysfunction. Arterioscler Thromb Vasc Biol 2002;22:1996 –2002. Cipolla MJ, Williamson WK, Nehler ML, Taylor LM, Porter JM: The effect of elevated homocysteine levels on adrenergic vasoconstriction of human resistance arteries: the role of the endothelium and reactive oxygen species. J Vasc Surg 2000;31:751–759. Mujumdar VS, Hayden MR, Tyagi SC: Homocyst(e)ine induces calcium second messenger in vascular smooth muscle cells. J Cell Physiol 2000;183:28 –36. Werner A, Rosa NR, Oliveira AR, Fernandes TG, Bello AA, Irigoyen M: Changes in blood pressure control in aged rats. Braz J Med Biol Res 1995;28:603– 607. Zhang B, Sannajust F: Diurnal rhythms of blood pressure, heart rate, and locomotor activity in adult and old male Wistar rats. Physiol Behav 2000;70:375–380. Guazzi M, Brenner DA, Apstein CS, Saupe KW: Exercise intolerance in rats with hypertensive heart disease is associated with impaired diastolic relaxation. Hypertension 2001;37:204 –208.
2006; 19:94 –97
Cardiovascular Effects of Hyperhomocysteinemia in Conscious Unrestrained Rats Richard H. Kennedy, Russell B. Melchert, and Jacob Joseph Background: Experiments were designed to determine whether hyperhomocysteinemia (Hhe) affects cardiovascular function when monitored in conscious unrestrained animals. Methods: Adult, male Sprague-Dawley rats were fed a homocystine-supplemented diet for 6 months. Blood pressure (BP), heart rate, and pulse pressure were monitored continuously, 24 h a day, using biotelemetry techniques. Results: The resulting intermediate level of Hhe was not associated with significant changes in heart rate, diastolic BP, systolic BP, or the circadian variation in heart rate. In spite of the lack of significant changes in systolic
S
and diastolic BP, there was a slight but statistically significant increase in pulse pressure after 4 months of treatment that returned toward control levels after 6 months. Conclusions: Current results indicate that Hhe alone does not have significant effects on BP. Furthermore, they suggest that the previously reported Hhe-induced adverse cardiac remodeling and diastolic dysfunction in this animal model are not the result of pressure overload. Am J Hypertens 2006;19:94 –97 © 2006 American Journal of Hypertension, Ltd. Key Words: Blood pressure, chronic hyperhomocysteinemia, heart rate, male rats, pulse pressure.
everal studies in both humans and animal models have suggested that hyperhomocysteinemia (Hhe) is associated with hypertension. For example, survey data from the Third National Health and Nutrition Examination Survey indicated that elevations in plasma homocysteine are associated with increases in blood pressure (BP) and the risk of hypertension,1 whereas other investigators reported that increased homocysteine levels are linked to hypertension in adolescents2 and type I diabetic patients.3 Similarly, studies in normotensive and spontaneously hypertensive (SHR) rats4 as well as in minipigs5 showed that experimentally induced Hhe results in elevated BP. In contrast, other investigators have suggested that hypertension is not related to Hhe. A report from the Framingham Heart Study showed no relationship between plasma homocysteine levels and the incidence of hypertension when adjusted for other factors such as age and gender,6 and recent work in our laboratory7,8 and others9 using normotensive and SHR rats showed no difference in
BP when compared between control and Hhe animals after chronic treatment of 6 weeks or longer. The cause of this disparity, especially in animal models of Hhe, is unknown but could be related to differences in species or strain, level or duration of Hhe, age of the animal, method of inducing the hyperhomocysteinemic state, or techniques used to measure BP. Current experiments were conducted using telemetered rats to examine the effects of Hhe during a 6-month exposure on BP, heart rate, and pulse pressure in a conscious unrestrained animal model. The Hhe, which was in the intermediate range, was induced by addition of homocystine to the chow. Results showed no significant changes in BP or heart rate; however, there was a slight effect on pulse pressure.
Received January 29, 2005. First decision July 7, 2005. Accepted July 16, 2005. From the Departments of Pharmaceutical Sciences (RHK, RBM, JJ), Internal Medicine (JJ), and Pharmacology & Toxicology (RHK, RBM), University of Arkansas for Medical Sciences, Little Rock, Arkansas; Department of Physiology (RHK), Loyola University of Chicago Stritch School of Medicine, Maywood, Illinois; and Department of Internal Medicine (JJ), VA Boston Healthcare System
and Boston University School of Medicine, West Roxbury, Massachusetts. This work was supported in part by a Grant-in-Aid from the American Heart Association, Heartland Affiliate (JJ). Address correspondence and reprint requests to Dr. Richard H. Kennedy, Research Services 120-400, Loyola University of Chicago Stritch School of Medicine, 2160 S. First Avenue, Maywood, IL 60153; e-mail: [email protected]
0895-7061/06/$30.00 doi:10.1016/j.amjhyper.2005.07.008
Methods Animal Model All procedures in this study were approved by the Institutional Animal Care and Use Committee at the University
© 2006 by the American Journal of Hypertension, Ltd. Published by Elsevier Inc.
AJH–January 2006 –VOL. 19, NO. 1
of Arkansas for Medical Sciences. Four-month-old male Sprague-Dawley (SD) rats (375 – 400 g; n ⫽ 5) were purchased from Harlan Sprague Dawley (Indianapolis, IN) and maintained in our institutional Division of Laboratory Animal Medicine on a 12:12 light-to-dark cycle (lights on at 6 AM) with free access to water and a purified amino acid diet (Harlan Teklad, Indianapolis, IN). After 7 to 10 days of acclimatization to this facility, the animals were anesthetized, and telemetry transmitters were surgically implanted as described later. After surgical recovery, baseline cardiovascular parameters were recorded, and the animals were placed on a Hhe-inducing diet (Harlan Teklad) for 6 months. As described previously,8 the Hhe diet was designed by supplementing the amino-acid-defined control diet with 9 g/kg homocystine (the disulfide form of homocysteine) to produce intermediate (30 to 100 mol/L) Hhe in the rats. Homocysteine levels were monitored in plasma. Blood was drawn into heparinized syringes from a tail vein before the animals were placed on the Hhe-inducing diet and at the end of the study, and total homocysteine in the plasma samples was measured by the fluorescence polarization immunoassay technique, which is routinely used in the clinical laboratories at the Central Arkansas Veterans Healthcare System Facility. Briefly, homocysteine and bound homocysteine were reduced to free homocysteine with dithiothreitol. The free homocysteine was converted to S-adenosyl-L-homocysteine (SAH) with S-adenosyl-Lhydrolase, and the concentration of SAH was determined by competition with fluorescein-tagged SAH for binding to an SAH-specific mouse antibody. Binding was assessed with an Abbott IMX System (Abbott Laboratories, Abbott Park, IL). This assay is comparable to chromatographic techniques.10 Telemetry Techniques for Monitoring Cardiovascular Function in Conscious Animals Cardiovascular function was monitored in conscious unrestrained rats using biotelemetry techniques. Animals were anesthetized with sodium pentobarbital (50 mg/kg, intraperiteonally), and biotelemetry transmitters (PA-C40, Data Sciences International, St. Paul, MN) were implanted in the abdominal cavity with the pressure-sensing catheter of the transmitter being inserted into the descending aorta just below the renal artery. After recovering from anesthesia, the rats were transferred to plastic cages that were positioned above telemetry receivers. Values for heart rate, diastolic BP, systolic BP, and pulse pressure were recorded continuously at 10-min intervals. Hourly values were obtained by averaging the six 10-min values within each hour. All data measurements were obtained using the Dataquest IV program (Data Sciences International). Postoperative recovery, which routinely required 7 to 10 days, was monitored through stabilization of BP and heart rate, as well as by the return of normal circadian variation in
HYPERHOMOCYSTEINEMIA AND BP
95
heart rate. After recovery was complete, baseline (control) parameters were captured for 1 week before the animals were placed on the Hhe diet for 6 months. Reported values are averages of the 3 days surrounding each interval. The telemetry transmitters were calibrated against standard pressure transducers before and after the study to insure that there were no significant drifts in calibration. Statistical Evaluation Data were evaluated by repeated measures ANOVA with a Student-Newman-Keuls post-hoc test or by t test as appropriate using Sigmastat (SPSS Inc., Chicago, IL). The criterion for significance was a P value ⬍ .05. Data are reported as means ⫾ SEM.
Results Animal Model Hyperhomocysteinemia did not appear to affect body weight in these male SD rats. Body weight before placing the animals on the Hhe diet (approximately 5 months of age) was 452 ⫾ 11 g (n ⫽ 5). At the end of the 6-month treatment period body weight was 533 ⫾ 15 g. These values are similar to those reported by the vendor for naïve animals (www.harlan.com/us/index.htm). The Hhe diet elicited a Hhe that was in the intermediate range (30 to 100 mol/L). Plasma homocysteine levels before treatment with the Hhe diet were 6.0 ⫾ 1.0 mol/L (n ⫽ 5). Values at the end of the 6-month treatment period were 32.7 ⫾ 1.4 mol/L. Similar plasma levels were observed in previous studies.7,8 Blood Pressure As shown in Fig. 1, chronic Hhe had no effect on diastolic or systolic BP in male SD rats at any time of day. Twentyfour-hour daily average values for systolic BP were 128 ⫾ 4, 130 ⫾ 4, 129 ⫾ 4, 131 ⫾ 4, and 130 ⫾ 5 mm Hg before and after 2 weeks, 6 weeks, 4 months, and 6 months on the Hhe diet, respectively (n ⫽ 5). Corresponding values for diastolic BP were 95 ⫾ 3, 95 ⫾ 3, 93 ⫾ 4, 92 ⫾ 4, and 92 ⫾ 5 mm Hg. Heart Rate As with BP, Hhe had no significant effect on heart rate at any time of day (Fig. 2). Twenty-four-hour daily average values for heart rate were 340 ⫾ 12, 333 ⫾ 12, 336 ⫾ 11, 339 ⫾ 13, and 325 ⫾ 14 beats/min before and after 2 weeks, 6 weeks, 4 months, and 6 months on the Hhe diet, respectively (n ⫽ 5). Maximum daily values (observed at 7 to 10 PM) were 379 ⫾ 13, 365 ⫾ 16, 378 ⫾ 11, 375 ⫾ 16, and 361 ⫾ 18 beats/min before and after 2 weeks, 6 weeks, 4 months, and 6 months treatment, respectively, with corresponding minimum daily values (observed between 7 AM and noon) being 290 ⫾ 8, 295 ⫾ 9, 297 ⫾ 14, 300 ⫾ 14, and 291 ⫾ 12 beats/min.
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FIG. 1. Mean hourly values for diastolic (dotted lines) and systolic (solid lines) blood pressure in conscious unrestrained male Sprague-Dawley rats before (closed circles) and after 2 weeks (open circles), 6 weeks (closed squares), 4 months (open squares), and 6 months (closed triangles) treatment with the hyperhomocysteinemia diet (n ⫽ 5). Values for blood pressure were recorded continuously at 10-min intervals with hourly values for each animal being obtained by averaging the six 10-min values surrounding each hour. Time of day is presented as a 24-h clock. Vertical bars represent SEM.
Pulse Pressure In contrast to BP and heart rate, pulse pressure showed slight but statistically significant changes over time (Fig. 3). Twenty-four-hour daily average values for pulse pressure increased gradually from the control level of 33 ⫾ 2 mm Hg to 35 ⫾ 2, 36 ⫾ 2, and 39 ⫾ 1 mm Hg after 2 weeks, 6 weeks, and 4 months on the Hhe diet (P ⬍ .05 control v 4 months; n ⫽ 5). At 6 months, the 24-h daily average returned
FIG. 2. Mean hourly values for heart rate in conscious unrestrained male Sprague-Dawley rats before (closed circles) and after 2 weeks (open circles), 6 weeks (closed squares), 4 months (open squares), and 6 months (closed triangles) treatment with the hyperhomocysteinemia diet (n ⫽ 5). Values for heart rate were recorded continuously at 10-min intervals with hourly values for each animal being obtained by averaging the six 10-min values surrounding each hour. Time of day is presented as a 24-h clock. Vertical bars represent SEM.
FIG. 3. Mean hourly values for pulse pressure in conscious unrestrained male Sprague-Dawley rats before (closed circles) and after 2 weeks (open circles), 6 weeks (closed squares), 4 months (open squares), and 6 months (closed triangles) treatment with the hyperhomocysteinemia diet (n ⫽ 5). Values for pulse pressure were recorded continuously at 10-min intervals with hourly values for each animal being obtained by averaging the six 10-min values surrounding each hour. Time of day is presented as a 24-h clock. Vertical bars represent SEM.
to 37 ⫾ 1 mm Hg, which did not differ from either the control or 4-month values. As shown in Fig. 3, changes in the hourly values generally reflect those in the 24-h averages.
Discussion Previous studies in animal models examining the effects of Hhe on BP have resulted in conflicting results. For example, a correlation between Hhe and elevated BP was observed in anesthetized normotensive and SHR rats after 12 weeks of intermediate Hhe (⬃30 mol/L) induced by adding homocysteine to the drinking water,4 as well as in conscious minipigs as measured by tail plethysmographic techniques after 4 months of mild Hhe (⬃10 mol/L) induced by dietary methionine.5 In contrast, Hhe was found to have no effect on BP in anesthetized normotensive or SHR rats after 10 weeks of intermediate (30 to 45 mol/L) or severe (⬃200 mol/L) Hhe induced by dietary homocystine,7,8 and another study in SHRs made mildly hyperhomocysteinemic (⬃12 mol/L) by dietary methionine showed that tail cuff-measured values for BP were not increased after 6 weeks treatment.9 Current experiments were designed to monitor the cardiovascular effects of Hhe in conscious, unrestrained animals during a 6-month exposure period with the Hhe being induced by addition of homocystine (the disulfide form of homocysteine) to the chow. Previous studies in this model of Hhe have shown that it results in a significant diastolic cardiac dysfunction with adverse remodeling after as little as 10 weeks of exposure.7,8 Current results indicated that intermediate Hhe (⬃33 mol/L) is not associated with elevations in BP after 2 weeks to 6 months of exposure. This does not entirely clarify the cause of the disparity in previous
AJH–January 2006 –VOL. 19, NO. 1
studies, but it does provide an evaluation in an animal model that is not anesthetized or subjected to the stress of plethysmographic techniques during hemodynamic measurement. In addition, current data show that Hhe does not influence heart rate or the circadian variation in heart rate, as there were no differences in minimum daily values, maximum daily values, or the times of day at which these minimums and maximums were observed. The absence of a difference in heart rate conflicts with the study by Rolland et al5 in minipigs, which showed that Hhe is associated with an increase in rate when monitored by tail cuff techniques. The absence of an observed effect of Hhe on BP in the current study is somewhat surprising in light of the numerous studies showing that Hhe is associated with decreases in endothelium-mediated vasodilation.11–13 However, this diminished endothelial function may be offset by direct effects of Hhe on vascular smooth muscle. For example, homocysteine has been shown to diminish the sensitivity of isolated vascular preparations to the vasoconstrictor action of norepinephrine,14 although other investigators found that homocysteine enhances the intracellular calcium response to angiotensin II.15 It is interesting to note that pulse pressure was increased after 4 months of Hhe but then decreased to levels not significantly different from control after 6 months. This slight, but statistically significant, increment in pulse pressure (an increase of ⬃6 mm Hg between control values and those at 4 months) was the result of a combined 3 mm Hg increase in systolic BP and 3 mm Hg decrease in diastolic BP. Because available studies have suggested that pulse pressure, like BP, does not change in rats during the age range included in current experiments,16,17 the observed increase in pulse pressure may suggest that Hhe is affecting vascular compliance or cardiac systolic function. In fact, our previous research using echocardiographic techniques showed that Hhe in normotensive rats is associated with ventricular hypertrophy and an increase in fractional area change, which is suggestive of increased contractility.7 Guazzi et al18 reported that ventricular hypertrophy can be associated with supernormal contractile function. Future studies are obviously required to ascertain the cause of this increased pulse pressure. In summary, current results demonstrate that chronic intermediate Hhe alone does not increase BP in a rodent model when monitored under conscious unrestrained conditions during a 6-month period. The absence of changes in BP in this model support the concept that the adverse cardiac remodeling and diastolic dysfunction caused by Hhe is not mediated by pressure overload.
HYPERHOMOCYSTEINEMIA AND BP
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Acknowledgments We thank Kerrey Roberto and Nathan Petty for their excellent technical assistance.
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Lim U, Cassano PA: Homocysteine and blood-pressure in the Third National Health and Nutrition Examination Survey, 1988 –1994. Am J Epidemiol 2002;156:1105–1113. Kahleová R, Palyzová D, Zvára K, Zvárová J, Hrach K, Nováková I, Hyánek J, Bendlová B, Kožich V: Essential hypertension in adolescents: association with insulin resistance and with metabolism of homocysteine and vitamins. Am J Hypertens 2002;15:857– 864. Neugebauer S, Tarnow L, Stehouwer C, Teerlink T, Baba T, Watanabe T, Parving H-H: Total plasma homocysteine is associated with hypertension in Type I diabetic patients. Diabetologia 2002; 45:1315–1324. Miller A, Mujumdar V, Palmer L, Bower JD, Tyagi SC: Reversal of endocardial endothelial dysfunction by folic acid in homocysteinemic hypertensive rats. Am J Hypertens 2002;15:157–163. Rolland PH, Friggi A, Barlatier A., Piquet P, Latrille V, Faye MM, Guillou J, Charpiot P, Bodard H, Ghiringhelli O, Calaf R, Luccioni R, Garcon D: Hyperhomocysteinemia-induced vascular damage in the minipig. Circulation 1995;91:1161–1174. Sundstrom J, Sullivan L, D’Agostino RB, Jacques PF, Selhub J, Rosenberg IH, Wilson PWF, Levy D, Vasan RS: Plasma homocysteine, hypertension incidence, and blood pressure tracking: the Framingham Heart Study. Hypertension 2003;42:1100 –1105. Joseph J, Joseph L, Shekhawat NS, Devi S, Wang J, Melchert RB, Hauer-Jensen M, Kennedy RH: Hyperhomocysteinemia leads to pathologic ventricular hypertrophy in normotensive rats: Am J Physiol 2003;285:H679 –H686. Joseph J, Washington A, Joseph L, Koehler L, Fink LM, HauerJensen M, Kennedy RH: Hyperhomocysteinemia leads to adverse cardiac remodeling in hypertensive rats. Am J Physiol 2002;283: H2567–H2574. Yen C-H, Lau Y-T: Vascular responses in male and female hypertensive rats with hyperhomocysteinemia. Hypertension 2002;40: 322–328. Blanco-Vaca F, Arcelus R, Gonzalez-Sastre F, Ordonez-Llanos J, Queralto-Compano JM: Comparison of the Abbott IMX and a high-performance liquid chromatography method for measuring total plasma homocysteine. Clin Chem Lab Med 2000;38:327–329. Ungvari Z, Csiszar A, Bagi Z, Koller A: Impaired nitric oxidemediated flow-induced coronary dilation in hyperhomocysteinemia: morphological and functional evidence for increased peroxynitrite formation. Am J Pathol 2002;161:145–153. Symons JD, Mullick AE, Ensunsa JL, Ma AA, Rutledge JC: Hyperhomocysteinemia evoked by folate depletion: effects on coronary and carotid arterial function. Arterioscler Thromb Vasc Biol 2002; 22:772–780. Dayal S, Brown KL, Weydert CJ, Oberley LW, Arning E, Bottiglieri T, Faraci FM, Lentz SR: Deficiency of glutathione peroxidase-1 sensitizes hyperhomocysteinemic mice to endothelial dysfunction. Arterioscler Thromb Vasc Biol 2002;22:1996 –2002. Cipolla MJ, Williamson WK, Nehler ML, Taylor LM, Porter JM: The effect of elevated homocysteine levels on adrenergic vasoconstriction of human resistance arteries: the role of the endothelium and reactive oxygen species. J Vasc Surg 2000;31:751–759. Mujumdar VS, Hayden MR, Tyagi SC: Homocyst(e)ine induces calcium second messenger in vascular smooth muscle cells. J Cell Physiol 2000;183:28 –36. Werner A, Rosa NR, Oliveira AR, Fernandes TG, Bello AA, Irigoyen M: Changes in blood pressure control in aged rats. Braz J Med Biol Res 1995;28:603– 607. Zhang B, Sannajust F: Diurnal rhythms of blood pressure, heart rate, and locomotor activity in adult and old male Wistar rats. Physiol Behav 2000;70:375–380. Guazzi M, Brenner DA, Apstein CS, Saupe KW: Exercise intolerance in rats with hypertensive heart disease is associated with impaired diastolic relaxation. Hypertension 2001;37:204 –208.