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Metabolic and Hemodynamic Markers of Endothelial Dysfunction in Patients With Hypertension and Patients With Type 2 Diabetes During the Cold Pressor Test

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Metabolic and Hemodynamic Markers of Endothelial Dysfunction in Patients With Hypertension and Patients With Type 2 Diabetes During the Cold Pressor Test
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  Metabolic and Hemodynamic Markers ofEndothelial Dysfunction in Patients WithHypertension and Patients With Type 2 DiabetesDuring the Cold Pressor Test Christian Fouillioux, MD, 1 Freddy Contreras, MD, 1 Mary Lares, MD, 1 Raquel Cano, MD, 2 Elliuz Leal, MD, 2 Nailet Arraiz, MgS, PhD, 2 Valmore Bermu´dez, MD, MPH, PhD, 2 * and Manuel Velasco, MD, PhD 1 Leptin, a 167-amino acid peptidic hormone secreted by adipose tissue, acts mainly in the arcuatehypothalamus nucleus as a satiety signal, but given its closed connections with inflammatory andendothelial systems, a probable regulatory role in blood pressure (BP) control by interaction withnitric oxide (NO) and C-reactive protein (CRP) has also been described. The cold pressor test (CPT) isa simple test that indirectly determines endothelial dysfunction. In this work, biochemical indicators(CRP, leptin, and NO) and hemodynamic indicators (systolic and diastolic BP) were performed andevaluated in patients with hypertension, patients with type 2, and control subjects during a singleCPT for assessment of endothelial dysfunction. A total of 43 subjects aged 25 to 60 years weredivided into three groups: 15 healthy volunteers, 13 patients with hypertension, and 15 patients withtype 2 diabetes were included in the study. A complete clinical history was obtained from eachsubject and a complete physical examination, including an electrocardiogram, was carried out.During the 30-minute assay, 0.9% saline solution was infused intravenously. CPT was performed toassess the cardiovascular reactivity at 15 minutes. The cardiovascular variables (systolic and diastolicBP) were measured at 0, 16, and 30 minutes. In addition, serum variables were extracted at the beginning and at the end of the experiment and statistical analysis was performed. CPTcaused in allsubjects a significant increase in BP and pulse. There were no significant differences in CRP or leptinin all groups, although we observed significant differences for NO ( P  ,  0.05). Sensibility andspecificity for all biochemical variables resulted in nonsignificant statistical or clinical importance asmarkers of endothelial dysfunction; however, a positive association was found when leptin and NOwere evaluated together (sensibility, 0.2; specificity, 0.8). CRP, leptin, and NO did not show any director significant association with the hemodynamic variables in this study, although a relationship wasobserved in NO according to group and among biochemical variables when studied together. Keywords:  hypertension, type 2 diabetes, leptin, C-reactive protein, nitric oxide INTRODUCTION Leptin is a 167-amino acid protein discovered throughpositional cloning of the ob-gene 1 and is expressedprimarily in white adipose tissue, 2–4  but also inhypothalamus, 5 hypophysis, stomach, 6 placenta, skel-etal muscle, and mammary gland. 7,8 Growing evidencesuggests that leptin is not mainly an ‘‘antiobesity 1 Clinical Pharmacology Unit, Vargas Medical School, CentralUniversity of Venezuela, Caracas, Venezuela; and  2 Endocrine and Metabolic Diseases Research Center, University of Zulia, School of  Medicine, Maracaibo, Venezuela.*Address for correspondence: La Universidad del Zulia, Facultadde Medicina, Escuela de Medicina, Centro de InvestigacionesEndocrino-Metabo´licas ‘‘Dr. Fe´lix Go´mez,’’ Caracas, Venezuela.E-mail: vbermudez@hotmail.com; fago@medscape.com American Journal of Therapeutics 15, 389–396 (2008) 1075–2765    2008 Lippincott Williams & Wilkins  hormone’’; moreover, it plays an important role in theregulation of several physiological processes. Leptin ismainly secreted by white adipose tissue in directproportion to the amount of energy stored in fat andacts by binding to specific leptin receptors (LR).Leptin # s actions in the hypothalamus and a varietyof peripheral organs are mediated by the long isoformof the leptin receptor (OB-R). 9,10 Multiple LR isoforms exist; all are products of a single LR gene and result from alternative mRNAsplicing and/or proteolytic processing. LRisoformsfallinto three classes: secreted, short, and long. Thesecreted forms are alternative splice products orproteolytic cleavage products of membrane-boundLR forms. These contain only extracellular leptin- binding domains and complex with circulating leptin,perhaps regulating free leptin concentrations. Short-and long-form (LRb) receptors contain identicalextracellular and transmembrane domains as well asthe same first 29 intracellular amino acids and divergein sequence secondary to alternative splicing. LRb ishighly conserved among species and possesses anintracellular domain of approximately 300 residues.Much of leptin # s action is attributable to effects in thecentral nervous system (CNS), especially in the basomedial hypothalamus, the site of highest LRbexpression. Here, leptin acts on neurons that regulatelevels of circulating hormones (eg, thyroid hormone,sex steroids, growth hormone). Leptin action on thesehypothalamic neurons also regulates the activity of theautonomic nervous system, although direct leptinaction on brainstem LRb-expressing neurons may playa role in this and other leptin actions. Leptin action onthe immune system appears to result from direct actionon LRb-expressing T cells. Leptin also may regulateglucose homeostasis (independently of effects onadiposity) at least partly through the CNS, but alsomay directly regulate some metabolic tissues.Serum leptin is highly correlated with adipose tissuemass in humans. 11–13 This hyperleptinemia results ina ‘‘leptin-resistance hypothesis’’ in which alterations atthe receptor level or leptin–signal transduction path-ways block leads to defective leptin action. However,there is a significant amount of variation in serumleptin for a given amount of body fat in the generalpopulation. This variation indicates that leptin isinfluenced by factors in addition to the amount of adipose tissue. Gender is a major predictor of leptin,and serum leptin levels are significantly higher infemales than in males of equivalent fat mass. 13–16 Gender may influence serum leptin through a directstimulatory effect of estrogen, or an inhibitory effectof testosterone, on leptin synthesis. 1 The gender-dependent distribution of adipose tissue into eithersc or omental depots 1 may also contribute to thevariability in serum leptin, because ob gene expressionis greater in sc than omental adipose tissue. 2–4 Oneadditional recently proposed possibility to explain theeffect of gender on serum leptin is that adipose tissuefrom males and females may exhibit a differentialresponse to stimulatory or inhibitory signals for leptinsynthesis. 4 It is well recognized that increased body weight isoften associated with metabolic disorders (hyperinsu-linemia and glucose intolerance) as well as increased blood pressure. Indeed, obesity activates both thesympathetic nervous and renin–angiotensin systemsand causes insulin resistance and hyperinsulinemia, allof which have been thought to raise blood pressure.The association between obesity and hypertensionsuggests that the adipose mass may serve as animportant tissue in the regulation of blood pressure.For example, plasma leptin concentration is increasedin animals with dietary-induced obesity and in the vastmajority of obese humans probably through leptinsympathetic nervous system activation1 15,16 and, if administered chronically, elevates arterial pressure. 1 In addition, hypertension is observed in transgenicmiceoverexpressingleptin,although their bodyweightis lower than in wild-type littermates. 2 Indeed, obesityis accompanied by hypertension in hyperleptinemicagouti yellow obese mice but not in leptin-deficientob/ob mice. 3 Finally, some studies indicate that plasmaleptin concentration correlates with blood pressure inhypertensive humans, whether or not obese. 4 These data suggest that hyperleptinemia is involvedin the pathogenesis of obesity-associated hypertension.In fact, when leptin penetrates the blood–cerebrospinalfluid barrier, it seems likely that leptin activates sym-pathetic nerve activity in the CNS. The link betweenplasma leptin and the autonomic nervous systemis strengthened by evidence of a direct relationship between musclesympathetic nerve activity and plasmaleptin concentrations. 17 Moreover, a direct relation-ship between plasma leptin and heart rate has beenobserved in hypertensive patients. 5,18 This relation-ship seems to be independent of body mass index,plasma insulin, blood pressure, smoking, and physicalactivity, 5 suggesting that leptin may influence car-diovascular neuronal control. Previous studies havedemonstrated that leptin stimulates nitric oxide (NO)production in vivo 18–20 and in isolated endothelialcells 8 through a mechanism similar to insulin, ie, byactivating protein kinases which in turn phosphor-ylates eNO synthase, increasing its activity even at lowcalcium concentrations 7,8,21 as well as inducing NO-dependent blood vessel dilatation both in vitro andin vivo. 6,22,23  American Journal of Therapeutics (2008)  15 (4) 390  Fouillioux et al   Obesity is characterized by hyperleptinemia andhypothalamic satiety center resistance to the anorecticeffect of adipose tissue hormones. 14 Because leptinexerts many effects outside the CNS, 13 it is of interestwhether these peripheralactions aremaintained or alsoimpaired in obesity. Recently, some examples of ‘‘peripheral leptin resistance’’ have been described.The stimulatory effect of leptin on fatty acid oxidationand inhibitory on triglyceride synthesis in skeletalmuscle are impaired in obese rodents 24 and humans. 25 This study suggests that the vasculature may beanother site of leptin resistance. Although decreasedability of leptin to stimulate NO generation could beaccounted for by the previously mentioned generalmechanismsofobesity-relatedendothelialdysfunction,the involvement of additional mechanisms, morespecific for leptin, cannot be excluded. In particular,chronic hyperleptinemia, which accompanies obesity,could lead to the downregulation of leptin receptors 26 and postreceptor signaling mechanisms 27 or couldinduce antagonistic signaling pathways such assuppressor of cytokine signaling-1 in target cells. 28 Therefore, the aim of this study was to evaluate bio-chemistry (High Sensitivity C-reactive protein, leptin,NO) and hemodynamic (diastolic and systolic blood pressure) during the cold pressor test (CPT) inpatients with hypertension and patients with type 2diabetes. MATERIAL AND METHODS Patient selection Forty-two subjects, of both genders, ranging from 18 to60 years of age (14 healthy, 13 patients with hyperten-sion, and 15 patients with type 2 diabetes) wererandomly selected from patients who attended thecardiovascular risk factor and diabetes outpatientservice at the Victorino Santaella # s Hospital InternalMedicine Department. People with type 2 diabetesmellitus were diagnosed according to the AmericanDiabetes Association criteria and hypertension accord-ing to the seventh report of the Joint NationalCommittee on prevention, detection, evaluation, andtreatment of high blood pressure (VIIJNC). All patientswere diabetic and hypertensive over 5 years sincediagnosis. Patients with alcoholism (stage 3 or more),malnutrition, endocrinologic disorders other than type2 diabetes, autoimmune diseases, coronary arterydisease, severe high blood pressure (with recent targetorgan damage history), and current drug therapy withlevothyroxine, glucocorticoids, insulin, sildenafil, beta- blockers, bromocriptine, and adrenergic agonists wereexcluded from the study.Each patient underwent a complete physical exam-ination (including anthropometric variables) andsurvey to record personal and family history of acuteand chronic diseases related to the inclusion andexclusion criteria described previously. Avenous bloodsample was drawn for routine laboratory tests likefasting blood glucose, urea, creatinine, and hemoglo- bin/hematocrit. Once each patient was admitted to thestudy, three groups were organized as follows: controlgroup constituted by healthy volunteers (n = 14),hypertensive patient group constituted by people withclass 1 and 2 high blood pressure (n = 13), and type 2diabetic patient group (n = 15). Experimental protocol All individuals were admitted at 7:00  AM  with previous14-hour fasting condition with accomplishment of pharmacologic treatment suppression in the preceding5 days to initiate the experimental phase, namely theCPT, as follows (Fig. 1):1. Assessment of blood pressure, cardiac frequencyand rhythm by mercury sphygmomanometer anddigital electrocardiograph;2. Catheterization of two venous accesses with yelko(Angiocath  , Becton and Diekinson, Franklin FIGURE 1.  Study protocol.  American Journal of Therapeutics (2008)  15 (4) Endothelial Dysfunction During the Cold Pressor Test   391  Lakes, NJ) catheters No. 20 in the right arm andNo. 18 in the left arm. The right one was used toinfuse isotonic saline solution at 20 drops perminute by infusion pump. The left one wasconnected to a trilumen catheter to collect bloodsamples during the experiment;3. The patient was committed to rest on a stretcher(during the entire process) and then connected toa vital sign monitor and Dinamap;4. At minute zero (0 # ), 20 mL of blood was extractedand placed in tubes with EDTA. Blood pressureandelectrocardiogramwastakeninthispointalso;5. At 15 minutes, the CPTwas performed subjectingthe right arm to immersion in cold water at 0  C to4  C for 60 seconds. After 1 minute, this procedurewas followed by electrocardiogram and bloodpressure determination; and6. At 30 minutes, 20 mL of venous blood was takenagain and another electrocardiogram and bloodpressure determination was carried out, conclud-ing at this time the experimental protocol. Sample processing and biochemicaldeterminations Blood sample was immediately centrifuged at 4500 rpmand the plasma obtained was stored at –70  C. Allsamples were processed no more than 1 week from itsextraction to determine at time 0 # and 30 #  plasma leptin by the IRMA method (Diagnostic Systems Laboratories,Webster, TX), HS-PCR by turbidimetric assay (Tina-quant CRP; Boehringer Mannheim Systems, Germany),and NO by the colorimetric method (Cayman ChemicalCompany, Ann Arbor, MI). Cold pressor test cut points The patients were grouped according to changes in blood pressure during the CPT in reactive or non-reactive patients. When the difference between D SBP 0–15 15 mm Hg or greater and/or  D DBP 0–15  12 mm Hg orgreater, the individual was considered as reactive andif   D SBP 0–15  less than 15 mm Hg and/or  D DBP 0–15  lessthan 12 mm Hg was considered negative reactivity. Statistical analysis Qualitative and quantitative variables are expressed asmeans (with standard deviations) and/or percentages.Mean differences were assesses by analysis of variancetest and dichotomic variable relationships was ex-plored by chi-squared test. Sensitivity and specificitycalculations were performed by Bays #  theorem andstatistical significance was assumed when  P # 0.05. Allstatistical procedures were carried out with SPSSversion 13 (SPSS Inc., Chicago, IL).This study was approved according to the rules of the Bioethics Board at the Fondo Nacional de Ciencia yTecnologı´a and Centro Nacional de Bioe´tica (NationalScience and Technology Fund and National BioethicalCenter), and each participant was required to completean informed consent. RESULTS AND DISCUSSION No statistical differences were observed among timeand biochemical parameters in this study, but therewere differences in NO levels among groups ( P , 0.05)(Table 1).The accepted standard method at the present timefor diagnosis of endothelial dysfunction is the mea-surement of arterial diameters with ultrasound of the brachial artery. This is obtained evoking a reaction of reactive hyperemia using acetylcholine and measuringthe diameter change of the artery. 6 This test, neverthe-less, is not very practical because of its complexity andhighcostofequipment.Forsometime,measurementof  biochemical parameters has been used with thepurpose of avoiding this test and to count on a simplemethod that shows the presence of endothelialdysfunction and alterations in the metabolic routes.In this study, NO, C-reactive protein, and leptin weretested as biochemical indicators of endothelial dys-function in a population of patients with hypertensionand patients with diabetes using the CPTas an indirectindicator of endothelial dysfunction (Table 1).The studied sample presented characteristics thatlimited its analysis, like age differences and high bodymass index. The average body mass index was 28.7kg/m 2 , without statistical significant differencesamong the three groups, confirming that the controlgroup as well as the other groups have ‘‘overweight’’members according to the existing classification of obesity. 13 Likewise, significant differences for age wereappraised, which makes the sample heterogeneous andnot accurate for other comparisons. Even so, somevaluable findings can be obtained.As for hemodynamic variables, significant differenceswereobservedbetweengroupsforallthehemodynamicvariables ( P  ,  0.01) and for systolic blood pressure intime ( P  = 0.05). When correlating time with the group,asignificantdifference( P , 0.05)wasobservedinallthevariables. Changes in the values of blood pressure weremoremarkedinpatientswithhypertension,asshowninFigure 2A. The average difference was positive in allgroups. These were the expected results and match theresults described in the literature.The distribution according to reactivity to the CPT(Fig. 2B) draws a uniform proportion among the  American Journal of Therapeutics (2008)  15 (4) 392  Fouillioux et al   healthy, diabetic, and hypertensive groups, the lattergroupwith 7% morepatients with positive reactivity tothe test when compared with the other two groups.It was expected that the control group would displaya greater amount of subjects with negative reactivity tothe CPT, and this little difference can be attributed tothe limitations mentioned previously. The chi-squaredtest was preformed to this distribution and a value of  x 2 = 0.803 was obtained with no statistical significantdifferences among groups ( P  = 0.669).Otherstudieshave shown that upto 29.7% ofnormalsubjectshaveCPTpositivereactivity. 29 Thisistheresultof endothelial dysfunction in healthy subjects andparticularly in those with hypertensive relatives, whichpoints to a genetic predisposition. 30,31 Biochemical indicators were then compared withhemodynamic indicators, the latter ones measured asreactivity to the CPT. As for differences between groups,leptin as well as NO showed notorious numericdifferences as far as their averages, but with non-statistical significantly high deviations.Leptin showed an inverse distribution to pressure inthe CPT. As mentioned previously, leptin tends to beelevated in obese persons and diminished in normalweight persons.As can be observed, no statistically significantdifferences were found in body mass index for thisdistribution; despite having slightly unequal values, both groups are overweight.No significant correlation was observed amongvalues of biochemical indicators and blood pressurechanges during the CPT (Table 1). Slightly negativecurves were found for leptin and positive for NO,which confirms previous findings in which leptinserum levels diminished in patients with more positivechanges in blood pressure. As for NO, it was observedthat in patients considered ‘‘nonreactive,’’ values of NO were slightly lesser than in patients with positive Table 1.  General linear model (GLM) for measurements at 0 # , 15 #  and 30 minutes for heart rate (HR), systolic bloodpressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), nitric oxide (NO), leptin and highsensitivity C reactive protein (HS-CPR) in healthy, type-2 diabetic patients and hypertensive patients.VariablesGroups GLMHealthy(n = 14)Type-2 diabetes(n = 15)Hypertensives(n = 13)Time(F)Groups(F)Time/Groups(F)HR (hb/min) 1,125 ( P   = 0,295) 1,282 ( P   = 0,289) 3,133 ( P   = 0,054)0 #  71,3 6  11,2 74,9 6  13,6 66,6 6  815 #  68,1 6  9,8 70,7 6  12,1 62,6 6  9,930 #  68,7 6  9 71 6  13,8 69,5 6  10,8SBP (mm Hg) 4,009 ( P   = 0,052) 18,722 ( P   = 0,000) 6,541 ( P   = 0,003)0 #  121,5 6  10,5 134  6 15 149,8 6  20,115 #  127,3 6  11,7 141,8  6 16,3 162 6  20,530 #  121,6 6  12,5 132,2  6 16,1 160 6  16,5DBP (mm Hg) 0,208 ( P   = 0,651) 11,663 ( P   = 0,000) 3,338 ( P   = 0,046)0 #  74,2 6  9,8 80,4 6  9,1 88,6 6  10,515 #  78,3 6  11,2 83,9 6  11,5 93,7 6  1230 #  72,9 6  8 79 6  9,1 92,7 6  10,4MAP (mm Hg) 2,809 ( P   = 0,102) 15,243 ( P   = 0,000) 3,703 ( P   = 0,033)0 #  89,8 6  10,3 97,8 6  12,3 107,2 6  10,715 #  96,7 6  9,4 104,9  6 14 117,9 6  1330 #  91,9 6  9 95,7  6 9,8 113,3 6  12,4NO ( m M) 0,773 ( P   = 0,385) 4,195 ( P   = 0,022) 1,058 ( P   = 0,357)0 #  18,9 6  5,3 21,4 6  6,4 29,3 6  17,430 #  18,1 6  6,5 23,8 6  8,8 30,3 6  16Leptin (ng/mL) 0,247 ( P   = 0,622) 0,935 ( P   = 0,401) 0,079 ( P   = 0,924)0 #  22,9 6  23,3 20,3 6  19,4 13  6 12,530 #  23,2 6  24,7 20,4 6  20 13,2 6  13,1HS-CRP (mg/dL) 0,172 ( P   = 0,680) 1,520 ( P   = 0,231) 4,736 ( P   = 0,014)0 #  0,41 6  0,20 0,48 6  0,25 0,64 6  0,6530 #  0,42 6  0,20 0,44 6  0,23 0,68 6  0,61 Values are expressed as arithmetic mean 6  standard deviation.  American Journal of Therapeutics (2008)  15 (4) Endothelial Dysfunction During the Cold Pressor Test   393
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