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Serum contents of endocannabinoids are correlated with blood pressure in depressed women Ho, WS V; Hill, Matthew N; Miller, Gregory E; Gorzalka, Boris B; Hillard, Cecilia J Feb 28, 2012

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RESEARCH Open AccessSerum contents of endocannabinoids arecorrelated with blood pressure in depressedwomenWS Vanessa Ho1,3, Matthew N Hill2,4*, Gregory E Miller2, Boris B Gorzalka2 and Cecilia J Hillard1AbstractBackground: Depression is known to be a risk factor for cardiovascular diseases but the underlying mechanismsremain unclear. Since recent preclinical evidence suggests that endogenous agonists of cannabinoid receptors(endocannabinoids) are involved in both cardiovascular function and depression, we asked whetherendocannabinoids correlated with either in humans.Results: Resting blood pressure and serum content of endocannabinoids in ambulatory, medication-free, femalevolunteers with depression (n = 28) and their age- and ethnicity-matched controls (n = 27) were measured. Infemales with depression, both diastolic and mean arterial blood pressures were positively correlated with serumcontents of the endocannabinoids, N-arachidonylethanolamine (anandamide) and 2-arachidonoylglycerol. Therewas no correlation between blood pressure and endocannabinoids in control subjects. Furthermore, depressedwomen had significantly higher systolic blood pressure than control subjects. A larger body mass index was alsofound in depressed women, however, it was not significantly correlated with serum endocannabinoid contents.Conclusions: This preliminary study raises the possibility that endocannabinoids play a role in blood pressureregulation in depressives with higher blood pressure, and suggests an interrelationship among endocannabinoids,depression and cardiovascular risk factors in women.Keywords: Depression, Systolic blood pressure, Diastolic blood pressure, Cardiovascular risk, Anandamide,2-arachidonoylglycerolBackgroundRecently, the lipid signaling molecules endocannabinoids(eCBs) have been linked to depression and anxiety dis-orders [1-5]. The eCBs are endogenous agonists of can-nabinoid CB1 and/or CB2 receptors and the prototypicalexamples are N-arachidonylethanolamine (anandamide;AEA) and 2-arachidonoylglycerol (2-AG). These hydro-phobic molecules are produced ‘on demand’ in the cen-tral and peripheral system by stimuli, such asdepolarization, increases in intracellular Ca2+ and activa-tion of G protein-coupled receptors linked to phospholi-pase cascades [6,7]. Interestingly, cannabinoid CB1receptor knock-out mice display biochemical andbehavioral changes that are often seen in depression,including increased activity of the hypothalamus-pitui-tary-adrenal axis, anxiety, anhedonia, reduced feedingand weight loss [2,8,9]. Alterations in eCB contents and/or CB1 receptor signaling have also been associated withstress and depression in laboratory animals, dependingon the context, stressor and brain region [3-5]. Itremains to be established whether or not pathologicalchanges in eCB contents worsen or alleviate symptomsof depression. In an attempt to investigate this, we havepreviously examined the serum content of eCB inwomen diagnosed with depression [10,11]. We observedthat serum content of AEA and 2-AG was increased inminor depression but decreased in major depression,suggesting that the endocannabinoid system may be dis-turbed in depressive disorders. There is also indicationthat circulatory eCBs levels are correlated with exposure* Correspondence: mnhill@ucalgary.ca2Department of Psychology, University of British Columbia, Vancouver V6T1Z4, BC, CanadaFull list of author information is available at the end of the articleHo et al. Lipids in Health and Disease 2012, 11:32http://www.lipidworld.com/content/11/1/32© 2012 Vanessa Ho et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.to stress [11], anxiety ratings and duration of depressiveepisode [10]. The role of eCBs in depression is furthersupported by the recent findings that variations of thegene encoding CB1 receptors is associated with depres-sive symptoms [12] and a reduction in antidepressanttreatment response in major depression, especially inwomen with high anxiety level [13].Depression is not only a persistent mood disorder, butalso a risk factor for cardiovascular diseases. It is preva-lent in heart failure patients and predicts poor prognosisfor those with ischemic heart disease or following myo-cardial infarction [14,15]. The underlying mechanisms ofthis association are unclear but the higher occurrence ofhypertension and changes in cardiac and arterial func-tions in depressed patients could play a role. Forinstance, depression has been associated with impairedautonomic control, enhanced platelet reactivity, upregu-lation of proinflammatory mediators and developmentof atherosclerotic plaques [14-17]. AEA and 2-AG areknown to modulate cardiovascular functions [18]. Inrodents, AEA and/or 2-AG can cause arterial dilation[19,20], reduced cardiac contractility [21] and hypoten-sion [22,23]. Furthermore, AEA and other CB1 receptoragonists modulate autonomic transmission [24,25], pla-telet reactivity [26,27] and inflammatory processes [28],all of which have been implicated in the cardiovascularrisks associated with depression in humans. These datalead us to hypothesize that eCBs are involved in theinteraction between depression and cardiovascular func-tion. Using the same cohort of volunteers as in ourrecent study [10], we have examined the relationshipbetween serum eCB content and resting blood pressurein depressed women.ResultsCorrelations between serum eCBs and blood pressure indepressed individualsThe average systolic blood pressure was significantlyhigher in depressed individuals than their matched con-trols (Table 1). There were no significant overall differ-ences in their averaged diastolic pressure, mean arterialpressure or heart rate (Table 1); however further analy-sis found that these parameters were significantlyincreased in minor as compared to major depression(Table 1).Pooling data from all depressed subjects masked thebidirectional changes in serum eCB contents in minorversus major depression [10], such that the averagedserum AEA and 2-AG contents were similar indepressed women (AEA: 0.8 ± 0.1 pmol/ml; 2-AG: 18.5± 2.7 pmol/ml) and in control subjects (AEA: 0.7 ± 0.1pmol/ml; 2-AG: 19.0 ± 2.4 pmol/ml). However, wefound that both serum AEA and 2-AG were positivelycorrelated with diastolic and mean arterial pressure inthe combined group of women diagnosed with majorand minor depression (Table 2). Based on Pearson’sCorrelation tests (r), it was estimated that about 20% to38% of variance in eCB content could be explained byvariation in diastolic pressure or mean arterial pressurein women with depression, and vice versa. In addition,AEA tended to correlate positively with systolic pressureor heart rate (Table 2); it might account for > 11% ofthe variance in systolic pressure and heart rate. In con-trast, no correlations between serum eCBs and bloodpressure or heart rate were found in the control subjects(Table 2). The distinct distributions of serum eCBs inrelation to blood pressure readings in control anddepressed subjects are shown in Figure 1 (for AEA) andFigure 2 (for 2-AG). Importantly, significant correlationsbetween eCBs and diastolic pressure, and between eCBsand mean blood pressure, were also obtained after con-trolling for a third variable; use of tobacco (% daily smo-kers, Depressives, 25%; Control: 0%), use of oralcontraceptives (% user, Depressives, 18%; Control: 30%),alcohol consumption (number of alcoholic drinks perweek, Depressives, 2.6 ± 0.9; Control, 1.3 ± 0.7), BMI(see below), waist-to-hip ratio (see below) or serum totalcholesterol levels (data not shown).BMI values were significantly elevated in depressedwomen (Table 1). It was noted that 2 of the depressedpatients would be considered for hypertensive treatment(with systolic/diastolic: 152/91 and 140/84 mmHg) andhad a BMI of 59 and 29 kg/m2 respectively. In both thecontrol and depressed subjects, BMI was positively cor-related with systolic pressure (Depressive: r = .41, P =0.03; Control: r = .38, P = 0.05) but not diastolicTable 1 Cardiovascular parameters and body mass indexMAP (mmHg) SBP (mmHg) DBP (mmHg) HR (beats/min) BMI(kg/m2)Control 81.4 ± 1.6 108.6 ± 1.5 67.9 ± 1.5 70.0 ± 1.8 25.4 ± 1.0Depressed patients 85.6 ± 1.7 115.1 ± 2.1* 69.8 ± 1.3 69.0 ± 2.0 31.5 ± 1.8**Minor depression 89.7 ± 3.3*† 118.2 ± 4.0* 73.7 ± 2.3*†† 71.9 ± 3.2†† 31.9 ± 3.2**Major depression 82.6 ± 1.4 112.9 ± 2.1 66.9 ± 1.2 66.8 ± 2.4 31.2 ± 2.2*** P < 0.05, ** P < 0.01 vs control; † P < 0.05, †† P < 0.01 vs patients with major depression; MAP, mean arterial blood pressure; SBP, systolic blood pressure; DBP,diastolic blood pressure; HR: heart rate; BMI, body mass indexHo et al. Lipids in Health and Disease 2012, 11:32http://www.lipidworld.com/content/11/1/32Page 2 of 9pressure (Depressive: r = .27, P = 0.17; Control: r = -.01,P = 0.97). In depressed subjects, BMI was also positivelycorrelated with mean arterial pressure (Depressive: r =.40, P = 0.03; Control: r = .15, P = 0.46). However, BMIwas not correlated with serum eCBs in either group(Depressives, AEA: r = .28, P = 0.15; 2-AG: r = .03, P =0.88; Control, AEA: r = -.02, P = 0.93; 2-AG: r = .05, P= 0.82).As in the case for BMI, waist-to-hip ratio, which is anindicator of abdominal obesity, of depressed subjects(0.82 ± 0.01) was also significantly larger than that ofcontrol subjects (0.79 ± 0.01; P = 0.01). In control sub-jects, 2-AG was significantly and positively correlatedwith waist-to-hip ratio but no other correlations weresignificant (Depressives, AEA: r = -.03, P = 0.90; 2-AG: r= .23, P = 0.24; Control, AEA: r = .28, P = 0.17; 2-AG: r= .41, P = 0.03).Table 2 Correlation between serum endocannabinoidsand blood pressure in depressionAEA 2-AGr* P† r PControl n = 27 MAP -.07 0.72 -.06 0.78SBP -.17 0.40 .04 0.83DBP -.05 0.81 -.04 0.84HR -.02 0.92 -.13 0.54Depressed patientsn = 28MAP .50 < 0.01‡ .46 0.01‡SBP .35 0.07 .22 0.26DBP .46 0.01‡ .62 < 0.01‡HR .34 0.08 .14 0.48* correlation coefficient as determined by Pearson correlation tests; †probability value; ‡ statistical significance; MAP, mean arterial blood pressure;SBP, systolic blood pressure; DBP, diastolic blood pressure; HR: heart rateFigure 1 Scatter plots of serum content of N-arachidonylethanolamide (AEA) and mean or diastolic blood pressure in control (a, b) ordepressed subjects (c, d). n = 27-28. Serum AEA is positively correlated with mean (r = .50) and diastolic blood pressure (r = .46) in depressedsubjects (c.f. Table 2).Ho et al. Lipids in Health and Disease 2012, 11:32http://www.lipidworld.com/content/11/1/32Page 3 of 9Total cholesterol levels in serum were not differentbetween controls (183.1 ± 6.9 mg/dL) and depressedsubjects (180.7 ± 5.8 mg/dL). There was a tendency for2-AG, but not AEA, to be positively correlated withserum cholesterol in both groups (Depressives, AEA: r =.08, P = 0.68; 2-AG: r = .33, P = 0.09; Control, AEA: r =.03, P = 0.90; 2-AG: r = .33, P = 0.09). No correlationbetween cholesterol and blood pressure was detected(data not shown).Correlations between serum eCBs and blood pressure inmajor versus minor depressionWe further examined if correlations between serumeCBs and blood pressure reported herein depend on theseverity of depression. The mean arterial (P = 0.04) anddiastolic blood pressure (P = 0.01) in minor depressionwas significantly greater than that in major depression(Table 1).In subjects with minor depression, 2-AG, but notAEA, was strongly and positively correlated with diasto-lic blood pressure (Table 3). Based on Pearson’s Correla-tion tests, it was estimated that almost 51% of variancein 2-AG content could be explained by variation in dia-stolic pressure and vice versa. In addition, AEA waspositively correlated with heart rate in minor depres-sives, accounting for about 32% of variance (Table 3). Insubjects with major depression, AEA, but not 2-AG, waspositively correlated with diastolic pressure (Table 3). Itwas estimated that about 23% to 29% of variance ofAEA content could be explained by variation in diastolicpressure and vice versa. AEA also tended to correlatepositively with mean arterial blood pressure (Table 3).Figure 2 Scatter plots of serum content of 2-arachidonoylglycerol (2-AG) and mean or diastolic blood pressure in control (a, b) ordepressed subjects (c, d). n = 27-28. Serum 2-AG is positively correlated with mean (r = .46) and diastolic pressure (r = .62) in depressedsubjects (c.f. Table 2).Ho et al. Lipids in Health and Disease 2012, 11:32http://www.lipidworld.com/content/11/1/32Page 4 of 9The correlations between eCBs (AEA or 2-AG) and dia-stolic pressure remained significant after controlling fora third variable, including: use of tobacco, use of oralcontraceptives, alcohol consumption, BMI, waist-to-hipratio or serum total cholesterol levels (data not shown).BMI values were significantly increased in both minorand major depression (Table 1). Interestingly, BMI waspositively correlated with systolic and mean arterialblood pressure in individuals with minor (MAP: r = .63,P = 0.02; SBP: r = .59, P = 0.03), but not major (MAP: r= .00, P = 0.99; SBP: r = .09, P = 0.73), depression. BMIwas not significantly correlated with diastolic pressure(Minor depression, r = .44, P = 0.15; Major depression,r = .02, P = 0.95). In addition, no correlation betweeneCBs and BMI was detected in either depression group(Minor depression, AEA: r = .39, P = 0.21; 2-AG: r =-.04, P = 0.89; Major depression, AEA: r = .13, P = 0.64;2-AG: r = .19, P = 0.48). It was noted that there was atendency for 2-AG, but not AEA, to be positively corre-lated with serum cholesterol in minor depression(Minor depression, AEA: r = -.05, P = 0.89; 2-AG: r =.57, P = 0.06; Major depression, AEA: r = .10, P = 0.70;2-AG: r = -.05, P = 0.86).DiscussionThe major finding in this study was that serum contentof both AEA and 2-AG were positively correlated withboth diastolic and mean arterial pressure in ambulatory,medication-free, women with depression. We also foundthat diastolic pressure was positively correlated with 2-AG in minor depression but with AEA in major depres-sion, hinting at a differential role of the eCBs in depres-sion. Our data also confirm previous suggestions thatdepression is accompanied by elevated blood pressure,which is a major risk factor for cardiovascular diseases.To our knowledge, this is the first report of acorrelation between eCBs and blood pressure in clinicaldepression. This correlation, however, does not necessa-rily imply a casual relationship. It is also important tonote that it is too early to generalize our data to a largerpopulation. Notably, the current data were obtained inrelatively young, female volunteers. Therefore it remainsto be determined if similar results also occur in oldersubjects and in men.By still unknown mechanisms, depression seems tohave a negative impact on basic cardiovascular func-tions. For instance, reductions in heart rate variability[15] and baroreflex sensitivity [17] as well as increasesin blood pressure or hypertension [29] have beenreported in depressed patients. These changes mightexplain why depression is a risk factor for cardiovasculardiseases, and increases mortality and morbidity inpatients with myocardial infarction or heart failure[14,15]. In this study, we found that depressed womenhave significantly higher systolic pressure (by an averageof 6.5 mmHg) and also tend to have a higher meanarterial pressure (by an average of 4.2 mmHg). Interest-ingly, increases in systolic and mean arterial blood pres-sure are more evident in individuals with minor, ascompared to major, depression. For the majority ofdepressed subjects, their systolic and diastolic pressuresremain within the normotensive range [30]. Neverthe-less, it is noteworthy that several epidemiological studieshave suggested a continual and gradual increase in car-diovascular risks with increased systolic and diastolicblood pressure, even when pressures remain within nor-mal range [31,32].There is strong evidence from laboratory animals thateCBs are involved in the regulation of blood flow andblood pressure, especially in pathophysiological condi-tions such as hypertension [18]. Exogenous addition ofAEA or 2-AG can reduce cardiac contraction, vasculartone and arterial blood pressure [19-23]. Importantly,Kunos and colleagues [22,33,34] have demonstrated thatthe hypotensive effects of AEA are more pronounced inhypertensive, anaesthetized animals. Indeed, in Sponta-neously Hypertensive Rats, a commonly used model forgenetic hypertension, inhibition of enzymatic degrada-tion of endogenous AEA is sufficient to reduce bloodpressure [33]. Notably, modulation of eCB signaling, byusing AEA enzyme inhibitors or CB1 receptor antago-nist, regulates blood pressure in hypertensive, but notnormotensive rats. These results suggest that an eCBstone limits the extent of hypertension under pathophy-siological conditions. This might explain our observedcorrelations of eCBs and blood pressure in thedepressed (hypertensive) but not in healthy subjects. Onthe other hand, increases in blood pressure haverecently been shown to elevate AEA content in the reg-ulatory center for baroreceptor reflex [35]. It has beenTable 3 Correlation between serum endocannabinoidsand blood pressure in minor versus major depressionAEA 2-AGr* P† r PMinor depression n = 12 MAP .44 0.15 .44 0.15SBP .36 0.25 .22 0.49DBP .30 0.34 .71 < 0.01‡HR .57 0.05‡ .11 0.74Major depression n = 16 MAP .48 0.06 -.20 0.47SBP .21 0.43 -.23 0.40DBP .54 0.03‡ -.30 0.26HR -.06 0.84 -.22 0.41In the same cohort of females, serum AEA is significantly increased in minordepression, whereas 2-AG is decreased in major depression 11. * correlationcoefficient as determined by Pearson correlation tests; † probability value; ‡statistical significance; MAP, mean arterial blood pressure; SBP, systolic bloodpressure; DBP, diastolic blood pressure; HR: heart rateHo et al. Lipids in Health and Disease 2012, 11:32http://www.lipidworld.com/content/11/1/32Page 5 of 9proposed that AEA enhances baroreflex function, whichmight be compromised in depression [17], by increasingneuronal activity at the nucleus tractus solitarius [35,36].Our data that serum contents of AEA and 2-AG arestrongly and positively correlated with diastolic andmean arterial blood pressure in depressed but nothealthy women, suggest that serum eCBs could functionto compensate for an elevated blood pressure in depres-sion. If this is true, the data also suggest that this feed-back mechanism fails to normalize the systolic pressure,which remains elevated in depressives. This hypothesisis consistent with the animal data available; however,the possibility that eCBs somehow contribute to theincrease in blood pressure cannot be ruled out. It isinteresting that serum eCB content and blood pressureare not correlated in control subjects. Furthermore,despite opposing changes of averaged, serum eCB con-tent in minor and major depression [10], either AEA or2-AG remains positively correlated with diastolic bloodpressure in both depression groups. Taken together, thepresent findings suggest that a positive correlationbetween serum eCBs and blood pressure could be acommon feature of depression at different levels ofseverity. It is conceivable that biochemical changes indepression, or its associated increase in blood pressure,play a facilitatory role in the positive correlationbetween eCBs and blood pressure.Emerging evidence suggests changes in eCB signalingare involved in depression and anxiety disorders. In thesame cohort of female subjects as in the current study, weobserved that serum 2-AG content is negatively correlatedwith the duration of current depressive episode and AEAcontent is negatively correlated with Hamilton ratings forcognitive and somatic anxiety in depressed subjects [10].However, there is no correlation between these parametersand blood pressure (data not shown). Together, our resultspoint to the complex interrelationships among serum con-tent of eCBs, depression and blood pressure. In this study,there is also evidence that AEA and 2-AG could play a dif-ferential role in minor vs major depression since diastolicpressure was positively correlated with 2-AG in minordepression and with AEA in major depression. The synth-esis and degradation of the two eCBs involve distinctenzymes [6,7] and eCB levels are often differentially regu-lated in stress and anxiety [3,10,11]. Alterations of eCBssignaling in depression and hypertension of differentseverity remain unclear but our data might indicate that 2-AG is the predominant eCB involved in blood pressurecontrol in minor depression, whereas AEA plays a moreimportant role in major depression. Such differential rolesalso highlight the need for measurements of both eCBs infuture studies on subtypes of depression.Depression is sometimes associated with an increasedprevalence of obesity and smoking, which could act asconfounding factors in the association between bloodpressure and eCBs. In this study, depressed subjects aremore likely to be obese (with BMI ≥ 30 kg/m2) com-pared to their matched controls. Whilst this might con-tribute to the increased blood pressure in depressives, adirect link between BMI and serum eCBs is not evident.In addition, tobacco smoking, the waist-to-hip ratio,serum total cholesterol also fail to explain the correla-tions between eCBs and blood pressure in depression. Itis noteworthy that the absence of correlation betweenBMI and eCBs in the current study (in women; averageage of 29 years) contrasts with the observation that BMIis positively correlated with plasma 2-AG, but not AEA,in men with an average age of 42 years [37]. The discre-pancy could also be due to the limited number of sub-jects with advanced obesity in our sample [38].Interestingly, however, there is indication that 2-AG, butnot AEA, is positively correlated with serum total cho-lesterol levels in our female subjects. Since subfractionsof cholesterol content were not determined in thisstudy, it is unknown if high-density lipoproteins (HDL)-cholesterol or low-density lipoproteins (LDL)-choles-terol, or both, are involved. This is of particular interestin light of the finding that the CB1 receptor antagonist,rimonabant, improves serum HDL-cholesterol of over-weight or obese subjects in a randomized, double-blinded clinical trial [39]. Further analysis also revealedthat correlations between 2-AG and total serum choles-terol, and between BMI and blood pressure, occurredpredominantly in individuals with minor depression. Atpresent, the significance of these subtle differences ofcardiovascular and metabolic variables between minorand major depression is unclear.To conclude, our study shows that serum contents ofeCBs are positively correlated with blood pressure indepressed women but not in their matched control sub-jects. We speculate that eCBs play a role in regulationof blood pressure in depression, perhaps acting to bufferan increase in blood pressure. In this study, depressedwomen also have a higher systolic blood pressure, bodymass index, waist-to-hip ratio and higher prevalence oftobacco smoking, all of which are associated withincreased risk of cardiovascular diseases. Since eCBs arelikely involved in both cardiovascular and neuronalfunctions, the interrelationships among these agents,cardiovascular parameters and depression warrantfurther attention.Given the negative impact of depression on patientswho have ischemic heart disease or have recently hadmyocardial infarction, it has been suggested that effec-tive treatment of depression can not only improve thequality of life but also the clinical outcome of patientswith heart diseases. A few, although not all, clinicaltrials have indeed shown that treating depression withHo et al. Lipids in Health and Disease 2012, 11:32http://www.lipidworld.com/content/11/1/32Page 6 of 9selective serotonin reuptake inhibitors (SSRI) signifi-cantly improve the prognosis of patients recoveringfrom myocardial infarction [40-42]. In this regard, it isinteresting to note that inhibitors of fatty acid amidehydrolase have been proposed to reduce the symptomsof anxiety and depression [43,44]. Perhaps increasingeCBs signaling by these inhibitors could also help nor-malize blood pressure homeostasis in depression. How-ever, this approach could also be detrimental todepressive individuals with obesity-related co-morbid-ities since a recent clinical study has reported thatplasma eCBs is inversely related to coronary blood flowin advanced obesity [38]. More clinical studies examin-ing the role of eCBs in depression and cardiovascularfunctions are required.MethodsSubjectsA total of 55 ambulatory, adult women from SaintLouis, MO, USA participated in the study; the samecohort of volunteers was used in our recent study [10].The protocol was approved by the Institutional ReviewBoard of Washington University USA and all subjectsprovided written informed consent. Depressed subjects(n = 28, age 29.0 ± 1.7) met the diagnostic criteriadefined by the forth edition of the Diagnostic and Statis-tical Manual (DSM-IV) [45] for clinical depression. Theyincluded subjects with Major Depressive Episode (n =16) and Minor Depressive Episode (n = 12). Theirmatched control subjects (based on age and ethnicity; n= 27, age 28.8 ± 1.6) had no lifetime history of psychia-tric illness. Each group had 12 Caucasian, 13/14 AfricanAmerican, 1 Hispanic and 1 Asian subjects. All subjectswere in good health, defined as having (a) no history ofchronic medical illness, (b) no indications of acute infec-tious disease at study entry, as evidenced by self-reportof symptoms and a normal complete blood count, and(c) no prescribed medication regimen, other than oralcontraceptives, in the past 6 months including anti-depressants. Candidates were excluded if they wereolder than 55; had been pregnant in the past year; weremenopausal, postmenopausal, had irregular menses;were undernourished as evidenced by serum albumin ≤3.3 g/dL; or with reported abuse of illicit substancesincluding cannabis, cocaine and heroin. More detailedinformation on the recruitment of volunteers, subse-quent assessment of clinical depression and demo-graphic characteristics of the sample population haspreviously been reported [10].Blood pressure measurementSubjects were seated in a comfortable chair with fore-arm on the armrest. Using appropriate cuff size, 3 bloodpressure readings, spaced 2 min apart, were collected byan automated oscillometric device (Dinamap Pro 100;Critikon Corporation). The averaged systolic, diastolicand mean arterial blood pressure were obtained. Thiswas followed by blood draw, via antecubital venipunc-ture, in the morning hours (0900 h - 1200 h). Bloodsamples were centrifuged for 15 min at 1000 g and theserum aspirated and frozen at -70 to -80°C until use. Allserum samples were frozen by 120 min following veni-puncture. Total serum cholesterol was also assessedusing enzymatic methods on a Hitachi 747 instrument(Kyowa Medex) in the Washington University Centerfor Clinical Studies. Upon completion of the study, par-ticipants were compensated US$150. These procedureswere approved by the Institutional Review Board ofWashington University, USA.Detection of serum endocannabinoidsSerum eCBs were extracted by using Bond Elut C18 solid-phase extraction columns (1 ml; Varian Inc, Lake Forest,CA). Serum samples (0.5 ml each) were thawed and madeup to 15% ethanol, to which the internal standards [2H8]-AEA (16.9 pmol) and [2H8]-2-AG (46.5 pmol) (CaymanChemicals, Ann Arbor, MI) were added. Samples werevortexed and centrifuged at 1000 × g for 4 min. Thesupernatant was loaded on C18 columns, which have beenconditioned with 1 ml redistilled ethanol and 3 ml of dou-ble distilled water (ddH2O). The remaining pellet waswashed with 100 μl of 15% ethanol and centrifuged againfor 3 min. The resulting supernatant was also loaded ontothe C18 column. Columns were washed with 5 ml ddH2Oand eluted with 1 ml of ethyl acetate. The ethyl acetatelayer in the resulting elute was removed and dried underN2. Lipids in the residual ddH2O phase were extracted bymixing with an additional 1 ml of ethyl acetate, which wasadded to the original ethyl acetate solution. Once dried,samples were resuspended in 20 μl of methanol and storedat -80°C. AEA and 2-AG were quantified using isotope-dilution, atmospheric pressure, chemical ionization liquidchromatography/mass spectrometry (LC-APCI-MS) asdescribed previously [46].Data and statistical analysisWe have recently detected a bidirectional change ofserum contents of AEA and 2-AG in minor versusmajor depression in the same cohort of female subjects[10]. To reveal potential, underlying relationshipbetween serum eCB content and resting blood pressure,we therefore first examined data obtained from alldepressed subjects (with either major or minor depres-sion). Further analysis was then performed to explorethe impact of the severity of depression on correlationsbetween serum eCBs and blood pressure.Data are given as mean ± SEM. Blood pressure, heartrate and BMI in individuals with depression and theirHo et al. Lipids in Health and Disease 2012, 11:32http://www.lipidworld.com/content/11/1/32Page 7 of 9matched controls were compared by Student’s unpaired t-tests or one-way analysis of variance (Prism 4.02; Graph-Pad Software Inc). Correlations between eCB content andblood pressure were analyzed by bivariate, Pearson’s corre-lation tests (SPSS 14.0; SPSS Inc.). Influences of potentialconfounders on these correlations were tested by control-ling for a third variable, for example tobacco use, bodymass index (BMI) and serum cholesterol level, using par-tial correlation tests (SPSS 14.0; SPSS Inc). P ≤ 0.05 wasconsidered statistically significant.Abbreviations2-AG: 2-arachidonoylglycerol; AEA: Anandamide; BMI: Body mass index; CB1:Cannabinoid receptor type 1; eCB: Endocannabinoids; DSM-IV: Diagnosticand Statistical Manual; HDL: High-density lipoprotein; LDL: Low-densitylipoproteins.AcknowledgementsOperating grants from the American Heart Association and the CanadianInstitute of Health Research (CIHR) to GEM and BBG; a Young InvestigatorAward from NARSAD and a Michael Smith Foundation for Health Research(MSFHR) Scholar Award to GEM; an Independent Investigator NARSADAward and a NIH grant NS41314 to CJH; a Natural Sciences and EngineeringResearch Council of Canada (NSERC) operating grant to BBG; and a MSFHRpostgraduate trainee award and a NSERC Canadian Graduate Scholarship toMNH. The authors would like to thank Craig Roelke for his technicalassistance.Author details1Department of Pharmacology & Toxicology, Medical College of Wisconsin,Milwaukee WI 53226, USA. 2Department of Psychology, University of BritishColumbia, Vancouver V6T 1Z4, BC, Canada. 3Division of Biomedical Sciences,St George’s, University of London, Cranmer Terrace, London SW17 0RE, UK.4Department of Cell Biology & Anatomy and Psychiatry, The Hotchkiss BrainInstitute, University of Calgary, Calgary, Canada.Authors’ contributionsMNH, GEM, BBB and CJH designed the study; WSVH, MNH and GEMperformed the experiments; WSVH, MNH and CJH wrote the paper. Allauthors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Received: 10 January 2012 Accepted: 28 February 2012Published: 28 February 2012References1. 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American Psychiatric Association, Washington (DC);1994, revised.46. Patel S, Carrier EJ, Ho WS, Rademacher DJ, Cunningham S, Reddy DS, et al:The postmortal accumulation of brain N-arachidonylethanolamine(anandamide) is dependent upon fatty acid amide hydrolase activity. JLipid Res 2005, 46:342-349.doi:10.1186/1476-511X-11-32Cite this article as: Ho et al.: Serum contents of endocannabinoids arecorrelated with blood pressure in depressed women. Lipids in Health andDisease 2012 11:32.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitHo et al. Lipids in Health and Disease 2012, 11:32http://www.lipidworld.com/content/11/1/32Page 9 of 9


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