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Prostaglandins & other Lipid Mediators 97 (2012) 43– 49

Contents lists available at SciVerse ScienceDirect

Prostaglandins and Other Lipid Mediators

AF and its metabolic enzymes in healthy volunteers: Interrelations andorrelations with basic characteristics�

araskevi Detopouloua,1, Tzortzis Nomikosa, Elizabeth Fragopouloua, George Stamatakisb,emosthenes B. Panagiotakosa, Smaragdi Antonopouloua,∗

Department of Nutrition -Dietetics, Harokopio University, 70 El. Venizelou Street, 17671, Athens, GreeceDepartment of Chemistry, National and Kapodistrian University of Athens, Panepistimioupolis, 15771, Athens, Greece

r t i c l e i n f o

rticle history:eceived 15 July 2011eceived in revised form 16 October 2011ccepted 25 October 2011vailable online 3 November 2011

eywords:AF

a b s t r a c t

PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), a potent inflammatory mediator, is synthesizedvia the remodeling and the de novo route, key enzymes of which are acetyl-CoA:lyso-PAF acetyltransferase(lyso-PAF-AT) and DTT-insensitive CDP-choline:1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase(PAF-CPT), respectively. PAF-acetylhydrolase (PAF-AH) and its extracellular isoform lipoprotein-associated phospholipase-A2 (Lp-PLA2) catabolize PAF. This study evaluated PAF levels together withleukocyte PAF-CPT, lyso-PAF-AT, PAF-AH and Lp-PLA2 activities in 106 healthy volunteers. Men had lowerPAF levels and higher activity of both catabolic enzymes and lyso-PAF-AT than women (P-values <0.05).

cetyl-CoA: lyso-PAF acetytransferaseDP-choline: 1-alkyl-2-acetyl-sn-glycerolholinephosphotransferaseAF-acetylhydrolasep-PLA2

umans

Age was inversely correlated with PAF levels in men (r = −0.279, P = 0.06) and lyso-PAF-AT in women(r = −0.280, P = 0.05). In contrast, Lp-PLA2 was positively correlated with age (r = 0.201, P = 0.04). More-over, PAF-CPT was positively correlated with glucose (r = 0.430, P = 0.002) in women. In addition, PrincipalComponent Analysis revealed three PAF metabolic patterns: (i) increased activities of PAF-CPT and PAF-AH, (ii) increased activities of PAF-CPT and lyso-PAF-AT and (iii) increased activity of Lp-PLA2. The presentstudy underlines the complexity of PAF’s metabolism determinants.

. Introduction

Platelet-activating factor (PAF), a phosphoglycerylether lipid (1--alkyl-2-acetyl-sn-glycero-3-phosphocholine) [1], constitutes aotent inflammatory mediator implicated in a plethora of patho-hysiologic conditions, such as reproduction [2], atherosclerosis3], allergic disorders [4], cancer [5] and other diseases. PAF is syn-hesized constitutively or under appropriate stimuli by various cellsncluding platelets, leukocytes and endothelial cells and it exerts itsctions through its G-protein coupled receptors [6]. Moreover, PAF

ntagonists of synthetic (e.g. rupatadine currently used in asthmareatment) or natural origin (e.g. ginkgolides) may suppress itsctions [6].

Abbreviations: PAF, platelet-activating factor; lyso-PAF-AT, acetyl-CoA:lyso-AF acetyltransferase; PAF-CPT, DTT-insensitive; CDP-choline, 1-alkyl-2-acetyl-n-glycerol cholinephosphotransferase; PAF-AH, PAF-acetylhydrolase; Lp-PLA2,ipoprotein-associated phospholipase-A2; BMI, Body mass index; PCA, Principalomponent Analysis.� The study was funded by the Hellenic Atherosclerosis Society.∗ Corresponding author. Tel.: +30 2109549230; fax: +30 2109577050.

E-mail address: antonop@hua.gr (S. Antonopoulou).1 Fellowship from the State Scholarship Foundation (IKY) and the Hellenictherosclerosis Society.

098-8823/$ – see front matter © 2011 Elsevier Inc. All rights reserved.oi:10.1016/j.prostaglandins.2011.10.003

© 2011 Elsevier Inc. All rights reserved.

The quantity of PAF in cells, tissues and biological fluidsis regulated via its enzymatic synthesis and catabolism. Withrespect to PAF metabolism, PAF can be synthesized by two dif-ferent enzymatic routes, namely the remodeling and the denovo pathway [7,8]. The remodeling pathway is believed toproduce PAF under inflammatory conditions and involves a struc-tural modification of ether-linked membrane phospholipids. Morespecifically, the action of cytoplasmic phospholipase A2 yieldslyso-PAF which is then acetylated by acetyl-CoA:lyso-PAF acetyl-transferases (lyso-PAF-AT, EC 2.3.1.67) leading to the formationof PAF [9]. The de novo pathway appears to be responsiblefor the constitutive production of PAF, maintaining its physio-logical levels in various tissues and blood. A key step in thisroute is the conversion of 1-O-alkyl-2-acetyl-glycerol to PAF bya specific dithiothreitol-insensitive CDP-choline:1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (PAF-CPT, EC 2.7.8.16) [10].As far as the catabolism of PAF is concerned, the most impor-tant enzyme involved is a PAF-specific acetylhydrolase (PAF-AH,EC 3.1.1.47), which cleaves the short acyl chain at the sn-2 posi-tion and forms lyso-PAF [11]. The plasma isoform of PAF-AH is

known as lipoprotein-associated phospholipase A2 (Lp-PLA2) dueto its attachment to lipoproteins and mainly LDL-particles [12].

PAF metabolic pathways have been mainly explored in vitroin various cell cultures as well as in animal models of disease

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4 P. Detopoulou et al. / Prostaglandins

6]. However, in humans there is a striking dearth of evidencen the field of PAF metabolism mainly due to the complexity ofhospholipids biochemistry and the lack of high throughput tech-iques for the measurement of PAF and its metabolic enzymes.p-PLA2 constitutes an exception, as it has attracted the attentionf the scientific community representing a promising cardiovas-ular risk marker [13]. Our group has recently shown that ineart failure patients both PAF biosynthetic routes are correlatedith well-established inflammatory markers [14]. To our knowl-

dge, there is no other study assessing simultaneously PAF and itsiosynthetic and catabolic enzymes in an appreciable number ofolunteers.

Therefore, the aim of the present work was to evaluate the dis-ribution of PAF levels as well as a panel of its key biosynthetic andatabolic enzymes activities in apparently healthy volunteers ando assess the contributing role of various biological variables (i.e.,ge, gender, body mass index (BMI), leukocytes, glycemic and lipidrofile), to the measured values.

. Materials and methods

.1. Materials and instrumentation

All reagents were of analytical grade and were supplied byigma (St. Louis, MO, USA). Analytical grade solvents, liquid-hromatography grade solvents and silicic acid 60 (0.2–0.5 mm),sed for column chromatography, were supplied from MerckDarmstadt, Germany). 1-O-hexadecyl-2-O-acetyl-sn-glycerol wasurchased from Biomol International (Plymouth Meeting, PA, USA).-O-hexadecyl-2-[3H] acetyl-sn-glycerol-3-phosphocholine ([3H]AF) with a specific activity of 10 Ci/mmol was obtained from Newngland Nuclear (Boston, MA). The purification of PAF by HPLC waserformed at room temperature on a HP HPLC Series 1100 liquidhromatography model (Hewlett Packard, Waldbronn, Germany)quipped with a Partisil 10SCX, WCS Analytical Column (What-an 4.6 mm × 250 mm), a 100 �L loop Rheodyne (7725i) loop valve

njector, a degasser G1322A, a quat gradient pump G1311A and HP UV spectrophotometer G1314A as a detection system. Thepectrophotometer was connected to a Hewlett-Packard (Hewlettackard, Waldbronn, Germany) model HP-3395 integrator-plotter.AF-induced platelet aggregation was measured in a Chrono-og (Havertown, PA, USA) aggregometer (model 400-VS) coupledo a Chrono-Log recorder (Havertown, PA, USA) at 37 ◦C withonstant stirring at 1200 rpm. Centrifugations were performedn a refrigerated superspeed Avanti 30 centrifuge (Beckman).omogenizations were conducted with the Bandelin Sonoplus son-

cator GM 2070 (Germany). Radioactivity was measured with aiquid scintillation counter (1209 Rackbeta, Pharmacia, Wallac,inland).

.2. Subjects

This was an observational study. One hundred and six sub-ects (n = 48 men, 44 ± 13 years and n = 58 women, 44 ± 14 years),rom the Athens greater area, were studied in our Institution.y the design of the study men were age- and BMI-matched toomen. Exclusion criteria were medical treatment, history of car-iovascular or any other inflammatory disease, cold or flu, acuteespiratory infection, dental problems and renal/hepatic abnormal-ties. The protocol was approved by the Bioethics Committee of the

arokopio University and was in accordance with the Declarationf Helsinki (1989) of the World Medical Association. All participantsave their informed written consent in order to participate in thetudy.

er Lipid Mediators 97 (2012) 43– 49

2.3. Anthropometric measurements

Weight and height were measured in light clothing and withoutshoes. Body mass index was then calculated as weight (kg) dividedby height2 (m2). Waist circumference was measured between thesuperior iliac crest and the lower rib margin in the midaxillary lineand hip circumference as the maximal horizontal circumference atthe level of the buttocks. Both circumferences were measured tothe nearest 0.1 cm and waist-to-hip ratio was calculated.

2.4. Isolation of leukocytes from heparinized blood

Five milliliters of heparinized blood was obtained from each vol-unteer and leukocytes were isolated as previously described [14].Protein concentrations of all preparations were determined accord-ing to the Bradford method [15] with the use of BSA as proteinstandard.

2.5. Biochemical and hematological measurements

Serum glucose, triacylglycerols, total-cholesterol and HDL-cholesterol were determined enzymatically in fasting samples(ACE analyzer, Schiapparelli Biosystems, Inc, NJ, USA) usingreagents from Alfa Wassermann (Woerden, The Netherlands).LDL-cholesterol was calculated with the Friedewald Formula.Leukocytes, leukocytes subpopulations as well as platelet count andmean platelet volume were measured with Cell Dyn 1700 Hema-tology Analyzer (Abbott Diagnostics, USA).

2.6. Determination of PAF levels in blood

PAF was isolated from blood, purified and determined bya previously published method [16]. Briefly, 5 mL of wholeblood were poured into plastic tubes containing 20 mL of abso-lute ethanol. Then, the blood with ethanol was centrifugedat 500 × g for 20 min and the resulting supernatant (contain-ing free-PAF) and pellet (containing bound-PAF) were extractedaccording to the Bligh–Dyer method [17]. Subsequently, two stepsof purification followed: silicic acid column chromatography andhigh-performance liquid chromatography. PAF levels were thendetermined by measuring the aggregatory activity towards washedrabbit platelets. The quantification of PAF was based on a stan-dard curve constructed with the use of known concentrations ofsynthetic PAF [1].

2.7. Determination of lyso-PAF-AT, PAF-CPT, PAF-AH activities inleukocyte homogenates and serum Lp-PLA2

The determination of lyso-PAF-AT and PAF-CPT activities inleukocyte homogenates was based on the measurement of theproduced PAF, after TLC separation, by the washed rabbit plateletaggregation assay according to previously published methods[18,19]. For the determination of lyso-PAF-AT activity, leukocytehomogenates, containing 12 �g of total protein, were incubatedwith 4 nmol of lyso-PAF and 40 nmol of acetyl-CoA for 10 min at37 ◦C in a final volume of 200 �L of 50 mM Tris/HCl buffer (pH7.4) containing 0.25 mg/mL of BSA. PAF-CPT was assayed at 37 ◦Cfor 5 min in a final volume of 200 �L of a reaction mixture con-taining 20 �g of leukocyte homogenate protein, 100 mM Tris–HCl(pH 8.0), 15 mM DTT, 0.5 mM EDTA, 20 mM MgCl2, 1 mg/mL of BSA,100 �M of CDP-choline and 100 �M of 1-O-hexadecyl-2-Oacetyl-sn-glycerol (added in the mixture in 2 �L of DMSO). Both reactions

were stopped by adding 1.11 mL of cold chloroform: methanol (2%acetic acid) and 0.3 mL of cold water in order to extract PAF bythe acid Bligh–Dyer method. Enzymatic activities were expressedas specific activities in nmol/min/mg protein for lyso-PAF-AT and

P. Detopoulou et al. / Prostaglandins & other Lipid Mediators 97 (2012) 43– 49 45

Table 1Selected biochemical and anthropometric characteristics of the participants.

Total (n = 106) Men (n = 48) Women (n = 58) P

Age (years) 44 ± 13 44 ± 13 44 ± 14 0.8BMI (kg/m2) 27 ± 5 28 ± 4 26 ± 6 0.2Waist circumference (cm) 85.2 ± 14.8 92.7 ± 9.2 78.7 ± 15.8 <0.001Hip circumference (cm) 104.6 ± 12.1 104.7 ± 7.0 104.5 ± 15.2 0.9Waist-to-hip ratio 0.81 ± 0.12 0.88 ± 0.06 0.75 ± 0.12 <0.001Total cholesterol (mmol/L) 5.5 ± 1.1 5.6 ± 0.9 5.4 ± 1.1 0.3HDL-cholesterol (mmol/L) 1.2 ± 0.3 1.1 ± 0.2 1.3 ± 0.3 <0.001LDL-cholesterol (mmol/L) 3.8 ± 0.9 3.9 ± 0.7 3.7 ± 0.9 0.3Triacylglycerols (mmol/L)a 0.9 (0.7–1.4) 1.3 (0.9–1.7) 0.7 (0.6–1.0) <0.001Glucose (mmol/L) 5.1 ± 0.6 5.2 ± 0.4 5.0 ± 0.7 0.01Leukocytes (K/�L) 5.9 ± 1.6 6.3 ± 1.9 5.6 ± 1.2 0.02Lymphocytes (K/�L) 2.0 ± 0.5 2.2 ± 0.5 1.9 ± 0.4 0.002Monocytes, eosinophils, baseophils (K/�L)a 0.5 (0.4–0.6) 0.5 (0.4–0.6) 0.5 (0.4–0.6) 0.8Granulocytes (K/�L) 3.4 ± 1.1 3.6 ± 1.3 3.2 ± 0.9 0.1Platelets (×109/L) 243 ± 56 238 ± 58 246 ± 54 0.4Mean platelet volume (fL) 10.2 ± 1.4 10.0 ± 1.7 10.4 ± 1.2 0.2

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ata are presented as mean ± standard deviation for normally distributed variable-test was used to compare means.

a These variables were log-transformed prior to statistical comparisons.

mol/min/mg for PAF-CPT. PAF-AH and Lp-PLA2 activities in leuko-yte homogenates and serum, respectively, were determined byhe trichloroacetic acid precipitation method using [3H] PAF as aubstrate [14]. Briefly, leukocyte homogenates (containing 25 �gf total protein) or 2 �L of serum were incubated with 4 nmol of

3H] PAF (20 Bq/nmol) for 30 min or 15 min, respectively at 37 ◦C in final volume of 200 �L of 100 mM Tris/HCl buffer (pH 7.2) con-aining 1 mM EGTA. The reaction was terminated by the additionf cold trichloroacetic acid (10% final concentration). The samplesere then placed in an ice bath for 30 min and subsequently cen-

rifuged for 2 min. The [3H]-acetate released into the aqueous phaseas measured on a liquid scintillation counter. All assays were per-

ormed in duplicate. The enzyme activity was expressed as nmol ofAF degraded per min per mL of serum or pmol of PAF degradeder min per mg of leukocyte homogenate protein. All assays wereerformed in duplicate.

.8. Statistical analysis

Normality was tested with the Kolmogorov–Smirnof criterion.ormally distributed continuous variables are presented as meanalues ± standard deviation, while skewed variables as median and5th–75th quartiles. Categorical variables are presented as relativerequencies (%). For the comparisons between genders the indepen-ent samples t-test for normally distributed variables was applied.oreover, log transformations were used in order to achieve nor-ality for the following variables: free-PAF, bound-PAF, total-PAF,

riacylglycerols and monocytes, eosinophils, baseophils. Correla-

ions were evaluated using the Pearson correlation coefficient andge-, gender-, BMI-adjusted correlations were evaluated with theartial correlation coefficient. A multivariate analysis using Princi-al Component Analysis (PCA) was also applied to assess patterns of

able 2AF levels and activities of its key biosynthetic and catabolic enzymes in apparently healt

Total

Free-PAF (fmol/mL)a 19 (9–91)

Bound-AF (fmol/mL)a 36 (10–410)

Total-PAF (fmol/mL)a 119 (34–578)

lyso-PAF-AT (nmol/min/mg) 8.18 ± 5.44

PAF-CPT (pmol/min/mg) 120 ± 830

Lp-PLA2 (nmol/min/mL) 22 ± 6PAF-AH (pmol/min/mg) 334 ± 118

ata are presented as mean ± standard deviation for normally distributed variables. Othea These variables were log-transformed prior to statistical comparisons. Student t-test

erwise data are presented as median (lower–upper quartile) (25th–75th). Student

the distributions of PAF metabolic enzymes. To decide the numberof components to retain from the PCA, the eigenvalues that derivedfrom the correlation matrix of the standardized variables wereexamined (the eigenvalue evaluates the proportion of the varianceexplained by each extracted component). Based on the principlethat the component scores are interpreted similarly to correlationcoefficients, the PAF metabolic patterns were defined in relation toscores of variables that correlated most with the component (scores>0.6 were used). It is thus noted that higher score absolute valuesindicate that an enzymatic activity contributes most to the formu-lation of a component. The orthogonal rotation with varimax optionwas used to derive optimal, non-correlated PAF metabolic patterns.The information was rotated in order to increase the representationof each enzyme to a component. All reported P-values were basedon two-sided tests and compared to a significance level of 5%. SPSS18 (SPSS Inc., Chicago, IL, USA) software was used for statisticalanalysis.

3. Results

3.1. Basic characteristics of the apparently healthy volunteers,PAF levels and activities of its key metabolic enzymes

In Table 1 the basic anthropometric and clinical characteris-tics of the participants are presented. There was no significantdifference between men and women regarding age, BMI and hipcircumference, whereas differences in waist circumference, waist-to-hip ratio as well as certain lipid and glycemic parameters were

documented. Moreover, men had higher leukocyte and lympho-cyte count compared to women. The levels of PAF and the activitiesof its metabolic enzymes for the total sample as well as for menand women, separately, are reported in Table 2. Due to the skewed

hy volunteers.

Men Women P

19 (9–38) 19 (9–115) 0.220 (8–313) 53 (16–651) 0.0382 (29–372) 152 (43–944) 0.019.39 ± 6.28 7.21 ± 4.46 0.04140 ± 100 120 ± 670 0.125 ± 4 20 ± 5 <0.001386 ± 127 292 ± 91 <0.001

rwise data are presented as median (lower–upper quartile) (25th–75th).was used to compare means.

46 P. Detopoulou et al. / Prostaglandins & other Lipid Mediators 97 (2012) 43– 49

Table 3Pearson partial correlation coefficients between PAF, its enzymes and clinical characteristics after adjustments for age and BMI.

free-PAF bound-PAF total-PAF lyso-PAF-AT PAF-CPT Lp-PLA2 PAF-AH

(pmol/mL)a (pmol/mL)a (pmol/mL)a (nmol/min/mg) (pmol/min/mg) (nmol/min/mL) (pmol/min/mg)

Men (n = 48)Glucose (mmol/L) 0.140 0.117 0.198 −0.151 −0.045 −0.002 −0.264

(P = 0.3) (P = 0.4) (P = 0.2) (P = 0.3) (P = 0.7) (P = 0.9) (P = 0.09)Triacylglycerols(mmol/L)a

0.128 0.139 0.225 0.005 −0.015 0.413 <0.001(P = 0.4) (P = 0.3) (P = 0.1) (P = 0.9) (P = 0.9) (P = 0.006) (P = 0.9)

Total-cholesterol(mmol/L)

−0.097 0.110 0.065 0.009 −0.115 0.610 −0.151(P = 0.5) (P = 0.4) (P = 0.6) (P = 0.9) (P = 0.4) (P < 0.001) (P = 0.3)

HDL-cholesterol(mmol/L)

−0.141 −0.094 −0.207 −0.033 −0.200 0.124 0.176(P = 0.3) (P = 0.5) (P = 0.1) (P = 0.8) (P = 0.2) (P = 0.4) (P = 0.2)

LDL-cholesterol(mmol/L)

−0.119 0.109 0.060 0.025 −0.085 0.553 −0.243(P = 0.4) (P = 0.4) (P = 0.7) (P = 0.8) (P = 0.5) (P < 0.001) (P = 0.1)

Leukocytes (K/�L) 0.405 0.043 0.321 0.222 0.169 0.113 −0.225(P = 0.01) (P = 0.7) (P = 0.04) (P = 0.1) (P = 0.2) (P = 0.4) (P = 0.1)

Women (n = 58)Glucose (mmol/L) −0.219 0.153 0.117 −0.043 0.430 0.105 0.173

(P = 0.1) (P = 0.2) (P = 0.4) (P = 0.7) (P = 0.002) (P = 0.4) (P = 0.2)Triacylglycerols(mmol/L)a

−0.195 0.005 −0.006 −0.150 −0.143 0.396 0.045(P = 0.1) (P = 0.9) (P = 0.9) (P = 0.2) (P = 0.3) (P = 0.007) (P = 0.7)

Total-cholesterol(mmol/L)

−0.100 0.075 0.062 −0.250 0.003 0.630 0.267(P = 0.4) (P = 0.6) (P = 0.6) (P = 0.07) (P = 0.9) (P < 0.001) (P = 0.06)

HDL-cholesterol(mmol/L)

0.033 0.108 0.012 −0.241 0.084 −0.083 −0.144(P = 0.8) (P = 0.4) (P = 0.9) (P = 0.08) (P = 0.5) (P = 0.5) (P = 0.3)

LDL-cholesterol(mmol/L)

−0.069 0.061 0.078 −0.169 0.020 0.646 0.313(P = 0.6) (P = 0.6) (P = 0.5) (P = 0.2) (P = 0.8) (P < 0.001) (P = 0.02)

Leukocytes (K/�L) −0.005 0.266 0.211 −0.065 0.121 0.256 0.027(P = 0.9) (P = 0.05) (P = 0.1) (P = 0.6) (P = 0.3) (P = 0.08) (P = 0.8)

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he values displayed are Pearson partial correlation coefficients and P-values (in paa These variables were log-transformed prior to statistical comparisons.

istribution of PAF its median values and quartiles (25th–75th) arehown in Table 2. It is noticed that men had lower total- and bound-AF levels, higher PAF-AH and Lp-PLA2 in parallel with higheryso-PAF-AT activities (P < 0.05) as compared with women (Table 2).

.2. Correlations of PAF levels and its key metabolic enzymesctivities with age and anthropometric variables

In the overall sample, age was positively correlated to Lp-LA2 activity (r = 0.201, P = 0.04). Moreover, age was negativelyorrelated to free-PAF levels in men (r = −0.279, P = 0.06) and toyso-PAF-AT in women (r = −0.280, P = 0.05). As far as anthropomet-ic variables are concerned, waist circumference was negativelyorrelated to PAF (r = −0.217, P = 0.03) and positively correlatedo Lp-PLA2 and PAF-AH (r = 0.286, P = 0.004 and r = 0.236, P = 0.01,orrespondingly). Moreover, waist-to-hip ratio was negatively cor-elated to PAF (r = −0.195, P = 0.05) and positively correlated top-PLA2 (r = 0.312, P = 0.002). No correlation was observed betweenMI and PAF or its enzymes.

.3. Correlations of PAF levels and its key metabolic enzymesctivities with lipid parameters, glucose and blood cells

In the overall sample PAF-CPT was positively correlated tolucose (r = 0.197, P = 0.05). Moreover, Lp-PLA2 was positively cor-elated to triacylglycerols (r = 0.384, P < 0.001), total-cholesterolr = 0.620, P < 0.001) and LDL-cholesterol (r = 0.609, P < 0.001). Theelation of Lp-PLA2 to lipidemic parameters is in line with the facthat lipoproteins constitute the carrier of the circulating enzyme inlasma [12]. In Table 3 the correlations of PAF and its enzymes withlinical parameters are displayed for men and women, separately.pecifically, in men, free- and total-PAF were positively correlated

o leukocyte count. In women, bound-PAF was positively corre-ated to leukocytes count (borderline significance) and PAF-CPT wasositively correlated to glucose concentration. It is also notewor-hy that the correlations of Lp-PLA2 with lipid parameters were

eses).

evident in both men and women. All the above reported correla-tions have been adjusted for age, gender (where applicable) andBMI. Moreover, age-adjusted correlations of free-PAF with glucose(r = −0.359, P = 0.007) and triacylglycerols (r = −0.279, P = 0.04) inwomen, were evident. However, the aforementioned correlationsbecame insignificant after further adjustment for BMI of the partic-ipants. No correlation was observed between counts of leukocytessubpopulations with neither PAF nor its enzymes (data not shown).

3.4. Inter-correlations of PAF levels and its key metabolicenzymes activities

In the overall sample free- and total-PAF were positivelycorrelated to PAF-CPT (r = 0.198, P = 0.05 and 0.213, P = 0.03, respec-tively). Moreover, PAF-CPT was positively correlated to bothlyso-PAT-AT (r = 0.295, P = 0.003) and PAF-AH (r = 0.202, P = 0.04). InTable 4 the inter-correlations of PAF and its enzymes are presentedfor men and women, separately. As it is shown, in men PAF-CPTwas positively correlated to free-PAF, total-PAF (borderline sig-nificance), lyso-PAF-AT and negatively correlated to Lp-PLA2. Inwomen, a borderline positive correlation was observed betweenthe two PAF catabolic isoenzymes, namely PAF-AH and Lp-PLA2.All the above correlations were adjusted for age and BMI.

3.5. Multivariate analysis

Taking the strong interrelations of PAF enzymes, the major PAFmetabolic patterns were identified using PCA (Table 5). Specifically,three PAF metabolic patterns were recognized in the studied sam-ple: (i) the first pattern was consisted of PAF-CPT and PAF-AH, (ii)the second pattern was consisted of PAF biosynthetic enzymes (i.e.,PAF-CPT and lyso-PAF-AT) and (iii) the third pattern represented

Lp-PLA2, all of which explained 85% of the total variation. We thentested the correlations between the above patterns and PAF levelsin the blood in order to identify which metabolic pattern is morelikely to affect its levels. As it can be seen in Table 6, the increased

P. Detopoulou et al. / Prostaglandins & other Lipid Mediators 97 (2012) 43– 49 47

Table 4Pearson partial correlation coefficients between PAF and its enzymes after adjustments for age and BMI.

Free-PAFa bound-PAFa total-PAFa lyso-PAF-AT PAF-CPT Lp-PLA2

(pmol/mL) (pmol/mL) (pmol/mL) (nmol/min/mg) (pmol/min/mg) (nmol/min/mL)

Men (n = 48)lyso-PAF-AT(nmol/min/mg)

0.075 −0.012 0.077 –(P = 0.6) (P = 0.9) (P = 0.6)

PAF-CPT(pmol/min/mg)

0.392 0.116 0.303 0.378 –(P = 0.01) (P = 0.4) (P = 0.05) (P = 0.01)

Lp-PLA2

(nmol/min/mL)−0.234 −0.105 −0.136 −0.207 −0.308 –(P = 0.1) (P = 0.4) (P = 0.3) (P = 0.1) (P = 0.04)

PAF-AH(pmol/min/mg)

0.216 0.179 0.231 −0.040 0.246 −0.201(P = 0.1) (P = 0.2) (P = 0.1) (P = 0.7) (P = 0.1) (P = 0.1)

Women (n = 58)lyso-PAF-AT(nmol/min/mg)

0.233 −0.034 −0.004 –(P = 0.08) (P = 0.8) (P = 0.9)

PAF-CPT(pmol/min/mg)

0.113 0.106 0.231 0.139 –(P = 0.4) (P = 0.4) (P = 0.09) (P = 0.3)

Lp-PLA2

(nmol/min/mL)−0.061 −0.065 −0.047 −0.122 −0.011 –(P = 0.6) (P = 0.6) (P = 0.7) (P = 0.4) (P = 0.9)

PAF-AH(pmol/min/mg)

0.049 0.141 0.184 0.100 0.148 0.267(P = 0.7) (P = 0.3) (P = 0.1) (P = 0.4) (P = 0.2) (P = 0.06)

The values displayed are Pearson partial correlation coefficients and P-values (in parentha These variables were log-transformed prior to statistical comparisons.

Table 5Component loadings derived using Principal Component Analysis for the identifica-tion of PAF metabolic patterns.

Components

1 2 3

Lp-PLA2 (nmol/min/mL) 0.112 0.002 0.947PAF-AH (pmol/min/mg) 0.880 -0.056 0.261lyso-PAF-AT(nmol/min/mg) 0.021 0.960 0.029PAF-CPT (pmol/min/mg) 0.628 0.481 −0.343% variance explained 36% 30% 19%

Component 1: increased PAF-AH and PAF-CPT activities. Component 2: increasedPAF-CPT and lyso-PAF-AT activities. Component 3: increased Lp-PLA2 activity.

Table 6Partial correlation coefficients between PAF metabolic patterns identified throughPrincipal Component Analysis and PAF levels, after adjustment for age, gender andBMI.

Dependent outcome Components

1 2 3

Overall sample (n = 106)Free-PAFa 0.144 0.146 −0.128

(P = 0.1) (P = 0.1) (P = 0.2)Bound-PAFa 0.130 −0.057 −0.083

(P = 0.2) (P = 0.5) (P = 0.4)Total-PAFa 0.213 0.019 −0.119

(P = 0.04) (P = 0.8) (P = 0.2)

Men (n = 48)Free-PAFa 0.353 0.106 −0.322

(P = 0.02) (P = 0.5) (P = 0.04)Bound-PAFa 0.198 −0.022 −0.097

(P = 0.2) (P = 0.8) (P = 0.5)Total-PAFa 0.312 0.092 −0.180

(P = 0.05) (P = 0.5) (P = 0.2)

Women (n = 58)Free-PAFa −0.001 0.256 −0.078

(P = 0.9) (P = 0.07) (P = 0.6)Bound-PAFa 0.118 −0.060 −0.107

(P = 0.4) (P = 0.6) (P = 0.4)Total-PAFa 0.185 −0.001 −0.153

(P = 0.2) (P = 0.9) (P = 0.3)

The values displayed are Pearson partial correlation coefficients and P-values (inparentheses). Component 1: increased PAF-AH and PAF-CPT activities. Component2: increased PAF-CPT and lyso-PAF-AT activities. Component 3: increased Lp-PLA2

activity.a These variables were log-transformed prior to statistical comparisons.

eses).

PAF-CPT and PAF-AH pattern was positively related to PAF levels(in the overall sample and in men). Moreover, the pattern that wascharacterized by Lp-PLA2 was inversely associated with free-PAFlevels, only in men. All the above associations were adjusted forage, gender (where applicable) and BMI.

4. Discussion

Although PAF is considered as a key player in the field ofinflammation and related diseases, such as cardiovascular dis-ease [14], its measurement in human studies is restricted tosmall numbers of participants and pathological conditions untiltoday [20–24]. In the present study a relatively high number ofapparently healthy volunteers were recruited and the relation ofPAF metabolism to anthropometric and clinical parameters wasaddressed, which attaches an additive interpretative value to ourresults. The determination of PAF levels in biological fluids isa time- and effort-consuming procedure. Additional difficultiesarise from the fact that the concentration of PAF in the blood isvery low and its half-life is short. In the present study medianvalues of PAF levels were reported. However, for comparabilityreasons, it should be mentioned that the mean value of total-PAFlevels measured with a biological assay of high sensitivity [16]were found 490 ± 850 fmol/mL (256 ± 445 pg/mL), which are lower[20,21,23–26] or higher [27,28] than other studies (control groupof healthy volunteers) using similar or different methodologicalapproaches.

As far as PAF metabolic enzymes are concerned, no dataexist with the exception of Lp-PLA2. The activity of Lp-PLA2 islower than that reported for Greek subjects in the ATTICA Study(22 ± 5 nmol/min/mL versus 39 ± 10 nmol/min/mL) [29] and otherstudies [13], a difference which may be explained by the factthat participants in the aforementioned studies had an unfavor-able cardiovascular risk profile as they were under hypoglycemic,hypolipidemic or anti-hypertensive treatment. Unfortunately,comparisons of leukocyte lyso-PAF-AT, PAF-CPT and PAF-AH activi-ties with other reports are difficult, since most studies are restrictedto cell cultures and different methodological procedures have been

followed [30,31].

The gender-dependent nature of the observed parametersdeserves special attention. Indeed, men had lower levels of bound-PAF and higher activities of lyso-PAF-AT, Lp-PLA2 and PAF-AH.

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p-PLA2 activity has been previously reported to be higher in menn various studies [13,29]. In this context, higher activities of PAFatabolic enzymes in men may lead to lower levels of PAF in thelood. Higher lyso-PAF-AT activity in men may be related to the

ncreased cardiovascular risk observed in them [32], which in turns associated with increased inflammatory molecules and leukocytectivation [33].

In the total sample age was positively correlated to Lp-PLA2ctivity, an observation, which is accordant with other studies13,34]. Moreover, age was negatively associated with free-PAFevels in men and lyso-PAF-AT activity in women. With respecto PAF there is only one study indicating a positive relation ofAF with age [35]. This discrepancy may be explained by the dif-erent ethnic background of the populations studied and/or theifferent methods used for PAF determination. In the present work,owever, the increases of Lp-PLA2 activity across age groups mayccount for the decreased PAF levels in the blood. Besides, inen we were able to document a negative relation of PAF with

pattern of increased Lp-PLA2 activity. A cursory glance at thenverse association of age with lyso-PAF-AT activity seems to indi-ate a strange regulatory pattern of lyso-PAF-AT activity in vivo.ndeed, the available experimental data for lyso-PAF-AT activityeriving mainly from cell cultures support that the enzyme isctivated in an inflammatory milieu [8]. Following this rational,t would be expected the activity of lyso-PAF-AT increases withgeing. Another point that should be raised is that two recentlydentified isoforms of lyso-PC acyltransferase, namely LPCAT1 andPCAT2 possess lyso-PAF acetylating activity and are thought toe responsible for PAF synthesis under basal conditions and in

nflammatory states [8,9]. It is also reported that LPCAT2 is acti-ated by its phosphorylation [36]. Under our lyso-PAF AT assayonditions no protein phosphatase inhibitor, such as vanadate,as included in the reaction buffer, therefore a possible involve-ent of dephosphorylation in the measured lyso-PAF acetylating

ctivity could not be excluded. Based on the above data, theelation of age with PAF and lyso-PAF-AT deserves further clari-cation.

The clinical relevance of PAF and its metabolic enzymes can beurthermore illustrated through their associations with other bio-hemical parameters. Apart from the strong correlations observedetween Lp-PLA2 and LDL-cholesterol, biologically attributed tohe transport of Lp-PLA2 mainly with LDL particles, a positiveorrelation was observed between free- and total-PAF levels andeukocytes count in men. The positive relation of PAF with leuko-ytes should be considered in the context of activation of leukocytesn inflammatory conditions.

It is also noteworthy that the activity of PAF-CPT was posi-ively related to glucose concentration in the total population andomen after adjustment for age and BMI. Moreover, an inverse age-

ndependent relation was identified between free-PAF and bothlucose and triacylglycerols levels in women, which was lost afterMI adjustment. The confounding effect of BMI to the above cor-elations may be due to its positive relation with glycemia andipidemia. Limited data exist on the relation of PAF metabolismnd glucose levels. More particularly, hyperinsulinemia (which, ateast in non diabetic states, goes hand in hand with hyperglycemia)nhibits PAF biosynthesis in vitro [37], while PAF has been found tonduce glucogenolysis in animals (thus increasing glucose levels)38] and is increased in Type 1 diabetes [23]. The relation of tria-ylglycerols to PAF has not been extensively studied. At the cellularevel (human fibroplasts) PAF treatment was found to enhance tri-cylglycerol synthesis [39], whereas intravenous injection of PAF

eads to increases of lipoprotein lipase and hydrolysis of circulat-ng triacylglycerols [40]. It is obvious that the relation of PAF andAF-CPT to both triacylglycerols and glucose levels needs furtherlarification in humans.

er Lipid Mediators 97 (2012) 43– 49

It is worthy commenting on the three identified PAF metabolicpatterns, i.e., (i) the one characterized by highly correlated PAF-AH and PAF-CPT, (ii) the one characterized by highly correlatedPAF-CPT and lyso-PAF-AT and (iii) the one characterized only byLp-PLA2. As far as the positive relation of PAF-CPT and PAF-AH isconcerned, it can be postulated from a biochemical perspective thatPAF-CPT synthesizes PAF, which in turn induces the productionof its catabolic enzyme (PAF-AH) as a compensatory mechanism.More particularly, it has been shown that PAF can transcription-ally regulate PAF-AH synthesis in monocytes through binding to itsreceptor [41], which may in part also explain the positive relationof this pattern to PAF levels per se.

The positive correlation of leukocyte PAF-CPT and lyso-PAF-AT has been previously underlined by our group in heart failurepatients [14]. It is thus further suggested that the biosyntheticroutes of PAF are not as independent as once believed, at least inleukocytes, and that both enzymes may respond to common stim-uli [42]. For example, hypoxia influences both PAF’s biosyntheticenzymes [42]. Phorbol esters, activators of protein kinase C, are ableto induce PAF-CPT [43], while inflammatory and oxidative markers(such as TNF-�, H2O2), also being able to act through protein kinaseC, increase lyso-PAF-AT synthesis [44,45].

With respect to the pattern of increased Lp-PLA2, it is remark-able that the particular enzyme constitutes on its own a singularPAF metabolic pattern, which was also reversely correlated withfree-PAF levels in men. Although a negative association wasobserved between PAF-CPT and Lp-PLA2, it was not strong enoughto form a distinct pattern. Instead, the increased Lp-PLA2, as one ofthe predominant patterns may represent a relative independenceof the enzyme or its dependence on different parameters. Indeed,Lp-PLA2 is secreted by blood cells and various tissues and serves asa “sensor” of atherosclerotic lesions or other peripheral signals. Forinstance, we have previously shown that the activity of Lp-PLA2 isstrongly determined by abdominal fat in males [46].

5. Conclusions

In conclusion, this is the first study to assess a panel of PAFmetabolic enzymes (leukocyte PAF-CPT, lyso-PAF-AT, PAF-AH andserum Lp-PLA2) in healthy volunteers and to substantiate therelation of PAF with its enzymes, anthropometric and clinicalparameters in the investigated sample. Despite the cross-sectionalnature of the present study, which can only prepare the ground fornew hypothesis generation and not provide causal explanations,gender, age, serum lipids, glucose and leukocytes count seem todetermine PAF levels and some of its enzymes’ activities. The natureof the observed relationships as well as the possible mechanismsthat can direct or influence them remain to be examined.

Conflict of interest

The authors declare that they have no competing interests.

Authors’ contributions

Paraskevi Detopoulou wrote the manuscript, performed bio-chemical analysis and the interpretation of the results; TzortzisNomikos performed biochemical analysis, interpreted the resultsand critically reviewed the manuscript; Elizabeth Fragopoulou per-formed biochemical analysis, interpreted the results and criticallyreviewed the manuscript; George Stamatakis performed bio-

chemical analysis; Demosthenes Panagiotakos critically reviewedthe manuscript and advised on statistical analysis; SmaragdiAntonopoulou designed the study, interpreted the results and crit-ically reviewed the manuscript.

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ole of funding source

The funding source was not involved in the study design nor theollection, analysis, and interpretation of data; in the writing of thiseport; and in the decision to submit the paper for publication.

cknowledgements

The authors would like to thank Dr. M. Xanthopoulou and Mrs.. Letsiou for their assistance in blood handling, Mrs. O. Kounari forer support in data collection, Mrs. M. Christea for her support inlood collection.

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