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Haptoglobin inhibits phospholipid transfer protein activity in hyperlipidemic human plasma Henderson, Ryan J; Wasan, Kishor M; Leon, Carlos G Jul 23, 2009

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ralssBioMed CentLipids in Health and DiseaseOpen AcceResearchHaptoglobin inhibits phospholipid transfer protein activity in hyperlipidemic human plasmaRyan J Henderson, Kishor M Wasan and Carlos G Leon*Address: Division of Pharmaceutics and Biopharmaceutics, Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, V6T 1Z3, CanadaEmail: Ryan J Henderson - hendersonrx@gmail.com; Kishor M Wasan - kwasan@interchange.ubc.ca; Carlos G Leon* - cleon@interchange.ubc.ca* Corresponding author    AbstractBackground: Haptoglobin is a plasma protein that scavenges haemoglobin during haemolysis.Phospholipid Transfer Protein (PLTP) transfers lipids from Low Density Lipoproteins (LDL) to HighDensity Lipoproteins (HDL). PLTP is involved in the pathogenesis of atherosclerosis which causescoronary artery disease, the leading cause of death in North America. It has been shown thatApolipoprotein-A1 (Apo-A1) binds and regulates PLTP activity. Haptoglobin can also bind to Apo-A1, affecting the ability of Apo-A1 to induce enzymatic activities. Thus we hypothesize thathaptoglobin inhibits PLTP activity. This work tested the effect of Haptoglobin and Apo-A1 additionon PLTP activity in human plasma samples. The results will contribute to our understanding of therole of haptoglobin on modulating reverse cholesterol transport.Results: We analyzed the PLTP activity and Apo-A1 and Haptoglobin content in six hyperlipidemicand six normolipidemic plasmas. We found that Apo-A1 levels are proportional to PLTP activity inhyperlipidemic (R2 = 0.66, p < 0.05) but not in normolipidemic human plasma. Haptoglobin levelsand PLTP activity are inversely proportional in hyperlipidemic plasmas (R2 = 0.57, p > 0.05). Whenthe PLTP activity was graphed versus the Hp/Apo-A1 ratio in hyperlipidemic plasma there was asignificant correlation (R2 = 0.69, p < 0.05) suggesting that PLTP activity is affected by the combinedeffect of Apo-A1 and haptoglobin. When haptoglobin was added to individual hyperlipidemicplasma samples there was a dose dependent decrease in PLTP activity. In these samples we alsofound a negative correlation (-0.59, p < 0.05) between PLTP activity and Hp/Apo-A1. When weadded an amount of haptoglobin equivalent to 100% of the basal levels, we found a 64 ± 23%decrease (p < 0.05) in PLTP activity compared to basal PLTP activity. We tested the hypothesis thatadditional Apo-A1 would induce PLTP activity. Interestingly we found a dose dependent decreasein PLTP activity upon Apo-A1 addition. When both Apo-A1 and Hpt were added to the plasmasamples there was no further reduction in PLTP activity suggesting that they act through a commonpathway.Conclusion: These findings suggest an inhibitory effect of Haptoglobin over PLTP activity inhyperlipidemic plasma that may contribute to the regulation of reverse cholesterol transport.Published: 23 July 2009Lipids in Health and Disease 2009, 8:27 doi:10.1186/1476-511X-8-27Received: 22 June 2009Accepted: 23 July 2009This article is available from: http://www.lipidworld.com/content/8/1/27© 2009 Henderson et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 8(page number not for citation purposes)Lipids in Health and Disease 2009, 8:27 http://www.lipidworld.com/content/8/1/27BackgroundHaptoglobin is an acute phase protein that scavenges hae-moglobin released into the circulation [1]. Haptoglobin,the plasma protein with highest binding affinity to hae-moglobin, is mainly expressed in the liver [2]. It plays ananti-oxidant role by binding free haemoglobin and form-ing a complex that is taken up by hepatocytes and macro-phages [3]. The human haptoglobin gene encompassesthree alleles: Hp1F, Hp1S and Hp2 [4]. The Hp2 allele isthe fusion product of the Hp1F and Hp1S alleles. Hap-toglobin presents as a dimer of two of these alleles whichbinds to one haemoglobin dimer [2]. Haptoglobinexpression is induced several fold in the event of inflam-mation triggered by infection, injury or cancer develop-ment [1,5]. Haptoglobin has been shown to play anantioxidant/anti-inflammatory role, to contribute to neu-trophil activation [6], to maintain reverse cholesteroltransport [7] and to modulate the inhibition of cyclooxy-genase and lipooxygenase [8], amongst other functions.In particular, haptoglobin has been shown to inhibit Lec-ithin-Cholesterol Acyltransferase (LCAT) in human ovar-ian follicular fluid [9]. LCAT is involved in the removal ofcholesterol excess from peripheral tissues [10]. LCATtransfers an acyl chain from high density lipoprotein(HDL) lecithin to cellular cholesterol. This activity is stim-ulated by the presence of Apo-A1, the main protein con-stituent of HDL. Balestrieri et al [9] demonstrated thatLCAT activity is negatively correlated with the Hp/Apo-A1ratio in human follicular fluid. The mechanism of actionof haptoglobin inhibition of LCAT activity has beendescribed [11]. The binding site of Haptoglobin on Apo-A1 has been mapped and it was demonstrated that theinteraction of haptoglobin to Apo-A1 is independent tothe binding of haptoglobin and haemoglobin. A peptidedesigned based on the sequence in Apo-A1 that putativelyinteracts with Haptoglobin was shown to restore LCATactivity inhibited by Hp demonstrating that the Apo-A1-Hp interaction is responsible for the inhibition of LCATactivity. Based on this evidence it has been speculated thathaptoglobin may play a role in the inhibition of reversecholesterol transport.In the present study we investigated the effect of hap-toglobin on the activity of another enzyme involved inreverse cholesterol transport, phospholipid transfer pro-tein (PLTP). PLTP is a plasma protein that transfers phos-pholipids from triglyceride-rich lipoproteins such as verylow-density lipoproteins (VLDL) and low-density lipo-proteins (LDL) to high density lipoproteins (HDL)[12,13]. PLTP occurs in plasma as two main forms: a highactivity PLTP (HA-PLTP) and a low activity PLTP (LA-PLTP). HA-PLTP is associated with the majority of plasmaPLTP activity. PLTP activity has been shown to be affecteddevelopment [16]. Moerland et al., [17] showed in atransgenic mouse model of PLTP expression that anacutely increased PLTP expression resulted in a highlyatherogenic lipoprotein profile. Shelly et al., [18] foundthat the phospholipid transfer protein deficiency amelio-rated diet-induced hypercholesterolemia and inflamma-tion in mice. There is evidence that even a 10% reductionon PLTP activity can lead to a significant reduction ofatherosclerosis progression [19], highlighting the role ofPLTP on the development of cardiovascular disease.In the present study we hypothesize that haptoglobininhibits PLTP activity. This is based on the fact that PLTPactivity is dependent on its binding to Apo-A1 [15] andthat haptoglobin has been shown to bind Apo-A1 [11]and to inhibit LCAT activity [9]. This work will furthercontribute to our understanding of the role of hap-toglobin on modulating reverse cholesterol transport aswell as the development of atherosclerosis.ResultsHaptoglobin and Apolipoprotein A1 levels in normolipidemic and hyperlipidemic human plasmaHaptoglobin and Apo-A1 levels were determined for eachone of the plasma samples (Table 1). When the Hp levelswere compared between the two groups (hyperlipidemicvs. normolipidemic) no difference was found. Likewise,when the levels of Apo-A1 were compared amongst thetwo groups they were not different.Inverse association between PLTP activity and haptoglobin levels in hyperlipidemic plasmaWe determined the PLTP activity as described elsewhere[20]. When we graphed the PLTP activity vs. the hap-toglobin levels we found a trend of a correlation (FigureTable 1: Apolipoprotein A1 (μg/mL) and Haptoglobin (μg/mL) levels in hyperlipidemic (H1-H6) and normolipidemic (N1-N6) plasmas used in this study (mean ± SD). Plasma # Apo-A1 (μg/mL) Haptoglobin (μg/mL)H1 3.9 ± 0.1 5897.7 ± 527H2 23.2 ± 1.9 5240.4 ± 110H3 275.7 ± 13 1245.7 ± 28H4 265 ± 26 4579.9 ± 259H5 185.8 ± 9 318.6 ± 14H6 167.3 ± 7 3891.1 ± 231N1 161.2 ± 4.9 500.5 ± 41N2 186.2 ± 16 6104.2 ± 134N3 201 ± 2.1 1480 ± 99N4 54.8 ± 3 4126.5 ± 124N5 269.9 ± 34 1322.7 ± 48N6 337.7 ± 28 3125.5 ± 48Page 2 of 8(page number not for citation purposes)by its association to Apo-A1 [14,15]. There is increasingevidence supporting the role of PLTP on atherosclerosisApo-A1 and haptoglobin were measured as described in Materials and Methods.Lipids in Health and Disease 2009, 8:27 http://www.lipidworld.com/content/8/1/271a R2 = 0.57, Correlation coefficient -0.75) albeit not sig-nificant (p = 0.08). In the case of the normolipidemicplasma there was no indication of a correlation (Figure1b).Direct association between PLTP activity and Apo-A1 levels in hyperlipidemic plasma. Correlation between the PLTP activity and the Hp/Apo-A1 ratio in hyperlipidemic plasmaWhen we compared the PLTP activity vs. the Apo-A1 levelswe found a positive correlation between these two varia-bles (Figure 2a, R2 = 0.66, Correlation coefficient 0.81, p <0.05) in hyperlipidemic plasma but not in normolipi-demic plasma (Figure 2b).Based on the model of LCAT inhibition by haptoglobin,we determined the relationship between PLTP activity andHt/Apo-A1 ratio (Figure 2c) in hyperlipidemic plasma. Anegative linear correlation was found (R2 = 0.69, p < 0.05)suggesting an inhibitory role of haptoglobin on PLTPactivity in this group of plasmas. When a semi logarithmicnon linear regression was used, we obtained a higher cor-relation (R2 = 0.998) than with the linear model.Inhibition of PLTP activity by the addition of increasing concentrations of haptoglobinTo further examine the inhibition of PLTP activity,increasing amounts of haptoglobin were added to our setof plasma samples (Figure 3a). Five out of six hyperlipi-demic plasma showed a decreased PLTP activity with anincreasing dose of added haptoglobin, irrespective of thebasal PLTP activity. On the other hand, the three normol-ipidemic plasma samples analyzed didn't show any cleartrend which is in agreement with our previous resultswhich do not indicate a role of haptoglobin inhibition inthis group. We further analyzed the data expressing it aspercentage inhibition of PLTP activity and found a signif-icant decrease on PLTP activity in the hyperlipidemicplasma after 5 min of haptoglobin addition (Figure 3b).This effect was reduced after 60 min of haptoglobin addi-tion (Figure 3c). When we analyzed the PLTP activity vs.the Hp/Apo-A1 ratio, we also found a negative correlation(Figure 3d, -0.697, p = 0.0217 and n = 11). Based on theinitial amount of haptoglobin in each plasma sample, weadded this specific amount of haptoglobin to each sam-ple. The effect was a 64% reduction in PLTP activity com-pared to untreated controls (Figure 4, p < 0.05). This factfurther supported an inhibitory role of haptoglobin overPLTP activity in vitro.Inhibition of PLTP activity by the addition of increasing concentrations of haptoglobinWe further explored the possible role of Apo-A1 on thehyperlipidemic plasmas (Figure 5a). We also confirmedthe Hp inhibitory effect on PLTP. When both Apo-A1 andHp were added, no additive effect was observed (Figure5b) suggesting that their inhibitory effect occurs througha common mechanism.DiscussionHaptoglobin genotype has been shown to regulate reversecholesterol transport in diabetes in vitro and in vivo [21]. Ithas been proposed that an enhanced oxidative modifica-tion of serum lipoproteins (LDL and HDL) in individualswith the Hp2 genotype is an important determinant ofaccelerated atherosclerosis in these individuals [22]. Inter-estingly, PLTP has been shown to efflux cholesterol Apo-A1 in murine macrophages [23].Another mechanism by which haptoglobin may regulatereverse cholesterol transport is by inhibiting LCAT [9].Since this inhibition is mediated through the Hp-Apo-A1interaction [11], we propose that other enzymatic activi-ties regulated by Apo-A1 may be affected by haptoglobinlevels. In particular, we are interested in PLTP which is animportant enzyme involved in reverse cholesterol trans-port [13] and its activity has been shown to be dependenton its association with Apo-A1 [15]. The positive correla-tion between PLTP activity and Apo-A1 levels in hyperlip-idemic plasma persisted. However, we didn't observe thiscorrelation in normolipidemic patients. A positive corre-lation was found between PLTP activity and Apo-A1 inplasma from patients with type 1 diabetes [24] which isconsistent with our observations. These authors had pre-viously demonstrated that patients with type 1 diabeteshave a significantly elevated PLTP activity and that thisactivity is correlated with HDL levels [25]. One of the dif-ferences between the two plasma groups that we used wasthe HDL content. We found a negative correlation (p =0.018) between PLTP activity and HDL levels. This corre-lation was specific to HDL as it was not found with totalcholesterol and triglycerides. Colhoun et al., [24] showeddifferences between the correlation of PLTP activity andHDL particle size. PLTP activity negatively correlated withsmall HDL while it positively correlated with large HDL.Soro et al., [26] also showed a negative correlationbetween HDL2 and PLTP activity. Since there is an associ-ation between reduced HDL particle size and hyperlipi-demia [27], it is possible that in our hyperlipidemicpatient group there is a higher small HDL/large HDL ratiothan in normolipidemic controls and this contributes to anegative correlation between PLTP activity and HDL.One of the limitations of our study is the sample size.However the correlations that we observed in the basalstate were maintained even when exogenous Hp and Apo-Page 3 of 8(page number not for citation purposes)haptoglobin inhibition of PLTP activity. Interestingly, wefound that Apo-A1 inhibited the PLTP activity in the sixA1 were added to the system. Nevertheless, Salvatore etal., [28] found a correlation between the cholesteryl ester/Lipids in Health and Disease 2009, 8:27 http://www.lipidworld.com/content/8/1/27cholesterol ratio (a measure of LCAT activity) and Hp/[Apo E +Apo-A1] ratio in a small number of multiple scle-rosis patients (n = 9).When we compared haptoglobin levels with PLTP activity,the correlation was insignificant (p = 0.08). However,when we changed our analysis to compare PLTP activitywith Hp/Apo-A1 ratio, we did find a negative correlationsuggesting a) that haptoglobin may inhibit PLTP activityand b) that Apo-A1 levels may affect this inhibitory inter-action.We further confirmed the Hp inhibitory effect by addingtoglobin addition in a dose-dependent way. This effectwas not seen in normolipidemic plasma. This effect couldalso be related to the differences in low activity and highactivity PLTP in hyperlipidemic and normolipidemicplasma and the differential association of Apo-A1 to thesetwo forms of PLTP [14]. The fact that PLTP activity is neg-atively correlated with the Hp/Apo-A1 ratio in hyperlipi-demic plasma has not been previously reported.ConclusionPLTP activity was inhibited by Haptoglobin and Apo-A1addition. Haptoglobin, HDL and PLTP activity correlationdata suggests the potential to use haptoglobin as abiomarker for the development of atherosclerosis as wellas a tool to understand the role of PLTP activity and hap-toglobin levels in reverse cholesterol and atherosclerosis.Materials and methodsChemicalsPLTP Activity Assay Kit's were obtained from Roar Bio-medical (New York, NY, USA). Purified Hpt (at least 95%pure by SDS-PAGE) was purchased from Calbiochem(San Diego, CA). Apolipoprotein A1 was purchased fromSigma-Aldrich (St Louis, MI).Plasma SamplesTwelve different human plasma samples (purchased fromBioreclamation [East Meadow, NY, USA]) were obtainedfrom donors representing both normolipidemic plasma(N = 6) and hyperlipidemic plasma (N = 6) based on thestandards set by the Ministry of Health and Welfare ofJapan (cholesterol <220 mg/dl and triglycerides <150 mg/dl) [29]. The cholesterol and triglyceride content of eachone of these twelve samples has been reported previously[20].Haptoglobin determinationHaptoglobin levels were determined using a two siteHuman Haptoglobin ELISA kit from ICL (Newberg, OR)as per the manufacturer instructions. Briefly, the control,standard and patient samples were added to the wellswhich had previously adsorbed the anti-Hp antibodies.The unbound proteins were removed by washing, andthen anti-Hp antibodies conjugated to horseradish perox-idase were added. These enzyme-labeled antibodies formcomplexes with the previously bound plasma Hp. Follow-ing washings, a chromogenic substrate was added andabsorbance was read at 450 nm. The concentration of Hpwas determined using a standard curve of purified Hp.Apo-A1 determinationThe human Apo-A1 EIA kit was purchased from CaymanChemical Company (Ann Arbor, MI). Briefly each well ofa. Association between PLTP activity and haptoglobin levels in hyperlipidemic plasma (R2 = 0.5733,  = 6)Figur  1a. Association between PLTP activity and hap-toglobin levels in hyperlipidemic plasma (R2 = 0.5733, n = 6). PLTP activity after 60 min was determined as described in Materials and Methods. Haptoglobin levels were determined using a two site Human Haptoglobin ELISA kit. Each plasma sample was analyzed in two independent experi-ments, with at least two replicates per experiment. b. Asso-ciation between PLTP activity and haptoglobin levels in normolipidemic plasma (R2 = 0.0007, n = 6). PLTP activity after 60 min was determined as described in Materials and Methods. Haptoglobin levels were determined using a two site Human Haptoglobin ELISA kit. Each plasma sample was analyzed in two independent experiments, with at least two replicates per experiment.a PLTP activity and Hp levels in hyperlipidemic plasma0102030405060700 1000 2000 3000 4000 5000 6000 7000Haptoglobin (ug/mL)PLTP activity (pmoles)PLTP activity and Hp levels in normolipidemic plasma0102030405060700 1000 2000 3000 4000 5000 6000 7000Haptoglobin (ug/mL)PLTP activity (pmol)baPage 4 of 8(page number not for citation purposes)exogenous haptoglobin to the plasma samples. In hyperl-ipidemic plasma, PLTP activity was inhibited by hap-the plate provided with the kit was coated with an Apo-A1specific antibody. When the samples and controls wereLipids in Health and Disease 2009, 8:27 http://www.lipidworld.com/content/8/1/27Page 5 of 8(page number not for citation purposes)a. Association between Apolipoprotein A1 levels and PLTP activity (60 min) in hyperlipidemic plasmaFigure 2a. Association between Apolipoprotein A1 levels and PLTP activity (60 min) in hyperlipidemic plasma. Apolipo-protein levels were determined using an EIA kit as outlined in Materials and Methods. Each plasma sample was analyzed in two independent experiments, with at least two replicates per experiment. The graph depicts the mean ± standard deviation (R2 = 0.6635, n = 6, p < 0.05). b. Association between Apolipoprotein A1 levels and PLTP activity (60 min) in normolipidemic plasma (R2 = 0.2732, n = 6, p > 0.05). c. Association between PLTP activity (60 min) and Hp/Apo-A1 ratio in hyperlipidemic plasma (R2 = 0.69, n = 6, p < 0.05).a PLTP activity and Apo-A1 levels in hyperlipidemic plasma0102030405060700 50 100 150 200 250 300 350Apolipoprotein A1 (ug/mL)PLTP activity (pmoles)PLTP activity and Apo-A1 levels in normolipidemic plasma0102030405060700 50 100 150 200 250 300 350 400Apolipoprotein A1 (ug/mL)PLTP activity (pmoles)bc PLTP vs Hp/Apo-A10102030405060700 200 400 600 800 1000 1200 1400 1600Hp/Apo-A1PLTP Activity (pmol)acLipids in Health and Disease 2009, 8:27 http://www.lipidworld.com/content/8/1/27Page 6 of 8(page number not for citation purposes)a. Effect of haptoglobin addition (2, 10 and 20 μg per sample) on PLTP activity (5 min) of individual hyperlipidemic (H, n = 6) nd normolipidemic (N, n = 2) plasma samplesFigure 3a. Effect of haptoglobin addition (2, 10 and 20 μg per sample) on PLTP activity (5 min) of individual hyperlipi-demic (H, n = 6) and normolipidemic (N, n = 2) plasma samples. From left to right: control, addition of 2 μg, 10 μg and 20 μg of Hp, respectively. One of two representative experiments with duplicate measurements per treatment. b. Effect of haptoglobin addition (2, 10 and 20 μg per sample) on PLTP activity (5 min) in hyperlipidemic plasma as a percentage of basal PLTP activity. Each plasma sample was analyzed in two independent experiments, with at least two replicates per experiment. The graph depicts the mean ± standard deviation of each group of six hyperlipidemic plasma (n = 6, p < 0.05). c. Ibidem, except that PLTP activity was analyzed after 60 min. d. Correlation between PLTP activity and Hp/Apo-A1 ratio in hyperlipidemic plasma samples with added haptoglobin (n = 11, Correlation coefficient -0.679, p < 0.05).Lipids in Health and Disease 2009, 8:27 http://www.lipidworld.com/content/8/1/27added to the wells any Apo-A1 would bind to these anti-bodies. After washings a new anti Apo-A1 antibody wasadded to detect the captured Apo-A1. Washings were fol-lowed by the addition of horseradish peroxidase conju-gate that will recognize the complex. Upon washings andaddition of a chromogenic substrate, the reaction wasstopped with acid and absorbance was read at 450 nm.The intensity of the color is proportional to the concentra-tion of Apo-A1 which was determined using a standardcurve.PLTP AssayEach plasma sample was tested for PLTP activity using anactivity assay that measures in vitro phospholipid transferactivity (Roar Biomedical, New York, NY). The PLTP assaywas carried as per manufacturer instructions as describedpreviously [20]. Initially, the PLTP activity test was studiedfor different concentrations of plasma protein and it wasdecided to be 25 μg as the PLTP activity was linear in thisrange. PLTP activity was determined for plasma samples(25 μg). Controls were added in the form of a picomolstandard to quantify PLTP activity and a blank control.Plates also measured plasma activity as a single entity inthe PLTP kit. Measurements were taken for 6 differentplasma samples in the hyperlipidemic range and 6 differ-ent plasma samples in the normolipidemic range. Thesesamples were done in duplicate on each 96-well test plateand each test plate was repeated at least twice for eachplasma sample.Statistical AnalysisThe groups tested in this study were compared againsteach other by applying a repeated measure analysis of var-iance (ANOVA) test and blocking results in set plasmas toconsisted of an N = 6 of which each experiment had atleast a replicate value of 2.The strength and direction of a linear relationshipbetween two random variables was measured by the Pear-son's coefficient of correlation as determined using Sigm-aStat™.AbbreviationsHp: Haptoglobin; PLTP: Phospholipid transfer protein;Apo-A1: Apolipoprotein A1; HDL: High density lipopro-tein; LDL: Low density Lipoprotein.a. Effect of Apo-A1 addition to the rate of PLTP activity within he first 60 minutes of reaction in hyperlipidemicplasmaFigure 5a. Effect of Apo-A1 addition to the rate of PLTP activity within the first 60 minutes of reaction in hyperlipidemic plasma. Two and four micrograms of Apo-A1 were added to the plasma samples and PLTP activity was measured within the first two and 60 minutes to calcu-late the rate of activity (nmoles product/min). One of two representative experiments. b. Effect of Apo-A1 and Hap-toglobin addition to the rate of PLTP activity within the first 60 minutes of reaction in hyperlipidemic plasma. Two micro-grams of Apo-A1 and/or the equivalent of 100% of basal hap-toglobin were added to the plasma (Pl: plasma alone, Pl+Apo-A1: plasma plus Apo-A1, Pl+Hp: plasma plus haptoglobin and Pl+Apo-A1+Hp: plasma plus Apo-A1 plus Haptoglobin) and PLTP activity was measured within the first two and 60 min-utes to calculate the rate of activity (nmoles product/min). One of three representative experiments.Effect of Haptoglobin addition on PLTP activity (5 min) in hyperlipidemic plasmaFigu e 4Effect of Haptoglobin addition on PLTP activity (5 min) in hyperlipidemic plasma. The equivalent of 100% of basal haptoglobin levels was added to each plasma sample and PLTP activity was measured as described previously (n = 6, p < 0.05).Page 7 of 8(page number not for citation purposes)account for base PLTP activity variance. Statistical differ-ences in the data was considered significant if the p valuefound was < 0.05. Data added for each plasma measureCompeting interestsThe authors declare that they have no competing interests.Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central Lipids in Health and Disease 2009, 8:27 http://www.lipidworld.com/content/8/1/27Authors' contributionsRJH conceived the study and carried out the experimentsand revised the manuscript. KMW participated in thedesign of the study, data analysis and helped to draft themanuscript. CGL participated in the design and coordina-tion of the study, and drafted the manuscript. 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