research article correlation between mitochondrial...

Research ArticleCorrelation between Mitochondrial Reactive Oxygen andSeverity of Atherosclerosis

Gabriel G Dorighello1 Bruno A Paim2 Samara F Kiihl3 Mocircnica S Ferreira2

Rodrigo R Catharino2 Anibal E Vercesi2 and Helena C F Oliveira1

1Department of Structural and Functional Biology Biology Institute State University of Campinas 13083-862 Campinas SP Brazil2Department of Clinical Pathology Faculty of Medical Sciences State University of Campinas 13083-887 Campinas SP Brazil3Department of Statistics Institute of Mathematics Statistic and Scientific Computation State University of Campinas13083-859 Campinas SP Brazil

Correspondence should be addressed to Helena C F Oliveira ho98unicampbr

Received 24 April 2015 Revised 7 June 2015 Accepted 22 June 2015

Academic Editor Celia Quijano

Copyright copy 2016 Gabriel G Dorighello et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Atherosclerosis has been associated with mitochondria dysfunction and damage Our group demonstrated previously thathypercholesterolemic mice present increased mitochondrial reactive oxygen (mtROS) generation in several tissues and lowNADPHNADP+ ratio Here we investigated whether spontaneous atherosclerosis in thesemice could bemodulated by treatmentsthat replenish or sparemitochondrial NADPH named citrate supplementation cholesterol synthesis inhibition or both treatmentssimultaneously Robust statistical analyses in pooled group data were performed in order to explain the variation of atherosclerosislesion areas as related to the classic atherosclerosis risk factors such as plasma lipids obesity and oxidative stress including livermtROS Using three distinct statistical tools (univariate correlation adjusted correlation and multiple regression) with increasinglevels of stringency we identified a novel significant association and amodel that reliably predicts the extent of atherosclerosis due tovariations inmtROSThus results show that atherosclerosis lesion area is positively and independently correlated with liver mtROSproduction rates Based on these findings we propose that modulation of mitochondrial redox state influences the atherosclerosisextent

1 Introduction

Oxidative stress seems to be a commondenominator unifyinga variety of classic risk factors mode of action that leads toatherosclerosis [1] The cellular sources of reactive oxygenspecies (ROS) are multiple including NAD(P)H oxidasexanthine oxidase lipoxygenase and cyclooxygenase systemsmitochondrial electron transport chain and autoxidationof diverse substances Previous studies found that ROSspecifically derived from mitochondria (mtROS) are rele-vant in the context of atherosclerosis For instance mtROSinduce endothelial dysfunction infiltration and activation ofinflammatory cells and apoptosis of endothelial and vascularsmooth muscle cells [2 3] In addition a role for mitochon-drialDNAdamage and dysfunction in atherogenesis has beenproposed in the last decade [4ndash9]

Our group have previously shown that mitochondriafrom various tissues of the hypercholesterolemic atheroscle-rosis-prone LDL receptor knockout mice (LDLrminusminus) releasemore ROS than wild type controls [10] In addition thismodel presents reduced content of mitochondrial NADPH[10] the main reducing power for the antioxidant sys-tem glutathione and thioredoxine reductaseperoxidase [11]This LDLrminusminus mitochondrial prooxidant state rendered theorganelle more susceptible to membrane permeability tran-sition (MPT) It has been well recognized that mitochondrialdysfunctions such as opening of the permeability transitionpore directly promote inflammation and oxidative stressphenomena involved in several cardiometabolic diseases [89 12] The mitochondrial intrinsic pathway of cell death canbe triggered by cholesterol accumulation in macrophages[13] an early event in atherogenesis In addition oxidative

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 7843685 10 pageshttpdxdoiorg10115520167843685

2 Oxidative Medicine and Cellular Longevity

damage of mtDNA is correlated with human and miceseverity of atherosclerosis [4 9]

In a previous study we verified that besides NADPHdeficiency LDLrminusminus mitochondria contained less Krebs cycleintermediates such as isocitrate and that treating the organellewith isocitrate (in vitro) and the LDLrminusminus mouse model withcitrate (in vivo) resulted in decreased mtROS productionand increased NADPH content [14] Major processes thatconsume cell NADPH and Krebs cycle intermediates arelipid and cholesterol biosynthesis which are elevated in thisanimal model [10] Therefore we hypothesized that eithercitrate supplementation or cholesterol synthesis inhibitioncould increase mitochondria NADPH availability decreasemtROS production and decrease atherosclerosis develop-ment in LDLrminusminus mouse

2 Material and Methods

21 Animals Mating pairs of LDLrminusminus (B6129S7-Ldlrlttm1HerJ homozygous for Ldlrlttm1Her stock number002207) and wild type controls (C57BL6J stock number000664) were purchased from The Jackson Laboratory(Bar Harbor ME) in 2009 and both colonies have beenestablished and maintained in strictly SPF conditions inThe Multidisciplinary Center for Biological Investigationin the Area of Laboratory Animal Science (CEMIB) ofthe State University of Campinas (Unicamp) The CEMIB-Unicamp is part of the International Council for LaboratoryAnimal Science (ICLAS) The colonies are maintainedthrough exclusive sibling mattings in order to maintain thehomogeneous genetic background When mice reach the ageof 4 weeks they are transferred to the Conventional AnimalFacility in the Department of Structural and FunctionalBiology at Unicamp Mice are then maintained in atemperature-controlled room (22 plusmn 1∘C) with 12 h lightdarkcycle with 15 cycles of air changes per hour and withfree access to food (standard laboratory rodent chow dietNuvital CR1 Colombo PR Brazil) and filtered water Fourto five mice are maintained per cage They are then enrolledin the experimental protocol which was approved by theEthical Committee for the Use of Animal (CEUAUnicampprotocol 1101-2) and by the Internal Biosecurity Committee(CIBio-IBUnicamp protocol 200802) One-month-oldmale LDLrminusminus mice were randomly separated into fourgroups control (CON) citrate (CIT) pravastatin (PRA)and citrate + pravastatin (CIT + PRA) The solutions ofcitrate (134mM citric acid + 11mM sodium citrate) andpravastatin (67mgL) equivalent to 10mgKg of body weightwere offered as the only source of drinking water Thesolutions were provided in dark bottles and changed everyother day during 3 months Additional experiments wereperformed in untreated LDLrminusminus and wild type controls forchecking differences in oxidative susceptibility status andoxidative lipid damage in their plasma VLDL particles aswell as for determining anti-inflammatory systemic markersControl of the colony phenotype is done by measuringfasting plasma cholesterol levels (LDLrminusminus gt 200mgdLand WT lt 80mgdL) and genotype by PCR in tail tip DNAsamples according toThe Jackson Laboratory protocol

22 Plasma Biochemical Analysis Blood samples were col-lected from either the retroorbital plexus or the tail tip ofanesthetized and overnight fasted mice Total cholesteroltriglycerides and nonesterified fatty acids were measured infresh plasma using standard commercial kits (Roche-HitachiGermany and Wako Germany) Glucose levels were mea-sured using a hand-held glucometer (Accu-Chek AdvantageRoche Diagnostic Switzerland)The plasma urea creatininealkaline phosphatase (ALP) alanine aminotransferase (ALT)and aspartate transaminase (AST) were determined usingan automated modular analyzer PP (Hitachi) with Rochereagents (RocheDiagnostic Germany)Theplasma cytokinestumoral necrosis factor alpha (TNF120572) interleukin 2 (IL-2)interleukin 4 (IL-4) and Interferon gamma (IFN120574) weremeasured with ELISA commercial kit (eBioscience SanDiego CA) according to the manufacturerrsquos instructionsPlasma oxidized LDL antibodies were measured in plasmaas described by Zaratin et al [15] Polystyrene 96-well plateswere coatedwith human copper-oxidized LDL and incubatedovernight at 4∘C Plates were then blocked with 1 gelatinin PBS for 48 h at room temperature After washing withPBS plasma samples (1 200) were added and incubated for2 h at room temperature After washing with PBS containing02mLL Tween 20 plates were incubated with 50 120583L ofperoxidase conjugated rabbit anti-mouse IgG antibody atroom temperature for 1 h Finally 75 120583L peroxidase substratesolution was added incubated for 15min and ended by25 120583L of 2M sulfuric acid The optical density (OD) wasthen measured at 450 nm The results are presented as theOD readings (arbitrary units) CuSO

4

-induced plasma thio-barbituric acid reactive substances (TBARS) concentrationswere determined by first exposing plasma to 01mM CuSO4at 37∘C for 5 hours Then 100 120583L of oxidized plasma wasincubated with 200120583L of 07 thiobarbituric acid in 005MNaOH and 60120583L of 50 trichloroacetic acid Samples wereincubated in boiling water for 30 minutes followed bycentrifugation at 664 g for 15 minutes The standard curvewas prepared using several dilutions of 005mM 1133-tetramethoxypropane The optical densities of samples andstandard curve were measured in a microplate reader at532 nm

23 Body Composition and Tissue Lipids Analyses The epi-didymal adipose tissue and liver and spleen fresh masseswere determined gravimetrically Mice carcass compositionwas determined by weighing carcass before and after waterdrying and before and after lipid extraction of the driedcarcass with petroleum ether as previously described bySalerno et al [16] Liver lipids were extracted using the Folchmethod [17]The liver content of cholesterol and triglycerideswas determined using colorimetric-enzymatic assays (Roche-Hitachi Germany) after dissolving the lipid extracts in atriton-containing buffer (01M potassium phosphate pH 74005M NaCl 5mM cholic acid and 01 Triton X-100)

24 Isolation of Mouse Liver Mitochondria Mitochondriawere isolated by conventional differential centrifugation at4∘C as previously described [18] The livers were homoge-nized in 250mM sucrose 1mM EGTA and 10mM Hepes

Oxidative Medicine and Cellular Longevity 3

buffer (pH 72) The mitochondrial suspension was washedtwice in the same medium containing 01mM EGTA andthe final pellet was resuspended in 250mM sucrose to afinal protein concentration of 80ndash100mgmL The proteinconcentration was determined by a modified Biuret assay

25 Reactive Oxygen Species Production (ROS) ROS produc-tion by mitochondria was monitored using the membrane-permeable fluorescent dye 2101584071015840-dichloro-dihydro-fluo-rescein diacetate (H

2

DCF-DA) according to Garcıa-Ruizet al [19] Briefly mitochondria (05mgmL) were addedto HANKS medium pH 72 containing 1 120583M H

2

DCF-DAand a 5mM mixture of NAD-linked substrates (malate +glutamate + 120572-ketoglutarate + pyruvate) in a 1mL cuvettewith constant stirring at 37∘C The fluorescence signal wasrecorded at the excitationemission wavelength pair of488525 nm using a fluorometer (Hitachi model F4500)Calibration was performed with known concentrations ofdichlorofluorescein (DCF) which is the product of H

2

-DCFoxidation

26 NADPH Oxidation Rates The redox state of pyridinenucleotides was measured as previously described [10] inthe mitochondrial suspension (1mgmL) containing 100 120583MEGTA 5mM succinate and 5 120583M rotenone by followingthe fluorescence signal in a Hitachi F-4010 spectrofluorom-eter using excitation and emission wavelengths of 366 and450 nm respectively and a slit width of 5 nm The extent ofpyridine nucleotide oxidation was calculated as a functionof the fluorescence increase induced by isocitrate additionInternal calibration was done by the addition of knownamounts of NADH

27 Histological Analysis of Atherosclerosis In situ perfusedhearts were excised and embedded in Tissue-Tek OCTcompound (Sakura USA) frozen at minus80∘C cut in 10 120583msections along 480 120583m aorta length from the aortic valveleaflets and stained by Oil Red O according to Paigen et al[20] The lipid-stained lesions were quantified as describedby Rubin et al [21] using the ImageJ (145 h) software

28 VLDL Oxidation Susceptibility Plasma VLDL fractionswere obtained from 12 h fasted mice by ultracentrifugationFive hundred 120583L of plasma was added to the bottom of theultracentrifuge tube and carefully overlaid with 300 120583L ofsaline solution (150mM NaCl 1mM EDTA and density of1006) The samples were centrifuged at 140000 rpm (1 times106 g) 16∘C during 50min in a microultracentrifuge Hitachi(model CS150GXL) Two hundred 120583L from the top layercontaining the VLDL fraction was collected The amountof VLDL used in the oxidation assay was normalized bythe triglyceride concentration (100mgmL) CuSO

4

-inducedoxidation (40 120583M 37∘C) was measured by detecting theformation of conjugated dienes at 234 nm over time (Spec-trophotometer Fusion Packard) according to a previousreport [22]

29 Lipid Chemical Markers Identification VLDL sampleswere submitted to a Bligh-Dyer extraction [23] Lipid extracts

were resuspended in 50 120583L of methanol H2

O milliQ (1 1)and 10 120583L of the latter was diluted in 990 120583L of methanol and01 formic acid Data acquisition was performed in an LTQ-XL Orbitrap Discovery Instrument (30000 FWHMThermoScientific Bremen Germany) in the positive ionmode and atthe 119898119911 range of 600ndash1000 for complex lipid identificationMass and intensity values for each spectrum were includedin the Principal Component Analysis (PCA) which wasperformed using Unscrambler v97 CAMOSoftware (Trond-heim Norway) After the discrimination of PCA potentialchemical markers were selected for identification For thisstructural propositions were performed using high resolutionas the main parameter Mass accuracy was calculated andexpressed in terms of ppm shifts according to Machado etal [24] Error values were considered with the assistanceof online database Lipid MAPS (University of CaliforniaSan Diego CA httpwwwlipidmapsorg) for guiding thechoice of potential lipid markers

210 Statistical Data Analyses The results are presented asthe means plusmn SE The comparisons between the groups wereanalyzed by one-way ANOVA with posttest of Tukey Forcorrelation analyses we performed Spearmanrsquos univariatecorrelation test partial (adjusted) correlation and multiplelinear regressions models using respectively GraphPadPrism 50 SPSS Statistics 140 and R package 29 In themultiple linear regressions analyses variable selection in eachmodelwas performedusing the regsubsets tool available in theleaps package for R [25 26] which identifies the exploratoryvariables that create the best-fitting linear regression modelsaccording to Bayesian Information Criterion (BIC)The levelof significance was set at 119875 lt 005

3 Results

In a pilot experiment diet-induced atherosclerosis wasevaluated in LDLrminusminus treated or not with 120mM citratedrinking solution [14] during two weeks The high citrateconcentration used in this study caused a marked increase inplasma lipid levels without changes in mtROS productionand resulted in 80 increase in aortic atherosclerosis lesionarea in the citrate treated LDLrminusminus mice (data not shown)Therefore for the next studies we employed a 50-fold lowercitrate dose (24mM) during 12 weeks and determined thespontaneous (not diet-induced) atherosclerosis developmentThis protocol did not cause elevation of plasma lipid levelsFor the cholesterol inhibition group (statins treatment)we chose a low dose of a hydrophilic statin (pravastatin10mgKgday) because we had previously shown that highstatin dose can directly damage mitochondria [27]This doseis proven to be still effective to reduce atherosclerosis inLDLrminusminus mice without important plasma lipid changes [28]

Control (CON) citrate (CIT) pravastatin (PRA) andcitrate + pravastatin (CIT + PRA) mice had similar bodycomposition and plasma lipid levels (Table 1) although PRAgroup of mice presented lower body weight at the end ofthe treatment The liver and renal functions were similarin all groups as verified through quantification of plasma


Table 1 Body composition plasma lipids and glucose levels renal and hepatic function markers systemic and mitochondrial redoxparameters of LDL receptor knockout mice treated during 3 months with citrate and pravastatin

Parameters Control Citrate Pravastatin Citrate + pravaBody weight1 197 plusmn 02 177 plusmn 03 173 plusmn 04lowast 182 plusmn 02Carcass fat mass2 141 plusmn 07 156 plusmn 16 142 plusmn 10 131 plusmn 19Carcass lean mass2 654 plusmn 09 645 plusmn 05 638 plusmn 06 649 plusmn 07Visceral fat mass3 091 plusmn 008 093 plusmn 011 074 plusmn 007 084 plusmn 012Liver mass3 468 plusmn 009 472 plusmn 008 463 plusmn 011 478 plusmn 004Spleen mass3 027 plusmn 001 027 plusmn 001 025 plusmn 002 025 plusmn 000Liver Cholesterol4 45 plusmn 06 51 plusmn 06 44 plusmn 06 59 plusmn 08Liver Triglycerides4 79 plusmn 137 97 plusmn 178 693 plusmn 108 1023 plusmn 156Glucose5 85 plusmn 41 77 plusmn 41 85 plusmn 33 79 plusmn 42Triglycerides5 103 plusmn 67 104 plusmn 66 110 plusmn 83 115 plusmn 88Cholesterol5 234 plusmn 133 238 plusmn 84 238 plusmn 121 252 plusmn 91Fatty acid6 080 plusmn 009 083 plusmn 006 075 plusmn 006 076 plusmn 003LDLox antibodies7 034 plusmn 007 054 plusmn 008 042 plusmn 009 054 plusmn 007TBARS6 2585 plusmn 103 2486 plusmn 78 2345 plusmn 66 2455 plusmn 152ALT8 360 plusmn 21 487 plusmn 42 448 plusmn 45 387 plusmn 31AST8 1177 plusmn 114 1381 plusmn 124 1550 plusmn 279 1300 plusmn 147ALP8 1581 plusmn 8 1456 plusmn 157 1731 plusmn 112 1724 plusmn 77Urea5 770 plusmn 24 847 plusmn 15 943 plusmn 54lowast 818 plusmn 43Creatinine5 012 plusmn 001 012 plusmn 001 012 plusmn 001 012 plusmn 001mtROS production9 171 plusmn 019 202 plusmn 032 166 plusmn 008 183 plusmn 018NADPH oxidation10 095 plusmn 013 080 plusmn 02 092 plusmn 021 073 plusmn 022Atherosclerotic lesion area11 316 plusmn 88 438 plusmn 48 611 plusmn 128 1168 plusmn 250lowast

Data are mean plusmn SE (119899 = 8ndash10group) Citrate (134mM citric acid + 11mM sodium citrate in the drinking water) Pravastatin (10mgKg of body weight) inthe drinking water (67mgL) 1g 2 related to the dry carcass 3 related to the body weight 4mgg of liver 5mgdL 6120583M 7absorbance 8UL 9nMDCFmgprotein minminus1 10120578MNADPHmg protein minminus1 11120583m2

times 103 lowast119875 lt 005

alkaline phosphatase (ALP) alanine aminotransferase (ALT)aspartate transaminase (AST) and creatinine plasma levelsHowever plasma urea levels in PRA group were 22 higherthan in the other groups (Table 1) The average values ofoxidative stress markers such as susceptibility to lipid perox-idation (copper sulfate-induced thiobarbituric acid reactivesubstances) titles of plasma oxidized LDL antibodies livermitochondrial NADPH oxidation rates and reactive oxygenspecies production rates (mtROS) were not significantlymodified by the three treatments (Table 1) The developmentof spontaneous atherosclerosis as measured by the areaof lipid stained lesions in the aortic root did not changesignificantly in CON CIT and PRA groups of mice Sur-prisingly the group CIT + PRA presented marked enhancedatherosclerosis (Table 1 and Figure 1)

Since the treatments did not alter the average valuesof classic atherosclerosis risk factors such as plasma lipidsobesity and oxidative stress statistical correlation analyses inpooled group data were performed in an attempt to explainthe differences observed in the variation of atherosclerosislesion areas Spearmanrsquos univariate correlation test showedthat the atherosclerotic lesion areas were positively correlatedwith plasma cholesterol (119903 = 039 119875 = 0046) triglycerides

(119903 = 043 119875 = 0026) ALP (119903 = 045 119875 = 002) andliver mitochondria ROS production (mtROS) (119903 = 054119875 = 0031) (Figure 2) We further tested the existenceof linear multiple regression models to explain the extentof atherosclerosis Three groups of variables were tested(1) associations with body composition variables (carcasslean and fat mass visceral fat mass and body weight) (2)associations with plasma variables (glucose triglyceridescholesterol free fatty acids LDLox antibodies and TBARS)and (3) associationswith hepatic variables (livermass choles-terol and triglycerides content and mtROS) For each modelvariables that create the best-fitting linear regression areselected by the software [25 26] to explain the atherosclerosisvariation (Table 2) The body composition variables did nothave any relevance in determining atherosclerosis variation(no significant associations were found) For the plasmavariables the software selected two significant models (a)association with plasma triglyceride level and (b) associationwith plasma triglyceride and glucose level For the hepaticvariables also two significant models were found (a) associ-ation with mtROS and (b) association with mtROS and livertriglycerides content (Table 2) According to these regressionmodels the impact of these variables on the atherosclerosis


Table 2 Multiple linear regression models for atherosclerosis and partial correlation adjusted by plasma triglycerides

Statistical analyses Significant parameters Correlation coefficients 119875 value(1) Body composition model1 None 006 012

(2) Plasma model2 (2a) Triglycerides 022 0006(2b) Triglycerides and glycemia 025 001

(3) Liver model3 (3a) mtROS 023 0035(3b) mtROS and liver triglycerides 028 0047

Partial correlation adjusted by plasma triglycerides4 mtROS 056 0047Parameters tested in each model 1carcass fat and lean mass visceral fat mass and body weight 2plasma glucose triglycerides cholesterol free fatty acidsLDLox antibodies and TBARS 3liver mass hepatic cholesterol and triglyceride contents and liver mitochondria ROS production 4plasma cholesterol plasmaalkaline phosphatase and liver mitochondria ROS production were tested after adjustment by plasma triglycerides

(a) (b)

(c) (d)

Figure 1 Representative images of aorta root with atherosclerosis lesions of LDL receptor knockout mice control (a) treated with citrate(b) pravastatin (c) and citrate + pravastatin (d) The aortic root was cryosectioned and stained for lipids with Oil Red O and counterstainedwith light green

extent can be interpreted as follows Ten-unit increase inplasma triglycerides (10mgdL) is expected to increase lesionsize by 17 (plasma model (2a)) and one unit increase inmtROS (nmol DCFmg protein) is expected to increase theatherosclerotic lesion size by 370 (hepatic model (3a))

In order to verify whether mtROS correlation withatherosclerosis was independent of the plasma triglyceridelevels we also performed the partial (adjusted) correlationtest This adjusted analysis included all variables that weresignificantly correlated with atherosclerosis by Spearmanrsquoscorrelation test (triglycerides cholesterol plasma ALP and

mtROS) After adjusting for plasma triglyceride levels theonly variable that remained significantly correlated withatherosclerosis was mtROS (119903 = 056 119875 = 0047) (Table 2)Therefore mtROS positive correlation with atherosclerosisis confirmed by three different statistical analyses and isindependent of the variations of classical risk factors theplasma lipid levels

These statistical correlations between liver mtROS andatherosclerosis suggest a possible mechanistic link betweenliver and artery disease The most likely connections aremetabolic (for instance secretion of oxidized VLDL) andor


9 10 11 12 13

160

200

240

280

320

Log-transformed lesion area

Chol

este

rol (

mg

dL)

(a)

9 10 11 12 13

60

90

120

150

180


Trig

lyce

rides

(mg

dL)

(b)

9 10 11 12 1380

120

160

200

240


ALP

(UL

)

(c)

9 10 11 12 13

12

16

20

24


MtR

OS

(120578m

ol D

CFm

glowastm

inminus1)

(d)

Figure 2 Spearmanrsquos correlations between log-transformed atherosclerosis lesion area (120583m2) and plasma cholesterol 119899 = 27 (a) plasmatriglycerides 119899 = 28 (b) plasma alkaline phosphatase 119899 = 26 (ALP) (c) and liver mitochondria ROS production 119899 = 16 (d) I = control += citrate = pravastatin 998771 = citrate + pravastatin

an oxidative induction of proinflammatory cytokines There-fore we compared LDLrminusminus and wild type mice regardingVLDL susceptibility to oxidation (Figure 3) and lipidmarkers(Table 3) as well as the levels of plasma cytokines (Table 4)Figure 3 shows that VLDL secreted by LDLrminusminus livers ismarkedly more susceptible to an oxidative insult (CuSO

4

)than the wild type VLDL compared at the same TG contentbasisWild type VLDL shows a 5-fold greater lag time for oxi-dation initiation and a 50 lower rate of oxidation thanVLDLfrom LDLrminusminus mice In addition mass spectrometry analysesof VLDL lipid extracts identified oxidized lipid markers

mainly phospholipids in VLDL from LDLrminusminus comparedwith wild type mice (Table 3) Both groups were clearly sepa-rated with an accuracy of 95 and mass errors of less than2 ppm Regarding systemic inflammatory markers Table 4shows that three proinflammatory cytokines (TNF120572 IL-2and IFN120574) were augmented in the plasma of LDLrminusminus mice

4 Discussion

Several studies have evidenced the relevance of mitochon-dria functionality to the development of atherosclerosis in


Table 3 Lipid chemical markers identified by high resolution electrospray ionization mass spectrometry (ESI-MS) analysis of plasma VLDLfrom LDL receptor knockout and wild type mice

Molecule Theoretical mass Experimental mass Error (ppm) LM IDlowast

Wild type

[PE(151224)+K]+ 7904784 7904769 minus18976 LMGP02010503[TG(140161201)+K]+ 8696995 8697010 17247 LMGL03014259[PG(180130)+Na]+ 7154884 7154889 06988 LMGP04030029[PC(130182)+H]+ 7165225 7165238 18143 LMGP01011348

[PC(130184)+Na]+ + OH 7514764 7514763 minus01331 LMGP01011351[PI(120205)+Na]+ 8234368 8234379 13359 LMGP06010031

[PE-Cer(152200)+Na]+ + OH 7115048 7115059 15460 LMSP03020077[PE-Cer(141221)+K]+ 7254994 7255002 11027 LMSP03020009

[PE-Cer(151200)+K]+ + 2OH 7294943 7294934 minus12337 LMSP03020074[PA(204200)+K]+ 7914988 7914975 minus16425 LMGP10010636

LDLrminusminus

[PC(161181)+H]+ + OH 7595778 7595791 17115 LMGP01090011[PC(190151)+H]+ 7605851 7605839 minus15777 LMGP01011732[PG(191182)+H]+ 7875484 7875472 minus15237 LMGP04010493

[PS(180160)+H]+ + OH 7815469 7815477 10236 LMGP03010888[PC(183181)+H]+ + OH 7835778 7835765 minus16591 LMGP01090030[PC(205181)+H]+ + OH 8075778 8075766 minus14859 LMGP01090051[PA(222160)+K]+ + OH 7855093 7855103 12731 LMGP10010761[PS(160172)+H]+ + 2OH 7624916 7624901 minus19672 LMGP03030011[PS(160205)+H]+ + OH 7824967 7824976 11502 LMGP03030022

[PI(140226)+Na]+ 8774838 8774845 07977 LMGP06010892PE phosphatidylethanolamine TG triacylglycerols PG phosphoglycerol PC phosphatidylcholine PI phosphatidylinositol PS phosphatidylserine Cerceramide PA phosphatic acid lowastLM ID Lipid MAPS identity Identification is based on exact mass of each compound and LipidMAPS databaseThe assignedIDs are for general structures and can be any of the positional isomers

LDLrminusminus

0 150 300 450 600

000

025

050

075

100

Wild type

Time (min)

ABS

Figure 3 Representative curve of oxidation susceptibility of plasmaVLDL obtained from LDL receptor knockout (LDLrminusminus) and wildtype mice The VLDL oxidation was induced by incubation withCuSO

4

(40120583M) Formation of conjugated dienes was followed byabsorbance changes at 234 nm along time The VLDL lag-time tostart oxidation was 73 plusmn 48 and 382 plusmn 799 minutes for LDLrminusminusand wild type mice respectively (119875 = 00007) The VLDL oxidationrate determined by the curve slopes was about 2-fold greater (119875 lt00001) in the LDLrminusminus than wild type VLDL The 119899 used in thisexperiment was of 6ndash8 mice

Table 4 Plasma cytokines of LDL receptor knockout and controlwild type mice

Cytokines (pgmL) LDLrminusminus Wild type 119875 value 119873

TNF120572 1364 plusmn 255 467 plusmn 53 0007 8ndash10IL-2 588 plusmn 107 281 plusmn 27 002 7-8IFN120574 1553 plusmn 238 819 plusmn 124 ns 8ndash10Data are mean plusmn SE 119875 value from Studentrsquos 119905-test ns nonsignificant

humans and animals [2 3 7 8] Most studies agree thatmitochondria ROS overproduction is likely implicated inatherogenesis and disease progression although bettermech-anistic understanding is still needed [29]

Because we had previously found thatmitochondria fromatherosclerosis-pronemousemodel (LDLrminusminus) had decreasedcontent of Krebs cycle intermediates reducing equivalentpower and increased ROS release we hypothesized that atleast part of these mitochondrial features could be attributedto an elevated cholesterol synthesis which consume bothKrebs cycle intermediates and cell reducing equivalentsTherefore in the present work we tested whether citratesupplementation combined or not with inhibition of choles-terol synthesis could decreasemitochondrial ROS release andatherosclerosis development In contrast to our hypothesisany of the treatments affected significantly the average values


of mtROS and the combined citrate supplementation withpravastatin treatment actually increased atherogenesis

Pravastatin alone increased plasma urea levels and ure-mia per se appears to be proatherogenic [30] Increased ureaconcentrations may increase the rate of isocyanate synthesisand increase carbamylation of lipids and proteins includingLDL [31] Previous studies showed that carbamylated LDLdose dependently promotes proliferation of human coronaryartery smoothmuscle cells endothelial cell death and expres-sion of adhesion molecules [32 33] On the other hand inthe group CIT + PRA urea plasma levels were normal Itis possible that citrate treatment may have counteracted theeffect of pravastatin on uremia since it was previously shownthat citrate reduced uremia induced by high fat diet [34]Concerning atherosclerosis extent it is difficult to explainwhy citrate + pravastatin would increase the progression ofspontaneous atherosclerosisWe postulate that an interactionbetween these factors may have a local vascular wall harmfulaction which we have not addressed in this study butpossibly related to mtROS production

A limitation of this study is the low number (119899) of miceand high variability of the responses Therefore we appliedstatistical methods to infer possible links between atheroscle-rosis and allmeasured parameters in pooled data Correlationand regression analyses in pooled data help to identify whichparameters related to risk factors (plasma lipids obesity andoxidative stress) have significant impact on the variation ofatherosclerosis As expected plasma cholesterol and triglyc-erides were positively correlated to aortic lesion size evenwithin a very narrow range Furthermore new positive corre-lations were identified named liver derived ALP andmtROSIncreasing levels of ALP suggest a correlation between liverdamage and cardiovascular disease which has already beenshown by others regarding liver enzymes [35] and C-reactiveprotein levels [36] However after adjusting the analysisby plasma triglycerides only mtROS remained positivelyand independently correlated with atherosclerosis Multiplelinear regression analysis also indicated that liver mtROSproduction could explain the extent of aortic atherosclerosiseither alone (Table 2 model (3a)) or in combination withliver triglycerides content (Table 2 model (3b)) Thereforeby using three distinct statistical tools (univariate correlationadjusted correlation and multiple regression) with increas-ing levels of stringency we confirm a significant associa-tion between variables and further quantify the amount ofvariation in the dependent variable (atherosclerosis) thatcan be explained by the independent variable (mtROS)Although these statistical analyses do not imply causationthese results together with the whole body of evidences inthe current literature lead us to propose that mechanisticlinks are conceivable Liver mtROS could be linked to thearterial disease by different ways The first one is through ametabolic connection that is by secreting altered amountsandor quality of VLDL the precursors of LDL Secondlyliver mitochondrial oxidative stress results in activation ofinflammatory pathways that may contribute to a systemicinflammation which in turn is strongly correlated withatherosclerosis [36] Finally liver could be a surrogatemarker

of the main events occurring in the arterial wall particularlylipid accumulation and mitochondrial oxidative stress Insupport for the metabolic connection we found that VLDLsecreted by LDLrminusminus livers is markedly more susceptibleto an oxidative insult (CuSO

4

) than the wild type VLDLcompared at the same TG content basis In addition theseLDLrminusminus VLDL are already secreted with several oxidizedlipids compared to the wild type VLDL At this point it isrelevant to recall and quote Davis and Hui in their GeorgeLyman Duff Memorial Lecture ldquoAtherosclerosis is a liverdisease of the heartrdquo [37] In that occasion the authorsreferred specifically to the complex processes involved in theliver assembly and secretion of apoB-containing lipoproteinsthe precursors of the atherosclerosis culprit the LDL

In conclusion our results revealed that mitochondrialreactive oxygen is a novel positive and independent riskfactor for atherosclerosis We propose that modulation ofmitochondrial redox state results in significant variation ofatherosclerosis extent These findings provide support forproposed strategies of using mitochondria-targeted antiox-idants as potential therapy for treatment of cardiovasculardiseases

Disclosure

The authors alone are responsible for the content and writingof the paper

Conflict of Interests

The authors report no conflict of interests

Acknowledgments

This study was supported by grants obtained by HelenaC F Oliveira and Anibal E Vercesi from the Brazilianagencies Fundacao de Amparo a Pesquisa do Estado de SaoPaulo (FAPESP 201150400-0 and 201151349-8) and Con-selho Nacional de Desenvolvimento Cientıfico e Tecnologico(CNPq) Gabriel G Dorighello and Bruno A Paim were sup-ported by CNPq and FAPESP fellowships respectively Theauthors thank Ms Natalia Mayumi Inada Edilene de SouzaSiqueira Santos and Gabriela C Tonini for the technicalassistance

References

[1] N R Madamanchi A Vendrov and M S Runge ldquoOxidativestress and vascular diseaserdquo Arteriosclerosis Thrombosis andVascular Biology vol 25 no 1 pp 29ndash38 2005

[2] M Hulsmans E van Dooren and P Holvoet ldquoMitochondrialreactive oxygen species and risk of atherosclerosisrdquo CurrentAtherosclerosis Reports vol 14 no 3 pp 264ndash276 2012

[3] VMVictor N Apostolova R Herance AHernandez-Mijaresand M Rocha ldquoOxidative stress and mitochondrial dysfunc-tion in atherosclerosis mitochondria-targeted antioxidants as


potential therapyrdquo Current Medicinal Chemistry vol 16 no 35pp 4654ndash4667 2009

[4] S W Ballinger C Patterson C A Knight-Lozano et al ldquoMito-chondrial integrity and function in atherogenesisrdquo Circulationvol 106 no 5 pp 544ndash549 2002

[5] S W Ballinger ldquoMitochondrial dysfunction in cardiovasculardiseaserdquo Free Radical Biology and Medicine vol 38 no 10 pp1278ndash1295 2005

[6] C M Harrison M Pompilius K E Pinkerton and S WBallinger ldquoMitochondrial oxidative stress significantly influ-ences atherogenic risk and cytokine induced oxidant produc-tionrdquo Environmental Health Perspectives vol 119 no 5 pp 676ndash681 2011

[7] N R Madamanchi and M S Runge ldquoMitochondrial dysfunc-tion in atherosclerosisrdquo Circulation Research vol 100 no 4 pp460ndash473 2007

[8] E P K Yu and M R Bennett ldquoMitochondrial DNA damageand atherosclerosisrdquo Trends in Endocrinology and Metabolismvol 25 no 9 pp 481ndash487 2014

[9] Y Wang G Z Wang P S Rabinovitch and I Tabas ldquoMacro-phage mitochondrial oxidative stress promotes atherosclerosisand nuclear factor-120581B-mediated inflammation in macropha-gesrdquo Circulation Research vol 114 no 3 pp 421ndash433 2014

[10] H C F Oliveira R G Cosso L C Alberici et al ldquoOxidativestress in atherosclerosis-prone mouse is due to low antioxidantcapacity of mitochondriardquoThe FASEB Journal vol 19 no 2 pp278ndash280 2005

[11] A J Kowaltowski R F Castilho and A E Vercesi ldquoMitochon-drial permeability transition and oxidative stressrdquo FEBS Lettersvol 495 no 1-2 pp 12ndash15 2001

[12] T R Figueira M H Barros A A Camargo et al ldquoMito-chondria as a source of reactive oxygen and nitrogen speciesfrom molecular mechanisms to human healthrdquo Antioxidantsand Redox Signaling vol 18 no 16 pp 2029ndash2074 2013

[13] PM Yao and I Tabas ldquoFree cholesterol loading ofmacrophagesis associated with widespread mitochondrial dysfunction andactivation of the mitochondrial apoptosis pathwayrdquoThe Journalof Biological Chemistry vol 276 no 45 pp 42468ndash42476 2001

[14] B A Paim J A Velho R F Castilho H C F Oliveira and AE Vercesi ldquoOxidative stress in hypercholesterolemic LDL (low-density lipoprotein) receptor knockout mice is associated withlow content of mitochondrial NADP-linked substrates and ispartially reversed by citrate replacementrdquo Free Radical Biologyamp Medicine vol 44 no 3 pp 444ndash451 2008

[15] A Zaratin M Gidlund P Boschcov L Castilho and E CottaDe Faria ldquoAntibodies against oxidized low-density lipoproteinin normolipidemic smokersrdquoThe American Journal of Cardiol-ogy vol 90 no 6 pp 651ndash653 2002

[16] AG Salerno T R SilvaM E CAmaral et al ldquoOverexpressionof apolipoprotein CIII increases and CETP reverses diet-induced obesity in transgenic micerdquo International Journal ofObesity vol 31 no 10 pp 1586ndash1595 2007

[17] J Folch M Lees and G H S Stanley ldquoA simple method for theisolation and purification of total lipides from animal tissuesrdquoThe Journal of Biological Chemistry vol 226 no 1 pp 497ndash5091957

[18] R S Kaplan and P L Pedersen ldquoCharacterization of phosphateefflux pathways in rat liver mitochondriardquo The BiochemicalJournal vol 212 no 2 pp 279ndash288 1983

[19] C Garcıa-Ruiz A Colell M Marı A Morales and J CFernandez-Checa ldquoDirect effect of ceramide on the mitochon-drial electron transport chain leads to generation of reactiveoxygen species role of mitochondrial glutathionerdquoThe Journalof Biological Chemistry vol 272 no 17 pp 11369ndash11377 1997

[20] B Paigen A Morrow P A Holmes D Mitchell and R AWilliams ldquoQuantitative assessment of atherosclerotic lesions inmicerdquo Atherosclerosis vol 68 no 3 pp 231ndash240 1987

[21] E M Rubin R M Krauss E A Spangler J G Verstuyft and SMClift ldquoInhibition of early atherogenesis in transgenicmice byhuman apolipoprotein AIrdquo Nature vol 353 pp 265ndash267 1991

[22] M-F Hau A H M Smelt A J G H Bindels et al ldquoEffects offish oil on oxidation resistance ofVLDL in hypertriglyceridemicpatientsrdquoArteriosclerosisThrombosis andVascular Biology vol16 no 9 pp 1197ndash1202 1996

[23] E G Bligh and W J Dyer ldquoA rapid method of total lipidextraction and purificationrdquo Canadian Journal of Biochemistryand Physiology vol 37 no 8 pp 911ndash917 1959

[24] D Machado S M Shishido K C S Queiroz et al ldquoIrradiatedriboflavin diminishes the aggressiveness of melanoma in vitroand in vivordquo PLoS ONE vol 8 no 1 Article ID e54269 2013

[25] T Lumley Leaps Regression Subset Selection R Package Version29 CRAN 2009

[26] R-Core-Team R A Language and Environment for StatisticalComputing R Foundation for Statistical Computing ViennaAustria 2013

[27] J A Velho H Okanobo G R Degasperi et al ldquoStatinsinduce calcium-dependent mitochondrial permeability transi-tionrdquo Toxicology vol 219 no 1ndash3 pp 124ndash132 2006

[28] B R Kwak N Veillard G Pelli et al ldquoReduced connexin43expression inhibits atherosclerotic lesion formation in low-density lipoprotein receptor-deficient micerdquo Circulation vol107 no 7 pp 1033ndash1039 2003

[29] Y Wang and I Tabas ldquoEmerging roles of mitochondria ROSin atherosclerotic lesions causation or associationrdquo Journal ofAtherosclerosis andThrombosis vol 21 no 5 pp 381ndash390 2014

[30] C Albert P RMertens and P Bartsch ldquoUrea and atherosclero-sis-evidence for a direct link involving apolipoprotein B proteinmodificationsrdquo International Urology and Nephrology vol 43no 3 pp 933ndash936 2011

[31] A G Basnakian S V Shah E Ok E Altunel and E O Apos-tolov ldquoCarbamylated LDLrdquoAdvances in Clinical Chemistry vol51 pp 25ndash52 2010

[32] E Ok A G Basnakian E O Apostolov Y M Barri and S VShah ldquoCarbamylated low-density lipoprotein induces death ofendothelial cells a link to atherosclerosis in patientswith kidneydiseaserdquo Kidney International vol 68 no 1 pp 173ndash178 2005

[33] E O Apostolov D Ray A V Savenka S V Shah and A GBasnakian ldquoChronic uremia stimulates LDL carbamylation andatherosclerosisrdquo Journal of the American Society of Nephrologyvol 21 no 11 pp 1852ndash1857 2010

[34] K M Surapaneni and M Jainu ldquoPioglitazone quercetin andhydroxy citric acid effect on hepatic biomarkers in Non Alco-holic Steatohepatitisrdquo Pharmacognosy Research vol 6 no 2 pp153ndash162 2014

[35] A Kerner O Avizohar R Sella et al ldquoAssociation betweenelevated liver enzymes andC-reactive proteinmdashpossible hepaticcontribution to systemic inflammation in the metabolic syn-dromerdquo Arteriosclerosis Thrombosis and Vascular Biology vol25 no 1 pp 193ndash197 2005


[36] P Libby ldquoInflammation in atherosclerosisrdquo ArteriosclerosisThrombosis and Vascular Biology vol 32 pp 2045ndash2051 2012

[37] R A Davis and T Y Hui ldquo2000 George Lyman Duff memoriallecture atherosclerosis is a liver disease of the heartrdquo Arte-riosclerosis Thrombosis and Vascular Biology vol 21 no 6 pp887ndash898 2001

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


damage of mtDNA is correlated with human and miceseverity of atherosclerosis [4 9]

In a previous study we verified that besides NADPHdeficiency LDLrminusminus mitochondria contained less Krebs cycleintermediates such as isocitrate and that treating the organellewith isocitrate (in vitro) and the LDLrminusminus mouse model withcitrate (in vivo) resulted in decreased mtROS productionand increased NADPH content [14] Major processes thatconsume cell NADPH and Krebs cycle intermediates arelipid and cholesterol biosynthesis which are elevated in thisanimal model [10] Therefore we hypothesized that eithercitrate supplementation or cholesterol synthesis inhibitioncould increase mitochondria NADPH availability decreasemtROS production and decrease atherosclerosis develop-ment in LDLrminusminus mouse

2 Material and Methods

21 Animals Mating pairs of LDLrminusminus (B6129S7-Ldlrlttm1HerJ homozygous for Ldlrlttm1Her stock number002207) and wild type controls (C57BL6J stock number000664) were purchased from The Jackson Laboratory(Bar Harbor ME) in 2009 and both colonies have beenestablished and maintained in strictly SPF conditions inThe Multidisciplinary Center for Biological Investigationin the Area of Laboratory Animal Science (CEMIB) ofthe State University of Campinas (Unicamp) The CEMIB-Unicamp is part of the International Council for LaboratoryAnimal Science (ICLAS) The colonies are maintainedthrough exclusive sibling mattings in order to maintain thehomogeneous genetic background When mice reach the ageof 4 weeks they are transferred to the Conventional AnimalFacility in the Department of Structural and FunctionalBiology at Unicamp Mice are then maintained in atemperature-controlled room (22 plusmn 1∘C) with 12 h lightdarkcycle with 15 cycles of air changes per hour and withfree access to food (standard laboratory rodent chow dietNuvital CR1 Colombo PR Brazil) and filtered water Fourto five mice are maintained per cage They are then enrolledin the experimental protocol which was approved by theEthical Committee for the Use of Animal (CEUAUnicampprotocol 1101-2) and by the Internal Biosecurity Committee(CIBio-IBUnicamp protocol 200802) One-month-oldmale LDLrminusminus mice were randomly separated into fourgroups control (CON) citrate (CIT) pravastatin (PRA)and citrate + pravastatin (CIT + PRA) The solutions ofcitrate (134mM citric acid + 11mM sodium citrate) andpravastatin (67mgL) equivalent to 10mgKg of body weightwere offered as the only source of drinking water Thesolutions were provided in dark bottles and changed everyother day during 3 months Additional experiments wereperformed in untreated LDLrminusminus and wild type controls forchecking differences in oxidative susceptibility status andoxidative lipid damage in their plasma VLDL particles aswell as for determining anti-inflammatory systemic markersControl of the colony phenotype is done by measuringfasting plasma cholesterol levels (LDLrminusminus gt 200mgdLand WT lt 80mgdL) and genotype by PCR in tail tip DNAsamples according toThe Jackson Laboratory protocol

22 Plasma Biochemical Analysis Blood samples were col-lected from either the retroorbital plexus or the tail tip ofanesthetized and overnight fasted mice Total cholesteroltriglycerides and nonesterified fatty acids were measured infresh plasma using standard commercial kits (Roche-HitachiGermany and Wako Germany) Glucose levels were mea-sured using a hand-held glucometer (Accu-Chek AdvantageRoche Diagnostic Switzerland)The plasma urea creatininealkaline phosphatase (ALP) alanine aminotransferase (ALT)and aspartate transaminase (AST) were determined usingan automated modular analyzer PP (Hitachi) with Rochereagents (RocheDiagnostic Germany)Theplasma cytokinestumoral necrosis factor alpha (TNF120572) interleukin 2 (IL-2)interleukin 4 (IL-4) and Interferon gamma (IFN120574) weremeasured with ELISA commercial kit (eBioscience SanDiego CA) according to the manufacturerrsquos instructionsPlasma oxidized LDL antibodies were measured in plasmaas described by Zaratin et al [15] Polystyrene 96-well plateswere coatedwith human copper-oxidized LDL and incubatedovernight at 4∘C Plates were then blocked with 1 gelatinin PBS for 48 h at room temperature After washing withPBS plasma samples (1 200) were added and incubated for2 h at room temperature After washing with PBS containing02mLL Tween 20 plates were incubated with 50 120583L ofperoxidase conjugated rabbit anti-mouse IgG antibody atroom temperature for 1 h Finally 75 120583L peroxidase substratesolution was added incubated for 15min and ended by25 120583L of 2M sulfuric acid The optical density (OD) wasthen measured at 450 nm The results are presented as theOD readings (arbitrary units) CuSO

4

-induced plasma thio-barbituric acid reactive substances (TBARS) concentrationswere determined by first exposing plasma to 01mM CuSO4at 37∘C for 5 hours Then 100 120583L of oxidized plasma wasincubated with 200120583L of 07 thiobarbituric acid in 005MNaOH and 60120583L of 50 trichloroacetic acid Samples wereincubated in boiling water for 30 minutes followed bycentrifugation at 664 g for 15 minutes The standard curvewas prepared using several dilutions of 005mM 1133-tetramethoxypropane The optical densities of samples andstandard curve were measured in a microplate reader at532 nm

23 Body Composition and Tissue Lipids Analyses The epi-didymal adipose tissue and liver and spleen fresh masseswere determined gravimetrically Mice carcass compositionwas determined by weighing carcass before and after waterdrying and before and after lipid extraction of the driedcarcass with petroleum ether as previously described bySalerno et al [16] Liver lipids were extracted using the Folchmethod [17]The liver content of cholesterol and triglycerideswas determined using colorimetric-enzymatic assays (Roche-Hitachi Germany) after dissolving the lipid extracts in atriton-containing buffer (01M potassium phosphate pH 74005M NaCl 5mM cholic acid and 01 Triton X-100)

24 Isolation of Mouse Liver Mitochondria Mitochondriawere isolated by conventional differential centrifugation at4∘C as previously described [18] The livers were homoge-nized in 250mM sucrose 1mM EGTA and 10mM Hepes




2


2


2

-DCFoxidation




4






3 Results

















(a) (b)

(c) (d)







9 10 11 12 13

160

200

240

280

320


Chol

este

rol (

mg

dL)

(a)

9 10 11 12 13

60

90

120

150

180


Trig

lyce

rides

(mg

dL)

(b)

9 10 11 12 1380

120

160

200

240


ALP

(UL

)

(c)

9 10 11 12 13

12

16

20

24


MtR

OS

(120578m

ol D

CFm

glowastm

inminus1)

(d)



4



4 Discussion





Wild type





LDLrminusminus




LDLrminusminus

0 150 300 450 600

000

025

050

075

100

Wild type

Time (min)

ABS


4












4



Disclosure




Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2


2


2

-DCFoxidation




4






3 Results

















(a) (b)

(c) (d)







9 10 11 12 13

160

200

240

280

320


Chol

este

rol (

mg

dL)

(a)

9 10 11 12 13

60

90

120

150

180


Trig

lyce

rides

(mg

dL)

(b)

9 10 11 12 1380

120

160

200

240


ALP

(UL

)

(c)

9 10 11 12 13

12

16

20

24


MtR

OS

(120578m

ol D

CFm

glowastm

inminus1)

(d)



4



4 Discussion





Wild type





LDLrminusminus




LDLrminusminus

0 150 300 450 600

000

025

050

075

100

Wild type

Time (min)

ABS


4












4



Disclosure




Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























(a) (b)

(c) (d)







9 10 11 12 13

160

200

240

280

320


Chol

este

rol (

mg

dL)

(a)

9 10 11 12 13

60

90

120

150

180


Trig

lyce

rides

(mg

dL)

(b)

9 10 11 12 1380

120

160

200

240


ALP

(UL

)

(c)

9 10 11 12 13

12

16

20

24


MtR

OS

(120578m

ol D

CFm

glowastm

inminus1)

(d)



4



4 Discussion





Wild type





LDLrminusminus




LDLrminusminus

0 150 300 450 600

000

025

050

075

100

Wild type

Time (min)

ABS


4












4



Disclosure




Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















9 10 11 12 13

160

200

240

280

320


Chol

este

rol (

mg

dL)

(a)

9 10 11 12 13

60

90

120

150

180


Trig

lyce

rides

(mg

dL)

(b)

9 10 11 12 1380

120

160

200

240


ALP

(UL

)

(c)

9 10 11 12 13

12

16

20

24


MtR

OS

(120578m

ol D

CFm

glowastm

inminus1)

(d)



4



4 Discussion





Wild type





LDLrminusminus




LDLrminusminus

0 150 300 450 600

000

025

050

075

100

Wild type

Time (min)

ABS


4












4



Disclosure




Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Wild type





LDLrminusminus




LDLrminusminus

0 150 300 450 600

000

025

050

075

100

Wild type

Time (min)

ABS


4












4



Disclosure




Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















4



Disclosure




Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article correlation between mitochondrial...

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