Research Article Metabolomics Profiling to Investigate the ...downloads.hindawi.com/journals/ecam/2015/897914.pdf · eicosatrienoic acid, docosatrienoic acid, and eicosenoic acid

Research ArticleMetabolomics Profiling to Investigate the PharmacologicMechanisms of Berberine for the Treatment of High-FatDiet-Induced Nonalcoholic Steatohepatitis

Jian Li1 Zezhou Liu1 Mingxing Guo2 Kejia Xu1

Miao Jiang3 Aiping Lu34 and Xiaoyan Gao2

1School of Basic Medical Sciences Beijing University of Chinese Medicine Beijing 100029 China2Center of Scientific Experiment Beijing University of Chinese Medicine Beijing 100029 China3Institute of Basic Research of Clinical Medicine China Academy of Chinese Medical Sciences Beijing 100700 China4School of Chinese Medicine Hong Kong Baptist University Kowloon Hong Kong

Correspondence should be addressed to Aiping Lu lap64067611126com and Xiaoyan Gao gaoxiaoyan0913sinacom

Received 8 September 2014 Revised 6 December 2014 Accepted 7 December 2014

Academic Editor Jia Wei

Copyright copy 2015 Jian Li et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objective Berberine has been used to treat nonalcoholic steatohepatitis (NASH) which has been addressed in many studiesIn this study we investigated the molecular pharmacology mechanisms of berberine using metabolomic techniques MethodsSprague-Dawley rats were randomly divided into three groups (10 rats in each group) (i) normal control group (ii) high-fatdiet- (HFD-) induced NASH model group and (iii) HFD berberine-treated group (id 200mgkg) The handling procedurelasted eight weeks Then UPLC-Q-TOFMS techniques coupled with histopathology and biochemical analyses were adopted toexplore the mechanisms of berberine on the protective effects against NASH Key Findings (i) According to conventional testresults berberine treatment plays a fighting role in HFD-induced NASH due to its beneficial effects against insulin resistanceinflammation and lipid metabolism (ii) Based on UPLC-Q-TOFMS techniques metabolic profiles that involved sphingomyelin(SM) phosphatidylcholine (PC) lysophosphatidylcholine (LysoPC) 13-hydroperoxy-9 11-octadecadienoic acid (13-HpODE)eicosatrienoic acid docosatrienoic acid and eicosenoic acid could provide potential metabolic biomarkers to address thepharmacological mechanisms of berberine Conclusions The parts of molecular pharmacological mechanisms of berberine forNASH treatment are related to the regulation of metabolic disruption involving phospholipid and unsaturated fatty acids in ratswith NASH

1 Introduction

Nonalcoholic fatty liver disease (NAFLD) is an increasinglyrecognized disease state in which lipids (triglycerides) accu-mulate in the hepatocytes in the absence of excessive alcoholconsumption [1 2] The earliest stage of NAFLD is hepaticsteatosis which is often contained However it can progressto nonalcoholic steatohepatitis (NASH) if inadequate treat-ment or poor prognosis occurs The operational definitionsfor NASH have remained uncertain until now Thus NASHis distinguished from simple steatosis by the presence of hep-atocyte ballooning cell death inflammatory infiltrate andorcollagen deposition in liver tissue [3] As the worldwide

trend currently continues towards an increased prevalenceof NAFLD [4ndash7] the pathogenesis diagnosis and specifictherapeutic strategies have duly gained the critical attentionof researchers

NASH is closely related tometabolic syndromes linked toobesity insulin resistance metabolic disorders free radicalreactions and dyslipidemia among others [8 9] Differentkinds of pharmacologic agents such as insulin sensitizersantioxidants n-3 polyunsaturated fatty acids ursodeoxy-cholic acid lipase inhibitors and lipid-lowering drugs aretherefore thought to have beneficial effects on the treatmentof NASH [10] However due to undesirable side effectsand the limited effectiveness of current chemical drugs for

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 897914 9 pageshttpdxdoiorg1011552015897914

2 Evidence-Based Complementary and Alternative Medicine

NAFLD researchers have focused on the development ofnatural drugs from herbs

Berberine (C20H18NO4) an isoquinoline alkaloid is one

of the main bioactive constituents of Rhizoma coptidis whichhas been used to treat diabetes in China for over a thousandyears [11] At present many derivatives of berberine forexample berberine hydrochloride berberine sulfate andberberine citrate have been developed for a patented drugused as an antimicrobial drug for treating gastroenteritisand bacillary dysentery in clinics [12] Moreover some novelpharmacological properties were discovered which mainlyconcern metabolic diseases such as obesity [13ndash15] type 2diabetes [16ndash18] and NAFLD [19ndash21] Although the pharma-cological mechanisms of berberine have been inferred frommany studies the data were mostly obtained from routineexaminations such as mediating insulin resistance regulat-ing the adenosine monophosphate-activated protein kinase(AMPK) pathway and modifying the gut microenvironment[19] The detailed metabolic mechanisms of berberinefortreating NASH have not been reported to date

Compared with other conventional methods is a betterapproach for studying the pathogenesis of diseases and phar-macological mechanisms by monitoring many endogenousldquolow-molecular-weightrdquo metabolites using data acquiringmethods such as LCMS NMR and GC [22 23] Herewe report a metabolomics study on a high-fat diet- (HFD-)induced NASH animal model using UPLC-Q-TOFMS tech-niques coupled with histopathology and biochemical analysisto reveal the mechanisms of berberine on the protectiveeffects against NASH

2 Materials and Methods

21 Chemicals and Reagents Standards and HFD were pur-chased from Charles River Company The feed formula ofHFD consists of 88 basic feed (58 fat 124 protein17 carbohydrate and 06 vitamin and mineral) 10 lardand 2 cholesterol Berberine was ordered from NanjingZe-Lang Pharmaceutical Technology Co Ltd (NanjingChina) Colorimetric kits of triglyceride HPLC grade formicacid were obtained from Sigma Chemical Co Ltd (StLouis MO USA) Methanol and acetonitrile (HPLC grade)were acquired from Fisher Corporation (Michigan USA)Ultrahigh purity water was prepared by Millipore-Q SAS67120MOLS HEIM (France)

22 Animal Treatment and Sampling A total of 30 maleSprague-Dawley rats (150plusmn10 g) were commercially obtainedfrom the Charles River Company (Beijing China Rodentlicense number SYXK 11-00-0039) Rats were randomlydivided into three groups (i) a normal control group witha standard diet and vehicle per os by gavage (NC) (ii) anHFD group with a vehicle (M) and (iii) an HFD group withberberine-treated group receiving HFD coupled intragastricadministration with berberine (Ber id 200mgkg bodyweight) The handing procedure lasted eight weeks Bloodsamples were collected using an abdominal aorta punctureand obtaining serum Parts of liver tissue were excised and

formalin-fixed immediately and some liver tissue was frozenby liquid nitrogen

23 Biochemical Assays Fasting serum alanine aminotrans-ferase (ALT) aspartate aminotransferase (AST) triglyceride(TG) total cholesterol (TC) blood glucose and low densitylipoprotein cholesterol (LDL-C) were measured by auto-mated procedures according to the manufacturersrsquo proto-cols (BioSino Bio-tech and Science Inc) TNF-120572 and IL-6 concentrations were analyzed using commercially avail-able ELISA kits (Shanghai Yanji Bio-tech Co Ltd) Fast-ing insulin concentrations were measured by a rat insulinradioimmunoassay kit and whole body insulin sensitivitywas estimated using the homeostasis model assessment ofinsulin resistance (HOMA-IR) using the following formulaHOMA-IR = fasting glucose (mmolL) times fasting insulin(120583UmL)225

24 Histopathological Evaluation Liver tissue paraffin sec-tions (4120583m)were stainedwith hematoxylin and eosin (HampE)The frozen section was dyed using red O oilThe pathologicaldiagnosis standard of NASH depends on the NAFLD activityscore (NAS) The diagnosis of NASH is clear with a NAS ge 5and is excluded when NAS lt 3 and values in between havethe possibility of NASH [24]

25 Metabolomic Analysis The global unbiased metabolicprofiling platform was based on a Waters ACQUITY UltraPerformance Liquid Chromatography (UPLC) system cou-pled to a Xevo G2-Q-TOFMS This platform was describedin detail in our previous publication [25] In brief 200120583Lof each serum sample was thawed on ice and extracted bymethanol protein precipitation The supernatant (400120583L)was transferred to a clean tube and evaporated until dry undera gentle stream of nitrogen The residue was reconstitutedwith 100 120583L of ultrahigh purity water and transferred to anautosampler vial UPLC-Q-TOFMSwas carried out using anACQUITYUPLCHSST3 column (21times 100mm 18 120583mUK)maintained at 45∘C The gradient program commenced with100 of a 01 formic acid in water for 1min at a flow rate of030mLmin which then changed to 60 methanol linearlywithin 8min and changed to 100 methanol linearly within2min This was then held for 2min before finally revertingback to 100 of 01 formic acid in water Once these initialsettings were reached the column was reequilibrated for2min The injection volume was 1 120583L The ESI ionizationsource parameters used were as follows capillary voltage3000V for positive ion mode and 2500V for negative ionmode cone voltage 30V for positive ion mode and 25V fornegative ion mode collision energy 6 eV desolvation gas750 Lh cone gas 50 Lh desolvation temperature 350∘Cand source temperature 100∘C Full scanmodewas employedin the mass range of 50ndash1200 amu Leucine-enkephalin wasused as the lock mass The collision energy was set at20 eV The UPLCMS raw data were preprocessed usingthe Micromass MarkerLynx Applications Manager version40 (Waters Corp Milford USA) The area of each peak

Evidence-Based Complementary and Alternative Medicine 3

after being recognized and aligned was normalized to thesummed total ion intensity of each chromatogram

The resulting data were then exported into EZinfo20 software (Waters Corporation Milford MA USA)for PCA and OPLS-DA Studentrsquos t-test and randomforest analysis were performed with MetaboAnalyst 20(httpwwwmetaboanalystca) to test the ability of themetabolomic data in correctly classifying the samples intotheir respective groups The MSE technique was used toassign the metabolite peaks Some available biochemicaldatabases such as HMDB (httpwwwhmdbca) KEGG(httpwwwgenomejpkegg) METLIN (httpmetlinscrippsedu) LIPID MAPS (httpwwwlipidmapsorg)and ChemSpider (httpwwwchemspidercom) were usedto analyze and explain potential biomarkers To assess theability to classify subjects as equivalents to the normalcontrol with NASH model and with berberine treatment arandom forest analysis was performed using the completemetabolomic data

26 Statistical Analysis The statistical analysis of the relativeintensity of biomarkers was performed by SPSS 170 Theintegration areas of the detected metabolites with high VIPvalues were first tested for the normality of the distribution Ifthe distribution followed the normality assumption paramet-ric Studentrsquos t-test was applied otherwise a nonparametricMann-Whitney 119880 test was performed to detect statisticallysignificant metabolites that were increased or decreasedbetween groups Differences were considered significant at avalue of 119875 lt 005 Statistical analysis of one-way ANOVAwasalso performed on the biochemical analysis data

3 Results and Discussion

31 Effects of Berberine on Liver Steatosis Inflammationand Serum Parameters Compared with the normal controladministration of HFD to rats caused a significant increase inbody weight However we observed a marked reduction inbody weight gain following berberine treatment Moreoverberberine was well tolerated in all rats without any adverseeffects (data not shown) To explore whether berberineexerted beneficial effects on liver histopathology paraffin-embedded specimens and frozen tissues were analyzed byHampE and red O oil staining The results showed that HFDcaused amarked accumulation of fat in hepatocytes (redOoilstaining demonstrated steatosis affected most of the hepato-cytes Figure 1(b)) and an evident infiltration of inflammatorycells in foci or in surrounding groups of hepatocytes asevidenced by arrows (Figure 1(a)) Treatment with berberineresulted in a general improvement of steatosis and inflam-mation associated with the HFD (Figures 1(a) and 1(b)) Noalterations were shown in the livers of rats fed with the stan-dard diet The results of histopathological scores indicatedthat berberine treatment plays a fighting role inHFD-inducedNASH In HFD-fed rats TNF-120572 the cytokines involved inthe development of inflammatory responses was significantlyhigher in serum but lower in the concentration in thetreatment of berberine (119875 lt 001 Figure 1(c)) Similarly IL-6

Table 1 Change in serum parameters of rats fed on a standard diet(NC) high-fat diet (M) or HFD with the berberine (Ber) treatmentfor eight weeks (119909 plusmn 119904 119899 = 10)

Index NC M BerALT (UL) 1949 plusmn 468 4067 plusmn 947lowastlowast 2092 plusmn 556

AST (UL) 19988 plusmn 3074 39060 plusmn 5913lowastlowast 28157 plusmn 7086

CHO(mmolL) 168 plusmn 019 281 plusmn 079lowast 207 plusmn 022

TG (mmolL) 056 plusmn 004 165 plusmn 012lowast 065 plusmn 013

LDL-C(mmolL) 016 plusmn 002 081 plusmn 042lowast 054 plusmn 038

Glucose(mmolL) 351 plusmn 067 474 plusmn 220lowast 324 plusmn 117

Insulin(mIUL) 1667 plusmn 40 1709 plusmn 240 1577 plusmn 353

HOMA index 26 plusmn 068 36 plusmn 302lowast 23 plusmn 075

Note lowast119875 lt 005 compared with NC group lowastlowast119875 lt 001 compared with NCgroup 119875 lt 005 and

119875 lt 001 compared with M group

another inflammation-related cytokine showed higher levelsin HFD rats and was downregulated by berberine treatment(119875 lt 005 Figure 1(d)) Biochemical serum parameters arereported in Table 1 The results showed that serum levels ofALT AST CHO TG and LDL-C were significantly higherin HFD-fed rats (119875 lt 005 or 119875 lt 001) However all theseparameters (except LDL-C) were lower in berberine-treatedrats (119875 lt 005 or 119875 lt 001) Compared to the normal controlHFD-fed rats showed a remarkable increase in fasting glucose(119875 lt 005) Interestingly the glucose alteration could beaffected by the treatment of berberine without changes infasting insulin levels The homeostasis model assessment forinsulin resistance (HOMA-IR) was lower in the berberine-treated group compared with that of HFD group (119875 lt 005)Our results are in agreement with most studies in whichberberine has a positive therapeutic effect on NAFLD due toits beneficial effects against insulin resistance inflammationand lipid metabolism [19 26ndash28]

32 Metabolomic Analysis A total of 1494 ions peaks wereobtained from UPLC-Q-TOFMS spectra (870 in positivemode and 624 in negative mode data not shown) To gain anoverview of the serummetabolic profile PCA and OPLS-DAwere used in the subsequent data analysis The score plots ofPCA showed well-delineated clusters and separation trendsof the normal control group the HFD group and HFD com-binedwith berberine-treated group in both positive ionmodeand negative ion mode highlighting the disease diagnosticpotential and drug intervention effect (Figure 2) Whenloading the plot we considered and selected key metabo-lites that predominantly accounted for variability along twovectors in the HFD-induced NASH model group relativeto the normal control group based on the VIP values (VIPgt 1) by two component OPLS-DA models (Figure 3) Theunivariate statistical method Studentrsquos t-test was performedon all serum features derived from calculations betweenthe HFD-induced NASH group and normal control group


NC M Ber

(a)

NC M Ber

(b)

0

5

10

15

20

25

NC M Ber

(ng

mL)

TNF-120572lowastlowast

(c)

0

10

20

30

40

50

60

70

80

NC M Ber

(pg

mL)

IL-6lowastlowast

(d)

Figure 1 Effects of berberine on hepatic pathological and the level of serum proinflammatory (TNF-120572 IL-6) (a) Liver tissue paraffin sectionand HampE staining (b) frozen section and red O oil staining (c) serum TNF-120572 and IL-6 level detected by ELISA

and berberine-treated group and model group respectivelyThe variables selected were those with statistical significance(119875 lt 005) A total of 72 metabolites (45 in positivemode and 27 in negative mode) were identified and listed(see supplementary data Table S1 in Supplementary Materialavailable online at httpdxdoiorg1011552015897914) Toaddress the perturbation degrees we further used ldquorandomforestrdquo analysis to filter significantly distinguished metabo-lites among three groups Figure 4 showed the importantfeatures ranked by random forest The features were rankedby themean decrease in classification accuracy when they arepermuted In the context of them we selected the potentialserum metabolic biomarkers and listed them in Table 2In brief the levels of sphingomyelin (SM 341 342 361423) and phosphatidylcholine (PC 341 374 383 384 386405 406 408) were significantly depressed (119875 lt 001119875 lt 005) while the levels of lysophosphatidylcholine (LPC

140 171 181 202) 13-hydroperoxy-911-octadecadienoicacid (13-HpODE) eicosatrienoic acid phytomonic aciddocosatrienoic acid and eicosenoic acid were significantlyhigher in HFD rats than those of the normal control rats(119875 lt 001 119875 lt 005) On the contrary berberinecould cause a systemic recovery from the HFD-inducedmetabolic perturbation in rats In the context of changedmetabolic profiles we sketched the perturbed metabolicnetwork associated with NASH (Figure 5) Figure 5 showsthat themetabolic pathways of phospholipid and unsaturatedfatty acid (UFA) correlated metabolites were involved inHFD-induced metabolic disorders

According to the traditional view 3-indoxyl-sulfuric acida renal toxin that accumulates in blood is derived fromtryptophan degradation via indole and indoxyl and conju-gated with sulfate PC is an essential phospholipid in mam-malian cells and tissue that is synthesized via the choline or


minus80

minus60

minus40

minus20

0

20

40

60

80minus

120

minus10

0

minus80

minus60

minus40

minus20 0 20 40 60 80 10

0

120

t[2]

t[1]ES+ model

Scores comp[1] versus comp[2] colored by sample group

(a)

minus40

minus20

0

20

40

minus10

0

minus80

minus60

minus40

minus20 0 20 40 60 80 10

0

t[2]

t[1]ESminus model


(b)

Figure 2 PCA scores plots discriminating HFD-fed rats from normal control rats and berberine treatment rats (a) positive ion mode (b)negative ion mode Note ◼ showed normal control rats 998771 showed HFD-induced NASH rats e showed NASH combined with berberinetreatment rats

S-plot (group 1 = minus1 group 2 = 1)10

05

00

minus05

minus10

p(c

orr)

[1]P

(cor

relat

ion)

minus02 minus01 00 01

p[1]P (loadings)

(a)


05

00

minus05

minus10

p(c

orr)

[1]P

(cor

relat

ion)

p[1]P (loadings)minus05 minus04 minus03 minus02 minus01 00 01 02

(b)

Figure 3 The results of multiple pattern recognition of serum biomarkers between normal control rats and HFD-induced NASH rats (a)OPLS-DA score plot under positive ion mode (b) OPLS-DA score plot under negative ion mode

phosphatidylethanolamine pathway The liver is principallyinvolved in themetabolism and release of PC into circulationUsually plasma PC is rapidly metabolized by phospholipaseA2 to release the fatty acid and LPC into the plasma poolfor distribution to extrahepatic tissues LPC is an importantsignaling molecule with diverse biological functions Reportsby other investigators have shown thatNAFLDwas associatedwith downregulated PC and upregulated LPC level whichsupported that PC and LPC might be effectors involvedin mediating cellular inflammatory response and insulinresistance [29 30] SM is a type of sphingophospholipidfound in animal cell membranes and is derived from PCand hydrolyzed by sphingomyelinases [31 32] It has beenshown that dietary supplementation with SM could decreasetotal hepatic cholesterol and triglyceride level and reduce

intestinal cholesterol absorption helping to maintain lipidhomeostasis [33] Our data agree with the mentioned reportand the results suggested that berberine could be usedagainst HFD by inducing NASH via directly perturbingthe abnormal phospholipid metabolic pathway involving therelative content of PC LPC and SM

In this experiment multiple increased metabolites ofUFA (13-HpODE eicosatrienoic acid phytomonic aciddocosatrienoic acid and eicosenoic acid) were involved inthe pathogenesis of HFD-induced NASH rats As knownthe mentioned UFAs are derived from linoleic acid (LA)[34 35] Except for the dietary resource the increased serumlevel of the abovementioned metabolites of UFAs suggestedupregulated endogenous LA which is paradoxically associ-ated with insulin sensitivity some researchers showed that


PC (405)LysoPC (202)

PC (341)PC (374)SM (423)

LysoPC (181)SM (342)SM (361)

CreatinePC (384)PC (386)

LysoPC (171)PC (383)SM (341)PC (352)

002 003 004 005

Mean decrease accuracy

N M B High

Low

ES+

(a)

Eicosenoic acidPhytomonic acid

SM (423)SM (342)SM (341)

Docosatrienoic acidSM (401)PC (386)

LysoPC (140)13-HpODE

PC (408)Indoxylsulfuric acid

PC (406)Eicosatrienoic acid

PC (384)

Mean decrease accuracy002 004 006 008 010

N M B High

Low

ESminus

(b)

Figure 4 Random forest importance plot for each subjects (a) positive ion mode (b) negative ion mode Note N normal control rat MNASH rat B berberine treatment rat

Table 2 Altered metabolites in serum samples of the nonalcoholic steatohepatitis model (M) and berberine (Ber) treatment group (PCphosphatidylcholine LysoPC lysophosphatidylcholine SM sphingomyelin 13-HpODE 13-hydroperoxy-octadecadienoic acid)

tR 119898119911 VIP ESI model Formula Metabolites Fold changes ClassifyMNC BerM

845 4683151229 217134 [M+H]+[M+FA-H]minus C22H46NO7P LysoPC (140) uarr darr Glycerophospholipids846 31122 109 [M-H]minus C18H32O4 13-HpODE uarr darr Linoleic acids873 50834 122 [M+H]+ C25H50NO7P LysoPC (171) uarr darr Glycerophospholipids888 52236 493 [M+H]+ C26H52NO7P LysoPC (181) uarr darr Glycerophospholipids896 54837 184 [M+H]+ C28H54NO7P LysoPC (202) uarr darr Glycerophospholipids925 30525 405 [M-H]minus C20H34O2 Eicosatrienoic acid uarr darr Fatty acids950 29526 119 [M-H]minus C19H36O2 Phytomonic acid uarr darr Fatty acids956 33328 106 [M-H]minus C22H38O2 Docosatrienoic acid uarr darr Fatty acids968 30928 228 [M-H]minus C20H38O2 Eicosenoic Acid uarr darr Fatty acids1003 7455570155 148193 [M+FA-H]minus[M+H]+ C39H77N2O6P SM (342) darr uarr Sphingolipids1030 8305787456 117104 [M+H]+[M+FA-H]minus C48H80NO8P PC (408) darr uarr Glycerophospholipids1037 74757 308 [M+FA-H]minus C39H79N2O6P SM (341) darr uarr Sphingolipids1038 70358 474 [M+H]+ C39H79N2O6P SM (341) darr uarr Sphingolipids1052 80657 508 [M+H]+ C46H80NO8P PC (386) darr uarr Glycerophospholipids1053 85056 255 [M+FA-H]minus C46H80NO8P PC (386) darr uarr Glycerophospholipids1092 79658 187 [M+H]+ C45H82NO8P PC (374) darr uarr Glycerophospholipids1094 73161 156 [M+H]+ C41H83N2O6P SM (361) darr uarr Sphingolipids1115 8346087859 445203 [M+H]+[M+FA-H]minus C48H84NO8P PC (406) darr uarr Glycerophospholipids1140 83662 227 [M+H]+ C48H86NO8P PC (405) darr uarr Glycerophospholipids1160 81262 625 [M+H]+ C46H86NO8P PC (383) uarr darr Glycerophospholipids1205 8116785566 381224 [M+H]+[M+FA-H]minus C47H91N2O6P SM (423) darr uarr SphingolipidsNote The data were calculated using the integrated peak areas The value represents fold change VIP was obtained from OPLS-DA with a threshold of 10The up and down arrows indicate a respective increased or decreased concentration of each metabolite in the HFD group versus normal control (MNC) andberberine-treated versus HFD group (BerM)


CDP-diacylglycerol + choline

Phosphatidylcholine (PC) darr

LysoPC (LPC) uarr

Phospholipid

SM darr

Linoleic acid

Eicosatrienoic acid uarr

Arachidonic acid


13-HpODE uarr

NASH

UFA

met

abol

ismPh

osph

olip

id m

etab

olism

Triglyceride uarrCholesterol uarr

uarr Eicosenoic acid

Figure 5 The metabolic network profile The map was gained byanalyzing known metabolic pathways

the increased proportion of LA in serum could improveinsulin sensitivity [36ndash39]while others held the opposite view[40] Although further studies might be required to clarifythe role of UFA in NASH altering the metabolism of UFAmight provide an interesting system to study pharmacologicalmechanisms of berberine

4 Conclusion

In this study we investigated the effect of berberine onNASHin anHFD-fed rat model using in vivoUPLC-MS techniquesOur HFD model showed serum hypertriglyceridemia andhypercholesterolemia confirming insulin resistant condi-tions Moreover the histopathological results from HFD-fed rats showed excessive accumulation of triglycerides withthe lipid vacuoles occupying the hepatocytes with enhancedinflammatory responses We demonstrated that berberine asa supplement could alleviate hepatic steatosis and patholog-ical grade reduce serum cholesterolemia and triglycerideand prevent the development of insulin resistance in HFD-fed rats In our strategy as proposed in this study globalmetabolic profiling was detected suggesting a metabolicdisruption associated with phospholipid and unsaturatedfatty acids in the HFD-induced NASH rats In contrastberberine has led to a network switch in the metabolicprofiles involving SM PC LPC 13-HpODE eicosatrienoicacid docosatrienoic acid and eicosenoic acidThese markersmatched with the metabolic pathways of phospholipid andunsaturated fatty acid (UFA) correlated metabolites whichpartly revealed the pharmacological mechanisms on NASHtreatment using berberine

Through our study in this paper alone we cannot yetidentify potential metabolites As widely known metabolite

identification is a complex process in metabolomics anddoes not provide 100 coverage [41] Currently there arestill many chromatographic peaks that cannot be identifiedin metabolomic data sets among the chromatography MSacquired data The reasons are that (1) the metabolites havenot been completely and experimentally characterized yetand the libraries and databases of experimental data appliedfor identification are not yet completed to reflect all knownmetabolites and that (2) not all metabolites known to bepresent can be purchased to construct mass spectral librariesto aid in the identification processes Therefore differentlevels of identification are available and the reportingprocedures for metabolite identification in metabolomicshave been described by the metabolomics standardsinitiative In this study the molecular formula was identifiedby searching the public databases such as HMDB(httpwwwhmdbca) KEGG (httpwwwgenomejpkegg)ChemSpider (httpwwwchemspidercom) and METLIN(httpmasspecscrippsedu) Therefore identification of alldetected metabolites is not currently achievable and isa significant limitation in metabolic profiling Becauseobtaining standards for precise identification andquantification is a complex and time-consuming processthis part of the experiment will be our focus in a future study

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Jian Li and Zezhou Liu contributed equally to this work

Acknowledgment

This work was partially supported by the 111 Project (Grantno B07007)

References

[1] P Angulo ldquoMedical progress nonalcoholic fatty liver diseaserdquoThe New England Journal of Medicine vol 346 no 16 pp 1221ndash1231 2002

[2] P Angulo ldquoNonalcoholic fatty liver diseaserdquo Revista de Gas-troenterologıa deMexico vol 70 supplement 3 pp 52ndash56 2005

[3] J C Cohen J D Horton and H H Hobbs ldquoHuman fatty liverdisease old questions and new insightsrdquo Science vol 332 no6037 pp 1519ndash1523 2011

[4] ZWang B Xia C Ma Z Hu X Chen and P Cao ldquoPrevalenceand risk factors of fatty liver disease in the Shuiguohu district ofWuhan city central Chinardquo Postgraduate Medical Journal vol83 no 977 pp 192ndash195 2007

[5] J-G Fan J Zhu X-J Li et al ldquoPrevalence of and risk factors forfatty liver in a general population of Shanghai Chinardquo Journalof Hepatology vol 43 no 3 pp 508ndash514 2005

[6] M Hamaguchi T Kojima N Takeda et al ldquoThe metabolicsyndrome as a predictor of nonalcoholic fatty liver diseaserdquoAnnals of Internal Medicine vol 143 no 10 pp 722ndash728 2005


[7] J D Browning L S Szczepaniak R Dobbins et al ldquoPrevalenceof hepatic steatosis in an urban population in the United Statesimpact of ethnicityrdquo Hepatology vol 40 no 6 pp 1387ndash13952004

[8] H Tilg and A R Moschen ldquoEvolution of inflammationin nonalcoholic fatty liver disease the multiple parallel hitshypothesisrdquo Hepatology vol 52 no 5 pp 1836ndash1846 2010

[9] L Pacifico V Nobili C Anania P Verdecchia and C ChiesaldquoPediatric nonalcoholic fatty liver disease metabolic syndromeand cardiovascular riskrdquoWorld Journal of Gastroenterology vol17 no 26 pp 3082ndash3091 2011

[10] K J P Schwenger and J P Allard ldquoClinical approaches to non-alcoholic fatty liver diseaserdquoWorld Journal of Gastroenterologyvol 20 no 7 pp 1712ndash1723 2014

[11] J-C Li X-F Shen and X-L Meng ldquoA traditional Chinesemedicine JiuHuangLian (Rhizoma coptidis steamed with ricewine) reduces oxidative stress injury in type 2 diabetic ratsrdquoFood and Chemical Toxicology vol 59 pp 222ndash229 2013

[12] C-H Chang W-Y Huang C-H Lai et al ldquoDevelopment ofnovel nanoparticles shelled with heparin for berberine deliveryto treatHelicobacter pylorirdquo Acta Biomaterialia vol 7 no 2 pp593ndash603 2011

[13] X Zhang Y Zhao M Zhang et al ldquoStructural changes of gutmicrobiota during berberine-mediated prevention of obesityand insulin resistance in high-fat diet-fed ratsrdquo PLoS ONE vol7 no 8 Article ID e42529 2012

[14] Y Hu and G E Davies ldquoBerberine inhibits adipogenesis inhigh-fat diet-induced obesity micerdquo Fitoterapia vol 81 no 5pp 358ndash366 2010

[15] W S Kim Y S Lee S H Cha et al ldquoBerberine improveslipid dysregulation in obesity by controlling central andperipheral AMPK activityrdquo American Journal of PhysiologymdashEndocrinology and Metabolism vol 296 no 4 pp E812ndashE8192009

[16] X Xie X Meng X Zhou X Shu and H Kong ldquoResearchon therapeutic effect and hemorrheology change of berberinein new diagnosed patients with type 2 diabetes combiningnonalcoholic fatty liver diseaserdquo Zhongguo Zhong Yao Za Zhivol 36 no 21 pp 3032ndash3035 2011

[17] Y Gu Y Zhang X Shi et al ldquoEffect of traditional Chinesemedicine berberine on type 2 diabetes based on comprehensivemetabonomicsrdquo Talanta vol 81 no 3 pp 766ndash772 2010

[18] H Zhang J Wei R Xue et al ldquoBerberine lowers blood glucosein type 2 diabetes mellitus patients through increasing insulinreceptor expressionrdquo Metabolism Clinical and Experimentalvol 59 no 2 pp 285ndash292 2010

[19] Y Liu L Zhang H Song and G Ji ldquoUpdate on berberine innonalcoholic Fatty liver diseaserdquo Evidence-Based Complemen-tary and Alternative Medicine vol 2013 Article ID 308134 8pages 2013

[20] X Chang H Yan J Fei et al ldquoBerberine reduces methylationof the MTTP promoter and alleviates fatty liver induced by ahigh-fat diet in ratsrdquo Journal of Lipid Research vol 51 no 9 pp2504ndash2515 2010

[21] M F Abdelmalek S O Sanderson P Angulo et al ldquoBetainefor nonalcoholic fatty liver disease results of a randomizedplacebo-controlled trialrdquo Hepatology vol 50 no 6 pp 1818ndash1826 2009

[22] J T Bjerrum O H Nielsen F Hao et al ldquoMetabonomicsin ulcerative colitis diagnostics biomarker identification andinsight into the pathophysiologyrdquo Journal of Proteome Researchvol 9 no 2 pp 954ndash962 2010

[23] J C Lindon E Holmes M E Bollard E G Stanley and J KNicholson ldquoMetabonomics technologies and their applicationsin physiologicalmonitoring drug safety assessment and diseasediagnosisrdquo Biomarkers vol 9 no 1 pp 1ndash31 2004

[24] D E Kleiner E M Brunt M van Natta et al ldquoDesign andvalidation of a histological scoring system for nonalcoholic fattyliver diseaserdquo Hepatology vol 41 no 6 pp 1313ndash1321 2005

[25] X Gao M Guo L Peng et al ldquoUPLC Q-TOFMS-basedmetabolic profiling of urine reveals the novel antipyreticmechanisms of qingkailing injection in a rat model of yeast-induced pyrexiardquo Evidence-Based Complementary and Alterna-tive Medicine vol 2013 Article ID 864747 8 pages 2013

[26] L-J Xing L Zhang T Liu Y-Q Hua P-Y Zheng and G JildquoBerberine reducing insulin resistance by up-regulating IRS-2mRNA expression in nonalcoholic fatty liver disease (NAFLD)rat liverrdquo European Journal of Pharmacology vol 668 no 3 pp467ndash471 2011

[27] J Y Zhou S W Zhou K B Zhang et al ldquoChronic effectsof berberine on blood liver glucolipid metabolism and liverPPARs expression in diabetic hyperlipidemic ratsrdquo Biologicaland Pharmaceutical Bulletin vol 31 no 6 pp 1169ndash1176 2008

[28] Y S Lee W S Kim K H Kim et al ldquoBerberine a naturalplant product activates AMP-activated protein kinase withbeneficial metabolic effects in diabetic and insulin-resistantstatesrdquo Diabetes vol 55 no 8 pp 2256ndash2264 2006

[29] B M Arendt D W Ma B Simons et al ldquoNonalcoholic fattyliver disease is associated with lower hepatic and erythrocyteratios of phosphatidylcholine to phosphatidylethanolaminerdquoApplied Physiology Nutrition andMetabolism vol 38 no 3 pp334ndash340 2013

[30] M S Han Y-M LimWQuan et al ldquoLysophosphatidylcholineas an effector of fatty acid-induced insulin resistancerdquo Journal ofLipid Research vol 52 no 6 pp 1234ndash1246 2011

[31] Z Li L B Agellon and D E Vance ldquoA role for high den-sity lipoproteins in hepatic phosphatidylcholine homeostasisrdquoBiochimica et Biophysica Acta Molecular and Cell Biology ofLipids vol 1771 no 7 pp 893ndash900 2007

[32] R Testi ldquoSphingomyelin breakdown and cell faterdquo Trends inBiochemical Sciences vol 21 no 12 pp 468ndash471 1996

[33] R W S Chung A Kamili S Tandy et al ldquoDietary sph-ingomyelin lowers hepatic lipid levels and inhibits intestinalcholesterol absorption in high-fat-fed micerdquo PLoS ONE vol 8no 2 Article ID e55949 2013

[34] P D Klein ldquoLinoleic acid and cholesterol metabolism in therat II Effects of dietary cholesterol on plasma and liver estercompositionrdquo Archives of Biochemistry and Biophysics vol 81no 2 pp 382ndash389 1959

[35] P D Klein ldquoLinoleic acid and cholesterol metabolism in the ratI The effect of dietary fat and linoleic acid levels on the contentand composition of cholesterol esters in liver and plasmardquoArchives of Biochemistry and Biophysics vol 76 no 1 pp 56ndash64 1958

[36] H Kahleova M Matoulek M Bratova et al ldquoVegetarian diet-induced increase in linoleic acid in serum phospholipids isassociated with improved insulin sensitivity in subjects withtype 2 diabetesrdquo Nutrition amp Diabetes vol 3 article e75 2013

[37] P Parra A Palou and F Serra ldquoModerate doses of conjugatedlinoleic acid reduce fat gainmaintain insulin sensitivitywithoutimpairing inflammatory adipose tissue status in mice fed ahigh-fat dietrdquo Nutrition and Metabolism vol 7 article 5 2010

[38] B Ahren AMari C L Fyfe et al ldquoEffects of conjugated linoleicacid plus 119899-3 polyunsaturated fatty acids on insulin secretion


and estimated insulin sensitivity in menrdquo European Journal ofClinical Nutrition vol 63 no 6 pp 778ndash786 2009

[39] F Moloney T-P Yeow A Mullen J J Nolan and H M RocheldquoConjugated linoleic acid supplementation insulin sensitivityand lipoprotein metabolism in patients with type 2 diabetesmellitusrdquoTheAmerican Journal of Clinical Nutrition vol 80 no4 pp 887ndash895 2004

[40] Y Kawashima K Musoh and H Kozuka ldquoPeroxisome prolif-erators enhance linoleic acid metabolism in rat liver Increasedbiosynthesis of 1205966 polyunsaturated fatty acidsrdquo Journal ofBiological Chemistry vol 265 no 16 pp 9170ndash9175 1990

[41] W B Dunn D Broadhurst P Begley et al ldquoProcedures forlarge-scale metabolic profiling of serum and plasma using gaschromatography and liquid chromatography coupled to massspectrometryrdquo Nature Protocols vol 6 no 7 pp 1060ndash10832011

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


NAFLD researchers have focused on the development ofnatural drugs from herbs

Berberine (C20H18NO4) an isoquinoline alkaloid is one

of the main bioactive constituents of Rhizoma coptidis whichhas been used to treat diabetes in China for over a thousandyears [11] At present many derivatives of berberine forexample berberine hydrochloride berberine sulfate andberberine citrate have been developed for a patented drugused as an antimicrobial drug for treating gastroenteritisand bacillary dysentery in clinics [12] Moreover some novelpharmacological properties were discovered which mainlyconcern metabolic diseases such as obesity [13ndash15] type 2diabetes [16ndash18] and NAFLD [19ndash21] Although the pharma-cological mechanisms of berberine have been inferred frommany studies the data were mostly obtained from routineexaminations such as mediating insulin resistance regulat-ing the adenosine monophosphate-activated protein kinase(AMPK) pathway and modifying the gut microenvironment[19] The detailed metabolic mechanisms of berberinefortreating NASH have not been reported to date

Compared with other conventional methods is a betterapproach for studying the pathogenesis of diseases and phar-macological mechanisms by monitoring many endogenousldquolow-molecular-weightrdquo metabolites using data acquiringmethods such as LCMS NMR and GC [22 23] Herewe report a metabolomics study on a high-fat diet- (HFD-)induced NASH animal model using UPLC-Q-TOFMS tech-niques coupled with histopathology and biochemical analysisto reveal the mechanisms of berberine on the protectiveeffects against NASH

2 Materials and Methods

21 Chemicals and Reagents Standards and HFD were pur-chased from Charles River Company The feed formula ofHFD consists of 88 basic feed (58 fat 124 protein17 carbohydrate and 06 vitamin and mineral) 10 lardand 2 cholesterol Berberine was ordered from NanjingZe-Lang Pharmaceutical Technology Co Ltd (NanjingChina) Colorimetric kits of triglyceride HPLC grade formicacid were obtained from Sigma Chemical Co Ltd (StLouis MO USA) Methanol and acetonitrile (HPLC grade)were acquired from Fisher Corporation (Michigan USA)Ultrahigh purity water was prepared by Millipore-Q SAS67120MOLS HEIM (France)

22 Animal Treatment and Sampling A total of 30 maleSprague-Dawley rats (150plusmn10 g) were commercially obtainedfrom the Charles River Company (Beijing China Rodentlicense number SYXK 11-00-0039) Rats were randomlydivided into three groups (i) a normal control group witha standard diet and vehicle per os by gavage (NC) (ii) anHFD group with a vehicle (M) and (iii) an HFD group withberberine-treated group receiving HFD coupled intragastricadministration with berberine (Ber id 200mgkg bodyweight) The handing procedure lasted eight weeks Bloodsamples were collected using an abdominal aorta punctureand obtaining serum Parts of liver tissue were excised and

formalin-fixed immediately and some liver tissue was frozenby liquid nitrogen

23 Biochemical Assays Fasting serum alanine aminotrans-ferase (ALT) aspartate aminotransferase (AST) triglyceride(TG) total cholesterol (TC) blood glucose and low densitylipoprotein cholesterol (LDL-C) were measured by auto-mated procedures according to the manufacturersrsquo proto-cols (BioSino Bio-tech and Science Inc) TNF-120572 and IL-6 concentrations were analyzed using commercially avail-able ELISA kits (Shanghai Yanji Bio-tech Co Ltd) Fast-ing insulin concentrations were measured by a rat insulinradioimmunoassay kit and whole body insulin sensitivitywas estimated using the homeostasis model assessment ofinsulin resistance (HOMA-IR) using the following formulaHOMA-IR = fasting glucose (mmolL) times fasting insulin(120583UmL)225

24 Histopathological Evaluation Liver tissue paraffin sec-tions (4120583m)were stainedwith hematoxylin and eosin (HampE)The frozen section was dyed using red O oilThe pathologicaldiagnosis standard of NASH depends on the NAFLD activityscore (NAS) The diagnosis of NASH is clear with a NAS ge 5and is excluded when NAS lt 3 and values in between havethe possibility of NASH [24]

25 Metabolomic Analysis The global unbiased metabolicprofiling platform was based on a Waters ACQUITY UltraPerformance Liquid Chromatography (UPLC) system cou-pled to a Xevo G2-Q-TOFMS This platform was describedin detail in our previous publication [25] In brief 200120583Lof each serum sample was thawed on ice and extracted bymethanol protein precipitation The supernatant (400120583L)was transferred to a clean tube and evaporated until dry undera gentle stream of nitrogen The residue was reconstitutedwith 100 120583L of ultrahigh purity water and transferred to anautosampler vial UPLC-Q-TOFMSwas carried out using anACQUITYUPLCHSST3 column (21times 100mm 18 120583mUK)maintained at 45∘C The gradient program commenced with100 of a 01 formic acid in water for 1min at a flow rate of030mLmin which then changed to 60 methanol linearlywithin 8min and changed to 100 methanol linearly within2min This was then held for 2min before finally revertingback to 100 of 01 formic acid in water Once these initialsettings were reached the column was reequilibrated for2min The injection volume was 1 120583L The ESI ionizationsource parameters used were as follows capillary voltage3000V for positive ion mode and 2500V for negative ionmode cone voltage 30V for positive ion mode and 25V fornegative ion mode collision energy 6 eV desolvation gas750 Lh cone gas 50 Lh desolvation temperature 350∘Cand source temperature 100∘C Full scanmodewas employedin the mass range of 50ndash1200 amu Leucine-enkephalin wasused as the lock mass The collision energy was set at20 eV The UPLCMS raw data were preprocessed usingthe Micromass MarkerLynx Applications Manager version40 (Waters Corp Milford USA) The area of each peak





















NC M Ber

(a)

NC M Ber

(b)

0

5

10

15

20

25

NC M Ber

(ng

mL)


(c)

0

10

20

30

40

50

60

70

80

NC M Ber

(pg

mL)

IL-6lowastlowast

(d)






minus80

minus60

minus40

minus20

0

20

40

60

80minus

120

minus10

0

minus80

minus60

minus40

minus20 0 20 40 60 80 10

0

120

t[2]

t[1]ES+ model


(a)

minus40

minus20

0

20

40

minus10

0

minus80

minus60

minus40

minus20 0 20 40 60 80 10

0

t[2]

t[1]ESminus model


(b)



05

00

minus05

minus10

p(c

orr)

[1]P

(cor

relat

ion)


p[1]P (loadings)

(a)


05

00

minus05

minus10

p(c

orr)

[1]P

(cor

relat

ion)


(b)







PC (341)PC (374)SM (423)

LysoPC (181)SM (342)SM (361)


LysoPC (171)PC (383)SM (341)PC (352)

002 003 004 005


N M B High

Low

ES+

(a)


SM (423)SM (342)SM (341)





PC (384)


N M B High

Low

ESminus

(b)








LysoPC (LPC) uarr

Phospholipid

SM darr

Linoleic acid


Arachidonic acid


13-HpODE uarr

NASH

UFA

met

abol

ismPh

osph

olip

id m

etab

olism





4 Conclusion








Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































NC M Ber

(a)

NC M Ber

(b)

0

5

10

15

20

25

NC M Ber

(ng

mL)


(c)

0

10

20

30

40

50

60

70

80

NC M Ber

(pg

mL)

IL-6lowastlowast

(d)






minus80

minus60

minus40

minus20

0

20

40

60

80minus

120

minus10

0

minus80

minus60

minus40

minus20 0 20 40 60 80 10

0

120

t[2]

t[1]ES+ model


(a)

minus40

minus20

0

20

40

minus10

0

minus80

minus60

minus40

minus20 0 20 40 60 80 10

0

t[2]

t[1]ESminus model


(b)



05

00

minus05

minus10

p(c

orr)

[1]P

(cor

relat

ion)


p[1]P (loadings)

(a)


05

00

minus05

minus10

p(c

orr)

[1]P

(cor

relat

ion)


(b)







PC (341)PC (374)SM (423)

LysoPC (181)SM (342)SM (361)


LysoPC (171)PC (383)SM (341)PC (352)

002 003 004 005


N M B High

Low

ES+

(a)


SM (423)SM (342)SM (341)





PC (384)


N M B High

Low

ESminus

(b)








LysoPC (LPC) uarr

Phospholipid

SM darr

Linoleic acid


Arachidonic acid


13-HpODE uarr

NASH

UFA

met

abol

ismPh

osph

olip

id m

etab

olism





4 Conclusion








Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NC M Ber

(a)

NC M Ber

(b)

0

5

10

15

20

25

NC M Ber

(ng

mL)


(c)

0

10

20

30

40

50

60

70

80

NC M Ber

(pg

mL)

IL-6lowastlowast

(d)






minus80

minus60

minus40

minus20

0

20

40

60

80minus

120

minus10

0

minus80

minus60

minus40

minus20 0 20 40 60 80 10

0

120

t[2]

t[1]ES+ model


(a)

minus40

minus20

0

20

40

minus10

0

minus80

minus60

minus40

minus20 0 20 40 60 80 10

0

t[2]

t[1]ESminus model


(b)



05

00

minus05

minus10

p(c

orr)

[1]P

(cor

relat

ion)


p[1]P (loadings)

(a)


05

00

minus05

minus10

p(c

orr)

[1]P

(cor

relat

ion)


(b)







PC (341)PC (374)SM (423)

LysoPC (181)SM (342)SM (361)


LysoPC (171)PC (383)SM (341)PC (352)

002 003 004 005


N M B High

Low

ES+

(a)


SM (423)SM (342)SM (341)





PC (384)


N M B High

Low

ESminus

(b)








LysoPC (LPC) uarr

Phospholipid

SM darr

Linoleic acid


Arachidonic acid


13-HpODE uarr

NASH

UFA

met

abol

ismPh

osph

olip

id m

etab

olism





4 Conclusion








Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















minus80

minus60

minus40

minus20

0

20

40

60

80minus

120

minus10

0

minus80

minus60

minus40

minus20 0 20 40 60 80 10

0

120

t[2]

t[1]ES+ model


(a)

minus40

minus20

0

20

40

minus10

0

minus80

minus60

minus40

minus20 0 20 40 60 80 10

0

t[2]

t[1]ESminus model


(b)



05

00

minus05

minus10

p(c

orr)

[1]P

(cor

relat

ion)


p[1]P (loadings)

(a)


05

00

minus05

minus10

p(c

orr)

[1]P

(cor

relat

ion)


(b)







PC (341)PC (374)SM (423)

LysoPC (181)SM (342)SM (361)


LysoPC (171)PC (383)SM (341)PC (352)

002 003 004 005


N M B High

Low

ES+

(a)


SM (423)SM (342)SM (341)





PC (384)


N M B High

Low

ESminus

(b)








LysoPC (LPC) uarr

Phospholipid

SM darr

Linoleic acid


Arachidonic acid


13-HpODE uarr

NASH

UFA

met

abol

ismPh

osph

olip

id m

etab

olism





4 Conclusion








Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















PC (341)PC (374)SM (423)

LysoPC (181)SM (342)SM (361)


LysoPC (171)PC (383)SM (341)PC (352)

002 003 004 005


N M B High

Low

ES+

(a)


SM (423)SM (342)SM (341)





PC (384)


N M B High

Low

ESminus

(b)








LysoPC (LPC) uarr

Phospholipid

SM darr

Linoleic acid


Arachidonic acid


13-HpODE uarr

NASH

UFA

met

abol

ismPh

osph

olip

id m

etab

olism





4 Conclusion








Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















LysoPC (LPC) uarr

Phospholipid

SM darr

Linoleic acid


Arachidonic acid


13-HpODE uarr

NASH

UFA

met

abol

ismPh

osph

olip

id m

etab

olism





4 Conclusion








Acknowledgment


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Metabolomics Profiling to Investigate the ...downloads.hindawi.com/journals/ecam/2015/897914.pdf · eicosatrienoic acid, docosatrienoic acid, and eicosenoic acid