Lipidomics Identification of Metabolic Biomarkers ... - ACS Publications

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Lipidomics Identification of Metabolic Biomarkers in Chemically Induced Hypertriglyceridemic Mice Hiu Yee Kwan,†,§ Yong-Mei Hu,†,§ Chi Leung Chan,† Hui-Hui Cao,† Chi Yan Cheng,† Si-Yuan Pan,‡ Kai Wing Tse,† Yiu Cheong Wu,† Zhi-Ling Yu,*,† and Wang Fun Fong*,† †

Centre for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China ‡ Department of Pharmacology, Beijing University of Chinese Medicine, Beijing, China S Supporting Information *

ABSTRACT: In this study, we aim to identify the potential biomarkers in hTG pathogenesis in schisandrin B-induced hTG mouse model. To investigate whether these identified biomarkers are only specific to schisandrin B-induced hTG mouse model, we also measured these biomarkers in a high fat diet (HFD)-induced hTG mouse model. We employed a LC/MS/MS-based lipidomic approach for the study. Mouse liver and serum metabolites were separated by reversed phase liquid chromatography. Metabolite candidates were identified by matching with marker retention times, isotope distribution patterns, and high-resolution MS/MS fragmentation patterns. Subsequently, target candidates were quantified by quantitative MS. In the schisandrin B-induced hTG mice, we found that the plasma fatty acids, diglyceroids, and phospholipids were significantly increased. Palmitic acid and stearic acid were increased in the plasma; oleic acid, linoleic acid, linolenic acid, arachidonic acid, and docosahexaenoic acid were increased in both the plasma and the liver. Acetyl-CoA, malonyl-CoA, and succinyl-CoA were increased only in the liver. The changes in levels of these identified markers were also observed in HFD-induced hTG mouse model. The consistent results obtained from both hTG models not only suggest novel biomarkers in hTG pathogenesis, but they also provide insight into the underlying mechanism of the schisandrin B-induced hTG. KEYWORDS: reversed phase liquid chromatography (RPLC), high-resolution MS/MS fragmentation, quantification, malonyl CoA

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INTRODUCTION Hypertriglyceridemia (hTG) is defined as a condition in which serum triglyceride (TG) reaches 150 mg/dL. In severe clinical cases, serum TG levels may exceed 500 mg/dL.1 By dry weight, TGs make up approximately 86%, 55%, and 23% of chylomicrometers, very low density lipoproteins (VLDLs), and intermediate density lipoproteins, respectively. hTG is a subtype of hyperlipidemia. On the basis of clinical and biochemical findings, elevation of TG levels in the serum occurs in all five hyperlipidemia subtypes except type II a.2 hTG is becoming prevalent in modern affluent societies and is a risk factor for nonalcoholic fatty liver disease3−5 and atherosclerosis, which may lead to coronary artery diseases.1 Moreover, it also increases the risk of pancreatitis6 and has an adverse effect on bone regeneration and strength.7 TG in the blood mainly comes from two sources. TG may come from dietary fat and eventually acquired by chylomicrometers. TG may also be derived by de novo fatty acid synthesis in the liver and then transported to the circulation by VLDL. Over-secretion of VLDL from liver may cause hTG condition in mice.8 © 2013 American Chemical Society

A number of hTG mouse models have been reported including high fat diet (HFD)-induced,8,9 sucrose-induced,10 fructose-induced,11 Western diet12(0.06% cholesterol/21% mild fat)-induced, and gene knockout models.13,14 Drug- or chemical-induced hTG models may offer alternative hTG models. Schisandrin B, a bioactive dibenzocycloocatdiene isolated from the dried fruits of Schisandrin chinensis, is known to decrease hepatic triglyceride levels in mouse model of hypercholesterolemia.15 However, if mice received high doses of Schisandrin B after being fasted overnight, hTG was developed,16,17 although the exact mechanism has not yet been delineated. We also found that the schisandrin B-induced hTG condition can be reversed by fenofibrate treatment.17 Therefore, this hTG model might be used in studying actions of TG lowering drugs and in understanding hTG pathophysiology. Metabolomics is emerging as an important and powerful approach for probing the relationship between complex metabolite compositions and a particular medical condition.18 Received: November 2, 2012 Published: January 22, 2013 1387

dx.doi.org/10.1021/pr3010327 | J. Proteome Res. 2013, 12, 1387−1398

Journal of Proteome Research

Article

Table 1. Chromatographic and Mass Spectrometric Parameters for FFA, Glycerolipids, Phospholipids, and Coenzyme Analysisa total FFA column

Agilent Eclipse plus C18 RRHD (2.1 mm × 50 mm, 1.8 μm)

mobile phase

A: H2O with 10 mM formate acetate, pH = 4.0 adjust with acetic acid B: ACN with 0.1% formic acid, 5−60%B (0−4 min) 60−80% B (4−4.5 min) 80−100% B (4.5−14 min)

gradient

flow rate injection volume ESI parameters polarity capillary voltage sheath gas flow rate sheath gas temp drying gas flow rate drying gas temp nebulizer gas fragmentor voltage nozzle voltage scanning range a

glycerolipids/phospholipids

coenzyme

Agilent Eclipse plus C18 RRHD (2.1 mm × 50 mm, 1.8 μm) A: ACN/H2O (4:6) with 10 mM ammonia formate B: ACN/isopropanol (1:9) with 10 mM ammonia formate 15−25% B (0−5 min) 25−40% B (5−7 min) 40−80% B (7−7.5 min) 80−85% B (7.5−24 min) 85−100% B (24−26 min) 400 μL 5 μL

210 μL 5 μL

negative 4.2 kV

positive 4.0 kV

positive 4.5 kV

10 L/min

10 L/min

10 L/min

350 °C

400 °C

400 °C

7 L/min

6 L/min

10 L/min

300 °C

300 °C

280 °C

30 psig 175 V

40 psig 150 V

50 psig 150 V

0V 100−1500 m/z

300 V 150−2000 m/z

0V refer to Table 5B

350 μL 5 μL

Agilent Eclipse plus C18 RRHD (2.1 mm × 50 mm, 1.8 μm) A: H2O with 5 mM ammonium acetate, 5 mM TEA, pH 5.6 adjust with acetic acid B: ACN with 0.1% formic acid 2.5−2.5% B (0−0.5 min) 2.5−12.5% B (0.5−5 min) 12.5−25% B (5−10 min)

Abbreviation: Electrospray ionization (ESI).

Technology Co. Ltd. (Kunming, China), and its purity was determined to be 98% by HPLC.

This method is particularly valuable for studying multicomponent, multitarget systems. The LC/MS/MS-based method has been applied to lipidomics studies because of its sensitivity, high resolution, and versatility.19,20 In this study, we aim to identify the potential biomarkers in hTG pathogenesis in the schisandrin B-induced hTG mouse model using a lipidomic approach. To investigate whether the identified biomarkers are specific to the schisandrin B-induced hTG model, we also measured these biomarkers in a HFDinduced hTG mouse model. The results obtained in this study not only suggest novel biomarkers in hTG pathogenesis, they may also provide information to suggest the underlying mechanism of the schisandrin B-induced hTG.

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Animal Models

Imprinting control region (ICR) male mice were used for the schisandrin B-induced hTG mouse model. C57BL/6 (C57) male mice were used for the HFD-induced hTG mouse model. Both mouse species were purchased from Hong Kong Chinese University Animal Facilities, and animal maintenance and tissue collections were carried out in accordance with the Abbott Laboratories guidelines on animal care and usage. All experimental protocols were endorsed by the University Committee on Research Practice in the Hong Kong Baptist University. Before the experiment, mice were allowed a 7-days habituation period in the laboratory in which the experiments were carried out. To establish schisandrin B-induced acute hTG mouse model, ICR mice were fasted overnight with free access to water and were randomly separated into five groups of 6 mice. The five groups included one vehicle control group (feed with olive oil) and four treatment groups (administrated with schisandrin B at four concentrations in olive oil). These high dose groups were later established as acute hTG animal model in this study. To establish HFD-induced hTG mouse model, C57 mice were fed HFD prepared by mixing 80 g of regular diet purchased from LabDiet (# 5001 LabDiet USA) with 20 g of lard21 for 15 weeks. Control mice were fed with regular diet (LabDiet #5001 USA) only. Both diet and water were supplied ad libitum during the experiment.

EXPERIMENTAL SECTION

Chemicals

Linolenic acid, linoleic acid, oleic acid, palmitic acid, stearic acid, hepteric acid, arachidonic acid (AA), docosahexaenoic acid (DHA), hexadecanoic-15,15,16,16,16-d5 acid, phosphatidylcholine-C 42 H 80 NO 8 P (PC1), phosphatidylethanolamineC9H18NO8P (PE1), phosphatidylserine-C13H24NO10P (PS1), sphingomyelin-C41H84N2O6P (SM1), CoASH, acetyl-CoA, malonyl-CoA, n-propionyl-CoA, succinyl-CoA, ammonia acetate, trimethylamine (TEA), and trichloroacetic acid (TCA) were all purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). The assay kit for measuring triglycerides and all organic solvents are HPLC grade from Sigma-Aldrich Chemical Co. (St. Louis, MO). Schisandrin B was purchased from Ningli 1388



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Sample Preparation

1290 Infinity UHPLC for specific quantification of targeted bioactive lipids and lipid metabolites. Different chromatographic gradient programs were used for different metabolites analyses as listed in Table 1. Chromatographic separation of total FFA and total lipids was performed on an Agilent Eclipse plus C18 RRHD column (2.1 mm × 100 mm, 1.8 μm) eluted with a gradient of solvent FAA-A (Milli-Q water contains 10 mM formate acetate, pH value 4.0 adjusted with acetic acid) and solvent FAA-B (acetonitrile contains 0.1% formic acid). Total lipids (glycerolipids and phospholipids) were separated with a gradient mobile phase comprising solvent TL-A (40% ACN with 10 mM ammonium acetate) and solvent TL-B (acetonitrile:isopropanol, 1:9) with 10 mM ammonium acetate. Chromatographic separation for the four short-chain acyl-CoA was performed on an Agilent Eclipse plus C18 RRHD column (2.1 mm × 50 mm, 1.8 μm) eluted with a gradient consisted of solvent AC-A (5 mM ammonium acetate, 5 mM TEA, pH values 5.6) and solvent AC-B (acetonitrile contains 0.1% formic acid). There was a pre-equilibration period of 10 min between individual runs. The electrospray ionization mass spectrometry (ESI−MS) spectra were acquired in both negative and positive ionization modes. Ultrapure N2 was used as the nebulizer and sheath gas. Product ion scanning experiments were conducted using ultrahigh-purity N2 as collision gas, and the collision energy was optimized for each representative analyte to generate the most abundant production ions. The quality and quantity analyses of total lipids were conducted in negative ion mode, while the quantification of acyl-CoA was carried out in positive mode. Resulted ion spectra were further used to select the precursor−product ion pairs for the development of MRM assays. The mass analyzer was scanning from 100 to 2000 (m/ z). The electrospray ionization (ESI) parameters were set as in Table 1.

For the triglyceride (TG) biochemical assay, blood samples were withdrawn from mice by heart puncture, and samples were frozen at −20 °C. For the comprehensive lipidomics investigation, blood samples were collected in heparin tubes and centrifuged immediately at 500g to get the plasma. For lipid extraction, to each 0.2 mL plasma sample were added 0.3 mL of 0.5 M KH2PO4, 1.5 mL of chloroform, and 0.5 mL of methanol. After vortex for 2 min and centrifugation at 2000g, the lower phase was collected and evaporated under a nitrogen stream. The residue was reconstituted in 10 μL of isopropanol, diluted with 90 μL of methanol, and subjected to FFA and phospholipid analyses. For the total glycerolipids analysis, the residue was reconstituted with 100 μL of isopropanol− acetonitrile (1:9, v/v), mixed well, and stored at −20 °C prior to LC/MS/MS analysis. Fresh mouse livers were quickly removed and snap frozen in liquid nitrogen. For chemical analyses, liver samples (0.2 g) were homogenized using Polyton PT 6600 in 750 μL of prechilled 10% trichloroacetic acid (TCA) supplemented with 500 ng/mL of mixed internal standard. The tissue homogenates were centrifuged at 14 500g for 10 min at 4 °C. The resulting supernatants were then extracted twice with 5 mL of chloroform/methanol solution (v/v = 2:1, Folch Reagent). After being centrifuged at 5000g for 5 min, the upper aqueous phase was carefully collected, combined, and loaded onto an Oasis HLB extraction column (Waters Oasis HBL 1 cm3) preconditioned with 1 mL of methanol followed by 1 mL of water. The loaded column was then washed with 1 mL of water and eluted with 2 mL of methanol. The methanol effluent was collected, dried under nitrogen stream, and reconstituted with 100 μL of ACN/H2O (containing 10 mM ammonia acetate and 0.1% formic acid) (2:8) for the short-chain acyl-CoAs assay. The lower layer of the centrifuged extraction solvent was dried under a N2 stream to get residue A. To exhaustively extract the total lipids from mouse livers, the remaining tissue residue in an eppendorf tube was further extracted by adding 1 mL of Folch Reagent, agitated for 1 min, incubated for 30 min on ice, and centrifuged. The supernatant was then dried under a N2 stream to get residue B. Residues A and B were combined and reconstituted in 200 μL of isopropanol/methanol (1:10, v/v), and 100 μL of the sample was used for total tissue lipids (FFA and phospholipids) analyses. Another 100 μL of the mixture was dried under a N2 stream, and the residue was dissolved in isopropanol−acetonitrile, 1:9, v/v for total glycerolipids analysis.

Calibration Study

To establish the standard curves, calibration solutions of selected FFA were diluted from their stock solutions (1 mg/mL in methanol) with its mobile phase (FAA-A:FAA-B, 2:8). Phospholipids calibration solutions were prepared in its mobile phase (TL-A:TL-B, 2:8) from their stock solutions (1 mg/mL in isopropanol/methanol, 1:9). For the construction of standard curves for acyl-CoA analysis, 1 mg/mL stock solution was prepared in methanol/water (1:1) for each short-chain acyl-CoA, and it was then diluted with ACN/H2O (10 mM ammonia acetate and 0.1% formic acid, 2:8) before LC/MS/ MS analysis. The calibration curves were calculated using leastsquares linear regression.

Serum Triglyceride Bioassay

TG was assayed by a commercial kit (Sigma-Aldrich Co.) following the manufacturer’s instruction in the 96-well format. A standard curve of TG was constructed. Serum samples were prepared in assay buffer and distributed to each well in which standard lipase solution (2 μL) was added and mixed. After 20 min at room temperature, O.D. 570 nm was read.

Recovery, Precision, and Accuracy Assay from Mouse Plasma and Liver Samples

Because all metabolites under consideration are endogenous in mice, it is impossible to obtain a real “blank” control sample for extraction recovery assay. Hence, pooled plasma and liver samples were used to evaluate the recovery of all tested analytes.22 Six aliquots of each plasma or liver tissue sample were spiked with their respective internal standards at the concentration of 1 μg/mL, and processed as described above. Mouse plasma was spiked with a lipid mixture containing standard FFA or phospholipids at three known concentrations. Metabolites were extracted from spiked and nonspiked blood samples, and the estimated recovery (R) was calculated as follows:

LC/MS/MS Analysis

An Agilent 6540 UHD Accurate-Mass Q-TOF LC/MS mass spectrometer (Agilent Technologies) was connected to an Agilent 1290 Infinity UHPLC via an ESI ion source for the analysis of total lipids. An Agilent 6450 Triple Quadrupole LC/ MS system accompanied with MassHunter Workstation software (version B.04.00 Qualitative Analysis, Agilent Technologies, Santa Clara, CA) was connected to an Agilent 1389



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dose of schisandrin B at 0.8g/kg caused a 3-fold increase in serum triglyceride, and a dose of 1.6g/kg caused a 4-fold increase in serum triglyceride. It is thus confirmed that high doses of schisandrin B increased triglyceride in the mouse serum.16,17

R = [(extracted spiked − extracted nonspiked) /nonextracted standards)] × 100%

At the same time, both mixed lipids standards (FFA and phospholipids) and lipid metabolites (short-chain acyl-CoAs) were spiked into mouse liver extracts at three different concentrations before further extraction. The inter- and intraday reproducibility of quantitative measurements was ascertained by performing repeated sample preparations and analyses in triplicates in five consecutive days. The control pooled mouse plasma was divided into several aliquots and stored at −20 °C. Quality control (QC) samples were prepared from stored aliquots by spiking the FFA, phospholipids, or sphingolipid at two different concentrations, and their internal standards at 1 μg/mL. Samples obtained at different times were evaluated and compared by the relative standard deviation (RSD) values of the recoveries. The mouse liver QC samples were prepared in the same way as the plasma QC samples except that they were also spiked by the five shortchain CoAs.

Schisandrin B Treatments Altered Principal Component Analysis (PCA) of Mouse Plasma and Liver Metabolites

Thousands of lipid metabolites were detected in the mouse liver and plasma samples by the Q-TOF LC/MS. Data were sent to MassHunter Workstation software (version B.04.00 Qualitative Analysis, Agilent Technologies, Santa Clara, CA) and Mass Profiler Professional software for analysis (version 2.2, Agilent Technologies, Santa Clara, CA) as described in the method. Entities with high scores for a particular PCA component following the expression pattern are shown in the PCA. In the schisandrin B-induced hTG mouse model, we found that plasma (Figure 2A and C) and liver (Figure 2B and D) metabolites profiles showed distinct clustering between the vehicle control group and the schisandrin B-induced (high dose 1.6g/kg or medium dose 0.4g/kg) hTG groups, suggesting that the hTG mice have a lipidomic profile different from that of the control mice.

LC/MS Data Processing and Statistical Analysis

The raw data were first processed by MassHunter Workstation software (version B.04.00 Qualitative Analysis, Agilent Technologies, Santa Clara, CA). Ions were extracted by molecular features characterized by retention time (RT), intensity in apex of chromatographic peak, and accurate mass. These results were then analyzed by Mass Profiler Professional (MPP) software (version 2.2, Agilent Technologies, Santa Clara, CA). We first set up a filtration platform to further filter the initial entities before doing Principal Component Analysis (PCA). Only entities with abundances above 3000 cps were selected. These entities were then passed a tolerance window of 0.15 min and 2 mDa chosen for alignment of RT and m/z values, respectively. The data were also normalized by internal standard. We employed one-way ANOVA to do the statistical analysis. The p-value of ANOVA was set to 0.05 (corresponding with the significance level of 95%). We also performed fold change (FC) analysis to further filter the entities, and only those entities with FC > 2 were selected.

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Schisandrin B Treatment Changed the Profiles of Differential Lipid Categories in the hTG Mouse Model

To achieve sufficient separation of the numerous isobaric and isomeric species and to semiquantify these compounds, the classification of these metabolites according to their calculated masses was carried out. We have used reference standards to semiquantify mouse plasma of the schisandrin B-induced hTG mice and the vehicle control mice. We further confirmed that the plasma triglyceride level was increased in the schisandrin Btreated hTG mouse model (Table 2). We also found that FFA level was increased by 58.04% after schisandrin B treatment (Table 2). The kinetic changes of the total plasma FFA levels as obtained by LC/MS/MS methods in various treated mouse groups were shown in Figure 3A, which indicated that different concentrations of schisandrin B resulted in different plasma fatty acid profiles. The negative extracted ion chromatograms (EICs) of untargeted plasma FFA in vehicle control blank group and schisandrin B-induced hTG mice groups are shown in Figure 3B. Moreover, we found that the diglyceroid level was increased by 79.5% after schisandrin B treatment, that of PE by 86.8%, PC by 80.7%, PS by 59.1%, PG by 41.1%, PA by 85.7%, and PI by 33.9% (Table 2). The negative EICs of untargeted plasma phospholipid in vehicle control group and schisandrin B-induced hTG mouse groups are shown in Figure 3C. Schisandrin B treatment did not have a significant effect on the levels of sphingolipids and sterol lipids (Table 2). These results clearly show a distinct lipid profile in the hTG mouse model.

RESULTS

Schisandrin B Treatments Induced Elevation in Serum Triglyceride Levels in the hTG Mouse Model

As shown in Figure 1, high doses of schisandrin B significantly elevated mouse serum triglyceride levels in the mouse model; a

Identification and Quantification of FFA, Phospholipids, and Short-Chain CoAs by Rargeted Lipidomics Analysis

Targeted lipidomics was performed on Q-TOF and triple quadruplole LC/MS to identify the specific lipid species that showed significant changes in their concentrations after schisandrin B treatment. Ion spectra of the lipid species were used to select the precursor−product ion pairs for the development of multiple reaction monitoring (MRM) assays. The electrospray ionization mass spectrometry (ESI−MS) spectra for MRM assay of selected FFAs and short-chain CoAs are shown in Supporting Information Figure S1.

Figure 1. Serum triglyceride levels in schisandrin B-induced hTG mouse model measured by biochemical assay. Mice were intragastrically treated with schisandrin B at increasing doses (0.2, 0.4, 0.8, 1.6 g/ kg in olive oil); vehicle control group received olive oil at 10 mL/kg. Values are shown as the means ± SEM, with n = 6. ***p < 0.001. 1390



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Figure 2. The Principal Component Analysis (PCA) of total lipids in (A) the plasma and (B) livers, and FFA in (C) the plasma and (D) livers of schisandrin B-induced hTG mice.

As shown in Table 3, phosphatidylserine, 3-sn-phosphatidylethanolamine, L-α-phosphatidylcholine, and sphingomyelin were significantly increased in the mouse plasma but not in liver of the schisandrin B-induced hTG mice. Oleic acid, linoleic acid, linolenic acid, arachidonic acid, and docosahexaenoic acid were all significantly increased in both plasma and liver of the schisandrin B-induced hTG mice, while palmitic acid and stearic acid were increased only in the plasma of the hTG mouse model (Table 3). Representative negative EICs of targeted FFA in the liver and plasma of the mice are shown in Figure 4A and B, respectively. Summaries of these results are presented in Figure 4C and D, respectively. Considering the acyl-CoAs are important mediators related to lipids metabolism, four major short-chain CoAs including CoASH, acetyl-CoA, malonyl-CoA, and succinyl-CoA were measured using n-propionyl CoA as the internal standard. As shown in Table 4, short-chain CoAs such as acetyl-CoA, malonyl-CoA, and succinyl-CoA were all significantly increased in the schisandrin B-induced hTG mouse livers. Summaries of these results are presented in Figure 5.

Table 2. Semiquantifications of Various Categories of Lipids in Schisandrin B-Induced hTG Mice and Vehicle Control Micea

lipid category Fatty Acids free fatty acids Glycerolipids triglyceroids (TG) diglyceroids (DG) total Glycerophospholipids PE PC PS PG PA PI total Sphingolipids sphingomyelins (SM)

control mouse plasma

hTG mouse plasma

chemical species studied

chemical species studied

numberb

concn (μg/ mL)

numberb

concn (μg/ mL)

12

71.08

12

120.21**

45 28 73

995.21 36.27 1031.48

64 33 97

1866.2 65.11 1931.31**

19 48 5 12 6 6 89

228 853 5.63 4.77 2.24 25.13 1118.77

24 64 9 15 8 6 123

426 1542 8.96 6.73 4.16 33.65 2021.5**

31

248.7

25

204.1

Investigation of the Identified Potential Biomarkers in HFD-Induced hTG Mouse Model

To investigate whether the identified biomarkers are only specific to the schisandrin B-induced hTG mouse model, we also measured these biomarkers in a HFD-induced hTG mouse model. To establish a HFD-induced hTG mouse model, we fed C57 mice HFD21 for 15 weeks and found that the serum triglyceride level was 2.8-fold higher when compared to control diet-fed

a

The standard FFAs and phospholipids were used as corresponding internal standards, respectively. n = 6, **p < 0.01. bOnly counted those compounds that were semiquantified in the experiment.

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Figure 3. (A) Plasma FFA kinetic changes in different treatment groups; (B) negative extracted ion chromatogram (EIC) of untargeted plasma FFA in vehicle control and hTG mouse groups; and (C) EIC of the phospholipids assayed in vehicle control and hTG mouse plasma.

mice (Figure 6A). We also found that the plasma and liver samples of control diet-fed mice and HFD-fed mice were

significantly clustered by the three principal components as shown in Figure 6B and C, respectively. 1392



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Table 3. Concentrations of the Selected FFAs and Phospholipids in Schisandrin B-Induced hTG Mice and Vehicle Control Micea plasma concn (μg/mL) compounds PS1 PE1 PC1 SM1 palmitic acid stearic acid oleic acid linoleic acid linolenic acid arachidonic acid docosahexaenoic acid

control mice 5.49 66.80 277.47 29.12 20.56 6.25 14.56 19.50 1.07 0.34 3.03

± ± ± ± ± ± ± ± ± ± ±

0.05 10.01 13.09 2.33 3.79 0.69 1.67 2.64 0.27 0.08 0.66

liver concn (μg/g)

hTG mice 8.54 102.38 334.46 41.40 36.98 15.70 29.13 24.97 3.18 0.51 4.94

± ± ± ± ± ± ± ± ± ± ±

0.45 12.34 22.34 2.79 5.38 2.05 4.49 2.09 0.91 0.09 0.53

control mice 13.77 137.71 386.41 23.42 27.76 34.17 19.20 16.42 4.40 0.36 2.89

± ± ± ± ± ± ± ± ± ± ±

0.48 4.12 8.09 3.95 1.12 3.67 2.24 2.72 0.09 0.09 0.36

hTG mice 13.58 134.92 380.96 22.87 24.53 34.95 27.48 39.63 4.93 0.63 5.23

± ± ± ± ± ± ± ± ± ± ±

0.63 3.98 9.13 3.01 2.91 6.30 2.72 3.92 0.16 0.08 0.50

Mean ± standard deviation, n = 6. PS1: Phosphatidylserine (C13H24NO10P). PE1: 3-sn-Phosphatidylethanolamine (C9H18NO8P). PC1: L-αPhosphatidylcholine (C42H80NO8P). SM1: Sphingomyelin (C41H84N2O6P).

a

2.91−6.78% in plasma and 3.02−6.91% in liver (Table 9). The chemical structures of phospholipids and short-chain CoAs analyzed in the study were shown in Supporting Information Figure S2A and S2B, respectively.

To examine if the changes in the levels of palmitic acid, stearic acid, oleic acid, and arachidonic acid in the HFDinduced hTG mice were the same as those changes in schisandrin B-induced hTG mice (Figure 4C and D), we performed targeted lipidomics to measure these FFA levels in the HFD-fed hTG mice. We found that oleic acid and arachidonic acid were significantly increased in both the plasma and the liver of the HFD-fed hTG mice when compared to control diet-fed mice (Table 5). Similarly, palmitic acid and stearic acid were increased only in the plasma but not in the livers of these mice (Table 5). Summaries of these results are presented in Figure 7A and B, respectively. We have also measured the malonyl-CoA level in the HFDfed hTG mouse livers. Similar to the finding obtained in schisandrin B-induced hTG mouse model, the level of malonylCoA was significantly increased in the HFD-fed hTG mouse livers when compared to the control diet-fed mice (Table 6). Summaries of these results are presented in Figure 7C. These results showed that in both the schisandrin B-induced and the HFD-induced hTG mouse models, malonyl-CoA levels were significantly increased, oleic acid and arachidonic acid were significantly increased in both the plasma and the livers, while palmitic acid and stearic acid were only significantly increased in the plasma but not the liver of these mice.

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DISCUSSION Different hTG mouse models have been reported including diet-induced8−12 and gene knockout13,14 models. In this study, we used schisandrin B-induced hTG mouse model16,17 to identify potential biomarkers in hTG pathogenesis. In the schisandrin B-induced hTG mouse model, we found that palmitic acid and stearic acid were increased in the plasma; oleic acid, linoleic acid, linolenic acid, arachidonic acid, and docosahexaenoic acid were increased in both the plasma and the liver. Acetyl-CoA, malonyl-CoA, and succinyl-CoA were increased only in the liver. The changes in the levels of these potential biomarkers were also observed in HFD-induced hTG mouse model. Schisandrin B is relatively nontoxic at the doses we used in the study,23 and the reversible pathogenic condition of the schisandrin B-induced hTG mouse model17 makes it a useful model for studying hTG pathogenesis and for the screening of TG lowering drugs. Schisandrin B-induced hTG mouse model is established by feeding fasting mice high doses of schisandrin B.16,17 In these mice, we detected approximately 3−4-fold increases in serum TG levels and a significant increase in FFA levels. It is known that the clinical parameter for hTG is an abnormal high concentration of TG in the blood. The normal TG level in human is less than 150 mg/dL, and TG levels exceeding 500 mg/dL, which is around a 3−4-fold increase, would be considered as a severe clinical hTG case.1 We have also detected individual FFAs and found that arachidonic acid and oleic acid significantly increased in the plasma and the liver, while palmitic acid and stearic acid significantly increased in the plasma. The changes in the levels of these FFAs observed in the schisandrin B-induced hTG mouse model were also observed in the HFD-induced hTG mouse model. The consistency of these results further suggests that these FFAs play an important pathogenic role in hTG. Indeed, FFA promotes the formation of reactive lipid moieties.5 An upset of the balance between oxidative stress and antioxidant response may trigger apoptosis or mitochondrial damage,5 which may explain the association between clinical cases of hTG and nonalcoholic fatty liver diseases.5 Further studies will be performed to verify the

Recovery, Intra-, and Interday Precision and Accuracy Test

Table 7 shows the molecular formula, m/z values in positive and negative ionization modes, and the MRM transition of these targeted lipid species in the Q-TOF and QQQ analysis. Using the developed UPLC-triple quadrupole and Q-Tof MS method, 7 FFAs, three phospholipids, 1 sphingolipid, 4 shortchain acyl-CoAs, and the corresponding internal standards (IS) were prepared at six concentrations, respectively. For each of the analytes, a calibration curve was generated using ratios of reference standards and their corresponding IS. Good correlation was obtained in both electrospray negative and positive ion modes with all R2 values being higher than 0.99 (Table 8). Eight batches of calibration solutions for each standard were used to determine limits of detection (LODs) and limits of quantification (LOQs). The LODs were assayed by using signal-to-noise ratios of 3, and the LOQ was defined as the concentration value above which the measured precisions were ≤15% RSD (Table 8). The RSD for the interday and intraday variations was also calculated to be within the range of 1393



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Table 4. Concentration of Short-Chain CoAs (μg/g) in Vehicle Control and Schisandrin B-Induced hTG Mouse Liversa short-chain acyl-CoA CoASH acetyl-CoA malonyl-CoA succinyl-CoA a

concn in vehicle control mice 20.09 83.69 0.32 11.91

± ± ± ±

0.52 8.52 0.02 0.85

concn in hTG mice 5.57 96.12 0.81 22.36

± ± ± ±

0.50 7.88 0.06 3.21

Mean ± standard deviation, n = 6.

Figure 5. Relative malonyl Co-A levels in schisandrin B-induced hTG mice. Values are shown as the means ± SEM, with n = 6. *p < 0.05.

Figure 6. (A) Serum triglyceride levels in HFD-induced hTG mouse model measured by biochemical assay. PCA of total lipids in (B) the plasma and (C) livers of HFD-induced hTG mice.

pathogenic roles of these FFAs in hTG and its associated diseases. Interestingly, the short-chain CoA such as malonyl-CoA was increased in both the schisandrin B-induced and HFD-induced hTG mouse livers when compared to their corresponding control groups. Up to the present, specific hTG biomarkers have not been clearly defined. Malonyl-CoA is a known potent inhibitor of carnitine acyl transferase-I, which is a mitochondrial membrane-associated enzyme responsible for transporting long-chain acyl-CoA into mitochondria, a rate-limiting step for β-oxidation.24,25 An increase in malonyl-CoA may inhibit βoxidation of FFA. In addition, malonyl-CoA is also a substrate for fatty acid synthesis, and itself is formed by the addition of a

Figure 4. The representative negative EIC of targeted FFAs in control and hTG mouse (A) liver and (B) plasma: (1) linolenic acid, (2) DHA, (3) AA, (4) linoleic acid, (5) IS1, (6) palmitic acid, (7) oleic acid, (8) heptadecanoic acid (IS2), (9) stearic acid. Relative (C) hepatic and (D) plasma FFA levels in schisandrin B-induced hTG mice. Values are shown as the means ± SEM, with n = 6. **p < 0.01, *p < 0.05. 1394



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Table 5. Concentrations of the Selected FFAs in Control Diet-Fed Mice and HFD-Fed hTG Micea plasma concn (μg/mL) compounds

control mice

palmitic acid stearic acid oleic acid arachidonic acid a

2.36 3.18 1.28 0.46

± ± ± ±

liver concn (μg/g)

hTG mice

0.50 0.66 0.06 0.03

6.90 8.66 4.68 0.88

± ± ± ±

control mice

1.53 1.72 0.66 0.07

8.36 15.56 1.10 0.41

± ± ± ±

hTG mice

2.05 2.62 0.08 0.01

8.91 12.54 3.26 3.52

± ± ± ±

3.10 2.85 0.50 0.55


Table 7. (A) Selected Ions of Targeted Lipids and FFA in QTOF Analysis; and (B) MRM Transition Ions of Targeted Coenzymes in QQQ Analysisa (A) compounds PS1 PE1 PC1 SM1 palmitic acid

positive mode [M + H]+ (m/z)

negative mode [M − H]− (m/z)

386.3041 300.0764 758.5700 732.6046

384.3054 298.0773 756.5103 730.6058 255.2324

stearic acid

283.2637

oleic acid

281.2481

linoleic acid

279.2324

linolenic acid

277.2168

arachidonic acid

303.2324

docosahexaenoic acid heptadecanoic acid IS1 IS2

327.2324 269.2481 260.4483

molecular formula C13H24NO10P C9H18NO8P C42H80NO8P C41H84N2O6P C16H32O2 (16:0) C18H36O2 (18:0) C18H34O2 (18:1) C18H32O2 (18:2) C18H30O2 (18:3) C20H32O2 (20:4) C22H32O2 (22:6) C17H34O2 (17:0) C16H27D5O2

(B) compounds CoASH acetyl-CoA malonylCoA succinylCoA n-propionylCoA

Figure 7. Relative (A) plasma and (B) hepatic FFA levels in HFDinduced hTG mice. (C) Relative malonyl Co-A level in HFD-induced hTG mice. Values are shown as the means ± SEM, with n = 6−8. **p < 0.01, *p < 0.05.

a

concn in control mice

concn in hTG mice

malonyl-CoA

0.07 ± 0.002

0.31 ± 0.019

MRM scan (m/ z) MSn

molecular formula

768.1 810.2 854.1

768.1→261.1 810.2→303.1 854.1→347.1

C21H36N7O16P3S C23H38N7O17P3S C24H38N7O19P3S

868.1

868.1→361.2

C25H40N7O19P3S

824.2

824.2→317.2

C24H40N7O17P3S

a

PSI: Phosphatidylserine. PEI: 3-sn-Phosphatidylenthanolamine. PCI: SMI: Sphingomyelin. IS2: Hexadecanoic15,15,16,16,16-d5 acid. L-α-Phosphatidylcholine.

Table 6. Concentration of Short-Chain CoAs (μg/g) in Control Diet-Fed Mice and HFD-Fed hTG Mouse Liversa short-chain acyl-CoA

positive mode [M + H]+ (m/z)

considered as the key “metabolic effector” and can impact many metabolic diseases.26 Moreover, elevation of malonylCoA may trigger apoptosis.27 Therefore, it is reasonable to speculate that malonyl-CoA plays an important role in the clinical pathogenesis of hTG, and malonyl Co-A could be a potential novel hTG biomarker. Because hTG is a disturbance in lipid metabolism, we had to monitor a huge number of bioactive lipids and their isomers with similar physicochemical properties and existing at very low physiological levels in biological samples. To overcome this problem, we employed a high-throughput analysis of the metabolites in different biological samples with a combined UHPLC-UHD Q-TOF MS/MS for untargeted lipidomics and


carboxyl group to acetyl-CoA in an enzymatic reaction catalyzed by acetyl-CoA carboxylase. An increase in intracellular malonyl-CoA, as shown in both the schisandrin B-induced and the HFD-induced hTG mouse models, is expected to increase TG synthesis in lipogenic tissues, such as the liver. The synthesized TG will be exported from the liver and found in circulating VLDL. Indeed, elevated lipoprotein such as VLDL is another clinical parameter for hTG.4 Malonyl-CoA is 1395



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Table 8. Linearity, Correlation Coefficient (R2), and Limit of Detectiona standards PS1 PE1 PC1 SM1 palmitic acid stearic acid oleic acid linoleic acid linolenic acid arachidonic acid docosahexaenoic acid CoASH acetyl-CoA malonyl-CoA succinyl-CoA

calibration curves

coefficient (R2)

linear range (μg/mL)

LOD (ng/ mL)

LOQ (μg/ mL)

y = 17.5x − 7.05 y = 33.9x − 3.08 y = 43.3x − 2.10 y = 40.5x − 11.49 y = 10.73x + 1.51 y = 11.62x + 1.52 y = 169.9x + 0.47 y = 157.2x + 0.50 y = 97.9x − 2.71 y = 114.8x − 2.77 y = 70.0x − 0.30 y = 94.25x − 1.98 y= 126.91x − 1.04 y = 11.22x + 0.05 y= 171.02x + 1.03

0.99

0.1−100

50

0.1

0.99

0.1−100

50

0.1

0.99

0.5−100

50

0.5

0.99

0.1−100

50

0.1

0.99

0.1−50

50

0.1

0.99

0.1−50

50

0.1

0.99

0.05−20

20

0.05

0.99

0.05−20

20

0.05

0.99

0.05−50

20

0.05

0.99

0.1−20

20

0.1

0.99

0.1−20

20

0.1

0.99

0.01−50

20

0.01

0.99

0.01−50

20

0.01

0.99

0.01−20

20

0.01

0.99

0.02−50

20

0.02

UHPLC-QQQ-MS/MS for targeted lipidomics. Also, our study demonstrated a clear discrimination of the lipidomic profiles between control mice and the hTG mice, and identified several potential hTG biomarkers. However, there are limitations in this study. We have suggested a number of hTG biomarkers with the mouse models, but these biomarkers are not yet confirmed in human clinical hTG cases. Cell experiments and animal experiments are needed to investigate how the levels of these biomarkers are elevated in hTG condition and the pathogenic roles of these biomarkers. Besides, hTG pathogenic conditions in mouse and human are not identical; application of hTG mouse model for the study of human hTG pathogenesis has limitations. For example, lack of active cholesteryl ester transfer protein (CETP) in mouse28 may hinder the study of the role of CETP in hTG.

■

CONCLUSION We have delineated the global lipidomic profiles and suggested potential biomarkers in hTG pathogenesis in schisandrin Binduced hTG mice. These biomarkers were also observed in HFD-induced hTG mouse model. Information shown here can serve as a basis for future studies of the roles of these individual FFAs and short-chain acyl-CoA in hTG pathogenesis. Pharmacological agents and other approaches that reduce their expressions could be effective anti-hTG therapeutics.

■

ASSOCIATED CONTENT

S Supporting Information *

Additional figures. This material is available free of charge via the Internet at http://pubs.acs.org.

■

a

PS1: Phosphatidylserine (C13H24NO10P). PE1: 3-sn-Phosphatidylethanolamine (C 9 H 18 NO 8 P). PC1: L -α-Phosphatidylcholine (C42H80NO8P). SM1: Sphingomyelin (C41H84N2O6P).

AUTHOR INFORMATION

Corresponding Author

*Phone: (852) 34112928 (W.F.F.), (852) 34112465 (Z.-L.Y.). Fax: (852) 34112902. E-mail: wff[email protected] (W.F.F.), [email protected] (Z.-L.Y.).

Table 9. Recovery, Intra-, and Interday Precision and Accuracy for Mouse Plasma and Liver Lipid Analysisa intraday

interday

recovery

RSD (%)

recovery

RSD (%)

standards

plasma

liver

plasma

liver

plasma

liver

plasma

liver

PS1 PE1 PC1 SM1 palmitic acid stearic acid oleic acid linoleic acid linolenic acid arachidonic acid docosahexaenoic acid heptadecanoic acid CoASH acetyl-CoA malonyl-CoA succinyl-CoA

86.1 83.4 92.2 81.9 91.7 96.1 82.7 86.9 94.2 89.1 90.1 96.1

91.3 88.6 89.3 76.2 87.4 89.3 78.1 83.1 85.5 76.6 77.3 88.7 75.9 81.6 79.3 82.1

4.57 5.22 3.78 6.84 3.66 4.12 5.06 4.91 4.11 3.85 4.29 3.69

6.03 5.74 4.18 6.91 9.27 8.36 5.17 7.02 5.64 9.01 6.66 9.69 4.33 4.01 5.49 4.58

90.1 89.2 92.1 88.6 90.3 94.1 89.2 90.4 91.7 83.6 89.3 98.4

77.3 81.3 83.6 77.5 85.3 86.6 77.6 83.7 84.0 77.9 79.1 84.2 74.3 79.6 80.3 83.9

3.82 3.26 4.77 5.14 4.01 5.89 5.41 5.12 5.02 4.17 4.33 6.78

6.19 6.52 5.13 6.41 9.47 7.62 4.92 3.22 5.46 8.56 5.23 9.32 5.16 4.44 6.56 5.78

a

PS1: Phosphatidylserine (C13H24NO10P). PE1: 3-sn-Phosphatidylethanolamine (C9H18NO8P). PC1: L-α-Phosphatidylcholine (C42H80NO8P). SM1: Sphingomyelin (C41H84N2O6P). 1396



Article

Author Contributions

(9) Park, S. H.; Ko, S. K.; Choi, J. G.; Chung, S. H. Salicornia herbacea prevents high fat diet-induced hyperglycemia and hyperlipidemia in ICR mice. Arch. Pharm. Res. 2006, 29, 256−264. (10) Moon, Y. A.; Liang, G.; Xie, X.; Frank-Kamenetsky, M.; Fitzgerald, K.; Koteliansky, V.; Brown, M. S.; Goldstein, J. L.; Horton, J. D. The Scap/SREBP pathway is essential for developing diabetic fatty liver and carbohydrate-induced hypertriglyceridemia in animals. Cell Metab. 2012, 15, 240−246. (11) Taleb-Dida, N.; Krouf, D.; Bouchenak, M. Globularia alypum aqueous extract decreases hypertriglyceridemia and ameliorates oxidative status of the muscle, kidney, and heart in rats fed a highfructose diet. Nutr. Res. (N.Y.) 2011, 31, 488−495. (12) Hartvigsen, K.; Binder, C. J.; Hansen, L. F.; Rafia, A.; Juliano, J.; Hörkkö, S.; Steinberg, D.; Palinski, W.; Witztum, J. L.; Li, A. C. A dietinduced hypercholesterolemic murine model to study atherogenesis without obesity and metabolic syndrome. Arterioscler., Thromb., Vasc. Biol. 2007, 27, 878−885. (13) Weinstock, P. H.; Bisgaier, C. L.; Aalto-Setälä, K.; Radner, H.; Ramakrishnan, R.; Levak-Frank, S.; Essenburg, A. D.; Zechner, R.; Breslow, J. L. Severe hypertriglyceridemia, reduced high density lipoprotein, and neonatal death in lipoprotein lipase knockout mice. Mild hypertriglyceridemia with impaired very low density lipoprotein clearance in heterozygotes. J. Clin. Invest. 1995, 96, 2555−2568. (14) Hofker, M. H.; van Vlijmen, B. J. M.; Havekes, L. M. Transgenic mouse models to study the role of APOE in hyperlipidemia and atherosclerosis. Atherosclerosis 1998, 137, 1−11. (15) Pan, S. Y.; Dong, H.; Zhao, X. Y.; Xiang, C. J.; Fang, H. Y.; Fong, W. F.; Yu, Z. L.; Ko, K. M. Schisandrin B from Schisandra chinensis reduces hepatic lipid contents in hypercholesterolaemic miceet al. J. Pharm. Pharmacol. 2008, 60, 399−403. (16) Pan, S. Y.; Dong, H.; Han, Y. F.; Li, W. Y.; Zhao, X. Y.; Ko, K. M. A novel experimental model of acute hypertriglyceridemia induced by Schisandrin B. Eur. J. Pharmacol. 2006, 537, 200−204. (17) Pan, S. Y.; Dong, H.; Guo, B. F.; Zhang, Y.; Yu, Z. L.; Fong, W. F.; Han, Y. F.; Ko, K. M. Effective kinetics of schisandrin B on serum/ hepatic triglyceride and total cholesterol levels in mice with and without the influence of fenofibrate. Naunyn-Schmiedeberg’s Arch. Pharmacol. 2011, 383, 585−591. (18) Dennis, E. A. Lipidomics joins the omics evolution. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 2089−2090. (19) Meikle, P. J.; Christopher, M. J. Lipidomics is providing new insight into the metabolic syndrome and its sequelae. Curr. Opin. Lipidol. 2011, 22, 210−215. (20) Sommer, U.; Herscovitz, H.; Welty, F. K.; Costello, C. E. LCMS-based method for the qualitative and quantitative analysis of complex lipid mixtures. J. Lipid Res. 2007, 47, 804−814. (21) Chan, P. T.; Fong, W. P.; Cheung, Y. L.; Huang, Y.; Ho, W. K.; Chen, Z. Y. Jasmine green tea epicatechins are hypolipidemic in hamsters (Mesocricetus auratus) fed a high fat diet. J. Nutr. 1999, 129, 1094−1101. (22) Mojgan, M.; Nicolaou, A. Lipidomic analysis of twenty-seven prostanoids and isoprostanes by liquid chromatography/electrospray tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2006, 20, 3023−3029. (23) Ko, R. K. M.; Mak, D. H. F. Schisandrin B and other dibenzocyclooctadiene lignans. In Modern Herbal Medicine: Modern Basis in Health and Disease Management; Packer, L., Halliwell, B., Ong, C. N., Eds.; Marcel Dekker: New York, 2004; pp 289−314. (24) Prip-Buus, C.; Pegorier, J. P.; Duee, P. H.; Kohl, C.; Girard, J. Evidence that the sensitivity of carnitine palmitoyltransferase I to inhibition by malonyl-CoA is an important site of regulation of hepatic fatty acid oxidation in the fetal and newborn rabbit. Perinatal development and effects of pancreatic hormones in cultured rabbit hepatocytes. Biochem. J. 1990, 269, 409−415. (25) Rasmussen, B. B.; Holmback, U. C.; Volpi, E.; Morio-Liondore, B.; Paddon-Jones, D.; Wolfe, R. R. Malonyl coenzyme A and the regulation of functional carnitine palmitoyltransferase-1 activity and fat oxidation in human skeletal muscle. J. Clin. Invest. 2002, 110, 1687− 1693.

§

These authors contributed equally.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Drs. Guo-Yuan Zhu and William Chi Shg Tai from the School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China, for their help on the animal model experiments and TG assay. This work was partially supported by grants FRG2/10-11/019, FRG1/11-12/050, FRG2/11-12/057, and FRG1/11-12/053 from Hong Kong Baptist University, and grant 31071989 from the National Natural Science Foundation of China.

■

ABBREVIATIONS hTG, hypertriglyceridemia; DG, diglyceride; TG, triglyceride; VLDL, very low density lipoprotein; DHL, high density lipoprotein; IDL, intermediate density lipoprotein; FFA, free fatty acid; FACoA, fatty acid coenzyme A; ACC, acetyl-CoA carboxylase; MCD, malonyl-CoA decarboxylase; CPT, carnitine palmitoyltransferase; AMPK, adenosine monophosphate protein kinase; AA, arachidonic acid; DHA, docosahexaenoic acid; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; LPE, lyso-phosphatidylethanolamin; LPC, lyso-phosphatidylcholine; TCA, trichloroacetic acid; UHPLC-UHDQ-TOF MS, ultra high performance liquid chromatography with ultra high definition accurate mass quadrupole time of flight-mass spectrometer; UHPLC-QQQ-MS, ultra high performance liquid chromatography with triple quadrupole mass spectrometer; ESI−MS, electrospray ionization mass spectrometry; ESI EIC, electrospray ionization extracted ion chromatogram; PCA, Principal Component Analysis; MPP, Mass Profiler Professional

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REFERENCES

(1) Pejic, R. N; Lee, D. T. Hypertriglyceridemia. J. Am. Board Fam. Med. 2006, 19, 310−316. (2) Frederoclspm, D. S.; Lees, R. S. A system for phenotyping hyperlipoproteinemia. Circulation 1965, 32, 321−327. (3) Tilg, H.; Kaser, A. Treatment strategies in nonalcoholic fatty liver disease. Nat. Clin. Pract. Gastroenterol. Hepatol. 2005, 2, 148−155. (4) Khurram, M.; Ashraf, M. M. A clinical and biochemical profile of biopsy-proven non-alcoholic Fatty liver disease subjects. J. Coll. Physicans Surg. Pak. 2007, 17, 531−534. (5) Tarantino, G.; Colao, A.; Capone, D.; Conca, P.; Tarantino, M.; Grimaldi, E.; Chianese, D.; Finelli, C.; Contaldo, F.; Scopacasa, F.; Savastano, S. Circulating levels of cytochrome C, gamma-glutamyl transferase, triglycerides and unconjugated bilirubin in overweight/ obese patients with non-alcoholic fatty liver disease. J. Biol. Regul. Homeostatic Agents 2011, 25, 47−55. (6) Hegele, R. A.; Pollex, R. L. Hypertriglyceridemia: phenomics and genomics. Mol. Cell. Biochem. 2009, 326, 35−43. (7) Pirih, F.; Lu, J. X.; Ye, F.; Bezouglaia, O.; Atti, E.; Ascenzi, M. G.; Tetradis, S.; Demer, L.; Aghaloo, T.; Tintut, Y. Adverse effects of hyperlipidemia on bone regeneration and strength. J. Bone Miner. Res. 2012, 27, 309−318. (8) Krugner-Higby, L.; Shelness, G. S.; Holler, A. Heritable, dietinduced hyperlipidemia in California mice (Peromyscus californicus) is due to increased hepatic secretion of very low density lipoprotein triacylglycerol. Compens. Med. 2006, 56, 468−475. 1397



Article

(26) Folmes, C. D. L.; Lopaschuk, G. D. Role of malonyl-CoA in heart disease and the hypothalamic control of obesity. Cardiovasc. Res. 2007, 73, 278−287. (27) Pizer, E. S.; Thupari, J.; Han, W. F.; Pinn, M. L.; Chrest, F. J.; Frehywot, G. L.; Townsend, C. A.; Kuhajda, F. P. Malonyl-coenzymeA is a potential mediator of cytotoxicity induced by fatty-acid synthase inhibition in human breast cancer cells and xenografts. Cancer Res. 2000, 60, 213−218. (28) Masson, D.; Staels, B.; Gautier, T.; Desrumaux, C.; Athias, A.; Le Guern, N.; Schneider, M.; Zak, Z.; Dumont, L.; Deckert, V.; Tall, A.; Jiang, X. C.; Lagrost, L. Cholesteryl ester transfer protein modulates the effect of liver X receptor agonists on cholesterol transport and excretion in the mouse. J. Lipid Res. 2001, 45, 543−550.

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