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Untargeted Lipidomic Analysis of Plasma from High-fat Diet-induced Obese Rats Using UHPLC–Linear Trap Quadrupole–Orbitrap MS

relative quantification of multiple lipid classes study an ultra-high-performance liquid chromatography (UHPLC)–linear trap quadrupole (LTQ)– orbitrap mass spectrometry total lipids HFD-induced High-fat diet (HFD)-induced obesity is a primary risk factor for serious health problems. Although much research has been performed at the genomic level, lipidomic studies were limited. In this study, we aim to obtain a comprehensive profile of circulating plasma lipids, which are altered in rodent rat obesity by untargeted liquid chromatography –mass spectrometry. Rats fed with HFD for 8 weeks had increased body weight, liver and adipose tissue weight. The analysis results revealed that polyunsaturated fatty acids (PUFAs) and their corresponding phosphatidylcholine, phosphatidylinositol, and phosphatidylserine were significantly decreased in rats fed with HFD. In contrast, less unsaturated and ether type phosphatidylglycerols were increased. The triacylglycerides (TAGs) having saturated FA were increased in the HFD condition, whereas TAGs having PUFA were decreased. The levels of many plasma lipids were altered, and interestingly PUFA derived lipids were negatively associated with obesity. This signifies the importance of a PUFAs enriched diet to overwhelm obesity associated diseases.


Chemicals
The LC/MS-grade isopropanol, methanol, and chloroform were purchased from Wako Pure Chemical Industries, Ltd.

Plasma samples and extraction of total lipids
Male 3-week-old WKAH/HkmSlc rats were housed in an airconditioned room at 22 ± 2 C with 55 ± 5% humidity, and the light period was set from 8:00 AM to 20:00 PM. The rats were housed individually in standard wire-bottomed cages and allowed ad libitum access to food and water. During the 2-week acclimation period, the rats were fed a normal diet based on the AIN-93G formulation ( Table 1). The fatty acid compositions of the diet analyzed by LC/MS are given in Table 2. For long-term experiments, the acclimatized rats (n = 13) were divided into two groups and were fed either the normal diet (ND) (n = 6) or high-fat diet (HFD) (n = 7) for 8 weeks. To prepare the HFD about 230 g of lard was added to the normal diet per kg in place  of the dextrin. The same animal samples were used for another part of the study, in finding the association between bile acid and hepatic steatosis (S. Hori et al., unpublished data). Portal and aortic blood were collected from rats under anesthesia using sodium pentobarbital (Somnopentyl, 50 mg/kg body weight, Kyoritsu Seiyaku Corporation, Tokyo, Japan) at week 8 without food deprivation. An anticoagulant agent (heparin, 50 U/mL blood, Nacalai Tesque, Inc., Kyoto, Japan) and a protease inhibitor (aprotinin, 500 KIU/mL blood, Wako Pure Chemical Industries, Ltd., Osaka, Japan) were added to blood samples for the preparation of plasma. Thereafter, the rats were killed by exsanguination. Experimental procedures were in accordance with Japanese laws on the use of animals for experimentation and were approved by the Hokkaido University Ethics Review Committee on Animal Experiments with approval number 17-0050. Total lipids were extracted from the rat plasma by the method reported by Folch et al. 20 In brief, 50 μL of each plasma samples was extracted with 200 μL of ice-cold methanol having pre-mixed internal standards, and were vortexed for 30 s at 3500 rpm. Subsequently, 200 μL of chloroform (vortex, 5 min) and 50 μL of Milli-Q (vortex, 30 s) were added. The extracts were centrifuged at 4 C, 252g for 10 min and the residue was re-extracted with 200 μL of chloroform. The combined extracts were dried under vacuum and redissolved in 100 μL of methanol and were transferred to LC/MS vials. The lipids from liver and epididymal adipose tissue were extracted by the same protocol as described above after homogenization with methanol using beads for 15 s (Bead Mill 4, Fisher Scientific).
The untargeted lipidomic analyses were conducted on a Shimadzu Prominence UHPLC system (Shimadzu Corp., Kyoto, Japan) connected to an LTQ orbitrap mass spectrometer (Thermo-Fisher Scientific Inc.). Both ESI-positive and ESInegative ionization mode acquisitions have the identical MS conditions: capillary voltage (3.0 kV), nitrogen-sheath gas flow (50 units,) and nitrogen-auxiliary gas (5 units). The MS1 data were acquired in the Fourier transform mode with a resolving power of 60000 and a scan speed of 2 Hz. The tandem mass data were obtained using collision-induced dissociation in the ion-trap mode and data-dependent acquisition with MS2 and MS3 scans at a collision energy 35 and 45 V, respectively. The MS1 scan range was set at m/z 150 -1700 and was m/z 250 -1600 for the positive and negative modes, respectively. The alignment, peak extraction, and extracted ion chromatogram (EIC) peak area integration and identification of lipid molecules from the raw data were performed by the MS DIAL software Ver. 3.9 (prime.psc.riken.jp). 21 The lipids were relatively quantified by choosing a suitable internal standard for each class. Data were plotted in Excel 2016 and multivariate hierarchical cluster analysis was performed by the ClustVis web-based software tool (biit.cs.ut.ee). The student's t test was conducted and p < 0.05 was considered to be statistically significant. Data were presented as the mean ± standard deviation.

Body weight, adipose and liver tissue weight
The seven rats on a high-fat diet (HFD) had a body weight of 348.35 (312.8 -392.6) g, which was considerably higher compared to the six rats on a normal diet (ND) with 316.6 (308.2 -329) g ( Fig. 2A). Food intake by weight in the HFD group was markedly lower (p < 0.05) than the ND group given by 847.9 ± 14.4, 721.6 ± 57, respectively. The total tissue weight of liver and adipose tissue was significantly elevated in rats fed with fat ( Fig. 2B).

Untargeted analysis of the rat plasma lipids and statistical analysis
High-resolution MS data were acquired by an LTQ orbitrap mass spectrometer in both positive and negative ionization modes. The total ion chromatogram (TIC) of the lipid profile of rat plasma acquired in the negative mode (

Concentrations and hierarchical cluster analysis of FA and TAG
The concentrations of free FAs identified in rat plasma is shown in Fig. 4A. The PUFAs such as FA 18:3, FA 20:5, FA 22:5 and MUFAs, FA 24:1 and FA 26:1 are significantly decreased in the rats fed with HFD. Hierarchical cluster analysis (HCA) shows the distinct clustering between the ND and HFD groups. The red color indicates the positive association with high levels of FAs, whereas blue color indicates the negative association with their low levels. The significantly altered TAGs (p < 0.05) are plotted in Fig. 4B. TAGs with PUFA were shown to be decreased in the rats fed with high-fat, whereas TAGs having MUFA or SFA were increased drastically with the HFD. The HCA reveals the well-defined clustering between the ND and HFD groups. The red color indicates the positive association with high levels of TAGs, whereas blue color indicates the negative association with their low levels. As shown their SFA and MUFA containing TAGs were diminished in rats under ND conversely PUFA TAGs were increased.

Concentrations and hierarchical cluster analysis of PC, LPC, and PI
The

Concentrations and hierarchical cluster analysis of PS, PG, PE, LPE and other altered metabolites
The  (Fig. 6C) was greater than two fold higher in the ND group compare to the HFD group. CE having FA 22:6 could be a docosahexaenoic acid (DHA), an essential ω-3 FA with diverse biological benefits. As per our knowledge CE 22:6 is identified for the first time in plasma of HFD rats. Another new class of lipids such as alkyl acyl glycerol's (AAG), AAG 18:5e-24:1 and a sphingomyelin, SM d18:1-16:0 were also significantly increased in the plasma of rats fed with HFD.

Concentrations of free fatty acids in epididymal adipose tissue and liver
In the epididymal adipose tissue, the concentrations of SFA and MUFA were not significantly altered between the ND and HFD groups, except for FA 18:0, which a shows positive association with obesity, whereas PUFAs, such as

Discussion
Diet is a prominent factor to control various metabolic diseases. Particularly, a high-fat diet and physical idleness cause overweight or obesity, which is a foremost factor in the  pathogenesis of metabolic disorders. 22 The concurrence of obesity, insulin resistance, hypertension and dyslipidemia is generally referred to as the "metabolic syndrome". 23 This condition affects about 20 -40% of the population in the industrialized nations, and its prevalence is expected to rise further in the next decades. 24 Also, HFD-induced obesity leads to a harmful activation of the immune system, with the increasing risk of heart disease, diabetes, and cancer. 25 Lipids are a very distinct group of molecules concerning their structure and function, and it is known that impaired lipid metabolism plays a significant role in obesity-related disorders. 12,13 In clinical chemistry, blood-based lipid biomarkers for obesity are crucial for the diagnosis of the disease. Moreover advanced LC/MS techniques were widely applied for lipid biomarker discovery in various models. 26,27 In the present study, a rodent rat model was used, and rats were fed with ND and HFD (fat source: lard) for 8 weeks. Then, the plasma samples were collected and extracted for total lipids by Folch method and comprehensive lipidomics studies was performed by using LTQ-MS coupled to UHPLC.
The body weight measurement showed a significant difference between the ND and HFD groups soon after 4 weeks of the diet ( Fig. 2A). Rats fed with HFD have a significantly higher body mass, liver and adipose tissue weights (Fig. 2B), but lesser food intake compared to rats on ND. These are the key marker of the model and the results were consistent with previous studies. 17,18,28 The TIC profile of rat plasma in negative and positive mode (Figs. 2C and 2D) was clearly illustrate the acquired classes of lipid metabolites in each mode. In positive mode measurement TAGs are abundant whereas in negative mode fatty acids and phospholipids.
Statistical analysis of all identified lipid metabolites in both the modes was performed. Figure 3A shows the PCA analysis results of 94 identified lipid metabolites in plasma samples of ND and HFD groups. The data show that both groups have significantly different lipid levels, indicated by the well-defined separation between the ND and HFD groups in the PCA plot. On the other hand, the volcano plot (Fig. 3B) of -log 10 (p-value) versus log 2 (ND/HFD) shows the significantly altered lipid metabolites.
SFA and MUFA containing TAGs such as TAG 52:0, TAG 45:1, TAG 54:1 and PIs, PI 36:1, PI 34:1 were significantly increased under HFD and are positively associated with obesity. The relative TAG composition varies with the obesity model. For example, in a diet-induced weight loss model, weight reduction in mice was associated with decreased levels of TAG containing SFA 29 and are consistent with our results. Whereas in plasma samples of rats fed with HFD for 3 weeks shown decreased SFA and MUFA TAGs levels, while developing obesity. 28 Earlier studies show MUFA PIs (PI 34:1, PI 36:1) were significantly higher in serum of obese mice, 17 and the results are coherent with our results. In contrast, several MUFA and PUFA containing PCs such as PC 32:1, PC 34:4, PC 32:4, PC:36:5, polyunsaturated fatty acid containing TAGs; TAG 52:6, TAG 54:7, an unsaturated fatty acid (FA 20:5), and PI 36:4 were significantly decreased under HFD and negatively associated with obesity. These results are consistent with previous reports which show decreased SFA and MUFA PCs levels in the plasma 17 and liver samples of obese mice. 30 The increased PUFA TAG with HFD were observed in rats having developing obesity, 28 whereas decreased PUFA TAG was seen in plasma samples of human under HFD. 19 Decreased PUFA TAG under high-fat diet in our study is coherent with human studies. PI 36:4 was significantly decreased in the plasma of mice under HFD 17 and is identical with our results.
The PUFAs such as FA18:3, FA 20:5, FA 22:5 and MUFAs, FA 24:1 and FA 26:1 were negatively associated with obesity (Fig. 4A). Also, hierarchical cluster analysis also illustrates distinct clustering between the ND and HFD groups with a significant negative association of PUFAs and MUFAs with obesity. In a previous human study under HFD, the plasma ω-3 PUFAs was decreased, but increased in a low-fat diet 19 and also in the liver samples of obese mice shows the same trend with decreased ω-3 PUFAs. 30 In contrast, PUFAs were increased under HFD in obesity-prone rats. 28 Our experimental results showed increased PUFA, FA 22:4 level in liver, but decreased at epididymal adipose tissue of rats fed with HFD. In addition, other PUFAs such as FA 22:2 and FA 22:5 were also decreased in adipose tissue, indicating strong correlation with the PUFAs in plasma and suggesting PUFAs as potential biomarkers for obesity. The increased PUFA, FA 22:4 in liver is consistent with a previous study with the obesity-prone rats. 28 Moreover, the high-fat diet, itself, as high levels of SFA and MUFA compare to the normal diet, as demonstrated in Table 2, indicating the direct effect of diet on the plasma and tissue lipid profile. The outcomes of our analysis are highly interrelated with human and mouse studies and demonstrates their significance in diet to control obesity-associated disorders.
The total TAG levels in both liver and plasma largely increased in rodent models and human obesity. 31,32 Nevertheless, the relative composition of TAG varied with the type of obesity model. Most importantly, the effect of unsaturation is clearly observed in several lipid classes including TAG, PC, PI and PE. TAGs with PUFAs were decreased and TAGs with SFAs were increased under the HFD condition (Fig. 4B). Hierarchical cluster analysis also shows good clustering between the ND and HFD groups with a significant negative association of PUFA TAGs with obesity. These results were consistent with several other models, as for examples SFA containing TAGs were decreased in diet induced weight loss model 29 and increased in the serum of individuals with nonalcoholic fatty-liver disease (NAFLD). 33 In contrast, PUFA containing TAGs were decreased by 40% in ob/ob mice 34 and negatively associated with NAFLD. 33 Phosphatidylcholine's such as PC 32:1, PC 31:2, PC 15:1e-15:1 and FA 20:4, FA 18:3 containing PC, PI and oxidized PI were significantly decreased under HFD but LPCs were unchanged to the diet (Figs. 5A and 5B). These results are consistent with previous reports on the liver tissue lipid profile in NALFD model 33 and serum of obese mice, 17 where the several PUFA PC, PIs were negatively associated with obesity. However, some of the PIs having FA 18:1 shows a significant increase in our study and are similar to the results obtained in serum lipid profiles of obese mice where MUFA PIs such as PI 34:1, PI 36:1 were significantly higher in HFD. 17 To our knowledge OxPIs have not measured in rodent models of obesity so far. The OxPI 18:0-20:4+1O is decreased significantly in the plasma of rats fed with HFD, this could be due to the decreased levels of PIs having FA 20:4.
The other phospholipids such as PS, PG, PE, and LPE profile were described in Figs. 6A and 6B, along with their HCA results. There is a significant clustering between ND and HFD groups for PS and PG. But the levels of PE and LPE were not so significant and unaltered in our study. Several MUFA and PUFA derived PS were determined for the first time in rat plasma and are negatively associated with obesity. These results are consistent with liver tissue PS profiles of obese mice. 30 Moreover, it is known that the nutritional supply of PS can protect the brain against oxidative stress in HFD rats, 33 signifies the potential need of a PS-enriched diet to overcome obesity related disorders. Additionally, in this study, we measured several PG for the first time in rat plasma, and which are positively associated with the obesity and shows a completely different trend from other type of phospholipids. These results are also consistent with a previous report, where PS levels were positively allied with obesity on plasma lipidomic analysis in a rodent model of obesity by HFD. 35 Among, other detected molecular species, cholesterol ester CE 22:6 ( Fig. 6C) was determined for the first time in rat plasma, which shows a significant difference between the ND and HFD groups. This is also negatively associated with obesity, which is consistent with previous studies where several ω-3 FA derived CE were decreased under HFD. 17,19,30 An ether type lipid AAG 18:5e-24:1 was also determined in rat plasma for the first time, and is significantly elevated in the HFD group. A previous study shows the increased level of sphingomyelin molecular species under HFD, 17 and is consistent with our data. SM d18:1-16:0 showed a positive relation with obesity.
In conclusion, the plasma of ND and HFD rat models were compared and characterized using lipid profiles to provide new intuitions to lipid metabolism in obesity-associated disorders. Substantial differences in the relative concentration of lipid species were observed between ND and HFD rats. PUFAs and its derived plasma lipids, were significantly decreased and negatively associated with obesity, demonstrating the importance of PUFAs enriched diet to devastate obesity-associated diseases. Hence, decreased levels of PUFAs in plasma could be the novel biomarkers of obesity. This study disclosed that UHPLC/ LTQMS-based untargeted lipid profiling is an effective analytical tool for the depiction of global lipid profile in ND and HFD fed rats.