Quantitative Analysis of Polar Lipids in the Nanoliter Level of Rat Serum by Liquid Chromatography/Mass Spectrometry/Mass Spectrometry

Abstract

Polar lipids in serum, including lysophospholipids (LPLs) and free fatty acids (FFAs), have a broad range of biological activities and require a suitable method for their quantitative analysis. Conventional methods use multistep procedures to simultaneously purify and analyze polar lipids and non-polar lipids in serum. However, the methods could result in inaccurate quantifications of polar and/or non-polar lipids because compounds with different polarities have different behaviors in solvent extraction and mass spectrometric ionization. In this study, a method was designed to analyze polar lipids in serum based on the polarities of LPLs and FFAs. The method consisted of extraction without filtration and analysis of the crude extract without multistep purification. Fifty LPLs and 32 FFAs were detected in rat serum. The concentrations of LPLs (1272.1 μmole/L in female and 999.8 μmole/L in male) and FFAs (1910.9 μmole/L in female and 1651.4 μmole/L in male) were determined. Peak areas of MS ion in Extract Ion Chromatogram (EIC) were used for the quantification in this study. The approach of quantification should be perfectly suitable for precise quantification of a specific serum component by adding its isotope standard to the serum before extraction.

Introduction

LPLs are major lipid components in serum. Varied polar headgroups in LPL components result in different subclasses such as lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidic acid (LPA), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), and lysophosphatidylserine (LPS). LPC represents 5–20% of total serum phospholipids depending on the mammalian species (1). LPC is recognized as an important factor in processes of signal transduction (2) and plays a functional role in various diseases, including atherosclerosis, diabetes, and cancer mediated by LPC-specific G-protein-coupled receptors (3). The serum levels of LPCs have been reported to increase gradually in animals fed a high cholesterol diet (4). Okita et al. have demonstrated an abnormally high LPC concentration (by 43% overall, relative to normal controls) in the plasma of ovarian cancer patients (5). The plasma level of LPC has been found to be significantly raised in both asthma and rhinitis groups (6). Other subclasses of LPL have also been reported to show various biological activities. LPE affects the regulated exocytosis of pancreatic acinar AR42J cells (7). LPA and platelet-activating factor (PAF) have well-characterized effects on cell behavior that are mediated through specific receptor-coupled signaling systems (8–11). LPG inhibits LPA (12), and LPI shows mitogenic activity (13). LPS acts as a potent coactivator for mast cells (14). Furthermore, bioactivities of different components in the same LPL subclass are not the same. In the plasma of ovarian cancer patients, the percentages of palmitoyl- and stearoyl-LPC species are significantly higher, whereas oleoyl- and particularly linoleoyl-LPC are significantly lower than in control subjects (5). The relative concentration between regioisomers of LPL (sn-1or sn-2 lyso subspecies) is also important because generation of these isomers would influence their removal from blood, their uptake and acylation, and/or their catabolism in tissues (15, 16). In addition to LPL, FFA is also a major polar lipid class in serum and implicated in signal transduction (17). Elevated levels of circulating FFAs promote lipid accumulation and insulin resistance in target tissues, which is strongly linked to type 2 diabetes (18). These findings emphasize a need for analysis of individual polar lipid components in serum.

Solvent systems containing chloroform and methanol were used to simultaneously extract polar and non-polar lipids from serum and plasma in the majority of studies (1, 4, 5). Identification of individual lipid components involved separation by multistep column (19, 20), thin-layer (21, 22) or HPLC chromatographies (23–25) and identification by chemical derivatization and gas chromatographic analysis (1, 5) or nuclear magnetic resonance spectroscopy (26, 27). More recently, electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to analyze polar lipids, and results suggested that ESI-MS/MS is sensitive (detection limit was <1 μmole/L) and reproducible for quantitative analysis of polar lipid components (3, 28, 29).

In our previous studies, a method for analysis of polar lipids was developed and 95 relative polar lipids including 18 LPCs, 12 LPEs, 11 LPIs, 11 LPAs, 4 LPGs and 39 FFAs were identified and quantitated in the extract of soy protein isolate (30, 31). The aim of the current study was to develop a rapid, accurate, and reproducible method for analysis of polar lipid components in serum. The method only includes two steps: exhaustive extraction and direct analysis of crude extract by LC-MS/MS. Serum components were identified by LC retention time and both positive and negative-ion spectra from ESI-MS/MS, and peak areas of MS ion in EIC were used for the quantification. The method could be a valuable tool for analysis of polar lipids in serum.

Materials and Methods

Materials and Reagents.

Eighteen standards were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO) for LC-MS/MS analysis including LPLs, free fatty acids, and flavonoids. Three standard mixtures were L-α-lysophosphatidylethanolamine (prepared from egg yolk, contains primarily stearic and palmitic acids), L-α-lysophosphatidylcholine (prepared by the action of phospholipase A on soybean L-α-phosphatidylcholine, contains primarily C-18 unsaturated fatty acids) and L-α-lysophosphatidylinositol (prepared by the action of phospholipase A on soybean L-α-phosphatidylinositol, contains primarily palmitic and stearic acids). Three L-α-lysophosphatidylcholine standards were 1-palmitoyl-sn-glycero-3-phosphocholine (16:0a/lyso-PC), 1-oleoyl-sn-glycero-3-phosphocholine (18:1a/lyso-PC), and 1-stearoyl-sn-glycero-3-phosphocholine (18:0a/lyso-PC). Fatty acid standards included [5S,12R]-dihydroxy-[6Z,8E,10E,14Z]-eicosatetraenoic acid (leukotriene B₄), 13(S)-hydroxyoctadeca-9Z,11E-dienoic acid, 15(S)-hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid, 12(S)-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid, cis-4,7,10,13,16,19-docosahexaenoic acid, eicosa-5Z,8Z, 11Z,14Z-tetraenoic acid (arachidonic acid), cis-9, cis-12-octadecadienoic acid, hexadecanoic acid (palmitic acid), cis-9-octadecenoic acid (oleic acid), and octadecanoic acid (stearic acid). Flavonoid standards were genistein and biochanin A.

Animal Studies.

Animal use protocols were approved by the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences. Pregnant Spraque-Dawley rats were purchased from Charles Rivers (Willington, MA) and arrived at our facility on gestational day 4. Animals were housed in an approved facility and animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences. Diets were purchased from Harlan Teklad (Indianapolis, IN) and were made according to the published AIN-93G formulation (32). Groups of n = 8 male and female rats from our breeding colony were used. Rats at age 86 days were sacrificed and sera were collected and stored at −70°C.

Sample Preparation for LC-MS/MS.

A pool was made by combining sera from eight rats per group (0.5 mL/rat). The extraction was performed in two containers: an extraction tube (1.5 mL microcentrifuge tubes) and a LC-MS sample vial. Pooled serum (40 μL) in an extraction tube was added with 80 μL 100% methanol (final methanol concentration is 67%) containing 0.2 μg genistein and 0.4 μg biochanin A as internal standards (IS), and vortexed vigorously for 10 mins followed by sonication in ice water for 10 mins. The mixture was centrifuged at RCF 15339 ×g in Centrifuge 5415 C (Eppendorf) for 20 mins and the supernate as an extract was removed carefully to the LC-MS sample vial. The extraction process was repeated twice with 80 μL of 100% methanol and 80 μL of 90% aqueous methanol, respectively. The volume of extracts from three extractions in LC-MS/MS sample vial was brought to 300 μL with 100% methanol and mixed well for LC-MS/MS analysis.

In order to examine the efficiency of the extractions, the pellet from the 3rd extraction was extracted with 100% ethanol (80 μL) and methanol:methylene chloride (1:2) (80 μL). The combined extract from these two extractions was evaporated to dryness under N₂ followed by drying in a freeze-dryer. The dried extract was reconstituted with 150 μL of 95% methanol for LC-MS/MS analysis.

LC-MS/MS Analysis.

The extract of serum was directly analyzed without any purification. Fifteen μL of sample was loaded on LC-MS/MS for analysis, which was equivalent to 2.0 μL of serum containing 10 ng genistein and 20 ng biochanin A as IS. LC-MS/MS was performed using a Bruker Esquire-LC multiple ion trap mass spectrometer equipped with an Agilent 1100 series liquid chromatograph. An HP ChemStation was used for data collection and manipulation.

A 150 × 4.6 mm i.d. Eclipse XDB-C8 column (Agilent Technologies, Wilmington, DE) was used with LC solvent at a flow rate of 0.5 mL/min. The LC gradient was 0.1% formic acid/acetonitrile (solvent B) in 0.1% formic acid/H₂O (solvent A) as follows: 20–58% in 20 mins; 58–64% from 20–33 mins; 64–100% from 33–60 mins; held at 100% from 60–65 mins and finally back to 20% in 70 mins. A diode-array detector set at five wavelengths of 200 ± 10, 240 ± 10, 290 ± 10, 320 ± 10 and 355 ± 10 nm to monitor the constituents in the eluant, and the constituents in the eluant were analyzed by automatic MS/MS with both negative and positive ion modes. For optimum MS analysis, 10 mM ammonium acetate (for negative-ion mode) or 2% formic acid (for positive-ion mode) in methanol were used as ionization reagents and added at a flow rate of 0.1 mL/min via a tee in the eluant stream of the HPLC just prior to the mass spectrometer. Conditions for ESI-MS analysis in both negative- and positive-ion modes included a capillary voltage of 3200 V, a nebulizing pressure of 33.4 psi, a drying gas flow of 8 mL/min, and a temperature of 250°C. Parameters that control the API interface and the mass spectrometer were set via the Smart Tune with compound stability of 50% and trap drive level of 50%. Ion Charge Control (ICC) was On including: target, 5000; maximum accumulation time, 50.00 ms; scan, m/z 80.00 to 850.00; averages, 10; and rolling averaging, off. Conditions for automatic MS/MS were width of the isolation, 4.0; fragmentation amplitude, 1.00 V; and number of parents, 1.

Quantification of Serum Components.

Internal standards (IS) (5 ng genistein and 10 ng biochanin A in 1 μL serum) were added to serum to monitor the reproducibility of extractions and LC-MS/MS analyses. Quantification was performed by LC-MS using MS peak areas of anions generated from Extracted Ion Chromatogram (EIC). Deprotonated molecular ions were used for FFAs and LPEs, and adduct ions [M+COO]⁻ for LPCs. Calibration curves were constructed using a series of diluted standards (800, 400, 200, 100, 50, 25, 12.5, 6.25 and 3.125 ng). Thirteen standards were used to create calibration curves including 16:0a/lyso-PC (Std 1), 18:1a/lyso-PC (Std 2), 18:0a/lyso-PC (Std 3), cis-4,7,10,13,16,19-docosahexaenoic acid (Std 4), arachidonic acid (Std 5), linoleic acid (Std 6), palmitic acid (Std 7), oleic acid (Std 8), stearic acid (Std 9), 13(S)-hydroxyoctadeca-9Z,11E-dienoic acid (Std 10), 15(S)-hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid (Std 11), 12(s)-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid (Std 12) and leukotriene B₄ (Std 13). Calibration curves of Std 1 were used for quantification of compounds 28 and 30; Std 2 for 13, 24, 25, 26, 27, 29, 31, 32, 33, 35, 36, 37 and 40; Std 3 for 47, 49, 51 and 58; Std 4 for 64; Std 5 for 65; Std 6 for 63, 66, 67; Std 7 for 70; Std 8 for 72; Std 9 for 73; Std 10 for 34 and 44; Std 11 for 43, 48 and 52; Std 12 for 53, 56 and 57; Std 13 for 11, 12, 14, 17 and 20 (Supplementary Tables 3–5 online). Results are expressed as means ± SD for at least three replicate determinations for each assay.

Results

Extraction and LC-MS/MS Analysis Strategy.

Several methods were designed to establish a protocol for the exhaustive extraction of polar lipids from serum. The results indicated that methanol was more efficient than chloroform-containing solvent systems for extraction of polar lipids from serum. The levels of serum polar lipids in the extract from the method using vortex were lower than the method using vortex followed by sonication. The final extraction procedure consisted of methanol and aqueous methanol extractions by vortex followed by sonication and extraction repeated for three times (67%, 100% and 90% aqueous methanol). There were no detectable IS and polar lipid components in the extract from a fourth extraction with 100% ethanol and a fifth extraction with methanol:methylene chloride (1:2).

Polar lipids in the final sample (crude extract of serum) were directly analyzed by LC-MS/MS with Reverse Phase (RP) HPLC and electrospray ionization (ESI). LC-MS/MS analysis was considered very reproducible based on the recovery rates of standards. For example, the means of MS peak areas of anions of IS genistein and biochanin A in all LC-MS/MS analyses were 1.12 × 10⁸ ± 3.33 × 10⁶ and 1.88 × 10⁸ ± 2.89 × 10⁶ (mean ± SD, n = 8), respectively. LC-MS/MS profiles of serum polar lipids are shown in Figure 1. The peak of each component can be generated by Extracted Ion Chromatogram Program and do not have any interference with the peaks of other components in the extract of serum.

Identification of Serum Polar Lipids.

The HPLC retention time and ESI-MS/MS data of serum polar lipid components are presented in Supplementary Tables 1 and 2 (online). Direct comparison of twelve serum components with their corresponding standards in LC-MS/MS analyses are shown in Figure 1A and B . Fifty LPLs and 14 of 32 FFAs were identified by comparing their MS data with the MS data of their corresponding standards from either our analysis (30, 31) or from literature (33). Fifty LPLs included 38 LPCs, 3 LPEs, 5 LPIs and 4 LPAs (Supplementary Table 1 online). Fourteen identified FFAs were 14, 45, 48, 52, 56, 57, 61, 63, 64–66, 70, 72, 73 (Supplementary Table 2 online). The remaining 18 FFAs (1, 11, 12, 17, 20, 23, 34, 43, 44, 50, 53–55, 62, 67–69, 71) did not have corresponding standards nor literature data for comparison. They were instead characterized by their retention time and product ions from collision-induced dissociation (CID) in LC-MS/MS analysis (Supplementary Table 2 online). Carboxylate anions [M-H]⁻ were major molecular ion species in the negative ionization mass spectra of FFAs, and CID of [M-H]⁻ yielded abundant ions [M-H-H₂O]⁻. Interpretation of MS data led to the formula as well as the numbers of hydroxyl group(s) and double bond(s) for the structures of these 18 FFAs. However, positions of hydroxyl group(s) and double bond(s) for these 18 FFAs could not be determined because neither their NMR data nor their corresponding standards were available.

Quantification of Serum Polar Lipids.

Quantification was performed by LC-MS using MS peak areas of anions generated from Extracted Ion Chromatogram (EIC). Concentrations of thirteen serum components, including major serum components 30, 51, 64, 65, 66, 70, 72 and 73, were determined from calibration curves of their corresponding standards. Compounds were quantified by calibration curves of their structure-related standards when their corresponding standards were not available (Supplementary Tables 3–5 online). The levels of LPLs and FFAs in sera are summarized in Tables 1 and 2 , respectively. LPC is a major subclass of LPL and represents more than 99.37% of total LPL in the sera of both female and male rats. LPE is a minor LPL subclass, and LPA and LPI are present in trace amounts in the sera. Nonhydroxyl FFAs are major FFAs in sera of both female and male rats. Nineteen oxygenated FFAs components, including eleven oxygenated arachidonic acids, were detected and oxygenated FFAs is a minor FFA subclass in sera. Comparison of female and male rats revealed the different profiles of serum polar lipids from female and male rats. The level of total polar lipids in sera of female rats (3183.0 ± 113.6 μmole/L) is higher than male rats (2643.3 ± 24.3 μmole/L). The μmole/L ratios of female and male are 1272.1 ± 76.6 versus 999.8 ± 27.5 for serum LPL, and 1910.9 ± 40.0 versus 1651.4 ± 20.3 for serum FFA. It should be noted that there is higher level of oxygenated FFA components in male serum (4.9%) than in female serum (3.7%).

Discussion

Chloroform-containing solvent systems are the common solvent systems for simultaneous extraction of polar and non-polar lipids from serum and plasma in many studies (1, 4, 5). However, simultaneous extraction of polar and non-polar lipids from serum can not be efficient and complete because of their different polarities. In this study, the analysis of lipids in serum was focused on polar lipids and several extraction methods were designed based on the polarities of polar lipids in serum. The results indicate that methanol-aqueous methanol solvent systems are more efficient for extraction of polar lipids from serum than chloroform-containing solvent systems. Levels of serum polar lipids in extracts from methods using vortex were lower than vortex followed by sonication. This may be an indication that sonication is a critical step in releasing polar lipids from serum protein and exhaustively extracting polar lipids from serum. Extraction for three times (67%, 100% and 90% aqueous methanol) with vortex and sonication is considered nearly complete because there were no detectable IS and polar lipid components in extracts from the fourth extraction with 100% ethanol and fifth extraction with methanol:methylene chloride (1:2). The extraction was conducted in two containers and did not involve filtration and multistep separation. The extraction procedure avoided any step that may cause loss and contamination of serum components. In order to avoid generation of artificial compounds during extraction, the extraction was conducted at room temperature and completed in three hours without addition of any chemicals, such as acids. The extract in the LC-MS/MS vial stood in −20°C (less than one week) until LC-MS/MS analysis.

Reverse phase (RP) HPLC with C8 column yielded a good separation of polar lipids in our previous studies (30, 31, 34), which was used in this study. ESI-MS is a major breakthrough for biological MS because this technique does not require reactions (hydrolysis and/or derivatization) of polar lipids. Alone or coupled with HPLC, ESI-MS can be used to directly analyze polar lipid components in a complex matrix, and provides highly sensitive and reproducible results (3, 28–31). In our previous study, 56 LPLs and 39 FFAs in the crude extract of soy protein isolate were separated and identified by LC-MS/MS (30). Using same method, 50 LPLs and 32 FFAs were identified or characterized in rat serum in this study.

The peak areas of standards shown in Figure 1A indicate that LC-MS has different sensitivity to different compounds. For example, the LC-MS profiles of polar lipids in the sera (Fig. 1B and 1C ) suggest that major serum components are 30, 51 and 65; however, the values obtained from the calibration curves of their corresponding standard indicate that major serum components are 30, 51, 64, 65, 66, 70, 72 and 73 (Tables 1 , 2 ). It is important to note that the value of the compound, which was obtained from its structural related standard, did not exactly represent its concentration in serum. LPC is a dominant subclass of LPL in serum and LPE is present in a low level, while only trace amounts of PLA and PLI were detected. LPA is an extracellular signaling mediator with a broad range of cellular responses, and has clinical importance as a potential biomarker in various diseases (35). LPA has been reported to be present in mammalian serum at low levels (1–5 μM) (36). In this study, several methods were designed to find an exhaustive extraction procedure, and results suggest that a more exhaustive extraction results in a higher concentration of PLC in the extract. On the other hand, more exhaustive extraction led to lower levels of other LPL subclasses including LPA and PLI in the extract of serum.

In summary, this method consists of an exhaustive extraction and direct analysis of the crude extract of serum by LC-MS/MS. It is a simple and reliable tool for establishment of serum polar lipid profile and analysis of individual component of polar lipids in serum, which is important for analysis individual component because different components of the same LPL subclass in blood have different bioactivities (5, 15, 16). The quantification approach using peak areas of MS ions in this study should be perfectly suitable for the precise quantification of a specific serum component by adding its isotope standard to the serum before extraction.

Note: Supplementary information is available on the Experimental Biology and Medicine website.

Table 1.

Difference of the Levels of Serum Lysophospholipids in Female and Male Rats

No. in Figure 1 ^a	LPLs	In serum	Levels in female	Levels in male	Male/Female
^a LPCs 10, 15, 16, 18, 19, 21, 22, 38, 39, 41, 42, 59 and 60; LPE 46; LPI-1 to LPI-5; and LPA-1 to PLA-4 are present in trace amounts in the rat sera.
Oxygenated lysophosphatidylcholines (compound 2–9):
Subtotal:		μmol/L	4.59 ± 0.34	9.75 ± 0.39	2.12 ± 0.08
Lysophosphatidylcholines:
13	14:0a/lyso-PC	μmol/L	1.58 ± 0.27	3.08 ± 0.74	1.95 ± 0.47
24	lyso/18:2a-PC	μmol/L	8.87 ± 0.41	13.48 ± 0.96	1.52 ± 0.11
25	lyso/20:4a-PC	μmol/L	3.90 ± 0.29	4.36 ± 0.16	1.12 ± 0.04
26	18:2a/lyso-PC	μmol/L	55.17 ± 5.64	75.38 ± 3.19	1.37 ± 0.06
27	20:4a/lyso-PC	μmol/L	27.99 ± 0.98	32.90 ± 4.83	1.18 ± 0.17
28	lyso/16:0a-PC	μmol/L	53.24 ± 1.94	63.14 ± 3.15	1.19 ± 0.06
30	16:0a/lyso-PC	μmol/L	391.40 ± 25.64	465.22 ± 16.33	1.19 ± 0.04
31, 32	lyso/18:1a-PCs	μmol/L	3.93 ± 0.68	8.60 ± 0.47	2.19 ± 0.12
33	15:0a/lyso-PC	μmol/L	2.90 ± 0.27	1.73 ± 0.55	0.54 ± 0.19
35	22:5a/lyso-PC	μmol/L	2.14 ± 0.23	1.23 ± 0.10	0.59 ± 0.04
36, 37	18:1a/lyso-PCs	μmol/L	27.20 ± 0.99	59.53 ± 1.77	2.19 ± 0.07
40	17:0a/lyso-PC	μmol/L	3.87 ± 0.46	3.20 ± 0.53	0.88 ± 0.10
47	lyso/18:0a-PC	μmol/L	72.82 ± 2.71	33.50 ± 2.59	0.46 ± 0.04
51	18:0a/lyso-PC	μmol/L	601.62 ± 73.08	207.54 ± 19.52	0.34 ± 0.03
58	20:1a/lyso-PC	μmol/L	2.90 ± 0.37	3.01 ± 0.30	1.04 ± 0.10
Subtotal:		μmol/L	1259.53 ± 76.82	975.89 ± 28.21	0.77 ± 0.02
Lysophosphatidylethanolamines:
29	16:0a/lyso-PE	μmol/L	2.36 ± 0.37	2.02 ± 0.21	0.85 ± 0.09
49	18:0a/lyso-PE	μmol/L	5.57 ± 0.53	4.19 ± 0.52	0.75 ± 0.09
Subtotal:		μmol/L	7.93 ± 0.85	6.21 ± 0.69	0.78 ± 0.09
Total LPL in serum:		μmol/L	1272.05 ± 76.63	999.84 ± 27.47	0.77 ± 0.02

Table 2.

Difference of the Levels of Serum Free Fatty Acids in Female and Male Rats

No. in Figure 1 ^a	Free fatty acids	In serum	Levels in female	Levels in male	Male/Female
^a Components 23, 45, 50, 54, 55, 61, 62, 69 and 71 are present in trace amounts in the rat sera.
Fatty acids with 2 oxygen atoms (nonhydroxyl group):
63	16:1 (Palmitoleic acid)	μmol/L	12.54 ± 0.61	16.83 ± 3.88	1.34 ± 0.31
64	Cis-4,7,10,13,16, 19-docosahexaenoic acid	μmol/L	196.84 ± 8.02	91.25 ± 10.17	0.46 ± 0.05
65	20:4 (arachidonic acid)	μmol/L	268.08 ± 20.68	300.07 ± 9.40	1.12 ± 0.04
66	18:2 (linoleic acid)	μmol/L	373.02 ± 10.37	393.99 ± 18.93	1.06 ± 0.05
67	22:5	μmol/L	42.74 ± 2.86	23.64 ± 3.81	0.55 ± 0.09
68	20:3	μmol/L	7.63 ± 0.66	8.79 ± 0.59	1.15 ± 0.08
70	16:0 (palmitic acid)	μmol/L	355.74 ± 18.18	341.26 ± 33.32	0.96 ± 0.09
72	18:1 (oleic acid)	μmol/L	283.39 ± 10.15	226.66 ± 6.77	0.80 ± 0.02
73	18:0 (stearic acid)	μmol/L	278.45 ± 17.52	167.65 ± 17.80	0.60 ± 0.06
Subtotal:		μmol/L	1839.36 ± 22.09	1570.15 ± 23.00	0.86 ± 0.01
Monohydroxyoctadeca-dienoic acid:
34	OH-18:2	μmol/L	0.55 ± 0.13	0.43 ± 0.10	0.78 ± 0.18
44	9-OH-10,12–18:2	μmol/L	21.92 ± 2.63	24.65 ± 1.11	1.12 ± 0.05
Subtotal:		μmol/L	22.48 ± 2.58	25.08 ± 1.14	1.12 ± 0.05
Monohydroxy-eicosatetraenoic acid:
43	OH-20:4	μmol/L	0.42 ± 0.21	0.42 ± 0.20	0.99 ± 0.48
48	15-OH-5,8,11,13–20:4	μmol/L	7.62 ± 1.47	7.73 ± 0.75	1.01 ± 0.10
52	11-OH-5,8,12,14–20:4	μmol/L	3.90 ± 1.36	6.40 ± 0.71	1.64 ± 0.18
53	OH-18:3	μmol/L	1.48 ± 0.42	1.38 ± 0.41	0.93 ± 0.28
56, 57	12-OH-5,8,10,14–20:4	μmol/L	29.95 ± 3.12	33.65 ± 1.66	1.12 ± 0.06
Subtotal:		μmol/L	43.36 ± 5.56	49.58 ± 2.73	1.14 ± 0.06
LTB ₄ and its isomers:
11	Isomer of LTB₄	μmol/L	0.65 ± 0.25	0.65 ± 0.14	1.00 ± 0.21
12	Isomer of LTB₄	μmol/L	1.10 ± 0.06	0.40 ± 0.25	0.36 ± 0.23
14	LTB₄	μmol/L	0.64 ± 0.12	1.26 ± 0.29	1.96 ± 0.45
17	Isomer of LTB₄	μmol/L	0.35 ± 0.05	0.41 ± 0.13	1.18 ± 0.37
20	Isomer of LTB₄	μmol/L	2.96 ± 0.17	3.90 ± 0.18	1.32 ± 0.06
Subtotal:		μmol/L	5.70 ± 0.47	6.61 ± 0.54	1.16 ± 0.10
Total FFA in serum:		μmol/L	1910.90 ± 39.98	1651.42 ± 20.27	0.86 ± 0.01

Figure 1.

LC-MS total ion chromatograms (TIC) of the relative polar component in serum of rats (B–E) with comparison of TIC of the standard mixture (A); Figure 1A, the standard mixture contained: [G, genistein; B, biochanin A; FA 1, leukotriene B₄ (14 in B); FA 2, 13(S)-hydroxyoctadeca-9Z,11E-dienoic acid (45 in B); FA 3, 15(S)-hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid (48 in B); FA 4, cis-4,7,10,13,16,19-docosahexaenoic acid (64 in B); FA 5, arachidonic acid (65 in B); FA 6, cis-9, cis-12-octadecadienoic acid (66 in B); FA 7, palmitic acid (70 in B); FA 8, oleic acid (72 in B); and FA 9, stearic acid (73 in B)]; and [LPC 1, 16:0a/lyso-PC (30 in B); LPC 2, 18:1a/lyso-PC (37 in B); and LPC 32, 18:0a/lyso-PC (51 in B)]. Peak * is lyso/16:0a-PC, an impurity from 30; Figure 1B, negative MS TIC of the relative polar component in serum of female rats. The compound No. of the minor serum components 1, 2, 4, 5, 14–16, 21–23, 25, 29, 32–35, 38, 39, 41, 43, 45, 46, 49, 50, 51–55, 58–63, 68 and 69 are not shown in Figure 1B, but presented in Supplementary Table 1 and 2 (online). Figure 1C, negative MS TIC of the relative polar component in serum of male rats. Figure 1D, positive MS TIC of the relative polar component in serum of female rats. Figure 1E, positive MS TIC of the relative polar component in serum of male rats.

Footnotes

This work was supported in part by the USDA Arkansas Children’s Nutrition Center (#6251-51000-005-02S) and Hubei College of Traditional Chinese Medicine in China.

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