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Omics Technologies Applied to Agriculture and Food
Rapid evaporative ionization mass spectrometry based lipidomics tracking of grass carp (Ctenopharyngodon idellus) during in vitro multiple-stage digestion Yanan Lin, Haixing Wang, Wei Rao, Yiwei Cui, Xina Yu, Zhiyuan Dai, and Qing Shen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01644 • Publication Date (Web): 28 May 2018 Downloaded from http://pubs.acs.org on May 28, 2018
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Journal of Agricultural and Food Chemistry
Rapid evaporative ionization mass spectrometry based lipidomics tracking of grass carp (Ctenopharyngodon idellus) during in vitro multiple-stage digestion
Yanan Lin1, Haixing Wang2, Wei Rao3, Yiwei Cui1, Xina Yu1, Zhiyuan Dai1,4, Qing Shen1,4,*
1 Institute of Seafood, Zhejiang Gongshang University, Hangzhou, 310012, China 2 Zhejiang Province Key Lab of Anesthesiology, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, 325035, China 3 Waters Corporation, Shanghai, 201206, China 4 The Joint Key Laboratory of Aquatic Products Processing of Zhejiang Province, Hangzhou, 310012, China
Conflict of Interest: All authors declare that they have no conflict of interest.
* Corresponding Author Prof. Qing Shen Tel: +86 0571 88071024. Fax: +86 15968148458. E-mail:
[email protected];
[email protected] 1
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ABSTRACT
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A rapid evaporative ionization mass spectrometry (REIMS) method was
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developed for lipidomics tracking of Ctenopharyngodon idellus during in
4
vitro multiple-stage digestion. The REIMS conditions were optimized to be
5
the temperature of heating probe 500 ◦C, sample amount 30 mg, and flow rate
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of auxiliary solvent 100 µLmin–1. The results showed that the phospholipids
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were detected to be dominated with variety and quantity in the crude and
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multiple-stage digested samples. The enzymatic effect on the phospholipids is
9
varied depending on the phospholipid classes, and the hydrolysis rate of
10
phospholipids increased as the degree of unsaturation of the acyl chain
11
increased. The principal component analysis (PCA) indicated that the ion at
12
m/z 809.61, 811.63, and 857.52 were the most noticeable species digested
13
during the process. This method exhibited great potential in fast lipidomics
14
profiling for inspecting the characteristics of nutritional lipid absorption
15
digestion in human gastrointestin.
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KEYWORDS:
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multiple-stage digestion; rapid evaporative ionization mass spectrometry;
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phospholipids.
lipidomics;
Ctenopharyngodon
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idellus;
in
vitro
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INTRODUCTION
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Phospholipids are a class of amphiprotic biomolecules with important
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physiological and nutritional functions for human body, which include a
23
hydrophilic head and a hydrophobic tail comprising the hydrocarbon chains of
24
two fatty acids.1 The biological membrane is constituted of lipid bilayer, and
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its permeability and fluidity attribute to the composition and distribution of
26
phospholipids.2,3 Recently, researchers found that phospholipids can play a
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significant role in lipid absorption regulation, activating cells, enhancing the
28
immunity and regeneration, and reducing hepatic lipid levels and
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inflammation by clinical and biological experiments.4 In addition, with the
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natural biocompatible, biodegradable and amphiprotic characteristics,
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phospholipids have been widely utilized as emulsifier, surfactantas, liposome
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membrane, and drug coat in food, cosmetics and pharmaceutical industries.5-8
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Therefore, tracking the phospholipids in vitro digestion can simulate and
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estimate their behavior in the human gastrointestinal process, providing a
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basis for designing phospholipid containing products.
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In vitro digestion simulating human gastrointestinal environment is
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generally considered to be a safe, rapid, inexpensive method without ethical
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restriction for investigating the gastrointestinal behavior of food and
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pharmaceutical, and establishing the physicochemical phenomena that
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governs their behavior.9,10 Currently, researchers used a static method that
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simulate the mouth, stomach, and small intestine phases, since it is postulated
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that passage of a sample through the mouth and stomach will appreciably
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alter its subsequent behavior in the small intestine.11,12 Meanwhile,
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gastrointestinal tract model can accurately mimic the sophisticated conditions
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in vivo, such as salt, bile acids, variable digestive enzyme and pH, etc.9
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Therefore, multiple-stage digestion is an effective tool simulating the transit of
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phospholipids and analyzing their behavior through the human digestive
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tract.
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The rapid expanding research field, lipidomics, is built on the advances in
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multiple analytical technologies, such as liquid chromatography (LC), gas
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chromatography (GC), mass spectrometry (MS), and nuclear magnetic
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resonance (NMR) spectroscopy.13-15 One of the major analytical platforms in
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current lipidomics practice is a multidimensional mass spectrometry
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(MDMS)-based shotgun method.16,17 Hydrophilic interaction chromatography
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(HILIC) or reverse phase LC coupled to MS/MS is also commonly used for the
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separation and detection of phospholipids because of its resolution, sensitivity
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and reproducibility.18 However, the analytical procedure of these techniques is
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complex and labor-costing. Recently developed REIMS is a new and efficient
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technology which can realize real-time and accurate identification of tissues
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and allow in vivo, in situ tissue analysis.19,20 REIMS lipidomic profile displays
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many complex phospholipid molecular species originating from the cell
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membrane and make the analysis of lipids with minimal sample preparation 4
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and analysis time in a few seconds.21,22 By far, REIMS has been successfully
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applied to identify and classify the biological tissues,23 detect and resect tumor
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as the intelligent knife of medical surgery,21 characterize microorganisms,22
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and etc. Therefore, the application of REIMS in the research field of food
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science and technology has bright further, while related reports and
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publications are very rare.
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In this study, a REIMS method was developed and optimized for tracking
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the phospholipids in Ctenopharyngodon idellus during in vitro multiple-stage
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digestion. The results can contribute to revealing the characteristics of lipid
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nutrition absorption and the principle of release of phospholipid anchored
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compound in human gastrointestinal digestion.
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MATERIALS AND METHODS
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Materials and reagents.
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U·g–1), gastric pepsin (2000 U·mg–1) from porcine gastric mucosa, gastric
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lipase (3000 Ug–1), pancreatin (2500 Umg–1 based on lipase) from porcine
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pancreas and bile salts from bovine bile were obtained from Sigma Aldrich (St.
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Louis, MO, USA). Chloroform, methanol, propan-2-ol, and acetone were
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chromatography grade from Merck (Darmstadt, Germany). The other
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chemicals and reagents are standard analytical grade and purchased from
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Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
Salivary α-amylase from human saliva (1500
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Sample preparation and lipid extraction. The grass carp (C. Idellus)
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was selected as a representative sample because it is an important economic
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fish species in China containing medium content of fat. Fresh fish samples (C.
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Idellus, weight ca. 3 kg) were purchased from Wal-Mart Stores, Inc.
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(Hangzhou, China), placed in ice at a fish/ice ratio of 1:2 (w/w), and
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transported to the Institute of Seafood, Zhejiang Gongshang University,
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Hangzhou. Upon arrival, five fish samples without evident sign of parasite
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were collected and steamed. The white muscle from both sides of the fish
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sample was obtained by the mean of a mechanical deboner after removing the
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internal organs, heads, and tails of fish. Then, the muscle sample was ground
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to uniformity and stored at 4 ◦C. Five parallel samples from different fish
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bodies were prepared, and four technical replicates were applied.
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For extracting the crude lipid (including phospholipids, free fatty acids,
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glycerides, etc.), about 10 g of sample was weighed and homogenized with 30
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mL mixture of chloroform and methanol (2:1, v/v). Then, 10 mL of water was
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added, and the mixture was oscillated and centrifuged at 4 ◦C at 10000 r·min–1
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for 10 min. The organic phase (lower phase) was collected by pipette making
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sure to avoid the solid sample and upper phase. The residue was re-extracted
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with 20 mL of chloroform for another two times. Afterward, the collections of
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organic phase were combined and condensed by a rotary evaporator at 50 ◦C.
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After cooling down, 300 mL pre-cooled acetone (-20 ◦C) was poured into the
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mixture with vigorous shaking. The phospholipids were obtained by collecting 6
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the precipitation and nitrogen drying. The total amount of phospholipids at
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different digestion stage (g/100 g) was calculated using the equation, Quantity
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= Wp/Ws × 100, where Wp is the weight of phospholipids extracted from the
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digestion juice at different stage (g), while Ws is the weight of the initial fish
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sample (g).
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In vitro multiple-stage digestion.
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idellus during in vitro digestion, artificial simulated oral, gastric and intestinal
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digestion was conducted following the harmonized protocol published by
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Minekus et al.9 and Martínez-Las et al.24
To track the phospholipids of C.
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At the oral digestion stage, salivary α-amylase was added to the simulated
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salivary fluid (SSF) to a final concentration of 75 U·mL–1, and the mixture was
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combing with the prepared sample by a ratio of 1:1 (w/v). The mixture was
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properly homogenized in a 50 mL falcon tube and incubated with agitation for
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2 min at 37 ◦C. For gastric phase simulation, pepsin and gastric lipase were
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added into the simulated gastric fluid (SGF) to a final concentration of 2000
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U·mL–1 and 120 U·mL–1 respectively, and mixed with the sample after oral
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digestion at a ratio of 1:1 (v/v). The pH was adjusted to 3 with HCl (1 M), and
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the mixture was incubated at 37 ◦C for 2 h with continuous shaking (50 rpm).
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Pancreatin is a mixture of enzymes extracted from porcine pancreas
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containing trypsin, pancreatic amylase and pancreatic lipase. Because lipid
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digestion was the focus of this study, the addition of pancreatin was on the
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basis of the lipase activity. Finally, the simulated intestinal fluid (SIF) with
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2000 U·mL–1 of pancreatin and 10 mM of bile salts were added into the
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gastric mixture at 1:1 (v/v) ratio, and the pH was adjusted to 7 with NaOH (1
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M). Afterward, the mixture was incubated at 37 ◦C and shook at 50 rpm for 2 h.
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At the end of each stage, the samples were taken to the boiling water bath
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immediately for 6 min to inactivate the enzymes. The phospholipids were
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extracted and weighed by the aforementioned methods. The experiment was
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performed in triplicate.
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The content of phospholipids of C.
Instruments and parameters.
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idellus during in vitro multiple-stage digestion was investigated by REIMS
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(Waters Co., Ltd., Shanghai, China) which mainly contained the electric
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heating probe (WSD151, Weller, Germany) and a quadrupole time-of-flight
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mass spectrometer (Xevo G2-XS, Waters Co., Ltd., Shanghai, China) coupled
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with a commercial available REIMS interface. Mass spectrometer was
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calibrated with sodium formate before analysis every day. For REIMS analysis,
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the aerosol formed via the electric heating probe was aspirated into the MS via
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a PTFE tube by a Venturi pump driven by 2 bar nitrogen and mounted on the
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REIMS interface. In REIMS interface, the auxiliary solvent of propan-2-ol was
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introduced into the interface via a stainless steel capillary (1/16ʹʹ O.D., 0.002ʹʹ
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I.D.) with leucine enkephalin added for lock mass correction. The aerosol and
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solvent were mixed and guided to a heated helix collision surface operated at
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around 750 ◦C to improve signal intensity and avoid contamination. The 8
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REIMS spectra were obtained at the rate of 1 scan/second between m/z 50 –
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1200 in negative mode and accumulated for each analysi. The MS data were
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analyzed by the statistical software package Masslynxv4.1. Only the peaks with
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signal-to-noise higher than 10 were selected and analyzed. For mass spectrum
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processing, background subtraction was applied to reduce the interring noise,
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automatic peak detection was used to center the spetrum, and then lock mass
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(Leu-enkephalin, m/z 554.2615) was run to calibrate the spectrum for exact
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mass.
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Statistical analysis.
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statistical analysis. The relative content of each peak was calculated using a
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peak area normalization method. Statistical analysis and the calculation of
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mean, standard deviation, and level of significance were performed by using
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SPSS 16.0 software (SPSS Inc.,Chicago, IL, USA). The ions showing significant
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difference were examined with one-way ANOVA (p < 0.05). 20 samples for
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each digestion stage was applied to principal component analysis (PCA) for
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the determination of the main sources of variability (phospholipid peaks and
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relative contents) presented in the data sets to establish the relation between
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crude and digested samples with phospholipids.
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RESULTS AND DISCUSSION
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Optimization of REIMS conditions.
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was performed by online mass spectrometric analysis of the aerosol produced
MS spectral data in centroid mode was exported for
The REIMS study of phospholipids
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by electric heating evaporation.22 The temperature of sampling probe, sample
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volume, and flow rate of auxiliary solvent are critical and significant factors
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for the analysis of phospholipids. These factors were evaluated to optimize the
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REIMS conditions. The intensities of the peaks at m/z 783.52, 809.61, 835.54,
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857.52, and 883.54 were selected as inspection indicators.
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The temperature of sampling probe exerted significant influence on the
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intensity of phospholipid. In this study, the effect of temperature was
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evaluated in the range from 350 to 550 ◦C (Figure 1A). The results indicated
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that the sample can’t be sufficiently evaporated and ionized to produce aerosol
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at low temperature. With the increase of temperature, the signal intensities of
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the five selected peaks had a clear upward trend, and the maximum values
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were reached at 500 ◦C. When the temperature exceeded 500 ◦C, the intensity
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values decreased significantly, because high thermal energy can cause
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excessive sample evaporation and ionization and produce aerosol which
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contain many impurities. Therefore, the temperature of 500 ◦C was a superior
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condition which can be adopted for subsequent optimization tests.
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Sample volume is an important parameter for REIMS study, because a
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small amount of sample makes insufficient aerosol during evaporation, while
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excessive sample volume will dilute the thermal energy, leading to reduced
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peaks of phospholipids. The effect of sample volume on the ionization of
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phospholipids was depicted in Figure 1B, which indicated that there is a
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positive correlation between the signal intensity of phospholipid and the
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sample volume. The intensities of the selected peaks increased in the range
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from 15 to 30 mg of sample, and kept steady when the sample volume further
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increased to 35 mg. Therefore, 30 mg digestive sample is enough for REIMS
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analysis.
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In addition, a steady flow of isopropyl was used as auxiliary solvent and
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introduced directly into the atmospheric interface to enhance the signal
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intensity of phospholipid and wash the ion transfer pipeline. The effect of the
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loading flow rate of auxiliary solvent (10, 20, 50, 100 and 150 µLmin–1) on the
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signal intensity and stability of phospholipids were investigated. As shown in
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Figure 1C, the signals of phospholipid became more and more intensive when
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the flow rate was increased gradually from 10 to 100 µLmin–1, and the highest
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values were obtained at 100 µLmin–1. When the flow rate of auxiliary solvent
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was continuously increased, a light drop of the signals was occurred, and the
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signals of phospholipid became unstable and fluctuated dramatically.
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Therefore, the flow rate of auxiliary solvent was set at 100 µLmin–1 for
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promising a series of intensive and high resolution signals.
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Total volume of phospholipids at different digestion stage.
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prerequisite to compare the total quantity of phospholipids at different
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digestion stages for tracking the changes of phospholipids. The digestion
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efficiency and quantity of phospholipids in vitro oral, gastric and intestinal
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digestion were evaluated and compared with that of non-digested sample and
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the results were presented in Figure 2. The phospholipid digestion efficiency
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of non-digested sample was set as the start point (0%). There is a slight
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increase of digestion efficiency in the oral digestion stage (5.89%), and this
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upward trend became significant during the gastric digestion stage (25.22%).
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The highest digestion efficiency was obtained at the intestinal digestion stage
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(61.75%). The absolute phospholipid content was also depicted in the Figure.
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This value decreased in sequence during the oral, gastric and intestinal
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digestion, and the trend of drop became more and more fierce along with the
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digestion process. In particular, the absolute phospholipid content fell from
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1.37 to 0.26 (%) in the intestinal digestion stage. This phenomenon could be
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explained by the diversity and plenty of phospholipase in intestinal tract. After
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the ingestion of food, phospholipids were mainly physically digested in the
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mouth and stomach. The function of the oral cavity was mainly the
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mechanical mastication and homogenization, and the stomach is subjected to
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primary emulsification of lipid by gastric acid and reduce the surface tension
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of lipids. In intestinal digestion, the SIF was composed of bile salts and
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pancreatin from porcine pancreas containing lipase, phospholipase and
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cholesterol esterase. Pancreatic lipase can catalyze the hydrolysis of fatty acids
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of phospholipids and release fatty acids and acyl lysophospholipids. In
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addition, bile salts and enzymes have a synergistic effect on the hydrolysis of
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phospholipids.25
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The phospholipids in fish tissue can be
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Identification of phospholipids.
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evaporated and ionized in both positive-ion and negative-ion modes. However,
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the signals of phospholipids in positive-ion mode were suppressed by
236
impurities, such as glycerides, because of the protonating competition during
237
the rapid evaporative ionization. The phospholipids in negative-ion mode are
238
obviously more abundant and informative, due to the substantial negative
239
charge of the headgroup.26 Therefore, negative-ion mode was used in the
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subsequent experiments, and the extract of phospholipids from non-digested
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C. idellus was used as a control group. The mass spectrum was recorded in
242
real-time as the electric heating probe touched the sample. The identities of
243
phospholipids were interpreted by investigating the characteristics of ions in
244
the database LIPID MAPS and comparing them with those in related
245
literature, and the results of phospholipids analysis at different digestion
246
stages were summarized in Table 1. Some ions with intensive signals were
247
confirmed by MS/MS analysis. For example, the ion of 885.5 could be
248
tentative identified as [PI(38:4)-H]- by exact mass value, which could be
249
confirmed by the characteristic fragments of m/z 283.1 (18:0) and m/z 303.1
250
(20:4), indicating the detailed structure [PI 18:0/20:4-H]-. The phospholipids
251
identified in non-digested sample, oral, gastric, and intestinal digested sample
252
majorly dominated in the m/z range of 700 to 950. A total of 35 phospholipid
253
molecular species were tentatively identified, which could be divided into
254
three phospholipid classes, including PC, PE, and PI. However, PS, PA and PG
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were not observed in this experiment, and it could be attributed to the low
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abundance of PS and PA in fish tissue and the rare existence of PG in animal
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muscle tissue.27 As shown in the spectrum, the m/z of the most phospholipid
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peaks came out as an odd number (such as 809.61), indicating that the
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nitrogen-containing moiety of PC or PE has lost during ionization. It is usually
260
a good rule to speculate the molecular formula of phospholipids. For example,
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the m/z 739.57 could be tentatively explained as [PC O-38:2-N(CH3)3]– or [PE
262
O-38:2-NH3]–, and the m/z of PC O-38:2 and PE O-38:2 became identical
263
after losing the nitrogen-containing moiety. This phenomenon is not
264
frequently observed in modern mass spectrometers which are usually
265
equipped with soft ionization techniques, such as electrospray ionization,
266
atmospheric pressure chemical ionization, and matrix assisted laser
267
desorption ionization. The intensive energy of electric heating probe make the
268
rapid evaporative ionization a kind of hard ionization, leading to the loss of
269
nitrogen-containing moiety possible. It is worth to mention that some peaks
270
were composed of more than one phospholipid species. As described, the
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overlap of [PI 34:4 - H]–, [PE 44:6 - NH3]– and [PC 44:6 - N(CH3)3]– peaks
272
produced the peak at m/z 829.53. This phenomenon could be attributed to the
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similarity of structure between PC with PE and the equivalent molecular mass
274
after losing some groups. Some phospholipid molecular species has positive
275
nutritional properties, such as m/z 795.51 (PC 42:9 & PE 42:9), m/z 929.51
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(PI 42:10), etc., whose sn-1 and sn-2 moieties involved eicosapentaenoic acid
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or docosahexaenoic acid regarded as important protective agents for health
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promotion and disease prevention.28
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Influence of digestion on the quantity of phospholipids.
280
spectra of phospholipids extracted from non-digested crude sample and oral,
281
gastric and intestinal digested samples were depicted in Figure 3. It was
282
obvious that in vitro simulated digestion had a great influence on
283
phospholipids, not only on the mass spectrometric profile, but also on the
284
specific intensity of phospholipids. Before digestion, the peaks of phospholipid
285
in crude sample had the best signal-to-noise (S/N) ratio and signal diversity
286
involving 35 phospholipids species. The ion at m/z 809.61 was the most
287
abundant, which could be tentatively interpreted as the overlap of [PC 42:2 -
288
N(CH3)3H]– and [PE 42:2 - NH3]–, followed by the ions at m/z 857.52 ([PI
289
36:4 - H]–) and m/z 811.63 ([PC 42:1 - N(CH3)3H]– & [PE 42:1 - NH3]–). After
290
oral digestion, although there is a reduction of the signal intensities from
291
1.35e7 to 2.57e6, the diversity of phospholipid molecular species didn’t
292
changed much, because of the absence of lipase in the oral environment. The
293
lingual lipase secreted by lingual serous glands in mouth wasn’t considered
294
here, because it works on medium- and long-chain triglycerides only but not
295
phospholipids. Along with the progress of multistage digestion, the
296
background noise of the mass spectra became more and more serious, and the
297
signal intensity of phospholipid decreased to 9.72e5 and 4.22e5 for gastric and
298
intestinal digestion stages, respectively. The specific dynamic changes of 15
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intensity were listed in detail in Table 1. After comprehensive analysis, it can
300
be seen that the absolute intensity (peak area, cps) were reduced for all the
301
phospholipids during the digestion process. The enzymatic effect on the
302
phospholipids is varied generally depending on the phospholipid classes. The
303
PI decreased more significantly than the PC and PE. For example, the most
304
intensive peak, m/z 857.52 ([PI 36:4 - H]–), decreased dramatically from
305
9.19% to 3.71% after gastrointestinal digestion, and some PI peaks were
306
digested completely, such as the m/z 879.50 ([PI 38:7 - H]–), 903.50 ([PI 40:9
307
- H]–), 931.53 ([PI 42:9 - H]–), etc. On the contrary, the relative percentage of
308
PC and PE increased gradually, attributing to the marked drop of PI. For
309
example, the relative percentage of m/z 811.63 at different stages were 6.03%
310
(oral phase), 10.21% (gastric phase), and 13.57% (intestinal phase). In
311
addition, it was observed that the phospholipids with high degree of
312
unsaturation are more easily digested. The polyunsaturated phospholipid
313
molecular species, 903.50 ([PI 40:9 - H]–), 931.53 ([PI 42:9 - H]–), and etc,
314
were digested completely. It has been reported that the hydrolysis rate of
315
phospholipids increased as the degree of unsaturated of the acyl chain
316
increased.25 The hydrolyzing selectivity of porcine pancreatic lipase is also
317
related to location of double bonds in the fatty acid, which appears to be
318
depending on the distance of the first double bond from the ester linkage
319
being hydrolyzed.29
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It is well known that PCA is an effective method
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PCA statistical analysis.
321
used for reducing the number of dimensions present in the whole data matrix
322
with only a minimum loss of original information. To determine the main
323
variability and establish the relation between phospholipids and digestion,
324
PCA was applied to analyze the data produced by REIMS. It normalized the
325
relative amounts of the phospholipids in crude and digested samples in a
326
reduced dimension plot for revealing the difference between each digestion
327
stage. As shown in Figure 4A, there are generally three major clusters
328
observed in the score plot. The lipidomic profile data from crude and orally
329
digested samples were mixed into a single cluster. It can be explained by the
330
limited digestive effect in the oral digestion stage due to the absent of
331
phospholipase. The other two samples of gastric and intestinal digestion
332
stages were distinctly separated by using only the first two PCA components.
333
The separation effect of the second principal component (PC2) was not as
334
good as that of PC1. In detail, the first principal component (PC1) separated
335
the three clusters by explaining 65.8 cumulative percent (cum %) of the
336
variance in the REIMS data set, and PC2 explained 11.7 cum %. The first two
337
PCA components with 77.5 cum % of the total variance could be considered to
338
be significant enough to demonstrate the variation of the digestion stages
339
among these samples. Figure 4B revealed the loadings for the variables in the
340
first two PCA components clearly, in which the most influential phospholipid
341
molecular species could found out. It showed that most of the phospholipid
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molecular species were clustered at the central zero line, whereas the rest were
343
dispersed far away from the center. The species with higher loading values
344
and maximum variance in the data were mostly influenced by the digestion
345
treatment. For instance, the ion at m/z 797.54 ([PG 38:4]–), m/z 809.61 ([PE
346
42:2 - NH3]– & [PC 42:2 - N(CH3)3]–), 811.63 ([PE 42:1 - NH3]– & [PC 42:1 -
347
N(CH3)3]–), 857.52 ([PI 36:4 - H]–), etc. were the most noticeable species, and
348
their content varied dramatically along with the digestion process. All of these
349
ions showing significant difference were examined with one-way ANOVA (p