TY - JOUR
AU - Miura, Shinji
AB - ABSTRACT Phosphatidylcholine (PC) is an essential component of the plasma membrane. Its profile varies with species and tissues. However, the PC profiles in meat have not been explored in depth. This study aimed to investigate the differences in PC profiles between various meat animal species and meat cut sites, along with the identification of characteristic PC molecules. The results demonstrated that the PC profiles of chicken meat differed from those of other species. Significant differences were also observed between the PC profiles of pork meat and the meat obtained from other species. The amount of PCs containing ether bonds was high in pork meat. PCs containing an odd number of carbon atoms were characteristic of beef and lamb meats. Furthermore, PC profiles differed based on the muscle location in chicken and pork. These results suggest that the PC profiles of skeletal muscles are indicators of animal species and muscle location. Graphical Abstract Open in new tabDownload slide Lipidomics of phosphatidylcholine molecules present in various meat animal species or muscle locations. Graphical Abstract Open in new tabDownload slide Lipidomics of phosphatidylcholine molecules present in various meat animal species or muscle locations. animal species, meat, muscle location, muscle type, phosphatidylcholine Lipids are indispensable molecules for cells that are involved in a wide variety of cellular functions, including functions of the cell membrane, energy storage, and signaling. Many studies have been conducted examining the biochemistry of lipids as it plays an important role in dietary nutrition (Alphonse and Jones 2016; Innes and Calder 2018; Shahidi and Ambigaipalan 2018; Unger, Torres-Gonzalez and Kraft 2019). However, fundamentally, we cannot measure the amount of all kinds of lipids simultaneously owing to the diversity and complexity of lipid molecules (Fathy, Subramaniam and Brown 2005). Thin-layer chromatography (Fuchs et al. 2011), gas chromatography (Brenna 2013), liquid chromatography (LC), Raman spectroscopy (Bruzas, Lum and Gorunmez 2018), Fourier-transform infrared spectroscopy (Li et al. 2019), nuclear magnetic resonance (Knothe and Kenar 2004), and mass spectrometry (MS) (Li et al. 2014) have been employed for the measurement of lipids. The selection of the method and its use are based on the chemical properties of target lipid molecules. With recent advances made in the design of the analysis apparatus, LC/electrospray ionization (ESI)-MS has been used for the precise analysis of phosphatidylcholine (PC) (Koivusalo et al. 2001; Brouwers 2011; Takahashi et al. 2018). Phospholipids are fundamental constituents of cell membranes and influence many physical properties associated with membrane functions, including fluidity, permeability, and the anchoring of membrane-related proteins. Glycerophospholipids are synthesized de novo from glycerol-3-phosphate via Kennedy pathway, and reconstructed into various phospholipids whose fatty acids can be removed or switched in the Lands cycle (Kennedy and Weiss 1956; Lands 1958; Wang and Tontonoz 2019). PC has a glycerol backbone with 2 fatty acids that are linked via an ester bond at the sn-1 and sn-2 position and a polar head. It is the most abundant class of phospholipids observed in the majority of eukaryotic cells (Vance 2015). The most abundant PC molecule varies in each species (Le Grandois et al. 2009). For instance, the most commonly observed molecular species are PC (16:0/18:2) and PC (16:0/18:1) in pork meat (Boselli et al. 2008), and PC (16:0/18:1), PC (16:0/18:2) and PC (18:0/18:2) in chicken breast meat (Takahashi et al. 2018). Ether phospholipids have the glycerol backbone with ether or vinyl-ether bond at the sn-1 position. These phospholipids account for approximately 20% of the total phospholipids in mammalian cell membranes (Dean and Lodhi 2018). In chicken breast, vinyl-ether phosphatidylethanolamine (PE) and PC accounted for 57% and 11% of the total PE and PC fractions, respectively (Takahashi et al. 2018). Most fatty acids are observed with an even number of carbon atoms in their carbon chains. However, fatty acids with an odd number of carbon atoms in their carbon chains have also been observed in rumen bacteria, namely, Ruminantia, dairy products, and fishes (Addison and Ackman 1970; Andrae et al. 2001; AbuGhazaleh et al. 2002). Pentadecanoic acid (15:0) and heptadecenoic acid (17:1) were found in the PC fraction of chicken breast (Takahashi et al. 2018). The amount and the percentage of each PC species in meat have already been reported; however, the comparison of the molecular species of PC in animal meats remains unexplored. The amount of fatty acids in the phospholipid fraction varies depending on the type of the skeletal muscle (Blackard et al. 1997). Dietary nutrition and exercise influence the PC profile by mediating alterations in hepatic and muscular fatty acid metabolism (Senoo et al. 2015; Inoue et al. 2017). In studies conducted with meat animals, dietary oil and rearing conditions with or without the mother influenced the PC profiles of chicken (Cui et al. 2020) and young goats (Czopowicz et al. 2020), respectively. In this study, we performed a lipidomic analysis of meat animal species and muscle locations using LC/ESI-MS in order to elucidate the characteristic PCs of meat. We observed that PC profiles were different between various animal species and their muscle locations. The results aided in the understanding of PC profiles in meat. Materials and methods Meat samples Chicken, pork, beef, and lamb meat cuts were purchased from the market. To compare PC profiles in different meat animal species, chicken (23 specimens grown in Japan), pork (21 and 3 specimens grown in Japan and Canada, respectively), beef (9, 8, 5, and 1 specimens grown in USA, Japan, Australia, and Republic of Nicaragua, respectively), and lamb (3 and 1 specimen grown in New Zealand and Australia, respectively) meats were used. All meats were refrigerated except for 2 meats obtained from beef, which were frozen. For comparison of PC profiles based on skeletal muscle location, breast, thigh, and drumstick of chicken, and loin and tenderloin of pork, which were all grown in Japan, were used. All meats were refrigerated. For the analysis of enzyme activity and protein expression, chicken (5 specimens grown in Japan), pork (5 specimens grown in Japan), beef (5 specimens grown in USA), and lamb (5 specimens grown in New Zealand) meats were used. All meats were refrigerated. Skin, fat, and tendons were removed from each type of meat. For analysis of lipids, meats were homogenized and boiled at 100 °C for 30 min, followed by cooling in iced water. After homogenization of the boiled meats, samples were freeze-dried and stored at −20 °C for lipid analysis. For the analysis of enzyme activity and protein expression, unboiled meats were frozen in liquid nitrogen and stored at −80 °C. The stored samples were homogenized under liquid nitrogen just before analysis. LC/ESI-MS Lipid extraction and LC/ESI-MS measurement were performed as previously described (Senoo et al. 2015). Total lipids were extracted from freeze-dried meats overnight using chloroform/methanol (2:1, v/v with 0.2 mg/mL butyl hydroxyl toluene), which was supplemented with 50 µg/mL PC (17:0/17:0) as an internal standard. The lipid fractions were evaporated to dryness under vacuum. Samples were reconstituted in an equal volume of acetonitrile/isopropanol/water (65:30:5, v/v/v). After solutions were diluted 10 times, 10 µL of samples were injected onto the LC/ESI-MS system. The lipidomic analysis was performed using an LCMS-8040 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan) and triple quad 4500 (Sciex, Framingham, MA, USA). For HPLC analysis, an Accucore RP-MS C18 column (2.6 µm, 2.1 × 50 mm, Thermo Fisher Scientific, Waltham, MA, USA) was used. Mobile phase A consisted of water/acetonitrile (60:40, v/v) and mobile phase B consisted of isopropanol/acetonitrile (90:10, v/v). Both mobile phases, A and B, were supplemented with 10 mM ammonium formate and 0.1% formic acid. The flow rate was maintained at 0.4 mL/min. The gradient conditions were set as follows: 40% B at 0 min, 40% B at 2 min, 52% B at 8 min, 60% B at 20 min, 100% B at 25 min, and 40% B at 30 min. To measure and quantify the PC profile comprehensively, a precursor ion scan was performed with the monitoring of m/z 600-1000 in Q1 and m/z 184 in Q3. The relative peak areas of each PC molecule were normalized by the peak areas of internal standard and sample weight. To determine the type of PC molecular species, a product ion scan was performed with the monitoring of m/z [M + HCOO]− in Q1 and m/z 100-1000 in Q3. Using the LIPID MAPS online MS tool, the MS spectrum was searched against a database of glycerophospholipid (https://www.lipidmaps.org/tools/structuredrawing/GP_p_form.php) precursor/product ions. To confirm the detected PCs, multiple reaction monitoring was performed with the transition of [M + HCOO]− to [RCOO]−. Catalase activity Preparation of samples and the measurement of catalase activity were performed using the Catalase Assay Kit (Cayman, Ann Arbor, MI, USA) following the manufacturer's instruction. Western blot After meat was homogenized under liquid nitrogen, protein was dissolved using RIPA Lysis Buffer (Merck, Darmstadt, Germany) containing protease and phosphatase inhibitors (Nacalai Tesque, Kyoto, Japan). The supernatant was collected after centrifugation (13 000 × g, 15 min, 4 °C). Forty micrograms of protein samples were used for SDS-PAGE analysis and subsequently transferred to nitrocellulose membranes. The transferred proteins were stained with Ponceau-S (Beacle, Kyoto, Japan) and quantified using Image Studio Software (LI-COR Biosciences, Lincoln, NE, USA). Blocking was performed with 5% bovine serum albumin in Tris-buffered saline with 0.1% Tween 20 for 1 h at room temperature. Membranes were incubated with primary antibody against catalase (C0979: MilliporeSigma, St. Louis, MO, USA) (1:4000) or alkylglycerone phosphate synthase (AGPS) (HPA030211: MilliporeSigma) (1:500) for 1 h at room temperature. Membranes were incubated in secondary antibody against mouse (#7076: Cell Signaling Technology, Danvers, MA, USA) (1:2000) or rabbit (#7074: Cell Signaling Technology) (1:1000) IgG conjugated to horseradish peroxidase (HRP) for 1 h at room temperature. The signals were detected using the chemiluminescence kit (RPN2236, GE Healthcare Japan, Tokyo, Japan), C-DiGit Blot scanner (LI-COR Biosciences), and Image Studio Software (LI-COR Biosciences). The detected signals were divided by the protein amount present in each lane. Statistical analysis Statistical analyses were performed as previously described (Senoo et al. 2015; Senoo et al. 2020). For lipidomic analysis, the detected peaks were aligned according to the m/z values and normalized retention time using Signpost MS (Reifycs, Tokyo, Japan). After the application of autoscaling, mean-centering, and scaling using standard deviation on a per-peak basis as pretreatment, a principal component analysis (PCA) was performed using JMP ver. 13 (SAS Institute, Cary, NC, USA). Statistical hypothesis tests of factor loading in PCA were performed to select PC molecules that had a statistically significant correlation with the principal component score. The P-value was calculated in accordance with a previous report (Yamamoto et al. 2014). Benjamini–Hochberg procedure was applied to determine the level of significance for multiple testing. The false discovery rate (FRD) was 5%. Hierarchical clustering analysis (HCA) was performed using the heatmap.2 function present in gplots package (Warners et al. 2019) of R (version 3.6.2) (R Core Team 2019). Autoscaling in which the relative peak area of each species was mean-centered and divided by the standard deviation was performed for the pretreatment. With the data obtained from the 2 groups, each group was compared with the other group using the Student's t-test (P < .05). The data obtained from 3 or 4 groups were analyzed using one-way ANOVA (P < .05). In the case of significant differences, each group was compared with the other groups by the Tukey–Kramer method (P < .05). These calculations were performed using JMP. Values are shown as the mean ± SE. Results PC profiles of the chicken, pork, beef, and lamb meats To examine the differences between the PC profiles of chicken, pork, beef, and lamb meats, lipidomic analyses were performed using LC/ESI-MS. Figure 1 shows the PCA scatter plots of the samples. The chicken was separated from the other species in the first principal component (Figure 1). This result showed that the aves exhibited a different PC profile from mammalian species. The pork was separated from the other species in the second principal component (Figure 1); although the cluster of pork and beef were closely located. PC profiles might have significantly differed owing to the differences in meat species. Multiple comparisons for factor loading in the first and second principal components were performed, but no PC demonstrated a statistically significant difference. In order to investigate the feature in PC molecules of each species, HCA was conducted. Characteristic PC molecules of each species were clustered (Figure 2). Cluster 1 reflected characteristic PCs in lamb, namely, PC (16:0/20:5), and PC (16:0/18:3). Cluster 2 reflected PC molecules whose amount was low in beef, namely, PC (16:0/18:1), PC (O-16:0/18:1), and PC (18:1/20:4). Cluster 3 reflected PC molecules whose amount was low in pork, namely, PC (16:0/16:1). Cluster 4 reflected characteristic PCs in beef, namely, PC (P-16:0/20:4), PC (15:1/16:1), PC (16:1/22:0), PC (16:1/18:2), and PC (14:1/18:2). Cluster 5 reflected characteristic PCs in pork, namely, PC (O-16:0/18:2), PC (16:0/18:2), and PC (P-18:0/18:2). Cluster 6 reflected characteristic PCs in chicken, namely, PC (16:0/20:4), PC (16:0/16:0), PC (18:0/20:4), and PC (16:0/22:4). Figure 1. Open in new tabDownload slide Differences in PC profiles of chicken, pork, beef, and lamb meats. Score plot of PCA for lipid species present in the PC fraction derived from chicken (n = 23), pork (n = 24), beef (n = 23), and lamb (n = 4) meats is shown. Figure 1. Open in new tabDownload slide Differences in PC profiles of chicken, pork, beef, and lamb meats. Score plot of PCA for lipid species present in the PC fraction derived from chicken (n = 23), pork (n = 24), beef (n = 23), and lamb (n = 4) meats is shown. Figure 2. Open in new tabDownload slide Heat map comparing PC profiles between chicken, pork, beef, and lamb meats. Dendrograms of hierarchical clustering analysis (HCA) of chicken (n = 23), pork (n = 24), beef (n = 23), and lamb (n = 4) meats and PC molecules were created based on PC profiles. Dendrograms and colors were assigned based on the autoscaled average value of each subject. N.D.; not determined. Figure 2. Open in new tabDownload slide Heat map comparing PC profiles between chicken, pork, beef, and lamb meats. Dendrograms of hierarchical clustering analysis (HCA) of chicken (n = 23), pork (n = 24), beef (n = 23), and lamb (n = 4) meats and PC molecules were created based on PC profiles. Dendrograms and colors were assigned based on the autoscaled average value of each subject. N.D.; not determined. The fatty acyl, alkyl, and alkenyl moiety in PCs observed in meat samples In order to determine the characteristic PC molecules of each species, the amounts of all PC molecules observed in chicken, pork, beef, and lamb were compared. A stacked bar graph demonstrating the amount of PC molecules was constructed (Figure 3a). PCs comprising more than 10% of the total PC fraction derived from the meat samples were highlighted in Figure 3a. PC (16:0/18:1), PC (16:0/18:2), and PC (18:0/18:2), which were the most abundant PCs in chicken, pork, beef, and lamb, accounted for more than 50% in all species. PC (16:0/18:1) was most abundantly observed in chicken, beef, and lamb samples, and relatively less in pork. With respect to the unsaturation of the fatty acid chain in PC, the amount of PCs in which at least one saturated fatty acid (SAFA), monounsaturated fatty acid (MUFA), or polyunsaturated fatty acid (PUFA) was present are shown in Figure 3b. Chicken and pork meat samples demonstrated high amounts of PCs with SAFA compared to the other species. Pork meat samples demonstrated a lower amount of PC molecules with MUFA and a higher amount of PC molecules with PUFA (Figure 3b). This result was observed as PC (16:0/18:2) and PC (O-16:0/18:2) in pork samples was abundantly present (Figure 3a and Table 1). With respect to the type of bond in the fatty acid chain, the amount of PCs with ether or vinyl-ether bonds are shown in Figure 3c. Pork samples demonstrated a high amount of PCs with ether bonds, namely, PC (O-16:0/18:2). Beef and lamb samples showed a high amount of PCs with a vinyl-ether bond (Figure 3c), namely, PC (P-16:0/18:1) (Figure 3a and Table 1). The amount of PCs with fatty acid chains containing an odd number of carbon atoms are shown in Figure 3d. Beef and lamb samples demonstrated a high amount of PCs with fatty acids containing an odd number of carbon atoms (Figure 3d), namely, PC (15:1/18:2) and PC (17:0/18:2) (Figure 3a and Table 1). Figure 3. Open in new tabDownload slide Amount of PC molecules in chicken, pork, beef, and lamb meats. (a) Stacked bar graph of the amount of PC molecules in the meats. PC molecules comprising more than 10% of the total PC fraction were shown with filled, dash, vertical line and diagonal line boxes. (b) Amount (%) of PC molecules with SAFA, MUFA, or PUFA in the total PC fraction. (c) Amount (%) of PC molecules with ether or vinyl-ether bond in the total PC fraction. (d) Amount (%) of PC molecules with fatty acids containing an odd number of carbon atoms in the total PC fraction. Chicken (n = 23), pork (n = 24), beef (n = 23), and lamb (n = 4) meats. Values are shown as mean ± SE. Values with different letters show significant differences (P < .05; chicken vs pork vs beef vs lamb). SAFA: saturated fatty acid. MUFA; monounsaturated fatty acid. PUFA; polyunsaturated fatty acid. Figure 3. Open in new tabDownload slide Amount of PC molecules in chicken, pork, beef, and lamb meats. (a) Stacked bar graph of the amount of PC molecules in the meats. PC molecules comprising more than 10% of the total PC fraction were shown with filled, dash, vertical line and diagonal line boxes. (b) Amount (%) of PC molecules with SAFA, MUFA, or PUFA in the total PC fraction. (c) Amount (%) of PC molecules with ether or vinyl-ether bond in the total PC fraction. (d) Amount (%) of PC molecules with fatty acids containing an odd number of carbon atoms in the total PC fraction. Chicken (n = 23), pork (n = 24), beef (n = 23), and lamb (n = 4) meats. Values are shown as mean ± SE. Values with different letters show significant differences (P < .05; chicken vs pork vs beef vs lamb). SAFA: saturated fatty acid. MUFA; monounsaturated fatty acid. PUFA; polyunsaturated fatty acid. Table 1. Characteristic PC molecules in chicken, pork, beef, and lamb meats . . . Amount in PCs (%) . . . . Chicken . Pork . Beef . Lamb . PC . RT (min) . m/z [M + H] + . (n = 23) . (n = 24) . (n = 23) . (n = 4) . 16:0/18:1 12.4 760.7 a36.80 ± 1.23 c18.88 ± 0.69 b25.60 ± 2.31 ab35.37 ± 3.28 16:0/18:2 10.8 758.7 b15.73 ± 1.45 a35.99 ± 1.73 b14.68 ± 2.02 b5.78 ± 1.26 18:0/18:2 13.0 786.7 a17.20 ± 0.89 b7.18 ± 0.65 b10.54 ± 1.45 b9.71 ± 1.33 O-16:0/18:2 12.2 744.7 b0.28 ± 0.05 a19.22 ± 1.18 b2.63 ± 0.41 b0.76 ± 0.76 P-16:0/18:1 13.6 744.7 b4.76 ± 0.42 c0.15 ± 0.07 a7.74 ± 0.90 a10.61 ± 1.65 P-16:0/20:4 11.6 766.7 b1.50 ± 0.29 b0.48 ± 0.13 a2.97 ± 0.45 b0.66 ± 0.19 P-18:0/18:2 12.6 770.7 b0.04 ± 0.02 a4.17 ± 0.55 b0.24 ± 0.08 b0.09 ± 0.05 15:1/18:2 11.8 742.7 c0.33 ± 0.04 b2.53 ± 0.41 a14.32 ± 0.88 b6.20 ± 1.45 17:0/18:2 11.7 772.7 b0.00 ± 0.00 b0.11 ± 0.04 a0.25 ± 0.02 a0.37 ± 0.12 . . . Amount in PCs (%) . . . . Chicken . Pork . Beef . Lamb . PC . RT (min) . m/z [M + H] + . (n = 23) . (n = 24) . (n = 23) . (n = 4) . 16:0/18:1 12.4 760.7 a36.80 ± 1.23 c18.88 ± 0.69 b25.60 ± 2.31 ab35.37 ± 3.28 16:0/18:2 10.8 758.7 b15.73 ± 1.45 a35.99 ± 1.73 b14.68 ± 2.02 b5.78 ± 1.26 18:0/18:2 13.0 786.7 a17.20 ± 0.89 b7.18 ± 0.65 b10.54 ± 1.45 b9.71 ± 1.33 O-16:0/18:2 12.2 744.7 b0.28 ± 0.05 a19.22 ± 1.18 b2.63 ± 0.41 b0.76 ± 0.76 P-16:0/18:1 13.6 744.7 b4.76 ± 0.42 c0.15 ± 0.07 a7.74 ± 0.90 a10.61 ± 1.65 P-16:0/20:4 11.6 766.7 b1.50 ± 0.29 b0.48 ± 0.13 a2.97 ± 0.45 b0.66 ± 0.19 P-18:0/18:2 12.6 770.7 b0.04 ± 0.02 a4.17 ± 0.55 b0.24 ± 0.08 b0.09 ± 0.05 15:1/18:2 11.8 742.7 c0.33 ± 0.04 b2.53 ± 0.41 a14.32 ± 0.88 b6.20 ± 1.45 17:0/18:2 11.7 772.7 b0.00 ± 0.00 b0.11 ± 0.04 a0.25 ± 0.02 a0.37 ± 0.12 Values in the same row with different letters show significant differences (P < .05; chicken vs pork vs beef vs. lamb). Values are shown as mean ± SE. Open in new tab Table 1. Characteristic PC molecules in chicken, pork, beef, and lamb meats . . . Amount in PCs (%) . . . . Chicken . Pork . Beef . Lamb . PC . RT (min) . m/z [M + H] + . (n = 23) . (n = 24) . (n = 23) . (n = 4) . 16:0/18:1 12.4 760.7 a36.80 ± 1.23 c18.88 ± 0.69 b25.60 ± 2.31 ab35.37 ± 3.28 16:0/18:2 10.8 758.7 b15.73 ± 1.45 a35.99 ± 1.73 b14.68 ± 2.02 b5.78 ± 1.26 18:0/18:2 13.0 786.7 a17.20 ± 0.89 b7.18 ± 0.65 b10.54 ± 1.45 b9.71 ± 1.33 O-16:0/18:2 12.2 744.7 b0.28 ± 0.05 a19.22 ± 1.18 b2.63 ± 0.41 b0.76 ± 0.76 P-16:0/18:1 13.6 744.7 b4.76 ± 0.42 c0.15 ± 0.07 a7.74 ± 0.90 a10.61 ± 1.65 P-16:0/20:4 11.6 766.7 b1.50 ± 0.29 b0.48 ± 0.13 a2.97 ± 0.45 b0.66 ± 0.19 P-18:0/18:2 12.6 770.7 b0.04 ± 0.02 a4.17 ± 0.55 b0.24 ± 0.08 b0.09 ± 0.05 15:1/18:2 11.8 742.7 c0.33 ± 0.04 b2.53 ± 0.41 a14.32 ± 0.88 b6.20 ± 1.45 17:0/18:2 11.7 772.7 b0.00 ± 0.00 b0.11 ± 0.04 a0.25 ± 0.02 a0.37 ± 0.12 . . . Amount in PCs (%) . . . . Chicken . Pork . Beef . Lamb . PC . RT (min) . m/z [M + H] + . (n = 23) . (n = 24) . (n = 23) . (n = 4) . 16:0/18:1 12.4 760.7 a36.80 ± 1.23 c18.88 ± 0.69 b25.60 ± 2.31 ab35.37 ± 3.28 16:0/18:2 10.8 758.7 b15.73 ± 1.45 a35.99 ± 1.73 b14.68 ± 2.02 b5.78 ± 1.26 18:0/18:2 13.0 786.7 a17.20 ± 0.89 b7.18 ± 0.65 b10.54 ± 1.45 b9.71 ± 1.33 O-16:0/18:2 12.2 744.7 b0.28 ± 0.05 a19.22 ± 1.18 b2.63 ± 0.41 b0.76 ± 0.76 P-16:0/18:1 13.6 744.7 b4.76 ± 0.42 c0.15 ± 0.07 a7.74 ± 0.90 a10.61 ± 1.65 P-16:0/20:4 11.6 766.7 b1.50 ± 0.29 b0.48 ± 0.13 a2.97 ± 0.45 b0.66 ± 0.19 P-18:0/18:2 12.6 770.7 b0.04 ± 0.02 a4.17 ± 0.55 b0.24 ± 0.08 b0.09 ± 0.05 15:1/18:2 11.8 742.7 c0.33 ± 0.04 b2.53 ± 0.41 a14.32 ± 0.88 b6.20 ± 1.45 17:0/18:2 11.7 772.7 b0.00 ± 0.00 b0.11 ± 0.04 a0.25 ± 0.02 a0.37 ± 0.12 Values in the same row with different letters show significant differences (P < .05; chicken vs pork vs beef vs. lamb). Values are shown as mean ± SE. Open in new tab Activity and expression of catalase and AGPS in meat Since the synthesis of PCs with ether or vinyl-ether bond is high in the peroxisome, the activity and amount of catalase, a peroxisomal marker, were measured. Furthermore, AGPS, which catalyzes the formation of ether or vinyl-ether bond at the sn-1 position by exchanging an acyl chain for alkyl group, was also detected. The activity and expression of catalase did not differ significantly between different meat species (Figures 4 and 5a). However, pork samples demonstrated a tendency of high activity (P = .13; Figure 4). AGPS expression was not detected in chicken and did not differ significantly between the 3 other meat species (Figure 5b). Figure 4. Open in new tabDownload slide Catalase activity in chicken, pork, beef, and lamb meats. Catalase activity was measured using the homogenate samples of chicken (n = 5), pork (n = 5), beef (n = 5), and lamb (n = 5) meats. Values are shown as mean ± SE and normalized by protein amount. Figure 4. Open in new tabDownload slide Catalase activity in chicken, pork, beef, and lamb meats. Catalase activity was measured using the homogenate samples of chicken (n = 5), pork (n = 5), beef (n = 5), and lamb (n = 5) meats. Values are shown as mean ± SE and normalized by protein amount. Figure 5. Open in new tabDownload slide The expression of catalase and AGPS protein in chicken, pork, beef, and lamb meats. The expressions of chicken (n = 5), pork (n = 5), beef (n = 5), and lamb (n = 5) meats were measured by Western blot. Values are shown as mean ± SE and normalized by protein amounts on the membrane. Values with different letters show significant differences (P < .05; chicken vs pork vs beef vs lamb). Figure 5. Open in new tabDownload slide The expression of catalase and AGPS protein in chicken, pork, beef, and lamb meats. The expressions of chicken (n = 5), pork (n = 5), beef (n = 5), and lamb (n = 5) meats were measured by Western blot. Values are shown as mean ± SE and normalized by protein amounts on the membrane. Values with different letters show significant differences (P < .05; chicken vs pork vs beef vs lamb). PC profiles based on muscle locations To determine the differences between PC profiles based on muscle location, PC molecules of meat cuts obtained from different parts were detected by LC/ESI-MS and analyzed using PCA (Figures 6 and 7). In chicken meat, the breast was separated from the thigh and the drumstick in the first principal component (Figure 6a). Five PC molecules, namely, PC (16:0/22:6), PC (18:0/18:1), PC (18:0/20:3), PC (O-16:0/18:2), and an unidentified PC, contributed to the differences between breast and thigh and drumstick samples (Figure 6b). In addition to these 5 PC molecules, the amounts of the other 5 PC molecules were significantly different between the breast, thigh, and drumsticks samples (Table 2). In pork, the loin was separated from the tenderloin in the first principal component (Figure 7a). Twelve PC molecules, namely, PC (16:0/18:1), PC (16:0/18:2), PC (16:0/20:4), PC (18:0/18:2), PC (18:1/18:2), PC (O-16:0/18:2), PC (O-16:0/20:4), PC (P-18:0/18:2), and PC (P-18:0/20:4), and 3 unidentified PCs contributed to the differences between loin and tenderloin samples (Figure 7b). In the loin, the amounts of PC (16:0/18:0), PC (O-18:0/18:2), and an unidentified PC were significantly higher than those in the tenderloin (Table 3). These results demonstrated that the PC profiles differed based on the locations of meat harvest in chicken and pork. Figure 6. Open in new tabDownload slide Differences in PC profiles of chicken meat cuts. (a) Score plot of PCA for lipid species in the phospholipid fraction derived from the chicken breast (n = 8) and the thigh and drumstick (n = 6) samples is shown. Thigh and drumstick meats are represented in closed (black) circles. Breast meats are represented in open (white) circles. (b) Loading plot of PCA for lipid species in the phospholipid fraction derived from the breast, thigh, and, drumstick samples. PC molecules with statistically significant differences are represented in closed circles. Figure 6. Open in new tabDownload slide Differences in PC profiles of chicken meat cuts. (a) Score plot of PCA for lipid species in the phospholipid fraction derived from the chicken breast (n = 8) and the thigh and drumstick (n = 6) samples is shown. Thigh and drumstick meats are represented in closed (black) circles. Breast meats are represented in open (white) circles. (b) Loading plot of PCA for lipid species in the phospholipid fraction derived from the breast, thigh, and, drumstick samples. PC molecules with statistically significant differences are represented in closed circles. Figure 7. Open in new tabDownload slide Differences in PC profile of pork meat cuts. (a) Score plot of PCA for lipid species in the phospholipid fraction derived from the pork loin (n = 7) and tenderloin (n = 3). Tenderloin meats are represented in closed squares. Loin meats are represented in open squares. (b) Loading plot of PCA for lipid species in the phospholipid fraction derived from the loin and tenderloin samples. PC molecules with statistically significant differences are represented in closed circles. Figure 7. Open in new tabDownload slide Differences in PC profile of pork meat cuts. (a) Score plot of PCA for lipid species in the phospholipid fraction derived from the pork loin (n = 7) and tenderloin (n = 3). Tenderloin meats are represented in closed squares. Loin meats are represented in open squares. (b) Loading plot of PCA for lipid species in the phospholipid fraction derived from the loin and tenderloin samples. PC molecules with statistically significant differences are represented in closed circles. Table 2. Amount of PC molecules showing significant differences between different muscle locations in chicken meat . . . Amount in PCs (%) . . . . Breast . Thigh and drumstick . PC . RT (min) . m/z [M + H]+ . (n = 8) . (n = 6) . 16:0/16:0 12.0 734.7 1.83 ± 0.19 1.05 ± 0.30 16:0/22:6 10.2 806.7 0.59 ± 0.13 0.09 ± 0.05 18:0/18:1 14.9 788.8 2.15 ± 0.19 0.78 ± 0.14 18:0/20:3 13.8 812.7 0.80 ± 0.12 0.11 ± 0.06 18:2/18:2 9.7 782.7 0.15 ± 0.05 0.00 ± 0.00 O-16:0/18:2 12.2 744.7 0.75 ± 0.07 0.21 ± 0.09 Unknown 11.9 731.7 2.09 ± 0.23 0.43 ± 0.07 18:0/18:2 13.0 786.7 11.09 ± 0.36 15.91 ± 0.68 18:0/20:4 12.9 810.7 3.12 ± 0.21 5.58 ± 0.45 Unknown 11.8 742.7 1.20 ± 0.08 1.89 ± 0.17 . . . Amount in PCs (%) . . . . Breast . Thigh and drumstick . PC . RT (min) . m/z [M + H]+ . (n = 8) . (n = 6) . 16:0/16:0 12.0 734.7 1.83 ± 0.19 1.05 ± 0.30 16:0/22:6 10.2 806.7 0.59 ± 0.13 0.09 ± 0.05 18:0/18:1 14.9 788.8 2.15 ± 0.19 0.78 ± 0.14 18:0/20:3 13.8 812.7 0.80 ± 0.12 0.11 ± 0.06 18:2/18:2 9.7 782.7 0.15 ± 0.05 0.00 ± 0.00 O-16:0/18:2 12.2 744.7 0.75 ± 0.07 0.21 ± 0.09 Unknown 11.9 731.7 2.09 ± 0.23 0.43 ± 0.07 18:0/18:2 13.0 786.7 11.09 ± 0.36 15.91 ± 0.68 18:0/20:4 12.9 810.7 3.12 ± 0.21 5.58 ± 0.45 Unknown 11.8 742.7 1.20 ± 0.08 1.89 ± 0.17 Amounts of PC are shown as mean ± SE. Open in new tab Table 2. Amount of PC molecules showing significant differences between different muscle locations in chicken meat . . . Amount in PCs (%) . . . . Breast . Thigh and drumstick . PC . RT (min) . m/z [M + H]+ . (n = 8) . (n = 6) . 16:0/16:0 12.0 734.7 1.83 ± 0.19 1.05 ± 0.30 16:0/22:6 10.2 806.7 0.59 ± 0.13 0.09 ± 0.05 18:0/18:1 14.9 788.8 2.15 ± 0.19 0.78 ± 0.14 18:0/20:3 13.8 812.7 0.80 ± 0.12 0.11 ± 0.06 18:2/18:2 9.7 782.7 0.15 ± 0.05 0.00 ± 0.00 O-16:0/18:2 12.2 744.7 0.75 ± 0.07 0.21 ± 0.09 Unknown 11.9 731.7 2.09 ± 0.23 0.43 ± 0.07 18:0/18:2 13.0 786.7 11.09 ± 0.36 15.91 ± 0.68 18:0/20:4 12.9 810.7 3.12 ± 0.21 5.58 ± 0.45 Unknown 11.8 742.7 1.20 ± 0.08 1.89 ± 0.17 . . . Amount in PCs (%) . . . . Breast . Thigh and drumstick . PC . RT (min) . m/z [M + H]+ . (n = 8) . (n = 6) . 16:0/16:0 12.0 734.7 1.83 ± 0.19 1.05 ± 0.30 16:0/22:6 10.2 806.7 0.59 ± 0.13 0.09 ± 0.05 18:0/18:1 14.9 788.8 2.15 ± 0.19 0.78 ± 0.14 18:0/20:3 13.8 812.7 0.80 ± 0.12 0.11 ± 0.06 18:2/18:2 9.7 782.7 0.15 ± 0.05 0.00 ± 0.00 O-16:0/18:2 12.2 744.7 0.75 ± 0.07 0.21 ± 0.09 Unknown 11.9 731.7 2.09 ± 0.23 0.43 ± 0.07 18:0/18:2 13.0 786.7 11.09 ± 0.36 15.91 ± 0.68 18:0/20:4 12.9 810.7 3.12 ± 0.21 5.58 ± 0.45 Unknown 11.8 742.7 1.20 ± 0.08 1.89 ± 0.17 Amounts of PC are shown as mean ± SE. Open in new tab Table 3. Amount of PC molecules showing significant differences between different muscle locations in pork meat . . . Amount in PCs (%) . . . . Loin . Tenderloin . PC . RT (min) . m/z [M + H] + . (n = 7) . (n = 3) . 16:0/18:0 18.9 762.7 8.50 ± 0.57 3.42 ± 0.49 O-18:0/18:2 19.1 772.8 1.55 ± 0.24 0.62 ± 0.18 Unknown 15.6 731.7 2.02 ± 0.11 1.47 ± 0.21 . . . Amount in PCs (%) . . . . Loin . Tenderloin . PC . RT (min) . m/z [M + H] + . (n = 7) . (n = 3) . 16:0/18:0 18.9 762.7 8.50 ± 0.57 3.42 ± 0.49 O-18:0/18:2 19.1 772.8 1.55 ± 0.24 0.62 ± 0.18 Unknown 15.6 731.7 2.02 ± 0.11 1.47 ± 0.21 Amounts of PC are shown as mean ± SE. Open in new tab Table 3. Amount of PC molecules showing significant differences between different muscle locations in pork meat . . . Amount in PCs (%) . . . . Loin . Tenderloin . PC . RT (min) . m/z [M + H] + . (n = 7) . (n = 3) . 16:0/18:0 18.9 762.7 8.50 ± 0.57 3.42 ± 0.49 O-18:0/18:2 19.1 772.8 1.55 ± 0.24 0.62 ± 0.18 Unknown 15.6 731.7 2.02 ± 0.11 1.47 ± 0.21 . . . Amount in PCs (%) . . . . Loin . Tenderloin . PC . RT (min) . m/z [M + H] + . (n = 7) . (n = 3) . 16:0/18:0 18.9 762.7 8.50 ± 0.57 3.42 ± 0.49 O-18:0/18:2 19.1 772.8 1.55 ± 0.24 0.62 ± 0.18 Unknown 15.6 731.7 2.02 ± 0.11 1.47 ± 0.21 Amounts of PC are shown as mean ± SE. Open in new tab The amount of PCs containing palmitic acid (16:0) or stearic acid (18:0) was compared between locations in chicken and pork. In chicken, the amount of PCs containing 18:0 was significantly lower in breast samples than that in the thigh and drumstick samples (Figure 8a). In pork, the amount of PCs containing 18:0 was significantly more abundant in the loin than that in the tenderloin (Figure 8b). For the comparison of the amounts of PCs with ether and vinyl-ether bonds between muscle locations, the total amount of PCs with ether and vinyl-ether bonds is shown in Figure 8c and d. No difference was observed in the amount of PCs with ether and vinyl-ether bonds various between muscle locations in chicken and pork. Figure 8. Open in new tabDownload slide Amount of PC molecules based on muscle locations in chicken and pork meats. (a) Amount (%) of PC molecules with at least one 16:0 or 18:0 in the total PC fraction derived from the chicken breast (n = 8) and thigh and drumstick (n = 6) samples. (b) Amount (%) of PC molecules with at least one 16:0 or 18:0 in the total PC fraction derived from pork loin (n = 7) and tenderloin (n = 3) samples. (c) Amount (%) of PC molecules with ether or vinyl-ether bond in the total PC fraction derived from the chicken breast (n = 8) and thigh and drumstick (n = 6) samples. (d) Amount (%) of PC molecules with ether and vinyl ether-bonds in the total PC fraction derived from pork loin (n = 7) and tenderloin (n = 3) samples. Tenderloin meats are represented in the closed (black) column. Loin meats are represented in the open (white) column. Values are shown as mean ± SE. * P < .05 (breast vs thigh-and-drumstick and loin vs tenderloin). Figure 8. Open in new tabDownload slide Amount of PC molecules based on muscle locations in chicken and pork meats. (a) Amount (%) of PC molecules with at least one 16:0 or 18:0 in the total PC fraction derived from the chicken breast (n = 8) and thigh and drumstick (n = 6) samples. (b) Amount (%) of PC molecules with at least one 16:0 or 18:0 in the total PC fraction derived from pork loin (n = 7) and tenderloin (n = 3) samples. (c) Amount (%) of PC molecules with ether or vinyl-ether bond in the total PC fraction derived from the chicken breast (n = 8) and thigh and drumstick (n = 6) samples. (d) Amount (%) of PC molecules with ether and vinyl ether-bonds in the total PC fraction derived from pork loin (n = 7) and tenderloin (n = 3) samples. Tenderloin meats are represented in the closed (black) column. Loin meats are represented in the open (white) column. Values are shown as mean ± SE. * P < .05 (breast vs thigh-and-drumstick and loin vs tenderloin). Discussion In the present study, we observed that the PC profiles of chicken, pork, beef, and lamb meats were different. We also determined characteristic PC molecules in each species. PC (16:0/18:1), PC (16:0/18:2) and PC (18:0/18:2) were present in the highest amounts in 4 meat species, which is in accordance with the results of previous studies conducted with chicken and pork (Boselli et al. 2008; Takahashi et al. 2018). Boiled meats were used in the analysis of PC to investigate the potential for quality evaluation of processed meats. Although boiling could change the PC profiles, our result concurs with previous reports using raw meats. Specifically, PC (16:0/18:1), PC (16:0/18:2), and PC (18:0/18:2) were the most common PCs found in boiled and raw meats (Boselli et al. 2008; Takahashi et al. 2018), suggesting that boiling did not affect the PC profiles of meats. PC (16:0/18:1), PC (16:0/20:5), and PC (16:0/22:6) are most abundant in marine products, which accounted for approximately 10%, 10%, and 20% of the total PC fraction, respectively (Boselli et al. 2012). Docosahexaenoic acid (22:6) and eicosapentaenoic acid (20:5) comprise 35% and 7%, respectively, of the phospholipid fraction derived from Pseudosciaena crocea (Liang et al. 2018). This finding indicates that PC (16:0/18:1) was a major molecule among a wide range of species, and land animals contained less ω-3 fatty acids in PCs than aquatic animals. The amount of phospholipids containing PUFAs declined with the decrease in body mass in aves (Hulbert et al. 2002; Szabó et al. 2006). However, the amount of PCs containing PUFAs did not correlate with body mass in this study, indicating that the PC profile might be influenced in taxonomy class and order. Ether phospholipids account for approximately 20% of total phospholipids in mammals (Dean and Lodhi 2018), which affects membrane fluidity and membrane fusion (Dean and Lodhi 2018). In this study, the amounts of PCs with ether bond, such as PC (O-16:0/18:2), were abundant in pork meat. This result indicated that the synthesis of PCs with ether bond was dependent on species. Initial steps for the synthesis of PCs with ether and vinyl-ether bonds take place in the peroxisome (Dean and Lodhi 2018). A fatty acyl-CoA is reduced to fatty alcohol by fatty acyl-CoA reductase 1 (FAR1) (Dean and Lodhi 2018). The fatty alcohol is catalyzed by AGPS, which forms an ether or vinyl-ether bond at the sn-1 position by exchanging an acyl chain (Dean and Lodhi 2018). We did not observe any differences in AGPS expression between different animal species. This result indicated that AGPS expression was not correlated with the amount of PCs with ether bond. AGPS was not detected in chicken proteins, which might be attributed to the lack of the primary antibody binding to AGPS; of note, the AGPS sequence was the least conserved in chicken and had a homology of 75%-79% with that of pork, beef, and lamb. The homology among the AGPS sequences of pork, beef, and lamb was 90%-98%. In contrast, primary antibody against catalase could detect the protein from all the above species. Among the analyzed species, the homology of catalase is relatively higher than that of AGPS. The catalase sequence was the least conserved in chicken and was 83%-84% homologous with that of pork, beef, and lamb. The homology among the catalase sequences of pork, beef, and lamb was 92%-98%. No significant differences were observed in catalase activity and corresponding protein expression, which could be due to the inconsistency among samples. The slaughter and storage conditions of meat samples may be inconsistent as they were obtained from the market. FAR1 is a potential rate-limiting enzyme in the synthesis of PCs with ether and vinyl-ether bonds, and is regulated by the negative feedback of cellular levels of vinyl-ether-linked PCs (Honsho and Fujiki 2017). However, FAR1 was not detected in meat proteins (data not shown). The underlying reason for the presence of a higher amount of PCs with ether bonds in pork requires further investigation. Furthermore, the amount of PCs with ether and vinyl-ether bonds in muscles is higher than that in intramuscular fat and transparent tissue in pork meat (Enomoto et al. 2020). Ether and vinyl-ether deficient mice demonstrate a severe abnormality in the neuromuscular junction and reduced muscle strength (Dorninger et al. 2017). In this study, the amount of PCs with ether and vinyl-ether bonds did not differ between various muscle locations within each species. Differences were observed in lipids but not proteins between meat species, suggesting that lipids might be considered as a target of the quality inspection of meat. Odd chain fatty acids, ie 15:0, 17:0, and 17:1, have been observed in the lipid molecules of ruminants that are derived from the bacterial species of the rumen, along with fishes and plants (Keeney, Katz and Allison 1962; French, Bertics and Armentano 2012). In addition to these fatty acids, 15:1 is observed in the bovine milk (Blaško et al. 2010), indicating that 15:0 is metabolized into 15:1 in vivo. In this study, the amounts of PC (15:1/18:2) and PC (17:1/18:2) were high in beef and lamb meats, indicating that the odd-chain fatty acids were absorbed from rumen metabolites, skim milk powder, and fish meal of the feed ingredient, and incorporated into PCs. In contrast, the mechanism of the biosynthesis of odd-chain fatty acid under in vivo conditions is known. Propionic acid, produced from the dietary fiber by intestinal bacteria, is a precursor for the synthesis of 17:0 in human and drosophila (Weitkunat et al. 2017; Sato et al. 2020). Fatty acid desaturase 2 catalyzes 17:0 to yield 17:1 in MCF-7 human cancer cells (Wang et al. 2020). Alpha-oxidation of 18:0 produced 17:0 in mouse (Jenkins et al. 2017). As the dietary total fat increased, the endogenous 15:0 decreased. However, 17:0 remained unchanged (Jenkins et al. 2018). Odd-chain fatty acids derived from the dietary ingredients and synthesized in vivo might have been incorporated into PCs. Breasts are primarily composed of fast-twitch muscle. The thigh and drumsticks are composed of higher amounts of slow-twitch muscle compared to the breast in chicken (Lilburn, Griffin and Wick 2019). In pork, the loin is composed of a higher amount of fast-twitch muscles than tenderloin (Velotto et al. 2010). We observed that the PC profiles of each muscle location were different among all species, and no common PC molecules were observed between the muscles of chicken and pork. Several studies suggested that phospholipid profiles might be associated with the physiological phenotype of the skeletal muscles (Andersson et al. 1998; Helge et al. 1999; Andersson et al. 2000; Helge et al. 2001; Senoo et al. 2015). For instance, the amount of 16:0 was lower in soleus muscle than that in the extensor digitorum longus (EDL) muscles, and 18:0 was higher in the soleus muscle than that in the EDL muscles (Blackard et al. 1997). Furthermore, in human skeletal muscles, the distribution of type I fibers in the muscles was correlated negatively with the amount of 16:0 and positively with the ratio of 18:0/16:0 in the phospholipid, respectively (Andersson et al. 2000). We reported that the overexpression of peroxisome proliferator-activated receptor coactivator 1α (PGC-1α) induced the switching of the fiber type from fast to slow-twitch muscles in mice. The PC profile of EDL was changed to a profile similar to that observed in slow-twitch fiber, which was accompanied by the increase in PC (18:0/22:6) in the fast-twitch muscle (Senoo et al. 2015). In this study, PC (18:0/22:6) was not detected in chicken breast and pork loin. This finding was not consistent with the previous reports (Andersson et al. 2000; Senoo et al. 2015). Phospholipid composition changes according to the body mass (Hulbert et al. 2002; Szabó et al. 2006). The amount of PCs binding 18:0 was higher in the chicken thigh and drumstick muscles containing a large proportion of slow-twitch muscle. On the contrary, the amount of PCs binding 18:0 was lower in the pork tenderloin. The genetic background and body mass might have influenced the composition of PCs. These findings suggested that the PC profile differed significantly depending on species, and the characteristic PC molecules present in the fast or slow-twitch muscles were different between chicken and pork meat. In conclusion, PC profiles were different between various meat species or muscle locations. PC molecules associated with different muscles were different between chicken and pork meats. Although further studies are required to determine the mechanism of alterations in the PC profile of various muscle types, PC profiles of the skeletal muscles might be indicators of meat quality. Acknowledgements We would like to thank Chizuru Toda for technical assistance in sample preparation and Shuhei Umebayashi and Takumi Akahori for technical assistance in LC/ESI-MS analysis. Data availability The data underlying this article will be shared on reasonable request to the corresponding author. Author contribution S.Y., S.K., N.S., A.M., and S.M. conceived and designed the study. S.Y., N.S., and N.M. performed lipidomic and statistical analyses. S.Y. and S.M. interpreted the results and analyzed the data. S.Y. prepared figures and tables. S.Y. and S.M. wrote the manuscript. 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TI - Differences in phosphatidylcholine profiles and identification of characteristic phosphatidylcholine molecules in meat animal species and meat cut locations
JF - Bioscience Biotechnology and Biochemistry
DO - 10.1093/bbb/zbab010
DA - 2021-01-20
UR - https://www.deepdyve.com/lp/oxford-university-press/differences-in-phosphatidylcholine-profiles-and-identification-of-44o26VgB9t
SP - 1
EP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -