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Profile of Lipids in Meat, Fat, and Egg of Siberian Sturgeon

Takeshi Naganuma

Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-hiroshima 739-8528, Japan

Abstract: Non-aqueous, lipophilic metabolites in the meat, fat, and eggs of a 4-year-old female Siberian sturgeon (Acipenser baerii) were analyzed. A total of 616 lipid molecular species were detected, and 373 were used for statistical analysis. The primary common metabolites among meat, fat, and egg were lysophosphatidylcholine (LPC) with fatty acid moieties of 18:1, 18:2, 20:5, and 22:6, and di-/tri-acylglycerolipids with fatty acid moieties of 16:0, 18:1, 18:2, and a few other unsaturated ones. The meat, fat, and egg tissues were separated according to their lipid composition by principal component analysis, and tissue-specific lipid biomarkers were detected by linear discriminant analysis. Representative biomarkers were lysoglycerophosphoserine with 22:6 fatty acid for meat, platelet-activating factor with 20:5 fatty acid for fat, and glycerophosphoethanolamine for eggs. The abundant LPC in this lipidomic study may be related to choline found in earlier aqueous metabolomic analysis using the same sturgeon. The coupling of lipidomics and aqueous metabolomics is thus suggested to be insightful and will help in the development of aquaculture for endangered sturgeons.

Keywords: sturgeon, lipidome, lipidomics, glycerolipids, glycerophospholipids, choline, fatty acids, EPA, DHA

Received September 01, 2022; Revised October 02, 2022; Accepted October 10, 2022

Citation: Takeshi Naganuma, Profile of Lipids in Meat, Fat, and Egg of Siberian Sturgeon. Journal of Food and Nutrition Research, 10 (10). 642-654. January 2022. doi:10.12691/jfnr-10-10-2

1. Introduction

The Siberian sturgeon (Acipenser baerii) is a species of the family Acipenseridae and is widely distributed in lakes and river basins in Siberia and surrounding regions [1,2]. Its natural populations are extant, but declining in Russia and Kazakhstan, and possibly extant in Mongolia and extinct in China. It is listed as critically endangered in the IUCN Red List of Threatened Species [2]. Females and males mature at 11-22 and 9-19 years old, respectively, with an average generation length of 54.6 years [2], which makes starting artificial reproduction less feasible; however, once F1 is obtained, F2 and following generations are reproduced stably. Siberian sturgeons are among the most common species in aquaculture, accounting for approximately 40% of aquaculture production and >30% of legal caviar production [3]. Genetic/genomic approaches to facilitate faster growth and sexual maturation may be applied in aquaculture [4] but not in Siberian sturgeons due to their high and variable chromosome counts [5].

Metabolomics, an alternative omics approach, has recently been used in several studies. NMR-based metabolomics, or metabonomics, was used for a toxicological survey of the Persian sturgeon (Acipenser persicus) [6]. Metabolomics based on liquid chromatography/mass spectrometry (LC/MS) of the Chinese sturgeon (Acipenser sinensis) revealed that female ovarian development is associated with energetic metabolism [7]. Lipidomics (a subset of metabolomics) combined with transcriptomics on the F2 generation of female Chinese sturgeons showed that dietary arachidonic acid promotes steroid hormone synthesis in the ovary [8]. Lipidomics provides detailed datasets of whole lipid molecules, such as phospholipids and triacylglycerols with two and three fatty acid moieties, respectively, such as EPA (eicosapentaenoic acid, 20:5) and DHA (docosahexaenoic acid, 22:6) in the caviar of Atlantic sturgeon (Acipenser sturio) [9].

Omics on Siberian sturgeons (Acipenser baerii) have not been performed, although many dietary experiments have been conducted [10]. An earlier study was the first to report the profile of aqueous (non-lipid) metabolites of Siberian sturgeon meat, fat, and egg [11]. As a follow-up, this study reports lipidomic profiles of the same sturgeon tissue samples. Lipidomics and aqueous metabolomics will hint at the metabolomic features of sturgeons.

2. Materials and Methods

2.1. Siberian Sturgeon

The studied Siberian sturgeon (Acipenser baerii), a four-year-old 3.5-kg female, was the same individual reported for aqueous metabolites [11]. It was fed with dry pellets containing fishmeal, flour, soybean oilcake and corn gluten meal. The fish was dissected on site, and the tissue samples (meat, fat and egg) were kept frozen at -20°C to -70°C until analysis.

2.2. Lipids Extraction and Detection

Three sub-samples (n = 3) for each >200-mg tissue, that is, a total of nine subsamples (3×3), were excised on dry ice and shipped in dry ice to the laboratory of the BGI (formerly Beijing Genomics Institute), China, for lipidomic analysis of lipophilic metabolites [12]. The following procedures were performed at the BGI. The thawed sample (25 mg) was placed in 800 μL of precooled dichloromethane/methanol (3:1 v/v) and 10 μL of SPLASH Lipidomix Mass Spec Standard solution (Avanti Polar Lipids, Birmingham, Alabama, USA) with two small steel balls. The mixture was then ground, sonicated, and centrifuged. The supernatant (600 μL) was lyophilized and reconstituted with 200 μL of isopropanol/acetonitrile/water (2:1:1), vortexed, sonicated, and centrifuged for liquid chromatography-mass spectrometry (LC-MS/MS).

An LC-MS/MS system consisting of an ACQUITY UPLC System with 2D LC Technology (Waters, Milford, Massachusetts, USA) and a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used for lipid separation and detection. The MS scan was performed in the range of m/z 200–2000.

2.3. Data Processing and Statistical Analysis

LC-MS/MS data were processed using LipidSearch 4.1 (Thermo Fisher Scientific) for automated peak extraction and lipid identification [13]. Identification levels or accuracy grades were divided into four degrees: A, lipid categories and all fatty acid chains completely identified; B, category-specific ions and fatty acid fragment ions detected; C, class-specific ions or fatty acid fragment ions detected; and D, lipid structures such as dehydrated ions unrecognized. Lipids identified at levels A and B were processed for statistical analyses, whereas lipids identified at levels C and D were not used. Principal component analysis (PCA) and agglomerative hierarchical clustering were performed using XLSTAT software (Addinsoft, New York, USA). Tissue-specific biomarker lipids were specified using the linear discriminant analysis (LDA) effect size algorithm (LEfSe; http://huttenhower.sph.harvard.edu/galaxy/) [14].

2.4. Lipids Classification and Nomenclature

The detected lipid molecules were represented and abbreviated according to the systematics developed and updated by the LIPID MAPS consortium (https://www.lipidmaps.org) [15,16,17].

3. Results and Discussion

3.1. Lipidomic Profiles of Meat, Fat and Egg of a Siberian Sturgeon

A total of 616 lipid molecular species belonging to 26 classes under five categories were commonly identified in meat, fat, and egg tissues. The numbers of lipid molecular species identified at levels A, B, C, and D were 239, 134, 227, and 16, respectively. A- and B-level data were used for further analyses, but C and D were not. The distribution of the number of lipid molecules with identification levels is shown in Figure 1.

Figure 1. Numbers of lipid molecule species identified at levels A, B, C and D in meat, fat, and egg of a Siberian sturgeon. Data from levels A and B were processed for statistical analyses, while lipids identified at levels C and D were not used

A total of 373 A- and B-level lipid molecules were commonly detected in meat, fat, and eggs of the studied Siberian sturgeons, and no tissue-specific lipid species were observed. Lipid populations of meat, fat, and eggs were distinctly separated on the PCA score plot, where PC1 and PC2 coordinates accounted for 58.92% and 22.10%, respectively, of the total variation in the dataset (Figure 2, left). Among the PCA-separated groups, eggs were distantly placed from the meat and fat, as shown in the dendrogram generated by agglomerative hierarchical clustering (Figure 2, right).

Figure 2. PCA plot (left) and dendrogram by agglomerative hierarchical clustering (right) on lipid compositions of meat, fat, and egg of a Siberian sturgeon. Meat, fat and egg were clustered separately

The separation of the PCA- and dendrogram-derived clusters was also contributed by such lipid molecular biomarkers, as detected by the linear discriminant analysis (LDA)-derived effect size algorithm (LEfSe) [14]. Tissue-specific lipid biomarkers with LDA scores >3.5 (Table 1) are shown in the LEfSe-generated cladogram (Figure 3).

Table 1. Lipid biomarkers of meat, fat, and egg of a Siberian sturgeon. Only the lipid biomarkers having LDA scores >3.5 are listed. The code “(rep)” of the lipid “LPC (18:4) (rep)”, for example, indicates that the fatty acid moiety “18:4” is repeated or duplicated, but the positions of carbon=carbon double bonds are not specified

Figure 3. Cladogram showing the lipid biomarkers that contributed to the PCA separation of meat, fat, and egg of a Siberian sturgeon. The code “(rep)” of the lipid “LPC (18:4) (rep)”, for example, indicates that the fatty acid moiety “18:4” is repeated or duplicated, but the positions of carbon=carbon double bonds are not specified

Lipid biomarkers detected in meat were lysoglycerophosphoserines (LPS) and lysophosphatidylcholine (LPC) of the glycerophospholipids (GP) and diacylglycerolipids (DG) of the category glycerolipids (GL); GP_LPS, GL_DG, and GP_LPC were characterized by 22:6, 18:1, and 14:0 fatty acid moieties, respectively.

Seven biomarkers of the category GP were detected for fat. Platelet-activating factors (PAF) in general showed the highest LDA score, and PAF with the 20:5 fatty acid moiety was the second highest among the fat biomarkers. Other fatty acid moieties such as 22:6, 18:4, and 16:2 were also found.

The biomarkers for eggs were glycerophosphoethanolamines (PE) in general and specific phosphatidylcholines (PC) of the category GP as well as triacylglycerolipids (TG) of the category GL, with fatty acid moieties of 22:6, 18:3, and 18:1.

The LEfSe-detected lipid biomarkers were partly characterized by polyunsaturated fatty acid moieties such as 18:3, 18:4, 20:5, and 22:6, which would be regarded as 18:3 (n-3/n-6), 18:4 (n-3), 20:5 (n-3), and 22:6 (n-3), respectively; that is, alpha-linolenic acid (ALA/GLA), stearidonic acid (SDA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), respectively, of human essential fatty acids, referring to previous studies on the fatty acid compositions of sturgeons [18,19,20,21,22].

3.2. Abundances of Major Lipid Molecules and Fatty Acid Moieties

Relative abundances, i.e., relative peak areas of individual molecules in gas chromatograms, of major lipid molecules are listed in Table 2; absolute concentration such as “μg/g tissue” was not determined by the non-targeted lipidomics in this study. The top 20 lipid molecules were dominated by the lipid categories_classes GP_LPC and GL_DG/TG, that is, lyso-phosphatidylcholines (LPC) of the category glycerophospholipids (GP) and di-/tri-acylglycerolipids (DG/TG) of the category glycerolipids (GL). The relative abundances of the 373 level-A and -B lipid molecules are listed in Table S1, in which GP_LPC and GL_DG/TG are dominant as well. An LPC, or a lyso-PC, is a phosphatidylcholine (PC) whose one of the two fatty acids is “lysed” or lost, possibly due to partial degradation (hydrolyzation) of a PC during storage, referring to the lipid class compositions of other fish roes in which PC overwhelms LPC [23]. The significant presence of choline in phospholipids suggests an essential role in lipid and betaine metabolism in the Siberian sturgeon lipidome.

In addition to the choline moiety in LPCs, moieties of 22:6 and 20:5 polyunsaturated fatty acids, or DHA and EPA, were detected in addition to mono- and di-unsaturated fatty acids 18:1 and 18:2, respectively.

Table 2. Relative abundances of significant lipid molecules in meat, fat, and egg of a Siberian sturgeon. The top 20 lipid molecules are listed in the order of relative abundance in meat. Relative abundances are based on peak areas of respective molecules in gas chromatograms. The highest relative abundance in each tissue is hatched. The code “(rep)” of the lipid “LPC (18:1) (rep)”, for example, indicates that the fatty acid moiety “18:1” is repeated or duplicated, but the positions of carbon=carbon double bonds are not specified. All the relative abundances of the total 373 A- and B-level lipid molecules are listed in Table S1

Regarding the glycerolipid DGs and TGs in Table 2, their fatty acid moieties consisted of saturated 16:0, mono/di-unsaturated 16:1, 18:1, and 18:2, polyunsaturated 18:3, 20:5, and 22:5 (DPA, docosapentaenoic acid). DHA (22:6) was not observed in the DGs or TGs (Table 2).

Focusing on EPA and DHA, the relative abundance of lipid molecules that contain EPA-/DHA-moieties in meat, fat, and eggs is shown in Table 3.

Table 3. Relative abundance (%) of lipid molecules that contain EPA and/or DHA as a moiety in meat, fat, and egg of a Siberian sturgeon

3.3. Lipidome and Aqueous Metabolome

A previous study reported that betaine (also known as N,N,N-trimethylglycine) and choline are aqueous biomarkers for the fat tissue of the same Siberian sturgeon individual analyzed in this study [11]. Betaine and choline are linked to folate-dependent one-carbon metabolism in humans [24]. They are reported to facilitate growth and lipid metabolism in Wuchang bream (Megalobrama amblycephala) [25] and thus are likely beneficial to the Siberian sturgeon. The abundance of lipid biomarkers of phosphatidylcholines (PC) and lysosphatidylcholine (LPC) in the studied sturgeon tissues (Table 1 and Table 2) may reflect their roles in storing choline as the central moiety of glycerophospholipids. The metabolic relationship among LPC, choline, and betaine may be presumed as follows:

PC/LPC (choline storage) à choline (oxidized to betaine) à betaine (as methyl-donor) ààà folate-dependent one-carbon metabolism.

Simultaneous targeted metabolomics and lipidomics would provide more insights for dissecting metabolic pathways driven in sturgeons and improving the growth and maturation of sturgeons in aquaculture.

4. Conclusions

This study produced the first lipidomic profile of Siberian sturgeons. The major common lipid molecules in meat, fat, and egg tissues are lysophosphatidylcholines (LPC) and di/triacylglycerolipids (DG/TG). LPC may be a storage molecule-form of choline oxidized to betaine and further linked to critical metabolic pathways, as inferred from separate aqueous metabolome analysis. Aqueous metabolomics and lipidomics can generate more insightful datasets if combined systematically.

Acknowledgments

Part of this study was supported by JSPS KAKENHI Grant Number 21K05782. Mr. Kazuyoshi Fujimoto of the surgeon farm Hiroshima-tyozame and Ms. Akiko Koi kindly provided technical advice and assistance. I would like to thank Editage (www.editage.com) for English language editing.

Statement of Competing Interests

The author declares that there is no conflict of interest.

List of Abbreviations

DG, diacylglycerolipid

DHA, docosahexaenoic acid

DPA, docosapentaenoic acid

EPA, eicosapentaenoic acid

GL, glycerolipids

GP, glycerophospholipid

IUCN, International Union for Conservation of Nature

LC/MS, liquid chromatography/mass spectrometry

LDA, Linear discriminant analysis

LdMePE, lysophosphatidyl-dimethyl-ethanolamine

LEfSe, LDA Effect Size algorithm

LPC, lysophosphatidylcholine

LPS, lysoglyerophosphoserine

PAF, platelet-activating factor

PC, phosphatidylcholine

PCA, principal component analysis

PE, glycerophosphoethanolamine

TG, triacylglycerolipid

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Table S1. Relative abundances of the total 373 A- and B-level lipid molecules in meat, fat and egg of a Siberian sturgeon. Top 20 lipid molecules in meat are listed in the order of relative abundances in meat. The highest relative abundance in each tissue is yellow-colored. The code “(rep)” of the lipid “LPC (18:1) (rep)”, for example, indicates that the fatty acid moiety “18:1” is repeated or duplicated but the positions of carbon=carbon double bonds are not specified

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