Composition of phospholipids of white muscle of six tuna species

Composition of Phospholipids of White Muscle of Six Tuna Species

Isabel Medina*, Santiago P. Aubourg, and Ricardo P~rez Martfn Instituto de Investigaciones Marinas del CSIC, 1:-36208 Vigo, Spain

ABSTRACT: A comparative study of the phospholipids of white muscle of six of the commercially utilized tuna species, including quantitative analyses of phospholipid classes and studies of the acyl composition of the major components. Plas- malogen compounds also were identified and quantified. Choline and ethanolamine glycerophospholipids were the most abundant classes in all the samples, as well as the only mole- cules containing plasmalogens (16:0, 18:0, and 18:1 alkenyl- ether chains). The patterns of fatty acid distribution within each of the phospholipid classes showed general similarities in the species analyzed. However, ratios between certain saturated and polyunsaturated fatty acids in different phospholipid classes showed remarkable differences. The high content of n-3 polyunsaturated fatty acids in the principal phospholipids, such as the plasmalogens, and taking into account the fatty acids possible importance in human nutrition, indicates that the white muscle of tuna species may be a potentially important dietary item. Lipids30, 1127-1135 (1995).

Tuna processing is an industry of well-known economic importance in many countries, including Spain, supporting a significant market demand and playing an important role in the field of human nutrition as components of the Spanish diet (1). One of the most important aspects in the quality of ma- fine foods concerns their lipid content and composition. Ma- fine lipids are known for their highly unsaturated fatty acid composition (2,3), which makes them very prone to oxidative damage (4,5). On the other hand, these lipids are now the sub- ject of a great deal of attention due to their high content of n- 3 polyunsaturated fatty acids (PUFA), which have potential roles to play against certain diseases (6,7).

Several papers describing the lipid compositions of tuna fish and their alterations during frozen storage or thermal processing have been published (8-10). Most of the work carried out has been focused on the composition of the simpler lipid classes containing two types of primary products per mole,

*To whom correspondence should be addressed at Instituto de Investiga- ciones Marinas del CSIC, Eduardo Cabello 6, E-36208 Vigo, Spain. Abbreviations: DMA, dimethylacetal; FAME, fatty acid methyl ester; GC, gas chromatography; HPLC, high-performance liquid chromatography; LPC, lysophosphaditylcholine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PL, phospholipid; PLA, plasmalogen; PS, phosphatidylserine; PUFA, polyunsaturated fatty acid; SPH; sphingomyelin; TLC, thin-layer chromatography.

such as triacylglycerides (11-13), and literature data related to complex lipids (classes that contain three or more different primary products per mole), such as phospholipids (PL) and, especially, plasmalogens (PLA), are quite limited (14-16).

The PL composition of fish species that are consumed as foods has attracted a great deal of attention due to several characteristics: (i) they possess a highly unsaturated fatty acid composition which makes them especially vulnerable to dam- ages during processing; (ii) some of the PL classes have been reported to possess antioxidant properties (17); and (iii) in contrast with triacylglycerides, the distribution of the PL classes with respect to their fatty acid composition is little in- fluenced by external and internal factors, an aspect that may be relevant for species characterization purposes (18-21). Further, the abundance in n-3 PUFA of these PL may give the fish muscle a supplementary nutritional value.

The aim of the present work was to study the PL composition in several of the tuna species most widely consumed in Spain prior to processing. Different chromatographic techniques, such as high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and gas chromatography (GC) have been combined to complete PL class identifications and quantification. The compositions of fatty acids and alkenyl ethers on the glycerol moieties were studied simultaneously using an acid derivatization procedure and subsequent analysis of the corresponding methyl esters and dimethylacetals (DMA) by GC. For identification purposes, GC coupled with mass spectrometry was employed.

MATERIALS AND METHODS

Raw material and lipid extraction. Six commercial tuna species were employed--big eye tuna (Thunnus obesus), bluefin (T. thynnus), bonito (Sarda sarda), frigate (Auxis thaz- ard), skipjack (Katsuwonus pelamis), and yellowfin (T. al- bacares). The fish came from the Atlantic Ocean and were purchased at a commercial market. All fish except bonito were landed frozen. Bonito fish were caught and kept on ice for three days. After arrival at our laboratory, the bonito were frozen at -40~ and all samples were stored at -20~ prior to analysis. Five individual fish for each species were used.

Fish were skinned, beheaded, and eviscerated, and the red muscle was removed. Lipids were extracted from the white muscle by the Bligh and Dyer method (22), and the muscle

Copyright �9 1995 by AOCS Press 1127 Lipids, Vol. 30, no. 12 (1995)

1128 I. MEDINA ETAL.

content was determined gravimetrically as described by Herbes and Allen (23). All organic solvents employed were reagent grade (E. Merck, Darmstadt, Germany).

Free fatty acid determination. Free fatty acids were measured using the Lowry and Tinsley (24) method based on the formation of a complex with (AcO)2Cu-pyridine (E. Merck).

Determination and purification of PL. Organic phospho- rus was determined on total lipid extracts according to the Ra- heja et al. method (25) based on a complex formation with ammonium molybdate (E. Merck). Lipid extracts were sub- jected to TLC on 20 x 20 cm glass plates coated with Silica Gel 60W (0.8 mm) (E. Merck) and were developed twice in the same direction with CHCI3/CH3OH/CH3COOH (100:15:2, by vol). The polar fraction remained at the bottom and was recovered from the silica gel by elution with CHCI3/CH3OH (2:1, voYvol).

Fractionation of PL classes. Total PL extracts were re- solved by high-performance TLC on I0 x 10 cm glass plates coated with silica gel 60W (0.25 mm) (E. Merck) and developed twice in the same direction in a solvent system of chlo- roform/methanol/ethyl acetate/isopropanol/KCl (0.25%) (30:9:25:6:18, by vol) (26). KCI and all organic solvents were reagent grade (E. Merck). Spots were visualized after spray- ing with cupric sulfate and subsequent heating at 160~ for 10 min. Individual PL classes were identified by comparison with PL standards (Matreya Inc., Pleasant Gap, PA). Optical densities of the charred spots were measured at 360 nm with a Shimadzu CS-910 dual-wavelength TLC scanner (Kyoto, Japan). In this way, sphingomyelin (SPH) and other minor PL classes, characterized by an essential saturated fatty acid composition, were quantified. Quantitation of other PL classes was not possible following this procedure as they showed highly unsaturated fatty acid composition, whereas the composition of the PL standards used was largely saturated (27). To determine the PLA content in choline and ethanolamine glycerophospholipids, elution on TLC plates (Silica Gel 60W, 20 x 20 cm, 0.25 mm) using the procedure described previously was performed. Spots corresponding to these classes, combining the diacyl and PLA forms [phosphatidylcholine (PC)-PLA, phosphatidylethanolamine (PE)-PLA], were scraped off and the lipid recovered for subsequent GC analysis.

Determination of PL fatty acid composition. To obtain the fatty acid composition of individual PL, a combination of HPLC and GC techniques was carried out. HPLC was performed with a Silica Gel 60 column (4.6 mm i.d x 25 cm.; Su- pelco, Inc., Bellefonte, PA), coupled with a Perkin Elmer Sol- vent Pump (Beaconsfield, Buckinghamshire, United King- dom), a Perkin Elmer LC-65 UV variable wavelength detector set at 205 nm, and a Hewlett-Packard 8380A integra- tor (Wotingham, Berkshire, United Kingdom). The separation was achieved with an isocratic elution in a solvent mixture of CH3CN/CH3OH/H3PO 4 (98:1: I, by vol). The flow rate was maintained at 1 mL/min. All solvents employed were of HPLC grade (E. Merck).

Polar lipids (150 lag) were injected into the HPLC system,

and the PL classes were separated under the conditions described previously. The identity of peaks was verified using PL standards. Diacyl forms of phosphatidylinositol (PI), phosphatidylserine (PS), PE, PC, and lysophosphatidylcholine (LPC) were separated completely, whereas other minor complex classes and PLA eluted with the eluent front. As a result of this separation, purified diacyl forms corresponding to the PC, PE, PI, PS classes and the monoacyl LPC class were obtained, and were transmethylated to determine their acyl composition. To eliminate the excess of H3PO 4 prior to the transmethylation, a washing step with an alkaline solution (6 % K2CO3) (E. Merck) was necessary.

Transmethylation oflipids. Purified diacyl PL classes coming from the above HPLC separation, and spots scraped from TLC plates containing both diacyl and alkenyl-acyl molecular species were converted into the corresponding fatty acid methyl esters (FAME) and DMA by the method of Lepage and Roy (28).

Quantification of PL classes. Transmethylated lipids containing mixtures of FAME and DMA were analyzed simultaneously by GC (Perkin Elmer 8700 chromatograph) employ- ing a fused silica capillary column SP-2330 (0.25 mm i. d. x 30 m; Supelco Inc.), programmed from 145 to 190~ at 1.0~ from 190 to 210~ at 5.0~ and then followed by a hold for 13.5 min at 210~ Nitrogen at 10 psig was the carrier gas, and a flame-ionization detector set at 250~ was used. A programmed temperature injector was used in the split mode (150:1) and heated from 45 to 275~ at 15~

Peaks were identified by comparison of their retention times with standard FAME mixtures (Supelco: PUFA No. 1 and No. 2; Larodan, Qualmix Fish). For quantitation purposes, peak areas were automatically integrated, and 19:0 fatty acid (Sigma, St. Louis, MO) was used as an internal standard.

Quantification of diacyl PL classes (PE, PC, PS, PI) and the monoacyl LPC was done knowing their respective fatty acid composition and the structure of the nonacyl moiety. The PLA content was obtained by doubling the molar percentage of total DMA in the mixture of DMA and FAME obtained after transmethylation of polar lipids, according to Visnaanathan et al. (29). Total contents corresponding to PC and PE were determined by combining the quantities of diacyl and PLA forms.

GC/mass spectrometry. A Hewlett-Packard model 5890 A chromatograph, equipped with on-column capillary injection, was used with a 5970 series mass selective detector. The separation was made using the same column and conditions described previously. Helium was used as carrier gas. The mass spectrometer was operated in the electron impact ionization mode (70 eV). The transfer line and ion source temperature were both 280~ Mass spectra of the different peaks were checked in order to differentiate FAME from DMA. The structures of the DMA components were investigated.

RESULTS AND DISCUSSION

Lipid and PL content. The lipid content for each species was rather constant, as can be observed from the low standard deviations calculated for five individual samples (Table 1).

Lipids, Vol. 30, no. 12 (1995)

WHITE MUSCLE PHOSPHOLIPIDS COMPOSITION OF SIX TUNA SPECIES 1129

TABLE 1 Lipid and Phospholipid Contents a in White Muscle of Six Tuna Species Tuna species Lipid h Phosphol ipid c

Bigeye 0.76 • 0.29 35.78 • 6.99 Bluefin 0.95 • 0.48 26.41 + 5.30 Bonito 1.94 • 0.19 1.60 • 0.16 Frigate 0.65 • 0.20 37.81 • 5.86 Skipjack 0.78 • 0.22 28.54 • 7.52 Yel lowf in 0.57 • 0.04 58.80 • 7~56

aResults are expressed as mean • standard deviation for five samples. bExpressed as gl100 g wet muscle. CExpressed as percent of total lipids.

Specimens corresponding to the bonito species showed the highest lipid content with results which confirmed those obtained for the other species. A great variability in the lipid content has been observed in migrating fish species, depending on both enviromental and endogenus factors (12,30-32).

On the other hand, possible decomposition of lipids during frozen storage of samples prior their acquisition also must be also considered (33,34). In order to clarify the extent of this reaction, the amounts of free fatty acids in all samples were calculated. Levels of free fatty acids were generally low, with values ranging between 0-5% of total lipid for all samples, the lowest values being those for bonito. The extent of lipid hydrolysis thus could not explain the low lipid content in respect to the bonito samples. Therefore, the lipid contents found must be considered as completely natural in these tunas, and data published for tuna species, such as skipjack or yellowfin (35,36), were very similar to those determined in this work,

All species except bonito showed high percentages of PL (Table 1). Because PL contain more of the higldy unsaturated fatty acids than do triacylglycerols, the white muscle of these species could be considered an important dietary source of PUFA.

PL content in muscle has proven to be relatively constant according to their presence in cellular membranes (3); this fact has led to the hypothesis of an inverse ratio between lipid content and PL proportion in total lipids (37). The regression between the lipid and the PL content in the present study has demonstrated an inverse correlation (correlation coefficient

= -0.75 l) probably related to the previously mentioned stability of PL content as opposed to the significant variations that may occur in the amount of simpler lipids in migrating fish species (3).

PL classes quantification. Table 2 shows the distribution of PL classes in each species. All species analyzed showed a similar qualitative and quantitative pattern of PL classes. In all cases, PC was the major class, followed by PE and LPC. PC and, secondly, PE usually have been measured as the main PL classes in fish species (11,38,39).

Minor classes such as phosphatidylglycerol and phospha- tidic acid also could be identified. In fact, some authors have described the presence of both PL classes in very low proportions in a wide variety of fish tissues (40,41).

The levels of LPC ranged between 15-20% in all tuna species studied. Previous researchers have described the presence of lyso-PL forms in commercially available fresh tuna muscle and the increase in their content during storage at low temperatures (9,14). In fact, it has been accepted that enzymatic PL hydrolysis can occur slowly during frozen storage or in the muscle of several fish species when held on ice (42). At this point, it seems clear that a certain level of hydrolysis in PL has taken place in all samples during its storage prior to acquisition. However, the extent of that hydrolysis did not ap- pear to be great as samples in which no free fatty acids (bonito) were detected showed proportions of LPC similar to those with higher levels (Table 2).

Other important differences have been recognized in PI and SPH contents among the species. Both PL have been found in several marine samples, although their concentra- tions vary depending on the tissue and organ considered (38,43).

PLA. Table 3 shows the PLA contents obtained in the different tuna species. Some papers have described the presence of alkenyl ethers in a wide variety of fish muscle (15,44). Levels of ether-linked PL in food have a particular interest from a dietary point of view, as its ingestion could contribute to the production of potent biochemical mediators (45). Fur- ther, the PLA content and composition have been shown to be relevant when studying changes in processed foods, as the content decreased as a result of thermal treatment (46). Both aspects may be important in this study, taking into account

TABLE 2 Percentage Distribution a of Phospholipid (PL) Classes in White Muscle of Six Tuna Species

Tuna species PL class b Bigeye Bluefin Bonito Frigate Skipjack Yel lowf in

PE 18.81 • I .I I 18.98 • 1.27 20.05 • 0.35 21.84 • 1.98 20.18 • 2.27 21.03 • 1.87 PI 5.81 • 0.86 6.71 • 2.18 2.25 • 0.05 10.88 • 2.17 4.90 • 0.70 8.46 • 1.70 PS 5.42 • 1.42 4.82 • 1.27 2.20 • 0.01 5.11 • 1.42 5.04 • 1.93 5.37 • 1,17 PC 42.13 • 3.58 42.18 • 5.50 53.85 • 0.35 47.36 • 3.78 51.52 • 5.56 37.88 • 3.26 SPH 3.33 • 0.96 5.55 • 0.59 7.55 • 0.45 2.98 • 1.70 0.50 • 0.04 3.98 • 1.63 LPC 22.13 • 3.85 15.40 • 3.21 13.80 • 0.40 12.02 • 3.22 18.27 • 0.94 21.54 • 3.95 Others 4.41 • 2.95 6.64 + 0.80 trace 1.73 • 0.50 1.52 • 0.37 2.75 • 0.36

aResults are expressed as mean • standard deviation for five samples. bPE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PC, phosphatidylcholine; SPH, sphingomyelin; LPC, lysophosphatidylcholine.

Lipids, Vol. 30, no. 12 (1995)


TABLE 3 Total Plasmalogen (PLA) Content a of Complex Lipids and of Phosphatidylcholine and Phosphatidylethanolamine in White Muscle of Six Tuna Species

Tuna species PL Content Bigeye Bluefin Bonito Frigate Skipjack Yel lowfin

% Total 10.54 • 2.18 15.92 • 2.68 17.01 • 1.40 11.93 + 2.10 20.50 • 2.73 13.07 + 2.52 PC-PLA b 7.77 • 1.24 12.37 • 2.23 37.89 • 1.18 7.80 • 0.40 17.99 • 2.26 6.53 • 0.36 PE-PLA b 38.87 • 5.88 41.37 + 1.83 62.11 + 1.18 40.51 • 3.81 56.85 • 3.28 49.02 • 5.80

aMeans and standard deviations for five samples. t'Results are expressed as percentage of each plasmalogen in the phospholipid class. Abbreviations as in Table 2.

TABLE 4 Alkenyl Ether Composition a of PE-PLA and PC-PLA in White Muscle of Six Tuna Species

PE PC Tuna species 16:0 b 18:0 b 18:1 n-9 b 16:0 b 18:0 b 18:1 n-9 b

Bigeye 46.54 + 1.04 33.85 _+ 1.99 19.61 +_ 1.14 71.65 + 2.43 17.02 ~: 2.10 10.75 + 2.10 Bluefin 65.58 + 0.51 26.10 + 0.77 8.32 _+ 0.26 77.17 + 1.72 33.85 • 1.99 19.61 + 1.14 Bonito 50.67 + 2.04 34.14 • 1.09 14.73 • 1.23 66.37 • 1.22 23.07 • 1.25 10.56 • 1.01 Frigate 40.03 • 2.16 51.57 • 1.61 8.40 • 0.69 76.15 • 2.25 17.88 • 1.77 5.97 • 0.78 Skipjack 53.22 • 0.88 35.59 • 0.98 11.28 • 1.21 79.54 _+ 1.49 13.98 • 1.25 6.98 • 0.77 Yellowfin 58.04 _+ 1.62 33.30 • 0.87 8.67 • 0.75 78.33 • 3.21 15.48 + 1 . 0 4 8.67 + 0.75

aMeans and standard deviations for five samples. Results are expressed as percentage of each fatty chain in the sn-1 position of the plasmalogen moiety. Ab- breviations as in Tables 2 and 3. bLong-chain alkenyl ethers.

that all species have shown high levels of PLA in the white muscle (Table 3).

The PLA proportion was found to have a wide range in the tuna species studied, but a simple relationship with the PL content could not be determined (Table 1). The comparison of both parameters in most of the species studied suggests that an inverse trend with respect to the PL concentration in muscle may exist, although the low PL content found in bonito is in disagreement with this possibility. In addition, some papers have proposed a possible mechanism of adaptation to lower temperatures in marine organisms which involves a de- crease in the PLA content with respect to diacyl forms of PL (44,47).

Finally, an enzymatic hydrolysis of PLA in marine invertebrates has been described during frozen storage, although it occurs more slowly than for diacylphospholipids (48). Be- cause no significant hydrolysis seems to take place in diacyl forms as a result of frozen storage, the extent of this reaction on PLA is likely to be insignificant.

From their mass spectra, DMA were identified as a result of the methanolysis of alkenyl ether chains corresponding to 16:0, 18:0, and 18:1n-9 fatty acids in accordance with previous results determined in Euthynnus pelamis (15,49). Other fatty chains, such as 17:0, 20:0, and 21:0, were detected as trace components.

Alkenyl ethers were found only as PC-PLA and PE-PLA forms for all the tuna species studied (Table 3). Both major PLA usually have been found in marine tissues in association with the major PL (3,36). As can be seen in Table 3, ethanolamine glycerophospholipids showed the largest concentration in PLA, reaching values to nearly 50% of the class.

With respect to the composition of the alkenyl ether chains, two different patterns were observed for each PLA class (Table 4). PC-PLA showed an important concentration in 16:0 (approximately 75%), whereas the levels of 18:0 and 18:1 n-9 in PE-PLA were higher than those found in its ho- mologue PC-PLA. This situation has been described previously for other marine organisms (50,51).

1-O-Alkyl-2-acylglycerophospholipids also have been found in some tissues of some marine organisms (3,14), but they were not detected at significant levels in this research.

Fatty acid composition. Fish PL are remarkable for their high content in PUFA, and substantial differences in their fatty acid composition could be found, depending on the muscle tissue or organism (3,39). Fatty acid compositions of the five major PL classes in six tuna species are shown in Tables 5-9. SPH was a minor class for all tuna species; it also showed a less complicated fatty acid composition with 16:0 (30-35%), 18:1n-9 (23-39%), and 24:1n-9 (12-15%) as major components, and low contents of PUFA (12% of 22:6n-3 approximately). This composition is similar to that obtained by Linares and Henderson (52) in trout muscle.

As can be inferred from Tables 5-9, the fatty acid composition exhibits certain discrepancies between classes and even between species. However, very characteristic patterns could be recognized between lipid classes originating in different samples. One of the most noteworthy was the preponderance of stearic acid over palmitic acid as the principal saturated acid in PE, and the high level of palmitic acid in PC. This distribution of 16:0 and 18:0 fatty acids seems to be a characteristic of the PL from a number of tissues (53), and it is diffi- cult to reconcile with the hypothesis that both PL come from

Lipids, Vol. 30, no. 12 (1995)

WHITE MUSCLE PHOSPHOLIPIDS COMPOSITION OF SIX TUNA SPECIES

TABLE 5 Fatty Acid Composition a of Phosphatidylcholine from White Muscle of Six Tuna Species

1131

Tuna species

Acid Bigeye Bluefin Bonito Frigate Skipjack Yel lowf in

14:0 1.11 • 0.22 3.78 _+ 1.06 1.12 • 0.43 1.48 • 0.57

15:0 0.50 • 0.07 1.24 • 0.19 0.61 • 0.12 0.73 • 0.12

16:0 20.20 • 1.94 20.05 • 1.94 23.22 • 2.02 25.46 • 0.97 17:0 1.19 • 0.246 1.20 • 0.15 1.26 • 0.08 1.07 • 0.18

18:0 3.03 • 0.41 5.96 • 1.44 4.19 • 1.25 4.51 • 0.71

20:0 0.35 • 0.08 2.97 _+ 2.07 0.80 • 0.20 0.37 • 0.13

24:0 0.46 • 0.38 1.34 • 0.22 0.47 • 0.22 0.46 • 0.21 16:1n-5 0.42 • 0.06 1.27 • 0.14 0.55 • 0.27 0.41 + 0.22

16:1n-7 0.79 • 0.17 2.47 • 0.20 0.79 + 0.28 0.93 • 0.19

16:1n-9 0.49 • 0.05 0.34 • 0.18 0.49 _+ 0.25 0.54 • 0.19 18:1n-7 1.23 • 0.243 1.58 • 0.19 0.97 + 0.39 1.27 • 0.22

18:1n-9 10.49 • 0.45 11.21 • 1.01 10.81 + 0.95 9.83 • 1.09

20:1 n-9 0.65 • 0.27 1.66 • 0.06 0.86 • 0.44 0.57 • 0.23 24:1n-9 4.15 • 0.77 2.05 • 0.42 2.57 + 0.73 3.36 • 0.41

18:2n-6 0.58 • 0.06 1.78 • 0.32 0.43 • 0.046 0.85 • 0.15

18:3n-3 0.58 • 0.12 0.76 • 0.12 0.64 • 0.22 0.50 • 0.09 18:4n-3 0.00 0.00 0.00 0.06 • 0.14

20:4n-6 5.63 • 1.66 2.26 • 0.62 1.27 • 0.32 4.83 • 0.30

20:4n-3 0.28 • 0.04 0.82 • 0.14 0.55 + 0.21 0.51 • 0.20

22:4n-6 0.76 + 0.20 0.74 _+ 0.25 0.24 • 0.07 0.30 • 0.07

20:5n-3 4.60 • 1.16 4.38 • 0.45 4.86 • 0.37 6.30 • 0.79

22:5n-3 1.22 • 0.13 2.24 _+ 0.96 1.09 • 0.13 1.20 • 0.31

22:6n-3 41.30 • 3.12 28.70 • 2.71 39.24 • 5.15 34.21 • 2.65

1.01 • 0.19

0.53 • 0.06

21.46 • 1.82

1.07 • 0.13

3.34 • 0.47

0.00 • 0.00 0.48 • 0.17 0.31 • 0.04 0.81 • 0.05

0.76 • 0.08

1.06 • 0.08

11.20 • 0.58

0.53 • 0.18

3.75 • 1.02

0.64 • 0.04

0.33 • 0.25

0.11 •

4.67 • 0.49

0.43 • 0.31 0.68 • 0.94

5.54 • 0.34

0.95 _ 0.30 39.81 • 2.53

1.43 • 0.24

0.68 • 0.14

19.26 • 1.20

1.23 • 0.13 3.60 • 0.40

0.47 • 0.27

0.77 • 0.24

0.62 • 0.28

0.92 • 0.19

0.55 + 0.25 1.26 • 0.07

11.64 • 0.74

0.64 • 0.39

2.99 • 0.84

0.70 • 0.13

0.49 • 0.06

0.00 4.27 • 0.71

0.66 • 0.49 0.44 • 0.18

4.77 • 0.97

0.98 • 0.26

41.63 + 3.1.9

aData are given in mol% as means and standard deviations for five different samples.

TABLE 6 Fatty Acid Composition a of Phosphatidylethanolamine from White Muscle of Six Tuna Species

Tuna species Acid Bigeye Bluefin Bonito Frigate Skipjack Yel lowf in

14:0 3.05 • 1.07 2.94 • 0.34 4.12 • 0.39 2.82 • 0.73

15:0 0.66 + 0.14 0.91 • 0.11 1.17 • 0.08 1.06 • 0.55 16:0 11.60 + 1.25 11.97 • 1.39 16.38 • 0.25 12.80 • 2.38

17:0 1.67 • 0.37 1.54 • 0.28 1.95 • 0.50 1.63 • 0.49

18:0 19.67 • 3.24 21.82 • 1.40 18.36 • 1.00 22.84 • 3.50

20:0 0.72 • 0.25 0.83 + 0.10 1.07 • 0.08 1.12 + 0.31

24:0 0.86 + 0.13 1.48 • 0.91 2.47 • 0.851 1.34 + 0.38

16:1n-5 0.65 • 0.43 0.91 • 0.52 2.41 + 0.23 0.89 • 0.50

16:1n-7 1.07 + 0.19 1.16 + 0.32 2.19 • 0.09 1.42 • 0.38

16:1n-9 0.76 • 0.49 0.77 _+ 0.64 1.23 • 0.61 0.66 • 0.49

18:1n-7 1.26 + 0.33 1.28 + 0.19 2.18 • 0.26 1.23 + 0.87

18:1 n-9 7.07 + 1.03 8.05 _+ 0.73 9.89 • 0.39 7.60 • 2.38

20:1 n-9 1.57 + 0.34 1.84 _+ 0.41 3.58 • 0.52 1.48 • 0.91

24:1n-9 4.73 • 1.40 3.09 + 0.89 2.54 + 0.78 4.14 • 1.18

18:2n-6 1.07 • 0.43 1.50 + 0.25 2.13 + 0.10 1.09 • 0.27

18:3n-3 0.58 • 0.12 0.76 + 0.12 0.64 + 0.22 0.50 • 0.09 18:4n-3 0.00 0.00 0.00 0.00

20:4n-6 3.98 • 0.57 4.33 + 0.63 2.48 • 0.097 4.77 • 1.62

20:4n-3 0.98 • 0.29 0.68 • 0.48 0.99 • 0.309 0.45 • 0.32

22:4n-6 2.93 • 4.72 1.27 • 0.83 1.57 • 0.739 1.44 • 2.31 20:5n-3 2.65 • 0.99 3.64 + 1.00 4.39 • 0.100 2.45 • 0.78

22:5n-3 1.33 • 0.28 1.88 • 0.40 1.93 • 0.11 2.00 • 0.74

22:6n-3 30.89 • 1.87 26.84 • 4.53 21.61 • 2.46 25.42 • 3.81

2.25 • 1.01

0.74 • 0.22

11.83 • 1.74 1.71 • 0.19

23.25 • 1.52

0.00 1.05 + 0.61

0.83 • 0.12

1.42 • 0.50

0.39 • 0.27

1.36 • 0.81

7.66 • 1.64

1.10 • 0.66

3.87 • 0.37

1.44 • 1.06 0.33 • 0.25

0.14 • 0.32

4.75 • 1.17

1.02 + 0.36 0.48 • 0.20

3.88 • 1.70

0.99 • 0.22 28.91 • 6.14

3.32 + 0.68

0.93 + 0.50 12.27 + 1.86

1.54 + 0.35

17.85 + 1.28

1.40 + 0.61

0.98 • 0.61

1.28 • 0.20

2.28 • 1.26

1.13 • 1.06

1.35 • 0.14

1 1 . 2 4 • 1.18

1.44 • 0.89

4.17 • 1.57

1.60 • 0.32

0.49 • 0.06

0.00

3.61 • 0.83

1.08 • 1.01

1.25 • 0.64

2.62 • 0.63

1.01 • 0.70

25.60 • 3.95


the same diglyceride precursor (54). On the other hand, this finding is supported by the alkenyl ether composit ion data mentioned previously (abundance of 18:0 in PE-PLA and of 16:0 in PC-PLA).

The preference of 16:0 over 18:0 as a saturated acid is re- peated among the PL classes with choline as the polar head group, such as SPH and LPC. On the contrary, PI shows the inverse situation with a great abundance of 18:0.

Lipids, Vol. 30, no. 12 (1995)


TABLE 7 Fatty Acid Composition a of Lysophosphatidylcholine from White Muscle of Six Tuna Species

Tuna species Acid Bigeye Bluefin Bonito Frigate Skipjack Yel lowfin

14:0 1.74 • 0.49 3.40 • 1.40 2.63 • 0.87 3.86 • 0.61 1.95 • 0.65 3.12 • 0.68

15:0 0.52 • 0.06 0.93 • 0.31 1.72 • 0.67 1.35 • 0.28 0,64 • 0.34 1.06 • 0.43

16:0 9.08 • 2,16 13.99 • 4.45 11.40 • 0,96 20.52 • 3.38 7.68 • 1.78 9.99 _+ 3.64

17:0 0.66 • 0,14 1.07 • 0.44 0.00 1.63 • 0.25 0.72 • 0.13 1.04 + 0.33

18:0 3.21 • 0.78 5.40 _+ 2.29 7.51 • 1.28 7.74 • 1.06 2.38 • 0.62 4.11 • 1.50

20:0 0.65 + 0.07 1.44 + 0.96 0.00 1.764 • 0.19 0.00 1.30 • 0.54

24:0 0.58 • 0,18 1.16 _+ 0.27 0.00 • 0.00 1.51 • 0.31 0.62 • 0.30 1.62 _+ 0.72

16:1n-5 0.66 • 0.14 1.23 • 0.69 0.93 • 0.25 2.10 • 0.38 0.78 _+ 0.21 0.87 • 0.27

16:1n-7 0.96 • 0.04 3.27 • 1.06 3.72 • 0.74 2.51 • 0.51 1.55 _+ 1.26 2.99 • 1.37

16:1 n-9 0.40 • 0.29 0.69 • 0.29 0.36 • 0.14 0.74 • 0.42 0.36 • 0.13 0.47 • 0.17

18:1 n-7 1.33 _+ 0.18 0.93 • 0.32 1.69 • 0.07 1.75 • 0.32 1.04 _+ 0.19 1.35 • 0.11

18:1 n-9 12.33 +_ 1,52 14.46 +_ 3.51 11.67 • 0.17 13.80 • I . I 3 15.91 _+ 1.97 13.35 -+ 2,30

20 : In -9 1.02 • 0,33 1.25 • 1.91 2.16 • 0.40 2.01 • I . I I 0.52 • 0.46 0.87 • 0.43

24:1 n-9 2.28 • 1,58 I . I I • I . I 5 1.36 • 0.17 1.81 • 0.98 0.57 _+ 0.300 1.20 • 0.38

18:2n-6 0.95 • 0.17 1.30 • 0.78 2.27 • 0.31 1.36 • 0.57 1.02 + 0.36 1.51 • 0.42

20:4n-3 0,56 • 0.43 0.49 • 0.27 0.29 • 0.06 1.15 • 0.52 0.68 • 0.45 0.57 • 0.36

20:4n-6 6.19 • 0,46 5.19 • 1.31 2.40 • 0.18 4.27 • 1.30 7,38 • 1.22 4.44 • 0.89

22:4n-6 4.89 • 0,32 3.65 _+ 1.07 3.69 • 0.88 4.26 + 0.5 4.83 • 0.37 4.33 • 0.84

20:5n-3 4.81 • 0.59 5.54 • 2.17 5.59 • 0.20 3.86 • 0.30 6.32 • 2.13 4.65 • 0.77 22:5n-3 1.21 • 0,14 1.18 • 0.37 3.71 • 1.33 1.58 • 0.41 1.36 • 0.16 1.48 • 0.46

22:6n-3 45.26 • 2.03 35.70 • 6.33 34.71 • 2.12 17.98 • 3.85 42,90 • 4,03 37.73 • 6.83

aData are given in tool% as means and standard deviations for five different samples.

TABLE 8 Fatty Acid Composition a of Phosphatidylinositol from White Muscle of Six Tuna Species


14:0 3.37 • I .I 4.57 • 1.41 3.16 • 0.55 3.86 • 1.55 2.83 • 0.71 2.77 • 0.04

15:0 0.77 • 0.18 1.62 + 0.46 1.09 • 0.09 1.28 _+ 0.43 0.93 • 0.16 1.27 + 0.55

16:0 12.98 • 1.94 15.52 + 3.14 12.29 • 1.42 13.91 • 0.77 11.29 _+ 1.38 12.66 • 1.41

17:0 1.96 • 0.86 2.15 • 0.48 2.13 • 0.24 2.05 • 0.06 1.99 • 0.19 2.02 • 0.12 18:0 21.14 • 4.64 14.70 • 4.12 16.54 • 0.56 24.02 • 2.31 25.60 • 1.73 24.42 • 2.98

20:0 1.45 • 1.04 1.12 • 0.34 1.58 • 0.72 0.47 • 0.48 0.00 • 0.00 0.75 • 0.53

24:0 0.96 • 0.00 1.73 + 0.86 1.66 • 0.72 1.28 • 0.78 0.85 • 0.87 1.55 • 0.72

16:1n-5 0.70 • 0.30 1.49 • 0.14 0.56 • 0.01 1.00 • 0.38 0.78 + 0.38 1.09 • 0.52

16:1 n-7 1.84 • 0.35 2.96 • 1.54 2.57 • 0.20 2.15 • 0.50 2.21 • 1.50 2.75 +_ 0.57

16:1n-9 0.54 • 0.43 1.18 • 0.76 1.62 • 0.42 0.48 • 0.15 0.98 _+ 0.53 0.46 + 0.18

18:1n-7 1.27 • 0.42 1.89 • 0.62 2.37 • 0.15 1.44 • 0.31 1.20 • 0.27 1.20 • 0.08

18:1n-9 8.32 • 1.08 12.04 • 3.35 17.78 • 0.82 7.66 • 1.15 8.13 • 0.05 8~77 • 1.82

20:1n-9 1.93 • 1.12 2.76 • 1.18 3.25 • 0.79 0.52 • 0.52 0.79 • 0.24 1.23 + 0.29

24:1 n-9 4.34 • 2.54 4,06 • 2.03 2.72 +_ 0.31 4.19 • 1.55 3.56 • 1.86 4.31 +_ I .I I

18:2n-6 0.91 • 0.06 1.04 • 0.06 2.09 • 0.05 1.39 • 0.28 1.01 • 0.03 1.25 _+ 0.19

18:3n-3 0.64 • 0.28 1.15 • 0.69 1.21 • 0.40 0.81 • 0.12 0.47 • 0.05 1.55 + 0.77

20:4n-6 5.09 • 2.35 5.17 • 1.18 1.51 • 0.07 3.60 • 0.37 5.53 • 1.20 4.64 _ 0.61

20:4n-3 0.43 • 0.11 0,37 • 0.16 0.43 • 0.01 0.68 • 0.25 0.38 • 0.66 1.01 _+ 0.57

22:4n-6 6.02 • 2.43 1,55 • 0.63 0.64 • 0.08 0.79 • 0.79 3.89 • 2.71 1.49 • 0.65

20:5n-3 2.18 • 1.29 4,34 • 3.04 4.85 • I . I 7 2.12 • 0.66 3.68 • 1.95 2.11 • 0.89

22:5n-3 1.00 • 0.59 0,79 • 0.23 1.34 • 0.16 1.23 • 1.06 0.80 • 0.01 1.69 • 0.84

22:6n-3 24.05 • 3.63 16.75 • 5.69 19.18 + 1.49 2.722 • 2.63 25.32 • 2.43 23.76 + 1.62

aData are given in mol% as means and standard deviations for five different samples,

The major monoene, 18: In-9, was found in similar proportions in all PL classes, although choline-type PLs presented higher levels. Other monoenes, like 20:1 n-9 and a hemologue in the elongation pathway, 24: ln-9, were found to be highly concentrated in PE, PI, and PS. This distribution was in agree-

ment with previous results reported in the case of the white muscles of albacore tuna and cod (39,46).

With respect to PUFA, 20:5n-3 and 22:6n-3 were especially concentrated in PC and LPC with levels around 5-6 and 30-45% of each fatty acid, respectively. Relatively high con-

Lipids, Vol. 30, no. 12 (1995)

WHITE MUSCLE PHOSPHOLIPIDS COMPOSITION OF SIX TUNA SPECIES

TABLE 9 Fatty Acid Composition a of Phosphatidylserine from White Muscle of Six Tuna Species

1133


14:0 3.80 • 1.20 4,90 • 1.26 4.59 _+ 1.12 5.31 + 1.72 15:0 1.21 • 0.24 1,37 + 0.22 1.76 • 0.47 1.64 • 0.18

16:0 16.70 • 3.12 16,61 • 1.08 19.29 • 1.63 17.98 • 1.72

17:0 1.89 • 0.67 1,73 • 0.44 3.42 • 1.29 1.88 • 0.42 18:0 16.79 • 2.48 15,39 + 2.48 12.77 • 0.60 17.46 • 3.09

20:0 2.25 • 1.02 1,13 • 0.28 1.34 • 0.69 1.69 • 0.56 24:0 1.01 + 0.68 2.33 • 1.89 1.41 • 0,324 2 .20 • 1.08 16:1n-5 3.33 • 3.78 1,41 • 1.81 1.82 • 0.28 1.64 • 0.65 16:1n-7 2.32 + 0.68 2,18 • 0.63 2.47 + 0.09 3,16 • 0.85 16:1n-9 1.51 • 0.93 1,17 • 0,59 0.56 • 0,08 0.70 _+ 0.09

18:1n-7 2.00 • 0.34 1,88 • 0,72 1.67 -+ 0.08 1.43 + 0.56

18:1n-9 10.93 • 1.57 12,65 + 1.23 13.77 • 1,31 11.42 • 1,40 20:1n-9 1.81 • 0.55 1,79 • 0.87 4.38 • 1.46 1.99 • 0.65

24:1n-9 5.02 • 1.52 3.44 • 1.00 2.65 • 0.73 3.47 • 2.49

18:2n-6 1.34 • 0.23 1.48 • 0.53 2.69 • 0.45 2.22 • 0.95

18:3n-3 1.48 • 1.10 2.12 • 0.41 0.94 • 0.19 1.51 • 0.59

20:4n-6 3.25 • 1.20 2.95 • 2.28 2.02 • 0.41 3.19 • 1.05

20:4n-3 0.15 • 0.34 0.37 • 0.44 0.91 + 0.08 0.49 • 0.45

22:4n-6 1.59 • 1.64 1,77 • 1.12 2.37 • 0.38 1.70 _+ 0.87 20:5n-3 2.97 • 2.36 2,89 • 1.96 3.12 • 1.29 2.71 • 1.12

22:5n-3 1.34 • 0.43 2,25 • 1.36 1.52 • 0.18 1.53 _+ 0.92

22:6n-3 17.44 • 5.71 14.62 • 2.30 I5.26 • 2.80 14.11 +_ 1.74

5,37 • 1.60

1,48 • 0.30

15,37 • 1.28

1,88 • 0.36

18.84 • 3.10

0,00 • 0.00 2,45 • 1.15

1,59 • 0,59

3,52 • 0.61

0,88 • 0.34 1,27 • 0.47

12,36 • 2.48

1,59 + 0.82

3,54 • 0.63

1,65 + 0.79

1,56 • 0.81

2.53 • 1.72

0.000 • 0.00 1,51 • 0.38

3.29 • 1.86

2,37 • 1.38

16,76 _+ 5.70

4.67 • 2.00 1.81 • 0.29

17.52 • 1.40

2.00 • 0.52 16.81 •

1.16 • 0.70

1.88 • 1.13

1.88 • 0.60 4.03 • 0.19

0.76 • 0,21 1.54 • 0.20

14.46 • 2.74

2.73 • 1.28

3.56 • 2.18

2.21 • 0.84

1.30 • 0.25

2.32 • 1.41 0.12 • 0.26

1.46 • 0.58

2.79 • 1.47

2.24 • 1.88

12.47 • 3.53


tents also were found in PE. Results obtained herein do not confirm the idea that PE should concentrate a higher PUFA content than any other PL class (18,38). PI and PS showed a more saturated composition with relatively low values of 22:6n-3 fatty acid (around 25 and 15%, respectively).

The high PUFA content found in LPC may be related to the enzymatic hydrolysis of PC occurring during storage at low temperatures. Some authors have found that this lipolysis takes place first with saturated fatty acids that are mainly esterified in s n - 1 position of the PL moiety, and in a second step with PUFA (14,55,56). As a result, a high concentration in PUFA could be obtained in the remaining lysophospholipids. The findings obtained in this research were in accordance with that notion.

However, a different composition was found in LPC of frigate as compared to the other species--saturated fatty acids (16:0 and 18:0) showed high contents, while n-3 PUFA (20:5 and 22:6) were present in lower contents. The proportion of these acids in PC was not significantly different from other species; hence, reasons related to a partial hydrolysis of PC could not explain the low content found in LPC. More complex factors surely may be considered, such as diet, temperature, salinity, or even internal conditions (18).

From a nutritional point of view, the enzymatic hydrolysis occurring in all samples did not seriously affect the n-3-composition, as can be inferred from the comparison of fatty acid composition of PC and PE in samples which showed higher levels of LPC, such as skipjack and yellowfin in contrast with bonito or frigate.

Finally, special emphasis often is given to the n-3/n-6

PUFA ratio for marine and freshwater fish actually consumed by humans (37,57). As could be expected, all PL classes described in this work were characterized by high ratios of n-3 to n-6 polyenes. Special attention must be given to the fatty acid composition of PI. Recently, several authors (52,58,59) have reported that PI from some marine fish tissues is especially enriched in 20:4n-6. In our case, PI showed a lower n-3/n-6 ratio than other PL classes, but arachidonic acid was not the major PUFA in any tuna species. This situation al- ready has been described in white muscle of cod (39), in contrast with previous reports.

A different situation could be observed in the relative contents of 20:5n-3 and 20:4n-6. In general, PE and PI showed a larger proportion of 20:4n-6 than that of 20:5n-3, and PC re- vealed the opposite (Tables 5, 6, and 8). Studies carried out on fish coming from Australian waters contained different relative contents of both acids, depending on the species (60,61). In addition, an important variation in the content of 20:4n-6 found in PC and LPC also was observed between the six tuna species (Tables 5 and 7). In these classes, bonito showed the highest values of the n-3/n-6 ratio.

The present research indicates that the PL of white muscle of fresh tuna species has a noteworthy nutritional value, sup- plying substantial amounts of PUFA and PLA. No significant differences were found in the distribution of the PL classes among the different tuna species studied. Furthermore, in terms of fatty acid and alkenyl ether compositions, PL classes showed some general similarities, although ratios between certain fatty acids were found to be different.

Lipids, Vol. 30, no. 12 (1995)


ACKNOWLEDGMENTS

We acknowledge financial support for Research Project ALl 90- 0773 provided by CICYT and Research Project CEE UP. 2.571 (of DG XIV).

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[Received January 5, 1995, and in final revised form September 28, 1995; Revision accepted October 10, 1995]

Lipids, Vol. 30, no. 12 (1995)

Composition of phospholipids of white muscle of six tuna species

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