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Lipid composition of blood platelets and erythrocytes of southern elephant seal (Mirounga leonina) and antarctic fur seal (Arctocephalus gazella)
Comparative Biochemistry and Physiology Part B 126 (2000) 39 – 47 www.elsevier.com/locate/cbpb
Lipid composition of blood platelets and erythrocytes of southern elephant seal (Mirounga leonina) and antarctic fur seal (Arctocephalus gazella) Christine Fayolle a, Claude Leray a, Philippe Ohlmann a, Genevie`ve Gutbier a, Jean-Pierre Cazenave a, Christian Gachet a,*, Rene´ Groscolas b a
INSERM U.311, Etablissement de Transfusion Sanguine, 10 Rue Spielmann, BP 36, 67065, Strasbourg, Cedex, France b Centre d’Ecologie et Physiologie Energetiques, CNRS, Strasbourg, France Received 4 November 1999; received in revised form 19 January 2000; accepted 31 January 2000
Abstract Erythrocyte and blood platelet phospholipid compositions were studied in three elephant seals and two fur seals, two species of marine mammals living in the Subantarctic region feeding on preys rich in (n-3) polyunsaturated fatty acids. Results were compared with those reported for related species and humans.In erythrocytes, the phospholipid (PL) and cholesterol (CHOL) contents were lower in pinnipeds than in humans. Phosphatidylcholine (PC) levels were higher in elephant seals than in fur seals, with a reverse trend for phosphatidylethanolamine (PE) and phosphatidylserine (PS). Both species had lower SM/PC ratios and PE plasmalogen concentrations than human. Erythrocytes were richer in (n-3) fatty acids (FA) in pinnipeds than in humans. In platelets, the PL content was lower and the CHOL content higher in elephant seals than in humans or in other phocid seal species studied to date. The SM/PC ratio was much higher than in other seal species or in man. In both species, the proportion of PE plasmalogens was higher in platelets than in erythrocytes. PL were more saturated in elephant seals than in fur seals. These results suggest that the erythrocytes and platelets of wild marine mammals may prove useful models to study the influence of dietary lipids on the structure and hemostatic function of these cells. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Marine mammals; Seals; Erythrocytes; Platelets; Cholesterol; Fatty acids; Phospholipids; Hemostasis
1. Introduction There is epidemiological and experimental evidence to suggest that in humans, diets rich in (n-3) polyunsaturated fatty acids (PUFA) of marine origin are associated with a reduction in thrombotic risk (Leaf and Weber, 1988; Connor * Corresponding author. Tel.: + 33-3-88212525; fax: +333-88212521. E-mail address: [email protected] (C. Gachet)
et al., 1993), possibly through lowering of the plasma triglyceride and cholesterol levels (Phillipson et al., 1985). It has long been known that dietary (n-3) PUFA are incorporated into blood cell phospholipids (PL) and are able to alter some of the functional properties of these cells. Thus, rheological studies have demonstrated that these fatty acids (FA) are efficient in altering cell deformability and filtrability and consequently in improving peripheral blood circulation (Cooper, 1977; Terano et al., 1983; Po¨schl et al., 1996). Platelet functions have also been extensively ex-
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amined and the inhibitory effect of ingested (n-3) FA on aggregation in response to agonists is thought to be mediated primarily by inhibition of cyclo-oxygenase, with a subsequent reduction in the conversion of arachidonic acid to thromboxane A2, a potent platelet aggregating agent (Lagarde et al., 1985; Scheurlen et al., 1993). The decrease in platelet reactivity was shown to be linked to incorporation of the two major (n-3) PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), into platelet membranes, in an acylated form in PL (Croset et al., 1992; Marangoni et al., 1993) or even in the free form (MacIntyre et al., 1984). Although the effects of feeding with fish, fish oils or mixtures of purified (n-3) PUFA on blood cell composition and function have been widely investigated in humans and laboratory animals, only a few observations exist in wild marine mammals like Phocidae or Otariidae living on natural preys rich in (n-3) PUFA. In fact, the previous studies of seal erythrocyte (Nelson, 1970) and platelet (Ahmed et al., 1989) lipids were carried out in captive animals fed either a diet of unspecified origin or an artificially controlled fish and shrimp diet, respectively. The two pinnipeds studied here, the southern elephant seal (Mirounga leonina) and antarctic fur seal (Arctocephalus gazella), forage in the same geographic area, mainly for cephalopods (elephant seal) or fish (fur seal) (Guinet et al., 1996; Cherel et al., 1997). Such preys are known to have a high content of (n-3) PUFA (Joseph, 1982; Raclot et al., 1998). It was therefore of interest to characterize the lipid composition of erythrocytes and platelets in elephant and fur seals living in their natural environment (subantarctic region) and to compare the results with those of previous reports in related species and humans. This information, together with further functional studies, should contribute to our knowledge of the relationships between marine food intake and hemostasis, not only in wild marine animals but also in man.
2. Materials and methods Only a few individuals were accessible in the study area at Possession Island in the Crozet Archipelago (southern Indian Ocean: 46° 26% S, 51° 52% E) in 1997, which explains the small size of the samples. Despite this small size, our study
provides relevant information defining lipid composition of cells involved in hemostatic function. However, our conclusions relating to comparisons between the two species should be regarded as provisional until larger populations are explored. Three female elephant seals 2–3 years of age and averaging 300 kg in weight were sampled in summer (January–February). One female fur seal of estimated weight 60 kg was sampled in summer (December) and another of approximate weight 50 kg in winter (August). All animals were nonbreeders resting ashore. The study was approved by the ethical committee of the ‘Institut Franc¸ais pour la Recherche et la Technologie Polaires’ and conformed to the agreed measures for the conservation of antarctic and subantarctic fauna. Blood was drawn from an intravertebral vein (lombar zone) of elephant seals after light anesthesia with Zoletil® (1 mg/kg i.m.) (Baker et al., 1990), or from a plantar vein (hind flipper) of fur seals under restraint. The samples were collected into plastic syringes containing acid-citrate-dextrose anticoagulant (1:7 volume ratio) and processed within one hour. Erythrocyte and platelet counts were determined with a hemacytometer after 1/200 and 1/20 dilution respectively and hematocrits by centrifugation in glass capillary tubes. The mean cell volumes (mm3) were calculated from hematocrits and red cell counts. Red blood cells and platelets were separated by low speed centrifugation (113×g, 15 min, 37°C). Red cell pellet was washed twice in saline prior to lipid analysis. Platelets were isolated from the platelet rich plasma layer by centrifugation (1000×g, 10 min, 37°C) and washed twice in Tyrode’s buffer containing human serum albumin (Cazenave et al., 1983) before lipid analysis. Total lipids were extracted immediately from washed red blood cells or platelets (Leray et al., 1987) and dissolved in 1 ml chloroform–methanol (2:1, v/v). After addition of ethyl gallate (0.3 mg/ml) as an antioxidant, the extracts were stored at −20°C for about 6 months until laboratory processing in France. Cholesterol (CHOL) and PL were separated by one-dimensional TLC on silicagel plates impregnated with boric acid and developed up to 1 cm below the top in a solvent system of chloroform–ethanol–water–triethylamine (35:30:7:35, vol/vol) (Leray et al., 1997). Lipid spots were located under UV light after a primuline spray, CHOL was quantified by colorimetry (Rudel and Morris, 1973) and PL were
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determined by phosphorus estimation (Rouser et al., 1970). Unfortunately, a technical incident during the processing of the SM fraction from the only available fur seal platelet sample prevented the determination of the FA composition of SM and the estimation of total lipids in this species. FA compositions of individual PL were obtained by transmethylation and gas-liquid chromatography after addition of a known amount of heptadecanoic acid as an internal standard (Leray et al., 1987). Plasmalogens were estimated by gas – liquid chromatography through the amounts of dimethyl acetals (DMA) obtained during the transmethylation process. Values are presented as the mean and range of the data, the small size of the samples preventing further statistical treatment.
3. Results and discussion Hematocrits, cell counts, size and the lipid composition of erythrocytes are given in Table 1. The hematocrits of the two species studied here do not differ greatly from values reported for a Phocidae of the northern hemisphere (Mirounga angustirostris) (Nelson, 1970), which is the only available literature data. PL and CHOL contents in southern elephant seals were about twice as high as in antarctic fur seals, which could be explained by an about twice higher cell volume. In comparison with human cells (respectively 300 and 125 mg/109 cells for PL and CHOL) (Van Deenen and De Gier, 1974), which are similar in size to that of fur seal cells, the PL and CHOL contents were two to three times lower in elephant seal and fur seal,
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respectively. In the absence of data on the protein content of erythrocyte plasma membrane in these marine mammals, no explanation could be proposed for these apparent discrepancies. The large size of elephant seal cells is in agreement with previous observations in northern phocid seals and this feature is thought to be linked to an adaptation to circulatory redistribution in these long-diving animals (Wickham et al., 1989). Despite these differences in lipid composition, only slightly smaller CHOL/PL ratios were observed in the species studied as compared to M. angustirostris (ratio of 0.95) (Nelson, 1970) and human cells (ratio of 0.87) (Van Deenen and De Gier, 1974). The distribution of erythrocyte membrane PL is shown in Table 2. The main difference between the two pinnipeds studied is the larger proportion of phosphatidylcholine (PC) in elephant seal (50%) as compared to fur seal cells (38%), which is compensated by a reverse trend for PE and PS. In both species, a low sphingomyelin (SM) content leads to low values of the SM/PC ratio as compared to humans (0.32, 0.49 and 0.93 in M. leonina, A. gazella and man, respectively). These relative SM contents suggest that the marine mammal erythrocytes are far more deformable (less viscous) than human red cells, which is probably a specific adaptation of the circulatory system in long-diving marine mammals (Kooyman, 1989). Previous rheological findings of reduced viscosity and flow resistance in seal blood corroborate this hypothesis (Smith et al., 1979; Wickham et al., 1989; Meiselman et al., 1992). Analysis of the FA composition of total erythrocyte PL (Fig. 1) reveals similar unsaturation levels in elephant and fur seals, despite noticeable
Table 1 Levels of cell counts, total phospholipid and cholesterol in erythrocytes and platelets of elephant seal and fur seala Erythrocytes
Hematocrit Cell counts Mean cell volume Phospholipid Cholesterol Cholesterol/phospholipid (mol/mol)
Platelets
Elephant seal (n= 3)
Fur seal (n = 2)
65.8% (38.9–72.6%) 4.5×106/ml blood (3.5–5.7) 150 mm3 (118–195) 174 mg/109 RBC (130–199) 63 mg/109 RBC (48–77) 0.73 (0.67–0.79)
49.0% (46.3–51.6%) – 6.2×106/ml blood (5.9–6.5) 125 000/ml blood (71 600–199 200) 78.5 mm3 (78–79) 92 mg/109 platelets (73–111) 337.5 mg/109 RBC (33–42) 0.82 (0.75–0.90)
Elephant seal (n = 3)
– 316 mg/109 platlets (269–342) 84 mg/109 platelets (72–91) 0.53 (0.53–0.54)
a Values are given as means two or three samples; in brackets: range. RBC, red blood cells. A technical incident prevented the determination of the amount of total phospholipids and cholesterol/phospholipid ratio in fur seal platelets.
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Table 2 Phospholipid composition of erythrocytes and platelets of elephant seal and fur seala Erythrocytes
Phosphatidylcholine (PC) Phosphatidylethanolamine (PE) Phosphatidylserine (PS) Phosphatidylinositol (PI) Sphingomyelin (SM)
Platelets
Elephant seal (n = 3)
Fur seal (n = 2)
Elephant seal (n =3)
50.0 (47.7–52.9) 22.0 (20.9–22.8) 8.6 (5.2–10.3) 3.3 (2.1–3.9) 16.1 (13.9–18.8)
38.2 (37.2–39.2) 27.7 (27.0–28.4) 12.4 (11.0–13.9) 3.0 (2.8–3.2) 18.7 (18.7–18.8)
33.1 (27.3–39.8) 15.4 (9.9–19.1) 15.9 (8.7–19.2) 10.4 (7.5–12.5) 25.3 (18.0–34.2)
a Values are means of the molar percentage of each phospholipid class; in brackets: range. A technical incident prevented the determination of the phospholipid composition in fur seal platelets.
differences at the levels of 20:4 (n-6) and 22:6 (n-3). There currently exist no other reports of the composition of erythrocyte PL in pinnipeds. As compared to humans (Van Deenen and De Gier, 1974), erythrocyte PL of the two marine mammals were more unsaturated (about 60% unsaturated FA vs. 50% in man). The main differences in composition were the lower linoleic acid content (1–2 vs. 10% in man), the higher arachidonic acid content in elephant seal (17.8 vs. 12.6% in man), and the importance of the (n-3) FA fraction (about 9, 17 and 5% in elephant seals, fur seals and man, respectively). Curiously, while similar amounts of 20:5 (n-3) and 22:6 (n-3) were found in fur seal PL, 20:5 (n-3) was three times more abundant than 22:6 (n-3) in elephant seal PL. These discrepancies, which were also present in plasma PL (unpublished data), could not be explained by differences in dietary habits, since cephalopods and myctophid fishes, the respective preys of elephant and fur seals (Guinet et al., 1996; Cherel et al., 1997), have similar PUFA contents with a predominance of 22:6 (n-3) over 20:5 (n-3) (Culkin and Morris, 1970; Raclot et al., 1998). Unexpectedly, levels of 22:6 (n-3) in the marine mammal erythrocytes lay in the range of values reported in humans, even without (n-3) FA supplementation, whereas levels of 20:5 (n-3) were more than ten times higher. Despite the very high intake of (n-3) fatty acids in the species studied, the presence in their erythrocytes of high 20:4 (n-6) levels together with 22:6 (n-3) levels similar to those found in man raises the question of the functional roles of these particular fatty acids which remain yet unresolved. The FA compositions of the individual PL purified from erythrocytes are given in Table 3. Among all glycerophospholipids, there is a gen-
eral trend towards a higher unsaturation index (unsaturated–saturated FA ratio) in elephant seals as compared to fur seals. SM displays the reverse trend with an unsaturation index three times higher in fur seals than in elephant seals. This balance of unsaturation features between glycero- and sphingolipids explains the small variations observed at the level of polyunsaturated FA (Fig. 1). Specific differences were detected in individual saturated FA contents. Thus, palmitic acid was less abundant in all glycerophospholipids in elephant seal as compared to fur seal erythrocytes, while stearic acid levels were similar among PL except for PS. In accordance with the global FA profiles, all erythrocyte PL contained a smaller proportion of (n-3) FA in elephant seals than in fur seals, the difference corresponding mainly to the difference in 22:6 (n-3) content. Conversely, the proportion of the most abundant (n-6) FA, arachidonic acid, was higher in all PL
Fig. 1. Fatty acid composition of erythrocyte phospholipids.Each bar corresponds to the mean and S.E.M. of three elephant seals and the mean of two fur seals. MUFA, monounsaturated fatty acids, SAT, saturated fatty acids.
PC
14:0 16:0 18:0 20:0 22:0 S. n-11 16:1 n-9 18:1 n-9 20:1 n-9 20:2 n-9 20:3 n-9 22:1 n-9 24:1 n-9 16:1 n-7 18:1 n-7 18:2 n-6 18:3 n-6 20:4 n-6 22:4 n-6 20:5 n-3 22:5 n-3 22:6 n-3 DMA UNSAT/SAT
PE
PS
PI
SM
Elephant seal
Fur seal
Elephant seal
Fur seal
Elephant seal
Fur seal
Elephant seal
Fur seal
Elephant seal
Fur seal
1.1 90.2 20.8 90.2 14.7 90.3 –
1.0 27.9 13.2 – 0.9 0.8 18.4 3.2 – – 0.5 – 1.1 5.7 1.2 0.5 11.3 – 6.4 2.0 4.4 – 1.4
0.7 9 0.2 7.1 9 0.2 16.0 9 0.3 –
1.3 14.1 17.3 – 1.3 0.8 9.9 5.1 – – 0.7 – – 2.4 0.9 0.5 16.7 1.7 9.4 3.6 9.0 3.1 1.9
0.8 90.1 3.6 9 0.3 19.3 91.0 –
1.6 7.6 35.0 – 2.2 1.0 6.5 4.3 – 0.6 1.9 – – 1.3 1.6 0.5 23.6 0.7 7.2 0.8 1.4 – 1.2
2.6 90.5 12.49 0.8 27.892.5 –
4.3 20.0 27.0 – 1.0 0.5 2.3 7.5 2.0 1.3 1.4 0.6 – 0.8 3.2 1.4 1.9 13.6 1.0 3.6 1.9 2.7 – 0.9
5.1 9 0.3 47.6 9 1.1 10.8 9 0.3 3.59 0.2 0.79 0.1 – 1.2 90.1 4.6 90.4 0.8 9 0.1 1.2 9 0.1 – 3.8 90.4 8.99 0.9 – 1.3 9 0.2 2.0 9 0.4 2.1 90.2 2.7 9 0.2 – 0.8 9 0.1 – – – 0.5 9 0.1
1.4 22.5 10.4 5.4 1.7 – 0.9 7.7 0.5 – – 1.3 38.9 – 0.7 0.8 1.9 0.9 0.6 0.5 – 1.7 – 1.4
1.1 90.1 0.6 90.1 17.8 90.3 2.9 90.3 – – 1.290.1 – 1.1 90.1 4.590.2 2.590.5 – 16.0 91.0 – 8.9 90.8 0.990.1 3.2 90.3 – 1.790.1
2.6 9 0.2 0.59 0.1 20.8 9 0.3 7.09 0.9 – – 2.4 9 0.2 – – 3.8 9 0.2 0.8 9 0.1 – 22.1 9 0.5 0.69 0.1 6.2 9 0.4 0.6 9 0.1 1.890.1 4.5 9 1.2 2.79 0.1
3.6 90.3 0.6 90.1 7.0 90.2 5.9 9 0.6 0.9 90.2 – 3.6 90.2 – – 2.5 9 0.2 1.3 9 0.4 – 44.5 91.5 – 4.1 90.2 – – – 3.2 90.2
0.79 0.1 2.19 0.4 7.39 0.7 2.7 9 0.3 1.8 9 0.3 – 4.89 1.4 – 0.5 90.1 4.6 90.8 5.49 1.5 1.09 0.2 20.491.7 – 2.4 90.6 0.5 90.2 – – 1.39 0.9
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Table 3 Fatty acid composition of phospholipids in erythrocytes of elephant seal and fur seala
a Values are means and S.E.M. +-+(mole%) of samples from three elephant seals and means of samples from two fur seals. Faty acidsB0.5% of the total are not shown. PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol; SM, sphingomyelin; S.n-11, sum of 20:1 and 22:1 n-11 fatty acids; DMA, dimethyl acetal derivatives formed from aldehydes; UNSAT/SAT, ratio of unsaturated to saturated fatty acids.
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Fig. 2. Fatty acid composition of platelet phospholipids.Each bar corresponds to the mean and S.E.M. of three elephant seals and to the value from one fur seal. MUFA, monounsaturated fatty acids; SAT, saturated fatty acids.
of elephant seal as compared to fur seal erythrocytes, the greatest difference being observed for PS (44.5 and 23.6%, respectively). PS nevertheless contained in both species about 23% of the total mass of membrane arachidonic acid. Although not accurately documented, the degree of unsaturation of PL fatty acid chains has been related to prothrombin activation in experimental models (Hecht, 1965; Govers-Riemslag et al., 1992). Hence it is possible that the high unsaturation of PS present in the erythrocytes of these two marine mammals and particularly its richness in 20:4 (n-6) play a role in the procoagulant activity of the circulating cells. This hypothesis deserves further investigation in studies of the influence of PL unsaturation on thrombin generation in marine and land mammals. Considering the proportion of dimethyl acetal derivatives (DMA) formed from the PE plasmalogen pool (3.1–4.5%), it was noticeable that in both species, plasmalogens represented only about 6 – 9% of ethanolamine PL, in contrast to about 43% in humans (Adosraku et al., 1994). This discrepancy points to a specific distribution of PE sub-classes in marine mammal erythrocytes and to the need for further exploration of their FA composition and metabolism. Important differences were detected in the length of the fatty acyl chain of SM. Whereas a 16 carbon chain (palmitic acid) was predominant in elephant seals (about 48%), a 24 carbon chain (nervonic acid) was predominant in fur seals (about 40%), as it is in the SM of human erythrocytes (Van Deenen and De Gier, 1974). Such
variations in the composition of SM could result in considerable differences in the membrane bilayer organization and contribute to a higher membrane fluidity of elephant seal as compared to fur seal erythrocytes. This effect would undoubtedly be enhanced by the lower SM/PC ratio and higher unsaturation index of elephant seal cells. There exist many studies of the relationships between the membrane lipids of erythrocytes and their functional properties. Data concerning diving mammals are however still fragmentary. Although the functional roles of erythrocyte PL remain poorly understood, we can nevertheless speculate that the differences in membrane properties between M. leonina and A. gazella may influence their erythrocyte deformability. The specific biochemical features revealed by this study of M. leonina erythrocytes (high unsaturation degree, high arachidonic acid content) may be invaluable considering the well known diving performances of this phocid mammal, which appears to be capable of extremely long dives of up to 2 h duration and 1595 m depth. The maximum dive duration and depth recorded for A. gazella are in comparison 10 min and 181 m, respectively (Butler and Jones, 1997). Interestingly, it has been suggested that an increase in red cell deformability may also limit platelet recruitment from flowing blood by reducing the release from erythrocytes of a potent platelet agonist, adenosine diphosphate (Turitto and Weiss, 1980), thus contributing to limit any thrombosis during diving. Cell counts and total PL and CHOL of elephant seal platelets are shown in Table 1. The elephant seal displays major differences in platelet lipid content as compared to the other phocid seal species studied to date (Ahmed et al., 1989). Thus, levels of PL were lower and CHOL levels higher in elephant seals than in other seals or humans, with a resultant higher CHOL/PL ratio in elephant seals. Table 2 gives the PL composition of elephant seal platelets. In these cells, a high SM content was accompanied by a low PC content, leading to a high SM/PC ratio (0.76), much higher than those reported in other seals (0.5) or in human platelets (0.45) (Ahmed et al., 1989). This would most likely tend to decrease the membrane fluidity. As shown in Fig. 2, elephant seal platelets contained higher levels of saturated FA than fur seal platelets, compensated by lesser
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amounts of monoenes. Similarly, greater amounts of (n-6) FA, predominantly represented by arachidonic acid, but lower levels of (n-3) FA were detected in elephant seal as compared to fur seal cells, despite comparable (n-3) FA levels in their preys. These relationships are similar but amplified in comparison with those observed in erythrocytes of the same species. It is interesting that in both species, 20:5 (n-3) and 22:6 (n-3) were more abundant in erythrocytes than in platelets, as was likewise the case in humans before and after prolonged administration of (n-3) FA (Marangoni et al., 1993). These observations are difficult to explain, since blood cells have different life spans and FA modifications could occur following recent ingestion, but also by prolonged exchange between tissues and circulating cells. Nevertheless, the present results suggest either that the two species had different chronic (n-3) FA ingestion rates, or that the fur seal in question had preyed on animals rich in (n-3) FA more
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recently than the elephant seals, platelets being a good cellular indicator of late (n-3) FA ingestion (Marangoni et al., 1993). Important differences were detected in the FA profiles of individual PL (Table 4) between the two species of the present study. An analysis of the arachidonic acid content of all glycerophospholipids revealed levels in fur seal platelets about half those in elephant seal platelets, as in the case of erythrocytes. This lower arachidonic acid content of fur seal platelets could infer lower thromboxane production, as was observed in humans after (n-3) FA supplementation (Prisco et al., 1995), with a consequent reduction in the sensitivity to agonists such as collagen, known to be eicosanoid dependent (Siess, 1989) and thus, leading to a potential lowering of thrombosis incidence. As in erythrocytes, the plasmalogen content of PE was in both our species less than half the values reported in other seal species (Ahmed et al., 1989) or in man (Natarajan et al.,
Table 4 Fatty acid composition of gylcero-phospholipids in platelets of elephant seal and fur seala PC
14:0 16:0 18:0 20:0 22:0 S. n-11 16:1 n-9 18:1 n-9 20:1 n-9 20:2 n-9 20:3 n-9 22:1 n-9 16:1 n-7 18:1 n-7 22:1 n-7 18:2 n-6 18:3 n-6 20:3 n-6 20:4 n-6 22:4 n-6 22:5 n-6 20:5 n-3 22:5 n-3 22:6 n-3 DMA UNSAT/SAT
PE
PS
PI
Elephant seal
Fur seal
Elephant seal
Fur seal
Elephant seal
Fur seal
Elephant seal
Fur seal
0.5 90.1 28.0 91.1 12.2 90.2 – – 0.8 90.1 1.4 90.1 16.0 91.1 4.291.5 0.6 90.1 – 1.1 90.2 0.7 90.1 6.9 90.6 – 1.6 90.2 1.190.2 1.190.2 18.9 91.2 – – 2.290.2 – 0.6 90.1 – 1.5 9 0.1
2.7 22.1 13.5 0.5 – 3.0 1.5 19.0 3.8 1.2 – 1.9 0.6 5.5 – 4.0 4.0 0.5 10.7 – – 1.7 1.1 0.9 – 1.6
1.29 0.6 5.89 0.7 16.0 9 0.1 – – – 1.39 0.4 9.09 0.7 2.69 0.3 1.69 0.8 – 1.39 0.1 0.69 0.2 3.09 0.4 0.69 0.1 0.79 0.1 1.79 0.2 0.59 0.2 35.79 5.1 – – 4.29 1.2 2.09 0.4 1.09 0.3 9.39 2.1 2.89 0.6
7.0 20.4 11.2 – – 1.4 5.1 10.2 0.8 3.7 – 1.0 – 5.1 – 2.3 0.9 – 16.1 0.6 – 1.8 1.0 1.0 9.6 1.2
1.1 90.4 4.7 90.8 41.2 96.8 – – 1.7 9 0.4 0.8 90.3 10.7 92.4 3.4 91.0 1.3 90.5 – 2.4 90.7 – 1.7 90.2 – – 2.0 9 0.2 0.59 0.2 23.4 95.1 – 0.5 90.1 1.1 90.5 0.6 9 0.3 – – 1.3 90.4
6.4 9.8 26.5 – – 2.7 0.9 17.1 4.2 3.8 0.8 2.9 – 3.4 – 1.8 4.0
1.7 9 0.7 5.7 9 0.5 37.0 94.3 – – – 1.7 9 0.2 4.2 9 0.5 1.4 9 0.4 2.5 9 0.9 – 1.4 90.2 0.5 9 0.2 1.5 9 0.1 0.6 90.3 0.5 9 0.2 2.8 9 0.5 0.7 90.6 35.3 9 5.3 0.6 9 0.1 – 0.8 90.2 – – – 1.3 90.3
2.0 10.7 27.5 2.5 0.8 1.3 – 11.1 2.4 0.8 1.1 5.7 0.5 0.9 0.8 3.1 4.1 0.5 15.7 0.8 2.7 1.2 1.4 2.1 – 1.3
10.5 1.1 – 1.0 0.6 – – 1.3
a Data are expressed as means and SEM (mole %) of samples from three elephant seals and one value from a sample from one fur seal. Fatty acids B0.5% of the total are not shown. Legends as in Table 3.
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1982). In view of the high concentrations of arachidonic acid in plasmalogens, our observations emphasize the need to examine the metabolism of these FA in order to assess the functional role of this apparent plasmalogen deficiency. In conclusion, the specific differences in the lipid compositions of the blood cells of these two seal species may reflect their taxonomical position or their natural history, which is not yet well known. It may therefore be of greater interest to conduct further comparative studies in wild animals sampled in their natural environment rather than in captive animals living in artificial conditions. Moreover, the present investigation of the lipid components of the membranes of pinniped erythrocytes and platelets illustrates the need for more research into the functional significance of such observations, in the fields of both hemostasis and circulatory dynamics.
Acknowledgements This work was supported by INSERM and the Institut Franc¸ais pour la Recherche et la Technologie Polaires (Programme 119). It received logistic support from the Terres Australes et Antarctiques Franc¸aises. The authors would like to thank VIRBAC for providing Zoletil® and J. Mulvihill for revising the English of the manuscript.
References Adosraku, R.K., Choi, G.T.Y., Constantino-Kokotos, V., Anderson, M.A., Gibbons, W.A., 1994. NMR lipid profiles of cells, tissues, and body fluids: proton NMR analysis of human erythrocytes lipids. J. Lipid Res. 35, 1925–1931. Ahmed, A.A., Celi, B., Ronald, K., Holub, B.J., 1989. The phospholipid and fatty acid compositions of seal platelets: a comparison with human platelets. Comp. Biochem. Physiol. 93C, 119–123. Baker, J.R., Fedak, M.A., Anderson, S., Arnbom, T., Baker, R., 1990. Use of a tiletamine-zolazepam mixture to immobilise wild grey seals and southern elephant seals. Vet Rec. 126, 75–77. Butler, P.J., Jones, D.R., 1997. Physiology of diving of birds and mammals. Physiol. Rev. 77, 837–899. Cazenave, J.P., Hemmendinger, S., Beretz, A., SutterBay, A., Launay, J., 1983. L’agre´gation plaquettaire:
outil d’investigation clinique et d’e´tude pharmacologique. Me´thodologı`e Ann. Biol. Clin. 41, 167 – 179. Cherel, Y., Guinet, C., Tremblay, Y., 1997. Fish prey of antarctic fur seals Arctocephalus gazella at Ile de Croy, Kerguelen. Polar Biol. 17, 87 – 90. Connor, W.E., DeFrancesco, C.A., Connor, S.L., 1993. N-3 fatty acids from fish oil: effects on plasma lipoproteins and hypertriglyceridemic patients. Ann. NY Acad. Sci. 683, 16 – 34. Cooper, R.A., 1977. Abnormalities of cell-membrane fluidity in the pathogenesis of disease. New Engl. J. Med. 297, 371 – 377. Croset, M., Bayon, Y., Lagarde, M., 1992. Incorporation and turnover of eicosapentaenoic and docosahexaenoic acids in human blood platelets in vitro. Biochem. J. 281, 309 – 316. Culkin, F.and, Morris, R.J., 1970. The fatty acids of some cephalopods. Deep-Sea Res. 17, 171 – 174. Govers-Riemslag, J.W.P., Janssen, M.P., Zwaal, R.F.A., Rosing, J., 1992. Effect of membrane fluidity and fatty acid composition on the prothrombinconverting activity of phospholipid vesicles. Biochemistry 31, 10000 – 10008. Guinet, C., Cherel, Y., Ridoux, V., Jouventin, P., 1996. Consumption of marine resources by seabirds and seals in Crozet and Kerguelen waters: changes in relation to consumer biomass. Antarct. Sci. 8, 23 – 30. Hecht, E.R., 1965. In: Lipids in blood clotting. Thomas, C.C. (Ed.), Springfield, USA. Joseph, J.D., 1982. Lipid composition of marine and estuarine invertebrates: part II: mollusca. Prog. Lipid Res. 21, 109 – 153. Kooyman, G.L., 1989. Diverse Divers. Springer-Verlag, Berlin. Lagarde, M., Drouot, B., Guichardant, M., Dechavanne, M., 1985. In vitro incorporation and metabolism of some icosaenoic acids in platelets: effect on arachidonic acid oxygenation. Biochim. Biophys. Acta 833, 52 – 58. Leaf, A., Weber, P.C., 1988. Cardiovascular effects of N-3 fatty acids. New Engl. J. Med. 318, 549 – 557. Leray, C., Andriamampandry, M., Gutbier, et al., 1997. Quantitative analysis of vitamin E, cholesterol and phospholipid fatty acids in a single aliquot of human platelets and cultured endothelial cells. J. Chromatogr. 696, 33 – 42. Leray, C., Pelletier, X., Hemmendinger, S., Cazenave, J.P., 1987. Thin-layer chromatography of human platelet phospholipids with fatty acid analysis. J. Chromatogr. 420, 411 – 416. MacIntyre, D.E., Hoover, R.L., Smith, M., et al., 1984. Inhibition of platelet function by cis-unsaturated fatty acids. Blood 3, 848 – 857.
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Marangoni, F., Angeli, M.T., Colli, S., et al., 1993. Changes of N-3 and N-6 fatty acids in plasma and circulating cells of normal subjects, after prolonged administration of 20:5 (EPA) and 22:6 (DHA) ethyl esters and prolonged washout. Biochim. Biophys. Acta 1210, 55–62. Meiselman, H.J., Castellini, M.A., Elsner, R., 1992. Hemorheological behaviour of seal blood. Clin. Hemorheol. 12, 657–675. Natarajan, V., Zuzarte-Augustin, M., Schmid, H.H.O., Graff, G., 1982. The alkylacyl and alkenylacyl glycerophospholipids of human platelets. Thromb. Res. 30, 119–125. Nelson, G.J., 1970. The lipid composition of the blood of marine mammals-I: young elephant seals, Mirounga angustirostris and harp seals, Pagophilus groenlandicus. Comp. Biochem. Physiol. 34, 109– 116. Phillipson, B.E., Rothrock, D.W., Connor, W.E., Harris, W.S., Illinworth, D.R., 1985. Reduction of plasma lipids, lipoproteins and apoproteins by dietary fish oils in patients with hypertriglyceridemia. New Engl. J. Med. 312, 1216–1219. Po¨schl, J.M.B., Leray, C., Groscolas, R., Ruef, P., Linderkamp, O., 1996. Dietary docosahexaenoic acid improves red blood cell deformability in rats. Thromb. Res. 81, 283–288. Prisco, D., Filippini, M., Francalanci, I., et al., 1995. Effect of N-3 fatty acidethyl ester supplementation on fatty acid composition of the single platelet phospholipids and on platelet functions. Metabolism 44, 562–569. Raclot, T., Groscolas, R., Cherel, Y., 1998. Fatty acid evidence for the importance of myctophid fish in the
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diet of king penguins, Aptenodytes patagonicus. Mar. Biol. 132, 523 – 533. Rouser, G., Fleicher, S., Yamamoto, A., 1970. Two dimensional thin layer chromatographic separation of polar lipids and determination of phosphorus analysis of spots. Lipids 5, 494 – 496. Rudel, L.L., Morris, M.D., 1973. Determination of cholesterol using O-phthalaldehyde. J. Lipid Res. 14, 364 – 366. Scheurlen, M., Kirchner, M., Clemens, M.R., Jaschnonek, K., 1993. Fish oil preparations rich in docosahexaenoic acid modify platelet responsiveness to prostaglandin-endoperoxide/thromboxane A2 receptor agonists. Biochem. Pharmacol. 46, 245 – 249. Siess, W., 1989. Molecular mechanisms of platelet activation. Physiol. Rev. 69, 58 – 178. Smith, J.E., Mohandas, N., Shohet, S.B., 1979. Variability in erythrocyte deformability among various mammals. Am. J. Physiol. 236, H725 – H730. Terano, T., Hirai, A., Hamazaki, T., et al., 1983. Effect of oral administration of highly purified eicosapentaenoic acid on platelet function, blood viscosity and red cell deformability in healthy subjects. Atherosclerosis 46, 321 – 331. Turitto, V.T., Weiss, H.J., 1980. Red cells: their dual role in thrombus formation. Science 207, 541 – 543. Van Deenen, L.L.M., DeGier, J., 1974. Lipids of the red cell membrane. In: Mac Surgenor, N. (Ed.), The Red Blood Cell, vol. 1. Academic Press, New York, pp. 147 – 164. Wickham, L.L., Elsner, R., White, F.C., Cornell, L.H., 1989. Blood viscosity in phocid seals: possible adaptations to diving. J. Comp. Physiol. 159, 153 – 158.
Lipid composition of blood platelets and erythrocytes of southern elephant seal (Mirounga leonina) and antarctic fur seal (Arctocephalus gazella) Christine Fayolle a, Claude Leray a, Philippe Ohlmann a, Genevie`ve Gutbier a, Jean-Pierre Cazenave a, Christian Gachet a,*, Rene´ Groscolas b a
INSERM U.311, Etablissement de Transfusion Sanguine, 10 Rue Spielmann, BP 36, 67065, Strasbourg, Cedex, France b Centre d’Ecologie et Physiologie Energetiques, CNRS, Strasbourg, France Received 4 November 1999; received in revised form 19 January 2000; accepted 31 January 2000
Abstract Erythrocyte and blood platelet phospholipid compositions were studied in three elephant seals and two fur seals, two species of marine mammals living in the Subantarctic region feeding on preys rich in (n-3) polyunsaturated fatty acids. Results were compared with those reported for related species and humans.In erythrocytes, the phospholipid (PL) and cholesterol (CHOL) contents were lower in pinnipeds than in humans. Phosphatidylcholine (PC) levels were higher in elephant seals than in fur seals, with a reverse trend for phosphatidylethanolamine (PE) and phosphatidylserine (PS). Both species had lower SM/PC ratios and PE plasmalogen concentrations than human. Erythrocytes were richer in (n-3) fatty acids (FA) in pinnipeds than in humans. In platelets, the PL content was lower and the CHOL content higher in elephant seals than in humans or in other phocid seal species studied to date. The SM/PC ratio was much higher than in other seal species or in man. In both species, the proportion of PE plasmalogens was higher in platelets than in erythrocytes. PL were more saturated in elephant seals than in fur seals. These results suggest that the erythrocytes and platelets of wild marine mammals may prove useful models to study the influence of dietary lipids on the structure and hemostatic function of these cells. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Marine mammals; Seals; Erythrocytes; Platelets; Cholesterol; Fatty acids; Phospholipids; Hemostasis
1. Introduction There is epidemiological and experimental evidence to suggest that in humans, diets rich in (n-3) polyunsaturated fatty acids (PUFA) of marine origin are associated with a reduction in thrombotic risk (Leaf and Weber, 1988; Connor * Corresponding author. Tel.: + 33-3-88212525; fax: +333-88212521. E-mail address: [email protected] (C. Gachet)
et al., 1993), possibly through lowering of the plasma triglyceride and cholesterol levels (Phillipson et al., 1985). It has long been known that dietary (n-3) PUFA are incorporated into blood cell phospholipids (PL) and are able to alter some of the functional properties of these cells. Thus, rheological studies have demonstrated that these fatty acids (FA) are efficient in altering cell deformability and filtrability and consequently in improving peripheral blood circulation (Cooper, 1977; Terano et al., 1983; Po¨schl et al., 1996). Platelet functions have also been extensively ex-
0305-0491/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 3 0 5 - 0 4 9 1 ( 0 0 ) 0 0 1 7 5 - 9
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amined and the inhibitory effect of ingested (n-3) FA on aggregation in response to agonists is thought to be mediated primarily by inhibition of cyclo-oxygenase, with a subsequent reduction in the conversion of arachidonic acid to thromboxane A2, a potent platelet aggregating agent (Lagarde et al., 1985; Scheurlen et al., 1993). The decrease in platelet reactivity was shown to be linked to incorporation of the two major (n-3) PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), into platelet membranes, in an acylated form in PL (Croset et al., 1992; Marangoni et al., 1993) or even in the free form (MacIntyre et al., 1984). Although the effects of feeding with fish, fish oils or mixtures of purified (n-3) PUFA on blood cell composition and function have been widely investigated in humans and laboratory animals, only a few observations exist in wild marine mammals like Phocidae or Otariidae living on natural preys rich in (n-3) PUFA. In fact, the previous studies of seal erythrocyte (Nelson, 1970) and platelet (Ahmed et al., 1989) lipids were carried out in captive animals fed either a diet of unspecified origin or an artificially controlled fish and shrimp diet, respectively. The two pinnipeds studied here, the southern elephant seal (Mirounga leonina) and antarctic fur seal (Arctocephalus gazella), forage in the same geographic area, mainly for cephalopods (elephant seal) or fish (fur seal) (Guinet et al., 1996; Cherel et al., 1997). Such preys are known to have a high content of (n-3) PUFA (Joseph, 1982; Raclot et al., 1998). It was therefore of interest to characterize the lipid composition of erythrocytes and platelets in elephant and fur seals living in their natural environment (subantarctic region) and to compare the results with those of previous reports in related species and humans. This information, together with further functional studies, should contribute to our knowledge of the relationships between marine food intake and hemostasis, not only in wild marine animals but also in man.
2. Materials and methods Only a few individuals were accessible in the study area at Possession Island in the Crozet Archipelago (southern Indian Ocean: 46° 26% S, 51° 52% E) in 1997, which explains the small size of the samples. Despite this small size, our study
provides relevant information defining lipid composition of cells involved in hemostatic function. However, our conclusions relating to comparisons between the two species should be regarded as provisional until larger populations are explored. Three female elephant seals 2–3 years of age and averaging 300 kg in weight were sampled in summer (January–February). One female fur seal of estimated weight 60 kg was sampled in summer (December) and another of approximate weight 50 kg in winter (August). All animals were nonbreeders resting ashore. The study was approved by the ethical committee of the ‘Institut Franc¸ais pour la Recherche et la Technologie Polaires’ and conformed to the agreed measures for the conservation of antarctic and subantarctic fauna. Blood was drawn from an intravertebral vein (lombar zone) of elephant seals after light anesthesia with Zoletil® (1 mg/kg i.m.) (Baker et al., 1990), or from a plantar vein (hind flipper) of fur seals under restraint. The samples were collected into plastic syringes containing acid-citrate-dextrose anticoagulant (1:7 volume ratio) and processed within one hour. Erythrocyte and platelet counts were determined with a hemacytometer after 1/200 and 1/20 dilution respectively and hematocrits by centrifugation in glass capillary tubes. The mean cell volumes (mm3) were calculated from hematocrits and red cell counts. Red blood cells and platelets were separated by low speed centrifugation (113×g, 15 min, 37°C). Red cell pellet was washed twice in saline prior to lipid analysis. Platelets were isolated from the platelet rich plasma layer by centrifugation (1000×g, 10 min, 37°C) and washed twice in Tyrode’s buffer containing human serum albumin (Cazenave et al., 1983) before lipid analysis. Total lipids were extracted immediately from washed red blood cells or platelets (Leray et al., 1987) and dissolved in 1 ml chloroform–methanol (2:1, v/v). After addition of ethyl gallate (0.3 mg/ml) as an antioxidant, the extracts were stored at −20°C for about 6 months until laboratory processing in France. Cholesterol (CHOL) and PL were separated by one-dimensional TLC on silicagel plates impregnated with boric acid and developed up to 1 cm below the top in a solvent system of chloroform–ethanol–water–triethylamine (35:30:7:35, vol/vol) (Leray et al., 1997). Lipid spots were located under UV light after a primuline spray, CHOL was quantified by colorimetry (Rudel and Morris, 1973) and PL were
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determined by phosphorus estimation (Rouser et al., 1970). Unfortunately, a technical incident during the processing of the SM fraction from the only available fur seal platelet sample prevented the determination of the FA composition of SM and the estimation of total lipids in this species. FA compositions of individual PL were obtained by transmethylation and gas-liquid chromatography after addition of a known amount of heptadecanoic acid as an internal standard (Leray et al., 1987). Plasmalogens were estimated by gas – liquid chromatography through the amounts of dimethyl acetals (DMA) obtained during the transmethylation process. Values are presented as the mean and range of the data, the small size of the samples preventing further statistical treatment.
3. Results and discussion Hematocrits, cell counts, size and the lipid composition of erythrocytes are given in Table 1. The hematocrits of the two species studied here do not differ greatly from values reported for a Phocidae of the northern hemisphere (Mirounga angustirostris) (Nelson, 1970), which is the only available literature data. PL and CHOL contents in southern elephant seals were about twice as high as in antarctic fur seals, which could be explained by an about twice higher cell volume. In comparison with human cells (respectively 300 and 125 mg/109 cells for PL and CHOL) (Van Deenen and De Gier, 1974), which are similar in size to that of fur seal cells, the PL and CHOL contents were two to three times lower in elephant seal and fur seal,
41
respectively. In the absence of data on the protein content of erythrocyte plasma membrane in these marine mammals, no explanation could be proposed for these apparent discrepancies. The large size of elephant seal cells is in agreement with previous observations in northern phocid seals and this feature is thought to be linked to an adaptation to circulatory redistribution in these long-diving animals (Wickham et al., 1989). Despite these differences in lipid composition, only slightly smaller CHOL/PL ratios were observed in the species studied as compared to M. angustirostris (ratio of 0.95) (Nelson, 1970) and human cells (ratio of 0.87) (Van Deenen and De Gier, 1974). The distribution of erythrocyte membrane PL is shown in Table 2. The main difference between the two pinnipeds studied is the larger proportion of phosphatidylcholine (PC) in elephant seal (50%) as compared to fur seal cells (38%), which is compensated by a reverse trend for PE and PS. In both species, a low sphingomyelin (SM) content leads to low values of the SM/PC ratio as compared to humans (0.32, 0.49 and 0.93 in M. leonina, A. gazella and man, respectively). These relative SM contents suggest that the marine mammal erythrocytes are far more deformable (less viscous) than human red cells, which is probably a specific adaptation of the circulatory system in long-diving marine mammals (Kooyman, 1989). Previous rheological findings of reduced viscosity and flow resistance in seal blood corroborate this hypothesis (Smith et al., 1979; Wickham et al., 1989; Meiselman et al., 1992). Analysis of the FA composition of total erythrocyte PL (Fig. 1) reveals similar unsaturation levels in elephant and fur seals, despite noticeable
Table 1 Levels of cell counts, total phospholipid and cholesterol in erythrocytes and platelets of elephant seal and fur seala Erythrocytes
Hematocrit Cell counts Mean cell volume Phospholipid Cholesterol Cholesterol/phospholipid (mol/mol)
Platelets
Elephant seal (n= 3)
Fur seal (n = 2)
65.8% (38.9–72.6%) 4.5×106/ml blood (3.5–5.7) 150 mm3 (118–195) 174 mg/109 RBC (130–199) 63 mg/109 RBC (48–77) 0.73 (0.67–0.79)
49.0% (46.3–51.6%) – 6.2×106/ml blood (5.9–6.5) 125 000/ml blood (71 600–199 200) 78.5 mm3 (78–79) 92 mg/109 platelets (73–111) 337.5 mg/109 RBC (33–42) 0.82 (0.75–0.90)
Elephant seal (n = 3)
– 316 mg/109 platlets (269–342) 84 mg/109 platelets (72–91) 0.53 (0.53–0.54)
a Values are given as means two or three samples; in brackets: range. RBC, red blood cells. A technical incident prevented the determination of the amount of total phospholipids and cholesterol/phospholipid ratio in fur seal platelets.
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Table 2 Phospholipid composition of erythrocytes and platelets of elephant seal and fur seala Erythrocytes
Phosphatidylcholine (PC) Phosphatidylethanolamine (PE) Phosphatidylserine (PS) Phosphatidylinositol (PI) Sphingomyelin (SM)
Platelets
Elephant seal (n = 3)
Fur seal (n = 2)
Elephant seal (n =3)
50.0 (47.7–52.9) 22.0 (20.9–22.8) 8.6 (5.2–10.3) 3.3 (2.1–3.9) 16.1 (13.9–18.8)
38.2 (37.2–39.2) 27.7 (27.0–28.4) 12.4 (11.0–13.9) 3.0 (2.8–3.2) 18.7 (18.7–18.8)
33.1 (27.3–39.8) 15.4 (9.9–19.1) 15.9 (8.7–19.2) 10.4 (7.5–12.5) 25.3 (18.0–34.2)
a Values are means of the molar percentage of each phospholipid class; in brackets: range. A technical incident prevented the determination of the phospholipid composition in fur seal platelets.
differences at the levels of 20:4 (n-6) and 22:6 (n-3). There currently exist no other reports of the composition of erythrocyte PL in pinnipeds. As compared to humans (Van Deenen and De Gier, 1974), erythrocyte PL of the two marine mammals were more unsaturated (about 60% unsaturated FA vs. 50% in man). The main differences in composition were the lower linoleic acid content (1–2 vs. 10% in man), the higher arachidonic acid content in elephant seal (17.8 vs. 12.6% in man), and the importance of the (n-3) FA fraction (about 9, 17 and 5% in elephant seals, fur seals and man, respectively). Curiously, while similar amounts of 20:5 (n-3) and 22:6 (n-3) were found in fur seal PL, 20:5 (n-3) was three times more abundant than 22:6 (n-3) in elephant seal PL. These discrepancies, which were also present in plasma PL (unpublished data), could not be explained by differences in dietary habits, since cephalopods and myctophid fishes, the respective preys of elephant and fur seals (Guinet et al., 1996; Cherel et al., 1997), have similar PUFA contents with a predominance of 22:6 (n-3) over 20:5 (n-3) (Culkin and Morris, 1970; Raclot et al., 1998). Unexpectedly, levels of 22:6 (n-3) in the marine mammal erythrocytes lay in the range of values reported in humans, even without (n-3) FA supplementation, whereas levels of 20:5 (n-3) were more than ten times higher. Despite the very high intake of (n-3) fatty acids in the species studied, the presence in their erythrocytes of high 20:4 (n-6) levels together with 22:6 (n-3) levels similar to those found in man raises the question of the functional roles of these particular fatty acids which remain yet unresolved. The FA compositions of the individual PL purified from erythrocytes are given in Table 3. Among all glycerophospholipids, there is a gen-
eral trend towards a higher unsaturation index (unsaturated–saturated FA ratio) in elephant seals as compared to fur seals. SM displays the reverse trend with an unsaturation index three times higher in fur seals than in elephant seals. This balance of unsaturation features between glycero- and sphingolipids explains the small variations observed at the level of polyunsaturated FA (Fig. 1). Specific differences were detected in individual saturated FA contents. Thus, palmitic acid was less abundant in all glycerophospholipids in elephant seal as compared to fur seal erythrocytes, while stearic acid levels were similar among PL except for PS. In accordance with the global FA profiles, all erythrocyte PL contained a smaller proportion of (n-3) FA in elephant seals than in fur seals, the difference corresponding mainly to the difference in 22:6 (n-3) content. Conversely, the proportion of the most abundant (n-6) FA, arachidonic acid, was higher in all PL
Fig. 1. Fatty acid composition of erythrocyte phospholipids.Each bar corresponds to the mean and S.E.M. of three elephant seals and the mean of two fur seals. MUFA, monounsaturated fatty acids, SAT, saturated fatty acids.
PC
14:0 16:0 18:0 20:0 22:0 S. n-11 16:1 n-9 18:1 n-9 20:1 n-9 20:2 n-9 20:3 n-9 22:1 n-9 24:1 n-9 16:1 n-7 18:1 n-7 18:2 n-6 18:3 n-6 20:4 n-6 22:4 n-6 20:5 n-3 22:5 n-3 22:6 n-3 DMA UNSAT/SAT
PE
PS
PI
SM
Elephant seal
Fur seal
Elephant seal
Fur seal
Elephant seal
Fur seal
Elephant seal
Fur seal
Elephant seal
Fur seal
1.1 90.2 20.8 90.2 14.7 90.3 –
1.0 27.9 13.2 – 0.9 0.8 18.4 3.2 – – 0.5 – 1.1 5.7 1.2 0.5 11.3 – 6.4 2.0 4.4 – 1.4
0.7 9 0.2 7.1 9 0.2 16.0 9 0.3 –
1.3 14.1 17.3 – 1.3 0.8 9.9 5.1 – – 0.7 – – 2.4 0.9 0.5 16.7 1.7 9.4 3.6 9.0 3.1 1.9
0.8 90.1 3.6 9 0.3 19.3 91.0 –
1.6 7.6 35.0 – 2.2 1.0 6.5 4.3 – 0.6 1.9 – – 1.3 1.6 0.5 23.6 0.7 7.2 0.8 1.4 – 1.2
2.6 90.5 12.49 0.8 27.892.5 –
4.3 20.0 27.0 – 1.0 0.5 2.3 7.5 2.0 1.3 1.4 0.6 – 0.8 3.2 1.4 1.9 13.6 1.0 3.6 1.9 2.7 – 0.9
5.1 9 0.3 47.6 9 1.1 10.8 9 0.3 3.59 0.2 0.79 0.1 – 1.2 90.1 4.6 90.4 0.8 9 0.1 1.2 9 0.1 – 3.8 90.4 8.99 0.9 – 1.3 9 0.2 2.0 9 0.4 2.1 90.2 2.7 9 0.2 – 0.8 9 0.1 – – – 0.5 9 0.1
1.4 22.5 10.4 5.4 1.7 – 0.9 7.7 0.5 – – 1.3 38.9 – 0.7 0.8 1.9 0.9 0.6 0.5 – 1.7 – 1.4
1.1 90.1 0.6 90.1 17.8 90.3 2.9 90.3 – – 1.290.1 – 1.1 90.1 4.590.2 2.590.5 – 16.0 91.0 – 8.9 90.8 0.990.1 3.2 90.3 – 1.790.1
2.6 9 0.2 0.59 0.1 20.8 9 0.3 7.09 0.9 – – 2.4 9 0.2 – – 3.8 9 0.2 0.8 9 0.1 – 22.1 9 0.5 0.69 0.1 6.2 9 0.4 0.6 9 0.1 1.890.1 4.5 9 1.2 2.79 0.1
3.6 90.3 0.6 90.1 7.0 90.2 5.9 9 0.6 0.9 90.2 – 3.6 90.2 – – 2.5 9 0.2 1.3 9 0.4 – 44.5 91.5 – 4.1 90.2 – – – 3.2 90.2
0.79 0.1 2.19 0.4 7.39 0.7 2.7 9 0.3 1.8 9 0.3 – 4.89 1.4 – 0.5 90.1 4.6 90.8 5.49 1.5 1.09 0.2 20.491.7 – 2.4 90.6 0.5 90.2 – – 1.39 0.9
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Table 3 Fatty acid composition of phospholipids in erythrocytes of elephant seal and fur seala
a Values are means and S.E.M. +-+(mole%) of samples from three elephant seals and means of samples from two fur seals. Faty acidsB0.5% of the total are not shown. PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol; SM, sphingomyelin; S.n-11, sum of 20:1 and 22:1 n-11 fatty acids; DMA, dimethyl acetal derivatives formed from aldehydes; UNSAT/SAT, ratio of unsaturated to saturated fatty acids.
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Fig. 2. Fatty acid composition of platelet phospholipids.Each bar corresponds to the mean and S.E.M. of three elephant seals and to the value from one fur seal. MUFA, monounsaturated fatty acids; SAT, saturated fatty acids.
of elephant seal as compared to fur seal erythrocytes, the greatest difference being observed for PS (44.5 and 23.6%, respectively). PS nevertheless contained in both species about 23% of the total mass of membrane arachidonic acid. Although not accurately documented, the degree of unsaturation of PL fatty acid chains has been related to prothrombin activation in experimental models (Hecht, 1965; Govers-Riemslag et al., 1992). Hence it is possible that the high unsaturation of PS present in the erythrocytes of these two marine mammals and particularly its richness in 20:4 (n-6) play a role in the procoagulant activity of the circulating cells. This hypothesis deserves further investigation in studies of the influence of PL unsaturation on thrombin generation in marine and land mammals. Considering the proportion of dimethyl acetal derivatives (DMA) formed from the PE plasmalogen pool (3.1–4.5%), it was noticeable that in both species, plasmalogens represented only about 6 – 9% of ethanolamine PL, in contrast to about 43% in humans (Adosraku et al., 1994). This discrepancy points to a specific distribution of PE sub-classes in marine mammal erythrocytes and to the need for further exploration of their FA composition and metabolism. Important differences were detected in the length of the fatty acyl chain of SM. Whereas a 16 carbon chain (palmitic acid) was predominant in elephant seals (about 48%), a 24 carbon chain (nervonic acid) was predominant in fur seals (about 40%), as it is in the SM of human erythrocytes (Van Deenen and De Gier, 1974). Such
variations in the composition of SM could result in considerable differences in the membrane bilayer organization and contribute to a higher membrane fluidity of elephant seal as compared to fur seal erythrocytes. This effect would undoubtedly be enhanced by the lower SM/PC ratio and higher unsaturation index of elephant seal cells. There exist many studies of the relationships between the membrane lipids of erythrocytes and their functional properties. Data concerning diving mammals are however still fragmentary. Although the functional roles of erythrocyte PL remain poorly understood, we can nevertheless speculate that the differences in membrane properties between M. leonina and A. gazella may influence their erythrocyte deformability. The specific biochemical features revealed by this study of M. leonina erythrocytes (high unsaturation degree, high arachidonic acid content) may be invaluable considering the well known diving performances of this phocid mammal, which appears to be capable of extremely long dives of up to 2 h duration and 1595 m depth. The maximum dive duration and depth recorded for A. gazella are in comparison 10 min and 181 m, respectively (Butler and Jones, 1997). Interestingly, it has been suggested that an increase in red cell deformability may also limit platelet recruitment from flowing blood by reducing the release from erythrocytes of a potent platelet agonist, adenosine diphosphate (Turitto and Weiss, 1980), thus contributing to limit any thrombosis during diving. Cell counts and total PL and CHOL of elephant seal platelets are shown in Table 1. The elephant seal displays major differences in platelet lipid content as compared to the other phocid seal species studied to date (Ahmed et al., 1989). Thus, levels of PL were lower and CHOL levels higher in elephant seals than in other seals or humans, with a resultant higher CHOL/PL ratio in elephant seals. Table 2 gives the PL composition of elephant seal platelets. In these cells, a high SM content was accompanied by a low PC content, leading to a high SM/PC ratio (0.76), much higher than those reported in other seals (0.5) or in human platelets (0.45) (Ahmed et al., 1989). This would most likely tend to decrease the membrane fluidity. As shown in Fig. 2, elephant seal platelets contained higher levels of saturated FA than fur seal platelets, compensated by lesser
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amounts of monoenes. Similarly, greater amounts of (n-6) FA, predominantly represented by arachidonic acid, but lower levels of (n-3) FA were detected in elephant seal as compared to fur seal cells, despite comparable (n-3) FA levels in their preys. These relationships are similar but amplified in comparison with those observed in erythrocytes of the same species. It is interesting that in both species, 20:5 (n-3) and 22:6 (n-3) were more abundant in erythrocytes than in platelets, as was likewise the case in humans before and after prolonged administration of (n-3) FA (Marangoni et al., 1993). These observations are difficult to explain, since blood cells have different life spans and FA modifications could occur following recent ingestion, but also by prolonged exchange between tissues and circulating cells. Nevertheless, the present results suggest either that the two species had different chronic (n-3) FA ingestion rates, or that the fur seal in question had preyed on animals rich in (n-3) FA more
45
recently than the elephant seals, platelets being a good cellular indicator of late (n-3) FA ingestion (Marangoni et al., 1993). Important differences were detected in the FA profiles of individual PL (Table 4) between the two species of the present study. An analysis of the arachidonic acid content of all glycerophospholipids revealed levels in fur seal platelets about half those in elephant seal platelets, as in the case of erythrocytes. This lower arachidonic acid content of fur seal platelets could infer lower thromboxane production, as was observed in humans after (n-3) FA supplementation (Prisco et al., 1995), with a consequent reduction in the sensitivity to agonists such as collagen, known to be eicosanoid dependent (Siess, 1989) and thus, leading to a potential lowering of thrombosis incidence. As in erythrocytes, the plasmalogen content of PE was in both our species less than half the values reported in other seal species (Ahmed et al., 1989) or in man (Natarajan et al.,
Table 4 Fatty acid composition of gylcero-phospholipids in platelets of elephant seal and fur seala PC
14:0 16:0 18:0 20:0 22:0 S. n-11 16:1 n-9 18:1 n-9 20:1 n-9 20:2 n-9 20:3 n-9 22:1 n-9 16:1 n-7 18:1 n-7 22:1 n-7 18:2 n-6 18:3 n-6 20:3 n-6 20:4 n-6 22:4 n-6 22:5 n-6 20:5 n-3 22:5 n-3 22:6 n-3 DMA UNSAT/SAT
PE
PS
PI
Elephant seal
Fur seal
Elephant seal
Fur seal
Elephant seal
Fur seal
Elephant seal
Fur seal
0.5 90.1 28.0 91.1 12.2 90.2 – – 0.8 90.1 1.4 90.1 16.0 91.1 4.291.5 0.6 90.1 – 1.1 90.2 0.7 90.1 6.9 90.6 – 1.6 90.2 1.190.2 1.190.2 18.9 91.2 – – 2.290.2 – 0.6 90.1 – 1.5 9 0.1
2.7 22.1 13.5 0.5 – 3.0 1.5 19.0 3.8 1.2 – 1.9 0.6 5.5 – 4.0 4.0 0.5 10.7 – – 1.7 1.1 0.9 – 1.6
1.29 0.6 5.89 0.7 16.0 9 0.1 – – – 1.39 0.4 9.09 0.7 2.69 0.3 1.69 0.8 – 1.39 0.1 0.69 0.2 3.09 0.4 0.69 0.1 0.79 0.1 1.79 0.2 0.59 0.2 35.79 5.1 – – 4.29 1.2 2.09 0.4 1.09 0.3 9.39 2.1 2.89 0.6
7.0 20.4 11.2 – – 1.4 5.1 10.2 0.8 3.7 – 1.0 – 5.1 – 2.3 0.9 – 16.1 0.6 – 1.8 1.0 1.0 9.6 1.2
1.1 90.4 4.7 90.8 41.2 96.8 – – 1.7 9 0.4 0.8 90.3 10.7 92.4 3.4 91.0 1.3 90.5 – 2.4 90.7 – 1.7 90.2 – – 2.0 9 0.2 0.59 0.2 23.4 95.1 – 0.5 90.1 1.1 90.5 0.6 9 0.3 – – 1.3 90.4
6.4 9.8 26.5 – – 2.7 0.9 17.1 4.2 3.8 0.8 2.9 – 3.4 – 1.8 4.0
1.7 9 0.7 5.7 9 0.5 37.0 94.3 – – – 1.7 9 0.2 4.2 9 0.5 1.4 9 0.4 2.5 9 0.9 – 1.4 90.2 0.5 9 0.2 1.5 9 0.1 0.6 90.3 0.5 9 0.2 2.8 9 0.5 0.7 90.6 35.3 9 5.3 0.6 9 0.1 – 0.8 90.2 – – – 1.3 90.3
2.0 10.7 27.5 2.5 0.8 1.3 – 11.1 2.4 0.8 1.1 5.7 0.5 0.9 0.8 3.1 4.1 0.5 15.7 0.8 2.7 1.2 1.4 2.1 – 1.3
10.5 1.1 – 1.0 0.6 – – 1.3
a Data are expressed as means and SEM (mole %) of samples from three elephant seals and one value from a sample from one fur seal. Fatty acids B0.5% of the total are not shown. Legends as in Table 3.
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1982). In view of the high concentrations of arachidonic acid in plasmalogens, our observations emphasize the need to examine the metabolism of these FA in order to assess the functional role of this apparent plasmalogen deficiency. In conclusion, the specific differences in the lipid compositions of the blood cells of these two seal species may reflect their taxonomical position or their natural history, which is not yet well known. It may therefore be of greater interest to conduct further comparative studies in wild animals sampled in their natural environment rather than in captive animals living in artificial conditions. Moreover, the present investigation of the lipid components of the membranes of pinniped erythrocytes and platelets illustrates the need for more research into the functional significance of such observations, in the fields of both hemostasis and circulatory dynamics.
Acknowledgements This work was supported by INSERM and the Institut Franc¸ais pour la Recherche et la Technologie Polaires (Programme 119). It received logistic support from the Terres Australes et Antarctiques Franc¸aises. The authors would like to thank VIRBAC for providing Zoletil® and J. Mulvihill for revising the English of the manuscript.
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