Fatty acid transport in cartilaginous fish: absence of albumin and possible

utilization of lipoproteins

V. J. Metcalf* and N. J. GemmellSchool of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand;*Author for correspondence: (Phone: +64-3-3642987 x 4848; Fax: +64-3-3642590; E-mail: victoria.metcalf@canterbury.ac.nz)

Accepted 11 November 2005

Key words: albumin, apolipoprotein, cartilaginous fish, Chondrichthyes, elasmobranch, fatty acid trans-port, high-density lipoprotein, lipid, low-density lipoprotein, very low-density lipoprotein

Abstract

There are conflicting reports over whether albumin is present in the Chondrichthyes. In addition, it isunknown whether given their low levels of extrahepatic b-oxidation, there is actually a need for a plasmaFFA transport system. We examined the plasma of several Chondrichthyan species, including represen-tatives of both the Holocephali and the Neoselachii using a combination of agarose gel electrophoresis,[14C]palmitic acid binding, SDS-PAGE, and Oil Red O staining following cellulose acetate electrophoresis.None of the eight species examined had a major acidic palmitate-binding protein with the characteristics ofalbumin and they all lacked proteins with the mass of albumin, 66 kDa. Palmitate was bound to specificplasma proteins in all elasmobranch species examined and these palmitate-binding proteins resembledlipoproteins based on their diffuse and variable mobilities and Oil Red O staining. These lipoproteins arepotentially LDL and VLDL based on their migration positions. Albumin appears to be absent fromChondrichthyan plasma and lipoproteins bind LCFA in its absence.

Abbreviations: AFP – alpha-fetoprotein; FAT – fatty acid transport protein; FFA – free fatty acid; HDL –high-density lipoprotein; LCAT – lecithin-cholesterol acyltransferase; LCFA – long chain fatty acid; LDL –low-density lipoprotein; VLDL – very low-density lipoprotein; SDS-PAGE – sodium dodecyl sulphatepolyacrylamide electrophoresis; TAG – triacylglycerol

Introduction

Fish primarily use lipid as a fuel to produce ATP,through the process of b-oxidation (Cowey andSargent 1977; Watanabe 1982). Most fish speciesare generally considered to be both hyperlipidae-mic and hypercholesterolaemic (Babin and Vernier1989), reflecting both the importance of lipid forenergy as well as for buoyancy in many fish spe-cies. They primarily obtain the neutral lipids, tri-acylglycerol (TAG) and wax esters, from their diet,

which is lipid rich, without having to rely heavilyon endogenous lipid biosynthesis.

In all vertebrates including fish, both exoge-nous (diet-derived) and endogenous lipid transportsystems operate (see Spector 1984; Babin andVernier 1989; Tocher 2003 for reviews). In theexogenous system, chylomicron or very low-den-sity lipoprotein (VLDL) particles containing die-tary-derived phosphoglycerides or TAG (Sargentet al. 1976), undergo successive hydrolysis of theTAG in circulation, resulting in a change from

Fish Physiology and Biochemistry (2005) 31: 55–64 � Springer 2006DOI 10.1007/s10695-005-5124-y

VLDL to low-density lipoprotein (LDL) viaintermediate density lipoprotein (IDL). Theresulting free fatty acid (FFA) is taken up by tis-sues for storage. Under conditions of stress such asstarvation, migration or reproduction, endogenouslipid stores are utilised. Many fish species lackadipose tissue and store TAG or wax esters ineither their muscle (Henderson and Tocher 1987)or their liver (Sargent et al. 1976). The main lipidtransport form are FFA, with the predominantFFA found in most vertebrates being long-chainfatty acids (LCFA). By themselves, LCFA arehighly insoluble (Spector and Fletcher 1978;Vorum et al. 1992; Vorum and Brodersen 1994)and in most vertebrates they are typically carriedby plasma albumin to tissues (Richieri et al. 1993)where they are used in b-oxidation.

The role of FFA as a transport form of lipid infish appears to be of varying importance, sug-gested by a range of plasma FFA concentrations(Larsson and Fange 1977). In addition, althoughalbumin is typically the major plasma protein inhigher vertebrates, the role of albumin in LCFAtransport in fish is unresolved. Albumin is presentin salmonids (Byrnes and Gannon 1990, 1992;Gong and Hew 1998; Metcalf et al. 1998a; Metcalfet al. 1998b), but some teleost fish species such asthe Antarctic toothfish Dissostichus mawsoni(Metcalf et al. 1999b), New Zealand eel speciesAnguilla dieffenbachii and Anguilla australisschmidtii, (Metcalf et al. 1999a) and carp (De Smetet al. 1998) lack albumin. In these species the roleof LCFA transport is assumed by high-densitylipoprotein (HDL). It is not known if this is acharacteristic of other fish species, includingnon-teleosts.

The Chondrichthyes or elasmobranchs are aprimitive group of cartilaginous fish that firstappeared over 400 million years ago and are cur-rently represented by some 800 species of sharks andrays (Neoselachii) and chimaeras (Holocephali).There are a number of unusual characteristics ofelasmobranchs, including their ability to osmo-conform, using a combination of urea and methyl-amines as solutes to maintain the extracellular andintracellular osmolarity close to that of the envi-ronment (Browning 1978). This solute system exertswidespread effects on the metabolism of lipids,ketone bodies and amino acids and the structure ofproteins and membranes (Ballantyne 1997).

While it has been claimed that elasmobranchshave little reliance on lipids as a fuel, they actuallydo figure prominently in their metabolism. Asmost elasmobranchs are carnivorous, dietaryenergy sources include both protein and lipid. Inaddition, they rely heavily on lipids, such as waxesters (Nevenzel 1970) and squalene (Nevenzel1989), deposited in large quantities in the liver toprovide static lift, aiding buoyancy, perhapsresulting from their lack of adipose tissue (Zammitand Newsholme 1979). Where they differ mostfrom teleost fish is in their substrate preferencesfor lipid catabolism of certain tissues. The abilityof elasmobranchs to undergo b-oxidation of eitherfatty acids or fatty alcohols is largely confined tothe liver and to a lesser extent, the kidney (Zammitand Newsholme 1979; Ballantyne and Moon 1986;Moyes et al. 1990; Richards et al. 2003). The high-urea concentrations decrease extrahepatic lipidcatabolism, presumably by affecting FFA trans-port. Extrahepatic tissues use other substratesinstead, usually ketone bodies, which themselvescan be oxidised in the mitochondria to provideenergy (Ballantyne 1997; Watson and Dickson2001; Richards et al. 2003). The hepatic lipids arestill important for this process, as the conversionof fatty acids in the liver to ketone bodies throughb-oxidation and ketogenesis provides the fuel thatcan then be distributed to other tissues for ketonebody oxidation (Watson and Dickson 2001). Ke-tone bodies are readily soluble and thus, a carrierprotein is not needed.

There has been considerable doubt over thepresence of the major FFA transporter, albumin,in Chondrichthyan fish. Some studies have foundno evidence for albumin in elasmobranchs (Irisawaand Irisawa 1954; Sulya et al. 1960; Bueker 1961;Gunter et al. 1961; Sulya et al. 1961; Fellows andHird 1981; Peters and Davidson 1991), whileothers have suggested that albumin is presentbased on bromocresol green binding (Fellows et al.1980), albumin/globulin ratios and starch gelelectrophoresis (Yanagisawa and Hashimoto1984a), automated spectrophotometry (Harmset al. 2002), or protein profiles (Rasmussen andRasmussen 1967; Yanagisawa and Hashimoto1984a; Harms et al. 2002). Albumin has beenreported in the blood of freshwater stingrays ofSouth America (Griffith et al. 1973). The fetalequivalent of albumin, alpha fetoprotein (AFP)

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has been described as present in fetal shark serumin ten shark species (Gitlin et al. 1973).

However, many of these studies were per-formed several decades ago using what are nowconsidered to be crude methodologies such asstarch gel and paper electrophoresis. In this studywe examined whether FFA binding, specificallyLCFA binding, occurs in the plasma of severalChondrichthyan species. We wished (1) to deter-mine whether albumin was present in any elas-mobranchs and (2) whether it was the FFAbinding protein in these species, or whether likesome teleost species, HDL takes over this role.Palmitate was bound to plasma proteins in allelasmobranch species examined, but thesepalmitate-binding proteins were not albumin andappeared to be lipoproteins (LDL- and VLDL-type). Albumin appears to be absent fromChondrichthyan plasma.

Materials and methods

Sample collection

Blood was collected into 5-ml tubes containing9 mg EDTA from the caudal vein of species rep-resentative of the Chondrichthyes. The speciesused in this study were: (1) Holocephali (Chimaera)– (a) elephant fish (Callorhinchus milii), n=5 (4female; 1 male); (2) Neoselachii (Skate, sharks,rays) – (a) carpet shark (Cephaloscyllium isabelli-um), n=2; (b) school shark (Galeorhinus galeus),n=3; (c) seven-gill shark (Notorynchus cepedi-anus), n=1; (d) dogfish (Squalus acanthias), n=1;(e) rig or spotted estuary smoothhound (Musteluslenticulatus), n=2; (f) rough skate (Dipturus nas-uta), n=2; and (g) smooth skate (Dipturus inno-minatus), n=1. Samples were then kept at 4 �Cprior to their delivery to the laboratory (withintwo days). Blood tubes were centrifuged and theplasma (supernatant) removed. Plasma sampleswere divided into aliquots, with the majoritystored at )80 �C and the remainder at 4 �C. Allexperiments were run on fresh samples stored at4 �C within 1 week of collection.

Electrophoretic analysis

Agarose gel electrophoresis was performed in 1%agarose in 38 mM Tris, 46 mM Na-Barbitone,

16 mM diethylbarbituric acid (pH 8.6) for 40 min(Brennan et al. 1988). Binding of [14C]palmitic acidand autoradiography was carried out at roomtemperature as previously described (Metcalf et al.1998a).

Reducing sodium dodecyl sulphate-polyacryl-amide electrophoresis (SDS-PAGE) at pH 8.8 wasperformed in 7.5% or 12% polyacrylamide gelswith a 3% stacking gel (Laemmli 1970). Sampleswere incubated in SDS reducing buffer; 4% SDS,10% 2-mercaptoethanol, 125 mM Tris, 40%glycerol, 0.002% bromophenol blue (BPB) at100�C for 5 min before electrophoresis. Gels werestained with 0.1% Coomassie Brilliant Blue R.

Cellulose acetate electrophoresis was per-formed on plasma samples (Lehmann andHuntsman 1966; Metcalf et al. 1999a, 1999b) inthe same buffer as that used for agarose gel elec-trophoresis. Plates were stained in: (a) amido blackin 10% acetic acid in 50% methanol for 10 min,followed by destaining in 10% acetic acid in 50%methanol; or (b) 30 ml 1.4% (w/v) Oil Red O inmethanol/7 ml 1 M NaOH for 2 h, followed bygently rinsing and wiping using distilled water. OilRed O stained plates were stored in glycerol atroom temperature.

Results

Agarose gel electrophoresis plasma protein profile

Plasma samples from a total of eight Chondri-chthyan species, including representatives of theHolocephali (Chimaeras) and the Neoselachii(Order Orectolobiformes, Hexanchiformes,Carcharhiniformes, Rajiformes, Squaliformes),were examined for the mode of LCFA transport(n=17). On agarose gel electrophoresis (pH 8.6),albumin is usually the predominant anionicplasma band as is the case with human plasma(Figure 1a–c, lane 1) and other albumin-contain-ing vertebrate plasmas, while lipoproteins such asHDL ( a lipoprotein), LDL ( b) and VLDL (pre-b)generally appear as broad diffuse bands, often withvariable mobility.

The five elephant fish samples had similarplasma profiles, with notably few plasma proteinsand in particular, a paucity of acidic proteins(Figure 1a, lanes 2–6). There were no sex-relateddifferences (cf. Figure 1a lanes 1–5 (female) with

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6 (male)). All elephant fish lacked a band witheither the mobility or shape characteristic ofalbumin. While there was a protein band atapproximately the same position as human albu-min, this was extremely faint and diffuse withvariable mobility, characteristic of an a-lipopro-tein, that is, HDL. Considerable quantities ofprotein were observed at both the b and pre-bpositions, indicating that elephant fish potentiallyhave both LDL and VLDL-like proteins, respec-tively. All the elephant fish examined had largeamounts of basic proteins in their plasma.

Likewise, other Chondrichthyan species lackedacidic proteins resembling albumin in theirplasma. The seven-gill shark (Figure 1b, lane 2)had a predominant plasma protein present with amobility less than that of human albumin( a-region). This species had little in the way of pre-b (VLDL-like) proteins but did have some b(LDL-like) material present. The school shark hada diffuse protein present with approximately thesame mobility as human albumin, its band mor-phology and position suggesting it was HDL(Figure 1b, lane 3). There was significant protein

Figure 1. Agarose gel electrophoresis of (a–c) Coomassie blue stain of plasma samples and (d–f) [14C]palmitic acid autoradiograph. (a)and (d) Lanes: 1, human; 2–5, individual female elephant fish; 6, male elephant fish. (b) and (e) Lanes: 1, human; 2, seven-gill shark; 3,school shark; 4, dogfish; 5, elephant fish. (c) and (f) Lanes: 1, 11, human; 2–3, individual rig; 4–5, individual school shark; 6–7,individual carpet shark; 8–9 individual rough skate; 10, smooth skate. All human plasma loadings were 0:3�l plasma, all fish plasmaloadings were 2�l. +, anode, – cathode. The human lipoproteins are not visible at the loading we have used and so their approximatemigration positions are indicated.

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present in the b and pre-b region. The dogfishlacked an acidic albumin-like protein but did havelow amounts of a protein resembling HDL inshape and mobility (Figure 1b, lane 4). However,the majority of plasma protein in this cartilaginousspecies was less acidic, with high concentrations ofprotein present in the b and pre-b region.

Both rig plasma samples lacked protein with themobility of human albumin, but had a very faint a-mobility protein (Figure 1c, lanes 2–3). The rigplasma protein profile was in general very diffuseand both b and pre-b region proteins were present.Two additional school shark samples had differentplasma profiles (Figure 1c, lanes 4–5) from theoriginal sample (Figure 1b, lane 3) in their lack of apredominant acidic band. This could potentially bedue to degradation of these plasma samples prior

to receipt or reflects collection of animals from aseparate area. The dominant b-protein was presentin both additional samples. The two carpet sharksamples had similar plasma profiles to each other(Figure 1c, lanes 6–7), with an extremely faint,highly acidic plasma protein as well as a diffuseprotein with mobility slightly less than that ofhuman albumin. Diffuse bands corresponding toproteins in the b and pre-b region were also presentat moderate concentrations. The two rough skatesamples had highly similar plasma profiles, lackinga protein similar to albumin but with a diffuseprotein band present with the same mobility ashuman albumin and extremely high concentrationsof b and pre-b (LDL and VLDL-type) proteinspresent (Figure 1c, lanes 8–9). The smooth skatehad low concentrations of plasma proteins in

Figure 2. Reducing 7.5% (a) (c) (e) and 12% (b) (d) (f) SDS-PAGE of plasma samples. (a) and (b) Lanes: 1, BioRad molecular massmarkers; 2, human; 3–6, individual female elephant fish; 7, male elephant fish. (c) and (d) Lanes: 1, BioRad molecular mass markers; 2and 7 ((c) only), human; 3, seven-gill shark; 4, school shark; 5, dogfish; 6, elephant fish. (e) and (f) Lanes: 1, BioRad molecular massmarkers; 2–3, individual rig; 4–5, individual school shark; 6–7, individual carpet shark; 8–9, individual rough skate; 10, smooth skate.All human plasma loadings are 0:1�l, all fish plasma loadings are 0:3�l. Protein marker sizes are indicated down the side.

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general with low amounts of an a-protein (HDL-like) band with the same mobility as humanalbumin as well as higher amounts of a pre-bprotein (VLDL-like) and some b material present(Figure 1c, lane 10).

Plasma protein long chain fatty acid binding

The ability of a protein to bind palmitate has beenused as a principal means of identifying albumin(Peters and Davidson 1991). The palmitate bind-ing capacity of the plasma of all the cartilaginousspecies studied was examined. None of the specieshad a protein present based on Coomassie stainingwith the characteristics of albumin (Figure 1a–c)and this result was upheld with the inclusion of[14C]palmitic acid in each plasma sample prior toelectrophoresis (Figure 1d–f). The protein whichmigrated in the a-region in elephant fish did notbind palmitate (Figure 1d, lanes 2–6). All thepalmitate binding in this species was to less acidicproteins, with binding to both the b and pre-bbands, although there was some individual vari-ability in binding. The binding pattern resembleddiffuse but specific binding to lipoproteins (LDL-and VLDL-like). Likewise, in all the other Chon-drichthyans examined, the band in the a-region didnot bind palmitate (Figure 1e, lanes 2–5 and 1f,lanes 2–10). Specific palmitate binding occurred toboth b and pre-b-lipoprotein-like bands; in otherwords LDL- and VLDL-type lipoproteins(Figure 1e and 1f).

Absence of albumin

Reducing SDS-PAGE was also used to analyseChondrichthyan plasma. In the five elephant fishsamples, there was no plasma protein present withthe molecular mass of albumin, 66 kDa(Figure 2a, lanes 3–7). All the elephant fishexamined had the same plasma profile, irrespectiveof gender. Major plasma proteins were present atapproximately 80 kDa and greater than 200 kDa.These may correspond to an 80 kDa apolipopro-tein unique to fish (Chapman et al. 1978; Babin1987a, b; Santulli et al. 1996; Metcalf et al. 1999b)and an apoB protein (Mills et al. 1977; Chapman1980). Analysis of the samples on a 12% gelshowed small amounts of a doublet protein bandat 28 kDa and a doublet band at approximately 14and 13 kDa (Figure 2b, lanes 3–7), and these may

be apoAI and 14 kDa fish apolipoproteinisoforms.

The protein profile of the other chondrichthyanspecies was highly similar to elephant fish (Fig-ure 2c–f). All had a major protein band atapproximately 80 kDa. There was no proteinpresent with a mass of albumin. All had significantmaterial of mass greater than 200 kDa. All hadprotein bands at approximately 28–31 kDa and adoublet band in the case of seven-gill shark (Fig-ure 2d, lane 3), dogfish (Figure 2d, lane 5), and rig(Figure 2f, lanes 2–3). Most had a large amount ofa protein present at approximately 12–14 kDa,except for seven-gill shark (Figure 2d, lane 3) witha doublet band present in both dogfish (Figure 2d,lane 5) and rig (Figure 2f, lanes 2–3). Carpet sharkhad much smaller amounts of this 14 kDa proteinpresent (Figure 2f, lanes 6–7). There were otherproteins present at 42–48 kDa in most species,except for skate (Figure 2e and 2f, lanes 8–10).

Lipid binding in the blood of cartilaginous fish

To ascertain whether the palmitate binding pro-teins in the Chondrichthyes species were lipopro-teins, cellulose acetate electrophoresis wasperformed followed by either amido black or OilRed O staining (data not shown). In humanplasma, binding of Oil Red O occurred both to a(HDL) and b (LDL) and pre-b (VLDL) lipopro-teins. In the Chondrichthyes, faint Oil Red Ostaining only occurred in the pre-b to b-region andnot in the a-region This showed that there wereLDL- and VLDL-type lipoproteins present in theChondrichthyes, but the a-region protein was notable to be identified.

Discussion

Traditionally albumin has been defined simply asthe major acidic plasma protein. This has lead toerroneous assignations of plasma proteins asalbumin; for example, carp ‘albumin’ (Nakagawaet al. 1976; Nakagawa and Kayama 1978;Yanagisawa and Hashimoto 1984b), was subse-quently shown to be HDL even though it bindsLCFA (De Smet et al. 1998). In the present studywe examined the plasma of eight Chondrichthyanspecies. None of the 17 individuals from the eight

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species that we examined appeared to have aplasma protein that resembled albumin based onelectrophoretic mobility and band morphology(Figure 1a–c), palmitate binding (Figure 1d–f) andmolecular mass (Figure 2). Thus, we conclude thatmarine elasmobranchs lack albumin.

There has been continued controversy overwhether elasmobranchs possess albumin. A surveyof vertebrate species using similar techniques tothe present showed that dogfish lacked a proteinwith the migration, palmitate binding and masscharacteristic of albumin (Peters and Davidson1991). In another study, paper electrophoresis andlack of BPB binding also indicated lack of albuminin a shark and skate species (Irisawa and Irisawa1954). The results of other studies indicating a lackof albumin have not been so conclusive (Sulyaet al. 1960; Bueker 1961; Gunter et al. 1961; Sulyaet al. 1961; Fellows and Hird 1981). The presentstudy is the most thorough study to date, both inthe methods used and the number of species used.

Other studies have claimed albumin is presentin elasmobranch plasma. One such study looked atalbumin/globulin ratios (Yanagisawa andHashimoto 1984a); another claimed trace amountsof albumin (Rasmussen and Rasmussen 1967); anda further early study stated that albumin is presentin some freshwater elasmobranchs (Griffith et al.1973). A recent study claimed that albumin waspresent in the bonnethead shark at 4 g/l (Harmset al. 2002), but their use of an automated clinicalspectrophotometer, designed for human samples,left the authors conceding that the values fellbelow the reliable limit of detection. A study ofbromocresol green binding found less than 1%serum albumin in seven species of elasmobranchs,but none of these bromocresol green-bindingproteins bound FFA (Fellows and Hird 1981). Webelieve all these studies have resulted in erroneousdetection of albumin in elasmobranchs, with thepossible exception of the freshwater stingrays.

Analbuminaemia seems to be a common statein fish, even those of different groups, such as somedistantly related teleosts (De Smet et al. 1998;Metcalf et al. 1999a, 1999b) and most elasmo-branchs. Loss of albumin expression is not aprimitive characteristic as the agnathan lampreypossesses albumin (Kuyas et al. 1983; Gray andDoolittle 1992; Filosa et al. 1998; Danis et al.2000). It has been hypothesised that loss of albu-min expression has occurred at least two times in

evolution, in the evolution of the teleost lineage(Metcalf et al. 1999b), and the present paper sug-gests an additional loss at least once in the elas-mobranch lineage. The lack of albumin expressionin elasmobranch plasma may be the result of genedeletion, mutational inactivation, or permanentdownregulation.

All vertebrate livers examined use b-oxidationof fatty acids as their primary energy source to fuelaerobic metabolism and with lipid generally storedextrahepatically, a lipid transport system isrequired to transport fatty acids back to the liverand other tissues for b-oxidation. Serum albuminmay have evolved to fill this role. Elasmobranchsare unusual in that they mainly utilise their liverfor lipid storage and as the major site for b-oxi-dation and thus, a fatty acid transport protein maynot be required, providing at least one reason whya lack of albumin in the plasma is tolerated.However, our results demonstrating specificin vitro binding of palmitate to plasma proteinssuggest that plasma LCFA transport may stilloccur in vivo. This may not be unexpected as a verylimited amount of muscle b-oxidation does stillappear to occur (Watson and Dickson 2001),necessitating fatty acids and at least some plasmafatty acid transport. It also may be that, if LCFAtransport does occur in vivo, that LCFA arerequired not just for energy metabolism, but forother roles such as signalling molecules.

There may be other reasons for the loss ofalbumin in elasmobranchs related to their coun-teracting solute strategy (Ballantyne 1997). Ureamay affect albumin formation or albumin bindingfatty acids in a way that is not counteracted bytrimethylamine oxide (Ballantyne and Moon 1986;Ballantyne 1997). Additional solutes, such asalbumin with bound FFA, may not be tolerated inhigh enough concentrations to be transported insufficient quantities to meet the energetic needs ofthe extrahepatic tissues (Watson and Dickson2001). The advantage to elasmobranchs insteadprimarily utilising ketones extrahepatically is thatthey are more soluble, do not require a transportprotein (Watson and Dickson 2001) and provideequivalent ATP energy (Voet and Voet 1995).

Although palmitate binding has often beenused as a means of identifying albumin in plasma,it is not a property which is restricted to albuminas we have shown in the present study (Figure1d–f) and elsewhere (Metcalf et al. 1999a, 1999b).

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In the previously published examples (De Smet etal. 1998; Metcalf et al. 1999a, 1999b), HDL bindsLCFA in some fish species in the absence ofalbumin. In the present study in contrast to aprevious study (Fellows and Hird 1981), whichclaimed that elasmobranchs are unique in thatthey do not possess serum fatty acid binding pro-teins, we observed strong and specific palmitatebinding to plasma proteins in all eight species andall the individuals that we examined (Figure 1d–f).

The marine Chondrichthyes, however, appearto use a distinct system from that of analbumi-naemic teleosts. Although some species appearedto have a protein that resembled HDL (diffuseacidic band of variable mobility present in the a-lipoprotein region) at very low to low concentra-tions (elephant fish, dogfish, rig, carpet shark,smooth skate), through to moderate to high con-centrations (seven-gill shark, school shark, roughskate), this protein did not bind LCFA(Figure 1d–f), nor did it bind Oil Red O.

For all the species, palmitate binding was toproteins in both the pre-b and b region. Theseproteins are lipoproteins (likely to be VLDL andLDL), indicated by their electrophoretic position,diffuse nature and variable mobility as well as OilRed O staining. These lipoproteins were generallyfound at high concentrations in elasmobranchplasma and represent major plasma proteins, inline with other studies showing high LDL andVLDL levels (Lauter et al. 1968; Mills et al. 1977;Chapman 1980; Babin and Vernier 1989). Theformer study also showed that 43% of FFA indogfish plasma are associated with the lipoproteinfraction, also supporting our results. Palmitic acidhas shown to be the second most abundant fattyacid of shark lipoprotein (Mills et al. 1977) andpalmitic acid accounted for 40% of all FFA in theplasma of all the elasmobranchs examined inanother study (Ballantyne et al. 1993), indicatingits choice as a LCFA for this study was appro-priate. The binding was specific binding to lipo-proteins as free [14C]palmitic acid migratestowards the anode in a very diffuse erratic manner(Metcalf et al. 1999b), unlike the binding observedhere. In addition, binding to distinct, clearlydefined ‘bands’ could be seen in many of the spe-cies, indicating that the binding was specific, ratherthan non-specific binding to a range of proteins inthe absence of a specific carrier. Thus, the Chon-

drichthyes appear to utilise non-HDL lipoproteinsto bind and potentially transport LCFA.

When plasma samples were analysed by SDS-PAGE on 7.5% gels, all Chondrichthyan specieswere found to possess similar plasma profiles(Figure 2a, c, e). All had a major, approximately80 kDa protein present. We observed significantamounts of protein of mass greater than 200 kDain all species, which may be apoB. Sharks, likeother fish species have been shown to possess anapoB-type apolipoprotein (Mills et al. 1977;Chapman 1980). On separation on 12% gels(Figure 2b, d, f) all species had small amounts ofmaterial at 28 kDa, possibly apoAI (Chapmanet al. 1978; Babin 1987a, b; Ando and Matsuzaki1996; Santulli et al. 1996). All the elasmobranchspecies, except for seven-gill shark, had moderateto high concentrations of an approximately14 kDa protein, which may correspond to a14 kDa apolipoprotein unique to fish (Kondoet al. 2001, 2005).

Marine elasmobranchs show no evidence foralbumin in their plasma and it is concluded thatthey have lost albumin expression, relating per-haps partly to their use of liver for lipid storage,their low levels of extrahepatic b-oxidation and totheir unusual solute system. In contrast to otheranalbuminaemic fish species and analogous toanalbuminaemic humans, lipoproteins that are notof the HDL class, are capable of LCFA binding inthese fish species. Future work should determinewhether the albumin gene can be identified in theChondrichthyes. In addition, detailed analysis ofthe lipoprotein and apolipoprotein components ofelasmobranch species is desirable.

Acknowledgements

We are grateful to Dave Tattle and Warrick Lyon,School of Biological Sciences, University ofCanterbury for the collection of blood samples; toProfessor Steve Brennan, Molecular PathologyLaboratory, Christchurch School of Medicine,University of Otago for allowing us the use of hisfacilities as well as his expertise; to the ProteinChemistry Laboratory, Canterbury Health Labo-ratories for performing the agarose gel electropho-resis experiments; and to the anonymous reviewerwho suggested improvements to the manuscript.

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