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Serum lipoproteins of the pacific sardine (Sardinops caerulea girard)
272
Sardine serum lipoproteins were separated into ultracmtrifugal density classw, and data on the lipid compositiow of these classes and the fatty acid distributions of cholesteryl esters, triglycerides, and pllospholipids are prescuted. The lipids present in the three densitv classes with llighest percentages were: cllolesteryl esters (ho’j,,) iu tlw \w-!. low densit)., phospholipids (35 YL) and triglycerides (30’)~~~)in the low dcnsit!., and plwspllolipids (jg’:{,) in the high densitv fractions. The fatti. acid conposition of the triglycerides from low aud high density fractions werr Get-!- similar, whereas marked differences \verr seen between the fatty acids of clwlester!-1 esters and pllospl~olipids for these two lipolmhh classes. Xgarose gel electroplmxGs of the sardine serum indicated the presence of three distinct bands with R-like mobility, but pre-,$ or p bands were not detected in whole serum or in any- densit!- fraction. The absciicc of lipoproteins with prc-p or /J niobilit~~ iu sardines suggests major differwcc5 h?tNwn fat translmrt iii manlrna~ian aud fish systems.
~1 great deal of information is available ou the properties of serum lipoproteins of various ~nanmmlsl~ IS, but JIO detailed study of MI serum lipoproteins has been I-Cported. ln this paper, we report on some of tlre’properties of sardine serum lipoproteins. Data on tlw lipid compositions of tllc ultracentrifugal classes’” and the fatt!. acid dstributions of cholestcryl esters, triglh-cerides, and I’llosl)lloliI’ids are presented. .\~arose gel rlectroplwresis was carried out on whle scfuni and tlir isolated ultraccntrifugal fractions. Our mvn interest iu sardiws resulted from earlier studies of the was esters of copepodsl”, a major source of food for the Pacific sardines Ii. l‘lle physical and chctnical properties trf tlw m-urn lipoprotein classes of these fish were charac~tctrizcd in order to
513RrM
LIPOPROTEINS
OF THE PACIFIC
learn more about the lipid metabolism
SARDINE
273
of an animal which ingests wax esters, and to
test the possibility that such a diet might result in the presence of long-chain or wax esters in sardine blood.
alcohols
METHODS
Live sardines were obtained from a bait barge in San Diego Bay within z days of capture from the open ocean. Blood (approximately 3 ml) was drawn from the dorsal aorta with a ro-ml syringe which had been rinsed with a I(),; heparin solution. The blood from the sardines clotted immediately and was centrifuged in an International Clinical Centrifuge at approximately IOOO xg for j min. Because the amount of heparin used was insufficient to prevent immediate clotting of the blood, it is unlikel!~ that tire concentration of the anti-coagulant was sufficient to produce any significant effect on the lipoproteins. Part of the total serum (40 ml) obtained from IOO sardines was used for total lipid studies while the remaining serum was placed in ice and shipped immediately to the Donner Laboratory at the ITniversitv of California for preparative ultracentrifugation of the serum lipoproteins using the techniques described b!F ITreeman cat~1.‘“. Electrophoresis of the different lipoprotein densit\. classes was done on agarose gels obtained from Bio-Kad Laboratories at pH 8.6 according to the procedure of Nobel ~‘t al.lQ. Lipids were separated by silicic acid columns into five fractions: hydrocarbons, cholesterol esters, triglycerides, polar lipids (free fatty acids, cholesterol, and digl!cerides), and phospholipids. Florisil columns were used to separate the polar lipid fraction into free fatty acids and cholesterol”‘. The purity of each fraction was tested by thin-layer chromatography, and where neccessary rechromatography on silicic acid columns was performed. -After weighing the different fractions the cholcsterol ester, triglyceride, and phospholipid fractions were transmethylated using 5()/o sulfuric acid in methanolzl. ITor gas-liquid chromatograplric analysis we used a Loenco Model 70 Hi-Flex apparatus (Loenco Inc., Mountain 1’iew, Calif.) fitted with a flame ionization detector and operated isothermally with 2.1 kg,‘cm” nitrogen carrier gas pressure. The two columns used were a 2.4 mx3.2 mm (outside diameter) column of 10”; diethylene glycol succinate polyester (DEGS) and a IS m x 2.3 mm column of 371, ()\‘-I on 6040 mesh GAS-Chrom P (all supplied by Applied Science Laboratories, Inc., State College, Pa.). Several different temperatures were used, depending on the compounds being
Ii. 1;. LEE,
27-l
11. L. P~PPIONE
analyzed. Hydrocarbons were analyzed at 150-170 “C on O\‘-I and DEGS; methyl esters of fatty acids at 16~1qo “C on DEGS and at 17+1qo “C on 01:-r. Triangulation was used to quantitate the results, which were normalized to total IOO”,,. Identities of fatty acids were determined from their retention times and a plot of total numlxr of carbons versus log retention times of howl saturated and unsaturated fatt!* acids. Hydrogenation of the fatty acid mixtures was carried out using platinum oxide to aid in the identification
of fattv
The concentrations
acids.
and chemical
composition
of the lipid moieties of tile three
classes ultracentrifugally isolated from sardine serum are given in Table I. The serum concentrations of lipids in each class were : very low density lipoproteins, 105 mg,“~oo 121 mg/loo ml; and high density lipoproteins, 560 ml; low density lipoproteins, mg/roo ml. The predominant lipids of the different classes were: cholester!4 esters (60(;4) in the total very low densit!, lipoprotein class, triglycerides (31 If/,)and phospl~olipid (35 YJ,) in the low den&v class and phospholipid (55’!‘“) in tire higIl tltwsity class.
0.
I
1 ..i
.3.7 2 ,j 2O.i
o., ‘I’ ‘1.
0.1 .
1
0.2 t i
‘7.1
SERUM
LIPOPROTEINS
Unesterfied
OF THE PACIFIC
cholesterol
SARDIIiE
was approximately
275
13”/~ of the lipid in low and high density
classes and HO{,in the very low density class. Separation of the polar lipid fraction by Vlorisil column chromatography indicated that the major part of this fraction was cholesterol while less than 15% was free fatty acid. Thin-layer chromatography of the polar lipid fraction showed only traces of mono- and diglycerides. Thin-layer chromatography of the phospholipid fraction showed that phosphatidyl choline was the major phospholipid with lesser amounts of phosphatidyl ethanolamine, lysolecithin, and sphingomyelin. No long chain alcohols or wax esters were found in any lipoprotein fraction. .I separate analysis on 40 ml of serum showed that total sardine serum lipid concentration was 790 mg/roo ml, which can be compared with serum lipid concentrations in ocean salnion1Q and herring (Lee, unpublished results) of zooo mg/roo ml and 813 mg/Ioo ml, respectively. The major lipids of the sardine serum (Table I) were phospholipid (do”/o), triglycerides (289;), and cholesteryl esters (22%). Patton ct al.>’ observed a similar lipid composition for the total blood serum of ocean salmon. Table II shows the fatty acid distributions of cholesteryl esters, triglycerides, and phospholipids of the low and high density classes. The principal fatty acids of cholesteryl esters were: zz:6 (52%), 20:s (zoo/o) and 16:o (16’j/,) in the low densit! class and zz:O (359/,), 16:o (23%), zo:5 (16~~6) and IS: I (12(j<,) in the high densit! class. In the phospholipid fraction, relatively higher amounts of 22: 6 and 20:5 were present in high density lipoproteins (370d1,and 170/;, respectively) than in low densit! lipoproteins (267; and IZ%, respectively). The percentage of 18: I was essentially the same in the phospholipids of both ultracentrifugal classes. Marked differences between the phospholipids of low and high density classes were noted in the amounts of 16: o (I’% in low and zq~/o in high den&v lipoproteins) and 16: I (35’j& in low and ;cy/, in high density lipoproteins). In the triglyceride fraction, the fatty acid distributions of the low and high density classes were very similar.
The electrophoretic distribution of sardine lipoproteins, along with a normal human distribution included for comparative purposes, is shown in Fig. I. Three bands with a-like mobility were noted in sardine serum, but the fi and pre-/? bands, present in normal human serum, were not detected in sardine serum. The three bands of sardine lipoproteins are referred to as z-like because they run ahead of human /3-lipoproteins. Electrophoretic distributions of the ultracentrifugal fractions indicated that a-like lipoproteins with the slowest mobility were present in the low density class and AGAROSE GEL ELECTROPHORESIS
0
Human
Fig.
I.
_
Sample Wet I
Sardine
.A~aroscgel elcctrophoretic
distnhution
of human
ant1 sardine
NioclriwL.
Blophys.
lipoproteins. Acta,
270 (1972)
272-278
I<. F.
276
LEE,
I). I.. PI-PPIOSli
those with the faster mobilities were present in the high density class. The total veq low density lipoproteins remained at the origin and their stainability was very poor. Similar results were obtained with paper electrophoresis.
Our studies of sardine serum lipoproteins
demonstrated
some marked differences
between the fat carriers of these fish and those of mammals. The percentages of triglycerides (30);) and phospholipids (I :io)in the total \‘er!: low density class are much less than reported values in mammalian studies1”-15,““s24. \‘cry low den+lipoproteins containing a high percentage of cholesteryl esters, as we lbave noted in the sardine, have only been observed in cholesterol-feel rabbitsZ5. The absence of pre-e and ~-lipoproteins in the electroplloretic clistrihutions of whole serum aud the ultracentrifugally isolated fractions of the sardine suggest furtller differences between these fish and mammals. The concentration of lipids in the total verv low densit), class (IOO mg/~oo ml) is sufficiently high to gi1.e rise to an exyrected pre:@ band instead of the poorly staining material at the origin. It is unlikely that the size of these lipoproteins was a factor because the serum was not turbid. Probabl\the negative
charge of the protein moiety \vas insufh
ieut to Jlermit these lipoproteins
to leave the starting well. Three distinct bands with a-like mobilit>,, observed in sardine seru.m, J~ave also been observed in salmon and trout (Puppjone, unpublished results). The low densit! lipoproteins of the sardines wercx found to have an electrophoretic mobilit!. which corresponded to the slowest migrating alphas. The presence of a-lipoproteins in the low densit?, class has also been observed in certain maIlimals”,‘“,13,~~,~7. The fatty acids among tllc different lipid: probabl!, are of dietar!. origin. The triglyceride fatt!r acids of thr sardine low and high density classes match very closely the was ester fatt!, acids of tile copepod, C‘al~us /zrl,nolr~~zr~icu~~~“, wllicII is probably in the diet of the Pacific sa.rclinel’. \\‘as esters are a major lipid of Calnnus localized in a large oil sac near the coJ)epocl gut. To what extent dietar). fatty. metabolized or modified in sardines can, of course, onlv he inferred from direct evidence. TJle lack of long-chain alcolrols and was esters in sardines
and are acids are sucll insuggests
that the was esters of the coJ>eJ)ods are h~drol~~zed, followed by oxidation of the acollol to the corresponding fatt>- acid. Kecent work h_ 1.1. Baker, J. C. Kevcnzel and K. 1’. Lee (unpublished results) sl~owed a verb- active ~vax ester lipasc in the p>-loric caecum of sardines and anchovies, but low wax ester lipasc activit!. in the liver, wllite muscle, and red muscle. The liigli Jxrcentagc of J)ol!renoic acids in tile cllol&eryl esters of botll Ion (72’jh) and Jligh densit>? lipoproteins (55”/;,) of the sardine is most interesting in terms of comparable mammalian data. Clrolesteryl linoleate is thr principal cholestcr\-I c,stcr of humans (j3 -_j()~~o)')9,'1", A still 1ngher content _ of pol\~enoic acid than this is pres;clnt in the cholestq.1 esters of rat (22 : 4, 46(jb and IS: 2, yj(To)“l and in bo\%x (It;: 2, 7(~‘?~) high densit!, lipoproteinsY. Although the combined percentages of the two principal cholesteq4 esters (20: j and 22 : 0) of sardine higIl density lipoproteins fall \vithill the range noted iu Imnlans, this \~tluc for low densit?, lipoJxoteins is as high as rat and bovine data. l’pon fractionation of sardine high densit), lipoproteins, IVY found tile major Jx)rtion to lx, in Fraction 3 (d I.12 I.21 g/ml) rather tlian in I;raction 2 (tl
SERUM
LIPOPROTEINS OF THE PACIFIC SARDINE
277
I.o~-1.12 g/ml). Interestingly, humans have a predominance of Fraction 3 (6oo80~/,), whereas, ratsG911 and bovines 32 have mostly Fractions z and I (d 1.09-1.04 g/ml). As a result of its hydrated density interval, Fraction I overlaps the low and high density lipoprotein classes. Because mammalian studies have shown that cc-lipoproteins of high density ITraction I (1555165 A) are larger than those of Fraction z (average diameter 05 A) and Fraction 3 (average diameter 65 A) 33, it is very likely that a similar size difference exists among sardine cc-like lipoproteins. The ultracentrifugal distribution data and the fatty acid data indicate that as the cc-like lipoproteins increase in size, the degree of unsaturation of the apolar lipids also increases. The studies of Scanu and co-workers31p36 indicate that apolar lipids occupy the high density lipoprotein core, and that alkyl chains of the cholesteryl esters are in a liquid state at temperatures below their melting point. Because the melting point of cholesteryl esters decrease with an increase in unsaturation of the alkyl chainsT, the sardine would be expected to have a high polyenoic acid content in their high density lipoprotein cholesteryl esters in order to exist at ocean temperatures (10~15 “C). The increase in the content of polyenoic acids among the apolar lipids of less dense a-like lipoproteins in the sardine might be an adjustment to extreme thermal conditions. However, a comparison with mammalian data suggest that apolar lipids of high densit lipoprotein Fraction I or 2 might preferentially contain a high amount of polyenoic acids. ACKKO\vLEDGMENTS The authors wish to acknowledge Professor A. \‘. Nichols of Donner Laborator!. for permitting us to use the ultracentrifugal equipment and also Dr 1;. T. Hatch for doing the agarose electrophoresis for us. The help of M. \‘rooman and Rlrs E. Baker in obtaining sardine blood is much appreciated. The work was supported by Sational Science Foundation Grant GB 24834 to professor A. A. Benson. REFERENCES I L. Evans, 1. I)azvy Sci., 47 (196.1) 46. 2 I,. Evans, s. I’atton and R. D. McCarthy, J. Dairy Ski., 11 (1961) 475. 3 M. Fried, H. c;. Wilcox, G. R. I~aloona, S. 1‘. Eoff, hl. S. Hoffman and 1). Zimmerman, Cwzfi. Hioclzon. Physiol., 25 (1968) 6.jr. .+ R. J. HaveI, H. r\. Eder and J. H. Bragdon, J. CZi?z. Invest., j.+ (X95.5)1345. 5 L. A. Hillyard, C. Entcnman, H. F&berg and 1. L. Chaikoff, J. Biol. CR?m., 214 (1955) 79. 6 S. Iioga, 1). L. Horwitz and A. M. Scanu, J. Lzpzd Rrs., IO (1969) 577. 7 L. A. Lewis, A. rl. Green and I. H. Page, Awz. 1. Physiol., 171 (19.52) 391. 8 Cc. L. Mills and C. I<. Taylour, Camp. 5’iOchlw. Physml., 40 (1971) 489. 9 G. J. Nelson, J. Lzpid Res., 3 (1962) 71. IO .I. \.. Nichols, Adz!. Biol. ,Wed. Phys., II (1967) 109. II D. L. Puppione, T.CKL-18821, LT. C. Berkeley, March (1969). 12 I). L. Puppione and ,2. \‘. Nichols, Physiol. Chrm. Phys., 2 (1970) 49. 13 1). I.. Iluppionc, C. Sardet, \V. Yamanaka, R. Ostuxld and A. V. Nichols, Blochim. Hlophys. .4cta, 231 (1471) 29.5. ~4 1). L. Puppione, T. I;ortc and A. V. Nichols, Cowp. 13iochenz. Physiol., 39B (1971) 673. 15 A. SGUIU, J. Lipid Hrs., 7 (196.5) 295. 1~) R. F. Lee, J. C. Nevenzel and G. A. Paffenhbfer, Science, 167 (1970) 1510. 17 C. H. Hand and L. Berner, U.S. Fish M’iZdZifp Sevv. Fish. Bull., 60 (1959) 175. 18 N. K. I~recman, F. T. Lindgren and .I. \-. Nichols in R. T. Holman, \f::. 0. Lundberg and T. Malkin, PYO~YPSS ilz the Chcnzistry of Fats alzd Other I.ipids, I’ergamon, Xew York, 1963, p. 11s. 1~ H. P. Noble. F. T. Hatch, H. A. Mazrimas, R. T. Lindgrcn, I,. C. Tensen and G. L. Xdamson, Lipids. .+ (1969) 5s. 20 Il. F. Lee, J. C. ?ievcnzel and G. A. I’affenhbfer, dlav. Biol., 9 (rg71) 99. Biochim.
Biophys.
Acta,
270
(1972)
272-278
27s
I<. F. LEE,
I). L. Pl’I’PlONE
Sardine serum lipoproteins were separated into ultracmtrifugal density classw, and data on the lipid compositiow of these classes and the fatty acid distributions of cholesteryl esters, triglycerides, and pllospholipids are prescuted. The lipids present in the three densitv classes with llighest percentages were: cllolesteryl esters (ho’j,,) iu tlw \w-!. low densit)., phospholipids (35 YL) and triglycerides (30’)~~~)in the low dcnsit!., and plwspllolipids (jg’:{,) in the high densitv fractions. The fatti. acid conposition of the triglycerides from low aud high density fractions werr Get-!- similar, whereas marked differences \verr seen between the fatty acids of clwlester!-1 esters and pllospl~olipids for these two lipolmhh classes. Xgarose gel electroplmxGs of the sardine serum indicated the presence of three distinct bands with R-like mobility, but pre-,$ or p bands were not detected in whole serum or in any- densit!- fraction. The absciicc of lipoproteins with prc-p or /J niobilit~~ iu sardines suggests major differwcc5 h?tNwn fat translmrt iii manlrna~ian aud fish systems.
~1 great deal of information is available ou the properties of serum lipoproteins of various ~nanmmlsl~ IS, but JIO detailed study of MI serum lipoproteins has been I-Cported. ln this paper, we report on some of tlre’properties of sardine serum lipoproteins. Data on tlw lipid compositions of tllc ultracentrifugal classes’” and the fatt!. acid dstributions of cholestcryl esters, triglh-cerides, and I’llosl)lloliI’ids are presented. .\~arose gel rlectroplwresis was carried out on whle scfuni and tlir isolated ultraccntrifugal fractions. Our mvn interest iu sardiws resulted from earlier studies of the was esters of copepodsl”, a major source of food for the Pacific sardines Ii. l‘lle physical and chctnical properties trf tlw m-urn lipoprotein classes of these fish were charac~tctrizcd in order to
513RrM
LIPOPROTEINS
OF THE PACIFIC
learn more about the lipid metabolism
SARDINE
273
of an animal which ingests wax esters, and to
test the possibility that such a diet might result in the presence of long-chain or wax esters in sardine blood.
alcohols
METHODS
Live sardines were obtained from a bait barge in San Diego Bay within z days of capture from the open ocean. Blood (approximately 3 ml) was drawn from the dorsal aorta with a ro-ml syringe which had been rinsed with a I(),; heparin solution. The blood from the sardines clotted immediately and was centrifuged in an International Clinical Centrifuge at approximately IOOO xg for j min. Because the amount of heparin used was insufficient to prevent immediate clotting of the blood, it is unlikel!~ that tire concentration of the anti-coagulant was sufficient to produce any significant effect on the lipoproteins. Part of the total serum (40 ml) obtained from IOO sardines was used for total lipid studies while the remaining serum was placed in ice and shipped immediately to the Donner Laboratory at the ITniversitv of California for preparative ultracentrifugation of the serum lipoproteins using the techniques described b!F ITreeman cat~1.‘“. Electrophoresis of the different lipoprotein densit\. classes was done on agarose gels obtained from Bio-Kad Laboratories at pH 8.6 according to the procedure of Nobel ~‘t al.lQ. Lipids were separated by silicic acid columns into five fractions: hydrocarbons, cholesterol esters, triglycerides, polar lipids (free fatty acids, cholesterol, and digl!cerides), and phospholipids. Florisil columns were used to separate the polar lipid fraction into free fatty acids and cholesterol”‘. The purity of each fraction was tested by thin-layer chromatography, and where neccessary rechromatography on silicic acid columns was performed. -After weighing the different fractions the cholcsterol ester, triglyceride, and phospholipid fractions were transmethylated using 5()/o sulfuric acid in methanolzl. ITor gas-liquid chromatograplric analysis we used a Loenco Model 70 Hi-Flex apparatus (Loenco Inc., Mountain 1’iew, Calif.) fitted with a flame ionization detector and operated isothermally with 2.1 kg,‘cm” nitrogen carrier gas pressure. The two columns used were a 2.4 mx3.2 mm (outside diameter) column of 10”; diethylene glycol succinate polyester (DEGS) and a IS m x 2.3 mm column of 371, ()\‘-I on 6040 mesh GAS-Chrom P (all supplied by Applied Science Laboratories, Inc., State College, Pa.). Several different temperatures were used, depending on the compounds being
Ii. 1;. LEE,
27-l
11. L. P~PPIONE
analyzed. Hydrocarbons were analyzed at 150-170 “C on O\‘-I and DEGS; methyl esters of fatty acids at 16~1qo “C on DEGS and at 17+1qo “C on 01:-r. Triangulation was used to quantitate the results, which were normalized to total IOO”,,. Identities of fatty acids were determined from their retention times and a plot of total numlxr of carbons versus log retention times of howl saturated and unsaturated fatt!* acids. Hydrogenation of the fatty acid mixtures was carried out using platinum oxide to aid in the identification
of fattv
The concentrations
acids.
and chemical
composition
of the lipid moieties of tile three
classes ultracentrifugally isolated from sardine serum are given in Table I. The serum concentrations of lipids in each class were : very low density lipoproteins, 105 mg,“~oo 121 mg/loo ml; and high density lipoproteins, 560 ml; low density lipoproteins, mg/roo ml. The predominant lipids of the different classes were: cholester!4 esters (60(;4) in the total very low densit!, lipoprotein class, triglycerides (31 If/,)and phospl~olipid (35 YJ,) in the low den&v class and phospholipid (55’!‘“) in tire higIl tltwsity class.
0.
I
1 ..i
.3.7 2 ,j 2O.i
o., ‘I’ ‘1.
0.1 .
1
0.2 t i
‘7.1
SERUM
LIPOPROTEINS
Unesterfied
OF THE PACIFIC
cholesterol
SARDIIiE
was approximately
275
13”/~ of the lipid in low and high density
classes and HO{,in the very low density class. Separation of the polar lipid fraction by Vlorisil column chromatography indicated that the major part of this fraction was cholesterol while less than 15% was free fatty acid. Thin-layer chromatography of the polar lipid fraction showed only traces of mono- and diglycerides. Thin-layer chromatography of the phospholipid fraction showed that phosphatidyl choline was the major phospholipid with lesser amounts of phosphatidyl ethanolamine, lysolecithin, and sphingomyelin. No long chain alcohols or wax esters were found in any lipoprotein fraction. .I separate analysis on 40 ml of serum showed that total sardine serum lipid concentration was 790 mg/roo ml, which can be compared with serum lipid concentrations in ocean salnion1Q and herring (Lee, unpublished results) of zooo mg/roo ml and 813 mg/Ioo ml, respectively. The major lipids of the sardine serum (Table I) were phospholipid (do”/o), triglycerides (289;), and cholesteryl esters (22%). Patton ct al.>’ observed a similar lipid composition for the total blood serum of ocean salmon. Table II shows the fatty acid distributions of cholesteryl esters, triglycerides, and phospholipids of the low and high density classes. The principal fatty acids of cholesteryl esters were: zz:6 (52%), 20:s (zoo/o) and 16:o (16’j/,) in the low densit! class and zz:O (359/,), 16:o (23%), zo:5 (16~~6) and IS: I (12(j<,) in the high densit! class. In the phospholipid fraction, relatively higher amounts of 22: 6 and 20:5 were present in high density lipoproteins (370d1,and 170/;, respectively) than in low densit! lipoproteins (267; and IZ%, respectively). The percentage of 18: I was essentially the same in the phospholipids of both ultracentrifugal classes. Marked differences between the phospholipids of low and high density classes were noted in the amounts of 16: o (I’% in low and zq~/o in high den&v lipoproteins) and 16: I (35’j& in low and ;cy/, in high density lipoproteins). In the triglyceride fraction, the fatty acid distributions of the low and high density classes were very similar.
The electrophoretic distribution of sardine lipoproteins, along with a normal human distribution included for comparative purposes, is shown in Fig. I. Three bands with a-like mobility were noted in sardine serum, but the fi and pre-/? bands, present in normal human serum, were not detected in sardine serum. The three bands of sardine lipoproteins are referred to as z-like because they run ahead of human /3-lipoproteins. Electrophoretic distributions of the ultracentrifugal fractions indicated that a-like lipoproteins with the slowest mobility were present in the low density class and AGAROSE GEL ELECTROPHORESIS
0
Human
Fig.
I.
_
Sample Wet I
Sardine
.A~aroscgel elcctrophoretic
distnhution
of human
ant1 sardine
NioclriwL.
Blophys.
lipoproteins. Acta,
270 (1972)
272-278
I<. F.
276
LEE,
I). I.. PI-PPIOSli
those with the faster mobilities were present in the high density class. The total veq low density lipoproteins remained at the origin and their stainability was very poor. Similar results were obtained with paper electrophoresis.
Our studies of sardine serum lipoproteins
demonstrated
some marked differences
between the fat carriers of these fish and those of mammals. The percentages of triglycerides (30);) and phospholipids (I :io)in the total \‘er!: low density class are much less than reported values in mammalian studies1”-15,““s24. \‘cry low den+lipoproteins containing a high percentage of cholesteryl esters, as we lbave noted in the sardine, have only been observed in cholesterol-feel rabbitsZ5. The absence of pre-e and ~-lipoproteins in the electroplloretic clistrihutions of whole serum aud the ultracentrifugally isolated fractions of the sardine suggest furtller differences between these fish and mammals. The concentration of lipids in the total verv low densit), class (IOO mg/~oo ml) is sufficiently high to gi1.e rise to an exyrected pre:@ band instead of the poorly staining material at the origin. It is unlikely that the size of these lipoproteins was a factor because the serum was not turbid. Probabl\the negative
charge of the protein moiety \vas insufh
ieut to Jlermit these lipoproteins
to leave the starting well. Three distinct bands with a-like mobilit>,, observed in sardine seru.m, J~ave also been observed in salmon and trout (Puppjone, unpublished results). The low densit! lipoproteins of the sardines wercx found to have an electrophoretic mobilit!. which corresponded to the slowest migrating alphas. The presence of a-lipoproteins in the low densit?, class has also been observed in certain maIlimals”,‘“,13,~~,~7. The fatty acids among tllc different lipid: probabl!, are of dietar!. origin. The triglyceride fatt!r acids of thr sardine low and high density classes match very closely the was ester fatt!, acids of tile copepod, C‘al~us /zrl,nolr~~zr~icu~~~“, wllicII is probably in the diet of the Pacific sa.rclinel’. \\‘as esters are a major lipid of Calnnus localized in a large oil sac near the coJ)epocl gut. To what extent dietar). fatty. metabolized or modified in sardines can, of course, onlv he inferred from direct evidence. TJle lack of long-chain alcolrols and was esters in sardines
and are acids are sucll insuggests
that the was esters of the coJ>eJ)ods are h~drol~~zed, followed by oxidation of the acollol to the corresponding fatt>- acid. Kecent work h_ 1.1. Baker, J. C. Kevcnzel and K. 1’. Lee (unpublished results) sl~owed a verb- active ~vax ester lipasc in the p>-loric caecum of sardines and anchovies, but low wax ester lipasc activit!. in the liver, wllite muscle, and red muscle. The liigli Jxrcentagc of J)ol!renoic acids in tile cllol&eryl esters of botll Ion (72’jh) and Jligh densit>? lipoproteins (55”/;,) of the sardine is most interesting in terms of comparable mammalian data. Clrolesteryl linoleate is thr principal cholestcr\-I c,stcr of humans (j3 -_j()~~o)')9,'1", A still 1ngher content _ of pol\~enoic acid than this is pres;clnt in the cholestq.1 esters of rat (22 : 4, 46(jb and IS: 2, yj(To)“l and in bo\%x (It;: 2, 7(~‘?~) high densit!, lipoproteinsY. Although the combined percentages of the two principal cholesteq4 esters (20: j and 22 : 0) of sardine higIl density lipoproteins fall \vithill the range noted iu Imnlans, this \~tluc for low densit?, lipoJxoteins is as high as rat and bovine data. l’pon fractionation of sardine high densit), lipoproteins, IVY found tile major Jx)rtion to lx, in Fraction 3 (d I.12 I.21 g/ml) rather tlian in I;raction 2 (tl
SERUM
LIPOPROTEINS OF THE PACIFIC SARDINE
277
I.o~-1.12 g/ml). Interestingly, humans have a predominance of Fraction 3 (6oo80~/,), whereas, ratsG911 and bovines 32 have mostly Fractions z and I (d 1.09-1.04 g/ml). As a result of its hydrated density interval, Fraction I overlaps the low and high density lipoprotein classes. Because mammalian studies have shown that cc-lipoproteins of high density ITraction I (1555165 A) are larger than those of Fraction z (average diameter 05 A) and Fraction 3 (average diameter 65 A) 33, it is very likely that a similar size difference exists among sardine cc-like lipoproteins. The ultracentrifugal distribution data and the fatty acid data indicate that as the cc-like lipoproteins increase in size, the degree of unsaturation of the apolar lipids also increases. The studies of Scanu and co-workers31p36 indicate that apolar lipids occupy the high density lipoprotein core, and that alkyl chains of the cholesteryl esters are in a liquid state at temperatures below their melting point. Because the melting point of cholesteryl esters decrease with an increase in unsaturation of the alkyl chainsT, the sardine would be expected to have a high polyenoic acid content in their high density lipoprotein cholesteryl esters in order to exist at ocean temperatures (10~15 “C). The increase in the content of polyenoic acids among the apolar lipids of less dense a-like lipoproteins in the sardine might be an adjustment to extreme thermal conditions. However, a comparison with mammalian data suggest that apolar lipids of high densit lipoprotein Fraction I or 2 might preferentially contain a high amount of polyenoic acids. ACKKO\vLEDGMENTS The authors wish to acknowledge Professor A. \‘. Nichols of Donner Laborator!. for permitting us to use the ultracentrifugal equipment and also Dr 1;. T. Hatch for doing the agarose electrophoresis for us. The help of M. \‘rooman and Rlrs E. Baker in obtaining sardine blood is much appreciated. The work was supported by Sational Science Foundation Grant GB 24834 to professor A. A. Benson. REFERENCES I L. Evans, 1. I)azvy Sci., 47 (196.1) 46. 2 I,. Evans, s. I’atton and R. D. McCarthy, J. Dairy Ski., 11 (1961) 475. 3 M. Fried, H. c;. Wilcox, G. R. I~aloona, S. 1‘. Eoff, hl. S. Hoffman and 1). Zimmerman, Cwzfi. Hioclzon. Physiol., 25 (1968) 6.jr. .+ R. J. HaveI, H. r\. Eder and J. H. Bragdon, J. CZi?z. Invest., j.+ (X95.5)1345. 5 L. A. Hillyard, C. Entcnman, H. F&berg and 1. L. Chaikoff, J. Biol. CR?m., 214 (1955) 79. 6 S. Iioga, 1). L. Horwitz and A. M. Scanu, J. Lzpzd Rrs., IO (1969) 577. 7 L. A. Lewis, A. rl. Green and I. H. Page, Awz. 1. Physiol., 171 (19.52) 391. 8 Cc. L. Mills and C. I<. Taylour, Camp. 5’iOchlw. Physml., 40 (1971) 489. 9 G. J. Nelson, J. Lzpid Res., 3 (1962) 71. IO .I. \.. Nichols, Adz!. Biol. ,Wed. Phys., II (1967) 109. II D. L. Puppione, T.CKL-18821, LT. C. Berkeley, March (1969). 12 I). L. Puppione and ,2. \‘. Nichols, Physiol. Chrm. Phys., 2 (1970) 49. 13 1). I.. Iluppionc, C. Sardet, \V. Yamanaka, R. Ostuxld and A. V. Nichols, Blochim. Hlophys. .4cta, 231 (1471) 29.5. ~4 1). L. Puppione, T. I;ortc and A. V. Nichols, Cowp. 13iochenz. Physiol., 39B (1971) 673. 15 A. SGUIU, J. Lipid Hrs., 7 (196.5) 295. 1~) R. F. Lee, J. C. Nevenzel and G. A. Paffenhbfer, Science, 167 (1970) 1510. 17 C. H. Hand and L. Berner, U.S. Fish M’iZdZifp Sevv. Fish. Bull., 60 (1959) 175. 18 N. K. I~recman, F. T. Lindgren and .I. \-. Nichols in R. T. Holman, \f::. 0. Lundberg and T. Malkin, PYO~YPSS ilz the Chcnzistry of Fats alzd Other I.ipids, I’ergamon, Xew York, 1963, p. 11s. 1~ H. P. Noble. F. T. Hatch, H. A. Mazrimas, R. T. Lindgrcn, I,. C. Tensen and G. L. Xdamson, Lipids. .+ (1969) 5s. 20 Il. F. Lee, J. C. ?ievcnzel and G. A. I’affenhbfer, dlav. Biol., 9 (rg71) 99. Biochim.
Biophys.
Acta,
270
(1972)
272-278
27s
I<. F. LEE,
I). L. Pl’I’PlONE