SERUM LIPOPROTEINS IN TWO SPECIES OF PHOCIDS (PHOCA VZTULZNA AND MIROUNGA ANGUSTIROSTRZS) DURING ALIMENTARY LIPEMIA DONALD L. PUPPIONE School of Public Health. University of California. Los Angeles. CA 90024 U.S.A.
(Received IS April 1977) Abstract--l. Serum lipoprotein distributions of harbor seals (Phoca oitulinn) and elephant seals (Mirounga angustirostris) during alimentary lipemia were measured by analytical ultracentrifugation. 2. Lipid compositions and concentrations of the total VLDL. LDL, and HDL classes were also determined. During alimentary lipemia increased serum triglycerides were associated with concentration changes in the total VLDL class. A postprandial increase in HDL phospholipid was observed in one animal. 3. Comparison of lipid concentration of the total VLDL class with the ST 2&400 data indicates that chylomicra are the principal triglyceride carriers during alimentary lipemia and that there are only slight changes among the S, 2&400 lipoproteins. 4. Electrophoretic analyses were carried out on sera obtained from elephant seals. Chylomicron and pre-beta bands were detected, but there was no discernible beta band. Consistent with the latter finding no Sy O-20 lipoproteins were observed in the analytical ultracentrifuge. 5. These data are discussed in terms of the clearance of chylomicron remnants.
INTRODUCTION In an earlier study of the serum lipoproteins of pinnipeds (Puppione & Nichols, 1970), the concentration of the triglyceride-rich lipoproteins was noted to be low in fasting animals. Because of their reliance on dietary intake to maintain their fat stores (Jangaard et al.. 1963). it was of interest to look for possible physical and chemical changes among the principle triglyceride carriers and the other serum lipoproteins during alimentary lipemia. In this study the serum lipoproteins of two species of phocids (Phoca h&a and Mirounga angustirostris) were characterized after the animals had been fed herring. Lipid compositions and concentrations of the major lipoprotein classes together with ultracentrifugal distributions are presented. Electrophoretic studies of whole serum were also carried out. The lipoprotein classes and their abbreviations as used in the text are defined as follows: (a) total very low density lipoproteins. VLDL, a combination of the chylomicron class (d < 0.94 g/ml; ST > 400) and the very low density lipoprotein class (do.961.006 g/ml; ST 2&400), (b) low density lipoproteins, LDL, (d l.OOG1.063 g/ml; SF O-20), (c) high density lipoproteins, HDL, (d 1.063-1.21 g/ml; F,,zo &20) and (d) HDL, (F, .zo 9-20). HDL, (F,,,, 359), and HDLJ (F,.,. O-3.5). S, rate is defined as Svedbergs of flotation, measured at 26°C in a medium of 1.745 molar NaCl (d 1.063 g/ml). Flotation rates corrected for the effects associated with concentration dependence are indicated by the symbol ST. F rate denotes a flotation rate measured at any other density, signified by a subscript. e.g. F, ,20. MATERIAL
AND METHODS
ent to 5% of their body weight, and blood was subsequently drawn at the 10th postprandial hour. Two weeks later, on two separate days, the feeding of harbor seals (I and II) was repeated. On one day, blood was drawn at the 9th and I Ith postprandial hours and on the next day at the 3rd. Sth, and 7th postprandial hours. A final set of samples was obtained from harbor seal II at the 7th. 9th. and I Ith postprandial hours to verify a secondary rise in triglyceride levels. At each bleeding. 60 ml of blood were obtained as described by Harrison & Tomlinson (1956). and the sera were obtained after the blood had clotted (DeLalla & Gofman. 1954). The appearance of each sample was noted as being either clear, turbid, or lactescent. The absence or presence of a creamy layer after the sample had been stored overnight in a refrigerator was noted the next day. Lipoprotein analyses
The procedures of Lindgren et al. (1967) were used for analytic ultracentrifugation of serum lipoproteins and the computer technique of Jensen et al. (1970) was employed for the graphic representation of fully corrected schlieren patterns (see Fig. I). The concentrations (mg/dl) of serum lipoproteins in the specific flotation rate intervals are shown in the lower portion of the distribution representations. The procedures for the isolation of the three lipoprotein classes and for the extraction, chromatography, and quantification of their lipids have been previously described (Freeman et al., 1963). Electrophoretic analyses were done on both paper (Hatch & Lees, 1968) and agarose (Noble et al.. 1969).
Table 1. Sex, weight and age of seals
Harbor seal
Feeding and bleeding procedures
Three harbor seals and two elephant seals (see Table
I for weight and age) were fed herring in amounts equival-
Elephant seal
I 11 III I II
Sex
Weight (kg)
Age
F F F M M
41 80 62 159 209
21 mo. 7-8 yrs 7-l 5 yrs 21 mo. 33 mo.
DONALD I.. PUPPIONF
I 2x
Table 2. Serum Ii~proteiR
concentration (mgidf)
10th hour data Harbor seal Efcphant seal
I fl III 1 II I
Harbor seal
Harbor seal
f
Harbor seal
II
Harbor seal
If
14 5 0 3 0 9th 11th
23
3rd %h 7th
18 33 31
3rd 5th
40 34
7th 9th
62 5x Xl
I Ith
16
2.1 8 4 0 0
9th and 1I th hour data 7 I7 24t? 3 17 222 3rd. 5th. and 7th hour data x 1s 210
I IO 718 5 8 214 3rd and 5th hour data 1X 17 163 9 9 is7 7th. 9th. and 1I ttt hour data 2
7
186
9 II
11 I?
199 X7
RESULTS
Samples obtained at the 5th and 7th hours were lactescent in both harbor seals studies. With the exception of the 10th and 1tth hour samples of harbor seal I, all the other samples were turbid and contained. after overnight storage in a refrigerator, a creamy chylomicron layer at the top.
The concentration of serum lipoproteins within various flotation rate intervals are given in Table 2. There was Iittle difference between these uttracentrifugal data and those previousIy reported for fasting pinni~ds ~Puppione & Nichols. 1970). On a given day, little concentration variatjons at different postprandial times were noted within the various flotation rate intervals. in samples obtained from harbor seals I and 11. Among the HDL subclasses, an apparent decrease in concentration was observed between the 3rd and 7th ~stprandia~ hours in harbor seal I and between the 3rd and 5th ~stprandia~ hours in harbor seal II. Repre~n~tive ultracent~fugal distributions of the serum lipoproteins of harbor seal I are shown in Fig. I. The two Schlieren peaks observed within the S; O-20 interval were also present in the patterns of the other two harbor seals. Average s; values for these two peaks were 9.2 and 2.2. It is Interesting to note that in the two elephant seals no S;: O-20 l~~proteins were detected. fn the HDL distributions of both species. the major peaks had an F,.zo value of approximately 2.5. Lipoprotein
229 Ilh 104 0 0
lipids
Table 3 shows the concentration and com~sitional changes which occurred in the total VLRL class and whole serum at different ~stprandiai times. Approximately 60% of the whole serum triglyceride was re-
141 11: 0 ii 0
I‘M I44
176 153 124 1;s H
7Y? 799
12x 126 12x
covered in the total VLDL class. and triglyceride concentration changes in serum were reflected in this i~~protein class. Peak triglyce~d~ levels were noted at the 7th hour in harbor seal I and at the 5th hour in harbor seal II. A secondary rise in the serum concentration of lipids of the total VLDL class of harbor seal 11 was observed in two separate experiments, i.e. the concentration was greater at the 11th than at the 9th ~stpmndial hour. In the total VLDL of both animals, increases or decreases in the triglyceride content of ii~proteins were associated with compensatory changes in the chofesteryl ester content. An increase in HDL phospholipids (ca. 80 mgidl) was observed in harbor seal II between the 3rd and 5th ~stprandia~ hours. Because there was little fluctuation in the lipid composition of LDL and HDL classes at the other times and because these values were essentially identical to those reported in fasting animals (Puppione & Nichols, 1970), these lipid data have been omitted. For each of the sampling times, the lipid concentrations of the total VLDL class was greater than the li~prote~n concentration of ST 20-400 class. This clearly indicates that the major lipid carriers within the total VLDL class have flotation rates greater than Sl; 400, i.e. they are chylomicra. Comparison of the data in Tables 2 and 3 shows that as the proportion of total VLDL lipid associated with the ST 20-400 lipoproteins increases, the t~giyceride content of Iipoproteins within the total VLDL class decreased.
Chylomicron bands at the point of sample application were clearly discernible on both paper and agarose when electrophoresis was done on elephant seal sera obtained at the i Ith and 13th ~stprandiaf hours. A faint pre-beta band was visible only on agarose. Con~nant with the uItmcentrifuga1 ST a-20 data in Table 2, no beta band was demonstrable on either
129
Serum li~prot~~ns in two species of phocids LOW-DENSITY
LlPOPROTEiN
HIGH-DENSITY
DlSTRl6UTlON BLEEDING
400
too
ST
SERIES
I
0
20
20
LlPOPROTElN
10
DISTRIBUTION
F 1 20
0
HARBOR SEAL I 3rd ht postprandial
BLEEDING
SERIES
II
9th hr ~ 1450
11th hr
11430
J
Fig. 1. Ultracentrifugal distribution of serum lipoproteins of harbor seal I at different postprandial times. agarose or paper. In the harbor
seal, the i~~pro~eins
with peak Sy value of 9.2 had primarily beta mobility and those with a value of 2.2 had mostly alpha mobiIity. DISCWSION The present study indicates that concentration and compositional variations in serum lipoproteins of seals in the postprandial state occur primarily in the total VLDL class as is the case in humans (Havel, 1957) and dogs (Hillyard et aL, 1958). The secondary
rises in triglycerides, noted twice in harbor seal II, are not unlike the observations of Gage & Fish (1924) who followed time changes in chylomicron levels in several species of mammals. Harlan & Be&her (1963) have shown that in normal human subjects the concentration increments within the S, 2@400 class tend to be equally distributed between the S, 20-100 and S, 100-400 subclasses (mean increases of 36 and 38 mg/dl, respectively). Assuming negligible fasting ieveIs for these two subclasses in seals, a comparable build up was found in the present study to occur only in the S; lOQ-400, with the major concentration
130
DONALD Table
3. Lipld concentrations
Elephant
Harbor Elephant
and composittons
of the total VLDL
Concentration (mg lipid/d1 serum)
Harbor
L. PIJPPION~
CF
10th hour VLDL 65 202 497 -7-w
seat
9th and I Ith hour VLDL II5 5.X x9 6.7 I28 5.5 I79 37
12.0 16.5 I I.4
9th I lth 9th 11th
and I I th hour W.S. I160 32.0 1I30 31.6 6X 35.1 661 34.5
4X.6 51.4 57.7 58.9
II.0 x.2
7.x s.1
0.5 0.5
5.9 5.X
3rd 5th 7th 3rd Sth 7th
3rd. Sth. and 7th hour VLDL I’X 4.7 IX8 4.3 ‘22 4.3 I65 3.4 201 4.6 171 5.5
X.X 7.5 X.0 11.2 I !.6 Il.6
7x.3 7X.4 75.3 76. I 7x.1 74. I
7.t; 9.1
29.0 27.7 28.0 38.2 34.2 34.6
4X.X 45.2 43.2 54.9 57.9 58.2
7th. 9th. and I I th hour VLDL 233 1.5 179 3.0 242 2.X
I220
9th
9th
I Ith
9th seal
Harbor
seal
Harbor
seal
Harbor
seal
Harbor
seal
Harbor
seal
3rd 5th 7th 3rd 5th 7th
Harbor
seal
7th 9th I Ith
Harbor
seal
7th 9th I Ith
3rd. 5th. and 7th hour 1250 1290 I280 701 805 742
7th. 9th. and 1430 I400
1500
I Ith
I I.0
0.4
X4.4
CJ0 X.I I
X3?
76
6.7 14.x 32.4 34.9
Xl 7.1 7.0 7.5
0.8 0.7
74.X 6X.3 6X.5 75.1
79 173 S.8 ‘J I
0.5 0.7 0.7 0.X:
Il.4 I 0
0.h
(I.6 0.7 0.7 0 .I
X.7 51 s,;
0.4 0 7 0.6 0 6 0.5 0.6
13.6
7.X
(I.9
I x.3
76
20.1 I.2 1.5 0.9
77 5‘
I ..3 I I 0.h
5.3 5.4
I.2 0.0
11.1 12.5 I I.6
75 7 73.2 75.7
9.9 IO.6 0 I
0.S 0.X 0.8
47.7 47.9 45.6
17.9 15.1 19.0
7.4 x.2 76
11.x
W.S.
hour W.S. 26. I 27.6 27 I
Abbreviations used in Table 3: CE. cholesteryl esters: PL. unesterified cholesterol; UFA. unestcrified fatty acids
changes taking place within the chylomicron class In both species of seals. In elephant seal I with a triglyceride level of 400 mg/dl at the 10th postprandial hour, the concentration of the Sy lOG400 was only 68 mg/dl. Consistent with these data. the agarose electrophorectogram contained a heavily staining chylomicron band at the origin and a very faint pre-beta band. Electron microscopic observation of the total VLDL class isolated from harbor seal serum has also demonstrated the presence of particles the size of chylomicra (T. Forte, personal communication). In an earlier study of alimentary lipemia in humans, Jones et al. (1951) noted that there was a transient increase in the concentration of lipoproteins with S, value greater than 60. and that within the VLDL class decreases in the concentration of lipoproteins of high S, value were associated with concomitant increases in the concentration of lipoproteins of
51.3
7x.7
I ‘F‘A
h’)
45.7 79, I 37.9
seal
Harbor
I
22.9 31.9 20.7 19.0
10th hour W.S. I160 I I60
seal
scat
h9.
Ii.2 X.6 6.3 7. I
I lth Harbor
I ‘C’ ..~_.._
X.3 3.7 I.? 1.3
seal
seal
T(;
PL ,___..
89X Harbor
and whole serum
phospholipid:
TG.
trlglyccrides:
I .o I? 0.7 L’C.
low S, value. These workers concluded that the clearance of triglycerides from the blood involves the conversion of larger lipoproteins into smaller ones. More recently it has been proposed that removal of triglycerides from chylomicra through the action of the enzyme extrahepatic lipase result in the formation of so-called “remnant particles” which can be subsequently cleared from the circulation by the liver (Redgrave, 1970). Using vitamin A as a marker, Hazzard & Bierman (1976) have been able to corroborate the earlier work of Jones and co-workers by demonstrating that chylomicron remnants, when not taken up by the liver. can be converted into a continuum of particles throughout the VLDL class. In comparing the turnover data of VLDL in humans and rats. Eisenberg & Levy (1975) have proposed that the marked difference in the LDL concentration of these two mammals may be related to the net uptake of
131
Serum lipoproteins in two species of phocids remnants containing the major apoprotein of LDL (i.e. apoprotein B), uptake being greater in rats than in humans. The absence of any appreciable build up in the Sy 20-100 subclass and the low concentration of beta lipoproteins would suggest that chylomicron remnants are rapidly and efficiently removed in seals, particularly in the elephant seals in which there are no detectable S, &20 or beta lipoproteins. The increase in HDL phospholipids of harbor seal II was the only change noted in the lipids moieties of LDL and HDL in this study. Similar changes have been reported in both humans (Havel, 1957; Nichols et al., 1962) and dogs (Hillyard et al., 1958). Have1 et al. (1973) have associated this change in HDL composition with the shedding of phospholipids from the surface of triglyceride carriers. They have postulated that this loss of polar lipids might facilitate in turn the uptake of apoprotein C from HDL by both chylomicra and VLDL. Apoprotein C consists of a group of peptides, one or more of which must be a component of a VLDL or a chylomicron to enable these triglyceride carriers to be suitable substrates for extrahepatic lipase. In the post-absorptive state, the C apoproteins are predominantly associated with HDL and in a sense HDL can be considered as an apoprotein C reservoir whose level rises and falls inversely to the concentration of the triglyceride-rich lipoproteins (Have1 et al., 1973). Among the subclasses of HDL,
HDL, apparently participates more than HDLJ in this exchange with the lipid carriers of the total VLDL class (Have1 et al., 1973). It is noteworthy that during peak triglyceride levels in the two harbor seals. the largest percent change in HDL concentration took place within the HDL, subclass. Whether or not alpha lipoproteins, more buoyant than HDL,, could be involved in this peptide exchange with lipoproteins in the total VLDL class remains to be demonstrated. Changes in the neutral lipid content of HDL have also been reported following fat ingestion (Nichols et al., 1962; Hillyard et al., 1958). Based on their in vitro studies, Nichols & Smith (1963) have associated the increase in the triglyceride content of HDL with the action of the serum enzyme, lecithin cholesterol acyl transferase (LCAT). In their studies, a reciprocal exchange of VLDL triglycerides for HDL cholesteryl esters was shown to be influenced by LCAT activity. This transfer of lipids undoubtedly takes place in the fasting condition as well. Lindgren et al. (1965) reported that in humans the triglyceride content of HDL increased as the fasting level of VLDL increased. In an earlier study of a patient with type III hyperlipoproteinemia (diagnosis then being xanthoma tuberosum), the cholesteryl ester content of VLDL and the triglyceride content of HDL were twice normal values (Lindgren et al., 1956). In patients with the so-called “type III” or “dysbetalipoprotein” condition, an abnormal beta lipoprotein often is found in the VLDL class. The protein moieties of these beta VLDL contain a high percentage of apoprotein E (the arginine-rich protein) (Have1 & Kane, 1973) which is present as a minor component of normal VLDL (Shore & Shore, 1973). The accumulation of these abnormal lipoproteins are generally considered to represent a defect in the conversion of remnant particles to beta LDL. Hazard & Bierman
(1976) have also noted that in subjects with endogenous hypertriglyceridemia clearance of remnant particles can be delayed. Utermann et al. (1975) have proposed that the neutral lipids, exchanged between HDL and VLDL, are in association with apoprotein E. It was noted above that the absence of an appreciable build up in the Sy 2CMOOduring alimentary lipemia as well as the low concentration of beta lipoproteins in both seals suggests that chylomicron clearance is very rapid in these animals. Although a certain amount of the exchange of neutral lipids probably takes place between intact triglyceride carriers and HDL, there was no evidence of it occurring in either species of seals during alimentary lipemia. Rather these findings, together with the above cited studies of type III subjects, would suggest that the remnant particles are the principal participants in this exchange of neutral lipid with HDL. Acknowledgements-The author wishes to thank Mr. R. Doyle and Mr. G. Adamson for their technical assistance and to acknowledge the support of Professor A. V. Nichols of the Donner Laboratory and Dr. R. Hubbard of the Biological Sonar Laboratory of the Stanford Research Institute. This work was supported by Research Grant HE 10878-04 from the National Heart Institute, U.S. Public Health Service and by the Atomic Energy Commission. Portions of this work, done by D. L. Puppione in partial fulfillment of the Ph.D. degree, was supported by a Public Health Service Biophysics Training Grant (5 TIBM 829).
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