© 1995 American Heart Association, Inc.
Articles |
Lipid Composition of HDL Subfractions in Dog Plasma and Lymph
From the Division of Lipoprotein Metabolism and Pathophysiology, Department of Physiology, Louisiana State University Medical Center, New Orleans.
Correspondence to Laurence Wong, Division of Lipoprotein Metabolism and Pathophysiology, Department of Physiology, Louisiana State University Medical Center, New Orleans, LA 70112.
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Abstract |
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Abstract We report the lipid composition of dog plasma and peripheral lymph lipoproteins as separated into pre-ß,
, and pre- fractions by agarose gel electrophoresis. Plasma
lipoproteins with mobility have a composition different from that
of plasma lipoproteins with pre- mobility, having 9% versus 11%
free cholesterol, 21% versus 17% cholesterol
ester, 1% versus 16% triacylglycerol, and 69%
versus 56% phospholipid. On the other hand, lymph and pre-
lipoproteins have compositions that are quite similar (9% versus 7%
free cholesterol, 17% versus 17% cholesterol
ester, 2% versus 4% triacylglycerol, and 71%
versus 71% phospholipid). The lipid compositions of plasma and lymph
lipoproteins are quite similar (9% versus 9% free
cholesterol, 21% versus 17% cholesterol
ester, 1% versus 2% triacylglycerol, and 70%
versus 72% phospholipid). The lipid compositions of plasma and lymph
pre- lipoproteins are different (11% versus 7% free
cholesterol, 17% versus 17% cholesterol
ester, 16% versus 4% triacylglycerol, and 56%
versus 71% phospholipid). Peripheral lymph lipoproteins
with pre-ß mobility contained 15% cholesterol, 13%
cholesterol ester, 10%
triacylglycerol, and 61% phospholipid. Compared
with plasma, peripheral lymph lipoproteins are free
cholesterol–enriched in all fractions. Calculated
stoichiometric ratios of lipid to apoA-I indicate that pre-ß
lipoproteins contain one molecule of apoA-I per particle,
lipoproteins have two molecules of apoA-I per particle, and pre-
lipoproteins have four molecules of apoA-I per particle.
Key Words: high density lipoproteins • phospholipids • cholesterol • cholesterol ester • triacylglycerol
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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It is well known that HDLs are heterogeneous. The heterogeneities could be based on flotation density, immunological composition, molecular size, or electrophoretic mobility. DeLalla and Gofman1 could detect three different HDLs on the basis of analytical ultracentrifugation. These different HDL subpopulations were termed HDL1, HDL2, and HDL3. Using antibodies, Alaupovic2 could isolate HDLs containing apoA-I only and HDLs containing apoA-I and apoA-II. These were called LpAI and LpAI:AII particles, respectively. There is recent evidence to suggest that LpA-I and LpA-I:A-II particles may have different metabolic properties.3 By using nondenaturing gradient polyacrylamide gel electrophoresis (NODEGRA-PAGE), Blanche et al4 have identified different HDLs on the basis of size. Castro and Fielding5 and Fielding and Fielding6 used a two-dimensional separation of lipoproteins combining agarose gel electrophoresis with NODEGRA-PAGE to detect at least six populations of apoA-I–containing lipoproteins in human plasma.
We have recently identified at least 12 populations of
apoA-I–containing lipoproteins in the human plasma7 by
using a two-dimensional system. Using this technique, we found that
the apoA-I subpopulations in dog plasma were somewhat simpler; there
were only six of them.8 In addition, there was only one
population of pre-ß particles (pre-ß1), as opposed to
the three populations that we and Castro and Fielding5
found (pre-ß1, pre-ß2, and
pre-ß3) in human plasma. Our recent observation that the
apoA-I distribution is changed in lymph (a model of
interstitial fluid) suggests that complex lipoprotein
interactions occur within the interstitial space as a
result of reverse cholesterol transport.8
There was an increase in the pre- and pre-ß populations with a
corresponding decrease in the population in the
lymph.8 Because the difference between plasma and lymph
lipoproteins probably represents a combination of (1)
interaction of lipoproteins with the peripheral cells, (2)
filtration through the interstitial matrix, and (3)
interactions of the lipoproteins with the capillary
endothelium, lipid analysis of these
lipoproteins may shed some light on these processes. Recently, the
existence of a pre- particle was confirmed independently by another
laboratory.9 Because of the observed differences in apoA-I
subpopulations between plasma and lymph, as well as our description of
a previously uncharacterized population of lipoproteins (pre-), we
have undertaken to characterize the subpopulations with respect to
their chemical composition.
In this article, we report that there were differences in chemical
composition between plasma and lymph pre- particles and differences
between plasma and plasma pre- particles. There was little
difference between plasma and lymph particles or between lymph
and pre- particles. By quantifying the apoA-I in these
subpopulations, we were able to estimate that pre-ß particles
contained one molecule of apoA-I, particles contained two molecules
of apoA-I, and pre- particles contained four molecules of
apoA-I.
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Methods |
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Materials
Purpose-bred male foxhounds were purchased from the Department of Veterinary Medicine, Louisiana State University, Baton Rouge. Unless otherwise stated, all solvents (eg, chloroform, methanol, hexane) were of HPLC grade and were purchased from Mallinckrodt (Baxter Scientific). Agarose was from FMC. Capillary gas chromatography columns (0.3 mm ID, 7.5 m) were from J&W Scientific. Lipid standards were from Serdary Research Laboratories. Silation reagent (BSTFA) with 1% (TMCS) was from Pierce Chemical. All other chemicals were from Sigma Chemical Co.
Procedures
Our methods for dog lymphatic cannulation and lymph collection
have been described.10 The technique for
two-dimensional gel electrophoresis of lipoproteins has also been
described.7 Agarose electrophoresis was performed as
previously described.8 We defined the apoA-I–containing
lipoproteins by their electrophoretic mobility on agarose gels.
Typically, 150 to 200 gels, in sets of four each, were run. A 0.5%
agarose ME (medium electroendosmotic) was used for isolation of plasma
and lymph lipoprotein fractions. After the electrophoresis, the gels
were aligned and the ß, pre-ß, , and pre- segments excised.
For evaluation of the completeness of separation of the fractions, one
of every four gels cut was routinely subjected to
re-electrophoresis on agarose gel and probed. This is shown in Fig 1
for a typical lymph sample. It can be seen in the
figure that contamination of the pre- from fractions is minimal.
The same is true of contamination of the pre-ß from fractions.
The fractions always contained some contamination with pre-
particles. It is estimated that contained 5% to 10%
pre-–derived apoA-I. The excised gels were then placed into
polycarbonate tubes and the lipoproteins were recovered by spinning of
the gels in a Ti 50.2 rotor (Beckman) at 40 000 rpm for 1 hour.
Recovery was typically from 90% to 95% as determined by the free
cholesterol concentration (Table 1). After
an aliquot was taken for apolipoprotein and phospholipid
analyses, the remaining lipoprotein fractions were lyophilized
by use of an Edwards lyophilizer (Edwards High Vacuum). Lipid was
extracted from the lyophilized fractions by use of the method of Folch
et al.11
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After solid-phase extraction of the phospholipids,12 total lipid analysis was performed by gas-liquid chromatography by use of the method of Kuksis and colleagues.13 14 The analysis was performed on a Hewlett-Packard model 5890 gas chromatograph with automatic on-column sample injection (Hewlett-Packard). Injector temperature was 40°C. Temperature programming was 40°C for 0.5 minutes, 30°C/min to 150°C, 20°C/min to 230°C, and 6°C/min to 340°C and holding at 340°C for 3.5 minutes. Hydrogen carrier pressure was 6 psi constant flow at 40°C.
Protein determination was by the method of Lowry et al.15 Apolipoprotein analyses were by slot blot (L.W. et al, unpublished data, 1995). Plasma and lymph fractions were diluted with PBS (0.01 mol/L sodium phosphate, pH 7.4, with 0.145 mol/L NaCl) containing 0.01% BSA. The samples were then applied in triplicate to a slot blot apparatus attached to a vacuum source. A standard curve was also constructed by use of a standard plasma of known apoA-I concentration. After 25 µL of each sample was applied to each slot, the membrane was removed, incubated for 10 minutes in PBS with 0.03% glutaraldehyde for 10 minutes, and washed twice and then incubated with PBS with 5% nonfat dry milk and 0.05% Tween-20 for another 10 minutes. Immunolocalization occurred when membrane was incubated in a solution containing PBS with 0.05% Tween (PBST) with 5% nonfat dry milk and the specific anti-dog apolipoprotein antisera. Incubation was at room temperature for 3 hours. The membrane was again washed three times for 5 minutes each time with PBST. The membrane was then incubated with iodinated anti-goat antibody, also for 3 hours, and washed as above. After the final wash, the membrane was completely dried and exposed on the PhosphorImager (Molecular Dynamics) cassette. Data were quantified on the phosphoimaging device. Phospholipid analyses were by the method of Zilversmit and Davis.16
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Results |
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Almost all of the cholesterol content of dog plasma is transported in particles with HDL densities. Cholesterol levels of VLDL+IDL in the dog are only 5% of the total plasma content.17 If we assume that most of the apoB-containing lipoproteins are in VLDL+IDL, we see that only 5% of the total plasma apoB is found in peripheral lymph.17 Therefore, the amount of VLDL+IDL in the interstitial fluid would be less than 0.5% of that in plasma. In addition, we have determined that no apoA-I exists in the peripheral lymph VLDL+IDL fraction.17 To determine the presence of other lipoproteins in the fractions isolated, we subjected the fractions to SDS–polyacrylamide gel electrophoresis followed by immunoprobing with antibodies to dog apoB, apoE, apoA-I, and apoA-IV (data not shown). We reasoned that the presence of apoB and apoE may signify the presence of VLDL+IDL. The plasma pre-ß fraction showed immunoreactivity to apoB, apoE, apoA-I, and apoA-IV. Therefore, it would appear that the plasma pre-ß fraction is contaminated with other lipoproteins. In contrast, lymph pre-ß lipoproteins showed immunoreactivity only to apoA-I. Plasma and lymph
and pre-
fractions also showed immunoreactivity only to apoA-I. We interpret
this to indicate that contamination with other lipoproteins is minimal
and not likely to interfere with our determinations. A typical plasma
and lymph profile of apoA-I is shown in Fig 2. The
experimental molecular weights are shown in Table 2. The
percent lipid distributions of the plasma and lymph lipoproteins are
shown in Fig 3. From this figure it can be seen that
there were both relative concentration and compositional differences
between plasma and lymph pre- lipoproteins. The plasma pre-
lipoproteins contained more triacylglycerol (16%
versus 4%) and less phospholipid (56% versus 71%) than the lymph
pre- lipoproteins. Interestingly, there were no major differences
between plasma and lymph -migrating particles (9% free
cholesterol, 20% cholesterol ester, 1%
triacylglycerol, and 70% phospholipid). There were
differences between plasma and lymph pre-ß particles. The plasma
pre-ß particles contained more cholesterol ester (30%
versus 13%) and less phospholipid (45% versus 61%) than lymph
pre-ß particles. Triacylglycerol (9% versus
11%) and free cholesterol (16% versus 15%) amounts were
similar between plasma and lymph pre-ß lipoproteins.
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We have previously shown that lymph had a higher weight ratio of free
to esterified cholesterol than did plasma,9
and we wondered where the increase in free cholesterol
would reside in the lymph lipoprotein fractions. A plot of the free
cholesterol–to–esterified cholesterol
ratios of the lipoprotein fractions, illustrated in Fig 4 (top), showed that all the lymph
fractions are enriched in free cholesterol, with lymph
pre-ß showing the greatest increase, followed by lymph particles
and lymph pre- particles. The ratio of free cholesterol
to phospholipid, illustrated in Fig 4 (bottom), showed
that both lymph and plasma pre-ß had the highest ratio of free
cholesterol to phospholipid. The and pre- fractions
had similar ratios, with lymph particles having slightly higher ratios
than plasma.
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For calculation of the stoichiometry of these particles, data from a
set of three dogs were used. The plasma and lymph lipoprotein fractions
were analyzed for lipid components and for apoA-I. The results
are shown in Table 1
. The majority of the lipid and apoA-I in plasma
resided in the fraction. Plasma pre-ß and pre- accounted for
10% and 3% of the total plasma apoA-I, respectively. In contrast,
lymph pre-ß and pre- accounted for 14% and 37% of the total
lymph apoA-I. By assuming a weighted average molecular weight for
phospholipid (645 D), cholesterol ester (813 D), and
triacylglycerol (886 D), we could calculate the
stoichiometric relationship of the plasma and lymph particles. The
results are presented in Table 3. Also
presented in Table 3 are the calculated molecular weights
of the lipoprotein fractions. Because molecular weight standards were
routinely run with NONDEGRA-PAGE, we could compare the calculated
molecular weight with observed average molecular weights of particles
in each mobility class (see Tables 2 and 3). The calculated molecular
weights of most particles were consistent with the actual
determined molecular weights. Plasma and lymph pre- particles
probably contain four molecules of apoA-I, and plasma and lymph
particles probably contain two molecules of apoA-I. Lymph pre-ß
particles probably contain one molecule of apoA-I. The calculated
molecular weight of the plasma pre-ß particle did not correspond to
that of the observed molecular weight. In view of our previous
observation that plasma pre-ß particles contained apoE and other
apolipoproteins, they were probably contaminated by other lipoproteins.
Therefore, the lipid composition of plasma pre-ß particles was most
likely a composite of different apoprotein-containing
lipoproteins.
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Discussion |
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Phillips et al18 and Wille et al19 have used agarose gel electrophoresis to identify a pre-
lipoprotein. The
lipoproteins identified by these two groups showed a high
lysophosphatidylcholine level, possibly because of the high
albumin content of the fraction. The lipoproteins that we
identified, however, migrate ahead of those they identified.
The isolation of lipoproteins from the plasma and lymph of dogs by
electrophoretic mobility would result in lipoproteins having limited
purity. Two sources of impurities are considered: contamination by
plasma proteins and contamination by other lipoproteins. In plasma,
apoA-I–containing pre-ß lipoproteins comigrated with VLDL+IDL to the
same electrophoretic mobility. In addition, in human plasma, Castro and
Fielding5 defined three populations of apoA-I–containing
pre-ß–migrating lipoproteins (pre-ß1,
pre-ß2, and pre-ß3). Dog plasma and
lymph lipoprotein patterns are simpler. Dogs have only one population
of pre-ß–migrating apoA-I–containing lipoproteins
(pre-ß1)8 and two molecular weight
populations of and pre- lipoproteins. However, contamination by
other lipoproteins cannot be ruled out. We tested for these
contaminations immunologically. Our tests showed that plasma pre-ß
fractions were indeed contaminated by other lipoproteins (VLDL+IDL and
apoA-IV–containing lipoproteins). However, lymph pre-ß lipoproteins
did not show such contamination by VLDL+IDL. Contamination by plasma
proteins was ignored in all fractions because the plasma proteins do
not bind significant amounts of lipid. We should also emphasize that
the calculated stoichiometric compositions of plasma and lymph
lipoproteins are averages of the two molecular weight populations of
and pre- lipoproteins.
This is the first report of the lipid composition of plasma and lymph
pre- and lymph pre-ß particles. The lipid composition of plasma
and pre- particles suggests that these two lipoprotein
populations are clearly distinct from each other. Plasma and lymph
particles have similar compositions (Fig 3). This makes
it likely that lymph lipoproteins with mobility are derived
directly from plasma lipoproteins with mobility. The similarity of
composition between plasma and lymph lipoproteins suggests that
either the lipoproteins did not pick up any lipids after they
crossed the interstitial space or that after interaction
with cells the lipoproteins acquire a different mobility. The lipid
composition of plasma pre- particles is significantly different from
lymph pre- particles. Plasma pre- lipoproteins contained more
triacylglycerol than lymph pre- lipoproteins.
The increased triacylglycerol content of plasma
pre- lipoproteins is probably caused by the action of
cholesterol ester transfer protein (CETP). The dog has low
but detectable CETP activity20 ; therefore, the
triacylglycerol could come from the exchange of
cholesterol ester for triacylglycerol.
The fate of the plasma pre- lipoprotein
triacylglycerol after it crossed the capillary wall
is enigmatic. Because the lymph pre- lipoprotein did not have high
triacylglycerol, it seems likely that the lipid is
either hydrolyzed by lipases in the interstitial space or
removed as the particles cross the capillary wall. Indeed, we have
found lipoprotein lipase but not hepatic lipase in the
peripheral lymph.21 One consideration is
whether lymph pre- particles could be derived from plasma pre-
particles. The concentration of apoA-I in peripheral lymph
is only 10% of that of plasma. Because the lymph pre- particles
have molecular weights similar to those of plasma and pre-
particles, we would expect the same amount of pre- particles to be
filtered through the interstitial space as that of
plasma lipoproteins (10%). By analyzing the absolute concentration
of apoA-I from plasma and lymph pre- fractions (plasma has 6 mg/dL
apoA-I and lymph has 8 mg/dL apoA-I), we conclude that only 0.6 mg/dL
of lymph pre- particles could come from plasma. The vast majority
(7.4 mg/dL) of the lymph pre- particles must be derived from other
particles. This conclusion leaves open the possibility that lymph
pre- particles may be generated in part within the interstitium. The
most likely source of lymph pre- particles is the lymph
particles. Evidence in support of this latter suggestion is our
observation that the lymph pre- and particles have very similar
lipid compositions (Fig 3). As to the fate of the
majority of plasma pre- particles (5.4 mg/dL), they could simply be
taken up by the liver. Bamberger et al22 have provided
evidence that hepatic lipase stimulates the uptake of HDL
cholesterol in liver cells. It is conceivable that the
plasma pre- lipoproteins, with their high
triacylglycerol content, are good substrates for
hepatic lipase.
Lymph lipoproteins have a higher ratio of free to esterified
cholesterol mass9 than plasma lipoproteins. An
analysis of the electrophoretically separated fractions showed
that pre-ß, , and pre- all had higher ratios of free to
esterified cholesterol mass (Fig 4), but
lymph pre-ß particles had the highest ratio of all. When data were
expressed as molecules per unit apoA-I, lymph pre-ß particles had 10
molecules of free cholesterol to 7 molecules of
cholesterol ester.
Several investigators have proposed that the ratio of free
cholesterol mass to phospholipid mass on a lipoprotein
particle is more important than the apolipoprotein species in
determining the ability of the particles to accept or donate
cholesterol from or to the cell.23 24 If the
ratio is high, then the net flux is from particle to cell membrane;
otherwise, it is the reverse. This hypothesis would predict that both
plasma and lymph pre-ß particles function as net donors of
cholesterol to cells, whereas the other particles are
cellular cholesterol acceptors. Our data for dog plasma
pre-ß lipoproteins' ratio of free cholesterol to
phospholipid is in excellent agreement with that reported by Castro and
Fielding5 for human pre-ß1 particles (0.19
versus 0.22). The observation that such a particle is in fact a good
cellular cholesterol acceptor suggests that either lymph
pre-ß particles result from the combination of cell-derived
cholesterol and another apoprotein A-I–containing particle
or that factors other than the ratio of free cholesterol to
phospholipid play a role in cellular cholesterol efflux.
Indeed, Fielding and Fielding6 have found a unique epitope
on pre-ß1 particles that may also be responsible for
cholesterol efflux. It should be pointed out that the
hypothesis concerning ratios of free cholesterol to
phospholipid was studied with recombinant HDLs, whereas our
experiments, and those of Castro and Fielding5 were
carried out with native lipoproteins. Even though lymph and pre-
lipoproteins have a higher ratio of free to esterified
cholesterol, their ratio of free cholesterol to
phospholipid may not be high enough to make them net
cholesterol donors. Although it is unlikely, we cannot rule
out the possibility that plasma and lymph pre-ß particles may be
generated in their respective compartments and that the two are
independently formed.
Once the lipid and apolipoprotein contents of these lipoproteins
are known (Table 1), it is not difficult to calculate
the stoichiometric relationship of apoA-I to other lipids. Our data
suggest that pre-ß particles contain one molecule of apoA-I. This is
similar to the observation of Castro and Fielding,5 who
reported partial lipid data for pre-ß1 particles. That
particles contained two molecules of apoA-I is consistent
with previous reports.25 26 Using chemical crosslinking,
Swaney26 and others have found that
HDL2 contained four molecules of A-I per particle.
The pre- particles, which contained four molecules of apoA-I per
particle, may very well be part of HDL2. Determined
on the basis of the calculated molecular weight, the data are within
experimental error of the results found by use of our NONDEGRA-PAGE
technique. However, as determined by the amount of apolipoprotein, this
particle is relatively lipid poor and may not be stable. We postulate
that this particle is formed by the combination of four pre-ß
particles or by the relative delipidation of particles. Whether
this particle is converted to other forms is also not known. Clearly,
more studies are needed to clarify these questions.
Because lymph pre- and particles have similar lipid
compositions, the difference in charge between the two particles is
most likely caused by a difference in phospholipid amount (56% versus
71%) and species (for example, an increase in phosphatidylethanolamine
and phosphatidylinositol). The amount of material we recovered is
insufficient for us to conduct a detailed phospholipid analysis
of the different particles.
In conclusion, we have provided lipid composition data on dog plasma and lymph lipoprotein particles separated by electric charge. Their differences and similarities with respect to cholesterol metabolism have also been discussed.
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Acknowledgments |
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This research was supported by NIH program project grant HL-25596. We wish to thank our colleagues Dr J.J. Thompson and Dr Lucille Lee for their many suggestions and discussions. We also wish to thank Dr A. Kuksis of the University of Toronto and Dr P. Dolphin of Dalhousie University for their help in setting up the gas-liquid chromatograph for lipid analyses. The expert technical assistance of Kati Horvath, Asya Shoichet, Chandra Tate, and Colleen Tierney is gratefully acknowledged.
Received April 24, 1995; accepted August 14, 1995.
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- Pieters MN, Castro GR, Schouten D, Duchateau P, Fruchart JC, Vanberkel TJC. Cholesterol esters selectively delivered in vivo by high-density-lipoprotein subclass lpA-I to rat liver are processed faster into bile acids than are lpA-I/A-II-derived cholesterol esters. Biochem J. 1993;292:819-823.
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