© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Concentrations of Electrophoretic and Size Subclasses of Apolipoprotein A-I–Containing Particles in Human Peripheral Lymph
From the Department of Cardiovascular Biochemistry (M.N.N., C.J.C., W.L.O., N.E.M.), St Bartholomew’s and the Royal London School of Medicine and Dentistry, London, UK, and the Department of Surgical Research and Transplantology (W.L.O.), Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland.
Correspondence to Prof Norman E. Miller, Department of Cardiovascular Biochemistry, St Bartholomew’s and the Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK. E-mail n.e.miller{at}mds.qmw.ac.uk
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Abstract |
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Abstract—When cultured cells are exposed to plasma, the initial acceptors of unesterified cholesterol are small lipid-poor apolipoprotein A-I (apoA-I)–containing high density lipoproteins (HDLs) with pre-ß electrophoretic mobility. These are converted by lecithin:cholesterol acyltransferase into larger spheroidal cholesteryl ester–rich HDLs with
mobility. To study the
determinants of the concentration of small pre-ß HDLs in tissue
fluids, we collected prenodal peripheral lymph from 34
fasted normal men. By crossed immunoelectrophoresis, the concentration
of pre-ß HDLs in lymph averaged 20% of that in plasma. On multiple
regression analysis, pre-ß apoA-I concentration in lymph was
directly related to pre-ß apoA-I concentration in plasma and
independently to apoA-I concentration in lymph. Similar results
were obtained when the same apoA-I–containing particles were
quantified by size exclusion chromatography. Lymph
pre-ß apoA-I concentration was low in a subject with familial
lecithin:cholesterol acyltransferase deficiency, despite a
normal plasma pre-ß apoA-I concentration, but was normal in a subject
with familial lipoprotein lipase deficiency. These results suggest that
the concentration of small pre-ß HDLs in human tissue fluids is
determined only in part by the transfer of pre-ß HDLs across
capillary endothelium from plasma. Local
production, by remodeling of spheroidal HDLs in tissue
fluids, may be equally important. Lipolysis of
triglyceride-rich lipoproteins by lipoprotein lipase
appears to have little effect.
Key Words: apoA-I • lymph • lipoprotein subclasses • LCAT deficiency • LPL deficiency
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Introduction |
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Apolipoprotein A-I (apoA-I) is the principal protein component of plasma HDLs and the cofactor of lecithin:cholesterol acyltransferase (LCAT).1 The HDLs are a heterogeneous family of lipoproteins that mediate reverse cholesterol transport from tissues.1 2 When cultured cells are exposed to plasma, the principal primary acceptors of cell-derived unesterified cholesterol (UC) are a minor subclass of small lipid-poor HDLs, whose only protein is apoA-I and which have pre-ß electrophoretic mobility.1 2 3 4 These are converted by LCAT into larger cholesteryl ester (CE)-rich spheroidal particles, which have
electrophoretic mobility and constitute the majority of plasma
HDLs.1 2 4 5 The origins of the pre-ß HDLs are not
certain, but similar particles are formed when lipid-free apoA-I
recruits phospholipids (PLs) from cultured cells.6 7
Lipid-free or lipid-poor apoA-I is released when spheroidal HDLs are
remodeled by PL transfer protein, CE transfer protein, or hepatic
lipase.8 9 10 There is evidence that pre-ß HDL
concentration is rate limiting for the specific apolipoprotein-mediated
component of UC efflux from cells. Thus, when human plasma samples were
preincubated, proportional reductions occurred in pre-ß HDL
concentration and cholesterol efflux from
fibroblasts.3 Others have found that
cholesterol efflux from hepatoma cells to transgenic rabbit
plasma was correlated with pre-ß HDL
concentration.11
Our knowledge of HDLs and their role in reverse cholesterol
transport is derived mostly from studies of plasma. However, most
peripheral cells are exposed not to plasma but to tissue
fluid. Studies in dogs,12 sheep,13 and
humans14 have shown that tissue fluid lipoproteins differ
from plasma lipoproteins. These differences reflect the differential
transfer of lipoproteins across endothelia and metabolic
events in the extravascular space. ApoA-I–containing particles with
pre-ß and mobilities have been demonstrated in
canine15 and human16 peripheral
lymph. Subclasses with pre- mobility17 and lipid-free
apoA-I15 have also been reported. However, there is no
information on the factors that determine the concentrations of these
different of apoA-I–containing particles in tissue fluid.
The difficulties of studying tissue fluid lipoproteins in humans are considerable. Suction blisters provide insufficient fluid and may be misleading, because the increase in capillary permeability must alter its composition.18 The only reliable matrix is prenodal peripheral lymph. Almost all published data on human peripheral lymph lipoproteins were obtained with the use of a collection procedure (from the foot) that has several major problems: a high failure rate, low flow rates, a short cannulation life, and the need to preinject a dye subcutaneously. To obviate these problems, we have adapted a procedure that uses a larger vessel in the leg.19 In the present study, we have partially characterized the major electrophoretic and size subclasses of apoA-I–containing particles in human lymph and investigated the factors that determine their concentrations.
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Methods |
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Subjects
There were 36 subjects: 34 healthy males, 1 male with familial LCAT deficiency, and 1 male with familial lipoprotein lipase (LPL) deficiency (Table 1
). Subjects
were excluded if they had renal, hepatic, endocrine, or
cardiovascular disease or if they were on a diet (other
than for LPL deficiency) or medication. In the subject with LCAT
deficiency, plasma LCAT activity, assayed by use of
apoA-I/lecithin/[14C]cholesterol
proteoliposomes, was 1.3 nmol · mL-1
· h-1 (normal, 39.4 to 112 nmol ·
mL-1 · h-1), the
cholesterol esterification rate in vitro was 6.5 nmol
· mL-1 · h-1
(normal, 18.0 to 31.4 nmol · mL-1
· h-1), and the plasma UC/CE ratio was 4.47
(normal, 0.39 to 0.55). The subject with LPL deficiency, described
elsewhere,20 had been on a low fat diet for several
years. The study was approved by an ethics committee. All subjects gave
informed consent.
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Clinical Procedures
Cannulations were performed between 8:00 and 11:00
AM under sterile conditions. The subjects had fasted
overnight but were allowed fat-free drinks. One leg was shaved to 20 cm
above the ankle, and the skin was sterilized (0.05% chlorhexidine). An
area of skin (
4 cm2) 4 to 8 cm above the ankle
over the anteromedial aspect of the tibia was anesthetized with
2% lignocaine in adrenaline (1:100 000). A 15- to 20-mm horizontal
incision was made in the center of this area. Under an operating
microscope (model M650, Wild Heerbrugg), the subcutaneous lymph vessels
were dissected, and 1 was selected for cannulation. A second incision
(3 mm wide) was made 10 mm above the first, through
which a cannula (Intramedic polyethylene tubing PE-60, catalogue No.
427416, Becton Dickinson & Co; ID 0.76 mm, OD 1.22 mm) was
passed into the wound. This had been tapered at 1 end, siliconized with
Sigmacote (Sigma Chemical Co), sterilized in 70% (vol/vol) ethanol for
24 hours, and flushed with sterile 0.15 mmol/L NaCl. The lymph
vessel was ligated proximally with silk (Mersilk, 5/0) and opened with
capsulotomy scissors. The first valve distal to the opening was
destroyed with the use of curved blunt forceps. The cannula was
inserted for 5 to 10 mm (toward the foot) and secured with a silk
ligature (Mersilk, 5/0). The skin wound was then closed with 2 sutures
(Prolene, 3/0). The other (untapered) end of the cannula was passed
into a 2-mL screw-topped polypropylene cryovial (Nunc A/S) containing 2
mg Na2EDTA. A gauze dressing was applied, and the
cannula and collection tube were secured with tape. Lymph was collected
for 3 to 6 hours, during which time the subject was ambulatory and
given a light fat-free meal and water. A blood sample was taken from an
antecubital vein into Na2EDTA (final
concentration 1 mg/mL).
Laboratory Procedures
Blood and lymph samples were centrifuged
(1500g for 15 minutes at 4°C), and the supernatants were
transferred to polypropylene tubes. Lymph volumes were determined by
weighing. In all analyses, plasma-lymph pairs from the same
subject were processed together. All assays were performed in
duplicate.
Lipids and Apolipoproteins
Total cholesterol, total
triglycerides (TGs), UC, and total choline-containing PLs
were quantified by use of commercial enzymes (Sigma) in a microtiter
plate spectrophotometer.21 CEs were calculated by
difference. TG measurements were not corrected for free glycerol.
Precinorm L (Boehringer-Mannheim GmbH) was used as a
calibrator. Plasma HDL cholesterol was measured with the
use of polyethylene glycol 8000 (final concentration 8% [wt/vol]).
Apolipoproteins were quantified by liquid-phase double-antibody
radioimmunoassays or by rocket immunoelectrophoresis with the use of
Tween 20 (final concentration 0.2% [vol/vol]) to expose cryptic
epitopes.21 The primary antisera were goat polyclonal IgGs
against delipidated human apolipoproteins (International Immunology
Corp). In the radioimmunoassays, the precipitating antibody was donkey
anti-goat IgG (Chemicon). Radioiodinated tracers were
prepared with the use of delipidated human apolipoproteins or LDL
(density 1.020 to 1.055 g/mL). Radioactivity was quantified to 
0.1%
error. All assays were standardized with the use of Precinorm L.
Nonlipoprotein Proteins
Several proteins were assayed to examine the general
relationship of molecular weight to the lymph/plasma (L/P)
concentration ratio. 1-Acid
glycoprotein (38 kDa),
1-antitrypsin (54 kDa), albumin (67
kDa), transferrin (76 kDa), complement C3 (180 kDa), and
2-macroglobulin (750 kDa) were quantified by
immunoelectrophoresis with the use of polyclonal antisera
(International Immunology Corp).
Preparative Electrophoresis
In 4 subjects, the distribution of apoA-I, apoA-II, apoB, and
lipids among subclasses of lipoproteins isolated by preparative
electrophoresis was studied. Samples (500 µL) were electrophoresed at
2°C to 4°C in 18 cmx20 cmx2 mm-thick slab gels composed of
1% (wt/vol) low electroendosmosis agarose (without albumin)
for 3 to 4 hours at 150 V in a barbital/EDTA buffer system (50
mmol/L barbital, 20 mmol/L barbituric acid, and 1 mmol/L
Na2EDTA, pH 8.6) by use of a vertical submerged
electrophoresis chamber (model GE-2/4 LS, Pharmacia LKB). Plasma
samples were diluted 10-fold in electrophoresis buffer. Paired lymph
and plasma samples were processed in a single gel. After
electrophoresis, the gels were sliced transversely into 22 strips,
extending from the origin to 125% of the migration of the
bromophenol blue dye front. Lipoproteins were recovered from each strip
by ultracentrifugation (15 minutes at 4°C, 50 000
rpm) in a Beckman 50.4Ti rotor. Recoveries of apoA-I and
cholesterol were >95%. Lipids and apolipoproteins were
assayed as described above.
Crossed Immunoelectrophoresis
ApoA-I concentrations in pre-ß and migrating particles
were quantified by crossed immunoelectrophoresis.21
Lymph-plasma pairs were run in the same gel. Lymph was run undiluted,
but plasma was diluted 1:4 with electrophoresis buffer, so as to
provide similar masses of apoA-I. The first dimension was through a 1%
(wt/vol) low electroendosmosis agarose slab gel (SeaKem LE, FMC
Bioproducts) at 30 V/cm for 2 hours at 4°C with the use of
63 mmol/L Tris, 27 mmol/L tricine, 1 mmol/L calcium
lactate, and 3 mmol/L sodium azide (pH 8.6) as electrolytes. The
second dimension was through the same gel impregnated with 0.5%
(vol/vol) goat polyclonal anti-human apoA-I serum (INCStar Corp), 0.2%
(vol/vol) Tween 20, and 3% (wt/vol) polyethylene glycol 8000, at 15
V/cm for 4 to 6 hours at 4°C with use of the same buffer.
Electrophoresis was always run to equilibrium. After they were soaked
in 150 mmol/L NaCl for 18 hours at ambient temperature,
antigen-antibody complexes were stained for 1 hour with 0.5% (wt/vol)
Coomassie blue R250 in ethanol/acetic acid/water (9:2:9 [vol/vol]),
and the relative proportions (area under the curve) of apoA-I in
pre-ß and regions were quantified. Absolute apoA-I concentrations
in the subclasses were calculated by reference to the total apoA-I in
the sample. The intra-assay coefficient of variation for pre-ß apoA-I
concentration was 11% (n=8, mean 5 mg/dL).
Size Exclusion Chromatography
In 18 normal subjects and the LPL-deficient subject,
high-performance size-exclusion chromatography
(HP-SEC) was used to separate apoA-I–containing particles into 3 size
subclasses. Aliquots (50 µL) of 4-fold diluted plasma or undiluted
lymph were passed through Superdex 200 and Superdex 75 columns (HR
10/30, Pharmacia) in series at ambient temperature.22 A
degassed solution of 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L
NaCl, 0.1% (wt/vol) Na2EDTA, and 0.1% (wt/vol)
sodium azide was used as an eluant (0.75 mL/min). After excluding the
void volume, 200 µL fractions were collected and assayed for apoA-I.
Recoveries were >90%. Particle sizes were determined with the use of
a protein mixture (catalogue No. MW-GF-1000, Sigma; molecular
mass 29 to 700 kDa). This procedure separates apoA-I–containing
particles in plasma into a major population of 70 to 500 kDa (fractions
16 to 41), a minor population of larger particles (>500 kDa, fractions
11 to 15), and a minor population of small particles (40 to 60 kDa,
fractions 42 to 56).21 22 The major population is composed
of CE-rich spheroidal HDLs. The small particles appear to be a
mixture of lipid-poor apoA-I (pre-ß1 HDLs) and
lipid-free apoA-I dimers.22
Statistical Analyses
Data from plasma and lymph samples from the same subjects were
compared statistically by the Student paired t test.
Associations were examined by the Pearson coefficient of linear
correlation. A value of P<0.05 was considered to be
statistically significant.
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Results |
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The total volume of lymph varied from 0.10 to 8.2 mL (mean±SEM, 1.71±0.28 mL). Flow rates averaged 0.68±0.14 mL/h (range 0.03 to 4.09 mL/h). In the normal subjects, total apoA-I concentration in lymph averaged 21.4±1.3% of that in plasma. Concentrations in the 2 matrices had a weak positive association (r=0.31, P=0.08). In 3 subjects, the ratio of apoA-I mass in HDLs with and without apoA-II (determined by immunoprecipitation of apoA-II) was similar in lymph and plasma (2.43±0.34 versus 2.17±0.09).
Preparative Electrophoresis
In both matrices, apoA-I migrated as a minor pre-ß band
and a major band, whereas apoA-II migrated only as an band
(Figure 1). The apoA-I and apoA-II of
particles migrated slightly faster in lymph than in plasma. ApoB
migrated mostly as a ß band in lymph and plasma, with no difference
in mobility between the matrices. All lipids eluted in 2 peaks
corresponding to the major and ß regions. Lymph contained
relatively more of each lipid in the region and, within this
region, relatively more in the faster migrating particles.
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) and lymph (•) separated by
preparative agarose electrophoresis. Results are from a
representative normal subject. The continuous lines
represent the L/P ratios in corresponding fractions.
Crossed Immunoelectrophoresis
No major differences were observed between the 2 matrices in the
mobilities of the pre-ß or peaks (Figure 2). Concentrations of pre-ß and
migrating apoA-I are summarized in Table 2; the mean pre-ß/ ratio in lymph
was not significantly different from that in plasma. The concentration
of pre-ß apoA-I in lymph was positively correlated with that in
plasma (Figure 3). The correlation
between the apoA-I concentration in lymph and that in plasma was
not statistically significant (Figure 3). Within both matrices,
there were significant positive correlations between the pre-ß apoA-I
and apoA-I concentrations (Figure 3).
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). Results obtained in the subjects with LCAT
deficiency (LCAT DEF., 
) are also
shown. The correlation coefficients are for the normal subjects.
Size Exclusion Chromatography
Typical size exclusion profiles of apoA-I appear in Figure 4. The results in normal plasma were as
previously described.21 22 The size distributions of the
small particles were similar in the 2 matrices. However, the
distribution of apoA-I within the major population of particles was
shifted toward the larger species and often also (to a lesser degree)
toward the smaller species, in lymph compared with plasma. In lymph,
the concentration of apoA-I in the small particles but not in the major
population of particles was significantly positively correlated with
its counterpart in plasma (Figure 5).
Within both matrices, the concentration of apoA-I in the small
particles was positively correlated with that in the major population
of particles (Figure 5). The ratio of apoA-I in the small
particles to that in the major population was similar in lymph and
plasma (Table 2
).
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Nonlipoprotein Proteins
In normal subjects, the L/P ratios of other proteins were
inversely related to their molecular masses (Figure 6). The mean L/P ratio of apoA-I in large
HDLs was close to that predicted for a macromolecule of 200 kDa
(the approximate mean molecular mass of most plasma HDLs). The mean L/P
ratio of apoA-I in small pre-ß HDLs was lower than expected for
particles of 40 to 60 kDa.
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) and pre-ß migrating (
) HDLs
are shown at 50 kDa. The curve was fitted by least-squares regression
(excluding apoA-I).
Familial LPL Deficiency
In plasma and lymph from the LPL-deficient subject, the
concentrations of pre-ß and migrating apoA-I were within the
normal ranges (Figure 3). Concordant results were obtained by
HP-SEC (Figure 5).
Familial LCAT Deficiency
The LCAT-deficient subject had a normal plasma pre-ß apoA-I
concentration but extremely low concentrations of apoA-I in plasma
and of pre-ß apoA-I and apoA-I in lymph (Figure 3).
Insufficient lymph was available from this subject for HP-SEC.
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Discussion |
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The present study is the first to examine the associations between the concentrations of apoA-I in the major electrophoretic and size subclasses of particles in human peripheral lymph and plasma. Reichl and coworkers16 23 24 characterized the distribution of apoA-I in human foot lymph by gel filtration, gradient gel electrophoresis, and starch block electrophoresis. However, because those studies were mostly semiquantitative and because each included only 3 to 6 subjects, they provided no information on the relationships of the different subclasses with each other or with their counterparts in plasma.
The vessel that we cannulated collects lymph from skin and subcutaneous tissues.18 In animals, the protein composition of subcutaneous lymph closely resembles that of local tissue fluid collected by micropuncture or by insertion of wicks or capsules.25 26 27 The dynamics of fluid flow in the interstitium favor the lymphatic over the venous capillary route for the return of macromolecules to plasma.18 Although regional differences in the lipoproteins of peripheral lymph have been described in animals, these have not been large.28 29 Therefore, our data are probably representative of most peripheral tissue fluids in humans. However, because all lymph was collected during the morning after an overnight fast, our results cannot be extrapolated to other times of the day or to the nonfasted state.
The lipids and apolipoproteins of peripheral lymph lipoproteins are derived largely from plasma. It is probable that lipoproteins cross endothelia mostly by filtration,30 31 although transcytosis may also contribute.32 Filtration favors small over large particles.30 31 33 Once outside the plasma compartment, the smallest particles will have the greatest probability of interacting with peripheral cells because of the sieving effect of the extracellular matrix.34
By preparative electrophoresis, we found that (as in plasma) the
pre-ß HDLs of lymph were devoid of apoA-II. Because of the proximity
of the pre-ß band to the apo B–containing lipoproteins, we could not
accurately determine the lipid content of the former. Within the
migrating species, apoA-I, UC, CE, and PLs were shifted toward the more
negatively charged particles relative to their profiles in plasma.
These results confirm and extend the observations of Reichl et
al.16 Possible explanations for this charge difference
include differences in apoA-I conformation and PL composition.
By crossed immunoelectrophoresis, pre-ß apoA-I concentration in lymph
averaged 20% of that in plasma, and there was no difference between
the matrices in the pre-ß/ ratio. Lymph pre-ß apoA-I
concentration was positively correlated with lymph apoA-I and
plasma pre-ß apoA-I concentrations but was not significantly
correlated with plasma apoA-I concentration. Although we failed to
identify any apoA-I with pre- mobility, as reported in canine
lymph,17 this may have reflected the lower resolving power
of our procedure.
In view of the likely importance of the small pre-ß particles as
acceptors of cell-derived UC in tissue fluids, we examined by multiple
linear regression the independent effects of the other fractions on
lymph pre-ß apoA-I concentration. This showed that lymph pre-ß
apoA-I was independently positively related to the plasma pre-ß
apoA-I and lymph apoA-I concentrations (P<0.0005 for
each coefficient) as follows: lymph pre-ß apoA-I=0.007+(0.075
· plasma pre-ß apoA-I)+(0.042 · lymph apoA-I).
Changes of 1 SD in the plasma pre-ß and lymph apoA-I
concentrations were associated with changes in lymph pre-ß apoA-I of
0.53 and 0.51 SD, respectively. Fifty-eight percent of the
interindividual variance in lymph pre-ß apoA-I concentration could be
explained in this way (P<0.0001).
Our HP-SEC results showed that the population of small
apoA-I–containing particles, previously shown in plasma to have
pre-ß electrophoretic mobility,22 had the same size
profile in lymph as in plasma. The distribution of apoA-I within the
major population, previously shown to be CE-rich
HDLs,22 was shifted toward larger particles in lymph. This
accords with reports that canine peripheral
lymph,35 sheep lung lymph,13 and human foot
lymph16 23 are enriched in large HDLs.
When we examined the correlations between the concentrations of apoA-I
in the 2 principal size subpopulations in lymph and plasma, the outcome
was analogous to that obtained for apoA-I in pre-ß and migrating
species. Multiple regression analysis also yielded analogous
results: the apoA-I concentration in the small particles of lymph was
positively related to that in the small particles of plasma and
independently to that in the large particles of lymph (not shown).
Thus, in normal fasted humans, the concentration of small pre-ß HDLs
in peripheral tissue fluid appears to be determined not
only by the concentration of pre-ß HDLs in plasma, presumably
reflecting their transport across endothelium, but also
by the concentration of spheroidal HDLs in the tissue fluid.
The positive association of lymph pre-ß apoA-I with lymph apoA-I
probably reflects local production of the former particles from
the latter particles in tissue fluid rather than interconversion in the
opposite direction. First, we have found that when lymph is incubated
at 37°C in vitro, pre-ß apoA-I concentrations increase without the
initial decrease that invariably occurs when normal plasma is
incubated.3 4 5 36 Second, LCAT activity, which catalyzes
the conversion of pre-ß HDLs to CE-rich HDLs,4 5
appears to be very low in tissue fluid.37 38 We have
confirmed that human lymph has an extremely low cholesterol
esterification rate in vitro (M.N.N. et al, unpublished data, 1999). In
the present study, we found in 3 subjects that the UC/CE and
pre-ß apoA-I/ apoA-I ratios in samples of lymph collected at
ambient temperature were identical to those in samples collected from
the same vessels into tubes kept at 0°C to 4°C (data not
shown).
Thus, our results support the notion that extravascular remodeling of spheroidal HDLs may play an important role in reverse cholesterol transport by generating small partially lipidated apoA-I–containing particles with high affinity for UC in the vicinity of peripheral cells. This might involve the release of lipid-free apoA-I from spheroidal HDLs, followed by the recruitment of PLs from cell membranes by the apoA-I.6 7 Further work is in progress in our laboratory into the mechanism of production of small pre-ß HDLs in human lymph.
In the subject with LPL deficiency, the concentration of apoA-I in
small pre-ß HDLs in lymph did not differ greatly from that predicted
by the concentrations of pre-ß apoA-I in plasma and apoA-I in
lymph (based on the regression equation in normal subjects). This
suggests that neither the fractional rate of transfer of pre-ß HDLs
from plasma across endothelium nor their generation in
tissue fluids from HDLs is greatly influenced by the lipolysis of
TG-rich lipoproteins at the blood-endothelium
interface.
The L/P ratio of pre-ß apoA-I by crossed immunoelectrophoresis was
essentially identical to the L/P ratio of apoA-I. Likewise, the L/P
ratio of apoA-I in small particles by HP-SEC was similar to the L/P
ratio of apoA-I in the major population of particles. This is of
interest, because one would expect the pre-ß HDLs, by virtue of their
smaller size, to cross endothelium from plasma more
readily than HDLs, giving them a greater L/P
ratio.30 31 When we measured the L/P ratios of several
nonlipoprotein proteins of different molecular masses, the L/P ratio of
pre-ß apoA-I was lower than expected for particles of 40 to 60 kDa.
In contrast, the L/P ratio of apoA-I was as expected for a
macromolecule of 200 kDa average molecular mass. For reasons already
discussed, the lower than expected pre-ß apoA-I concentration is
unlikely to have been a consequence of LCAT-mediated conversion of
pre-ß HDLs to HDLs either in vivo or in the collection tube. This
suggests that there are metabolic factors in
peripheral tissues that either degrade pre-ß HDLs or
convert them into other particles by an LCAT-independent mechanism.
Theoretically, these might include catabolism by cells, although tissue
culture studies have indicated that this is unlikely to be
quantitatively significant.39 Because pre-ß HDLs are
more susceptible to proteolysis than are other HDLs,40
they might be degraded by enzymes released into tissue
fluid,41 42 although no apoA-I fragments were detected in
our HP-SEC fractions of lymph. A third possibility is that some small
pre-ß HDLs are converted in tissue fluid into larger discoidal
species that are incompletely resolved by our laboratory procedures, as
a consequence of uptake of cell-derived PL and
UC.2 13 35 38
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Acknowledgments |
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This work was supported by the British Heart Foundation. This article is dedicated to the late Drago Reichl, PhD.
Received October 28, 1999; accepted April 3, 2000.
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