Vol. 80, No. 1, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
January 13, 1978
Pages
Identification Containing
of Nascent Arginine-Rich
81-88
High Density Lipoproteins Protein in Human Plasma
James 8. Ragland,
Phillip D. Bertram and Seymour M. Sabesin Division of Gastroenterology of Tennessee Center for the Health Sciences and Veterans Administration Hospital Memphis, Tennessee
University
Received
November 22,
1977
Summx A high density lipoprotein fraction accumulates in the plasma of pasts with alcoholic hepatitis when a severe 1ecithin:cholesterol acyltransferase (EC 2.3.1.43) deficiency is present. The major apoprotein present in this fraction is arginine-rich protein, the fraction is a preferred substrate for 1ecithin:cholesterol acyltransferase, and by electron microscopy appears as stacked bilayer discs. It is proposed that the lipoprotein represents the accumulation of nascent high density lipoprotein and is the principal pathway through which arginine-rich protein is secreted by the liver in man. The results also suggest that apoprotein AI is acquired by normal high density lipoprotein during the course of lipoprotein metabolism. INTRODUCTION Circulating nascent
plasma
lipoproteins
showed
that
lipoproteins
of hepatic
nascent
contained
was enrimzhed
in unesterified
bilayer
stained
particles
25 H, frequently
constituent
(2).
It
preferred
forming
with
of circulating that purified
perfusates
et al
in the presence esters
HDL revealed
relatively
apoprotein small
and protein
disc-shaped
lipid
human LCAT.
Marsh
(3)
of apo AI,
plasma
added
of 190 -+
in rat
activator
than
and
microscopy
amounts
HDL rather
of
Electron
present
HDL and a required
of
(1)
of 46 -+ 5 5: and diameter
The major
nascent
the metabolism
Hamilton
and phospholipid.
plasma rat
from
of cholesteryl
of nascent
rouleaux.
protein
for
levels
liver
a mean edge thickness
was demonstrated substrate
origin.
in rat
cholesterol
preparations
HDL was arginine-rich major
decreased
with
to be derived
or intestinal
HDL accumulating
LCAT inhibitor
of negatively
appear
nascent the
of LCAT HDL was the
labeled
amino
acid
HDL, high density The following abbreviations will be used in this paper: VLDL, very low density lipoprotein; lipoprotein; LDL, low density lipoprotein: LCAT, 1ecithin:cholesterol acyltransferase (EC 2.3.1.43). ape, apoprotein; 0006-291X/78/0801-0081$01.00/0 81
Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
BIOCHEMICAL
Vol. 80, No. 1, 1978
precursors
to a nonrecirculating
apoproteins
secreted
were
ape B, and apo C with rich
protein
with
hereditary
protein
perfusion protein
It
has also
been
contain
and found
(highest
most of the radioactivity
LCAT deficiency
injury
appears
hepatitis
the nascent
(6),
shown that
that
specific
of nascent
bilayer
ester
further
that
the major activity),
HDL in arginine-
HDL fractions
discs
of patients
and arginine-rich
as bilayer
characterized that
it
from
rat
with
LCAT deficiency,
stacked
similar liver
Further,
alcohol-induced very
low
composition In the present
subjects
in composition
perfusates.
lipid
discs.
the HDL from several is
acute
and HDL of abnormal
microscopy
and find
patients
have an extreme
concentrations,
HDL isolated for
we reported
hepatitis)
by electron
we have
substrate
study
(alcoholic
cholesteryl
study, holic
liver
(4,5).
plasma which
rat
arginine-rich
and apo C.
In a recent liver
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
with
alco-
and ultrastructure it
to
is a preferred
human LCAT. MATERIALS
AND METHODS
Six patients with alcoholic hepatitis were selected for study on the basis of clinical and laboratory findings previously described (6). They were admitted to the Clinical Research Center and studied throughout the course of their illness until recovery (4-12 weeks). All blood samples for lipoprotein studies were collected in EDTA following a 14 hour fast. Dithionitrobenzoic acid was added to all samples to inhibit LCAT activity. Lipoprotein density classes were isolated by sequential ultracentrifugation and lipid and total protein content determined as previously described. HDL fractions studied represent d b 1.063, < 1.21. Lipoproteins were solubilized by boiling in 1% sodium dodecyl sulfate for one minute and apoproteins separated by polyacrylamide gel electrophoresis, essentially according to the method of Laeammle (7) using 5% stacking gel and 15% running gel. Apoprotein composition was estimated by densitometry of the Coomassie Blue stained gels without correction for differences in dye binding between different apoproteins. Arginine-rich protein was isolated from alcoholic hepatitis HDL by gel filtration on Sephadex G-200 and preparative electrophoresis. Analytical electrophoresis of the preparation revealed a single band corresponding to argininerich protein of normal VLDL. Amino acid analyses were performed through the courtesy of Drs. A.H. Kang and J.M. Seyer, Veterans Administration Hospital, Memphis, Tennessee. LCAT activity was assayed essentially as described by equivalent to 5pg of unesterified cholesterol HDL fragtions with [ C] cholesterol and assayed in a total volume of 0.2 plasma fraction of d > 1.225 as enzyme source. Blanks were enzyme was added and also in which no HDL source was added from the results reported.
82
Hamilton et al (1). were equilibrated ml using a normal run in which no and were subtracted
BIOCHEMICAL
Vol. 80, No. 1, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE
1
Percent Composition of HDL from Patients with Alcoholic Hepatitis on Admission and Following Recovery Unesterified Cholesterol
Cholesteryl Esters
Admission n=6
13.7 9 1.1-
1.5 + 0.4-
Recovery n=4*
4.4 + 4.0-
18.0 + 2.6 -
< .OOOl
< .OOOl
p** i/ * **
Triglycerides
Phospholipid
11.3 + 2.95.1
+
4.6 .36
Total Protein
45.8 + 3.5-
27.8 + 2.1
27.4 + 4.1
43.1 + 2.8-
< .OOl
< .OOOl
Values in the Table are the mean percentages of each component found in all patients + 1 standard error of the mean. Of six patients admitted to the study, two did not recover. Probability of difference between mean of admission and recovery groups.
RESULTS The composition icantly
different
of HDL from patients from
that
essentially
normal
(Table
cholesteryl
esters
were
protein
was strikingly
icantly
elevated. Apoprotein
hepatitis protein
revealed
yet
in normal were
or near
bands
to obtain not
adequately
cholesterol were
elevated, were
from patients
of unusually
amounts
absence
of apo AI,
the gels
invariably molecular
2 as other
separation
between
apo C from
a3
protein. arginine-rich
total
not
signif-
with
alcoholic
the predominant
contained weight,
and
of arginine-rich
normally
apo AII.
HDL was
was elevated,
Triglycerides
large
was signiftime
2) of HDL isolated
in Table
separate
at which
phospholipids
of higher
maximal
hepatitis
recovery
an HDL fraction.
In addition,
protein
alcoholic
unesterified
absent,
(Table
HDL and designated
selected
AI and did
nearly
the presence
in HDL.
unidentified
Thus,
low for
and a deficiency
apoprotein
of HDL following 1).
analysis
with
one or more as
not ordinarily The gel
seen
conditions protein
and apo
BIOCHEMICAL
Vol. 80, No. 1, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE Soluble with
2
Apoprotein Content of HDL from Patients Alcoholic Hepatitis on Admission and Following Recovery APOPROTEIN
AI Admission
35.4 5 it 8.9
I-L=6
Recovery n=4
80.4 4.3
P*
+ -
Other
36.7 + 3.3-
14.3 + 4.a
2.4 .9
.92
11.2 + 2.0-
+ -
3.0 2 2.3
< .OOl
< .OOOl
Values in the Table are mean percentages of dye bound by each apoprotein + 1 standard error as determined by densitometric scanning of gels. Probability of difference between mean of admission and recovery.
ii *
In order
to establish
in the HDL fractions normally
found
whether
were
primarily
In every
protein
was obtained The amino
one subject protein
from normal
ilarity
was best
thousand
co-electrophoresis
and alanine
absent;
all
by electron
only
similar,
characteristic stained
microscopy.
patient
not
next
acid
highest
features preparations In every
arginine-rich
case,
content
in
from HDL from
of arginine-rich This
of 104 parts amino equal,
acid
sim-
per
present,
and $ cystine
protein.
of the HDL fractions
the HDL obtained
a4
isolated
(8,9,10).
and approximately
of all
to arginine-rich
to that
the major
of arginine-rich
HDL with
apo AI was not detectable
investigators
was
protein
of the two lipopro-
protein
by a mean arginine
protein
of the abnormal
identical,
by other
Glutamic
with
band corresponding
In this
though
of arginine-rich
of proportions
of arginine-rich
VLDL reported
were
range
a single
1).
demonstrated
leucine
Negatively
at a wide
composition
by two analyses.
amounts
in VLDL,
(Figure
was very
large
to or identical
instance,
acid
the
similar
VLDL was performed
teins.
HDL.
15 + 7.1
< .OOOl
normal
Arginine-Rich Protein
C + AI1
from
were
examined
the patients
was
Vol. 80, No. 1, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Other t -ARP
Figure 1. Polyacrylamide gel electrophoresis of HDL from alcoholic hepatitis and normals and normal VLDL. The apoproteins are identified on the right side of the figure. ARP=arginine-rich protein; A, 27ng alcoholic hepatitis HDL protein; B, 57ng normal VLDL protein; C, A mixed with B; D, l/2 A mixed with l/5 B, E, 16ng normal HDL protein mixed with l/2 B. Figure 2. Electron micrographs of negatively from a patient with severe alcoholic hepatitis (right). Magnification=148,000.
when first (Figure These tron
studied 2),
approximately
preparations micrographs
HDL fractions Since substrate from normal lyzed
consisted
it for
primarily 150-240
were
nascent
of rouleaux
i in diameter
practically
of rat
HDL (1).
of stacked with
microscopy
had been
established
(1)
purified
LCAT, we prepared
that
nascent
all
spherical
of the patients HDL.
HDL was a preferred
The fraction
of d > 1.225
This
8.
from elec-
preparation
LCAT.
a5
recovery,
discs
of 30-50
a lipoprotein-free
by ultracentrifugation. purified
bilamellar
an edge width
as normal rat
of HDL recovery
in appearance
Following
by electron
and used as partially
preparations and following
indistinguishable
appeared
plasma
stained (left)
preparation
contained
of LCAT was diaonly
BIOCHEMICAL
vol. 80, No. 1, 1978
traces
of free
alone
with
plasma from
cholesterol
[14C]
and showed
cholesterol.
was a better
insignificant
The nascent
substrate
the same patients
AND BIOPHYSICAL
for
LCAT activity
HDL isolated
LCAT than
following
RESEARCH COMMUNICATIONS
either
recovery
from
normal
(Table
when incubated
alcoholic
hepatitis
HDL or HDL obtained
3).
DISCUSSION The HDL isolated have
an extreme
accumulates
plasma
in rat
liver
nascent
human HDL.
These
In both greater
the production
quantities
are normally suggestion
the course into
(10)
from
liver
that
mainly
derived
from nascent
An important
question
the arginine-rich
a component
pathway
of nascent
HDL rich
primarily
with
which
apo AI. (1)
tends
the chains
to confirm
this
of bilayer
discs.
which
process
detec-
human
in
the rat.
of severe
LCAT deficiency,
much
in the HDL fraction
HDL.
This
is
fact
transferred
along
than with
pathway
of arginine-rich
nascent
HDL.
such is
the case,
found
in normal
the course
rat
If
human plasma
of lipoprotein
to be answered it
enters
liver
as a result
and normal
HDL which
of electron
micrographs
presented
The relative
86
spherical proportions
is
VLDL is
or not apo
by a different
protein,
some normal
it
metabolism.
is whether plasma
the
to VLDL
the main
may well
since
rat
is not if
perfusates
in arginine-rich An examination
circulating
HDL is much more rapid
the HDL fraction
from
of normal
accumulate
HDL or whether
HDL isolated
and human plasma
be explained
protein
remains
with
inhibited.
in rats
that
HDL during
and becomes associated Nascent
al
protein
of nascent
activity.
et
is via
HDL that
that
VLDL plus
suggests
who
than
arginine-rich
of metabolism
plasma
could
conditions protein
that
likely
AI is
under
LCAT is
protein
a more efficient
in human VLDL or rat
of others
protein
is
of arginine-rich
found
to the nascent
perfusates
of arginine-rich differences
hepatitis
in which
HDL in rat
of nascent
humans and rats,
alcoholic
The HDL fraction
protein. amounts
if
conditions
of nascent
(by LCAT)
with
to be analogous
under
significant
HDL metabolism
or conversely,
during
perfusates
to be arginine-rich
in normal
of patients
appears
apoprotein
contains
table
the plasma
LCAT deficiency
The characteristic appears
from
of LCAT be a mixture contains by Hamilton
HDL appeared of arginine-rich
together
BIOCHEMICAL
Vol. 80, No. 1, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 3 Activity
of LCAT* on HDL
Source Alcoholic
of Plasma
Hepatitis
Recovery
Control
.020
.027
. ot39** *
Expressed as pg [14 C] cholesterol incubation medium. Each value is the mean of duplicate
**
protein
and apo AI in the nascent
represent wide
mean values
range
nascent
of relative
HDL.
apo AI but bands
human nascent
from
proportions
relatively
large
et al
(1)
that
the interaction
with
In fact,
of peptides protein.
rat
of nascent
in These
This
HDL with
either
observed
a
and apo AI in
HDL containing
no detectable
the apo AI1 and apo C findings
supports
serum HDL rich
hepatitis
actually
protein
amounts
apo AI.
per ml of
alcoholic
we have
2) have nascent
contain
hour
determinations.
(Figure
normal
per
of arginine-rich
to arginine-rich
HDL does not
esterified
HDL of subjects
patients.
Some patients
in addition
Hamilton
of six
HDL
suggest
that
the proposal
pure of
in apo AI is probably
derived
LCAT and apo AI or a complex
of
apo AI and LCAT. Arginine-rich diet
(8),
ncreased
protein
a condition amounts
physiological cholesterol
which
role and/or
cholesterol.
protein
the same relationship and Connor
esters
protein This appears (11)
to the necessity
Our results
cholesteryl
esterified
in VLDL of rabbits
lead
of arginine-rich
of arginine-rich
cholesterol
might
of cholesterol.
quantity
Barter
accumulates
ARP is
in plasma.
associated
occurred to hold concluded
in human HDL following
also
with
with
in
that
there
injection
87
for
suggest
that
We always
are
of labeled
of plasma
of
a probable of unesterified
found
the presence
the rat
cholesterol
transport
the transport
any level
true
fed a high
a large
of excess
un-
cholesterol,
and
(1). two pools mevalonic
of esterified acid.
They
BIOCHEMICAL
Vol. 80, No. 1, 1978
speculate
that
the source
of cholesteryl
turns
more slowly.
over
to investigate block
those
in animal
esters
metabolized,
HDL which
in lipoprotein
accumulates
provides
as a result
The present
suggest
that
represents
in apo AI and cholesteryl
esters,
protein
metabolism
in the plasma
product
of the liver
formed
represents
LDL, and a larger hepatitis
metabolism.
investigations,
pool
in VLDL,
Human alcoholic
nascent
bolic
rapidly
a small,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
results
nascent pool
of HDL which
a unique
meta-
in man, as well normal
a metabolic
end-product
rather
opportunity
of a reversible
circulating
compartment
HDL and is
than
plasma
as
HDL, rich of lipo-
a secretory
or intestine.
This work was supported by research grants from the Acknowledgements: National Institutes of Health, AM-17398, the General Clinical Research Center, RR 05423, the American Heart Association and the Veterans Administration.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Hamilton, R.L., Williams, M.C., Fielding, C.J., and Havel, R.J. (1976) J. Clin. Invest. 58, 667-680. Fielding, C.J., Shore, V.G., and Fielding, P.E. (1972) Biochem. Biophys. Res. Commun. 46, 1493-1498. Marsh, J.B. (1976) J. Lipid Res. 17, 85-90. Forte, T., Norum, K.R., Glomset, J.A., and Nichols, A.V. (1971) J. Clin. Invest. 50, 1141-1148. Utermann, G.H., Menzel, J., and Langer, K.H. (1974) FEBS Lett. 45, 29-32. Sabesin, S.M., Hawkins, H.L., Kuiken, L., and Ragland, J.B. (1977) Gastroenterology 72, 510-518. Laemmli, V.K. (1970) Nature 227, 680-685. Shore, V.G., Shore, B., and Hart, R.G. (1974) Biochemistry 13, 1579-1585. Utermann, G. (1975) Hoppe-Seyler's Z. Physiol. Chem. 356, 1113-1121. Havel, R.S., and Kane, J.P. (1973) Proc. Natl. Acad. Sci. USA 70, 2015-2019 Barter, P.J. and Connor, W.E. (1976) J. Lab. Clin. Med. 88, 627-639.
88
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
January 13, 1978
Pages
Identification Containing
of Nascent Arginine-Rich
81-88
High Density Lipoproteins Protein in Human Plasma
James 8. Ragland,
Phillip D. Bertram and Seymour M. Sabesin Division of Gastroenterology of Tennessee Center for the Health Sciences and Veterans Administration Hospital Memphis, Tennessee
University
Received
November 22,
1977
Summx A high density lipoprotein fraction accumulates in the plasma of pasts with alcoholic hepatitis when a severe 1ecithin:cholesterol acyltransferase (EC 2.3.1.43) deficiency is present. The major apoprotein present in this fraction is arginine-rich protein, the fraction is a preferred substrate for 1ecithin:cholesterol acyltransferase, and by electron microscopy appears as stacked bilayer discs. It is proposed that the lipoprotein represents the accumulation of nascent high density lipoprotein and is the principal pathway through which arginine-rich protein is secreted by the liver in man. The results also suggest that apoprotein AI is acquired by normal high density lipoprotein during the course of lipoprotein metabolism. INTRODUCTION Circulating nascent
plasma
lipoproteins
showed
that
lipoproteins
of hepatic
nascent
contained
was enrimzhed
in unesterified
bilayer
stained
particles
25 H, frequently
constituent
(2).
It
preferred
forming
with
of circulating that purified
perfusates
et al
in the presence esters
HDL revealed
relatively
apoprotein small
and protein
disc-shaped
lipid
human LCAT.
Marsh
(3)
of apo AI,
plasma
added
of 190 -+
in rat
activator
than
and
microscopy
amounts
HDL rather
of
Electron
present
HDL and a required
of
(1)
of 46 -+ 5 5: and diameter
The major
nascent
the metabolism
Hamilton
and phospholipid.
plasma rat
from
of cholesteryl
of nascent
rouleaux.
protein
for
levels
liver
a mean edge thickness
was demonstrated substrate
origin.
in rat
cholesterol
preparations
HDL was arginine-rich major
decreased
with
to be derived
or intestinal
HDL accumulating
LCAT inhibitor
of negatively
appear
nascent the
of LCAT HDL was the
labeled
amino
acid
HDL, high density The following abbreviations will be used in this paper: VLDL, very low density lipoprotein; lipoprotein; LDL, low density lipoprotein: LCAT, 1ecithin:cholesterol acyltransferase (EC 2.3.1.43). ape, apoprotein; 0006-291X/78/0801-0081$01.00/0 81
Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
BIOCHEMICAL
Vol. 80, No. 1, 1978
precursors
to a nonrecirculating
apoproteins
secreted
were
ape B, and apo C with rich
protein
with
hereditary
protein
perfusion protein
It
has also
been
contain
and found
(highest
most of the radioactivity
LCAT deficiency
injury
appears
hepatitis
the nascent
(6),
shown that
that
specific
of nascent
bilayer
ester
further
that
the major activity),
HDL in arginine-
HDL fractions
discs
of patients
and arginine-rich
as bilayer
characterized that
it
from
rat
with
LCAT deficiency,
stacked
similar liver
Further,
alcohol-induced very
low
composition In the present
subjects
in composition
perfusates.
lipid
discs.
the HDL from several is
acute
and HDL of abnormal
microscopy
and find
patients
have an extreme
concentrations,
HDL isolated for
we reported
hepatitis)
by electron
we have
substrate
study
(alcoholic
cholesteryl
study, holic
liver
(4,5).
plasma which
rat
arginine-rich
and apo C.
In a recent liver
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
with
alco-
and ultrastructure it
to
is a preferred
human LCAT. MATERIALS
AND METHODS
Six patients with alcoholic hepatitis were selected for study on the basis of clinical and laboratory findings previously described (6). They were admitted to the Clinical Research Center and studied throughout the course of their illness until recovery (4-12 weeks). All blood samples for lipoprotein studies were collected in EDTA following a 14 hour fast. Dithionitrobenzoic acid was added to all samples to inhibit LCAT activity. Lipoprotein density classes were isolated by sequential ultracentrifugation and lipid and total protein content determined as previously described. HDL fractions studied represent d b 1.063, < 1.21. Lipoproteins were solubilized by boiling in 1% sodium dodecyl sulfate for one minute and apoproteins separated by polyacrylamide gel electrophoresis, essentially according to the method of Laeammle (7) using 5% stacking gel and 15% running gel. Apoprotein composition was estimated by densitometry of the Coomassie Blue stained gels without correction for differences in dye binding between different apoproteins. Arginine-rich protein was isolated from alcoholic hepatitis HDL by gel filtration on Sephadex G-200 and preparative electrophoresis. Analytical electrophoresis of the preparation revealed a single band corresponding to argininerich protein of normal VLDL. Amino acid analyses were performed through the courtesy of Drs. A.H. Kang and J.M. Seyer, Veterans Administration Hospital, Memphis, Tennessee. LCAT activity was assayed essentially as described by equivalent to 5pg of unesterified cholesterol HDL fragtions with [ C] cholesterol and assayed in a total volume of 0.2 plasma fraction of d > 1.225 as enzyme source. Blanks were enzyme was added and also in which no HDL source was added from the results reported.
82
Hamilton et al (1). were equilibrated ml using a normal run in which no and were subtracted
BIOCHEMICAL
Vol. 80, No. 1, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE
1
Percent Composition of HDL from Patients with Alcoholic Hepatitis on Admission and Following Recovery Unesterified Cholesterol
Cholesteryl Esters
Admission n=6
13.7 9 1.1-
1.5 + 0.4-
Recovery n=4*
4.4 + 4.0-
18.0 + 2.6 -
< .OOOl
< .OOOl
p** i/ * **
Triglycerides
Phospholipid
11.3 + 2.95.1
+
4.6 .36
Total Protein
45.8 + 3.5-
27.8 + 2.1
27.4 + 4.1
43.1 + 2.8-
< .OOl
< .OOOl
Values in the Table are the mean percentages of each component found in all patients + 1 standard error of the mean. Of six patients admitted to the study, two did not recover. Probability of difference between mean of admission and recovery groups.
RESULTS The composition icantly
different
of HDL from patients from
that
essentially
normal
(Table
cholesteryl
esters
were
protein
was strikingly
icantly
elevated. Apoprotein
hepatitis protein
revealed
yet
in normal were
or near
bands
to obtain not
adequately
cholesterol were
elevated, were
from patients
of unusually
amounts
absence
of apo AI,
the gels
invariably molecular
2 as other
separation
between
apo C from
a3
protein. arginine-rich
total
not
signif-
with
alcoholic
the predominant
contained weight,
and
of arginine-rich
normally
apo AII.
HDL was
was elevated,
Triglycerides
large
was signiftime
2) of HDL isolated
in Table
separate
at which
phospholipids
of higher
maximal
hepatitis
recovery
an HDL fraction.
In addition,
protein
alcoholic
unesterified
absent,
(Table
HDL and designated
selected
AI and did
nearly
the presence
in HDL.
unidentified
Thus,
low for
and a deficiency
apoprotein
of HDL following 1).
analysis
with
one or more as
not ordinarily The gel
seen
conditions protein
and apo
BIOCHEMICAL
Vol. 80, No. 1, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE Soluble with
2
Apoprotein Content of HDL from Patients Alcoholic Hepatitis on Admission and Following Recovery APOPROTEIN
AI Admission
35.4 5 it 8.9
I-L=6
Recovery n=4
80.4 4.3
P*
+ -
Other
36.7 + 3.3-
14.3 + 4.a
2.4 .9
.92
11.2 + 2.0-
+ -
3.0 2 2.3
< .OOl
< .OOOl
Values in the Table are mean percentages of dye bound by each apoprotein + 1 standard error as determined by densitometric scanning of gels. Probability of difference between mean of admission and recovery.
ii *
In order
to establish
in the HDL fractions normally
found
whether
were
primarily
In every
protein
was obtained The amino
one subject protein
from normal
ilarity
was best
thousand
co-electrophoresis
and alanine
absent;
all
by electron
only
similar,
characteristic stained
microscopy.
patient
not
next
acid
highest
features preparations In every
arginine-rich
case,
content
in
from HDL from
of arginine-rich This
of 104 parts amino equal,
acid
sim-
per
present,
and $ cystine
protein.
of the HDL fractions
the HDL obtained
a4
isolated
(8,9,10).
and approximately
of all
to arginine-rich
to that
the major
of arginine-rich
HDL with
apo AI was not detectable
investigators
was
protein
of the two lipopro-
protein
by a mean arginine
protein
of the abnormal
identical,
by other
Glutamic
with
band corresponding
In this
though
of arginine-rich
of proportions
of arginine-rich
VLDL reported
were
range
a single
1).
demonstrated
leucine
Negatively
at a wide
composition
by two analyses.
amounts
in VLDL,
(Figure
was very
large
to or identical
instance,
acid
the
similar
VLDL was performed
teins.
HDL.
15 + 7.1
< .OOOl
normal
Arginine-Rich Protein
C + AI1
from
were
examined
the patients
was
Vol. 80, No. 1, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Other t -ARP
Figure 1. Polyacrylamide gel electrophoresis of HDL from alcoholic hepatitis and normals and normal VLDL. The apoproteins are identified on the right side of the figure. ARP=arginine-rich protein; A, 27ng alcoholic hepatitis HDL protein; B, 57ng normal VLDL protein; C, A mixed with B; D, l/2 A mixed with l/5 B, E, 16ng normal HDL protein mixed with l/2 B. Figure 2. Electron micrographs of negatively from a patient with severe alcoholic hepatitis (right). Magnification=148,000.
when first (Figure These tron
studied 2),
approximately
preparations micrographs
HDL fractions Since substrate from normal lyzed
consisted
it for
primarily 150-240
were
nascent
of rouleaux
i in diameter
practically
of rat
HDL (1).
of stacked with
microscopy
had been
established
(1)
purified
LCAT, we prepared
that
nascent
all
spherical
of the patients HDL.
HDL was a preferred
The fraction
of d > 1.225
This
8.
from elec-
preparation
LCAT.
a5
recovery,
discs
of 30-50
a lipoprotein-free
by ultracentrifugation. purified
bilamellar
an edge width
as normal rat
of HDL recovery
in appearance
Following
by electron
and used as partially
preparations and following
indistinguishable
appeared
plasma
stained (left)
preparation
contained
of LCAT was diaonly
BIOCHEMICAL
vol. 80, No. 1, 1978
traces
of free
alone
with
plasma from
cholesterol
[14C]
and showed
cholesterol.
was a better
insignificant
The nascent
substrate
the same patients
AND BIOPHYSICAL
for
LCAT activity
HDL isolated
LCAT than
following
RESEARCH COMMUNICATIONS
either
recovery
from
normal
(Table
when incubated
alcoholic
hepatitis
HDL or HDL obtained
3).
DISCUSSION The HDL isolated have
an extreme
accumulates
plasma
in rat
liver
nascent
human HDL.
These
In both greater
the production
quantities
are normally suggestion
the course into
(10)
from
liver
that
mainly
derived
from nascent
An important
question
the arginine-rich
a component
pathway
of nascent
HDL rich
primarily
with
which
apo AI. (1)
tends
the chains
to confirm
this
of bilayer
discs.
which
process
detec-
human
in
the rat.
of severe
LCAT deficiency,
much
in the HDL fraction
HDL.
This
is
fact
transferred
along
than with
pathway
of arginine-rich
nascent
HDL.
such is
the case,
found
in normal
the course
rat
If
human plasma
of lipoprotein
to be answered it
enters
liver
as a result
and normal
HDL which
of electron
micrographs
presented
The relative
86
spherical proportions
is
VLDL is
or not apo
by a different
protein,
some normal
it
metabolism.
is whether plasma
the
to VLDL
the main
may well
since
rat
is not if
perfusates
in arginine-rich An examination
circulating
HDL is much more rapid
the HDL fraction
from
of normal
accumulate
HDL or whether
HDL isolated
and human plasma
be explained
protein
remains
with
inhibited.
in rats
that
HDL during
and becomes associated Nascent
al
protein
of nascent
activity.
et
is via
HDL that
that
VLDL plus
suggests
who
than
arginine-rich
of metabolism
plasma
could
conditions protein
that
likely
AI is
under
LCAT is
protein
a more efficient
in human VLDL or rat
of others
protein
is
of arginine-rich
found
to the nascent
perfusates
of arginine-rich differences
hepatitis
in which
HDL in rat
of nascent
humans and rats,
alcoholic
The HDL fraction
protein. amounts
if
conditions
of nascent
(by LCAT)
with
to be analogous
under
significant
HDL metabolism
or conversely,
during
perfusates
to be arginine-rich
in normal
of patients
appears
apoprotein
contains
table
the plasma
LCAT deficiency
The characteristic appears
from
of LCAT be a mixture contains by Hamilton
HDL appeared of arginine-rich
together
BIOCHEMICAL
Vol. 80, No. 1, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 3 Activity
of LCAT* on HDL
Source Alcoholic
of Plasma
Hepatitis
Recovery
Control
.020
.027
. ot39** *
Expressed as pg [14 C] cholesterol incubation medium. Each value is the mean of duplicate
**
protein
and apo AI in the nascent
represent wide
mean values
range
nascent
of relative
HDL.
apo AI but bands
human nascent
from
proportions
relatively
large
et al
(1)
that
the interaction
with
In fact,
of peptides protein.
rat
of nascent
in These
This
HDL with
either
observed
a
and apo AI in
HDL containing
no detectable
the apo AI1 and apo C findings
supports
serum HDL rich
hepatitis
actually
protein
amounts
apo AI.
per ml of
alcoholic
we have
2) have nascent
contain
hour
determinations.
(Figure
normal
per
of arginine-rich
to arginine-rich
HDL does not
esterified
HDL of subjects
patients.
Some patients
in addition
Hamilton
of six
HDL
suggest
that
the proposal
pure of
in apo AI is probably
derived
LCAT and apo AI or a complex
of
apo AI and LCAT. Arginine-rich diet
(8),
ncreased
protein
a condition amounts
physiological cholesterol
which
role and/or
cholesterol.
protein
the same relationship and Connor
esters
protein This appears (11)
to the necessity
Our results
cholesteryl
esterified
in VLDL of rabbits
lead
of arginine-rich
of arginine-rich
cholesterol
might
of cholesterol.
quantity
Barter
accumulates
ARP is
in plasma.
associated
occurred to hold concluded
in human HDL following
also
with
with
in
that
there
injection
87
for
suggest
that
We always
are
of labeled
of plasma
of
a probable of unesterified
found
the presence
the rat
cholesterol
transport
the transport
any level
true
fed a high
a large
of excess
un-
cholesterol,
and
(1). two pools mevalonic
of esterified acid.
They
BIOCHEMICAL
Vol. 80, No. 1, 1978
speculate
that
the source
of cholesteryl
turns
more slowly.
over
to investigate block
those
in animal
esters
metabolized,
HDL which
in lipoprotein
accumulates
provides
as a result
The present
suggest
that
represents
in apo AI and cholesteryl
esters,
protein
metabolism
in the plasma
product
of the liver
formed
represents
LDL, and a larger hepatitis
metabolism.
investigations,
pool
in VLDL,
Human alcoholic
nascent
bolic
rapidly
a small,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
results
nascent pool
of HDL which
a unique
meta-
in man, as well normal
a metabolic
end-product
rather
opportunity
of a reversible
circulating
compartment
HDL and is
than
plasma
as
HDL, rich of lipo-
a secretory
or intestine.
This work was supported by research grants from the Acknowledgements: National Institutes of Health, AM-17398, the General Clinical Research Center, RR 05423, the American Heart Association and the Veterans Administration.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Hamilton, R.L., Williams, M.C., Fielding, C.J., and Havel, R.J. (1976) J. Clin. Invest. 58, 667-680. Fielding, C.J., Shore, V.G., and Fielding, P.E. (1972) Biochem. Biophys. Res. Commun. 46, 1493-1498. Marsh, J.B. (1976) J. Lipid Res. 17, 85-90. Forte, T., Norum, K.R., Glomset, J.A., and Nichols, A.V. (1971) J. Clin. Invest. 50, 1141-1148. Utermann, G.H., Menzel, J., and Langer, K.H. (1974) FEBS Lett. 45, 29-32. Sabesin, S.M., Hawkins, H.L., Kuiken, L., and Ragland, J.B. (1977) Gastroenterology 72, 510-518. Laemmli, V.K. (1970) Nature 227, 680-685. Shore, V.G., Shore, B., and Hart, R.G. (1974) Biochemistry 13, 1579-1585. Utermann, G. (1975) Hoppe-Seyler's Z. Physiol. Chem. 356, 1113-1121. Havel, R.S., and Kane, J.P. (1973) Proc. Natl. Acad. Sci. USA 70, 2015-2019 Barter, P.J. and Connor, W.E. (1976) J. Lab. Clin. Med. 88, 627-639.
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