CUN. CHEM. 41/3, 405-412 (1995)
#{149}
Lipids
and
Lipoproteins
Measurement and Physiological Significance of Lipoprotein and Hepatic Lipase
Activities in Preheparin Plasma
Timothy D.G. Watson,’
Christopher
J. Packard
Chee-Eng
Tan,2 Michael
McConnell,
A radiochemical method for selective measurement of
postheparin lipase activities was adapted to analyze
lipoprotein lipase and hepatic lipase in preheparin
plasma. The assay sensitivity was increased about fourfold by doubling both the volume of plasma used and the
volume of lipolytic products taken for liquid scintillation
counting, and was further improved by increasing the
incubation period by 50% to 90 mm. Rabbit antiserum to
human hepatic lipase was unsuitable for the selective
measurement of lipoprotein lipase because of apparent
endogenous lipolytic activity. Preheparin hepatic lipase,
however, was sensitive to inactivation by sodium dodecyl
sulfate (SDS), the inhibition being greatest (>90%) for
plasma incubated with an equal volume of 40 mmoVL
SDS. Intra- and interassay CVs for the two enzymes were
12.5-14.6%
and 17.4-19.7%,
respectively. In a crosssectional study of 84 healthy subjects, pre- and posthep-
arm hepatic lipase activities were higher in men than
women, were correlated with indices of obesity, and were
significantly correlated with one another, which explained
the association of the former with plasma concentrations
of high-density lipoprotein (HDL), HDL2, and small, dense
low-density
lipoproteins.
There was no significant
rela-
tionship between pre- and postheparin lipoprotein lipase
activities, but the former were correlated with plasma
concentrations of free fatty acids (FFA) and very-lowdensity lipoprotein. Apparently, preheparmn activities of
hepatic lipase, but not of lipoprotein lipase, may be a
useful measure of the physiological function of “whole
body” enzyme activity in cross-sectional
and metabolic
studies, where heparinization is not possible. Preheparin
lipoprotein lipase activities, however, may reflect dis-
placement of the enzyme by FFA and subsequent binding to remnants of triglyceride-rich lipoproteins.
Indexing
Terms: radioass,ay/enzyme activity/Iipids/Iipoproteins/Iipo!ysis/sex-related differences/obesity/vanation,
soume of
Lipoprotein
responsible
by circulating
cans on the
via heparan
lipase, found
lipase and hepatic lipase (EC 3.1.1.3) are
for the hydrolysis
of triglycerides
carried
lipoproteins
and are bound to proteoglyluminal
surface of vascular
endothelium
sulfate binding domains
(1). Lipoprotein
predominantly
in the heart, skeletal mus-
Department
of Pathological Biochemistry,
Glasgow Royal Infirmary, University NHS Trust, Glasgow G4 OSF, UK.
‘Present
address (and address for correspondence):
Waltham
Centre for Pet Nutrition, Freeby Lane, Waltham-on-the-Wolds,
Melton Mowbray, Leics, LE14 4RS, UK. Fax +44(0)664 415440.
2Present
address:
Department
of Medicine,
Hospital, Singapore 0316.
Received August 15, 1994; accepted
Singapore
November
General
22, 1994.
Sarah K. Clegg, Lorne
F. Squires,
and
cle, and adipose tissue, is the rate-limiting
enzyme for
the hydrolysis
of triglycerides
carried by chylomicrons
and very-low-density
lipoproteins
(VLDL) (2 )3 Hepatic
lipase is largely
confined
to the liver, with smaller
amounts
in steroidogenic
organs, and is involved in the
intravascular
remodeling
of low- and high-density
lipoproteins (LDL and HDL) (3). Negligible amounts of
both enzymes
are found free in plasma,
and their
activities
are usually measured
in blood samples
collected after intravenous
injection of heparin or, uncommonly, in adipose
tissue
obtained
by percutaneous
biopsy (lipoprotein
lipase) (4).
The requirement
for heparinization
prevents
serial
measurements
during
short-term
metabolic
studies
and hinders
routine
screening
of lipoprotein
and hepatic lipase activities
in clinical and epidemiological
investigations.
There has, therefore,
been recent interest in whether
the amounts
of free enzyme in plasma
can be (a) measured
precisely
and (b) used as a measure of “whole body” enzyme
activity.
To date, two
assay systems have been described:
one that measures
total and hepatic lipase activity and calculated
lipoprotein lipase as the difference
(5), and another
that
measures
lipoprotein
lipase after inhibiting
hepatic
lipase with goat antiserum
to human hepatic lipase (6).
To increase the sensitivity
of these assays, the authors
either partially
purified
the enzymes
over heparinSepharose
(5) or reduced the background
activity by
repurifying
the substrate
and using an alternative
procedure
to extract the labeled lipoytic products
(6).
The metabolic
relevance
of these measurements
was
first studied in a group of 12 male subjects by Eckel et
al. (5), who found that the pre- and postheparin
activities of lipoprotein
lipase and hepatic lipase were related, and that the activities of both enzymes increased
after an oral glucose load. In a follow-up study of 93
men, Eckel et al. confirmed
that the pre- and postheparm activities
of hepatic,
but not lipoprotein,
lipase
were correlated;
they also showed that the preheparin
activities
of both enzymes
were associated
with LDL
cholesterol
concentration,
which they suggested
was
due to binding
of the enzymes to lipoprotein
remnants
(7). A significant
relationship
between pre- and postheparm hepatic lipase activities was also demonstrated
by
Karpe et al. (6) in a group of 44 men, either healthy
men or survivors
of a myocardial
infarction.
These
authors
also
showed
that
only
lipoprotein
lipase
activ-
Nonstandard abbreviations: VLDL, LDL, HDL, very-low-,
low-, and high-density lipoprotein, respectively; FFA, free fatty
acids; FA, fatty acids; and SDS, sodium dodecyl sulfate.
CLINICAL CHEMISTRY, Vol. 41, No. 3, 1995
405
increased after an oral fat load, and that this was in
parallel with changes in plasma triglycerides
and total
of the system,
which we subsequently
used to determine preheparin
lipase activities
in 84 healthy
subjects. To provide
further
insight
into the metabolic
significance
of preheparin
lipoprotein and hepatic activities,
we correlated
these measurements
with the
HDL3, in mgfL, were determined
by analytical
ultracentrifugation
(11).
Postheparin
lipoprotein
lipase and hepatic
lipase.
We measured
the activities
of lipoprotein
lipase
and
hepatic lipase in postheparin
plasma incubated
with a
gum arabic-stabilized
triglyceride
emulsion
containing
glycerol
tri[1-14C]oleate
(Amersham
International,
Aylesbury,
UK) at a specific activity
of 30 mCilmol
triglyceride
fatty acids (12). In each assay 10 L of
postheparin
plasma was diluted in 30 L of 0.15 mol/L
NaCl and then incubated
in duplicate
with 500 L of
substrate
at 28#{176}C
for 60 mm. The substrate
consisted of
24 .tg of triolein in 100 L of 50 gfL gum arabic, 100 pL
of 100 gfL bovine serum albumin,
250 L of 0.2 16 or
2.16 mol/L NaCl (all in 0.2 mol/L Tris-HC1, pH 8.4), and
either 50 L of serum or of 0.15 moIIL NaCl. The FFA
generated
by lipolysis
were extracted
from the glycerides (13) and the amount of radiolabel
was counted by
subjects’
liquid scintillation.
ity
and unsaturated
(18:2) free fatty acids (FFA). They
suggested
that these associations
reflected the dissociation of lipoprotein
lipase from the vascular
endothehum by chylomicron-derived
FFA, with subsequent
binding
of the enzyme to triglyceride-rich
lipoproteins
(6).
Here, we describe an alternative,
more readily applicable assay system that does not rely on afilnity
chromatography
or a supply of antiserum
to hepatic lipase
for measuring
lipoprotein
lipase and hepatic lipase in
preheparin
plasma.
plasma
We also report on the performance
concentrations
of lipids, lipoproteins,
The results
were expressed
as mil-
limoles of fatty acids (FA) released per liter of postheparm plasma in 1 h of incubation
(mmol/L FA per hour).
The activity of hepatic lipase was measured
by using
Materials and Methods
the high-salt
substrate
(final concentration
1.0 mol/L
NaC1) to inhibit lipoprotein
lipase; i.e., the 0.15 mol/L
Subjects and samples.
Blood samples were obtained
from 84 healthy subjects (46 men and 38 women) after
NaC1 was included instead of serum. Lipoprotein
lipase
was analyzed
in postheparin
plasma
that had been
an overnight
fast. Blood for plasma lipid and lipopropreincubated
with an equal volume
of 25 mmol/L
tein concentrations
was collected into tubes containing
K2EDTA (final concentration,
1 gIL); a separate
aliquot
sodium dodecyl sulfate (SDS) in 0.2 mol/L Tris, pH 8.2,
for preheparin
lipases
was placed in a chilled tube
at 26#{176}C
for 60 mm to inhibit hepatic lipase (14); the
containing
lithium heparin
and kept on ice until sepaincubation
mixture
contained
the low-salt
substrate
0.1 mol/L NaCl) and pooled serum
rated by centrifligation
at 2000g for 20 mm at 4#{176}C. (final concentration
Blood for postheparin
plasma was then collected into
as a source of apolipoprotein
C-Il. The intraassay
CVs
lithium
heparin
10 mm after intravenous
injection
of
for the lipoprotein
lipase and hepatic
lipase assays
70 IIJ of heparin
per kilogram
of body weight.
The
were 4-9% and 3-6%, respectively,
for pools of high
samples
were placed on ice, and the plasma
samples
and low enzyme activity (15).
were separated
as above. Both pre- and postheparin
Assay
modifications
for measuring
preheparin
Iisamples
were centrifuged
within 30 mm of collection
poprotein
lipase and hepatic lipase. We modified the
and stored at -70#{176}C
within 60 min. The study was
postheparin
lipase
assay
to improve its sensitivity
by
increasing
(a) the volume of plasma,
(b) the volume of
approved
by the Research
Ethics Committee
of GlasFA fraction taken for liquid scintillation
counting,
and
gow Royal Infirmary.
Plasma lipids and lipoproteins.
Cholesterol
and tri(c) the incubation
period. The effect of repurifying
the
substrate
by thin-layer
chromatography
was also evalglyceride concentrations
were measured
with commeruated,
as was the inhibition
of preheparin
hepatic
cially available kits (nos. 816302 and 816370; BCL,
Lewes, UK), as was FFA (NEFA C; Wako Chemicals,
lipase by rabbit polyclonal antiserum
to human hepatic
Osaka, Japan).
Lipoprotein
cholesterol
concentrations
lipase (16) and by SDS. The antiserum
was diluted in
were determined
after isolating VLDL by ultracentrifan equal volume of isotonic saline and 0-30 L of this
ugation
and precipitating
LDL from HDL in the inmixture was incubated
with 10 L of pre- and posthepfranate
with heparmn-manganese
chloride
(8). LDL
am plasma at 4#{176}C
for 1 h. The pre- and postheparin
plasma
samples
were also incubated
with an equal
subfractions
were isolated by nonequilibrium
densityvolume of 0-100 mmoIJL SDS in 0.2 mol/L Tris, pH 8.2,
gradient ultracentrifugation
as previously described
at 26#{176}C
for 1 h and analyzed for lipase activity as above
(9). The LDL profiles comprised
overlapping
or only
slightly separated
populations
of particles
from which
at low (total hipase) and high (hepatic
lipase)
salt
three distinct
subfractions
were resolved; these correconcentrations.
Finally, we examined
the performance
sponded
in size and density
to LDL-I (1.025-1.034
of the modified
assays
with respect
to intraand
interassay
precision.
kg/L), LDL-II (1.034-1.044
kg/L), and LDL-III (1.0441.060 kg/L), as originally
defined
by Krauss
and
Statistical
methods.
Statistical
manipulations
and
significance
testing
were performed
by using the PC
Blanche
(10). The individual
subfraction
areas were
version of Minitab Release
8 (Minitab
Inc., State Colquantified,
corrected
for previously
calculated
absorptivities,
and expressed as lipoprotein
concentrations
in
lege, PA). For two-group
comparisons
we used the
mgfL
plasma.
Plasma
concentrations
of HDL2 and
Mann-Whitney
two-sample
rank sum test. The distriand lipoprotein
subfractions
rin enzyme activities.
and with
their
406 CLINICAL CHEMISTRY, Vol. 41, No. 3, 1995
posthepa-
bution of each variable
was assessed
by drawing
normality plots; the data for pre- and postheparin
lipoprotein lipase, and plasma concentrations
of triglycerides,
VLDL cholesterol,
HDL2, and LDL-III were log-transformed for normal
distribution.
The association
between pairs of variables
was measured
with the Pearson product
moment
correlation
coefficient,
and the
significance
of the relationship
was tested by linear
regression
analysis.
Results
Modification
of Posthepann Lipase Assay
Preliminary
postheparin
activity
greater
analyses
of preheparin
assay conditions
in such
samples
than in the blank
plasma
indicated
was
only
with
that the lipase
0-15
counts/min
(i.e., saline) incubations.
We
therefore
modified the assay to increase
the gulf between results
for the sample and the blank incubations. Repurification
of the labeled
triolein
by thinlayer chromatography
did not significantly
decrease
the blank activity in incubations
at low- and high-salt
concentrations
for either 60 or 90 mm (mean ± SD,
counts/mm):
49 ± 25 for repurified
substrate
vs 52 ±
20 for nonrepurifled
substrate
(n = 16 each, P = 0.64).
The amount of sample radioactivity
in excess of that of
the blank was effectively
quadrupled
by using 20 L
instead of 10 L of plasma in the assay incubations
and
by taking 2 mL instead of 1 mL of the upper FA fraction
for scintillation
counting: 45 counts/mm
for 20 .tU2 mL
vs 10 counts/min
for 10 1.dJlO mL (mean of 4 samples
each).
Increasing
the duration
of incubation
produced
a
steady increase
in total preheparin
lipase activity for
the first 1.0 h, followed by a steep but linear increase
between
1.0 and 2.0 h, a peak at 2.5 h, and a steep
decline
to 3.0 h (Fig. 1). The time chosen
incubations
was taken from the middle
300
250
for all future
of the linear
part,
i.e., 1.5 h. These manipulations
masking
-
200
-
150
-
in linear
of the antihepatic
lipase effect by endogenous
lipase activity, we repeated
the experiment
after heating the antiserum
(56#{176}C
for 30 mm). The results were
compared
with those for untreated
antiserum
and for
“blank incubations”
of fresh and heat-inactivated
serum from a healthy human volunteer
(Fig. 3). Although
heat treatment
partially
restored the antiserum’s
antihepatic
lipase effect, the inhibition
was incomplete
(-40% at most) and was eliminated
when volumes >10
were used.
Incubation
of postheparin
plasma
with SDS produced a pattern
of hepatic lipase inhibition
similar
to
that obtained
with the antiserum:
Concentrations
of
SDS >25 mmol/L
reduced
the enzyme’s
activity
by
Postheparin plasma
100
Preheparin plasma
500
7
80
/1
400
80
.
resulted
increases
in total lipase activity up to a maximum
of
22.6 mmol/L FA per hour for pre- and postheparin
plasma.
Occasionally,
there was poor agreement
between assay duplicates,
usually with one outlier (“flier”) result for which the reported
value was on the
order of lOOs rather than lOs of units (e.g., duplicate
results of 315 and 83 counts/min);
this occurred
for,
e.g., one enzyme in one sample in every 2-3 assays and
was overcome by analyzing
each sample in triplicate
and eliminating
the “ifier” results.
Incubating
postheparin
plasma with the antiserum
to hepatic lipase resulted
in a progressive
inhibition
of
hepatic lipase activity; the inhibition
was obvious with
5 L of antiserum
and maximal
(>93%) with 20 L
(Fig. 2). Total lipase
activity
in this postheparin
plasma remained
constant
over the range where hepatic lipase was inhibited,
indicating
that lipoprotein
lipase activity
was not affected
by the antiserum.
Incubating
preheparin
plasma
with antiserum
produced a different
response,
in that total and hepatic
lipase activities
were only slightly inhibited
by 5 L of
antiserum
and then rose steeply with increasing
volumes (Fig. 2). To test whether
this effect was due to
70
60
50
a
a
100
0,
50
-
#{182}
I
40
//
30
20
-
10
0
0
0.0
If
0.5
II
1.0
1.5
I
2.0
2.5
1#{149}1#{149}11#{149}1#{149}1
0
3.0
Time (h)
Fig. 1. Effects of incubation duration on total preheparin lipase
activity.
Prehepasin plasma was collected from healthy volunteers and incubated with
the low-salt substrate, including serum as a source of apolipoprotein C-Il, for
0-3 h. The lipase activity measured represents the mean (±SEM) of four
separate expenments.
5
10
15
20
20
Volume
:.T5,
I
50
0
of antiserum
6
10
(oL)
Fig. 2. Effects of rabbit antiserum to human hepatic lipase on preand postheparin total lipase
and hepatic lipase
activities.
Pre- and postheparin plasma (10-pLaliquots) from a healthy volunteer was
incubated with 0-30 pL of antiserum diluted with an equal volume of 0.15
moVL NaCI at 4’C for 60 mm; lipase actvity was determined at low (total
lipase EDand high epatlo lipase
salt concentrations. Results are expressed as a percentage of the activity in the absence of antiserum.
CLINICAL CHEMISTRY, Vol. 41, No. 3, 1995 407
between
96% and 99% (Fig. 4). Incubation
of preheparm plasma
with SDS also resulted
in a progressive
200
inhibition
of hepatic
lipase,
by --90% at 40 mmol/L
(Fig. 4). At concentrations
150
-
100
-
50
-
0
0
5
10
Volume
15
of antiserum/serum
20
(pL)
Fig. 3. Effects
of heat treatment on the anti-hepatic lipase activity of
the rabbit antiserum to human hepatic lipase.
Allquots (10 4) of preheparin plasma from a healthy volunteer were incubated wIth 0-30 p1 of untreated antiserum ,
heat-inactivated antiserum
, fresh humanserum (#{149}),
and heat-inactivated humanserum (<>),all diluted
twofold in 0.15 moL/LNaCI,at 4’C for 60 mm; then hepatic lipase activity was
measured. The data represent the mean (±SEM) of three separate experiments (except fresh human serum, which was a single experiment) and are
expressed as a percentage of the activity in the absence of antiserum or
serum.
100
plasma
Pbsthepann
100
80
90
80
60
70
70
50
60
50
60
\
40
30
40
30
20
20
50
10
0
II
‘I
0
10 20
I
30
40
50
50
10
0
I
50 90100
0
10
20
30
40
50
of SDS (mmol/L)
Fig. 4. Inhibition of pre- and postheparin hepatic lipase activity by
Concentration
greater than this, however,
hepatic lipase activity rose sharply; therefore, we selected 35 mmol/L
SDS for all subsequent
analyses
of
preheparin
lipoprotein
lipase activity.
The intraassay
imprecision
(CV), determined
by replicate analysis
of six triplets
of a single sample,
was
12.5% for lipoprotein
lipase
and 14.6% for hepatic
lipase. Interassay
CVs, determined
from 14 separate
assays performed
over a 3-month
period, were 19.7%
and 17.4% for lipoprotein
lipase and hepatic
lipase,
respectively.
None of the assay variability
appeared
due to freeze-thawing:
We saw no significant
differences in total preheparin
lipase activities
in eight
samples analyzed fresh (69.8 ± 60 moI/L FA per hour)
and after a single freeze-thaw
cycle (57.3 ± 46 moI/L
per hour; P
0.33 by one-sample
=
Wilcoxon test).
Pre- and Postheparin Lipases and Plasma Lipids and
Lipoproteins
The pre- and postheparin
lipase activities
and respective correlations
are summarized
in Table 1. Both
pre- and postheparmn
hepatic
lipase
activities
were
significantly
greater
in men than women, the latter
being only 66% and 51%, respectively,
of the values for
the men. There was no significant
relationship
between
pre- and postheparin
lipoprotein
lipase but there was a
strong positive
correlation
between the two hepatic
lipase measurements
(Fig. 5). The correlation
was also
significant
between preheparin
lipoprotein
and hepatic
lipase activities.
The age, body mass index, waist:hip
ratio, and lipid and lipoprotein
concentrations
of the 84
subjects
are summarized
in Table 2 and correlated
with pre- and postheparin
enzyme activities.
Preheparin
lipoprotein
lipase was positively
correlated with VLDL cholesterol
and FFA concentrations
(Table 2); however, multivariate
analysis showed these
associations
were independent
of any relationship
be-
SDS.
tween VLDL and FFA (Table 3). Postheparin
s4iiquots of pre- and posthepann plasma were incubated with an equal volume
of SOS in 0.2 mol/LTfls,pH 8.2, at 26’C for 60 mm and assayed for hepatic
lipase activity. The prehepailn data represent the mean (±SEM) of three
separate experiments. Results are expressed as a percentage of the activity
In the presence of 0 SDS.
tom
Table 1. Pre- and posthepann
lipoprotein lipase and hepatic
lipase
activities
in healthy subjects.
Correlation coefficIent
with prehepatin
Mean ± SD Ilpase acty, imol/L FA per hour
All
Men
(n=84)
(n=46)
lipopro-
lipase
activity
was negatively
correlated
with
plasma triglycerides
and was positively
related to age
and the plasma concentrations
of LDL-I, HDL cholesterol, and HDL2 (Table 2).
Women
(n=38)
LPI.
HL
Lipoprotein
lipase
29.8 ± 15.9
Prehepann
4100 ± 1850
Posthepann
29.0 ± 14.9
3810 ± 1350
30.9 ± 17.3
4430 ±
2280
-
0.15
0.23
0.05
Hepatic lipase
33.3 ± 20.3
17 100±8990
Prehepann
Posthepann
0.05;
408
‘
P > 0.005;
P> 0.0001.
CLINICAL CHEMISTRY. Vol. 41. No. 3, 1995
39.5 ± 20.4
22110±8420
25.9k’ ± 17.7
11170±5320
0.23k
0.06
-
0.540
Preheparin
hepatic
lipase was correlated
with body
and intermediate
LDL subfractions
(LDL-I and LDL-II) (Table 2). On multivariate
analyses, in which pre- and postheparin
hepatic lipase activities were used as covariates,
the former was an independent predictor of body mass index, while the latter
was an independent
predictor of waist:hip
ratio, HDL
cholesterol,
and HDL2 concentrations
(Table 3). Neither variable was independently
predictive
of LDL-III.
metabolic
studies.
Such measurements
do, however,
require assays with appropriate
sensitivity
and precision, and application
demands
that their physiological
relevance is known.
With these objectives in mind, we
set out to develop a relatively uncomplicated
assay for
measuring preheparin
lipoprotein and hepatic lipase
and to investigate
the metabolic
significance of these
measurements.
The detection
limit of the assay was improved
by
doubling
the volume of plasma
used, taking
2 mL
rather than 1 mL of the FA fraction for liquid scintillation counting
(and thereby
reducing
counting
error
and increasing
precision) and extending
the incubation
period by 30 mm. Although
the apparent
enzyme
DIscussIon
activity
was much
greater
when longer incubations
were used, the response was not linear beyond 120 mm;
mass index, waist:hip
ratio, and the concentrations
of
HDL cholesterol,
HDL2, and the small dense LDL
(LDL-III)
(Table 2). These relationships
were also
present for postheparin
hepatic lipase, which was in
addition inversely
of large, buoyant,
related to the plasma concentrations
The measurement
of lipoprotein
and hepatic lipase
activities
in nonhepariized
plasma
is attractive
because it obviates the need for heparinization
and offers
the potential for monitoring
enzyme activities
during
this
suggests
that
nonenzymatic
release
of FA may
analyses
of preheparin plasma
lipoprotein Ilpase (LPL) and hepatic lipase (HL)
Table 3. Multivariate
activities.
45
o
-
Response variable
Preheparin LPL
-
o
a
0
30
Body mass index
-
o
o o
o
#{149}
#{149}
0
S
t#{149}#{176}
ot
,
-
S
#{149}
I
0
I
10
I
I
I
I
I
I
I
20
30
40
50
60
70
80
90
Preheparin hepatic lipase (pmol/L FA per hour)
Fig. 5. Correlation of pre- and postheparin hepatic Iipase activities in
46 men (0) and 38 women (S): r = 0.54, P < 0.0001.
Table 2. Correlations
P
5.7
8.0
12.5
0.026
0.008
0.1
0.001
0.753
Waist:hip ratio
Preheparin HL
2.9
0.596
Posthepann HL
10.9
LDL-IlI
Prehepann HL
3.6
0.001
0.073
S
-
105
0
C,
r2, %
Posthepann HL
o
0
15
Predictors
VLDL-C
FFA
Prehepann HL
Postheparin HL
3.2
0.090
HDL cholesterol
Preheparin HL
1.7
0.716
HDL2
Postheparin HL
Preheparin HL
Postheparin HL
16.3
0.0
19.9
0.000
0.886
0.000
The predictorswere includedas covanates in general linear models to
establish (a) their relativecontributionsto variance in the response vanable,
and (b) whetherthey were independently relatedto the response variable.
VLDL-C, very-low-density lipoprotein cholesterol.
of plasma lipid and lipoprotein concentrations and other variables with pre- and postheparin
lipoprotein lipase (LPL) and hepatic lipase (HL) activities.
Correlation (r)
Postheparin
Preheparin
Mean ± SD
(n = 84)
variables
Age, years
Body mass index, kg/m2
Waist:hip ratio, m/m
38
25.1
0.85
5.35
1.31
0.59
(C), mmoVL
Triglyceride mmoVL
VLDL-C, mmoVL
Cholesterol
LDL-C, mmoVL
LDL-I, mg/L
LDL-lI, mg/L
LDL-IlI, mVL
HDL-C, mmoVL
HOL2, mg/L
HDL3, mg/L
FFA, mmoVL
8P >0.05; b P >0.01;
3.52
780
1870
830
1.26
560
2250
0.51
C
P >0.005;
d
± 14
± 3.8
± 0.11
± 1.09
± 0.67
± 0.35
± 0.92
± 620
± 1080
± 910
± 0.35
± 380
± 650
± 0.23
LPL
HL
LPL
HL
0.16
-0.03
-0.13
-0.12
0.15
0.248
-0.01
0.17
0.03
-0.01
0.12
0.03
0.13
0.278
0.04
0.238
-0.12
0#{149}42d
0.278
-0.03
0.15
0.14
0.07
-0.01
0.03
0.32#{176}
-0.09
-0.04
0.12
-0.18
0.06
0.268
043d
-0.09
0.11
0.13
0.02
040d
0.17
-0.18
0.33#{176}
044d
-0.30#{176}
0.10
0.04
0.32C
0.16
0.16
-0.54#{176}
-0.13
-0.24
P >0.0001.
CLINICAL CHEMISTRY, Vol. 41, No. 3, 1995
409
become significant
with extended
incubations.
These
modifications
raised
the sample
results
to 10-80
counts/mm
above those for the blank incubations-an
effect comparable
to the performance
of the assay of
Eckel et al. (5). Repurifying
the substrate
did not,
however, significantly
reduce the background
activity.
We also emphasize
the need for considerable
care in
handling
and processing
samples; because the enzymes
are unstable,
the samples
must
be kept at 4#{176}C
at all
stages (except the incubations)
until after the solvent
extraction is complete.
The next step in the development
of the assay was to
secure a means
for inhibiting
hepatic
lipase in the
measurement
of lipoprotein
lipase. Although
Eckel et
a!. (5) had circumvented
this by calculating
lipoprotein
lipase
as the difference
between
total and hepatic
lipase activities,
this practice
means
that any errors in
the two separate
measurements
will be compounded
in
the calculation
of lipoprotein
lipase activity. Thus, we
consider
selective
measurement
of lipoprotein
lipase
more desirable
and usually accomplished
this for postheparin
plasma
by inhibiting
the hepatic lipase with
specific antiserum
or SDS. Although immunoinhibition
is often regarded
as the method of choice for this (4),
and was used by Karpe et a!. (6) to measure
lipoprotein
lipase in preheparin
plasma, this type of assay is
limited by access to, or the availability,
of antiserum.
As an alternative,
SDS provides
effective and specific
inhibition
of postheparin
hepatic
lipase (17) and is
appropriate
for postheparin
lipoprotein
lipase measurements
(14).
The antiserum
used here had characteristic
antihepatic lipase activity, as demonstrated
with posthepann plasma;
with preheparin
plasma,
however,
some
endogenous
lipolytic activity was apparent.
The endogenous activity was not obvious in postheparin
plasma
because
its hepatic
lipase activity
is some 500-fold
higher than in preheparin
plasma and overwhelms
any
antiserum-associated
lipase activity. We calculate
that
the endogenous
lipase activity in 20 L of antiserum
would reduce its inhibitory
effect on postheparin
hepatic lipase by only 0.5%. Heat inactivation
of the
antiserum
did partially
reduce its apparent
lipolytic
activity,
but this effect was lost with the addition
of
increasing
volumes
of the antiserum.
Any effect of
serum per se on the stability
of the substrate
during
the incubation
was discounted
by parallel experiments
in which fresh and heat-inactivated
human serum was
used. This antiserum-associated
lipase activity
was
previously
not apparent
when preheparin
lipolytic activity was measured
after inhibition
with goat antiserum
to human hepatic lipase and to human lipoprotein lipase
(5) or with goat antiserum
to human
lipoprotein
lipase (18). Whether the lipolytic activity of
the antiserum
used here was specific to rabbit serum
was not investigated,
but the results indicate the need
for caution in using antiserum
to measure
preheparin
lipases.
The reproducibility
of preheparin
lipase measurements is a major concern.
Eckel et al. (5) reported
410
CLINICAL CHEMISTRY. Vol. 41. No. 3, 1995
intraassay,
but not interassay,
CVs of 7.2% and 15.4%
for total lipase and hepatic
lipase,
respectively.
Although Karpe et al. (6) did not report on the intraassay
precision
of their method,
they did acknowledge
that
the interassay
CV for lipoprotein
lipase was 20-25%
and that this variation
should be taken in consideration when evaluating
their data. The methods
developed here gave within- and between-assay
precision for
both enzymes
slightly superior
to those of assays previously described.
The metabolic
significance
of preheparin
lipoprotein
and hepatic lipase activities
has been previously
studied both in fasted
subjects
and in response
to oral
glucose and fat loads. The relation between preheparin
lipoprotein
lipase and fasting
FFA concentrations
in
the present
study was not apparent
in the study by
Karpe et al. (6), although
they did find that these
analytes
were associated
postprandially
through
FFA
of chylomicron
origin. These observations
are consistent with the evidence
that fatty acids can dissociate
lipoprotein
lipase from its binding
sites (19, 20) and
support
the hypothesis
that FFA, through
release from
triglyceride-rich
lipoproteins,
exert negative-feedback
control
on lipoprotein
lipase.
The relation
between
postprandial
FFA
concentrations
and
lipoprotein
lipase may be complicated
by the glucose present
in a
mixed meal, which increases
preheparin
lipoprotein
lipase activity (5). Regardless,
the present
data indicate that under steady-state
conditions
(i.e., fasting)
the release
of FFA from VLDL undergoing
lipolysis
may play an important
role in regulating
the activity of
lipoprotein
lipase.
The association
of preheparin
lipoprotein
lipase with
VLDL cholesterol
may reflect either a precursor-product relationship
between
the circulating
concentrations
of VLDL and FFA or the binding of lipoprotein
lipase to
VLDL. In refutation
of the former, we saw no correlation between VLDL cholesterol
and FFA (r = 0.02) and
found by multivariate
regression
that these two analytes were independently
related to preheparin
lipoprotein lipase activity.
We therefore
consider
the second
explanation
to be more likely and consistent
with the
observation
that lipoprotein
lipase is coeluted
from
plasma with the remnants
of triglyceride-rich
lipoproteins (18). The association
of lipoprotein
lipase with
these lipoproteins
may be important
for the removal of
the enzyme
by the liver and may direct lipoprotein
remnants
for cellular
uptake,
either
by interacting
directly with the LDL-receptor-related
protein (21, 22)
or by binding to heparan
sulfate at cell surfaces
(2325).
The absence of any significant
correlation
between
pre- and postheparin
lipoprotein
lipase activities
in
this
and previous
studies
(6, 7) suggests
that the
plasma and endothelial
pools of the enzyme are not in
equilibrium.
This disequilibrium
arises because
the
amounts
of lipoprotein
lipase free in the circulation
are
kept low by avid uptake and degradation
of the enzyme
by the liver. In addition,
the administration
of heparin
alters
the enzyme’s
apparent
specific activity
(26),
either by posttranslational
activation
of the enzyme or
by increasing
the fraction of enzyme transferred
to the
vascular
endothelium
at the expense of that degraded
at the site of synthesis
(1).
In contrast, the plasma pool of hepatic lipase appears
to be in equilibrium
with the lipase released by heparim, which may be due to the fact that the binding sites
for hepatic
lipase in the liver do not mediate
rapid
degradation
of the enzyme
(1). Preheparin
hepatic
lipase activity is eluted on gel-filtration
chromatography in two peaks: one between
LDL and HDL, and a
second after HDL (18). Thus, the relationships
between preheparin
hepatic lipase and plasma lipoproteins probably
reflect the action of hepatic lipase on
LDL and HDL metabolism,
rather than the binding of
the enzyme to lipoprotein
particles.
The correlations
that we found with preheparin
hepatic
lipase
are
therefore
consistent
with the influence
of the enzyme
on HDL cholesterol
concentrations
(27), its role in the
lipolysis of HDL2 (28), and the intravascular
remodelling of large LDL to small, dense LDL subfractions
(29, 30). The origins of the associations
of pre- and
postheparin
hepatic lipase with body mass index and
waist:hip
ratio are unclear,
but postheparin
hepatic
lipase has been correlated
with these variables
and
with percent body fat and abdominal
and femoral fat
cell weights, in a previous study of obese women (31).
In conclusion,
we describe
a sensitive,
uncomplicated, and reproducible
assay for measuring
lipoprotein lipase and hepatic
lipase in preheparin
plasma.
This
study confirms
that preheparin
hepatic
lipase
activities
are related to the enzyme’s activity in postheparin
plasma and are a measure
of the role that it
plays in lipoprotein
metabolism.
Such measurements
may be of use in situations
where heparinization
is not
possible (although
the relative imprecision
of the preheparin
measurements
should be noted). Preheparin
lipoprotein
lipase activities
correlate with neither postheparin
activities
nor the metabolic
function
of this
enzyme
but may reflect the release of enzyme from the
vascular
endothelium
by plasma FFA and subsequent
binding
to the remnants
of triglyceride-rich
lipoproteins. Given that such remnant
particles
are resistant
to further
lipolysis
by lipoprotein
lipase,
and that
hepatic
lipase does not appear to be associated
with
lipoproteins
in plasma,
we think it unlikely
that the
amounts
of activity
for either lipase in preheparin
plasma play any significant
part in lipoprotein
metabolism.
T.D.G.W. was supported by a Fellowship from the Weilcome
Trust. C.-E.T. was the recipient of a Health Manpower Development Scholarship
from the Ministry of Health, Singapore. This
work was in part supported by a British Heart Foundation award
(BHF 190/1262) and a vacation research grant from the Scottish
Office Home and Health Department.
The technical assistance of
Carole Smith and Liz Murray is gratefully acknowledged.
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