CLIN. CHEM. 39/2, 218-223 (1993)
Hepatic and Lipoprotein Lipases Selectively Assayed in Postheparin Plasma
Alec D. Henderson, William Richmond, and Robert S. Elkeles
Sensitive, reliable procedures are reported for the selec-
tiveassay of lipoprotein lipase (LPL) and hepatic lipase
(HL) in postheparin plasma samples. LPL is inhibited in
the HL assay by inclusion of 0.76 mol/L sodium chloride in
the substrate. In the LPL assay, specificity is attained by
pretreating the sample with sodium dodecyl sulfate, which
selectively denatures HL. This LPL methodwas validated
by direct comparison with a procedure in which HL is
inactivated by an antiserum to human HL. We used the
described assays to quantify LPL and HL activity in 32
normal adults, demonstrating a clear sex difference for
both enzymes. On average, the men displayed higher HL
and lower LPL activities than did the women.
Indexing Terms: enzyme inhibitionassay
ences
sex-related differ-
immunoassaycompared
The hydrophobic nature of lipid molecules necessitates their intravascular transport as lipoprotein complexes. Two enzymes, lipoprotein lipase (LPL; EC
3.1.1.34) and hepatic lipase (ilL; EC 3.1.1.3), catalyze
lipoprotein triglyceride and phospholipid hydrolysis,
playing a key role in intravascular lipoprotein metabolism.’ Both enzymes are located on capillary endothehum, LPL being found on most if not all peripheral
capillary beds, in contrast to HL, which is almost exclusive to the hepatic sinusoids (1). The enzymes bind to
the endothelium via a heparin sulfate-like ligand, projecting into the bloodstream, where they interact with
their circulating substrate. However, because their affinity for heparin is higher than that for the heparin
sulfate-like ligand, injection of an intravenous heparin
bolus displaces both enzymes into postheparin plasma
(PHP), where their activity may be quantified.
The enzymes, although apparently similar with respect to their ability to catalyze lipoprotein triglyceride
hydrolysis, differ in their substrate requirements, pH
optima, activation, and inhibition. LPL, which displays
maximal catalytic activity at pH 8.2 (2), is inactivated
by 1 moIIL sodium chloride (3-6) and protarnine sulfate
(2, 3, 5, 7),agents to which HL is resistant
(4-6). In
addition, LPL displays a requirement for apoprotein
(apo) C-lI (5-10), which is supplied by adding heatinactivated human serum to the substrate emulsion. In
contrast, HL has a pH optimum of 8.8(2), shows reduced
activity in the presence of inactivated serum (11), and is
Unit of Metabolic Medicine, St. Mary’s Hospital Medical School,
Imperial College of Science, Technology and Medicine, Norfolk
Place, LondonW2 1PG, UK.
‘Nonstandard
abbreviations: LPL, lipoprotein lipase; HL, hepatic lipase; PHP, postheparin plasma; SDS, sodium dodecyl sulfate; ape, apoprotein; BSA, bovine serum albumin; and LSI,
light-scattering index.
Received June 2, 1992; accepted October 1, 1992.
218
CLINICAL CHEMISTRY, Vol. 39, No. 2.
1993
inhibited by the concentrations of sodium dodecyl sulfate (SDS) thought to stabilize LPL (7).
These differences have been exploited to achieve the
selective assay of HL by including 1 mol/L sodium
chloride in the substrate to inactivate LPL (12). The
LPL activity in the sample may then be indirectly
estimated as the difference between the total lipase and
HL activities. The assay of total lipase activity is,
however, unsatisfactory because the optimal substrate,
pH, and activator requirements of both enzymes cannot
be achieved in a single assay system.
Direct assay of LPL can be achieved by use of an
antiserum to inactivate HL (4). An alternative may be
offered by SDS, an agent known to selectively inactivate
ilL (7, 10), and a direct LPL assay based on this
observation has been reported (7). Despite the ready
availability of SDS, this procedure has not been widely
adopted, perhaps because of the laborious nature of the
anion-exchange procedure used to isolate the fatty acid,
and because of doubts regarding the efficacy of the HL
inactivation.
In the present study we have developed sensitive,
reliable procedures for the selective assay of LPL and ilL
activities, in which the fatty acid arising from the enzyme-catalyzed triglyceride hydrolysis is isolated by a
simpleextraction
procedure (13). We used SDS to inhibit
IlL and validated this procedure by comparing our results with those obtained with an anti-HL antiserum. In
addition, we evaluated the use of gum arabic and leci-
thin-stabilized
substrate
emulsionsand investigated the
influence of albumin and apo C-fl on assay specificity.
MaterIals and Methods
Materials
Glass scintillation
vials, methanol,
chloroform,
hep-
toluene,
and glacial
aceticacidwere obtained from
Fisons Scientific Equipment (Loughborough, UK). “Ecoscint” scintillation fluid was purchased
from National
Diagnostics (Manville, NJ). Sodium chloride, sodium
dodecyl sulfate, sodium carbonate, sodium bicarbonate,
and ferric chloride were purchased
from Merck Ltd.
(Dagenham, Essex, UK). Carbonate-bicarbonate
buffer
(0.1 mol/L), pH 10.5, was prepared by mixing 800 mL of
0.1 moIfL sodium carbonate with 200 mL of 0.1 mol/L
sodium bicarbonate.
Intralipid
10% was supplied by
KabiVitrum
Ltd. (Uxbridge, UK). Tholein, bovine serum
albumin
(BSA) (essentially
fatty
acid-free),
and Tris
(“Trizma
Preset 8.2 and 8.8”) were obtained from Sigma
ChemicalCo.,Ltd.(Dorset, UK). [‘4C]Triolein was purchased from Amersham
International
plc (Aylesbury,
tane,
Bucks, UK). Plain glass blood-collection tubes (10 mL)
were supplied by Sherwood Medical Industries (Crawley,
Sussex, UK). The anti-HL antiserum was a gift from T.
Olivecrona, University of Umea (Umea, Sweden).
containing 0.5 g of ferric chloride per liter we added 2
mL of the upper phase, swirled the contents, and mixed
this with 16 mL of Ecoscint:toluene
(7:1 by vol). The
Assay Principle
[‘4CIoleic
acid was quantifiedby liquidscintillation
The procedures described quantify LPL orHL activity counting with a Beckman (Brea, CA) LS 7500 scintillation counter (channel 397-655, counted over 5 mm with
in PHP by measurement of the oleic acid produced from
the enzyme-catalyzed
hydrolysis of a triolein emulsion
use of automatic quench correction based on Compton
curve). The LPL activity was calculated from the differcarrying a [‘4Cltriolein radiolabel. The liberated oleic
acid is isolated by a selective extraction
procedure and
ence in counts per minute between the “blank” and the
its mass determined
by reference to the 14C-labeled
sample vials.
tracer, which is quantified by liquid scintillation counting. Lipase activity is calculated in terms of micromoles
HL Assay Procedure
of oleic acid released per hour per milliliter of PHP.
The ML assay was identical to the LPL assay but with
thefollowing
modifications: The 2.75 mL of 200 g/L BSA
LPL Assay Procedure
was added to the triolein/emulsifler
mixtures
before
Substrate
preparation.
Two types of substrate emulsonication, the0.8mL of1.42mol/L NaCl was replaced
sion were produced by sonicating triglyceride with two
by 2.58 mL of 3.24 mol/L NaC1, and the solutions were
different emulsiflers (gum arabic and lecithin): The gum
prepared in 0.2 mol/L Tris buffer, pH 8.8; the substrates
arabic emulsion consisted of a mixture of 100 mg of
were used without addition of, or preincubation with,
unlabeled triolein, 5 pCi of [‘4C]triolein, and 5.68 mL of
inactivated
serum; and no sample pretreatment was
90 g/L gum arabic in 0.2 mol/L Tha buffer, pH 8.2; the
used, 10-/LL aliquots of PHP being added directly to the
lecithinemulsion consistedof a mixture of 1 mL of
substrate.
Intralipid,
5 pCi of [‘4C]triolein,
and 4.68 mL of 0.2
mol/L Ths buffer, pH 8.2. The mixtures were sonicated
Optimization of Assay Conditions
in 20-mL glass scintillation vials. The sonication was
performed
with an MSE Soniprep (Fisons Scientific
Substrate concentration. The substrate triglyceride
concentration required to attain zero-order kinetics was
Instruments,
Crawley, UK) at amplitude 10,with 24
cycles of 20 son, lOs off, and mixing the vial contents by
evaluated for the gum arabic and lecithin emulsions
described. We diluted aliquots of each triglyceride
soninversion after every 4th cycle. After sonication
we
added 2.75 mL of 200 g/L BSA and 0.8 mL of 1.42 mol/L
icate to 258 L with saline and supplemented these with
sodium chloride (both in 0.2 mol/L Tris buffer, pH 8.2)
BSA, NaC1, and human serum (in the same proportions
as the standard sonicate), such that final substrate
and mixed the emulsion by inversion.
Substrate preincubation. Aliquots of substrate emultriglyceride concentrations were in the range 0-8 g/L.
We used thesesubstrates
to investigate
the kineticsof
sion (0.42 mL) were preincubated for 90 mm at 37 #{176}C,
in
LPL and ilL activity in aliquots of a single sample of
20-mL plain glass blood-collection
tubes, with 80 L of
heat-inactivated pooled human serum as the source of
PHP.
Effect of sonication on lecithin (Intralipid)
and gum
apoC-il. The serum pool was prepared from blood donations from 50 subjects,heat-inactivated at 50 #{176}C
for 45
arabic-based emulsions. The effect of sonication on the
particle size distribution
of the gum arabic and lecithin
mm, and stored as 2-mL aliquots at -20 #{176}C
for 1 year
emulsions was assessed by nephelometry (14). Scintilbefore use.
lationvials containing either(a)100 mg of triolein and
Sample preincubation.
Thirty minutes before comple5.68 mL of 90 g/L gum arabic in 0.2 mol/L Tris buffer,
tion of the substrate preincubation, we mixed in an
pH 8.2, or (b) 1 mL of Intralipid diluted to 5.68 mL with
Eppendorftube equalvolumes (0.25mL) ofsample and
70 mmoh/L SDS in 0.2molJL Tris buffer, pH 8.2, and
0.2mol/L Ths buffer, pH 8.2,were subjectedtoO, 6, 12,
18, 24, or 30 sonication cycles (as described in LPL
kept this at room temperature
(20 #{176}C).
An aliquot of
Assay Procedure). The light-scattering index (LSI), an
isotonic saline was treated in the same fashion for use as
index of particle diameter, of each emulsion was detera blank.
mined with aliquota of emulsion diluted 100-fold with 9
Enzyme-catalyzed
triglyceride hydrolysis. On compleg/L sodium chloride solution by use of a Mark IV
tion of the substrate and sample preincubations,
we
micronephelometer (Scientific Furnishings
Ltd., Poynadded 10 L of sample to each of three tubes of activated
ton, Cheshire, UK).
substrate and allowed the LPL-catalyzed triglyceride
Effect of albumin incorporation on HL substrate. To
hydrolysis to proceed for 1 h at 28#{176}C.
Triglyceride
hydrolysis was stopped by adding 5.33 mL of methaassessthe effect on HL activity of adding the BSA before
nol:chloroform:heptane (56:50:40 by vol).
the triglyceride sonication, we analyzed 12 samples for
Extraction
of liberated fatty acid. After 20 s of vigorHL activity
by thestandardprocedureand by sonicating
the substrate before adding the BSA. To minimize the
ously shaking the hydrolysis mixture, we added 1.5 mL
influence of batch differences, we pooled four separate
of 0.1 mohJL carbonate-bicarbonate buffer, pH 10.5,
batches of emulsion for each substrate type.
shookthecontentsforlOs,and centrifuged
the contents
We also evaluated the effect of the presence of albufor 45 mm at 1500 x g with a swing-out rotor.
To a glass
scintillation
vialcontaining50 pL ofglacial
aceticacid min at substratesonication
on nonspecific lipase activ-
CLINICAL CHEMISTRY. Vol. 39, No. 2, 1993
219
ity-non-LPL
(i.e., salt-resistant), non-ilL (i.e., SDS-
resistant)
lipase activity-by
repeating
the above
experiment with six PHP samples, assaying both before
and after pretreatment with SDS (for inactivation of
ilL).
Comparison of lipase activities observed with gum
arabic and lecithin sonicates. We analyzed 10 samples
for LPL and HL activity, using the optimized gum
arabic- and lecithin-based emulsions described above.
Incorporation
of apo C-lI activator into LPL substrate.
The optimal serum volume and preincubation
time for
substrate activation
with ape C-il was determined by
assay of a single sample with aliquots
of the gum
arabic/triolein
substrate
at a triglyceride
concentration
of 9.1 g/L, preincubating
the substratewith 0-80 L of
heat-inactivated
pooled human serum for90 min at
37#{176}C.
Activationof the lecithin-based
substratewas
similarly investigated by using 80 L of serum and
preincubatingfor70 mm.
Comparison of SDS and anti-HL antiserum for inactivation of HL. To compare the use of SDS and anti-ilL
antiserum for the selective assay of LPL, we assayed 15
samples (a) by the standard LPL procedure, (b) by an
adaptationinwhich theSDS pretreatmentwas replaced
by a 2-h preincubation at 4#{176}C
of the PHP with goat
antiserum to human HL, and (c) by using the antiserum
preincubation but including SDS (0.7 mmol/L final concentration) in the substrate to assess any surfactant
effect
introduced by the SDS.
Assay Precision
To assess the extent of the within-assay imprecision,
we prepared five l0O-j.L aliquots from a singlesample of
PHP and treated these as individual samples (each
assayedintriplicate)
ina singlerun ofboththeLPL and
HL assay procedures. Because each sample was assayed
in triplicate, we were able to assess the within-assay
in Figure 1. Both emulsions saturate the availableenzyme at triglyceride concentrations >5 g/L. LPL
displays a higher maximal activity with the gum arabic
emulsion.ilL,although achieving the same maximal
activity
with both emulsions,attains saturation at a
lower triglyceride concentration with the lecithin emulsion. On the basis of these results, we adopted a triglyceride concentration of 9.1 g/L for routine assays.
trated
Effect of sonication on gum arabic- and lecithin-based
emulsions. The changes in LSI observed on sonicating
the gum arabic- and lecithin-based mixtures are illustrated in Figure 2. The gum arabic mixture gave an
increase in LSI with successive sonication reaching a
maximum after 15 cycles;in contrast, the lecithin emul-
sion displayed a small decrease in LSI.
Effect of albumin incorporation on HL substrate. The
ilL activities detected in the 12 samples assayed by
usingsubstratesonicated(x)inthe absenceofBSA and
(y) in its presence were found to be highly correlated (r2
=
0.97) around a line y = 1.06x - 1.05. Thus, use of
postalbuminated substrate led to a small increase in
mean ML activity (mean y = 12.00 ± 7.43, mean x =
12.28 ± 6.90). This increase was not,however,statistically significant (Student’s paired t-test, P = 0.258).
The salt-resistant
lipase (i.e., non-LPL) activity dotected in six samples pretreated with SDS to inactivate
ilL, when expressed as a percentage of the reported ML
activity,
decreasedfrom a mean 5.42% (range 4.0-7.3%,
median 5.25%) to 3.93% (range 1.2-7.3%, median
3.55%) when the prealbuminated
substrate was used.
This decrease was statistically significant in a one-sided
Student’s paired t-test (t = -2.232, P <0.05).
Comparison of triglyceride hydrolase activities
observed with gum arabic- and lecithin-based
emulsions.
Figure 3 summarizes the LPL and HL activities
observed in 10 samples assayed with the gum arabic and
lecithin substrates, at a triglyceride concentration of 9.1
precisionforsinglemeasurements, the means of two
replicates,
and the means of three replicates.
Between-assay CV was determined
by assaying l-mL
aliquotsofa single“control”
PHP (stored at -18 #{176}C)
in
20 runs of the LPL and ilL assay procedures.
25
a-
ReferenceRanges forLPL and HL Activities
a-
-I
E
Reference ranges for the LPL and ML procedures were
by obtaining
blood samples from 32 volun-
15
evaluated
teers with no known metabolic abnormalities. The samples were collected 15 mm after intravenous injection of
heparin (100 lU/kg body weight). These were allowed to
clotfor30 mm at room temperature
in 20-mL plain
glassblood-collection tubes,thencentrifuged
for15 miii
at1500 x g. Aliquots(1mL) ofeach PHP were stored for
1 month at -18 #{176}C,
before analysis for LPL and HL
activity.
0
E
=
0
10
a
a
0
a
0.
-I
Results
5
0
Optimization of Assay Conditions
Substrate concentration.
0
20
The variation of lipase activ-
ity with substrate triglyceride concentration observed
with the gum arabic and lecithin emulsions is illus220 CLINICAL CHEMISTRY, Vol. 39, No. 2, 1993
2
substrate triIyceride
4
concetratjon
6
S
(mg/mU
Influenceof substrateconcentrationon lipoprotein Iipase
) and hepatic lipase (-)
activitiesobtainedwiththe gum
arabic (0)- and Intralipid
(#{149})-based
substrates
Fig. 1.
400
a.
a.
-s
E 20
#{163}
300
S
0
E
C
4.
C
U
200
a
-I
a.
-J
a
0
-I
S
tO
C
0
4,
100#{149}
0
0
30
10
preincubation
10
20
30
Sonicatlon applied (cycles)
Fig.2. Changesin light-scatteringindex with sonication for gum
arabic (0)- and Intralipid (#{149})-based
emulsions
g/L. On average, both LPL and ML give a higher
lipolytic activity with the gum arabic emulsion. The
LPL activities
observed with the two substrates were
highly correlated (r2 = 0.93), as were those observed
with ML (r2 = 0.85). Use of the lecithin-based substrates
yielded a mean of 58% less LPL activity and 34% less
ML activity.
Incorporation of apo C-Il activator into LPL substrate.
Addition of heat-inactivated pooled human serum
greatly increased the LPL activity detected with the
gum arabic/triolein
and lecithin substrates. As illustrated in Figure 4, a further increase in LPL activity
was obtained when the gum arabic/triolein
emulsion
was preincubated
with the inactivated
serum; LPL
activity increased 3.6-fold when the substrate was pre-
50
length (minutes)
FIg. 4. Changes In LPL activity observed on preincubatirig the gum
arabic substratewith0 (0), 20 (#{149}),
40(0), 60 (s), and 80 ct. (E,) of
heat-inactivatedpooled human serum
(A) Changesobservedonpt’elncubatlng
theIntrailpid-based
substratewIth80
pL of heat-Inactivated
serum
for 90 mm with 80 pL of serum. The lecithin
substrate showed no preincubation requirement, the
maximal LPL activity being obtained on adding 80 i.&L
of serum to the substrate aliquots.
incubated
Comparison
of SDS and anti-HL antiserum
for inactivation of HL. SDS and a goat antiserum raised against
human IlL were evaluated for use in the selective assay
of LPL in PHI’ samples. The LPL activities detected
with (x) the antibody procedure and (y) the SDS procedure correlated (r2 = 0.84) around a linear-regression
limeofy = 0.96x + 1.70 (Figure 5, lower axis), indicating
that the same enzymatic activity is quantified by each
procedure. The increase in mean LPL activity from 6.54
mol h’
mL’
measured by the antibody
procedure
-
.
20
C
S
15
(“U.
15
y-1.13x+0.45
_
tr2 -
.
1
12
0.96, n
20)
-
.
I;!
-
0.44:
*“-0.44.
.
#{149}
L70
.101
SD 229)
-
0.44:
-
0.51
15#{149}
4’0.95.
_...-
0S
y0.96x+
10
U
a
-
1.70
0.54.
‘
-
20)
S
a
.9.
-J
3
.
l
0
0
5
10
Lipase activity
with gum
(pmoles/hr/ml.
15
20
25
arabic emulsion
PHPP
FIg.3. Comparisonof LPL (0) and HL (#{149})
activities obtained
withthe
gum arabic and Intralipid
emulsions
,
I
0
5
I
10
7.98. SD 2.08)
I
15
LPI. by
antibody pretreatment
limoios/hr/m(.
PHP)
Fig. 5. Comparison of LPL activities detected with the SOSandthe
anthody procedures, with (upper) and without (lowe,) SOS Included
In thelatterprocedure
CLINICAL CHEMISTRY, Vol. 39, No. 2,
1993
221
to 7.98 hmol h’ mL
with the SDS procedure was
statistically significant (Student’s t-test, t = -7.80, P =
0.000). The LPL activity measured by the antibody
procedure was increased when 0.7 mmol/L SDS was
included in the substrate (upper axis, Figure 5): The
linear-regression line of y = 1.13x + 0.48 obtained
closely resembles that observedwith the SDS procedure.
The increase in mean LPL activity from (6.54 to 7.85
jmol
h’ mL1)
was again statistically significant
(Student’s paired t-test, t = -11.02, P = 0.000). By
-
-
-
.
assess the statistical
significance
of the observed sex
differences in LPL and ML activity. By the MannWhitney U test, the data are consistent with a trend in
the women toward a higher LPL and lower HL activity
(mean women’s LPL rank 20.59 vs men’s LPL 11.87
mol
h
mL’,
P <0.01; mean women’s HL rank
12.18 vs men’s ML 21.40 pmol h’
mL’, P <0.01).
.
.
-
DIscussIon
The kinetic studies described show that both LPL and
paired Student’s t-test, the data obtained
with this
modified antibodyproceduredid not differfrom that
obtained with the SDS methodology (respective mean
LPL activityof 7.85 and 7.98 zmol h1 ‘mL’,
t =
-0.74,not significant).
HL require
Assay Precision
glyceride concentration with the lecithin emulsion. For
the 10 samples examined,
however, higher catalytic
activities were obtained for both enzymes when gum
arabic-based emulsions were used.
Gum arabic emulsions have previously been shown to
give a higher LPL-catalyzed
triglyceride hydrolysis
than do lecithin emulsions (15), perhaps reflecting a
preference for a larger-diameter substrate (16). In the
present studies, the gum arabic emulsion yielded a
higher 121 value (Figure 2) than did the lecithin emulsion, consistent with the formation of larger vesicles.
Furthermore, in the case of IlL, higher triglyceride
concentrations
were required with the gum arabic substrate. This is consistent with the production of an equal
number of larger-diameter
substrate particles. However, both observations could equally be attributable
to
nonspecific protein binding by the gum arabic emulsion,
leading to decreased triglyceride
substrate availability
attheoil-waterinterface (17). Substrate specificity (18,
19) may also contribute to the apparent preference of
both enzymes for the gum arabic emulsion. Intralipid is
a phospholipid-stabilized
soybean oil emulsion, comprising a complex mixture of triglycerides (20), in contrast
to the homogeneoustriolein content of the gum arabicstabilized emulsion. Any preferential hydrolysis of nontriolein substrates would result in an apparent decrease
in lipase activity (only triolein hydrolysis being detected
by reference to the “C label).
The contribution to the reported ML activity of a
salt-resistant
(non-LPL)
and SDS-resistant (non-IlL)
activity decreased on use of prealbuminated substrate.
On the basis that any increase in specificity is desirable
and that the sensitivity of the procedure appears unaffected, we advocate the use of prealbuminated substrates in the HL assay. Nonetheless, use of postalbuminated substrates appears to lead to little if any
alteration in ML activity.
In the present studies, optimal conditions for substrate activation were determined for a single substrate
triglyceride
concentration (9.1 g/L). LPL activity was
greatly increased when inactivated serum was added
and, when the gum arabic emulsion was used, was
further increased by preincubation at 37#{176}C.
In view of
the observed variation of particle size with somcation
-
For the gum arabic emulsified substrate, the withinassay CV, calculated from the SD around the mean of
five (triplicate) determinations of a single sample within
a single assay run, was 6% for the LPL assay and 3% for
the HL assay. When only two replicates were used,
analytical performance was unaffected. Use of single
determinations led to increased imprecision, intra-assay
CVs of 8% and 5% being observed with the LPL and HL
assays, respectively.
The between-assay CV, calculated on the basis of the
SD around the mean LPL and IlL activities observed in
a single sample assayed in 20 runs of each procedure,
was 12% for LPL and 9% for ML (mean activities of 10.8
and 12.4mol h’
mL’, respectively).
-
.
Reference Ranges for LPL and HL
Limited reference ranges for each procedure were
obtained by assay of PHP samples from 32 normal
volunteers. As Table 1 shows, the data forthe women
were skewed toward lower values, mean activities
(mo1 h1 mL’) of 13.21 and 9.03 and medians of
11.87and 7.53beingobtainedforLPL and ML, respectively. In contrast, the LPL data for men were skewed
toward the higher values (mean 13.05, median 13.39).
HL data in men’s samples showed a slight positive skew
(mean 20.47, median 19.64).
Given the distribution of the observed data and the
small sample size, we used nonparametric analysis to
-
.
Table 1. Llpoproteln Lipase and Hepatlc Lipase
Activities (mol h
mL) in 32 Normal Subjects
-
.
Women(n=17)
Range
Median
Mean
Men(n=15)
LPL
HI
6.44-23.42
11.87
13.21
5.12-16.91
LPL
3.33-24.41
7.53
9.03
13.39
19.64
13.05
20.47
7.87
0.62
2.73
SD
4.55
3.63
5.27
Skewness
0.57
2.27
0.71
0.24
2.13
2.62
Kurtosis
HI
8.45-38.91
Mann-Whitney U test:
LPL meanrank 20.59 vs 11.87 P = 0.0087.
HL mean rank 12.18 vs. 21.40 P = 0.0055
222 CLINICAL CHEMISTRY, Vol. 39, No. 2, 1993
a substrate
triglyceride
concentration
in
catalytic activity with the
gum arabic and lecithin
substrates. LPL displays a
higher maximum catalytic activity with the gum arabic
substrate; in contrast, IlL displays the same maximal
activity with both substrates, although at a lower tri-
excessof 5 g/L for maximal
(Figure 2), and the known influence of particle diameter
on apoprotein uptake (21), we emphasize that investigators should determine the optimal period of incubation and substrate:serum ratio for different sonication
procedures and for each serum pool used to supply
heat-inactivated
apo C-Il.
The observed interassay CVs of 12% and 9% for LPL
and HL, respectively, are consistent with those reported
by previous workers who used gum arabic emulsions (7,
22,23). In the present studies, using triplicate analysis,
we obtained intra-assay CVs of 6% and 3% for the LPL
and ML procedures, respectively. Use of duplicate analysis led to no decrease in analytical performance. On
this basis, we recommend the use of duplicate analysis
in routine assay.
The comparison between SDS and anti-HL antiserum
for selective assay of LPL confirms the efficacy of selective inhibition of ML by SDS (7, 10) and the use of SDS
for the selective assay of LPL (7). The increase in LPL
activity observed with the antiserum procedure on addition of 0.70 mmol/L SDS to the substrate is consistent
with a surfactant effect of SDS on the substrate. Although the data obtained by the SDS and antiserum
procedures are not directly interchangeable, they are
highly correlated. Thus, SDS pretreatment offers a convenient, consistent, off-the-shelf alternative to the use of
an anti-HL antiserum in the assay of LPL.
We have applied the described assaysin the diagnosis
of LPL deficiency and in investigation of the mochanisins underlying
hyperlipidemias
associated with anti-
hypertensive therapy (24), gestational hypertriglyceridemia (25), acute hyperinsulinemia
(26), obesity (27),
and insulin
insensitivity
(28).
We are indebted to Thomas Olivecrona, Department of Physiological Chemistry, University of Umea, for his kind gift of the
anti-HL antiserum used in these studies. The statistical advice of
Judith Dymond, Department of Public Health, St. Mary’s Hospital
Medical School, is gratefully acknowledged.
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