147

Original Contributions

Effect of Hepatic Lipase on LDL in Normal Menand Those With Coronary Artery Disease

Alberto Zambon, Melissa A. Austin, B. Greg Brown, John E. Hokanson, and John D. Brunzell

Hepatic triglyceride lipase (HL) is thought to play a role in the formation of low density lipoproteins(LDLs) from small very low density lipoproteins (VLDLs) and intermediate density lipoproteins (IDLs).To analyze the possible physiological role of HL in determining LDL buoyancy, size, and chemicalcomposition, HL activity and LDL were studied in 21 patients with coronary artery disease (CAD) and 23normolipidemic subjects. In both groups, LDL buoyancy and size were inversely associated with HLactivity levels. The effect of HL on LDL size was comparable in CAD patients and in normolipidemicsubjects. HL appeared to influence LDL lipid composition primarily by affecting the surface lipidcomponents. The free cholesterol content of LDL particles was highly correlated with HL activity in bothCAD and normolipidemic individuals. The free cholesterol to phospholipid ratio in LDL particlescorrelated with HL in both CAD and normolipidemic subjects. When the individuals were separatedaccording to their LDL subclass patterns, pattern B subjects had significantly higher HL than pattern Asubjects in both CAD and normolipidemic groups. The analysis of the cholesterol distribution profilesacross the lipoprotein density gradient confirmed that LDL buoyancy is affected by HL. These datasupport the hypothesis that HL modulates the physical and compositional properties of LDL andcontributes to the expression of the LDL subclass phenotype. suggesting a physiological role for HL inLDL metabolism. (Arteriosclerosis and Thrombosis 1993;13:147-153)

KEY WORDS • hepatic lipase • LDL • coronary heart disease • lipoprotein size • lipoproteindensity

The association between low density lipoprotein(LDL) cholesterol and coronary artery disease(CAD) is now believed to be causal.'-4 LDLs

have been characterized as heterogeneous lipoproteinsthat include particles differing in size, density, andprotein and lipid composition.5"7 A number of differentintravascular enzymes are involved in the lipoproteincascade leading to LDL formation. These include lipo-protein lipase (LPL), hepatic triglyceride lipase (HL),and lipid transfer protein. Although HL seems to beinvolved in the conversion of remnant particles initiallyformed by LPL action to LDL,8 the physiological role ofHL in LDL metabolism is not clear. HL appears to besynthesized by the liver cells and bound to the luminalsurface of the endothelial cells of the liver where theremodeling of small very low density lipoprotein(VLDL) and intermediate density lipoprotein (IDL) to

From the Division of Metabolism, Endocrinology and Nutrition(A.Z., J.E.H., J.D.B.) and the Division of Cardiology (B.G.B.),Department of Medicine, and the Department of Epidemiology(M.A.A.), School of Public Health and Community Medicine,University of Washington, Seattle.

Supported by National Institutes of Health (NIH) grant HL-30086 and NIH First Award HL-38760. A portion of this study wasperformed at the University of Washington Medical Center,Clinical Research Center (NIH RR-37). A.Z. is a recipient of agrant from the Italian National Research Committee (CNR,bando n. 203.04.13).

Address for correspondence: John D. Brunzell, MD, Division ofMetabolism, Endocrinology and Nutrition, Department of Medi-cine, University of Washington, Mailstop RG-26, Seattle, WA98195.

Received April 6, 1992; revision accepted October 26, 1992.

LDL may take place.9 The strongest evidence support-ing the role of HL in LDL formation comes from studiesperformed on human and animal models with HLdeficiency.810-14 In these studies, low HL activity wasassociated with larger and more buoyant LDL particles,often extending into the IDL density range.

To further examine the possible involvement of HL indetermining the physical characteristics of LDL, thecompositional and physical properties of LDL wereexamined in relation to HL activity levels in CADpatients and normolipidemic subjects.

MethodsSubjects

Twenty-one men aged 50.2 ±9 (mean±SEM) yearsand with elevated apolipoprotein (apo) B plasma levels(>125 mg/dl), a family history of CAD, and evidence ofcoronary atherosclerosis by coronary arteriography3

were selected. All subjects were participants in a clinicalintervention trial of intensive drug therapy for CAD;blood samples for the present study were drawn at aprotocol time point 6 weeks after discontinuing lipid-lowering therapy, at which time all LDL and apoB-related measurements had returned to pretreatmentbaseline levels.3 Two subjects were taking /J-blockers atthe time of the present study. Dietary advice was givenaccording to the American Heart Association's step Iand II diets.3 Patients with diabetes; severe hyperten-sion; cancer; or liver, thyroid, or kidney diseases wereexcluded.

An additional 23 Caucasian men aged 33 ±2 yearswere recruited by advertisement and were paid to

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participate in the present study. Plasma triglycerides(TGs), LDL cholesterol (LDL-C), high density lipopro-tein cholesterol (HDL-C), and apo B values werebetween the fifth and 95th percentiles as reported in theLipid Research Clinics Population Study (North Amer-ica) for white men.1516 None of these subjects wastaking any medication known to alter lipid metabolismnor was following particular dietary restrictions.

The present study was approved by the human sub-jects review committee of the University of Washington.Informed consent was obtained from all subjects.

Blood CollectionBlood samples were collected in 0.1% EDTA after a

12-hour fast. An intravenous heparin bolus (LyphoMedInc., Rosemont, 111.) of 60 IU/kg was then administered,and after 10 minutes blood was collected in iced lithium-heparin tubes for the measurement of HL activity.Plasma was immediately processed by centrifugation at4CC and stored at either 4 C for subsequent lipoproteinanalysis or at -70°C for later determination of HLactivity.

Single Vertical-Spin Density GradientUltracentrifugation for Apo B-Containing Particles

This technique17 was developed as a variation of thesingle vertical-spin ultracentrifugation method byChung et al18 to specifically improve the resolution ofapo B-containing lipoproteins. By layering 2 ml ofplasma adjusted to a density of 1.080 g/ml underneath12 ml of a 1.006 g/ml NaCl solution, a discontinuous saltgradient was produced in a Sorvall TV-865B (DuPontCo., Wilmington, Del.) rotor. Samples were centrifugedat 65,000 rpm for 90 minutes (total wt2=2.36xl0") at10°C; the tubes were then fractionated from the bottomat a flow rate of 1.7 ml/min, and 38 0.45-ml fractionswere collected. Total cholesterol was measured in eachfraction. The relative flotation rate (Rf), which charac-terized LDL peak buoyancy, was obtained by dividingthe fraction number containing the LDL-C peak by thetotal number of fractions collected. To evaluate theintra-assay coefficient of variation, density gradientultracentrifugation (DGUC) of paired samples was per-formed on plasma from nine subjects. The plasma fromeach subject had been frozen with ethanol/dry ice andstored at — 70°C for 1-29 months for replicate analysis.The Rf of the LDL peak was obtained as describedabove. The coefficient of variation of the Rf valuesobtained by replicate analyses19 of the same sampleswas 0.2%. Thus, the LDL Rf number determined bysingle vertical-spin DGUC was found to be highlyreproducible. The fractions selected to contain thematerial defining the sharp peak of LDL were pooledafter DGUC. The ranges characterizing these fractionsvaried among subjects and were based on the choles-terol profile of each sample to ensure that the pooledsamples contained the entire lipoprotein peak.

Nondenaturing Gradient Gel ElectrophoresisSix microliters of whole plasma was diluted 4:1 in

tracking solution (50% sucrose and 0.01% bromophenolblue) and electrophoresed at 12-14°C in 2-16% poly-acrylamide gradient gels (Pharmacia, Uppsala, Sweden)at 125 V for 24 hours using tris(hydroxymethyl)ami-nomethane (0.09 M)/boric acid (0.08 M)/Na2EDTA

(0.003 M) buffer, pH 8.3. Samples were then electro-phoresed as described.5 Gels were stained for lipid withoil red O and scanned at 490 nm. The moleculardiameter of peaks was determined using a calibrationcurve based on standards of known diameters. Thestandards included ferritin and thyroglobulin (Pharma-cia) with molecular diameters of 12.2 and 17.0 nm,respectively, and thyroglobulin dimer and carboxylatedlatex beads (Dow Chemical) with diameters of 23.6 and38.0 nm, respectively. To determine the intra-assaycoefficient of variation, fasting plasma was obtainedfrom 13 individuals with TG values ranging from 46 to602 mg/dl. These samples were collected in refrigeratedtubes containing 1 mg/ml EDTA, spun at 2,500 rpm toremove cells, and separated into several aliquots. Onealiquot was stored at 4°C for <1 week before electro-phoresis. The remaining aliquots were immediately fro-zen using ethanol/dry ice and stored at -70°C forreplicate analysis performed within 1-8 weeks. Themean diameter of the major LDL subspecies of the 13fresh samples was 26.0 nm. Replicate analysis of thesame samples that were frozen and then electropho-resed after thawing showed a mean diameter of 26.91nm. The coefficient of variation of the diameter of themajor LDL subspecies by replicate analysis19 of 13samples was 0.8%. Thus, the determination of LDLpeak particle diameter by gradient gel electrophoresiswas found to be highly reproducible.

Postheparin Plasma Lipase ActivityTotal lipolytic activity was measured in postheparin

plasma as described previously.20 The substrate contain-ing glycerol tri[l- Cjoleate (Amersham, ArlingtonHeights, 111.) and lecithin was incubated with aliquots ofpostheparin plasma for 60 minutes at 37°C, and theliberated free fatty acid (FFA) radioactivity was ex-tracted and counted. Lipase activity is expressed asnanomoles of fatty acid released per minute per milli-liter of plasma (nmol FFA/min per ml). LPL activitywas selectively eliminated by incubation with a specificmonoclonal antibody (5D2) against LPL; HL activitywas defined as the remaining activity detected in thepostheparin sample after incubation with the anti-body.21 For each assay, a bovine milk LPL standard anda human postheparin plasma standard were included toadjust for interassay variation.

Other AnalysesLipoproteins were isolated using standard sequential

ultracentrifugation.22 Plasma total cholesterol andVLDL cholesterol (VLDL-C), LDL-C, and HDL-Cwere determined by enzymatic methods (BoehringerMannheim, Indianapolis, Ind.).23 Plasma TG and LDLTGs were measured by use of an enzymatic kit (SigmaChemical Co., St. Louis, Mo.). LDL free cholesterol,esterified cholesterol, and phospholipid analyses wereperformed by the Northwest Lipid Research Laborato-ries following published procedures.24 Apos B and A-Iwere analyzed by immunonephelometry (Behring Diag-nostic, Sommerville, N.J.), as described.25 Briefly, eachassay was calibrated using frozen serum reference ma-terial with an assigned value traceable to primaryreference material. Frozen-serum quality-control poolsat three different levels (low, normal, and high) wereincluded in each run. The average intra-assay and

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Zambon et al Hepatic Lipase and LDL Cholesterol 149

TABLE 1. Clinical and Upoproteln Characteristics of CADPatients (n=21)

Age (years)BMI (kg/m2)HL (nmol FFA/min per ml)LPL (nmol FFA/min per ml)Total cholesterol (mg/dl)Total triglycerides (mg/dl)VLDL cholesterol (mg/dl)LDL cholesterol (mg/dl)HDL cholesterol (mg/dl)Apolipoprotein B (mg/dl)Apolipoprotein AI (mg/dl)

Mean±SEM

50±227±0.6

264±31210±23311±18202±2242±5

223±1845±2

209±14136±5

Range

35-6523-3035-563

106-476118-44864-4106-77

90-38129-67

129-334100-179

CAD, coronary artery disease; BMI, body mass index; HL,hepatic lipase; FFA, free fatty acids; LPL, lipoprotein lipase;VLDL, very low density lipoprotein; LDL, low density lipopro-tein; HDL high density lipoprotein.

interassay coefficient of variation for both apo A-I andapo B was <3%.

Statistical AnalysisStatistical analyses were performed with STATISTIX,

PC DOS version 2.0 (NH Analytical Software) andSIGMAPLOT version 4.1 (Jandel Scientific) computer pro-gTams. A linear regression model was used to determinecorrelation coefficients for the association of HL activitywith LDL physicochemical indices. Where appropriate,an unpaired two-tailed Student's t test was applied. Theresults are expressed as mean±SEM. The level ofstatistical significance for these experiments wasp<0.05.

ResultsIn CAD patients (Table 1), mean TG (p<0.001), total

cholesterol (p<0.001), VLDL-C (/xO.OOl), LDL-C(/?<0.001), and apo B (/><0.001) values were signifi-cantly higher than in normolipidemic subjects (Table 2),whereas HDL-C and apo A-I measurements were notsignificantly different between the two groups. No signif-icant differences were observed between CAD pa-

TABLE 2. Clinical and Lipoprotein Characteristics ofNormolipidemic Subjects (n=23)

Mean±SEM Range

Age (years)BMI (kg/m2)HL (nmol FFA/min per ml)LPL (nmol FFA/min per ml)Total cholesterol (mg/dl)Total triglycerides (mg/dl)VLDL cholesterol (mg/dl)LDL cholesterol (mg/dl)HDL cholesterol (mg/dl)Apolipoprotein B (mg/dl)Apolipoprotein AI (mg/dl)

33±223±0.7

271±2O170±ll181±688±917±2

113±650±2

100±5135±3

21-5920-33

154-55579-284

122-22342-2448-49

58-16030-6756-142

117-162

BMI, body mass index; HL, hepatic lipase; FFA, free fatty acids;LPL, lipoprotein lipase; VLDL, very low density lipoprotein;LDL, low density lipoprotein; HDL, high density lipoprotein.

tients and normolipidemic subjects for LPL activity(210±23 versus 170±ll nmol FFA/min per ml, respec-tively) and HL activity (264±31 versus 271±20 nmolFFA/min per ml). The range of HL activity was similarin CAD patients and normolipidemic subjects (Tables1 and 2). Age and body mass index (BMI) weresignificantly higher (p<0.005) in CAD patients than innormolipidemic subjects. Although these groups weredifferent primarily because of the disease status, thedifference in age may also have contributed to thevariation in various parameters. No significant associ-ation was found for either group between HL activityand age (CAD patients: r= -0.006,/?=0.98; normolip-idemic subjects: r= -0.19,^=0.38) or between HLandBMI (CAD patients: r=0.07, p=0.76; normolipidemicsubjects: r=0.34, p=0.ll).

In CAD patients, a significant inverse linear relationbetween HL activity and both the diameter of the majorLDL subclass (Figure 1A; r=-0M, p=0.036) and theLDL buoyancy (Figure IB; r=-0.80, /?<0.001) wasfound. When the same linear regression was performedwithout the two patients treated by 0-blockers, theinverse relation between HL and LDL physical proper-ties was even more evident (HL versus LDL diameter:r=-0.51, p=0.026; HL versus Rf: r=-0.83, /xO.OOl).In normolipidemic subjects a similar, inverse lineartrend between HL activity and the diameter of themajor LDL subclass was seen (Figure 2A; r=— 0.60,p<0.0025). A significant, strong inverse relation alsowas observed between HL and LDL buoyancy (Figure2B;r=-0.72,p<0.001).

Possible LDL compositional modifications related toHL activity and accounting for changes in size andbuoyancy were subsequently analyzed in the CAD pa-tients and normolipidemic subjects. Linear regressionanalysis was performed and correlation coefficients cal-culated between HL activity and LPL activity and 1) thelipid composition of each LDL fraction, 2) the LDLlipid composition per particle (obtained by dividingeach lipid value of the LDL fraction by the same LDLfraction's apo B content), and 3) the relative lipiddistribution within the surface of the LDL particle andbetween the surface and core (Table 3).

In CAD patients, HL activity was inversely andsignificantly associated with free cholesterol, cholesterylester, TG, and phospholipids of the LDL fraction. Innormolipidemic subjects the free cholesterol of the LDLfraction was significantly associated with HL activitywhile cholesteryl ester, TG, and phospholipids did notshow significant associations. There was no associationbetween LPL activity and any lipid component of theLDL fraction in either the CAD or normolipidemicgroup.

In CAD patients as well as in the normolipidemicgroup, HL activity was strongly and inversely related tothe free cholesterol content per LDL particle. In indi-viduals with CAD, the phospholipid content per LDLparticle was also significantly and inversely associatedwith HL activity, whereas neither group showed arelation between HL activity and cholesteryl ester andTG content per LDL particle. LPL activity in both theCAD and normolipidemic groups was not associatedwith the lipid composition per LDL particle. In CADpatients and in normolipidemic subjects, a markedinverse association was also seen between HL activity

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300 -,

aas 275 -o

h 250 -o

r=-0.46p<0.05

0 100 200 300 400 500 600

HEPATIC UPASE ACTIVITY (nmol/mln/ml)

0.40 -i

r=-0.80p<0.001

FIGURE 1. Plots show correlation by linear regressionanalysis between (panel A) hepatic lipase (HL) activity(nmol/min per ml) and low density lipoprotein (LDL) size(A) and (panel B) HL activity and LDL buoyancy (rela-tive flotation [Rf] value) in coronary artery diseasepatients.

100 200 300 400 500 600

HEPATIC LIPASE ACTIVITY (nmol/mln/ml)

and the free cholesterol to phospholipid ratio per LDLparticle (r=-0.81, p<0.005, CAD group; r=-0.58,/)<0.005, normolipidemic group). Conversely, LPL ac-tivity was not associated with the free cholesterol tophospholipid ratio of LDL particles.

The HL activity affected the overall lipid distributionbetween surface lipids (free cholesterol and phospho-lipids) and core lipids (TG and cholesteryl ester) ofLDL particles in individuals of both groups. A statisti-cally significant inverse relation was found between theHL activity and the LDL lipid surface to core ratio(r=-0.60,/><0.025, CAD patients; r=-0.59,p<0.005,normolipidemic subjects). The LPL activity was notassociated with the LDL lipid surface to core ratio ineither the CAD or normolipidemic group.

Gradient gel electrophoresis analysis of LDL wasused to classify subjects into two subgroups based on theLDL subclass patterns A and B according to criteriapreviously reported.26-27 In the present study both CADpatients and normolipidemic subjects characterized byLDL subclass distribution pattern B had significantlyhigher HL activity than pattern A individuals (HLactivity, 328 versus 160 nmol/min per ml, p< 0.005, CADpatients; 303 versus 197, p<0.05, normolipidemicsubjects).

DiscussionPrevious studies have mainly examined the effect of

HL on VLDL and HDL metabolism,28-32 whereas thepossible effect of HL on LDL physical properties andcomposition is less clear. The present study supports thehypothesis that HL is involved in determining LDLphysicochemical properties,10-12 suggesting also that thisfinding is observed despite marked differences in clini-cal characteristics and lipid levels of the subjectsexamined.

We studied two populations, normolipidemic menand those with CAD, with different clinical character-istics and lipoprotein values, to evaluate the physiolog-ical effect of HL action on LDL particles. In bothpopulations, subjects with low HL activity had relativelylarge (by gradient gel electrophoresis) and buoyant (byDGUC) LDLs, whereas high values of HL activity wereassociated with LDL particles that were smaller anddenser. In particular, linear regression analyses showedthat 64% and 52%, in CAD patients and normolipi-demic subjects, respectively, of the variation (calculatedas r2) in the LDL buoyancy was accounted for bydifferences in HL activity. The changes in LDL sizewere accounted for by differences in HL activity to theextent of 26% in CAD patients and 36% in normolipi-

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Zambon et al Hepatic Lipase and LDL Cholesterol 151

300 i

g 275

250 -

225 -

0

r= -0.60p< 0.0025

0 100 200 300 400 500 600

HEPATIC UPASE ACTIVITY (nmol/mln/ml)

0.40 -

2 0.35

s1CO

r= -0.72P< 0.001

0.30 -

0.25 -

0.20

0.0100 200 300 400 500 600

HEPATIC UPASE ACTIVITY (nmol/min/ml)

FIGURE 2. Plots show correlation by linear regression analy-sis between (panel A) hepatic lipase (HL) activity (nmollminper ml) and low density lipoprotein (LDL) size (A) and(panel B) HL activity and LDL buoyancy (relative flotation[Rf] value) in normolipidemic subjects.

demic subjects. These data confirm previous studies thatshowed the presence of more buoyant and larger LDLsin both familial10" and acquired12 forms of HL defi-

ciency. The low coefficients of correlation (r) obtainedfor the association between HL activity and LDL size inboth populations indicate that other factors, geneticallyand environmentally modulated,262733 also play an im-portant role in accounting for changes in LDL size. LPLactivity was not associated with the buoyancy or the sizeof LDL particles in either CAD patients or normolipi-demic subjects.

Compositional studies were performed on the LDLfraction to explain the physical changes associated withHL activity. HL activity was significantly and inverselyassociated with the four lipid components of the LDLfraction in CAD patients. In normolipidemic subjectsthe LDL free cholesterol content was significantly andinversely associated with HL activity. In CAD patientslow HL activity was associated with a significant in-crease in TG, phospholipids, and free and esterifiedcholesterol content of the LDL fraction, confirmingprevious studies on animal models.34-35 Changes in thedistribution of the lipid components in the LDL frac-tion, reported as absolute lipid values, provide a generalinsight into the effect of HL activity on this lipoproteinsubclass. To specifically examine the compositionalmodifications per LDL particle, the apo B value of thefraction containing the LDL peak was determined ineach subject, and the LDL lipid to apo B ratio for eachlipid component was calculated. Notably, in CAD pa-tients, high HL activity resulted in LDL particles thatwere relatively lipid depleted; in particular, a statisti-cally significant decrease in free cholesterol and phos-pholipid content per LDL particle was observed. Innormolipidemic subjects the free cholesterol contentshowed the same statistically significant trend. Jansen etal34 demonstrated that inhibition of HL activity by useof a specific antibody did not affect the disappearance oflabeled LDL protein from plasma. The present datasuggest that the HL-related removal of phospholipidsand free cholesterol from LDL particles occurs inde-pendently of the degradation of their protein moieties.Because of the phospholipase At activity possessed byHL, the decrease in phospholipids induced by highlevels of this enzyme is easily understood. The decreasein free cholesterol content per LDL particle associatedwith high levels of HL activity in both the CAD and

TABLE 3. Linear Regression Analysis (r Values) Between Hepatic Lipase and Lipoprotein Lipase and LDL Lipid Composition

LDL

Free cholesterol

Cholesteryl ester

Triglyce rides

Phospholipids

Free cholesterol/apo B

Cholesteryl ester/apo B

Triglycerides/apo B

Phospholipids/apo B

Free cholesterol/phospholipids

Surf/core

r

-0.64

-051

-0.57

-0.61

-0.72

-0.34

-0.4

-0.45

-0.81

-0.6

CAD

HL

P

< 0.005

<0.025

< 0.025

< 0.005

<0.005

NS

NS

<0.05

<0.005

<0.025

patients

LPL

r

0.29

0.32

0.11

0.32

-0.01

-0.34

-0.35

-0.42

0.26

0.18

P

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

r

-0.5

0.02

0.3

-0.02

-0.59

0.19

0.36

0.06

-0.58

-0.59

Normolipidemic

HL

P

<0.025

NS

NS

NS

< 0.005

NS

NS

NS

<0.005

< 0.005.

subjects

LPL

r

-0.12

-0.13

-0.01

-0.18

0.04

-0.02

-0.13

-0.06

-0.07

0.11

P

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

LDL, low density lipoprotein; CAD, coronary artery disease; HL, hepatic lipase; LPL, lipoprotein lipase; apo, apolipoprotein; Surf, LDLsurface lipids; core, LDL core lipids. Lipids and apolipoprotein in milligrams per deciliter and lipoh/tic activity in nanomoles per minute permilliliter were used for regression analyses.

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normolipidemic groups is likely consequent to the HLaction on LDL phospholipids, with increased efflux offree cholesterol from LDL to HDL particles. Indeed, amodification of the relative free cholesterol to phospho-lipid molar ratio of LDL particles was demonstrated asa major determinant of the free cholesterol net transferfrom LDL to HDL particles.36-37 The statistically signif-icant, inverse association between HL activity and theLDL free cholesterol to phospholipid ratio observed inboth groups can also be explained by this metabolicmodel.

The free cholesterol to phospholipid ratio measuredin whole plasma has been reported to be positivelycorrelated with the risk for ischemic vascular diseases.38

Whether HL activity affects the risk for ischemic vascu-lar diseases by modifying the free cholesterol to phos-pholipid ratio of LDL particles must be confirmed. HLactivity appeared primarily to influence the surface lipidcomponents of LDL particles. A statistically significantrelation between this enzyme and the LDL surface tocore lipid ratio was found in CAD patients and normo-lipidemic subjects.

The analysis of the DGUC cholesterol profiles of thetwo populations confirmed that LDL buoyancy is af-fected by HL activity. These results confirm previousstudies that used DGUC to analyze the lipoproteinprofile in subjects with HL deficiency.10-11 Decreasedbuoyant LDLs with an increase in dense LDL particlescharacterized the lipoprotein profile of subjects withhigh HL activity in both populations.

The LDL subclass size distribution appears to begenetically influenced, as shown by Austin et al.26-27 Inthose studies, gradient gel electrophoresis analysis ofLDL was used to classify subjects into two subgroupsbased on the LDL subclass patterns A and B accordingto criteria previously reported.26-27 In the present studyboth patients and control subjects with pattern B hadhigher HL activity than pattern A individuals, suggest-ing that HL may be involved in the expression of theLDL subclass patterns.

In summary, we have described studies of the effectof HL on LDL physical and compositional characteris-tics and on LDL-C distribution in two populations withmarkedly different clinical and lipid characteristics. Inboth groups HL seems to affect both LDL size andbuoyancy, and its action mainly involves the surfacelipid components of LDL particles. The cholesteroldistribution within the LDL density range also seems tobe influenced by HL activity, with increased denseLDLs and a reduction of buoyant LDL-C associatedwith high levels of HL activity. These data suggest thatHL, among other genetic and environmental factors, isimportant in determining LDL physical and composi-tional properties in both CAD and normolipidemicpopulations, supporting the hypothesis of a physiologi-cal role for HL in LDL metabolism.

AcknowledgmentsThe authors would like to express their thanks to Alegria

Aquino-Albers, Steve Hashimoto, Martha Kimura, CarrieNelson, Alem Nicodimos, and Jennifer Watanabe for theirexcellent technical assistance.

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A Zambon, M A Austin, B G Brown, J E Hokanson and J D BrunzellEffect of hepatic lipase on LDL in normal men and those with coronary artery disease.

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