Atherosclerosis, 95 (1992) 151- 170 0

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1992 Elsevier Scientific Publishers Ireland, Ltd. All rights reserved. 0021-9150/92/$05.00

Printed and Published in Ireland

ATHERO 04862

Apolipoprotein A-I and apolipoprotein B containing lipoprotein particles in coronary patients treated with extracorporal low density lipoprotein precipitation (HELP) Eugen Korena, Victor W. Armstrongb, Gudrun Muellerb, Paul R. Wilsona, Peter Schuff-Werner b, Joachim Thieryc, Thomas Eisenhauer b, Petar Alaupovica and Dietrich Seidel” “Lipoprotein and Atherosclerosis Research Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, Oklahoma 73104 (USA), bCenter of Internal Medicine, University of Giittingen and ‘Institut of Clinical Chemistry, Grosshadem Clinic, Ludwig-Maximilians University, Munich (FRG)

(Received 18 April, 1991) (Revised, received 10 April, 1992) (Accepted 4 May, 1992)

Summary Evidence for chemical and biological heterogeneity of human plasma lipoprotein density classes has been steadily accumulating over the last 15 years. Furthermore, several recent reports have indicated potential clinical significance of certain lipoprotein subspecies as either atherogenic or antiatherogenic. It is generally accepted that lipid lowering treatments can retard or even reverse development of atherosclerotic lesions. However, very little is known about effects of various lipid lowering treatments on specific lipoprotein particles. The purpose of this study was to explore the effects of heparin induced extracorporal low density lipoprotein precipitation (HELP) on various subspecies of plasma lipoprotein particles defined primarily by their apolipoprotein composition. Using particle specific enzyme immunoassays, the immediate changes in lipoprotein particle profiles were analyzed after a single HELP treatment in 12 patients with angiographically documented coronary artery disease. In a separate group of 6 patients, particles were repeatedly measured over a period of 96 h following a HELP treatment. Single HELP treatment caused an immediate and highly significant decrease (67%) in the concentration of simple lipoprotein particles containing apolipoprotein B (apo B) as a sole apolipoprotein (LP-B). Various subspecies of complex particles containing apo B and other apolipoproteins (Lp-B-complex) were also decreased although to a lesser degree (44-530/o). HELP treatment caused an insignificant, 3% decrease of lipoprotein particles containing apo A-I but no apo A-II (Lp-A-I) and a 6% decrease in the concentration of particles containing both apo A-I and apo A-II (Lp-A-I:A-II). During the 96-h period following HELP treatment various apo Correspondence to: Eugen Koren, Lipoprotein and Atherosclerosis Research Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104 USA.

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B containing particles recovered at different rates in different patients. In hypercholesterolemic patients Lp-B recovered at a high rate whereas the recovery of Lp-B-complex remained sluggish. In hypertriglyceridemic patients recovery of Lp-B-complex was very fast, significantly surpassing the recovery of Lp-B. These results confirm beneficial possibilities of HELP treatment which efficiently reduces atherogenic apo B containing lipoproteins and leaves antiatherogenic particles, especially Lp-A-I, virtually unaffected. It appears, however, that HELP treatment should be particularly suitable for patients with high levels of Lp-B because of its pronounced lowering effect on this, potentially most atherogenic, lipoprotein particle. In patients with high Lp-B-complex HELP should be more efficient in conjunction with hypotriglyceridemic medication as triglyceride lowering drugs tend to decrease Lp-B-complex and increase Lp-B particles.

Key words: LDL apheresis; Coronary artery disease; Lipoprotein

Introduction Heparin induced extracorporal low density lipoprotein precipitation (HELP) has been proven as an efficient cholesterol lowering treatment in patients with homozygous and heterozygous familial hypercholesterolemia [l-4]. The method is based on precipitation of apolipoprotein B (apo B)-containing lipoproteins with heparin at low pH followed by the removal of precipitate, restoration of pH and reinfusion of plasma into patient. Such a treatment significantly reduces plasma low density lipoproteins (LDL) and very low density lipoproteins (VLDL) both of which are associated with a risk of atherosclerosis [5-71. The concentration of high density lipoproteins (HDL), known as a negative risk factor [8-121, is much less affected [ 1,4]. None of the conventional lipoprotein density classes is chemically homogenous. It has been shown that both HDLz and HDLs can be subfractionated by immunoaffinity chromatography [ 13- 151, agarose electrophoresis [ 161,combination of ion exchange and hydroxylapatite column chromatography [ 171 and chromatofocusing [ 181. In all cases two major subfractions of high density lipoproteins were distinguished on the basis of apolipoprotein composition. A subfraction called lipoprotein A-I (Lp-A-I) contains apo A-I but no apo A-II [13,14,17]. The other subfraction, lipoprotein A-I:A-II (Lp-A-I:A-II), consists of particles containing both apo A-I and apo A-II [19]. Although some of the Lp-A-I particles could contain minor quantities of apo E an&or apo C

particles; Cholesterol; Triglycerides

peptides, the great majority contain apo A-I as a sole apolipoprotein [17,18]. A small fraction of Lp-A-I:A-II particles contains apo E, apo D and/or apo C peptides in addition to apo A-I and apo A-II [ 191. The two major HDL subfractions, i.e., Lp-A-I and Lp-A-I:A-II, are unequally distributed along the HDL density range. Lp-A-I is more abundant in HDL2, whereas Lp-A-I:A-II prevails in HDL3. In very high density lipoproteins (VHDL), Lp-A-I is again more abundant than Lp-A-I A-II [13]. These two subpopulations of HDL particles undergo separate metabolic pathways [20]. Lp-A-I also appears to have a higher capacity for reverse cholesterol transport [15,21]. It has also been suggested that Lp-A-I is a better negative predictor of CAD than the HDL cholesterol [22]. Apolipoprotein heterogeneity of low density lipoproteins (LDL) has also been demonstrated. Particles containing apo B as a sole apolipoprotein (Lp-B) are the most abundant population within LDL. However, up to 10% of LDL particles contain apo E in addition to apo B (Lp-B:E). Lp-B:E from LDL binds to LDL-receptor with higher affinity than Lp-B [23,24]. Very low density lipoprotein class (VLDL) comprises the most heterogenous population of particles not only in terms of size and density but also in terms of apolipoprotein composition. According to the lipoprotein families concept, tive different types of particles can be found across the entire VLDL density range (d = 0.94- 1.006 g/ml). These are Lp-B containing apo B as a sole apolipoprotein, Lp-B:E containing apo B and apo

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E, Lp-A-II:B-complex containing apolipoproteins A-II, B, C-I, C-II, C-III, D and E, Lp-B:C:E containing apolipoproteins B, C-I, C-II, C-III and E and Lp-B:C containing apolipoproteins B, C-I, CII and C-III. Apo C-III is the most abundant C apolipoprotein in the VLDL particles. In healthy donors, the most abundant VLDL subpopulations are Lp-B:C:E, Lp-A-1I:B complex and Lp-B:E particles accounting for 45-55% of the total VLDL. Lp-B comprise up to 20% and Lp-B:C up to 25% of all VLDL particles. However, these percentages are variable depending on the type of lipoprotein status. For example, in type V hyperlipoproteinemia, Lp-B:C:E is the most abundant VLDL subspecies (60%) whereas in Tangier’s disease Lp-A-II:B-complex represents the predominating (70%) population [ 19,251. Glycogen storage disease is characterized by an unusually high percentage (40%) of Lp-B:C particles. Although all of the described live apo B containing families are polydisperse and extend through the entire VLDL density range, their predominant density is shifted towards lower or higher values depending on the plasma triglyceride concentration [ 191. The importance of subclassification of VLDL into lipoprotein families defined by apolipoprotein composition is further emphasized by differences in metabolic and pathophysiological properties of various particles. Lipoprotein lipase has been shown io degrade Lp-A-II:B-complex at a slower rate compared to Lp-B:C:E [25]. Apo Econtaining VLDL particles bind to LDL receptor with higher affinity compared to those without apo E [26]. The apo B-containing lipoproteins also differ with respect to their atherogenic potential. In the Cholesterol-Lowering Atherosclerosis Study (CLAS), lipoprotein particles containing apo B and C-III were the only significant predictors of the progression of coronary artery disease (CAD) in patients treated with colestipol and nicotinic acid [27,28]. In our preliminary study of 84 CAD patients, triglyceride enriched particles in which apo B is associated with apo C and apo E were better predictors of the severity of CAD than VLDL-cholesterol or LDL-cholesterol. These particles appeared to be predictive of multiple, moderately stenosing coronary lesions found in elderly patients. On the other hand, cholesterol

rich Lp-B particles were more significant predictors of CAD than LDLcholesterol in younger patients with less frequent but highly stenosing lesions [29]. These data demonstrate that the conventional lipoprotein density classes contain metabolically and pathophysiologically distinct particles that can be defined, isolated and quantified on the basis of their apolipoprotein composition but not on the basis of their lipid composition or density. In view of these considerations, the purpose of this study was to explore the effects of HELP treatment on various apo B and apo A-I containing lipoprotein particles for two major reasons. First, it was of interest to ascertain whether the HELP treatment affects various lipoprotein particles in a selective or indiscriminate fashion. Second, HELP patients represent a unique human model that allows an insight into the recovery of lipoprotein particles after their removal from circulation. Materials and Methods Patients The twelve patients recruited into this study at the Center of Internal Medicine, University of Giittingen, had angiographically documented clinically symptomatic coronary heart disease (Table 1) and had been undergoing regular HELP treatment for a mean of 14.3 months (range: 2-28 months) at the time of the investigation. HELP-LDL-apheresis The procedure has been described in detail elsewhere [l-3]. Briefly, blood obtained from a forearm vein is circulated through a 0.45-pm filter to obtain plasma which is then mixed with an equal volume of 0.2 M acetate buffer (pH 4.85) containing 100 IU/ml heparin to effect precipitation of apo B-containing lipoproteins and a limited number of other plasma proteins including librinogen. The suspension is continuously recirculated through a 0.45~pm polycarbonate filter in order to separate plasma from the precipitate. Subsequently, excess heparin is completely adsorbed on a DEAE-cellulose membrane, physiologic pH is

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TABLE 1 LIPID AND LIPOPROTEIN

PROFILES OF CORONARY PATIENTS TREATED WITH HELP SYSTEM

Patients

Number of vessels showing 50% or higher stenosis

Total cholesterol

Triglycerides

LDL cholesterol

HDL cholesterol

Ltia)

I 2 3 4 5 6 7 8 9 IO II I2

3 I I 2 3 3 3 3 3 3 2 3

170 295 218 231 248 262 266 217 241 316 232 138

93 153 155 208 I09 I09 128 122 441 175 237 78

96 196 156 149 173 197 I90 I36 132 222 157 80

54 78 36 54 61 50 51 59 37 62 43 39

9.8 7.0 5.8 4.0 7.5 10.0 7.0 6.3 4.0 5.2 4.5 10.2

restored by bicarbonate dialysis and the excess fluid is removed by ultrafiltration. The treated plasma is finally remixed with the blood cells and returned to the patient by the other forearm vein. The complete procedure is monitored by a microprocessor-guided fully automated device (Plasmat Secura, B. Braun Melsungen, FRG). Blood flow rates are generally around 60-80 ml/mm yielding plasma flow rates of 20-30 ml/mm. Patients are generally treated every 7 days, with 3000 ml of plasma being cleared at each session. It should be also be mentioned that all patients receive an i.v. heparin anticoagulant bolus 10 min before the treatment (100 units/kg) and a heparin infusion (1000 units/h) during the treatment. Our pilot studies showed that there is an initial 20% decrease in plasma triglycerides which lasts for the first 30 min following the bolus injection. Triglycerides remain stable at that level for 30-40 min followed by a gradual return to the pretreatment levels during the last 30 min of the HELP procedure. Acute effects

To study the acute effects of HELP treatment on various plasma lipid and lipoprotein subpopulations, blood samples were withdrawn directly prior to the apheresis and again at the end of apheresis after treatment of 3000 ml of plasma.

Kinetic studies

The recovery of different lipoprotein particles was followed in two groups of patients. Fasting blood samples were taken immediately before and after an apheresis and at 4, 24, 48, 72, and 96 h after the end of the procedure. A group of 3 hypertriglyceridemic patients had triglyceride levels of 619 i 203 mg/dl (mean f S.D.), total cholesterol of 285 f 58 mg/dl, LDL-cholesterol (LDL-C) of 202 i 18 m@dl and HDL-cholesterol (HDL-C) of 32 * 12 mg/dl. The second group comprised 3 hypercholesterolemic patients with triglyceride levels of 125 i 56 mg/dl, total cholesterol of 366 f 102 mg/dl, LDL-C of 294 f 99 mg/dl and HDL-C of 56 * 2.9 mg/dl. Precipitation of lipoprotein particles in vitro The pH-dependent precipitation of the various lipoprotein particle subpopulations was explored in the in vitro experiments. Serum samples (0.1 ml) were mixed with an equal volume of 0.2 M acetate buffer containing 100 IU/ml heparin that had been adjusted to the following pH values: 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2 and 5.4. In the control experiments, heparin was omitted from the acetate buffer. After mixing and a IO-min incubation at room temperature the samples were centrifuged to remove the precipitate and the supernatant was analyzed.

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Analytical procedures

Total cholesterol and triglycerides were determined with commercially available enzymatic test kits (Boehringer, Mannheim, FRG). Serum LDL cholesterol was quantified by a precipitation procedure based on dextran sulfate (QuantolipLDL, Immuno, Heidelberg, FRG) and HDL-cholesterol by the phosphotungstate/MgClz precipitation procedure (Boehringer, Mannheim, FRG). Lp(a) was determined by the previously published method

t21. Enzyme-linked immunosorbent assay of lipoprotein particles containing apo A-I

The concentrations of Lp-A-I and Lp-A-I:A-II were determined by the previously described micro-ELISA [13]. Briefly, a microtiter plate was coated with antibody to apo A-II, blocked and incubated with plasma samples to immobilize the lipoprotein particles containing both apo A-II and apo A-I. The unbound plasma constituents were removed by washing, peroxidase-labeled antibody to apo A-I added, the plate rewashed, peroxidase substrate added and the resulting color measured. The optical density readings were used to determine the concentration of apo A-I associated with apo A-II. Another microtiter plate was coated with antibodies to apo A-I, blocked and incubated with identical plasma samples to immobilize all apo A-I containing particles. The optical density readings from this plate were used to determine the concentration of total apo A-I. By subtracting the concentration of associated apo A-I from the total apo A-I, the concentration of unassociated apo A-I (Lp-A-I) was derived. The method is specific, rapid and precise. Within- and between-assay coefficients of variation are 5.6% and 9.8%, respectively. The average normolipidemic concentration of apo A-I associated with apo A-II (Lp-AI:A-II) is 79 mg/dl in women and 78.8 mg/dl in men. The corresponding values for unassociated apo A-I (Lp-A-I) are 64.4 and 57.7 mg/dl, respectively [ 131. Enzyme-linked immunosorbent assays of lipoprotein particles containing apo B

Lipoprotein

particles containing

apo B were

quantified by a non-competitive, ‘sandwich’ micro-ELISA procedure using 96-well microtiter plates (No. 3590, serocluster, flat bottom EIA plate, Costar, Cambridge, MA). Biotinylated antibodies, prepared by the use of N-hydroxysuccinimidobiotin (Sigma, St. Louis, MO), were detected with the Streptavidin-horseradish peroxidase conjugate (Bethesda Research Laboratories, Gaithesburg, MD). Peroxidase substrate color reagent containing H202, (2,2,-azino-di(3_ethylbenzothiazoline)sulfonate) and bovine serum albumin (BSA) were purchased from Kirkegaard and Perry Laboratories Inc. (Gaithesburg, MD). Absorbance was measured at 605 nm with a microELISA reader MR600 (Dynatech Instruments, Inc., Torrance, CA) interfaced to an IBM XT computer. Standard curve parameters and concentrations of lipoprotein particles were calculated by the use of the ELISANALYSIS program (Jeff Peterman, Inc., Alabama, GA). Principle of the method The method consists of the following steps: (a)

measurement of total plasma apo B (first plate); (b) measurement of apo B associated with all other apolipoproteins or ‘total Lp-B-complex’ (second plate); (c) measurement of apo B associated with apo A-II, C-I, C-II, C-III, D and E or ‘Lp-A-1I:B’ (third plate); (d) calculation of apo B free of other apolipoproteins (Lp-B) by subtracting the total Lp-B-complex from total plasma apo B and (e) calculation of apo B associated with a mixture of lipoprotein particles comprising Lp-B:E, Lp-B:CI:C-II:C-1II:E and Lp-B:C-III (Lp-B:C:E+B:E+B: C-complex) by subtracting the value for Lp-A-1I:B from the value of total Lp-B-complex. Each plasma sample was analyzed on all three microELISA plates. On the first plate coated with ‘pan’ apo B monoclonal antibody, all apo B containing particles were retained and quantified by the use of biotinylated polyclonal antibody to apo B followed by streptavidin peroxidase. On the second plate, wells were coated with a mixture of monoclonal antibodies to apo A-II, apo C-III and apo E. After removal of the unbound plasma constituents by washing, biotinylated polyclonal antibody to apo B was applied followed by streptavidin-peroxidase. Apo B associated with other apolipoproteins, i.e., total Lp-B-complex,

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was quantified using this plate. Because the plate was coated with a mixture of monoclonal antibodies to apolipoproteins A-II, C-III and E, a variety of lipoprotein particles had been retained. However, only those containing apo B were quantified due to the use of biotinylated polyclonal antibody to apo B as the second antibody. The third plate coated with ‘pan’ monoclonal antibody to apo A-II was used for quantifying apo B associated with Lp-A-1I:B. The concentration of four types of lipoprotein particles, i.e., Lp-B, total Lp-B-complex, Lp-A-1I:B and Lp-B:C:E+B:E+B: C-complex, are expressed in terms of mg of apo B/d1 of plasma. For example, the concentration of 15 mg/dl measured for the total Lp-B-complex means that 15 mg/dl of plasma apo B is present in the form of this lipoprotein subfraction. Monoclonal ‘pan’ apo B antibody and biotinylated polyclonal antibody to apo B

Production and characterization of murine monoclonal ‘pan’ antibody to human apo B has been described elsewhere [30]. Briefly, this antibody (IgGt, Kappa) showed high and closely comparable affinities for all apo B containing lipoprotein density classes, apo B-100 and apo B48 and removed all apo B from normolipideniic and hyperlipidemic plasma samples. Pan-apo B antibody was purified from ascites by the use of a Protein-A column, dialyzed against phosphate buffered saline (pH 7.4; PBS), concentrated with dry sucrose to 1 mg IgG/ml, aliquoted and stored in tightly closed cryovials (Costar, Cambridge, MA) at -80°C. Antiserum to apo B was prepared in goats as described previously [31] and used as a source of polyclonal antibody to apo B. The polyclonal IgG was isolated by ammonium sulfate precipitation followed by ion exchange chromatography on a DEAE-agarose column at pH 8.0. The purified IgG fraction was biotinylated [32], dialyzed extensively against PBS, aliquoted and stored at -80°C in cryovials. Monoclonal antibodies to apolipoproteins A-II, CIII and E

Monoclonal antibodies to apolipoproteins and lipoproteins have been produced for the last 7 years at the Oklahoma Medical Research Foundation’s centralized Monoclonal Antibody Labor-

atory. So far, a library of nearly 100 monoclonal antibodies to LDL, HDL, apolipoproteins A-I, AII, B, C-III and E has been developed for the Lipoprotein and Atherosclerosis Research Program. Four monoclonal antibodies were selected for these assays. Using the criteria described for ‘pan’ B antibody two additional ‘pan’-antibodies were characterized and selected. Monoclonal ‘pan’ antibody to apo A-II, CdBs (IgGi, Kappa), shown to bind to all apo A-II containing lipoprotein density classes with high and closely comparable affinities allows a complete removal of apo A-II from normolipidemic and hyperlipidemic plasma samples [33]. Monoclonal antibody to apo C-III, XbA3 (IgGi, Kappa), also binds to all apo C-III containing density classes with high and closely comparable affinities and allows complete removal of apo C-III from normolipidemic and hyperlipidemic plasma. Two monoclonal antibodies to apo E, both of which are IgG,, Kappa, were selected. One of them, EfD3, binds predominantly to HDL and LDL with high affinity, whereas the other, EfB,, shows a higher affinity for VLDL. Together, these two antibodies allow a complete removal of apo E from normolipidemic as well as hyperlipidemic plasma. All antibodies were purified from ascites by the use of protein-A atTinity chromatography, dialyzed extensively against PBS, concentrated with dry sucrose to 1 mg IgG/ml, aliquoted and stored at -80°C in the cryovials. The four antibodies used in this study were selected on the basis of their ability to separate apo B associated with other apolipoproteins (total Lp-B-complex) from particles containing apo B as the sole apolipoprotein (Lp-B) as follows. Using an immunoaffinity column containing CdBS, XbA3, EfD3 and El& antibodies it was possible to completely adsorb total Lp-B-complex while the Lp-B passed through the column and was recovered in the unretained fraction. This experiment was successfully carried out with nine hyperlipidemic (total cholesterol > 220 mg/dl, triglycerides > 300 mg/dl) and five normolipidemic plasma samples. It should also be emphasized that the above antibodies have to be used in the following molar ratio to ensure complete retention of total Lp-B-complex; CdBs = 1, XbAs = 1.6, EfD3 = 2.6 and EfBi = 2.6. Deletion of either antibody results in an incomplete reten-

163

tion of complex apo B-containing lipoprotein particles particularly in hyperlipidemic plasma samples. Primary standards

Total Lp-B-complex, Lp-A-1I:B and Lp-B served as primary standards. They were isolated and characterized as follows. Total Lp-B-complex. A mixture of VLDL and from LDLi, isolated by ultracentrifugation hypertriglyceridemic plasma (triglycerides = 1500 mg/dl), was run over described immunoaffinity column containing monoclonal antibodies to apolipoproteins A-II, C-III and E. After washing, the column was eluted with 3 M NaSCN (pH 7.4) in order to recover the retained fraction as described previously [34]. Neutral lipid composition showed triglycerides as a predominant lipid (65%), while negative staining electron microscopy revealed a heterogenous population of spherical particles varying in size from 23 to 150 nm. When rerun over the same column, this fraction was completely retained without significant changes in its composition. Detailed immunodiffusion and electroimmunoassay studies against monospecific polyclonal antisera [35-381 showed that a great majority of lipoprotein particles in the retained fraction contained apo B, apo E, apo C-III and apo A-II. These results were consistent with our previous studies demonstrating the presence of LpB:C:E, Lp-B:E and Lp-A-1I:B:C:D:E as major and Lp-B:C-III and Lp-B:C-I:C-II:C-III as minor subspecies of complex apo B containing lipoprotein particles [23,39]. It was, therefore, concluded that the fraction retained by the column contained all constituents of the usually encountered mixture of complex apo B-containing lipoprotein particles. This fraction was used as a primary standard for the total Lp-B-complex. The concentration of apo B in the total Lp-B-complex standard was determined by electroimmunoassay [31]. Aliquots of the total Lp-B-complex were stored at -80°C without significant changes in composition for at least 12 months. Lp-A-II:B-complex. Isolation and characterization of this subpopulation of lipoprotein particles was described in detail elsewhere [25]. The concentration of apo B in Lp-A-II:B-complex was also determined by electroimmunoassay.

Lp-B. LDL2 isolated from normolipidemic plasma was run over the immunoaffinity column containing monoclonal antibodies to apolipoproteins A-II, C-III and E. Approximately 90% of the apo B applied to the column was recovered in the unretained fraction in which no other apolipoproteins, excluding ape(a), were detectable by double immunodiffusion or by the respective electroimmunoassays. Gradient-gel and SDS-polyacrylamide electrophoresis only revealed apo B-100, while electron microscopy showed a rather uniform population of spherical particles with an average diameter of 18-20 nm. Cholesterol ester was the predominant (70%) neutral lipid. Since these characteristics were consistent with those of previously described Lp-B [34,39], the LDLz fraction not retained by the mixture of monoclonal was considered as a suitable primary Lp-B standard. The concentration of apo B in the primary Lp-B standard was determined by electroimmunoassay. Storage at -80°C did not cause any significant changes in the properties of Lp-B. Secondary standards and controls

Using the total Lp-B-complex, Lp-A-1I:B complex and Lp-B as primary standards, the concentrations of these lipoprotein particles were determined in three selected plasma samples intended for the standard curve and high and low controls respectively. These plasma samples were stored in 5 ~1 aliquots at -80°C and used on a weekly basis. Details of the method

Three plates were coated with respective monoclonal antibody solutions (‘pan’ apo B, ‘pan’ apo A-II and a mixture of CDbs, XbAs, EfD3 and EfB,) for 18 h at 25°C in a humidified chamber (100 ~1 antibody solution per well) and blocked with 1% BSA in PBS for 1 h at 25°C. After three washes with PBS, plasma samples diluted in 1% BSA-PBS were pipetted in duplicates (100 @well) to all three plates following an identical pattern. The pattern consisted of seven dilutions (1:2000 through 1:128 000) of plasma selected for the standard curve and of four dilutions (1:8000 through 164 000) for each of the controls and unknown samples. Plates were then incubated for 18 h at 25°C in a humidified chamber and washed

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three times with PBS. After washing, 100 @well of the biotinylated polyclonal antibody to apo B was added to all plates followed by an incubation for 2 h at 25°C. The next step included 3 washes with PBS and a 2-h incubation at 25°C with streptavidin-peroxidase diluted lOOO-fold in 1% BSA. In the final step, the plates were washed 5 times with PBS and the peroxidase substrate (100 pi/well) was added to all wells. After 30 min the optical density was determined with a microELISA reader (MR600, Dynatech, Torrance, CA). All three assays, i.e., total Lp-B-complex, Lp-AII:B-complex and Lp-B, were satisfactory in terms of reproducibility because neither of the respective inter- and intra-assay coefficients of variation exceeded 10%. Verification of assay and quality control

To verify the specificity and recovery of the described assays, isolated apo B-containing lipoprotein particles were mixed and analyzed. Known quantities of isolated Lp-B were added to the known amounts of either total Lp-B-complex or Lp-A-II:B-complex in order to prepare the following mixtures: Lp-B/Lp-B-complex with 10% of total apo B in the form of Lp-B and 90% of apo B in Lp-B-complex followed by 20% Lp-B/80% Lp-B-complex; 40% Lp-B/60% Lp-B-complex and 90% Lp-B/10% Lp-B-complex. Each of the above mixtures was applied to ELISA plates immediately after mixing and analyzed in order to determine the recovery of both lipoprotein subspecies. This experiment was repeated four times; the average recoveries were 98% f 5% for Lp-B and 94% f 9% for total Lp-B-complex or Lp-A-II:Bcomplex. Additional validation of ELISA was carried out by the use of immunoprecipitation with purified polyclonal antibodies. Described mixtures of Lpand Lp-B/Lp-B-complex B/Lp-A-1I:Bcomplex were incubated, respectively, with polyclonal antibody to apo A-II and with a mixture of polyclonal antibodies to apo A-II, apo C-III and apo E for 6 h at room temperature. The incubation was followed by centrifugation (10 min in a microfuge) and separation of supernatant. Meanwhile, aliquots of the original lipoprotein mixtures with no antibodies added were diluted with PBS to the same volume recorded in lipoprotein/antibody mixtures. Concentrations of apo B were deter-

mined by the electroimmunoassay (EIA) in both supematants after immunoprecipitation and properly diluted lipoprotein mixtures. The concentrations of apo B in diluted lipoprotein mixtures represented total apo B values in each case. The concentration of apo B in the supematant determined after the precipitation with the mixture of polyclonal antibodies to apolipoproteins A-II, CIII and E represented Lp-B. The difference between total apo B and apo B found in the supernatant after the precipitation with the mixture of polyclonal antibodies represented Lp-B-complex. The difference between total apo B and apo B found in the supemate after the precipitation with polyclonal antibody to apo A-II represented the concentration of Lp-A-II:B-complex. The average recoveries from four separate experiments were 96% i 3% for Lp-B, 97% f 9% for Lp-Bcomplex and 92% f 7% for Lp-A-II:B-complex. Total apo B concentrations determined in lipoprotein mixtures by ELISA and by EIA correlated significantly (r = 0.97, P < 0.01, n = 16). Both of these methods were also applied to plasma samples with variable cholesterol and triglyceride concentrations resulting in significant correlation (r = 0.91, P c 0.05, n = 25). To establish criteria for accepting ELISA plates, standard curve and high and low controls were repeatedly analyzed on 30 plates for each of the described lipoprotein particle assays. Based on these data, an average slope, standard error and coefficient of correlation were calculated for each standard curve. Average values f S.D. for the high and low controls were also determined. These parameters were used as criteria for acceptance of ELISA plates as follows. The plate was accepted if (a) the coefficient of correlation for the standard curve was higher than 0.96, (b) the slope of the standard curve was within the range (* 1 S.E.) of the average slope and (c) high and low controls were within the respective ranges ( f 1 S.D.) of the high and low average values. To accept the value for an unknown sample the four described dilutions had to give an average value with a coefficient of variation (CV) of 10% or lower. If the CV exceeded lo%, the most extreme of the four values was eliminated. If the CV of the remaining three values still exceeded 10% the sample was reanalyzed. In summary, the assays used in this study allow-

165

ed quantitative determination of two types of apo A-I (Lp-A-I and Lp-A-I:A-II) containing lipoprotein particles and four subpopulations of apo B containing lipoprotein particles (Lp-B, total Lp-Bcomplex, Lp-A-II:B-complex and Lp-B:C:E+B: E+B:C complex). It should be pointed out that the Lp-B population determined in described fashion could also comprise particles containing ape(a) in addition to apo B, i.e., Lp(a):B. However, patients selected for this study were low in Lp(a) (Table 1) which minimized potential error in determination of Lp-B. Based on generally accepted 1:l molar ratio of ape(a) to apo B in Lp(a) and on the protein content of Lp(a) (30% of the total lipoprotein mass), the amount of apo B in Lp(a):B could not account for more than 3% of the Lp-B value even in patients with highest (10 mg/dl) measured Lp(a) levels. Results

Analysis of the supernatants after the in vitro precipitation with heparin at different pH-values revealed differences among the lipoprotein particles. All apo B containing lipoproteins displayed a pH-dependent precipitation similar to that already observed for total cholesterol and total apo B [40]. Maximum precipitation occurred at pH of 4.8 (Fig. lb). The Lp-B particles were precipitated more efficiently than any of the com-

plexes. In contrast to the apo B containing lipoproteins, there was a minimal precipitation of apo A-I containing lipoproteins. The lipoprotein Lp-A-I was not precipitated, whereas the Lp-AI:A-II complex showed around lo-20% precipitation over a rather broad pH range (Fig. la). Results of the removal of lipoprotein particles from patients’ plasma by the HELP treatment were in agreement with those of the in vitro precipitation. The concentrations of apo Bcontaining lipoprotein particles changed significantly after a single treatment with HELP. Lp-B was removed most efficiently, decreasing from an average of 96.5 mg/dl before treatment to 31.8 mg/dl after treatment. The apo B containing complexes decreased significantly as well although not as much as Lp-B (Table 2). Alterations in the levels of the apo A-I containing lipoprotein particles were much less pronounced and insignificant (Table 2). Plasma lipids changed concomitantly with particles. Total cholesterol and especially LDL-cholesterol decreased most significantly, reflecting the highest decrease observed in cholesterol rich Lp-B whereas the less pronounced decrease in triglycerides (Table 3) could be accounted for by the lower decrease observed in triglyceride rich apo B containing complexes. In a kinetic study the recovery of apo B containing particles was followed in two groups of patients over a period of 4 days after a single

A

.Ff

30.5 4.0

4.5

5.0

PH

5.5

6.0

.a

30.5 4.0

4.5

5.0

5.5

6.0

PH

Fig. 1. Precipitation of lipoprotein particles with heparin at low pH. Removal of apo A-I containing particles (A) was insignificant based on concentrations of Lp-A-I (A-A) and LpA-LA-II (O-O) in heparin supematant. Removal of apo B-containing particles (B) was significant in the case of all analyzed particles. However, Lp-B (A-A) was most efficiently removed followed by Lp B:C:E+B:E+B:C-complex (O-O) and Lp-A-IIB-complex (Cm. Each point represents average concentration (mean GZSD., n = 3) of the lipoprotein remaining in supematant after the removal of heparin precipitate. Concentrations of lipoprotein particles are expressed in percentages of the corresponding concentrations found in the native plasma.

166 TABLE 2 CHANGES IN APO B-CONTAINING AND APO A-I-CONTAINING HELP TREATMENT

LIPOPROTEIN PARTICLES CAUSED BY A SINGLE

All values represent mean * SD. from 12 patients.

Before treatment After treatment Percent decrease after treatment

Lp-B

Total Lp-B-complex

96.5 zk 20.4 31.8 ?? 1.4+’

15.3 f 4.8 8.4 ?? 3.0’

61

Lp-A-II:Bcomplex

Lp-B:C:E + B:E + B:Ccomplex

8.6 f 2.4 4.8 zt 1.9*

45

6.1 + 3.1 3.6 f 1.3*

44

53

Lp-A-I

Lp-A-I:A-II

39.2 ?? 14.3 31.4 f 13.8 (NS) 3

49.4 f 15.6 46.1 f 15.2 (NS) 6

Significantly different: *P < 0.01, **P < 0.005; NS, difference not significant.

HELP treatment. In hypercholesterolemic patients Lp-B showed the highest rate of recovery increasing from 25% to 90% of the original concentration within 4 days whereas apo B containing complexes recovered at significantly lower rate (Fig. 2a). The data on lipoprotein particle recovery were corroborated, in these patients, with the recovery of plasma lipids. LDL cholesterol recovered at the higher rate comparable to that of Lp-B whereas triglycerides recovered at significantly lower rate comparable to the recovery of apo B containing complexes (Fig. 2b). Hypertriglyceridemic patients were characterized by the rapid recovery of apo B containing complexes, especially Lp-B:C:E+B:E+B:C-complex, which occasionally even surpassed original before treatment concentration measured

(Fig. 3a). A very similar pattern was observed in recovery of triglycerides (Fig. 3b). Recoveries of Lp-B (Fig. 3a) and LDL cholesterol (Fig. 3b) were significantly slower in these patients. Interestingly, Lp-A-II:B-complex recovered at an intermediate rate in both hypercholesterolemic and hypertriglyceridemic patients. Diseusslon Cholesterol rich low density and triglyceride rich very low density lipoproteins are both significant predictors of coronary artery disease (CAD) 15-71. Conversely, high density lipoproteins are accepted as negative predictors of CAD and are considered antiatherogenic [8- lo]. Several recent reports, however, indicate that the concentration

TABLE 3 CHANGES IN PLASMA LIPIDS AND LIPOPROTEINS

CAUSED BY A SINGLE HELP TREATMENT

All values represent mean & S.D. from 12 patients.

Before treatment After treatment Percent decrease after treatment

Total Cholesterol

Triglycerides

LDL-C

HDL-C

Lp(a)

236 f 49 114 f 21** 51

167 f 98 102 zt 56 39 NS

157 f 42 42 f 12** 13

52 zk 12 44+ 9NS 15

6.8 f 2.3 3.6 zt 0.8** 41

Signilkant difference: **P < 0.005; NS, difference not significant.

167

24

0

48

72

96

Time after HELP (hours) Fig. 2. Recovery of apo B containing with HELP. Lp-B (A-A) recovered ( o-0). Lipid analyses demonstrated ( O-O). Each point represents mean

._. 1

0

24

48

72

96

Time after HELP (hours)

lipoprotein particles (A) and lipids (B) in the plasma of hyp-ercholesterolemic patients treated at the highest rate followed by Lp-A-II:B-complex (Cm and Lp-B:C:E+B:E+B:C-complex relatively fast recovery of LDL cholesterol (A-A) as well as slower recovery of triglycerides f SD. of three patients. Concentrations are expressed in percentages of the corresponding concentrations determined before HELP treatment.

of specific lipoprotein particles are potentially better predictors of CAD than conventional lipid and/or lipoprotein parameters. For example, the concentration of Lp-A-I was a more powerful negative predictor of CAD than HDL-cholesterol or apo A-I in 50 normolipidemic males with angiographically documented CAD 1221. This observation seems to be consistent with the hypothesis that Lp-A-I is the most efficient subfraction of HDL in reverse cholesterol transport [15,21]. In the Cholesterol Lowering Athero-

sclerosis Study (CLAS), lipoprotein particles containing both apo B and apo C-III were the only significant predictor of the progression of CAD in patients treated with cholestipol and nicotinic acid [27,28]. In our preliminary study of 84 CAD patients, triglyceride enriched particles in which apo B is associated with apolipoproteins C and E (Lp-B:C:E+B:E+B:C-complex) were better predictors of the severity of CAD than VLDLcholesterol or LDL-cholesterol. These particles appeared to be predictive of multiple, moderately

,/k /A 20. 0

40 72 96 24 Time after HELP Ihours)

0 0

24 48 72 96 Time after HELP (hours)

Fig. 3. Recovery of apo B containing lipoprotein particles (A) and lipids (B) in the plasma of hypertriglyceridemic patients treated with HELP. Lp-B:C:E+B:E+B:Ccomplex (0-O) recovered at the highest rate followed by Lp-A-1I:Bcomplex (cq and Lp-B ( A-A). Triglycerides recovered relatively quickly (O-O) whereas LDL cholesterol (A-A) recovered at much slower rate. Each point represents mean f SD. of three patients. Concentrations are expressed in percentages of the corresponding concentrations determined before treatment.

168

stenosing coronary lesions found in elderly patients. On the other hand, simple cholesterol rich particles i.e., Lp-B were more significant predictors of CAD than LDL-cholesterol in younger patients with less frequent but highly stenosing lesions [29]. In view of these reports it was of interest to explore the effects of HELP treatment on the different apo A-I and apo B containing lipoprotein particles. The results of this study clearly show that heparin precipitates Lp-B in vitro more efficiently than apo B-containing complexes. Accordingly, the removal of Lp-B from the patients’ circulation was more efficient compared to the complexes. Although the basis for this difference was not specifically addressed in this study it appears reasonable to assume that the heparin binding sites of apo B [41] are more exposed on Lp-B and less accessible in complex particles because of the presence of other apolipoproteins and more abundant lipid moiety. HELP treatment should, therefore, be regarded as most suitable for patients with increased Lp-B. This assumption is supported by the data from the kinetic study which demonstrated not only the less efficient initial removal of apo B containing complexes but also their higher regeneration rate during the five days of post-treatment period. Nonetheless, HELP treatment did acutely reduce complexes and triglyceride levels by an approximate 50%. Patients with elevated levels of apo B containing complexes would probably benefit from a combination of triglyceride lowering medication and HELP treatment. It has been shown, for example, that tibrate derivatives cause reduction of triglycerides and total Lp-B-complex particles with a concomitant increase in the concentration of Lp-B [42]. Therefore, the above combination of fibrates and HELP is likely to optimize the removal of both atherogenic lipoproteins. Analysis of apo A-I containing particles shows that the slight decrease in HDL cholesterol observed after HELP treatment [l] can be accounted for by the removal of Lp-AI:A-II. These data argue against any harmful effect of HELP treatment since the more antiatherogenic subfraction of HDL, i.e. Lp-A-I, remained unchanged. Recovery patterns of apo B containing lipoprotein particles (Figs. 2A and 3A) show some

similarities as well as differences between hypercholesterolemic and hypertriglyceridemic patients. Since Lp-B:C:E+B:E+B:C-complex is the most abundant family of particles in VLDL and Lp-B is the major particle of LDL, it is more than likely that Lp-B:C:E+B:E+B:C-complex is degraded into Lp-B as the final product of the lipolytic cascade. This assumption is supported by some direct evidence of formation of Lp-B during an in vitro lipolysis of VLDL [43]. On the other hand, Lp-A-1I:B complex is assumed to form in plasma compartment by association of Lp-A-II with LpB:C:E-complex [25,44]. Lp-A-1I:B complex is also a substrate for lipoprotein lipase [25] and could presumably give rise to Lp-B as a final product. Therefore, immediately after the HELP treatment there is a relative deficiency of the common precursor for both Lp-B and Lp-A-1I:B complex, i.e., Lp-B:C:E+B:E+B:C-complex. This should make generation of Lp-B and Lp-A-II:B-complex relatively slow. At the same time, removal of Lp-B via LDL receptor most likely proceeds at the usual rate. The net result is, obviously, a decrease in LpB concentration during the first 4 h after HELP. Relative deficiency of the common precursor, i.e., Lp-B:C:E+B:E+B:C-complex could affect recovery pattern of Lp-A-1I:B complex in a similar fashion. The slower generation combined with an unchanged lipolytic degradation of this family should also result with a decrease in the concentration of Lp-A-1I:B complex in plasma. The difference between hypercholesterolemic and hypertriglyceridemic patients can be explained by an increased secretion and slow lipolysis of VLDL particles both of which coexist in hypertriglyceridemia [45]. Such a combination of factors could certainly be responsible for the observed higher recovery rates of Lp-B:C:E+B:E+B:Ccomplex and Lp-A-1I:B complex as well as relatively lower recovery of Lp-B. In conclusion, HELP-LDL-apheresis leads to a marked improvement in the lipoprotein particle profile of patients, by drastically reducing the levels of atherogenic lipoproteins Lp-B and, to a lesser extent, apo B containing complexes while retaining HDL and in particular the Lp-A-I subfraction. These data also support the most recent report describing regression of atherosclerotic lesions in patients repeatedly treated with HELP [461.

169

Acknowledgements

This study was supported by the Oklahoma Center for the Advancement of Science and Technology and by the Oklahoma Medical Research Foundation. We thank Margo French for her patience and skillful preparation of the manuscript. References

11

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