Adapting pharmacokinetic properties of a humanized anti-interleukin-8 antibody for therapeutic applications using site-specific pegylation.

A neutralizing anti-interleukin-(IL-)8 monoclonal antibody was humanized by grafting the complementary determining regions onto the human IgG framework. Subsequent alanine scanning mutagenesis and phage display enabled the production of an affinity matured antibody with a >100-fold improvement in IL-8 binding. Antibody fragments can be efficiently produced in Escherichia coli but have the limitation of rapid clearance rates in vivo. The Fab' fragment of the antibody was therefore modified with polyethylene glycol (PEG) in order to obtain a more desirable pharmacokinetic profile. PEG (5-40 kDa) was site-specifically conjugated to the Fab' via the single free cysteine residue in the hinge region. In vitro binding and bioassays showed little or no loss of activity. The pharmacokinetic profiles of the 20 kDa, 30 kDa, 40 kDa, and 40 kDa branched PEG-Fab' molecules were evaluated in rabbits. Relative to the native Fab', the clearance rates of the PEGylated molecules were decreased by 44-175-fold. In a rabbit ear model of ischemia/reperfusion injury, all PEGylated Fab' molecules were as efficacious in reducing oedema as the original monoclonal antibody. These studies demonstrate that it is possible to customize the pharmacokinetic properties of a Fab' while retaining its antigen binding activity.

Mass spectrometry innovations in drug discovery and development.

This review highlights the many roles mass spectrometry plays in the discovery and development of new therapeutics by both the pharmaceutical and the biotechnology industries. Innovations in mass spectrometer source design, improvements to mass accuracy, and implementation of computer-controlled automation have accelerated the purification and characterization of compounds derived from combinatorial libraries, as well as the throughput of pharmacokinetics studies. The use of accelerator mass spectrometry, chemical reaction interface-mass spectrometry and continuous flow-isotope ratio mass spectrometry are promising alternatives for conducting mass balance studies in man. To meet the technical challenges of proteomics, discovery groups in biotechnology companies have led the way to development of instruments with greater sensitivity and mass accuracy (e.g., MALDI-TOF, ESI-Q-TOF, Ion Trap), the miniaturization of separation techniques and ion sources (e.g., capillary HPLC and nanospray), and the utilization of bioinformatics. Affinity-based methods coupled to mass spectrometry are allowing rapid and selective identification of both synthetic and biological molecules. With decreasing instrument cost and size and increasing reliability, mass spectrometers are penetrating both the manufacturing and the quality control arenas. The next generation of technologies to simplify the investigation of the complex fate of novel pharmaceutical entities in vitro and in vivo will be chip-based approaches coupled with mass spectrometry.

Modulating pharmacokinetics of an anti-interleukin-8 F(ab')(2) by amine-specific PEGylation with preserved bioactivity.

By covalently attaching biocompatible polyethylene-glycol (PEG) groups to epsilon-amino groups of the F(ab')(2) form of a humanized anti-interleukin-8 (anti-IL-8) antibody, we sought to decrease the in vivo clearance rate to give a potentially more clinically acceptable therapeutic. The in vivo clearance was modulated by changing the hydrodynamic size of the PEGylated antibody fragments. To achieve significant increases in the hydrodynamic size with minimal loss in bioactivity, high molecular weight linear or branched PEG molecules were used. Modification involved N-hydroxy-succinamide reaction of the PEGs with primary amines (lysines and/or the N-terminus) of the anti-IL-8 F(ab')(2). The process of adding up to four linear 20 kDa PEG, or up to two branched 40 kDa PEG, gave reproducible distribution of products. The components with uniform size (as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were purified by a single-step ion-exchange high-performance liquid chromatography and showed no significant loss of biological activity in ligand binding and cell-based assays. Addition of a single branched 40 kDa PEG to a F(ab')(2) (molecular weight (MW)=1.6 million Da) or up to two 40 kDa branched PEG (MW=1.9 million Da) increased the serum half-life to 48 h as compared with the unPEGylated F(ab')(2) with a half-life of 8.5 h. This study shows that by attaching high molecular weight PEGs at a one or two sites, bioactive antibody fragments can be made reproducibly with sizes tailored to achieve the desired pharmacokinetics.

Assessment of the myotoxicity of pharmaceutical buffers using an in vitro muscle model: effect of pH, capacity, tonicity, and buffer type.

The purpose of the present study was to investigate the myotoxicity of three buffers containing carboxylic acid groups (i.e., acetate, succinate, and citrate) as a function of their pH, capacity, and tonicity. The myotoxicity of these buffers in the range of pH 2-6 and 0.001-0.1 M buffer capacity was assessed using cumulative creatine kinase (CK) release from an isolated rodent muscle model following injection. Phenytoin and 0.9% NaCl injection were used as positive and negative controls, respectively. Buffer solutions were prepared. A lower pH and higher buffer capacity was linked to increased myotoxicity for the acetate buffers. However, for succinate and citrate buffers, pH appeared to influence the extent of myotoxicity, whereas buffer capacity did not seem to have an effect. When either NaCl or trehalose was used as a tonicity-adjusting agent at pH 6, isotonic 0.01 M buffer solutions dramatically lowered the cumulative CK release compared to those that were not isotonic. Isotonic succinate buffers displayed the lowest myotoxicity, whereas citrate buffers displayed the highest values. Citrate buffers containing three carboxylic acid groups showed higher myotoxicity than succinate buffers and acetate buffers at 0.001 and 0.01 M buffer capacities, whereas acetate buffer produced higher cumulative CK release than citrate and succinate buffers at 0.1 M buffer capacity. The myotoxicity of pharmaceutical buffers containing carboxylic acid groups appears to be directly affected by lowering the pH of the solution.

Safety and antitumor activity of recombinant soluble Apo2 ligand.

TNF and Fas ligand induce apoptosis in tumor cells; however, their severe toxicity toward normal tissues hampers their application to cancer therapy. Apo2 ligand (Apo2L, or TRAIL) is a related molecule that triggers tumor cell apoptosis. Apo2L mRNA is expressed in many tissues, suggesting that the ligand may be nontoxic to normal cells. To investigate Apo2L's therapeutic potential, we generated in bacteria a potently active soluble version of the native human protein. Several normal cell types were resistant in vitro to apoptosis induction by Apo2L. Repeated intravenous injections of Apo2L in nonhuman primates did not cause detectable toxicity to tissues and organs examined. Apo2L exerted cytostatic or cytotoxic effects in vitro on 32 of 39 cell lines from colon, lung, breast, kidney, brain, and skin cancer. Treatment of athymic mice with Apo2L shortly after tumor xenograft injection markedly reduced tumor incidence. Apo2L treatment of mice bearing solid tumors induced tumor cell apoptosis, suppressed tumor progression, and improved survival. Apo2L cooperated synergistically with the chemotherapeutic drugs 5-fluorouracil or CPT-11, causing substantial tumor regression or complete tumor ablation. Thus, Apo2L may have potent anticancer activity without significant toxicity toward normal tissues.

Stability of alkoxycarbonylamidine prodrugs.

PURPOSE: Alkoxycarbonylamidine prodrug modification was used to mask the positively-charged amidine moiety of an Arg-Gly-Asp peptidomimetic and enhance oral bioavailability. The aqueous stability of ethoxycarbonylamidine (ECA), ethanethiocarbonylamidine (ETCA) and phenoxycarbonylamidine (PCA) prodrugs was examined. METHODS: Degradation was followed by RP-HPLC and rate constants were determined from a degradation scheme defined by product analysis. RESULTS: ECA gave a pH of maximum stability at pH approximately 7 and was independent of pH below pH approximately 4. A novel degradation pathway of ECA, conversion to ethoxycarbonyl- aminocarbonyl, was observed below pH 7. The relative rates below pH 7 were ECA approximately ETCA < PCA, in the same order of decreasing pKa of the conjugate acid of the substituted amidino group. Base-catalyzed cleavage of ECA to yield the amidine derivative gave the relative rates ECA < ETCA < PCA, in agreement with the decreasing pKa of the leaving groups. CONCLUSIONS: The observed rate constants at all pHs were small enough that only 5-30% (depending on the substituent) undesirable degradation is predicted during transit time of the gut. The spontaneous post-absorptive conversion to the amidine drugs at neutral pH is predicted to be 6x greater for the PCA than the ECA prodrugs.

Flux measurements across Caco-2 monolayers may predict transport in human large intestinal tissue.

Confluent monolayers of Caco-2 cells, a human colonic carcinoma cell line, have been used extensively to predict intestinal absorption. A direct comparison of uptake characteristics, however, between cell monolayers and human tissue is missing in the literature. We have determined the flux for a series of small organic molecules, peptide and protein therapeutics, across Caco-2 monolayers and normal human colonic and rectal tissue in vitro to assess whether or not a predictive correlation of transport exists. Caco-2 cells were grown to confluency of Snapwells, and human tissue was obtained from patients undergoing surgery for localized tumors. Mucosa-serosa fluxes were measured by HPLC for small molecules and peptides, and proteins were analyzed by ELISA or RIA. Permeability coefficients were calculated from flux data and compared with previously published coefficients where possible. The permeability coefficients for the examined molecules were of a similar magnitude across Caco-2 cell monolayers and human tissues, ranging from 10(-7) to 10(-5) cm/s. A best-fit analysis of a log-log plot of transport measurements obtained in these two systems gave a good correlation (R2 = 0.991). From this limited data set it appears that uptake characteristics for human colon and rectum are similar to those of Caco-2 cell monolayers. Thus, flux measurements across Caco-2 monolayers may be predictive for permeabilities of human colon and rectum for different classes of therapeutics.

Approaches to analysis of aggregates and demonstrating mass balance in pharmaceutical protein (basic fibroblast growth factor) formulations.

Denaturation, aggregation, and precipitation, which are common events in protein aging, limit the development of pharmaceutical protein formulations. Using the example of basic fibroblast growth factor (bFGF), we describe a systematic approach for quantitative recovery of soluble and insoluble aggregates in aged samples to achieve mass balance in three analytical methods, UV spectroscopy, size exclusion HPLC (HP-SEC), and reverse phase HPLC. Soluble aggregates were evaluated by UV spectroscopy and HP-SEC; the latter method was modified to include 2 M guanidine hydrochloride (GnHCl) in the mobile phase in order to differentiate and simultaneously analyze native and denatured protein. Insoluble aggregates were first solubilized with GnHCl and then recovered quantitatively with the modified HP-SEC method. Chaotrope treatment did not affect the UV peak absorbance but altered the HPLC profiles. The changes were consistent with dissociation of disulfide-linked multimers to monomers with an intramolecular disulfide linkage. This phenomenon was used for estimation of the monomer-multimer distribution in the untreated aggregated sample. These methods established approaches for quantitative recovery and characterization of bFGF aggregates.

Probing the conformation of protein (bFGF) precipitates by fluorescence spectroscopy.

Aggregation and precipitation are major events in the handling and aging of most protein pharmaceuticals. We demonstrate the utility of fluorescence spectroscopy in determining protein conformation in precipitates using basic fibroblast growth factor (bFGF) as an example. Conversion of the native to the soluble denatured from by chaotropes was accompanied by an increase in tryptophan emission. The emission spectra of resuspended precipitates were as reproducible as the spectra of the soluble form. The sum of emission spectra of native soluble bFGF and denatured precipitated bFGF was superimposable on the spectrum of the unfractionated suspension, suggesting that quantitative analysis of denatured aggregates in turbid protein formulations is possible. The ratio of tryptophan to tyrosine emissions increased with increasing extent of denaturation both in solution and in suspension. For example, salting out by ammonium sulphate increased the fluorescence index (indicative of denaturation) which was reversible upon dissolution. In addition, aging (35 degrees C) of bFGF in the presence of sulphated ligands produced precipitates with native-like fluorescence index, in contrast to denatured precipitates formed without ligands.

Major degradation products of basic fibroblast growth factor: detection of succinimide and iso-aspartate in place of aspartate.

The degradation products of basic fibroblast growth factor (bFGF) were isolated by ion exchange HPLC (HP-IEC) and characterized. The predominant product at pH 5 was a succinimide in place of aspartate15 as determined by LC/MS, N-terminal sequencing, and susceptibility to degradation at pH > 6.5. The rate of appearance of the succinimidyl-bFGF at 22 degrees C was comparable to that reported for small peptides, consistent with a high flexibility predicted for asp15-gly. Tryptic mapping together with [3H]-methylation indicated that iso-aspartate was formed at the position of asp15. Size exclusion HPLC indicated the presence of intact and truncated dimers and trimers which associated through disulfide linkages. Two truncated monomer forms were found that co-eluted by HP-IEC; the cleavages were determined to be at asp28-pro and asp15-gly using LC/MS and N-terminal sequencing. These degradation products which occurred at sites that are away from receptor or heparin binding domains of bFGF remained bioactive in a cell proliferation assay.

Chromatographic methods for quantitative analysis of native, denatured, and aggregated basic fibroblast growth factor in solution formulations.

High-performance liquid chromatography (HPLC) methods were developed for evaluating stability of human recombinant basic fibroblast growth factor (bFGF) against denaturation and aggregation in solution formulations. Reversed-phase chromatography (RP-HPLC)-insensitive to bFGF tertiary structure--was used to measure total soluble protein; heparin affinity chromatography (HepTSK) provided quantitative analysis of native bFGF species. The folding state of bFGF was determined by fluorescence spectroscopy: Tryptophan emission, which was quenched in native protein, increased upon unfolding. Slow unfolding/refolding kinetics of bFGF in 2 M guanidine hydrochloride made possible the separation of native from denatured species by size exclusion chromatography (SEC). Although the tertiary structure affected bFGF retention times, it did not change the sample recovery by SEC. These chromatographic techniques, which quantitatively measure physical and chemical changes taking place in solution formulations, can be used in future investigations of bFGF stability.

Single photon radioluminescence. I. Theory and spectroscopic properties.

The excitation of a fluorescent molecule by a beta-decay electron (radioluminescence) depends upon the electron energy, the distance between radioactive 'donor' and fluorescent 'acceptor', and the excitation characteristics and solvent environment of the fluorophore. The theory for calculation of single photon radioluminescence (SPR) signals is developed here; in the accompanying paper, measurement methods and biological applications are presented. To calculate the three-dimensional spatial profile for electron energy deposition in an aqueous environment, a Monte Carlo calculation was performed incorporating theories of electron energy distributions, energy loss due to interactions with matter, and deflections in electron motion due to collisions. For low energy beta emitters, 50% of energy deposition occurs within 0.63 micron (3H, 18.5 keV), 22 microns (14C, 156 keV), 25 microns (35S, 167 keV), and 260 microns (36Cl, 712 keV) of the radioisotope. In close proximity to the beta emitter (100 nm, 3H; 10 microns, 14C) the probability for fluorophore excitation is approximately proportional to the inverse square of the distance between the beta emitter and fluorophore. To investigate the other factors that determine the probability for fluorophore excitation, SPR measurements were carried out in solutions containing 3H and a series of fluorophores in different solvents. In water, the probability of fluorescence excitation was nearly proportional to the integrated absorbance over a > 1,000-fold variation in absorbances. The probability of fluorescence excitation was enhanced up to 2,600-fold when the fluorophore was in a "scintillant" aromatic or hydrocarbon solvent. SPR emission spectra were similar to fluorescence emission spectra obtained with photon excitation. The single photon signal due to Bremsstrahlung increased with wavelength in agreement with theory. The distance dependence for the SPR signal predicted by the model was in good agreement with measurements in which a 14C donor was separated by known thicknesses of water from a fluorescently-coated coverglass. Quantitative predictions for radioluminescence signal as a function of donor-acceptor distance were developed for specific radioisotope-fluorophore geometries in biological samples.

Single photon radioluminescence. II. Signal detection and biological applications.

A quantitative theory for excitation of fluorescent molecules by beta decay electrons is reported in the accompanying manuscript; experimental detection methods and biological applications are reported here. The single photon signals produced by an excited fluorophore (single photon radioluminescence, SPR) provide quantitative information about the distance between radioisotope and fluorophore. Instrumentation was constructed for SPR signal detection. Photons produced in a 0.5-ml sample volume were detected by a cooled photomultiplier and photon counting electronics. To minimize electronic noise and drift for detection of very small SPR signals, a mechanical light chopper was used for gated-signal detection, and a pulse height analyzer for noise rejection. SPR signals of approximately 1 cps were reproducibly measurable. The influence of inner filter effect, sample turbidity, and fluorophore environment (lipid, protein, and carbohydrate) on SPR signals were evaluated experimentally. SPR was then applied to measure lipid exchange kinetics, ligand binding, and membrane transport, and to determine an intermolecular distance in an intact membrane. (a. Lipid exchange kinetics.) Transfer of 12-anthroyloxystearic acid (12-AS) from sonicated lipid vesicles and micelles to vesicles containing 3H-cholesterol was measured from the time course of increasing SPR signal. At 22 degrees C, the half-times for 12-AS transfer from vesicles and micelles were 3.3 and 1.1 min, respectively. (b. Ligand binding.) Binding of 3H-oleic acid to albumin in solution, and 3H-2,2'-dihydro-4,4'-diisothiocyanodisulfonic stilbene (3H-H2DIDS) to band 3 on the erythrocyte membranes were detected by the radioluminescence of the intrinsic tryptophans. The SPR signal from 5 microCi 3H-oleic acid bound to 0.3 mM albumin decreased from 13 +/- 2 cps to 3 +/- 2 cps upon addition of nonradioactive oleic acid, giving 2.7 high affinity oleic acid binding sites per albumin. The SPR signal from 1 microCi 3H-H2DIDS bound selectively to erythrocyte band 3 in erythrocyte ghosts (1.5 mg protein/ml) was 2.2 +/- 0.8 cps. (c. Membrane transport). Dilution of J774 macrophages loaded with 3H-3-O-methylglucose and BCECF gave a decreasing SPR signal with a half-time of 81 s due to methylglucose efflux; the SPR measurement of the efflux rate was in agreement with a conventional tracer efflux rate determination by filtration. 20 microM cytochalasin B inhibited efflux by 97%. (d. Distance determination.) The SPR signal from erythrocyte membranes labeled with 27 microCi 3H-oleic acid and 10 microM of fluorescein-labeled wheat germ agglutinin was 5.7 +/- 0.5 cps, giving an average glycocalyx-to-bilayer distance of 5 nm. The results establish methods for experimental detection of SPR signals and demonstrate the applications of radioluminescence to the measurement of lipid exchange kinetics, ligand binding, membrane transport, and submicroscopic distances in intact membranes in real time.

A novel photoactivatable cross-linker for the functionally-directed region-specific fluorescent labeling of proteins.

A cleavable cross-linking reagent, sulfosuccinimidyl-2(7-azido-4-methylcoumarin-3-acetamido)-ethyl-1,3'- dithiopropionate (SAED), was synthesized for the selective transfer of a coumarin fluorophore from a 'donor' protein to a position near the binding site of an interacting 'target' protein. SAED contains a terminal N-sulfosuccinimidyl ester for conjugation to the donor, a terminal photoactivatable azido-coumarin species for cross-linking with the interacting target, and a central disulfide spacer for the release of the labeled target after cleavage. To evaluate the effectiveness of this labeling reagent, soybean trypsin inhibitor (STI) was derivatized (approximately 0.5 mol/mol) with SAED and then photolyzed in the presence of trypsin. A single fluorescent cross-linked species (6-7 mol% of total STI) was observed by SDS/PAGE and, after reductive cleavage, was shown to be a 1:1 STI-trypsin complex. This complex was not detected without photolysis or with an inactivated cross-linker. Importantly, complex formation was inhibited by an excess of unmodified STI and prevented by substitution of a non-interacting protein for trypsin. Cleavage of the cross-linked complex revealed that the trypsin, but not the STI, was fluorescent; the uncomplexed trypsin fraction remained unlabeled. These results demonstrated the specificity of the labeling of trypsin by fluorescent-transfer cross-linking with SAED. An efficiency of about 15% for this cross-linking mediated labeling of trypsin was calculated. The short cross-linking span of SAED (less than or equal to 1.8 nm) strictly limited the labeling to the vicinity of the contact region of trypsin with STI. Thus, this novel cross-linker permits the region-specific targeting of a fluorophore near a functionally important binding site.

Conformational changes in the foot protein of the sarcoplasmic reticulum assessed by site-directed fluorescent labeling.

Ca2+ release from sarcoplasmic reticulum during excitation--contraction coupling is likely to be mediated by conformational changes in the foot protein moiety of the triadic vesicles. As a preparative step toward the studies of dynamic conformational changes in the foot protein moiety, we have developed a new method that permits specific labeling of the foot protein moiety of the isolated membranes with a fluorophore. A novel fluorescent cleavable photoaffinity cross-linking reagent, sulfosuccinimidyl 3-((2-(7-azido-4-methylcoumarin-3-acetamido)ethyl)dithio)propionate (SAED), was conjugated with site-directing carriers, polylysine (Ca(2+)-release inducer) and neomycin (Ca(2+)-release blocker). The conjugates were allowed to bind to polylysine- and neomycin-binding sites of the heavy fraction of SR (HSR). After photolysis, the cross-linked reagent was cleaved by reduction and the fluorescently labeled HSR was separated from the carriers by centrifugation. These procedures led to specific incorporation of the methylcoumarin acetate (MCA) into the foot protein. Polylysine and neomycin bound to different sites of the foot protein, since neomycin, at release-blocking concentrations, did not interfere with polylysine binding. The fluorescence intensity of the foot protein labeled with the carrier, neomycin, showed biphasic changes as a function of ryanodine concentration (increasing up to 1 microM ryanodine and decreasing above it), while with the carrier polylysine, ryanodine induced no change in fluorescence intensity. In contrast, the fluorescence intensity of the foot protein labeled with each of the two carriers, neomycin and polylysine, showed almost identical calcium dependence (first increasing from 0.1 microM to about 3.0 microM calcium concentration, and then decreasing at higher calcium concentrations).(ABSTRACT TRUNCATED AT 250 WORDS)

Distance between skeletal protein 4.1 and the erythrocyte membrane bilayer measured by resonance energy transfer.

To assess the molecular architecture of the human erythrocyte skeletal protein 4.1:bilayer interface, the distance between a donor sulfhydryl-specific fluorescent probe attached to a region near the glycophorin-binding domain of protein 4.1 and an acceptor lipophilic probe in the exposed leaflet of inside-out vesicles (IOVs) was measured by fluorescence resonance energy transfer. To prevent aggregation and loss of function, protein 4.1 was labeled in situ on the surface of IOVs, purified, and rebound onto fresh IOVs. The labeled protein 4.1 was similar to the native protein in its gel electrophoretic pattern and its binding affinity to stripped-IOVs (Kd 35 +/- 4 nM). Energy transfer was assessed using two donor-acceptor pairs, 5-[2-[(iodoacetyl)amino]ethyl] aminonaphthalene-1-sulfonic acid and 3,3'-ditetradecyloxacarbocyanine perchlorate, or 5-iodoacetamidofluorescein and tetramethylrhodamine phosphatidylethanolamine. Using both donor fluorescence intensity and lifetime quenching measurements, an average distance of 75 +/- 5 A between the probe on the protein and the surface of IOVs was found. In parallel fluorescence resonance energy transfer studies with protein 4.1 and liposomes with a phospholipid composition similar to the inner leaflet of the red cell membrane, a closer distance was found (49 +/- 5 A). Two control experiments validated energy transfer: (a) the spectrum of a mixture of IOVs separately labeled with donor and acceptor was different from the spectrum of the doubly labeled IOVs at identical donor and acceptor concentrations; and (b) no energy transfer was observed following detergent disruption of the geometric relationship between donor and acceptor. Taken together, these observations suggest that membrane-bound protein 4.1 is elongated and that the labeled site is located at a position deep in the 30-kDa N-terminal glycophorin-binding domain of the protein. The data are also consistent with the view that the cytoplasmic tail of glycophorin is interposed between protein 4.1 and the lipids. These experiments represent the first measurement of a distance between a skeletal protein and the lipid bilayer.

Factors influencing interaction of human plasma low-density lipoproteins with discoidal complexes of apolipoprotein A-I and phosphatidylcholine.

The effect of plasma components on the particle size distribution and chemical composition of human plasma low-density lipoproteins (LDL) during interaction with discoidal complexes of human apolipoprotein A-I and phosphatidylcholine (PC) was investigated. Incubation (37 degrees C, 1 h and 6 h) of LDL with discoidal complexes in the presence of the plasma ultracentrifugal d greater than 1.20 g/ml fraction (activity of lecithin-cholesterol acyltransferase inhibited) produces an increase in LDL apparent particle diameter two-to six-fold greater than that observed in the absence of the plasma d greater than 1.20 g/ml fraction. In incubation mixtures of LDL and discoidal complexes, both in the presence and absence of the plasma d greater than 1.20 g/ml fraction, the extent of LDL apparent particle diameter increase is: (1) approximately three-fold greater at 6 h than at 1 h, and (2) markedly greater for LDL with initially small (22.4-24.0 nm) major components than for LDL with initially large (26.2-26.8 nm) major components. The facilitation factor in the plasma d greater than 1.20 g/ml fraction is not plasma phospholipid transfer protein. Purified human serum albumin produces an apparent particle diameter increase comparable to the plasma d greater than 1.20 g/ml fraction. The discoidal complex-induced increase in LDL apparent particle diameter value by albumin is associated with an increase in phospholipid uptake by LDL and a decreased loss of LDL unesterified cholesterol. In preliminary experiments, high-density lipoproteins (HDL) reverse the apparent particle diameter increase originally induced by discoidal complexes. The presence of HDL (HDL phospholipid/LDL phospholipid molar ratio of 10:1) in the incubation (6 h) mixture of LDL and discoidal complexes also attenuates LDL apparent particle diameter increase. In vivo, the plasma LDL/HDL ratio may be a controlling factor in determining the extent to which phospholipid uptake and the associated change in LDL particle size distribution occurs.

Interaction of human-plasma low-density lipoproteins with discoidal complexes of apolipoprotein A-I and phosphatidylcholine, and characterization of the interaction products.

Interaction of human low-density lipoproteins (LDL) with discoidal complexes comprised of egg yolk phosphatidylcholine and human apolipoprotein A-I (molar ratio, 88:1, respectively) was investigated. The multicomponent gradient gel electrophoretic pattern of LDL is transformed to one that includes a predominant component with an apparent particle diameter larger than that of the initial major LDL but still in the size range of normal LDL. The apparent particle diameter increase (range, 0.2-3.5 nm) is proportional to the increase (range, 6-40%) in LDL phospholipid/protein weight ratio following incubation (37 degrees C; 6 and 24 h); the smaller the initial LDL diameter, the greater the apparent particle diameter increase and percentage of phospholipid uptake. The LDL unesterified cholesterol/protein weight ratio decreases (range, 33-39%), but does not correlate with the increase in apparent particle diameter value. Interaction products are round particles with intact apolipoprotein B and show no evidence of phospholipid degradation. The products appear more dense than expected from the size vs. density relationship observed for nonincubated LDL subspecies. In addition to products in the normal LDL size range, larger components (apparent particle diameter range, 29.0-41.2 nm) also form and may be association complexes of phospholipid-modified LDL. Our results indicate that phospholipid uptake by LDL may contribute to the particle size polydispersity observed in plasma LDL.