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Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1203-1214

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:1203-1214.)
© 1996 American Heart Association, Inc.

Articles

Effects of Intravenous Infusion of Lipid-Free Apo A-I in Humans

M.N. Nanjee; J.R. Crouse; J.M. King; R. Hovorka; S.E. Rees; E.R. Carson; J.-J. Morgenthaler; P. Lerch; N.E. Miller

the Department of Cardiovascular Biochemistry, St Bartholomew's Hospital Medical College, London, UK (M.N.N., N.E.M.); the Section on Endocrinology and Metabolism, Department of Medicine, The Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC (J.R.C., J.M.K.); the Centre for Measurement and Information in Medicine, Department of Systems Science, City University, London, UK (R.H., S.E.R., E.R.C.); and the Central Laboratory, Swiss Red Cross, Bern, Switzerland ((J.-J.M., P.L.).

Correspondence to Prof Norman E. Miller, Department of Cardiovascular Biochemistry, St Bartholomew's Hospital Medical College, Charterhouse Square, London EC1M 6BQ, UK.


*    Abstract
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 *Abstract
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 down arrowMethods
 down arrowResults
 down arrowDiscussion
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Apolipoprotein (apo) A-I is the principal protein component of the plasma high density lipoproteins (HDLs). Tissue culture studies have suggested that lipid-free apo A-I may, by recruiting phospholipids (PLs) and unesterified cholesterol from cell membranes, initiate reverse cholesterol transport and provide a nidus for the formation, via lipid-poor, pre-ß–migrating HDLs, of spheroidal {alpha}-migrating HDLs. Apo A-I has also been shown to inhibit hepatic lipase (HL) and lipoprotein lipase (LPL) in vitro. To further study its functions and fate in vivo, we gave lipid-free apo A-I intravenously on a total of 32 occasions to six men with low HDL cholesterol (30 to 38 mg/dL) by bolus injection (25 mg/kg) and/or by infusion over 5 hours (1.25, 2.5, 5.0, and 10.0 mg·kg-1·h-1). The procedure was well tolerated: there were no clinical, biochemical, or hematologic changes, and there was no evidence of allergic, immunologic, or acute-phase responses. The 5-hour infusions increased plasma total apo A-I concentration in a dose-related manner by 10 to 50 mg/dL after which it decreased, with a half-life of 15 to 54 hours. Coinfusion of Intralipid reduced the clearance rate. The apparent volume of distribution exceeded the known extracellular space in humans, suggesting extensive first-pass clearance by one or more organs. No apo A-I appeared in the urine. Increases in apo A-I mass were confined to the pre-ß region on crossed immunoelectrophoresis of plasma and to HDL-size particles on size exclusion chromatography. Increases were recorded in HDL PL, but not in HDL unesterified or esterified cholesterol. Increases also occurred in LDL PL and in very low density lipoprotein cholesterol, triglycerides, and PL but not in plasma total apo B concentration. These results can all be explained by combined inhibition of HL and LPL activities. Owing to the effects that this would have had on HDL metabolism, no conclusions can be drawn from these data about the role of lipid-free apo A-I in the removal of PL and cholesterol from peripheral tissues in humans. The kinetic data suggest that the fractional catabolic rate of lipid-free apo A-I exceeds that of spheroidal HDLs and is reduced in the presence of surplus PL.


Key Words: apo A-I • cholesterol • HDL • phospholipids • triglycerides


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Human apo A-I is a 243–amino acid residue protein, the gene for which is on chromosome 11.1 2 Apo A-I is secreted by the liver and small intestine as pro–apo A-I, which undergoes extracellular cleavage of a hexapeptide by a circulating peptidase to yield the mature apolipoprotein.3 Mature apo A-I is the major protein of plasma HDL, different subclasses of which contain one to four apo A-I molecules per particle.4 5 Apo A-I catabolism occurs principally in the kidney and liver.6 In the kidney glomerular filtration of small particles may be important,7 and in both organs cell surface receptors may play a role.8 9 There is evidence that the HDLs, specifically the apo A-I–containing HDLs, retard atherogenesis. Epidemiologically, low apo A-I and HDL cholesterol concentrations are associated with increases in atherogenesis and coronary heart disease,10 11 and failure to synthesize apo A-I owing to defects in the apo A-I/C-III/A-IV gene cluster is associated with premature atherosclerosis in humans.12 13 Gene transfer of human apo A-I confers protection against atherosclerosis in cholesterol-fed C57BL/6 mice,14 apo E–knockout mice,15 and human apo(a)–transgenic mice16 ; intravenous infusion of apo A-I or HDL has been found to inhibit atherogenesis in cholesterol-fed rabbits.17 18 However, in other studies infusion of apo A-I had no such effect,19 20 and knockout of the apo A-I gene failed to induce atherosclerotic lesions in mice.21 Furthermore, some mutations in the apo A-I gene that lower HDL cholesterol in humans may not increase the incidence of coronary heart disease.22 Such findings emphasize the need for more work on the relationships among apo A-I, atherogenesis, and cholesterol transport.

There is sound evidence that lipoproteins that contain apo A-I play a central role in the transport of cholesterol from tissues to the liver (ie, "reverse cholesterol transport"). Native and reconstituted HDLs have long been known to promote cholesterol efflux from cultured cells.23 24 When fibroblasts containing radiolabeled cholesterol are incubated with plasma, most of the released radioactivity is initially found in very small HDLs.4 25 These small HDLs normally contain only 2% to 5% of plasma apo A-I and are members of a family of particles that differ from most HDLs in that the former show pre-ß electrophoretic mobility on agarose gels. These so-called pre-ß1 LpA-Is may be composed of one molecule of apo A-I in association with PLs and UC.25 Cell-derived radiolabeled UC later appears in larger, PC-rich discs (pre-ß2 LpA-I) that contain three molecules of apo A-I and subsequently in still larger discs (pre-ß3), wherein the UC is esterified by LCAT.25 The resultant CEs are transferred by CETP to the cores of spheroidal {alpha}-migrating HDLs. Pre-ß2 LpA-I may be produced by fusion of pre-ß1 particles, whereas pre-ß3 particles may be complexes of pre-ß2 LpA-I, LCAT, and CETP.25 The concentration of pre-ß1 LpA-I may be the rate-limiting component for cholesterol efflux from fibroblasts,26 although it is unknown whether these events, which occur when cultured cells are exposed to plasma, accurately reflect those that occur in vivo.

The origin of pre-ß1 LpA-I particles is unknown, but there is increasing evidence from tissue culture studies that these particles may be formed by recruitment of PL and UC from the plasma membranes of cells by lipid-free apo A-I.27 28 29 30 31 32 Evidence has been presented for the presence of lipid-free apo A-I in human plasma33 and canine peripheral lymph.34 Lipid-free apo A-I may be released from spheroidal HDLs through the actions of CETP and HL35 36 and possibly also during lipolysis of TGRLs by LPL34 37 and fusion of spheroidal HDLs.38

The concept that lipid-free apo A-I recruits PL and UC from cell membranes and thereby both initiates reverse cholesterol transport and provides a nidus for HDL assembly has not been examined in vivo. The present study was undertaken to test this hypothesis. Lipid-free human apo A-I was infused intravenously into healthy humans with low plasma concentrations of HDL. Increases in the numbers of pre-ß LpA-I and HDL PL were consistently recorded but not in the numbers of {alpha}-LpA-Is or HDL cholesterol. The most striking effect, however, was a rise in plasma VLDL.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
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Apo A-I
Human apo A-I was prepared in the Central Laboratory of the SRC from pooled human plasma as already described.39 40 After isolation apo A-I was dissolved in 4 mol/L guanidine HCl, dialyzed, pasteurized (60°C for 10 hours), transferred to sterile bottles, and lyophilized. Each bottle contained 1 g protein and sufficient phosphate and sucrose or mannitol to ensure physiological pH and isosmolarity after addition of 40 mL water. No preservatives were used. The preparation was stored at 4°C prior to use. All blood donors had been tested for the absence of hepatitis B and C antigens and HIV antibodies.

A full description of the preparation has been published.39 40 By SDS-PAGE 95% to 98% of the protein was found to consist of apo A-I. The residual protein included traces of apo A-II, {alpha}1-antitrypsin, albumin, and transferrin. By isoelectric focusing and immunoblotting three isoforms of mature apo A-I were detected. As would be anticipated41 by HP-SEC, apo A-I was present as monomers, dimers, and oligomers. By use of a TSKG 3000 SW column, a flow rate of 0.7 mL/min, and a 10 mmol/L sodium phosphate (pH 7.4)/0.02% NaN3 buffer, 85% were monomers, l3% dimers, and 2% oligomers; on a Superose-6 column, however, the amount of oligomeric forms appeared to be >2%. The isolated and purified apo A-I had a circular dichroic spectrum similar to that of apo A-I isolated by other methods and retained 85% to 99% of its cofactor activity for LCAT in vitro. On crossed immunoelectrophoresis essentially all apo A-I demonstrated pre-ß mobility and readily associated with PC in vitro to form discoidal particles (Fig 1Down). These particles promoted cholesterol efflux from cultured mouse macrophages and human umbilical cord endothelial cells and accumulated CEs under these conditions in the presence of LCAT.43



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  		Figure 1. Negative-stain electron photomicrograph of discoidal complexes of SRC apo A-I and soybean PC (molar ratio, 1:150). Similar particles were formed with egg PC. Staining with phosphotungstate was performed according to Forte and Nordhansen.42

Subjects
All subjects were healthy men with low plasma HDL cholesterol levels, as shown in the TableDown. Subjects were excluded from the study if there was clinical or biochemical evidence of hematologic, hepatic, renal, or endocrine disease; if they were known to have allergies, including reactions to parenterally administered proteins; or if they had pronounced hyperlipidemia. All gave informed written consent.


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  		Table 1. Clinical Details of Study Subjects

Experimental Design
The study was approved by the Ethics Committee of The Bowman Gray School of Medicine and by the US Food and Drug Administration (BB-IND No. 3795). Two series of experiments were carried out, each involving four subjects. Two subjects participated in both series (TableUp). On different occasions and in a sequence predetermined by a Latin square, each series I subject was given apo A-I at a dose of 25 mg/kg in each of four modes: (1) by bolus injection (1.67 mg·kg-1·min-1 over 15 minutes); (2) by continuous infusion via a Harvard pump for 5 hours (5 mg·kg-1·h-1; 0.2 mL·kg-1·h-1); (3) by bolus injection started 0.5 hour after starting an infusion of 20% Intralipid (Kabi Vitrum; 0.5 mL/min for 0.5 hour followed by 1.0 mL/min for 4.5 hours); and (4) by continuous infusion over 5 hours, started 0.5 hour after starting a similar infusion of Intralipid. Intralipid was used to investigate whether additional PL from lipolyzed TGRLs would modulate the kinetics of infused apo A-I and its effects on HDL cholesterol. In series II the effects of varying the infusion rate of apo A-I were studied. In a sequence conforming to a Latin square, each subject was given four continuous infusions of apo A-I over 5 hours (without Intralipid) at rates of 1.25, 2.5, 5.0, and 10.0 mg·kg-1·h-1 (total doses of 6.25, 12.5, 25, and 50 mg/kg). During both series, consecutive experiments were separated by 14 to 28 days, during which time plasma apo A-I and lipoprotein levels returned to baseline.

Clinical Procedures
All clinical procedures were performed at the Prevention and Clinical Research Building or the General Clinical Research Center of The Bowman Gray School of Medicine. Subjects arrived in the morning after a 12- to 14-hour overnight fast. Sterile, distilled water (40 mL) was added to each bottle of apo A-I. The bottles were then sealed and gently swirled in a water bath at 37°C for 30 minutes to yield a clear solution (25 mg apo A-I per milliliter). Aliquots (1 mL) were transferred to plastic tubes and stored at -70°C for future reference. Thirty minutes before each infusion, an intradermal skin test for allergy to the preparation to be infused was performed on the subject's forearm. No positive reactions (swelling or erythema) were observed. Pulse rate, blood pressure, and body temperature (sublingual) were measured 15 minutes before the start of each infusion or bolus injection and at 30-minute intervals for 8 hours thereafter. When Intralipid was infused, it was administered into the arm not used for apo A-I infusion. Subjects were given a light, fat-free meal (bread, jam, fruit, and orange juice) 0.5 and 5 hours after the start of apo A-I infusion or bolus. All blood samples were collected from the arm not used for apo A-I infusion and at least 20 cm distal to the Intralipid infusion site. Blood for lipoprotein assays was collected into Na2EDTA (1 mg/mL) and immediately centrifuged at 4°C. During series I, blood was collected at -1, 0, 0.25, 1, 2.5, 5, and 8 hours relative to the start of the apo A-I bolus and also at 4, 6, and 7 hours after the start of the apo A-I infusion. The subjects were then discharged but returned the next morning to give a 24-hour blood sample. In series II, blood was collected at -96, -24, -1, 0, 1, 2, 3, 5, 8, 24, 48, and 72 hours. The -96-, -24-, 24-, 48-, and 72-hour samples were withdrawn after an overnight fast. Aliquots from each sample were stored at -70°C for measurements of acute-phase proteins. Blood samples for routine clinical chemistry (SMAC) and complete blood count were collected at -1 and 24 hours. Two weeks after each experiment, blood was collected for assay of serum antibodies to SRC apo A-I and human HDL. Urine samples were collected at 0 to 8 hours (at the research center) and at 8 to 24 hours (at home by the patient and kept at 4°C until they were brought to the research center the next day). The two collections were pooled and aliquots taken for apo A-I immunoassay.

Laboratory Procedures
Plasma Lipid Concentrations
Plasma concentrations of total cholesterol, UC, total TGs, and total choline-containing PLs were quantified by enzymatic assays using commercially available enzymes (Sigma Chemical Co) and Trinder-type colorimetric reagents (Research Organics) in a microtiter plate spectrophotometer (Ceres UVHDi, Bio-Tek Instruments). CE concentration was calculated by difference, and TG measurements were not corrected for endogenous free glycerol. A preparation of pooled, lyophilized human serum (Precinorm, Boehringer Mannheim Corp) was used as the calibration standard. Duplicate measurements were made, with CVs <=3%. HDL lipids were measured after precipitation of apo B–containing lipoproteins with unbuffered PEG 6000 (final concentration, 8% wt/vol).44

Plasma Apo A-I and Apo B Concentrations
Plasma apo A-I and apo B concentrations were quantified by liquid-phase, double-antibody radioimmunoassays. Both procedures used Tween 20 (final concentration, 0.2% vol/vol) to expose cryptic epitopes and reduce nonspecific binding and PEG 6000 (final concentration, 3% wt/vol) to enhance reaction kinetics. After a 3-hour incubation at 37°C and centrifugation at 3000g for 30 minutes at 4°C, the radioactivity in antibody-bound pellets was counted in a gamma spectrometer (IsoData 20/10, ICN) to <=0.1% counting error. The primary antisera were polyclonal IgGs raised in sheep against delipidated human apo A-I and apo B (Boehringer Mannheim). A common secondary antiserum, donkey anti-sheep IgG (Chemicon), was used as the precipitating antibody; 125I tracers were prepared by a modification of the ICl method45 with delipidated human apo A-I and LDL (d=1.020 to 1.055 g/mL). Both immunoassays were standardized by using dilutions of Precinorm. Samples and standards were assayed in duplicate with CVs of 4% to 9% for apo A-I and 3% to 11% for apo B.

Crossed Immunoelectrophoresis
Nonsieving, charge-based electrophoresis in the first dimension was performed through a 1% (wt/vol) low-electroendosmosis agarose slab gel impregnated with 0.5% (wt/vol) human albumin at 10 V/cm for 3 hours at 4°C with 50 mmol/L sodium barbital, 20 mmol/L barbituric acid, and 1 mmol/L Na2EDTA (pH 8.6) as the electrolyte in a Bio-Phoresis horizontal electrophoresis chamber (Bio-Rad). Electrophoresis in the second dimension was performed through the same gel matrix impregnated with 0.5% (vol/vol) goat polyclonal anti-human apo A-I serum (INCStar Corp), 0.2% (vol/vol) Tween 20, and 3% (wt/vol) PEG 6000 at 15 V/cm for 18 hours at 4°C with the same buffer system. After removal of soluble reactants by soaking in three 2-L changes of 150 mmol/L NaCl, insoluble antigen-antibody complexes were visualized by staining with 0.5% (wt/vol) Coomassie Brilliant Blue R250 in ethanol/acetic acid/water (9:2:9, vol/vol/vol).

HP-SEC
EDTA-plasma was subjected to HP-SEC at ambient temperature by passage of 100-µL aliquots through a 10x300-mm Superose-6 gel permeation column (HR 10/30, Pharmacia). A degassed solution of 50 mmol/L Tris HCl (pH 7.4), 150 mmol/L NaCl, 0.1% (wt/vol) Na2EDTA, and 0.1% (wt/vol) NaN3 was used as the eluant, which was pumped at 0.5 mL/min by a computer-driven high-performance liquid chromatography pump (Rainin). After the void volume was excluded, 200-µL fractions were collected into 96-well microtiter plates (Nunc) by use of a Gilson FC203 fraction collector, and aliquots were assayed for lipids and apolipoproteins as described above. Recovery of all constituents was >90%. In this procedure human lipoproteins elute in three peaks, corresponding to VLDLs (fractions 4 to 7), LDLs (fractions 9 to 15), and HDLs (fractions 17 to 23). Repeated HP-SEC of a plasma sample three times in 1 day followed by lipid analysis of individual fractions and summation of cholesterol, TGs, and PLs in the three lipoprotein size ranges yielded CVs of 4% to 7%.

Urinary Apo A-I Excretion
Apo A-I in urine was measured by immunoturbidimetry and immunoelectrophoresis. Immunoturbidimetry was performed at the lipid laboratory of The Bowman Gray School of Medicine (standardized by the Centers for Disease Control and Prevention) with a commercial kit (Sigma). Aliquots (0.5 µL) were incubated at 15°C with an IgG fraction of goat anti-human apo A-I with polymeric enhancers and detergent, followed 15 minutes later by measurement of light scatter at 340 nm in a Cobas Fara II centrifugal analyzer (Roche Diagnostics). For immunoelectrophoresis 5-µL aliquots were electrophoresed through an antibody-impregnated agarose gel matrix identical to that used for crossed immunoelectrophoresis (see above), and insoluble immune complexes ("rockets") were visualized by staining with Coomassie Brilliant Blue R250. Assays of urine samples that had been spiked with SRC apo A-I (final concentration, 50 mg/dL) and stored at 4°C or ambient temperature for 24 hours showed negligible loss of immunoreactive apo A-I.

Antibodies to Apo A-I and HDL
The presence of antibodies to SRC apo A-I and human HDL (isolated from pooled plasma by preparative ultracentrifugation) in serum samples that had been stored at -70°C until the end of the study was detected by immunoblotting, passive hemagglutination, and ELISA. In series I, serum was collected 60 minutes before each administration of apo A-I and 4 to 8 weeks after completion of the series (ie, five samples from each subject over 2 or 3 months, during which time four doses of 25 mg/kg were given). In series II, serum was collected from all subjects immediately before the first infusion and 1 week after the fourth infusion.

For the immunoblots antigens were separated by SDS-PAGE under reducing conditions on 5% to 20% gradient gels. Pyronin G was used as a marker dye. Transfer to nitrocellulose was performed in 25 mmol/L Tris, 192 mmol/L Gly, and 20% methanol for 1.5 hours at 90 V. Cathodic nitrocellulose was stained for protein (AuroDye Forte, Amersham) and anodic nitrocellulose was used for immunoblotting. After it was blocked with 3% (wt/vol) gelatin in 20 mmol/L Tris HCl (pH 7.5), 500 mmol/L NaCl, the nitrocellulose was incubated with serum samples diluted 1:10. Bands were visualized by using an alkaline phosphatase–conjugated goat anti-human IgG (Bio-Rad), diluted 1:2000, and developed with bromochloroindolyl phosphate/nitroblue tetrazolium according to the manufacturer's protocol. The SRC apo A-I preparation, a human serum pool, polio virus, and diphtheria toxin were used as antigens.

Passive hemagglutination was performed with human type O Rh-negative erythrocytes coated with CrCl3 and five different human HDLs. These were incubated in microtiter plates with serum diluted 1:4, 1:8, 1:16, and 1:32. The mixtures were examined for agglutination after 2 and 24 hours. Rabbit serum containing anti-human HDL and sheep serum containing antibodies to SRC apo A-I (diluted 1:16 to 1:512) were used as controls.

For the ELISA, microtiter plates were coated with SRC apo A-I (2 µg/mL), human HDL (2 µg apo A-I per milliliter), or tetanus toxoid (1 µg/mL). The wells were washed, filled with blocking solution (1% [wt/vol] casein hydrolysate 0.05% [vol/vol] Tween 20, 0.2% [wt/vol] NaN3 in phosphate buffer, pH 7.2), and washed again. Serum samples were added and the plates incubated at room temperature for 2 hours. After another wash, the wells were incubated with biotin-labeled sheep anti-human IgG (previously shown to react with human IgA, IgG, and IgM). The conjugate was visualized with a streptavidin–alkaline phosphatase conjugate and developed with 4-nitrophenylphosphate, and absorbances were recorded as OD405 minus OD490. Serum from a polytransfused patient (with auto-antibodies against determinants c, d, y, and z of the Ag system) and serum from a subject with type IV hyperlipoproteinemia served as controls.

Acute-Phase Proteins
During series II, plasma concentrations of TNF-{alpha} and CRP were assayed at each time point between -24 and 24 hours relative to the start of infusions. For these measurements plasma samples were stored at -70°C until completion of the study. TNF-{alpha} was determined by an immunoradiometric assay kit (Medgenix) and CRP was assayed by immunophelometry (Behring) with reagents provided by the manufacturer.

Kinetic Analyses
Concentration-versus-time curves for plasma total apo A-I were subjected to multicompartmental analysis. We assumed that the plasma concentration of endogenous apo A-I was constant and that the disposition kinetics of apo A-I was time-invariant during the experiments. Because coinfusion of Intralipid and apo A-I violated the second assumption, only experiments without Intralipid were subjected to kinetic analysis. A one-compartment and a two-compartment model were tested. For both models, the generic convolution integral provided a description of the underlying dynamic relationship

where c(t) is the plasma concentration of total apo A-I, cb the basal concentration of total apo A-I at t=0, u(t) the unit impulse response function, and r(t) the input function. For the one-compartment model, the unit impulse response had the form u(t)=Ae-kt, where A and k are the parameters of the model. For the two-compartment model, u(t)=A1e-k1t+A2e-k2t, where A1, A2, k1, and k2 are the model parameters. The input function r(t) represents the infusion or bolus of apo A-I. The convolution integral was solved analytically and the SAAM package was used to implement the analytic solution and estimate the relevant model parameters.46 The CV of error associated with the measurement of plasma apo A-I was assumed to average 5.5% (based on laboratory experience). The precision of parameter estimates was calculated from the inverse of the Fisher transformation matrix47 and expressed as the CV. Parameter estimates with CVs >100% were eliminated from further processing. On this basis the one-compartment model was adopted.

Statistical Analyses
Changes in plasma analysates relative to t=0 were examined by a repeated-measures two-way ANOVA and Fisher's protected least significant difference test; P<.05 was considered statistically significant.


*    Results
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 up arrowIntroduction
 up arrowMethods
 *Results
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No adverse clinical effects were observed during or after the infusions: the subjects experienced no symptoms; no rashes developed; all preinfusion skin tests with SRC apo A-I were negative; and there were no significant changes in pulse rate, blood pressure, or temperature. No significant changes were observed in plasma sodium, potassium, chloride, CO2, urea, glucose, calcium, phosphorus, urate, albumin, total bilirubin, direct bilirubin, alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase, creatinine, or iron levels. Leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean erythrocyte volume, and platelet count were also unaffected*.

Plasma Apo A-I
The plasma concentration–time curves for total apo A-I are illustrated in Fig 2Down. During series I the apparent half-life of exogenous apo A-I averaged 2.3±0.50 hours (mean±SEM) after bolus injection and 2.0±0.10 hours during infusions. The series II infusions gave much greater half-lives: 54.1±16.0, 22.0±8.7, and 14.6±4.8 hours at infusion rates of 2.5, 5.0, and 10 mg·kg-1·h-1, respectively (the half-life at 1.25 mg·kg-1·h-1 could not be determined with adequate precision). By ANOVA this decreasing trend for half-life as infusion rate increased was not significant (P>.05). The apparent Vdss of exogenous apo A-I could be estimated with precision only during the 5 and 10 mg·kg-1·h-1 infusions of series II, when they averaged 101±18.0 and 82±5.8 L, respectively (P<.05). The maximum increment in apo A-I concentration during the 5-hour infusion, normalized to a total dose of 25 mg/kg (Cmax), decreased as the dose increased: 43±9.0, 32±7.0, 29±5.5, and 21±3.5 mg/dL (ANOVA P<.05). Coinfusion of Intralipid reduced the clearance rate of apo A-I from plasma (Fig 2Down). However, this decrease was not quantifiable because the disposition of apo A-I was not time-invariant under these conditions.



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  		Figure 2. Plasma concentration–time curves for total apo A-I. Left panels (series I) show results obtained after administration of 25 mg apo A-I per kilogram of body weight IV by (a) bolus injection, (b) bolus injection with Intralipid coinfusion, (c) 5-hour infusion, and (d) 5-hour infusion with Intralipid coinfusion. Right panels (series II) show results obtained with 5-hour infusions (all without Intralipid) at rates of (e) 1.25 mg·kg-1·h-1, (f) 2.5 mg·kg-1·h-1, (g) 5.0 mg·kg-1·h-1, and (h) 10 mg·kg-1·h-1. Results are means±SEM of four subjects. Note that left and right panels have similar scales on the y axes (concentration) but different x axes (time).

The effects of apo A-I administration (without Intralipid) on the distribution of apo A-I in plasma fractions separated by HP-SEC are illustrated in Fig 3Down. All bolus injections and 5-hour infusions at all infusion rates increased apo A-I mass in a broad range of fractions, corresponding in size to HDLs. No consistent pattern was detected in the time courses of apo A-I in the different HDL size subclasses. No apo A-I was detected in particles of VLDL or LDL size. No such measurements were made during coinfusion of Intralipid.



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  		Figure 3. Distribution of apo A-I in plasma fractions separated by HP-SEC (Superose-6) collected before and various times after administration of apo A-I (25 mg/kg IV) without coinfusion of Intralipid: comparison of apo A-I delivery by bolus injection (left) and by 5-hour infusion (right). Profiles (a) and (f) show absolute apo A-I mass in the different fractions at t=0. Other panels show changes from t=0 values after 15 minutes (b and g), 5 hours (c and h), 8 hours (d and i), and 24 hours (e and j). All results are presented as micrograms apo A-I per fraction (note differences in scales). Results are from a single representative subject. Cholesterol mass peaks in VLDL-, LDL-, and HDL-size particles were in fractions 5, 11, and 20, respectively.

The effects of apo A-I administration alone on the distribution of apo A-I in particles with pre-ß and {alpha}-electrophoretic mobility are illustrated in Fig 4Down. After bolus injection and 5-hour infusion at all doses, the increase in plasma apo A-I was always confined to the pre-ß fraction. No increase in {alpha}-migrating apo A-I was observed, even during return of the pre-ß apo A-I concentration to baseline. No such measurements were made in samples collected during coinfusion of Intralipid.



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  		Figure 4. Distribution of apo A-I by two-dimensional agarose gel immunoelectrophoresis of whole plasma immediately before (t=0) and 15 minutes, 5 hours, 8 hours, and 24 hours after administration of exogenous apo A-I (25 mg/kg IV) without Intralipid: comparison of apo A-I delivery by bolus injection (upper panels) and by 5-hour infusion (lower panels). Left and right peaks in each case represent pre-ß– and {alpha}-migrating particles, respectively. Results are from a single representative subject.

Plasma Lipoprotein Lipids
The effects of bolus injections and 5-hour infusions of apo A-I alone on lipids in HDL and non-HDL fractions (separated by PEG 6000 precipitation) are illustrated in Figs 5 and 6DownDown. Bolus injection had no statistically significant effect on any cholesterol fraction (Fig 5Down). In contrast, significant increases were observed in non-HDL TG, non-HDL PL, and HDL PL levels, all of which were still elevated after 24 hours (P<.05). There was a nonsignificant increase in HDL TGs. Qualitatively similar effects were observed when apo A-I was infused over 5 hours. The most consistent changes were a progressive rise in HDL PLs (mean increase at 24 hours, 13%, P<.05) and an almost linear rise in non-HDL TGs during infusion (mean increase at 5 hours, 43%, P<.05; Fig 6Down). TG levels then decreased and returned to baseline by 48 or 72 hours. The increments in HDL PLs and non-HDL TGs were related to the apo A-I infusion rate (Fig 7Down).



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  		Figure 5. Changes in HDL lipids (left) and non-HDL lipids (right) separated by PEG 6000 precipitation before and after administration of exogenous apo A-I (25 mg/kg IV) by bolus injection (without Intralipid). Results are means±SEM of four subjects.



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  		Figure 6. Changes in HDL lipids (left) and non-HDL lipids (right) separated by PEG 6000 precipitation, before, during, and after administration of exogenous apo A-I (25 mg/kg IV) by 5-hour infusion (without Intralipid). Results are means±SEM of four subjects.



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  		Figure 7. Increments in HDL PLs (after 24 hours) and in non-HDL TGs (after 5 hours) relative to t=0 when apo A-I was infused (without Intralipid) over 5 hours at rates of 1.25, 2.5, 5.0, and 10 mg·kg-1·h-1. HDL and non-HDL lipoproteins were separated by PEG 6000. Results are means±SEM of four subjects.

Changes in lipid content of the subclasses of VLDL, LDL, and HDL were examined by HP-SEC. After bolus injection of apo A-I major increases were observed in all four lipids in VLDL-size particles. CE and PL but not TG decreased in LDL-size particles, particularly in the larger subclasses. In all fractions containing HDL-size particles the most striking change was an increase in PL mass. A 5-hour infusion of apo A-I produced qualitatively similar changes, except that the PL content of LDL-size particles increased more slowly than that of HDL-size particles and the CE content of HDL-size particles decreased slightly (Fig 8Down).



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  		Figure 8. Distribution of lipids by Superose-6 HP-SEC of whole-plasma samples collected before and various times after administration of 25 mg/kg IV apo A-I by 5-hour infusion (without Intralipid). Top row profiles (a, f, k, and p) show absolute masses (micrograms per fraction) at t=0. Other profiles show changes from t=0 values after 15 minutes (b, g, l, and q), 5 hours (c, h, m, and r), 8 hours (d, i, n, and s), and 24 hours (e, j, o, and t). Results are from a single representative subject.

When Intralipid was coinfused with apo A-I, major increases in plasma total TGs and PLs were observed (data not shown), which could be directly attributed to the lipids in the infusate. Plasma total UC (but not CE) concentration also increased, an effect previously reported during infusion of Intralipid alone.48 No measurements of lipids in PEG 6000 or HP-SEC fractions were made when apo A-I and Intralipid were coinfused.

Other Measurements
There was a trend for plasma total apo B to increase during and after the 5-hour infusions. However, these changes were small and not statistically significant (data not shown). No measurements were made of apo B in HP-SEC fractions. Passive hemagglutination, ELISA, and immunoblotting failed to detect antibodies to the SRC apo A-I preparation or human HDL in any serum sample (data not shown). In all assays the controls behaved as anticipated. No increases in either TNF-{alpha} or CRP were observed in any subject at any time point during or after the 5-hour infusions at 1.25, 2.5, 5.0, and 10 mg·kg-1·h-1 (Fig 9Down). In no urine sample was apo A-I detected by either immunoturbidimetry or immunoelectrophoresis. As discussed in "Methods," this could not be explained by loss of immunoreactivity of the infused apo A-I.



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  		Figure 9. Effects of 5-hour infusions of apo A-I at infusion rates of 1.25, 2.50, 5.0, and 10 mg·kg-1·h-1 on plasma concentrations of TNF-{alpha} and CRP in four men. Means±SEM.


*    Discussion
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The apo A-I used in this study had been isolated from pooled human plasma. Preliminary studies in vitro had confirmed its ability to associate with PL to form discoidal particles (Fig 1Up), to promote cholesterol efflux from cultured cells, and to activate LCAT.39 40 43 Its behavior during electrophoresis in agarose and its circular dichroic spectrum were as expected. When given intravenously apo A-I produced no evidence of an acute-phase response, and routine clinical, biochemical, and hematologic assays remained normal. No antibody response was detected in any subject. It is reasonable to assume, therefore, that apo A-I behaved in a physiological manner and that no immunologic or toxic effects contributed to the changes in lipoproteins.

The kinetics of apo A-I differed from those expected from previous studies that used radioiodinated HDL. Disposition of apo A-I conformed to a one-pool model, whereas 125I–apo A-I HDL behaves as two major pools, probably corresponding anatomically to the intravascular and extravascular spaces.49 50 51 52 53 Theoretically, our finding of a single pool could have reflected confinement of infused apo A-I to the intravascular space or its transfer between intravascular and extravascular spaces at rates great enough to create a single kinetic compartment. Our finding that Vdss greatly exceeded the total extracellular volume of normal adult males ({approx}160 mL/kg) indicated that this parameter must have been overestimated. The explanation for this is not apparent. Although free apo A-I adsorbs to plastic tubing, any such losses would have been small.28 The possibility that the plasma concentration of infused apo A-I was underestimated is unlikely: we used a detergent to ensure complete exposure of epitopes; SRC apo A-I, human HDL, and a commercial quality-control preparation (Precinorm, Boehringer Mannheim) showed identical immunoreactivity; and when SRC apo A-I was added to plasma in vitro, the predicted increments in total apo A-I were recorded (data not shown).

Overestimation of Vdss would have occurred if the concentration of endogenous apo A-I (assumed to be unchanged) had been reduced by the infusion, because the concentrations of exogenous apo A-I in the model would have been underestimated. This event could have occurred if exogenous apo A-I displaced endogenous apo A-I from HDLs, as can occur in vitro,54 followed by rapid catabolism of the displaced molecules. Although this happens when human apo A-l is infused into rats,55 it seems unlikely when homologous apo A-I is being infused. A more likely explanation for the large value of Vdss is that apo A-I was rapidly cleared before it had been uniformly distributed throughout the extracellular space. This suggestion would be compatible with the fact that the apo A-I residence time was much shorter than that anticipated from the known FCR of most endogenous apo A-I in humans: 0.09 to 0.42 pool per day.49 50 51 52 53

Values for apo A-I half-life obtained by using radioiodinated, lipid-free apo A-I are similar to those obtained with radioiodinated, autologous HDLs.52 56 57 This does not necessarily conflict with our findings. When injected in trace amounts, radioiodinated, lipid-free apo A-I exchanges rapidly with the much larger pool of exchangeable apo A-I in plasma spheroidal HDLs, so that most of the radioactivity becomes associated with the latter before significant clearance of the tracer has occurred. The pools of both lipid-free and total apo A-I are essentially unchanged. In contrast the mass of apo A-I that we infused was 10% to 120% of the preexisting plasma total apo A-I pool and would have expanded the pool of lipid-free apo A-I by several-fold. This is consistent with our finding that essentially all of the increment in plasma apo A-I was in the pre-ß–migrating fraction. Our failure to detect antibodies excludes the possibility that the SRC apo A-I was recognized as a foreign protein. Thus, our results support other evidence58 59 that the FCR of lipid-free (or lipid-poor) apo A-I greatly exceeds that of spheroidal HDLs. Lipid-poor apo A-I may correspond to the small, rapidly turning over fraction of plasma apo A-I that has recently been identified by Fisher et al60 using [2H]Leu. As we found no apo A-I in urine, any contribution to the high FCR by glomerular filtration6 7 must have been followed by complete reabsorption and catabolism by renal tubular cells.

There were no significant differences in apo A-I kinetics when the same total dose (25 mg/kg) was given by bolus injection (ie, over 15 minutes) or by slow infusion over 5 hours. The much shorter half-lives observed during series I than series II after the 5 mg·kg-1·h-1 infusions can probably be explained by the shorter duration of the first series, leading to underestimation of this parameter. The increase in residence time of SRC apo A-I when coinfused with Intralipid accords with other evidence that lipolysis of TGRLs reduces the FCR of HDL apo A-I7 50 51 52 53 and extends this effect to lipid-free apo A-I. This may reflect an association between infused apo A-I and PL released during lipolysis of the chylomicron-like particles in Intralipid, the PL liposomes that Intralipid contains, or both.48 The trend for half-life to decrease as apo A-I infusion rate increases (series II) can explain the associated decrease in Cmax, which may reflect saturation of PL uptake by apo A-I.

On HP-SEC of plasma, apo A-I content increased in all fractions that contained it at baseline. There was no consistent pattern of increase in the different fractions. We do not know how much of the apo A-I in each fraction was lipid free and how much was associated with lipids or HDLs. Apo A-I self-associates in vitro,41 61 and HP-SEC of the SRC preparation revealed some oligomers that eluted in the same fractions as spheroidal HDLs. It is possible that some oligomers were slow to dissociate in vivo. Other oligomers and dimers probably dissociated rapidly in vivo and, along with infused monomeric apo A-I, became associated with PL to produce pre-ß–migrating particles of different size (see below). Our failure to detect apo A-I in particles smaller than endogenous HDLs does not exclude the presence of circulating monomeric apo A-I, which was not resolvable by the Superose-6 system. Furthermore, such monomers would probably have crossed the capillary endothelia more rapidly than would have the oligomers.

The extent to which infused apo A-I is likely to associate with native plasma HDLs can be estimated from published data. Shepherd et al54 showed that addition of lipid-free apo A-I to human HDLs in vitro not only displaced endogenous apo A-I but also produced a net increase in apo A-I content that was related to the initial lipid-free apo A-I to HDL molar ratio. With these data it can be calculated that 15 minutes after bolus injection (when the greatest increments in plasma total apo A-I were observed, averaging 47 mg/dL), the concentration of apo A-I in spheroidal HDLs is likely to have increased by {approx}8% ({approx}7 mg/dL) as a result of this process. Apo A-I levels in {alpha}-migrating spheroidal HDLs might also be expected to rise as a consequence of their production from pre-ß HDLs.4 25 27 Our failure to detect an increase in {alpha}-migrating apo A-I might reflect the limited precision of crossed immunoelectrophoresis, the absence of any major conversion of pre-ß to spheroidal particles, or a concomitant decrease in the concentration of endogenous spheroidal HDLs. The extent to which pre-ß apo A-I was composed of lipid-free apo A-I and apo A-I/PL particles cannot be determined from our results.

When given by either bolus injection or 5-hour infusion, apo A-I increased the mass of total choline-containing PL in the HDL-containing fractions of plasma. This effect was evident earlier by HP-SEC than by PEG precipitation, probably reflecting the greater specificity of the former separation procedure. The effect on HDL PL increased with increasing infusion rate up to 5 mg·kg-1·h-1. In light of the evidence that lipid-free apo A-I recruits PL from cultured cells,28 29 30 31 this observation may reflect PL efflux from tissues. Although HDLs normally acquire PL from lipolyzed TGRLs, increased transfer from this source can probably be discounted because the rate-limiting factor appears to be lipolysis of TGRLs,62 which (as discussed below) seems to have decreased. At least part of the rise in HDL PLs was probably a consequence of their decreased hydrolysis by HL. PL and TG of HDLs are effective substrates for HL,63 and although there are no published data on the effects of lipid-free apo A-I on the phospholipase activity of HL, there is sound evidence that apo A-I inhibits its triglyceridase activity in vitro.64 65 66 A similar effect in vivo could have contributed (along with reduced LPL activity and increased CETP activity67 ) to the trend for HDL TG to increase.

No increases in HDL UC or HDL CE were recorded by either PEG precipitation or HP-SEC. On the contrary, by the more specific HP-SEC procedure small decreases in CE content of most HDL fractions were detected. This finding was unexpected, in view of the evidence that lipid-free apo A-I recruits UC from cultured cells.28 29 30 31 32 We have shown that SRC apo A-I/PL particles promote efflux of cholesterol from cultured mouse J774 macrophages and human umbilical vein endothelial cells43 and that lipid-free SRC apo A-I both promotes efflux of [3H]cholesterol from perfused rat spleens (M.A. Mindham et al, 1991, unpublished observations) and activates plasma LCAT in vitro.39 40 It is possible that increases in the uptake and esterification of tissue-derived cholesterol by HDLs occurred in our experiments but were masked by a concomitant increase in the transfer of CE from HDLs to VLDL and other lipoproteins. Lipid-free apo A-I has been shown to increase CETP activity in vitro,67 and this effect is likely to have been enhanced in vivo by the rise in VLDL concentration.68

Unexpectedly, the most striking responses to lipid-free apo A-I were increases in VLDL lipids. Increases in plasma TGs were observed when rats were given either rat or human apo A-I intravenously.55 This effect at least partially probably reflects inhibition of HL by apo A-I: small VLDL particles are effective substrates for HL69 ; anti-HL antibodies increase VLDL residence time in monkeys70 ; and slow lipolysis of small VLDLs, producing hypertriglyceridemia, is a feature of familial HL deficiency in humans.71 72 73 74 The fact that all size subclasses of VLDL, and not just the smaller particles, increased in our experiments suggests that inhibition of LPL may also have contributed. This accords with reports that lipid-free apo A-I inhibited lipolysis of Intralipid by purified LPL in vitro, though with less potency than for HL.75 76 Since we observed no reductions in glycerol release after SRC apo A-I (5 to 250 mg/dL) was added to postheparin human plasma in vitro (M.N.N. and N.E.M., 1993, unpublished observations), inhibition of lipolysis by lipid-free apo A-I may occur via a mechanism that precedes the association of enzyme and lipoprotein. This does not appear to be a consequence of apo A-I binding to lipoproteins, since Shepherd et al54 detected no binding of radioiodinated apo A-I to VLDL in vitro and we detected no apo A-I in VLDLs isolated by HP-SEC.

The rate of rise in plasma total TG content during apo A-I infusions at >=5 mg·kg-1·h-1 averaged 9 mg·dL-1·h-1 (range, 3.4 to 19). This value corresponded to a mean rate of increase in the plasma TG pool of {approx}4 mg·kg-1·h-1. Because the endogenous TG transport rate in human plasma averages {approx}8 mg·kg-1·h-1,77 78 it appears that lipolysis was reduced by {approx}50% at this dose.

Hypertriglyceridemia is a well-recognized component of the acute-phase response.79 80 81 Therefore, the possibility that increases in VLDL concentration in our subjects were due to this mechanism was considered. This possibility can probably be discounted because plasma TNF-{alpha} and CRP levels were unchanged, and no other clinical, biochemical, or hematologic features of the acute-phase response82 were observed.

The changes that occurred in LDL lipids were also compatible with combined inhibition of HL and LPL, the reduction in LDL cholesterol reflecting decreased conversion of VLDLs to LDLs and PL enrichment of LDL reflecting decreased HL activity.71 72 73 74 Our failure to detect apo A-I in HP-SEC fractions corresponding to LDLs excludes the possibility that PL increases in those fractions reflected the presence of large apo A-I/PL particles. The absence of any decrease in plasma apo B suggests that the FCR of LDL was not greatly affected.

In summary, the effects of lipid-free apo A-I on human lipoproteins in vivo can be explained by a combination of its known inhibitory effects on HL and LPL activities in vitro. The increase in HDL PL may also reflect greater recruitment of PL from tissues. Because the decrease in lipolysis will reduce the transfer of UC from VLDLs to HDLs and increase the transfer of CE in the reverse direction, the failure of HDL cholesterol to rise does not necessarily indicate that cholesterol efflux from peripheral tissues does not increase. Further studies will be needed to establish the effect of apo A-I infusion on reverse cholesterol transport in vivo.


*    Selected Abbreviations and Acronyms
 
CE(s) = cholesteryl ester(s)
CETP = cholesteryl ester transfer protein
Cmax = maximum increment in plasma concentration above baseline, adjusted to a total dose of 25 mg/kg, ie, measured increase in concentration (mgxdL)x25 mg/kg÷dose infused (mg/kg).
CRP = C-reactive protein
CV(s) = coefficient(s) of variation
ELISA = enzyme-linked immunosorbent assay
FCR = fractional catabolic rate
HL = hepatic lipase
HP-SEC = high-performance, size-exclusion chromatography
LCAT = lecithin-cholesterol acyltransferase
LpA-I = HDL containing apo A-I only
LPL = lipoprotein lipase
PAGE = polyacrylamide gel electrophoresis
PC = phosphatidylcholine
PL(s) = phospholipid(s)
SRC = Swiss Red Cross
TG(s) = triglyceride(s)
TGRL(s) = triglyceride-rich lipoprotein(s)
TNF-{alpha} = tumor necrosis factor-{alpha}
UC = unesterified cholesterol
Vdss = volume of distribution


*    Acknowledgments
 
This research was supported by the SRC Foundation, the British Heart Foundation, and the General Research Center of The Bowman Gray School of Medicine (grant No. MOI RR07122 from the National Institutes of Health, Bethesda, Md).


*    Footnotes
 
*The only uncertainty regarding clinical side effects concerns subject No. 3, who developed an exudative pleural effusion several days after his eighth infusion. No cause for this was found. The patient also complained of minor arthralgia in his fingers. No antibodies to apo A-I and no changes in TNF-{alpha} or CRP were detected. The effusion subsequently resolved.

Received July 21, 1995; revision received February 14, 1996;
*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
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