Quantitative measurement of lipoprotein particles containing both apolipoprotein AIV and apolipoprotein B in human plasma by a noncompetitive ELISA.
Several studies have shown that apo AIV can be found as the free form or associated mainly with HDL, but also with TRLs (3,14-21). Thus, apo AIV can be found in HDL (1.063 kg/L < d < 1.210 kg/L) as well as in TRL (d <1.006 kg/L), two species of plasma lipoproteins differing in density and lipid and apolipoprotein composition that can be distinguished by the absence or presence of apo B. Indeed, in the free state or in the HDL size range, apo AIV does not coexist with apo B, whereas in the chylomicrons, VLDL, or LDL size range, an association between these two apolipoproteins has been observed (17). In addition to apo AIV and apo B, other apolipoproteins could be present in these particles. For these reasons, we measured the total bulk of particles containing at least apo AIV and apo B, i.e., the lipoprotein B (Lp B):AIV family, and named them Lp B:AIVf. Particles free of apo B but containing apo AIV and possibly containing other apolipoproteins were named the Lp AIV non-B family (Lp AIV non-Bf).
The main goal of this study was to develop a reliable method for the quantification of particles containing both apo AIV and apo B (Lp B:AIVf), using a two-site differential ELISA. In addition, we measured Lp B:AIVf in different situations of impaired lipid metabolism to determine the effect of such situations on Lp B:AIVf concentrations.
Materials and Methods
PLASMA SAMPLES AND BIOCHEMICAL ANALYTES
Human plasma samples were obtained from normolipidemic (n = 71), hypercholesterolemic [total cholesterol (TC) >2000 mg/L; LDL-cholesterol (LDL-C) >1600 mg/L; triglycerides (TGs) <2000 mg/L; n = 21] and mixed hyperlipidemic (TGs and TC >2000 mg/L; n = 20) patients. Venous blood samples were collected from patients after an overnight fast into tubes containing EDTA. Plasma samples were separated by centrifugation and then divided into aliquots and immediately analyzed or kept at -20 [degrees]C until analysis.
Before measurement of Lp B:AIVf and total apo AIV, apo B, TC, TG, and HDL-cholesterol (HDL-C) concentrations were measured enzymatically on a multianalyte analyzer (Hitachi 747; Roche Molecular Biochemicals). LDL-C was calculated according to the Friedewald formula (22). apo AIV was determined using a classic sandwich ELISA, as adapted from Rosseneu et al. (23). Lp AIV non-Bf was calculated by subtracting Lp B:AIVf from total apo AIV.
Isolation and purification of LDL. Lipoprotein B of a narrow density range (d = 1.040-1.053 kg/L) was purified by preparative ultracentrifugation (24) from pooled plasma samples lacking apo(a) from healthy human donors, and dialyzed against 5 mmol/L ammonium bicarbonate. The purity of the lipoprotein B was determined by electrophoresis on polyacrylamide gels and immunoblotting, as described previously by Towbin et al. (25).
Obtention and purification of immunoglobulins (IgY). Antiapo B immunoglobulins were produced in the Ecole Nationale Veterinaire (Names, France). Antiserum to apolipoprotein B was obtained from laying hens. Briefly, 500 t~,g of LDL in Freund's complete adjuvant was administrated subcutaneously to laying hens. Hens then received other doses of LDL in Freund's incomplete adjuvant every 2 weeks: twice with 250 t~,g and four times with 100 N.g. In parallel, eggs were collected daily beginning the 7th day after the first injection, identified by appropriate labeling, and stored at 4 [degrees]C until the antibodies were extracted.
Preparation and purification of antibodies to apo B. hninunoglobulins (IgY) were extracted from egg yolk by the method described by Polson (26). After precipitation with polyethylene glycol, the remaining infranate was dissolved in 0.1 mol/L phosphate-buffered saline, pH 7.4 (PBS; Life Technologies), and stored at -20 [degrees]C. Pooled fractions of these total immunoglobulins suspended in PBS were applied to a LDL column, prepared by coupling CNBr-Sepharose (Pharmacia) with narrow-density range LDL and equilibrated with 0.1 mol/L PBS, pH 7.4.
Columns were run using a Biologic LP chromatography system (Bio-Rad). The retained fraction, corresponding to specific antibodies against apo B, was eluted with 0.2 mol/L glycine-HCI, pH 2.8. The collected active fractions were then successively pooled, dialyzed against PBS, concentrated in polyethylene glycol (Sigma), dialyzed once more, and finally stored at -20 [degrees]C in 0.5-mL aliquots (1.34 g/L) before use. Purified anti-apo B antibodies were checked by immunoblotting and did not react with apo AIV.
Polyclonal antibodies to apo AIV and horseradish peroxidase-conjugated polyclonal antibodies to apo AIV. Polyclonal antibodies to apo AIV and horseradish peroxidase-conjugated polyclonal antibodies to apo AIV were purchased from Institut Pasteur. Anti-apo AIV antibodies did not react with apo B, as verified by immunoblotting.
IMMUNOASSAYS FOR Lp B:AIVf AND TOTAL apo AIV
To measure total apo AIV and Lp B:AIVf, we coated 96-well Nunc hninuno Maxisorp microtiter plates (Roche Diagnostics) with anti-apo AIV or anti-apo B antibodies (0.1 mL/well; 20 mg/L protein, previously diluted in 0.1 mol/L PBS, pH 7.4; Life Technologies) and incubated the plates overnight at 4 [degrees]C. The plates were then washed four times with 0.2 mL of 0.1 mol/L PBS, pH 7.4. To minimize nonspecific binding, we added 0.2 mL of a 1 g/L solution of bovine serum albumin (Sigma) in PBS to each well and incubated the plates at 37 [degrees]C for 2 h. From this point, all reagents were diluted in 0.1 mol/L PBS, pH 7.4, containing 1 g/L bovine serum albumin (dilution buffer).
Plates were washed twice more with PBS, after which appropriate dilutions of calibrators, controls, or plasma samples in dilution buffer (0.1 mL/well) were added to the wells, and the reaction was allowed to incubate 2 h at 37 [degrees]C. Plates were then washed with PBS, and peroxidase-labeled anti-apo AIV antibody (0.1 mL/well; diluted 4000- and 1200-fold in dilution buffer for apo AIV or Lp B:AIVf assays, respectively) was added. After the addition of this antibody, the entire plate was incubated for 2 h at 37 [degrees]C for apo AIV and for 2.75 h at 37 [degrees]C for Lp B:AIVf. These different incubation times were chosen for practical reasons and convenience. Finally, after the plates were washed four times, ABTS(r) substrate solution (0.2 mL/well; Roche Diagnostics) was added, and the enzymatic reaction was allowed to proceed for 30 min in the dark at 37 [degrees]C. The absorbantes were read at 405 nm on a Spectra Max Pro spectrophotometer (Molecular Devices). Calibration curves for apo AIV and Lp B:AIVf were obtained using the four-parameter regression incorporated in the spectrometer software (Soft MAX Pro; Molecular Devices).
Calibrators. Lyophilized calibrators and controls were obtained from SEBIA, which provided the apo AIV concentration in these commercial samples, previously determined against pure apo AIV as primary standard. The Lp B:AIVf concentrations in the calibrators were determined by immunoprecipitation with anti-apo B (27). The absence of apo B in the supernatant was checked by ELISA. The Lp B:AIVf concentration was calculated by difference between the apo AIV concentration in the intact sample and in the supernatant after apo B precipitation. In both cases, the apo AIV concentration was determined by ELISA. This procedure was performed 10 times on the same lot of calibrators, and the mean of 60 mg/L was used to determine sample and control concentrations.
Influence of epitope exposure. To determine the influence of epitope exposure on Lp B:AIVf and apo AIV methodologies, we exposed 16 samples to 0.5 g/L Tween 20 (Sigma) before measurements and compared the results obtained between treated samples and crude samples. To compare slopes obtained in this method with slopes for the same samples untreated, we serially diluted six samples, pretreated or not with Tween 20, to concentrations of 4-20 and 20-150 [micro]g/L for Lp B:AIVf and apo AIV, respectively.
Specificity and precision. The specificity of the assay was checked by measuring the concentration of Lp B:AIVf after precipitation with an anti-apo B antibody.
Assay precision was estimated by the ANOVA method described in NCCLS recommendations (28). The within run CV was estimated by repeated measurements (n = 25) of Lp B:AIVf concentrations in three plasma samples containing high, medium, and low amounts of Lp B:AIVf. The between-run CV was assessed by analyzing (n = 10) three samples, containing high, medium, and low amounts of Lp B:AIVf, for 4 consecutive days.
[FIGURE 1 OMITTED]
Analytical recovery and methods comparison. To determine analytical recovery, we prepared a series of plasma samples containing increasing concentrations of Lp B:AIVf by adding five different amounts of a previously concentrated standard plasma (SEBIA) containing a known concentration of Lp B:AIVf to a plasma sample.
Analytical recovery was calculated as the ratio (as a percentage) between the measured and calculated Lp B: AIVf concentration. Results obtained by the proposed method (Lp B:AIVf ELISA) were compared with those obtained indirectly by the apo AIV ELISA, as described previously by Alsayed et al. (29). The apo AIV concentration was measured in the supernatants from microwell plates coated with anti-apo B, as for the determination of total apo B concentrations. This allowed us to determine Lp AIV non-Bf concentrations. These results were subtracted from the total apo AIV concentrations to obtain the calculated Lp B:AIVf concentrations.
Effects of freezing and TG concentrations. The effect of freezing (sample stability) was investigated by determining Lp B:AIVf concentrations in plasma samples stored 0, 3, and 9 months at -20 [degrees]C (n = 20).
To determine the effect of added TGs, we added five different amounts of a TG-rich plasma sample (plasma B) containing known concentrations of TGs and Lp B:AIVf to a plasma sample (plasma A), which yielded TG concentrations up to 5560 mg/L. Recovery was estimated as the ratio (as a percentage) between measured and calculated concentrations.
Multiple mean comparisons with ANOVA were used to compare results in normolipidemic, hypercholesterolemic, and mixed hyperlipidemic individuals. In case of significance, the Bonferroni test was used for multiple t-test comparisons between groups. Correlations between Lp B:AIVf and other lipids and lipoproteins were assessed by linear regression analysis.
OPTIMIZED ASSAY CONDITIONS
The optimal concentration of anti-apo B antibody for coating the microtiter plates was 20 mg/L. The optimal dilution of anti-apo AIV conjugate was ~1:1200 (i.e., the value giving a wider range of absorbantes with a lower blank value).
A typical calibration curve is shown in Fig. 1. Absorbance was plotted against Lp B:AIVf concentration (after a logarithmic transformation), as estimated by apo AIV bound to apo B in F[micro]g/L. Under these conditions, the Lp B:AIVf calibration curve was linear from 3.75 to 30 [micro]g/L.
INFLUENCE OF EPITOPE EXPOSURE
To determine the influence of epitope exposure on the Lp B:AIVf and apo AIV methods, we exposed 16 samples to 0.5 g/L Tween 20 (Sigma) before measurements and compared the results with those obtained with untreated samples. To compare slopes obtained in this method with slopes of the same samples untreated, six samples, pretreated or not with Tween 20, were serially diluted to give concentrations of 4-20 and 20-150 F[micro]g/L for Lp B:AIVf and apo AIV, respectively. The results of this comparison are presented in Table 1. A nonparametric paired test did not reveal any statistically significant differences in mean results and slopes between Tween 20-treated samples and untreated samples.
SPECIFICITY AND PRECISION OF THE ASSAY
The within-run CVs, estimated by analyzing three different human plasma samples 25 times, were 11%, 9.2%,and 9.1% for Lp B:AIVf concentrations of 33.3, 50.6, 76.5 mg/L, respectively.
The between-run CVs of 8.5%, 11%, and 10% were determined by measuring Lp B:AIVf in three specimens containing 40, 59, and 78 mg/L Lp B:AIVf, respectively, on 4 consecutive days (Table 2).
ANALYTICAL RECOVERY AND METHODS COMPARISON
The recoveries for five known amounts of Lp B:AIVf added to a plasma were 95-107% (Table 3).
Comparison of Lp B:AIVf concentrations measured in 20 plasma samples by both the Lp B:AIVf ELISA and the apo AIV ELISA yielded a statistically significant linear relationship: y = 1.10x + 1.12 (r = 0.81; P <0.01). The detected amounts of Lp B:AIVf (55.9 [+ or -] 14.9 vs 50.0 [+ or -] 10.9 mg/L, direct measurement vs apo AIV ELISA) indicated relatively satisfactory recoveries.
EFFECTS OF FREEZING AND TG ENRICHMENT
Lp B:AIVf concentrations in plasma samples (n = 20) stored at -20 [degrees]C for 0, 3, and 9 months were 50.7 [+ or -] 13.7, 47.1 [+ or -] 9.4, and 48.3 [+ or -] 12.8 mg/L, respectively, with no statistically significant difference. These results indicate that a storage period up to 9 months at -20 [degrees]C does not cause significant changes in Lp B:AIVf concentrations.
The recoveries for five different amounts of TGs added to plasma were 91-99%, as shown in Table 4, indicating that TGs up to 5560 mg/L had no effect on Lp B:AIVf concentrations.
QUANTIFICATION OF Lp B:AIVf IN NORMOLIPIDEMIC, HYPERCHOLESTEROLEMIC, AND MIXED HYPERLIPIDEMIC PLASMA SAMPLES
Shown in Table 5 are plasma Lp B:AIVf concentrations obtained for 112 normolipidemic, hypercholesterolemic, or mixed hyperlipidemic men and women (age range, 16-87 years) as well as their lipids, apo B, total apo AIV, and Lp AIV non-Bf concentrations. As shown in Table 5, the Lp B:AIVf concentration in normolipidemic samples (43 [+ or -] 12 mg/L) was statistically different from the Lp B: AIVf concentrations in hypercholesterolemic (53 [+ or -] 13 mg/L; P <0.001) and mixed hyperlipidemic (70 [+ or -] 18 mg/L; P <0.001) plasmas. Moreover, hypercholesterolemic and mixed hyperlipidemic plasma Lp B:AIVf concentrations were also statistically different from each other (P <0.01). As expected, apo B concentrations were statistically different in the three groups. In contrast, apo AIV and Lp AIV non-Bf concentrations were similar among these groups.
The correlations produced by linear regression analysis of Lp B:AIVf, apo AIV, and Lp AIV non-Bf concentrations against the other analytes in the whole population are given in Table 6. We observed a strong significant correlation between Lp B:AIVf concentrations and apo B (r = 0.634; P <0.0001), cholesterol (r = 0.588; P <0.0001), TGs (r = 0.699; P <0.0001), and LDL-C (r = 0.551; P <0.0001). On the other hand, we found no association between Lp B:AIVf and total apo AIV, Lp AIV non-Bf, or HDL-C. Moreover, we found no correlation between apo AIV or Lp AIV non-Bf and any of the tested analytes.
This report describes a new method for measurement of the plasma concentration of lipoproteins containing at least apo AIV and apo B (Lp B:AIVf). This method is reproducible and convenient enough to be applied to large sample studies.
Lipoprotein particles containing apo AIV have already been characterized in human plasma. In 1993, Duverger et al. (17) showed that there are mainly two apo AIV-containing particle subspecies: Lp AIV and Lp AI:AIV. In Lp AIV, they estimated that the amount of plasma apo B associated with apo AIV was ~1.9%. In addition, several studies have shown that 1-15% of apo AIV seems to be associated with TRLs (14,15,19-21, 30-33). These described distributions are lower than our results (6-45%; mean, 27%). These discrepancies could be partially explained by the techniques used. Ultracentrifugation for a long period of time (18-48 h) at high speed (150 000g) may have a disruptive effect, thus weakening the association between apo AIV and lipoproteins, and leading to an underestimation of bound apo AIV (1). Gel-permeation chromatography and affinity chromatography could cause dilution of the sample, which may also limit the capacity of Lp B:AIVf detection. Furthermore, apo AIV-containing lipoproteins are heterogeneous in size, and their plasma distribution is rather complex; apo AIV could be associated with lipoproteins distributed across the entire size spectrum. These subfractions could contain both apo AIV and apo B (17). Therefore, estimating the Lp B:AIVf concentration by measuring apo AIV in the VLDL size range may lead to underestimation. The reliability of our assay could be higher because plasma sample processing is simpler, without any sample pretreatment, avoiding any loss of Lp B:AIVf particles.
This Lp B:AIVf assay is fast, precise, specific, and reproducible. For the first time, Lp B:AIVf subspecies can be quantified directly in plasma samples. In the fasting state, this method clearly showed the existence of particles containing both apo AIV and apo B in normolipidemic individuals as well as in hypercholesterolemic or mixed hyperlipidemic individuals. Fasting hyperlipidemic patients have more apo AIV bound to apo B (Lp B:AIVf), i.e., in TRLs or in apo B-containing lipoproteins, than fasting normolipidemic individuals. In addition, the Lp B:AIVf concentration is clearly correlated with increases in cholesterol and TGs as well as apo B, whereas total apo AIV and Lp AIV non-Bf are not related to these lipoproteins. These results could be explained by a redistribution of apo AIV in favor of apo B-containing lipoproteins, in case of an accumulation of apo B-containing lipoproteins. This finding is in agreement with results obtained by Verges et al. in 1994 (30).
In conclusion, the method described here provides specific, reproducible, rapid, and direct determination of Lp B:AIVf particles in plasma. The concentrations of these lipoprotein particles are well correlated with apo B, cholesterol, TGs, and LDL-C. Our preliminary results suggest a redistribution of apo AIV in favor of apo B-containing lipoproteins when those accumulate in plasma. This redistribution of apo AIV toward apo B-containing lipoproteins could change the biological and metabolic properties of apo AIV. In particular, association of apo AIV to apo B-containing lipoproteins could confer atherogenic properties to this apolipoprotein. Therefore, the potential interest of Lp B:AIVf measurement as a risk marker for cardiovascular disease should be further evaluated.
We thank Pfizer Company (Paris, France) for the financial support of Fanny Ferrer in realizing this work.
Received December 31, 2001; accepted February 28, 2002.
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 Nonstandard abbreviafions: apo, apolipoprotein; TRL, triglyceride-rich lipoprotein; Lp B, lipoprotein B; TC, total cholesterol; LDL-C and HDL-C, LDL-and HDL-cholesterol, respectively; TG, triglyceride; and PBS, phosphate-buffered saline.
FANNY FERRER, [1,2] EDITH BIGOT-CORBEL,  PATRICK N'GUYEN  MICHEL KREMPF,  and JEAN-MARIE BARD [1,2] *
 INSERM U539, Centre de Recherche en Nutrition Humaine, CHU Hotel Dieu, 44035 Nantes, France.
 Laboratoire de Biochimie, UFR de Pharmacie, 44035 Nantes, France.
 Ecole Nationale Veterinaire, 44300 Nantes, France.
* Address correspondence to this author at: Laboratoire de Biochimie Fondamentale et Appliquee, UFR de Pharmacie, 1 rue Gaston Veil, 44093 Nantes Cedex 1, France. E-mail Jean-Marie.Bard@sante.univ-nantes.ir.
Table 1. Influence of Tween 20 pretreatment on Lp B:AIVf and apo AIV measurements and on slopes. Lp B:AIVf Crude Tween 20 P Concentration (mean 36 [+ or -] 9 36 [+ or -] 7 0.776 [+ or -] SD), mg/L (n = 16) Slope (mean [+ or -] 0.863 [+ or -] 0.792 [+ or -] 0.844 SD; n = 6) 0.194 0.126 apo AIV Crude Tween 20 P Concentration (mean 153 [+ or -] 70 155 [+ or -] 74 0.888 [+ or -] SD), mg/L (n = 16) Slope (mean [+ or -] 1.060 [+ or -] 0.956 [+ or -] 0.156 SD; n = 6) 0.166 0.222 Table 2. Between-run imprecision for the Lp B:AIVf ELISA. Day 1 2 3 4 Low (40 mg/L) Mean, mg/L 37.0 42.1 40.6 40.2 SD, mg/L 1.6 2.0 3.6 3.5 CV, % 8.5 Medium (59 mg/L) Mean, mg/L 52.4 63.7 63.7 58.1 SD, mg/L 3.8 4.0 3.0 6.0 CV, % 11 High (78 mg/L) Mean, mg/L 71.4 78.6 82.0 79.4 SD, mg/L 7.9 8.2 2.5 8.5 CV, % 10 Table 3. Analytical recovery as assessed by enrichment of a sample with plasma of a known Lp B:AIVf concentration. Calculated Lp B:AIVf, mg/L 54.8 64.1 70.8 75.8 82.9 Measured Lp B:AIVf, mg/L 58.5 68.2 69.3 72.3 86.1 Recovery, % 107 106 98 95 104 Table 4. Effect of TGs on the Lp B:AIVf measurement. Sample (a) A A/B 1/5 A/B 2/4 A/B 3/3 TGs, mg/L 1030 1940 2840 3750 Calculated Lp B:AIVf, mg/L 61.9 77.6 93.3 Measured Lp B:AIVf, mg/L 46.2 59.9 70.0 88.5 Recovery, % 97 91 95 A/B 4/2 A/B 5/1 B TGs, mg/L 4660 5560 6470 Calculated Lp B:AIVf, mg/L 109.0 124.7 Measured Lp B:AIVf, mg/L 106.7 124.0 36.2 Recovery, % 98 99 (a) A/B ratios are given as volume/volume. A represents a plasma sample with a TG concentration of 1030 mg/L progressively enriched in TG by addition of sample B with a TG concentration of 6470 mg/L. Table 5. Concentrations (a) of Lp B:AIVf and various lipids and lipoproteins in normolipidemic and dyslipidemic individuals. Normolipidemia Hypercholesterolemia (n = 71) (n = 21) Sex, F/M 27/44 10/11 Age, years 53 [+ or -] 15 56 [+ or -] 18 TC, mg/L 1950 [+ or -] 390 2600 [+ or -] 300 (b) TGs, mg/L 1180 [+ or -] 380 1350 [+ or -] 430 HDL-C, mg/L 550 [+ or -] 240 470 [+ or -] 140 LDL-C, mg/L 1120 [+ or -] 260 1860 [+ or -] 280 (b) apo B, mg/L 910 [+ or -] 220 1380 [+ or -] 230 (b) apo AIV, mg/L 217 [+ or -] 151 273 [+ or -] 249 Lp B:AIVf, mg/L 43 [+ or -] 12 53 [+ or -] 132 Lp B:AIVf/ 27 [+ or -] 16 35 [+ or -] 22 apoAIV, % Lp AIV nonBF, 174 [+ or -] 148 220 [+ or -] 246 mg/L Mixed hyperlipidemia P, ANOVA (n = 20) Sex, F/M 5/15 Age, years 56 [+ or -] 12 TC, mg/L 2680 [+ or -] 260 (b) <0.0001 TGs, mg/L 3250 [+ or -] 1300 (b,e) <0.0001 HDL-C, mg/L 390 [+ or -] 110 (c) <0.0113 LDL-C, mg/L 1550 [+ or -] 390 (b,f) <0.0001 apo B, mg/L 1420 [+ or -] 160 (b) <0.0001 apo AIV, mg/L 220 [+ or -] 105 <0.40 Lp B:AIVf, mg/L 70 [+ or -] 18 (b,f) <0.0001 Lp B:AIVf/ 37 [+ or -] 16 (d) <0.043 apoAIV, % Lp AIV nonBF, 150 [+ or -] 101 <0.38 mg/L (a) Concentrations of analytes are given as the mean [+ or -] SD. (b-d) Compared with normolipidemic individuals: (b) P <0.001; (c) P <0.01; (d) P <0.05. (e,f) Compared with hypercholesterolemic individuals: (e) P <0.001; (f) P <0.01. Table 6. Correlations of Lp B:AIVf, Lp AIV non-Bf, and total apo AIV with other plasma lipoprotein constituents in the whole population (n = 112). r Lp B:AIVf Apo AIV Lp AIV non-Bf TC 0.588 (a) 0.12 0.062 TGs 0.699 (a) -0.009 -0.061 HDL-C -0.217 0.22 0.245 LDL-C 0.551 (a) 0.049 0.001 apo B 0.634 (a) -0.049 -0.114 apo AIV 0.197 0.995 (a) (a) P <0.0001.
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|Title Annotation:||Lipids, Lipoproteins, and Cardiovascular Risk Factors|
|Author:||Ferrer, Fanny; Bigot-Corbel, Edith; N'Guyen, Patrick; Krempf, Michel; Bard, Jean-Marie|
|Date:||Jun 1, 2002|
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