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Clinical Chemistry 46: 224-233, 2000;
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(Clinical Chemistry. 2000;46:224-233.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

Flow Cytometric Assessment of LDL Ligand Function for Detection of Heterozygous Familial Defective Apolipoprotein B-100

Bent Raungaard1,a, Finn Heath2, Peter Steen Hansen2, Jens Uffe Brorholt-Petersen1, Henrik Kjærulf Jensen2 and Ole Færgeman1

1 Department of Internal Medicine and Cardiology, Aarhus Amtssygehus University Hospital, Tage Hansens Gade 2, DK-8000 Aarhus C, Denmark.

2 Department of Cardiology, Skejby Sygehus University Hospital, DK-8200 Aarhus N, Denmark.
a Author for correspondence. Fax 45-89-49-76-19; e-mail rau{at}dadlnet.dk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Familial defective apolipoprotein (apo) B-100 (FDB) is caused by a mutation in the apoB gene and characterized by decreased binding of LDL to LDL receptors because of reduced function of the apoB-100 ligand. FDB may be associated with severe hypercholesterolemia and cannot always be distinguished from familial hypercholesterolemia phenotypically.

Methods: We used a fluorescence flow cytometry assay with Epstein-Barr virus-transformed lymphocytes to detect reduced LDL ligand function by competitive binding with fluorescently conjugated LDL (DiI-LDL). The assay was tested and validated using LDL from patients heterozygous for the Arg3500-Gln mutation and their first-degree relatives. Knowing the actual apoB genotype of patients and relatives allowed us to assess the ability of the assay to predict the results of DNA analysis. The results were compared to measurements of LDL ligand function in unrelated healthy control subjects to characterize functionally the Arg3500-Gln mutation.

Results: Fluorescence was significantly increased in cells incubated with DiI-LDL in competition with unlabeled LDL from FDBR3500Q heterozygotes compared with cells incubated with DiI-LDL in competition with unlabeled LDL from relatives or unrelated healthy control subjects. Thus, patients heterozygous for the Arg3500-Gln mutation had significantly reduced LDL ligand function. The binding affinity of LDL from FDBR3500Q heterozygotes was 32% of that in non-FDB relatives and healthy controls. The assay had a diagnostic sensitivity of 0.95 and diagnostic specificity of 0.89.

Conclusions: The diagnostic accuracy of the assay was too low to allow reliable diagnosis of individual cases of heterozygous FDBR3500Q. However, fluorescence flow cytometry may supplement genetic identification of FDB and functionally characterize gene mutations associated with major reductions in LDL ligand function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Increased LDL-cholesterol in plasma is a major risk factor for atherosclerosis (1). It is well recognized, moreover, that risk factor modification can prevent, reduce, or reverse the atherosclerotic process, and can reduce fatal and nonfatal coronary events in patients with existing coronary heart disease (2)(3)(4) as well as in asymptomatic individuals with mild (5) or moderate (6) hypercholesterolemia. Thus, early diagnosis and intervention is important, especially in asymptomatic high-risk subjects, such as people with inheritable forms of hypercholesterolemia.

LDL concentrations in plasma depend on the balance between synthesis and catabolism. Usually, ~70% of LDL is removed from the blood by LDL receptors located on the surface of most cells, primarily hepatocytes (7)(8). Apolipoprotein (apo)1 B-100 is the major structural protein of LDL and acts as a specific ligand in the cellular binding and uptake of LDL by LDL receptors (9). The relevance of this catabolic pathway is illustrated by genetic disorders affecting the receptor or ligand. In familial hypercholesterolemia (FH), LDL catabolism is impaired because of mutations in the LDL-receptor gene (10), and in familial defective apolipoprotein B-100 (FDB) hypercholesterolemia is attributable to defects in the structure of apoB-100 caused by mutations in the gene encoding the apolipoprotein (11). FDB was first observed in individuals with moderate hypercholesterolemia (12). However, FDB patients may have markedly increased cholesterol concentrations similar to those in FH patients, and clinical findings may also be similar (13)(14)(15)(16)(17)(18). Accordingly, clinical findings and family history of premature atherosclerosis are inadequate to diagnose individuals with defects in LDL ligand function. More specific methods are required to separate FDB from other types of severe hypercholesterolemia.

Gene mutation detection has identified several hundred different mutations in the LDL-receptor gene that disrupt receptor function. In contrast, FDB has been associated with surprisingly few mutations (19)(20)(21)(22)(23)(24)(25)(26)(27)(28), of which only three have been linked with reduced LDL ligand function. Most common is the replacement of Arg at residue 3500 (Arg3500) of apoB-100 by Gln (FDBR3500Q) (19)(20)(23)(24)(25)(27). More infrequent is the substitution of Cys for Arg at residue 3531 (FDBR3531C) (21)(23)(24)(25)(27)(28) and the substitution of Trp for Arg3500 (FDBR3500W) (22).

Although molecular genetic methods are unsurpassed in the detection of recognized mutations, they are often time-consuming and unsuited for screening for unknown mutations. In addition, these methods cannot functionally characterize mutations in the LDL receptor or apoB gene to determine their impact on LDL catabolism. Functional tests of LDL-receptor activity and LDL ligand function can theoretically identify defects in receptor-mediated LDL catabolism in hypercholesterolemic patients and simultaneously distinguish between receptor and ligand defects. Studies of LDL-receptor activity classically have been based on the uptake of 125I-labeled LDL from healthy individuals by cultured patient fibroblasts (9). Similarly, LDL ligand function initially was studied by measuring binding of 125I-labeled patient LDL to cultured human fibroblasts from healthy individuals (19). Fluorescence flow cytometry (FFC) represents a newer and less hazardous approach to functional assessment of LDL ligand-receptor interaction.

We previously validated a FFC assay for functional evaluation of LDL-receptor activity in heterozygous FH patients (29)(30), and we have gained experience with FFC in measuring LDL ligand function (31). In the present study, we tested and validated a FFC assay with Epstein-Barr virus (EBV)-transformed B lymphocytes for functional evaluation of LDL ligand function. LDL was prepared from previously identified FDBR3500Q heterozygotes and their non-FDB relatives. Knowing the actual apoB genotype of the patients and relatives allowed us to assess the ability of FFC to predict the results of DNA analysis. Results were compared to measurements of LDL ligand function in unrelated healthy control subjects to functionally characterize the Arg3500-Gln mutation. We also looked for linear relationships between LDL ligand function and plasma lipid concentrations, and we determined the relative binding affinity of LDL from FDBR3500Q heterozygotes. Finally, we examined whether long-term storage of serum at -80 °C before preparation of LDL alters the binding properties of LDL.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
subjects and lipids
Twenty-one individuals heterozygous for the Arg3500-Gln mutation and 23 of their non-FDB first-degree relatives were included. All were members of three Danish families previously shown to have FDBR3500Q (32). We also studied 24 unrelated healthy individuals without dyslipidemia or family history of coronary heart disease as controls. Plasma total cholesterol, HDL-cholesterol, triglyceride, and apoB concentrations were measured by conventional techniques. Plasma LDL-cholesterol concentrations were calculated by the Friedewald equation (33). Any lipid-lowering medications were discontinued for 4 weeks, and all individuals fasted overnight before blood sampling. The study was approved by the local ethics committee as being in accordance with the Helsinki Declaration.

lipoprotein preparation
Serum was prepared by low-speed centrifugation and stored immediately at -80 °C for up to 2 years before preparation of LDL. LDL (1.019 < d < 1.063 kg/L) was prepared by density gradient ultracentrifugation and dialyzed extensively against Hanks’-buffered saline solution containing 5 µmol/L EDTA at 4 °C and pH 7.4. Immediately after dialysis, LDL-protein concentrations were determined by the method of Bradford (34), and LDL was stored at 4 °C for a maximum of 1 week before analysis of ligand function. Hereafter, LDL prepared from individuals heterozygous for the Arg3500-Gln mutation will be termed FDB-LDL, LDL from non-FDB relatives will be termed non-FDB-LDL, and LDL from unrelated healthy control subjects will be termed control-LDL.

cell preparation
B lymphocytes from a normolipidemic individual were EBV-transformed as described by Neitzel (35). A pool of proliferative EBV-lymphoblasts were harvested, divided into aliquots, and frozen at -135 °C until utilization. Before analysis, cells were thawed and preincubated for 2 weeks at 37 °C in a humidified carbon dioxide incubator suspended in RPMI 1640 (Life Technologies) with L-glutamine (290 mg/L), penicillin (100 000 units/L), streptomycin (100 mg/L), and 100 mL/L fetal calf serum. Twenty-four hours before analysis, RPMI 1640 was replaced by a serum-free medium (AIM V; Life Technologies) to stimulate LDL-receptor expression.

cell incubation
Stimulated EBV-lymphoblasts were incubated for 60 min at 37 °C with (a) pure LDL (30 mg/L) prepared from one of the study subjects (FDB-LDL, non-FDB-LDL, or control-LDL) to measure background fluorescence and cell autofluorescence; (b) pure 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-conjugated LDL (DiI-LDL, 10 mg/L; Molecular Probes Europe) to estimate maximum LDL binding and uptake; or (c) a 3:1 mixture (by volume) of LDL and DiI-LDL to measure the degree to which LDL successfully competed with DiI-LDL in binding to EBV-lymphoblasts. A comparable subpopulation of EBV-lymphoblasts was identified by a fluorescein isothiocyanate (FITC)-conjugated B-cell-specific monoclonal antibody (CD20-FITC) or an IgG1 isotype-FITC (both from Dako).

flow cytometric measurements
Fluorescence was measured with a FACSort flow cytometer (Becton Dickinson) equipped with a 15-mW, 488-nm, air-cooled argon laser and linked to a Macintosh Quadra 650 computer with CellQuest software (Becton Dickinson). Forward scatter and side scatter were adjusted to exclude debris and dead cells. FITC emission was measured at 530 nm ± 15 nm (FL1), and DiI emission was measured at 585 nm ± 21 nm (FL2). An acquisition gate that included morphologically homogeneous EBV-lymphoblasts was defined in a forward scatter vs side scatter dot-plot. The acquisition number for this gate was set at 40 000. A CD20-positive acquisition gate was defined in a forward scatter vs FL1 dot-plot using the IgG1 isotype-FITC to determine the 1% cutoff limit for CD20 positivity. Only cells recorded in these two gates simultaneously were included in measurements of FL2. Fluorescence signals of FL2 were recorded on a logarithmically transformed intensity scale in a histogram. The ability of FDB-LDL, non-FDB-LDL, and control-LDL to compete with DiI-LDL in binding to EBV-lymphoblasts was expressed as the ratio of the median fluorescence of cells incubated with a mixture of LDL and DiI-LDL to the median fluorescence of cells incubated with pure LDL (DiI-ratio). All DiI-ratios were based on measurements performed in triplicate. All DiI-ratios were divided by median fluorescence of cells incubated with pure DiI-LDL to take into account variations in LDL-receptor number on cell surfaces (DiI-ratiocorr).

calculation of relative binding affinity
The law of mass action states that the rate of a reaction is proportional to the product of the concentrations of the reactants, or in terms of receptors, the proportions of receptors that are free or occupied. Thus, at equilibrium, the proportion of occupied receptors is given by the Hill-Langmuir equation (36):

where pAR is the proportion of receptors occupied by ligand A, [A] is the concentration of ligand A, and KA is the dissociation equilibrium constant of ligand A for the receptors. This is true when one ligand is present at a constant concentration. When another ligand is added as a competitive antagonist to a single class of homogeneous and independent receptors, the concentration–response relationship is altered for the agonist. When both the agonist (A) and antagonist (B) combine with the receptors according to the law of mass action, the proportion of receptors occupied by ligand A at equilibrium is expressed by the Gaddum equation (36):

where [B] is the concentration of ligand B, and KB is the dissociation equilibrium constant of ligand B for the receptors. The inverse of the dissociation equilibrium constant gives the association equilibrium constant, also called the affinity constant. Thus, to evaluate the binding affinity of LDL and DiI-LDL to LDL receptors (i.e., the quantitative relationship between agonist and antagonist concentrations and the proportion of receptors occupied by agonist), we considered the ability of LDL to compete with DiI-LDL in binding to LDL receptors on EBV-lymphoblasts.

statistics
Significance was set at 0.05. To test for differences in LDL ligand function between groups of examined LDL (FDB-LDL, non-FDB-LDL, and control-LDL) we used the Mann–Whitney U-test to compare groups of DiI-ratioscorr. Linear relationships between LDL ligand function and plasma lipid concentrations were investigated by regression analysis and expressed as coefficients of determination (R2). The significance of the slopes of the regression lines was determined by the Fisher r to z transformation.

Performance characteristics of the FFC assay are given by calculations of precision and accuracy. To evaluate precision, we estimated the random analytical error given by within-run, between-run, and biological variation by making repeated measurements. Accuracy, as defined by Zweig and Campbell (37), was evaluated as the diagnostic correctness of the FFC assay when compared with DNA analysis. The decision threshold [best cutoff value for the DiI-ratiocorr to discriminate between normal (non-FDB) and reduced (heterozygous FDBR3500Q) LDL ligand function] was found using ROC curves (37)(38)(39). The decision threshold was set where the best possible sensitivity [equal to probability of true heterozygous FDBR3500Q detection (PTP)] and specificity [equal to probability of true non-FDB detection (1 - PFP)] for the FFC assay were obtained. To quantify the diagnostic accuracy of our assay, we calculated the area under the ROC curve as the sum of rectangles under the graph.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mean lipid values for all study subjects are given in Table 1 . Plasma cholesterol, LDL-cholesterol, and apoB were significantly higher in FDBR3500Q heterozygotes than in non-FDB relatives and healthy controls (P <0.001). Plasma HDL-cholesterol was 0.8 mmol/L in one FDBR3500Q heterozygote and 0.7 mmol/L in one non-FDB relative. One FDBR3500Q heterozygote and two non-FDB relatives were hypertriglyceridemic (2.8–3.6 mmol/L). All other individuals had plasma HDL-cholesterol >=0.9 mmol/L and plasma triglycerides <=2.5 mmol/L. None of the study subjects had liver, thyroid, or renal disease, as assessed by routine laboratory tests.


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  		Table 1. Plasma lipid concentrations (mean ± SD) in study subjects.

In preliminary incubation experiments, we studied concentration- and time-dependent binding and internalization of LDL by stimulated EBV-lymphoblasts, using DiI-LDL and LDL prepared from normolipidemic individuals. Thus, we established the optimal mixture of LDL and DiI-LDL (data not shown). We chose a 3:1 mixture of LDL and DiI-LDL, which gave a fluorescence signal well above the background fluorescence. The concentration of DiI-LDL was intentionally kept relatively low to prevent fluorescently conjugated LDL from dominating the LDL mixture and hence conceal changes in the binding affinity of unlabeled LDL. We then determined the ideal incubation time for a 3:1 mixture of LDL and DiI-LDL (data not shown). Incubation times longer than 60 min produced more dispersion of results.

Morphologically homogeneous and CD20-positive EBV-lymphoblasts were identified by FFC. Fluorescence signals of DiI emission from these cells were recorded on a logarithmically transformed intensity scale in a histogram as illustrated in Fig. 1 .



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  		Figure 1. Morphologically homogeneous and CD20-positive EBV-lymphoblasts identified by FFC.

The cells were incubated with pure LDL prepared from one of the study subjects (FDB-LDL, non-FDB-LDL, or control-LDL; LDL), pure DiI-LDL (DiI-LDL), or a 3:1 mixture of LDL and DiI-LDL (LDL + DiI-LDL) and were further analyzed by FFC to determine the binding and uptake of DiI-LDL in competition with LDL. Fluorescence signals of DiI emission from these cells were recorded on a logarithmically transformed intensity scale in a histogram. The ability of FDB-LDL, non-FDB-LDL, and control-LDL to compete with DiI-LDL in binding to EBV-lymphoblasts was expressed as the ratio of the median fluorescence of cells incubated with a mixture of LDL and DiI-LDL to the median fluorescence of cells incubated with pure LDL (DiI-ratio). All DiI-ratios were divided by median fluorescence of cells incubated with pure DiI-LDL to take into account variations in LDL receptor number on cell surfaces (DiI-ratiocorr).

Within each of the three groups of measurements, we found some interindividual variation in DiI-ratioscorr as illustrated in Fig. 2 . This indicates substantial variability of LDL ligand function among patients with the same apoB gene mutation (Arg3500-Gln) and in the normolipidemic population, in line with observations by others (40). Within each group, we measured a few high and low values (outliers) that led to a small overlap between the patient group and the two non-FDB groups. The fluorescence for cells incubated with FDB-LDL and DiI-LDL was increased significantly (mean increase, 1.6-fold) compared with measurements of cells incubated with non-FDB-LDL or control-LDL and DiI-LDL (P <0.0001). The fluorescence for cells incubated with non-FDB-LDL and DiI-LDL did not differ from the fluorescence for cells incubated with control-LDL and DiI-LDL.



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  		Figure 2. Ability of LDL from FDBR3500Q heterozygotes, non-FDB relatives, and unrelated healthy control subjects to compete with DiI-LDL in binding to EBV-lymphoblasts.

Values were expressed as the ratio of the median fluorescence of cells incubated with a mixture of LDL and DiI-LDL to the median fluorescence of cells incubated with pure LDL, divided by the median fluorescence of cells incubated with pure DiI-LDL (DiI-ratiocorr). The higher the DiI-ratiocorr, the lower the affinity of the sample LDL. There was some interindividual variation in DiI-ratioscorr within each of the three groups of measurements. A few high and low values in each group led to a small overlap between the patient group and the other two groups. The fluorescence for cells incubated with DiI-LDL and FDB-LDL (FDB heterozygotes) was significantly increased compared with measurements of cells incubated with DiI-LDL and non-FDB-LDL (nonFDB relatives) or control-LDL (unrelated healthy controls). Fluorescence for cells incubated with non-FDB-LDL and DiI-LDL did not differ from fluorescence for cells incubated with control-LDL and DiI-LDL.

To estimate precision of the FFC assay, we divided serum samples from the same individual into subsamples, all of which were prepared and analyzed simultaneously (within-run variation), made repeated measurements on samples from the same individual (between-run variation), and simultaneously analyzed samples obtained from an unrelated healthy study subject sampled once weekly over a 6-week period (biological variation). Estimates of within-run, between-run, biological, and total variance and the coefficients of variation are given in Table 2 . Within-run variance was based on simultaneous analysis of six serum subsamples, as indicated in Fig. 3 , and accounted for 16% of the total variance. Between-run variance was based on repeated measurements on serum samples from seven healthy study subjects, as indicated in Fig. 4 , and accounted for the largest variance for the assay (76%). Biological variance was based on simultaneous analysis of four serum samples from one healthy study subject sampled at weekly intervals, as indicated in Fig. 3 , and accounted for 8% of the total variance.


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  		Table 2. Precision of the FFC assay determined by ANOVA type I variance estimates.



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  		Figure 3. Variability over time of the affinity of LDL for the LDL receptor.

Each circle represents an analysis of the LDL ligand function of LDL prepared from an unrelated healthy study subject sampled once weekly over a 6-week period. Two serum samples (weeks 2 and 5) were divided into four subsamples each before freezing. LDL was prepared simultaneously from three subsamples obtained at week 2 and three subsamples obtained at week 5. LDL ligand function was subsequently determined for all six LDL preparations within the same run ({circ}), giving an estimate of within-run variance. LDL was prepared simultaneously from each of the weekly serum samples (weeks 1–6), and LDL ligand function was determined for all six preparations within the same run (•), giving an estimate of biological variation. Measurements for LDL obtained at weeks 5 and 6 were outliers (deviated >2 total SDs from the mean of the remaining measurements). The two values were excluded in the calculations of biological variance.



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  		Figure 4. Values for serum samples from eight unrelated healthy controls divided into two subsamples each before freezing.

LDL was prepared simultaneously from one subsample from each study subject, and the ligand function was analyzed for all eight preparations within the same run (run 1). This procedure was repeated for the second set of subsamples (run 2). Between-run variation was estimated by comparing measurements for each study subject from run 1 and run 2. We excluded the unrelated healthy control subject who presented an extreme difference (>2 total SDs) between runs 1 and 2 from the calculations of between-run variance. Excluded measurements are indicated by a dashed line.

An ROC curve for the diagnostic performance of the FFC assay based on measurements of LDL ligand function among the genetically identified FDB-family subgroup of study subjects (FDBR3500Q heterozygotes and non-FDB relatives) is given in Fig. 5 . The area under the ROC curve was 0.954.



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  		Figure 5. ROC curve.

Plot of all sensitivity vs (1 - specificity) pairs resulting from continuously varying the decision threshold [best cutoff value for the DiI-ratiocorr to discriminate between normal (non-FDB relatives) and reduced (FDBR3500Q heterozygotes) LDL ligand function] over the entire range of DiI-ratioscorr observed.

The values for the best decision threshold for the DiI-ratiocorr to discriminate between heterozygous FDBR3500Q and non-FDB, sensitivity (PTP), and specificity (1 - PFP) for the assay are given in Table 3 .


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  		Table 3. Performance of the FFC assay.

We found a positive relationship between values for DiI-ratiocorr and plasma LDL-cholesterol (Fig. 6 A; R2 = 0.35; P <0.0001) and plasma apoB (Fig. 6B ; R2 = 0.30; P <0.0001) in the total study population, indicating that as the LDL binding affinity for the LDL receptors decreases, the plasma LDL-cholesterol concentrations tend to increase. No correlation was observed between values for DiI-ratiocorr and plasma triglycerides (Fig. 6C ). We found identical trends in the FDB-family subgroup of study subjects (data not shown).



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  		Figure 6. Scatter diagrams of the relationships between values for DiI-ratiocorr and plasma LDL-cholesterol (A), plasma apoB (B), and plasma triglycerides (C) in the total study population.

R2 signifies coefficients of determination. p indicates the significance of the slope of the regression line as determined by the Fisher r to z transformation. N.S., not significant.

DiI-ratioscorr gave a measure of the amount of fluorescent LDL bound to EBV-lymphoblasts, i.e., the ability of LDL to compete with DiI-LDL in binding to LDL receptors, which mirrored LDL ligand function. With use of the Hill-Langmuir equation for measurements of EBV-lymphoblasts incubated with pure DiI-LDL and the Gaddum equation for measurements of EBV-lymphoblasts incubated with a 3:1 mixture of control-LDL, non-FDB-LDL, or FDB-LDL and DiI-LDL, we calculated that the binding affinity of FDB-LDL for LDL receptors was 32% of the healthy controls and non-FDB relatives (control-LDL and non-FDB-LDL), in agreement with binding studies of LDL from FDBR3500Q heterozygotes presented previously by us (31) and others (11)(19)(21)(28)(40)(41).

All serum samples were stored at -80 °C until analysis. The longest storage time was for serum samples obtained from the unrelated healthy control subjects, which were stored for up to 2 years. We previously have shown that storage of serum at -80 °C for 3 months before LDL preparation does not affect LDL ligand function (31), and others have found that serum can be stored at -20 °C for 12 months without alterations in LDL binding properties (42). To investigate whether storage at -80 °C for 2 years influenced ligand function of prepared LDL, we repeated blood samples on six of the unrelated healthy control subjects. Serum was prepared as described in Materials and Methods and frozen at -80 °C for <3 months before analysis was performed. FFC showed that storage for up to 2 years had no detectable effect on LDL ligand function when results were compared with samples stored <3 months (data not shown). This finding is important for future use of the assay because it improves the practical application of the assay. It allows multiple samples to be collected before analysis and makes repeated measurements practicable.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Mutations in the apoB gene occur at a frequency of ~1:500 in most Caucasian populations (11)(13)(14), occur more frequently in Switzerland (43) and less frequently among Scandinavians (27)(44)(45)(46)(47), and as such are as common as mutations in the LDL-receptor gene. Because FDBR3500Q may cause severe hypercholesterolemia and can imitate FH phenotypically, these two forms of inheritable hypercholesterolemia cannot be distinguished on clinical appearance alone. It has been estimated that perhaps as many as 5% of patients diagnosed as FH heterozygotes on clinical grounds actually have normal LDL receptors and instead are carriers of the FDB mutation (15). We previously identified five Danish families with FDBR3500Q. By screening the apoB gene for the most common mutation (Arg3500-Gln), we categorized 46 FDBR3500Q heterozygotes and 57 non-FDB relatives (32). In the present study, we examined the LDL ligand function of some of these genetically identified family members to test and validate an FFC assay. Knowing the apoB genotype of the study subjects allowed us to validate the assay’s ability to identify defects in LDL ligand function based on molecular genetics.

Several different methods have been used to provide evidence for the existence of abnormalities in the apoB-100 molecule. An inherent problem with techniques such as restriction fragment length polymorphism detection and immunophenotyping for identification of genetic polymorphisms of apoB-100 is that they give no information on apoB function. One method to determine LDL ligand function is 125I-labeling of LDL and analysis of its ability to bind to cultured fibroblasts in conventional displacement (9) or dual-label (40) assays. However, these methods are hazardous and difficult to use in clinical studies. Another method to specifically determine binding properties of LDL measures the proliferation rate of a human monocyte cell line (U937) (26)(28)(42)(48)(49). These cells lack the capacity for endogenous cholesterol synthesis and therefore have an absolute requirement for LDL-receptor-dependent uptake of extracellular LDL-cholesterol for growth (50). Culturing in lipid-deficient medium leads to depletion of intracellular cholesterol and arrest of cell growth within 48 h (51). Thus, the induced growth of U937 cells mirrors the ligand function of purified LDL added to a delipidated medium. In a study of 10 normolipidemic individuals and 16 patients with FDBR3500Q, Van den Broek et al. (42) found the method to have a diagnostic sensitivity of 87.5% and diagnostic specificity of 100% when U937 cells were incubated with whole serum. However, they also found that the subnormal proliferation of U937 cells incubated with serum from patients with FDBR3500Q was variable and could not be predicted from plasma LDL-cholesterol concentrations. An explanation for the lack of correlation may be that the growth-promoting effect of LDL on U937 cells depends on the content of cholesterol in lipoproteins and not the protein content (48)(52). Thus, a possible confounding influence on the assay could be high plasma triglyceride concentrations, which decrease the size of LDL, thus affecting its binding affinity (53). Because a variable cholesterol/apoB ratio in the LDL isolated from different individuals will influence the growth-promoting effect, it is not possible to compare results for LDL from different individuals with this assay without adjustment for cholesterol content. Others have found variation in U937 cell proliferation rates attributable to changes in experimental conditions (culture conditions, duration of cell starvation, and initial cell density) (49). When these factors are taken into consideration, however, the U937 proliferation assay seems to be an accurate test for the detection of reduced LDL ligand function.

FFC has advantages over radioligand assays used for characterization of LDL ligand function: it is nonhazardous; and it can simultaneously measure the fluorescence signals of several probes attached to a single cell. Furthermore, the use of gates in the acquisition and analysis processes of FFC can exclude fluorescence signals of the medium and cellular debris and can include or exclude various cell populations based on cell morphology and cell-surface marker labeling. In our assay, we used EBV-lymphoblasts identified morphologically and by labeling with a specific fluorescently conjugated surface marker antibody (CD20-FITC). EBV-lymphoblasts are mainly cells with B-cell lineage, are easily cultured, and are fast growing and immortal. They express high-affinity LDL receptors (54) that are regulated in a fashion similar to that in primary lymphocytes, i.e., down-regulated in the presence of LDL and up-regulated in a lipid-deficient environment. Thus, culturing EBV-lymphoblasts in a serum-free medium can induce a more than ninefold increase in LDL receptor expression when compared with primary lymphocytes (55). The EBV-lymphoblast population is heterogeneous, representing different stages of maturation and possible different functional subsets (55). To avoid the influence of potential variations in LDL-receptor expression on our measurements, we selected a morphologically and antigenically homogeneous subpopulation of EBV-lymphoblasts by FFC gating. The pan-B-cell antigen CD-20 is expressed on the surface of B cells at higher density compared with most other surface antigens (including CD-19), making it an attractive choice for reliable B-cell enumeration by FFC (56).

Our measurements indicated that the FFC assay was accurate and precise. However, this was true only when the occasional aberrant measurements of DiI-ratiocorr (outliers) were excluded in the calculations of within-run, between-run, and biological variance. When outliers were excluded, the dominant variability of the assay was between runs, as indicated in Table 2Up . Our findings underscore the importance of reanalysis of serum samples if the measured DiI-ratiocorr exceeds the acceptance range (mean of group ± 2 total SDs). At least two independent analyses of each serum sample should, therefore, be evaluated. This will increase the precision of the assay and prevent the acceptance of aberrant measurements. Reanalysis of serum samples must then be performed if the between-run difference of DiI-ratiocorr exceeds two total SDs.

apoB-100 is the only major protein in LDL and acts as a ligand for the LDL receptor. Thus, it is natural to expect that differences in LDL ligand function have a significant impact on plasma LDL-cholesterol concentrations. This expectation is supported by the negative correlation between LDL ligand function (inverse of DiI-ratiocorr) and plasma LDL-cholesterol concentrations observed in this study, and by the positive correlation between proliferation of U937 cells and LDL-cholesterol concentrations in healthy subjects and patients with type IIa hyperlipoproteinemia found by others (42). However, Mendel (40) demonstrated the opposite correlation and considered the LDL ligand function as minor compared with other factors involved in LDL catabolism.

Because LDL contains a single molecule, apoB-100, two LDL populations are present in individuals heterozygous for the Arg3500-Gln mutation: one with wild-type apoB-100 and one with mutant apoB-100. LDL with normal ligand function will be catabolized at normal rates, leaving behind LDL with reduced ligand function, especially small and dense (1.040 < d < 1.063 kg/L) LDL (57), to accumulate in plasma. Consequently, the plasma LDL in patients heterozygous for the Arg3500-Gln mutation typically is composed of 70% abnormal LDL and 30% normal LDL (12)(19)(41)(58)(59), and LDL prepared from these patients has approximately one-third of the binding affinity of normal LDL, as demonstrated by us previously (31) and in this study, and by others (11)(19)(21)(28)(40)(41). On the basis of these observations, it has been calculated that LDL carrying the Arg3500-Gln mutation has <10% of normal binding affinity for LDL receptors (11)(21)(59). Arnold et al. (59) confirmed this prediction by showing that the LDL-receptor binding ability of LDL containing the Arg3500-Gln mutation is 9% of the LDL-receptor binding ability of LDL containing the wild-type apoB.

In summary, we present a FFC assay for the detection of reduced LDL ligand function. The assay is simple, reproducible, and nonhazardous, but its diagnostic accuracy is too low to allow reliable diagnosis of individual cases of heterozygous FDBR3500Q, which is better achieved by mutational analysis. The assay is comparable to the established U937 proliferation assay, with a diagnostic accuracy in the same range. Our functional assay, being independent of variations in the cholesterol/apoB ratio of LDL, seems better able to predict the relative binding affinity of mutant apoB-100, however. We found a strong positive correlation between LDL ligand function and plasma LDL-cholesterol and apoB concentrations in a population of individuals heterozygous for the Arg3500-Gln mutation, their non-FDB first-degree relatives, and unrelated healthy controls. Furthermore, our data show that LDL from patients heterozygous for the Arg3500-Gln mutation has significantly lower ligand function than LDL from their non-FDB relatives and unrelated healthy control subjects: LDL from FDBR3500Q heterozygotes had one-third the binding affinity of the LDL from non-FDB individuals. Diagnosis of individual cases of heterozygous FDBR3500Q, therefore, still depends on mutational analysis, but FFC can extend such analysis by functionally characterizing this and any other apoB mutations that cause major reduction in LDL ligand function. The assay has the potential to detect and characterize defects in the ability of other ligands to bind to their respective receptors.


   Acknowledgments
 
This study was supported by the Danish Heart Foundation (Grants 981464a22605 and 981464b22606), the Danish Medical Research Council, and the Eva og Henry Frænkels Mindelegat. We thank Professor Steen Kølvraa for generously providing EBV-transformed B lymphocytes, and Dr. Lars Ulrik Gerdes for advice concerning the statistical analysis. We thank Pia Buchtrup Hornbek, Gitte Glistrup Nielsen, and Anette Thomsen for excellent technical assistance.


   Footnotes
 
1 Nonstandard abbreviations: apo, apolipoprotein; FH, familial hypercholesterolemia; FDB, familial defective apolipoprotein B-100; FFC, fluorescence flow cytometry; EBV, Epstein–Barr virus; DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; FITC, fluorescein isothiocyanate; DiI-ratio, ratio of the median fluorescence of cells incubated with a mixture of LDL and DiI-LDL to the median fluorescence of cells incubated with pure LDL; PFP, probability of false heterozygous FDBR3500Q detection; and PTP, probability of true heterozygous FDBR3500Q detection.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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