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The Journal of Lipid Research, Vol. 39, 987-998, May 1998
Copyright © 1998 by Lipid Research, Inc.

Original Article

Plasma and fibroblasts of Tangier disease patients are disturbed in transferring phospholipids onto apolipoprotein A-I

Correspondence to: Arnold von Eckardstein.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Discussion REFERENCES

Plasmas of patients with Tangier disease (TD) lack lipid-rich -HDL which, in normal plasma, constitutes the majority of high density lipoprotein (HDL). Residual amounts of apolipoprotein (apo)A-I in TD plasma occur as lipid-poor or even lipid-free preß-HDL. By contrast to normal plasma, TD plasma does not convert preß-HDL into -HDL. Moreover, fibroblasts of TD patients were found to be defective in secreting cholesterol or phospholipids in the presence of lipid-free apoA-I. We have therefore hypothesized that both defective conversion of preß-HDL into -HDL and defective lipid efflux from TD cells onto lipid-free apoA-I result from a disturbance in phospholipid transfer occurring in both cellular and extracellular compartments. To test this hypothesis we established an assay that measures the activity of plasma, cells, and cell culture media to transfer radiolabeled phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) from vesicles onto apoA-I, apoA-II, albumin, or reconstituted HDL. Plasmas, HDL, and lipoprotein-depleted plasma of normolipidemic probands as well as cell homogenates and culture media of normal fibroblasts were active at 37°C but not at 4°C in transferring radiolabeled PC, PI, and PE dose- and time-dependently onto either lipid-free apoA-I or reconstituted HDL. Transfer of glycerophospholipids onto apoA-II was much lower than onto apoA-I; transfer onto albumin was close to background. Compared to ten normolipidemic plasmas and four apoA-I-deficient plasmas, plasmas of six TD patients were significantly reduced by 40–50% in their glycerophospholipid transfer activities. Compared to eight normal fibroblast cell lines, homogenates and culture media of four TD fibroblast cell lines were reduced by 40–50% and 30–35%, respectively, in their activity to transfer PC, PI, or PE onto apoA-I.

Our data suggest that in TD the same mechanism underlies both defective conversion of preß-HDL into -HDL and impaired efflux of cellular lipids, namely a defective phospholipid transfer.—von Eckardstein, A., A. Chirazi, S. Schuler-Lüttmann, M. Walter, J. J. P. Kastelein, J. Geisel, J. T. Real, R. Miccoli, G. Noseda, G. Höbbel, and G. Assmann. Plasma and fibroblasts of Tangier disease patients are disturbed in transferring phospholipids onto apolipoprotein A-I. J. Lipid Res. 1998. 39: 987–998.

Supplementary key words: prebeta-HDL, familial HDL deficiency, reverse cholesterol transport, cholesterol efflux, phospholipid transfer proteins

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Discussion REFERENCES

Tangier disease (TD) is a rare, high density lipoprotein (HDL) deficiency syndrome that is inherited as an autosomal-codominant trait and whose metabolic and genetic basis is unknown. Clinical symptoms in homozygotes include hyperplastic orange tonsils, hepatosplenomegaly, and relapsing peripheral neuropathy, probably due to cytoplasmic cholesteryl ester deposition in macrophages and Schwann cells, respectively (1) (2). The biochemical hallmark of TD plasma is the absence of -migrating HDLs, i.e., -LpA-I, which in normal plasma contain approximately 95% of apolipoprotein (apo) A-I and whose concentration is reflected by HDL-cholesterol. Residual amounts of apoA-I of TD plasma are found in an HDL subclass, which in normolipidemic plasma accounts for less than 5% of total apoA-I and which because of its electrophoretic mobility is termed preß₁-LpA-I (3). By contrast to lipid-rich -HDL, which produce slow and unsaturable net cholesterol efflux only in the presence of lecithin:cholesterol acyltransferase (LCAT), this lipid-poor or even lipid-free particle promotes fast and saturable cholesterol efflux from certain but not all cell types, e.g., from fibroblasts and macrophages. This type of cholesterol efflux occurs independently of LCAT and in parallel with phospholipid efflux. It is sensitive to the treatment of cells with proteases and modulated by activation or inhibition of protein kinase C (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16). Therefore, it was suggested that cholesterol efflux mediated by lipid-free apoA-I depends on specific protein–cell surface interactions.

Interestingly, lipid-free apoA-I was found completely inactive in releasing cholesterol and phospholipids from TD fibroblasts (17) (18). Compared to normal fibroblasts, the unspecific cholesterol efflux from TD fibroblasts promoted by native or trypsinized HDL was reduced by only 30–50% or even unaffected. Oram and Yokoyama (6) and Francis, Knopp, and Oram (17) hypothesized that the failure of lipid-free apoA-I to pick up phospholipids and cholesterol from TD cells caused not only cellular lipid accumulation and foam cell formation but also a block in the maturation of HDL. Actually, we have previously demonstrated that in contrast to plasmas of normolipidemic subjects or patients with other forms of familial HDL deficiency, TD plasma lacks the activity to convert lipid-poor preß₁-LpA-I or lipid-free apoA-I into -LpA-I (19). Therefore, and as preß₁-LpA-I and -LpA-I differ by the content and composition of phospholipids (4) (20) (21) (22) (23), we hypothesize that plasmas and fibroblasts of TD patients are defective in transferring phospholipids onto apoA-I. To test this hypothesis we established an assay that measures the activities of plasmas, homogenized cells, and cell culture media to transfer glycerophospholipids from vesicles onto apoA-I and reconstituted HDL. By this test we were able to demonstrate significantly reduced phospholipid transfer activities in both plasmas and fibroblasts of TD patients.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Discussion REFERENCES

Subjects
Six patients with Tangier disease (TD), four patients with apoA-I deficiency, and ten normolipidemic volunteers participated in this study after informed consent and in accordance with the ethical standards laid down in the relevant version of the 1964 Declaration of Helsinki. Serum concentrations of lipids, lipoproteins, and apolipoproteins as well as plasma activities of lipid transfer enzymes are summarized in Table 1. Among the six TD patients, two German siblings (T1 and T2) and a man from Pakistan (T3) have been described previously (24) (25). T1 and T2 have clinically presented with hepatosplenomegaly, T3 with multiple peripheral motoric and sensory losses. Case reports of three other TD patients from Germany (T4), The Netherlands (T5), and Spain (T6) have not yet been described and are presented below. In all cases, isoelectric focusing of plasma and subsequent anti-apoA-I immunoblotting identified residual amounts of apoA-I and proapoA-I occurring with regular isoelectric points and at equal concentrations, which is a typical finding in TD (2).

The 60-year-old German TD patient (T4) suffered from myocardial infarction at the age of 49 years and underwent aortocoronary bypass surgery at the age of 50 years. In addition to HDL deficiency and hypertriglyceridemia (see Table 1), cardiovascular risk factors included overweight (body mass index = 29.0 kg/m²), arterial hypertension, and hyperuricemia. Gingiva was hyperplastic. Spleen and liver were not enlarged. Neither the history nor the physical examination of the patient gave indications of peripheral neuropathy. No corneal opacifications were found upon inspection. Routine laboratory examinations over the last 10 years repeatedly led to the detection of thrombocytopenia (100.000 ± 10.000 platelets/µl). The patient has two sisters who are also deficient of HDL and two children who have half-normal HDL-cholesterol levels. DNA sequence analysis of the apoA-I gene revealed wild-type (J. Geisel, K. Oette, H. Funke, A. von Eckardstein, and G. Assmann, unpublished results).

The Dutch TD patient (T5) suffered from myocardial infarction of the inferior wall at the age of 38 years. Coronary angiography revealed an 80% proximal stenosis of the right coronary artery and diffuse severe atherosclerosis of the left coronary artery. Subsequently, the patient underwent coronary bypass surgery and recovered uneventfully. Absence of HDL and severely reduced levels of apoA-I (Table 1) were the only coronary risk factors. Biopsy of both tonsil and rectal mucosa showed the presence of lipid-laden macrophages suggestive of TD. The patient's father and daughter had HDL-cholesterol levels below the 10th percentile of age-matched controls.

The 51-year-old Spanish TD patient (T6) came to medical attention in 1994 because of hepatosplenomegaly and thrombocytopenia (120.000 platelets/µl). Biopsies of bone marrow and spleen led to the identification of foamy histiocytes that were characterized as Niemann-Pick type. At the age of 20 years the patient underwent tonsillectomy because of yellowish and enlarged adenoid tissue. Physical examination revealed no signs of peripheral neuropathy. The patient's mother and four children had decreased HDL-cholesterol levels (<34 mg/dl).

Four patients from two families with apoA-I deficiency have been described previously. In one case a premature truncation at residue 28 led to apoA-I deficiency in a 32-year-old Italian woman (26). Two brothers and a sister from another Italian family are compound heterozygotes for an apoA-I null allele and an arginine for leucine substitution at residue 141, i.e., apoA-I(L141R)_Pisa (27) (28).

Collection of blood samples
Blood samples were collected after overnight fasting and cooled immediately on ice. Sera and EDTA-plasmas were obtained by centrifugation at 4°C (800 g, 15 min), aliquoted, and stored at -70°C. When not sampled in Münster, plasmas and sera were shipped on dry ice to this laboratory.

Cell culture of fibroblasts
Fibroblasts were cultivated from skin biopsies that were obtained from hip skin of eight unrelated healthy normolipidemic volunteers and four patients with Tangier disease (T1, T2, T4, and T5) as decribed previously (29). The cells were grown and maintained in DMEM that contained 10% FCS and 1% antibiotics/antimycotics (Sigma Chemical Co., St. Louis, MO). Experiments described below were performed after three to ten passages.

Fibroblasts of the same donor were cultured in 75-cm² flasks. At the state of near confluence, cells were washed three times with PBS (pH 7.4) and then incubated for 24 h in 9 ml DMEM that contained 1% antibiotics/antimycotics and 30 g/l bovine serum albumin (BSA) as the basic medium and was supplemented with either no additive or 100 µg/ml LDL or 10% FCS. Thereafter the cells were washed three times with PBS and incubated again for 24 h in 9 ml lipoprotein-free DMEM. This medium was removed and centrifuged in a microcentrifuge for 10 min at 15,000 rpm to precipitate cell debris. The supernatants were frozen at -80°C. The cells were washed three times with PBS and then removed into 6 ml PBS with a rubber policeman. The cell suspension was also frozen at -80°C.

Isolation of lipoproteins, apoA-I, and apoA-II
Very low density lipoproteins (VLDL, d < 1.006 g/ml), intermediate and low density lipoproteins (IDL and LDL, 1.006 < d < 1.061 g/ml), high density lipoproteins (HDL, 1.063 < d < 1.21 g/ml), and lipoprotein-depleted plasma (LPDP, d > 1.21 g/ml) were isolated from plasma by sequential isopycnic ultracentrifugation (30).

ApoA-I and apoA-II were isolated from HDL by fast performance liquid chromatography as described previously (31) using a MonoQ HR5/5 column as the stable phase (Pharmacia, Bromma, Sweden). The mobile phase was run with a gradient consisting of buffer A (15 mM Tris-HCl, 10 mM NaCl, 5 M urea) and buffer B (15 mM Tris-HCl, 1 M NaCl, 5 M urea). The gradient was eluted within 30 min with a flow of 0.5 ml/min. Purity of apoA-I and apoA-II was ascertained by reversed-phase high performance liquid chromatography (HPLC) (32) and SDS-PAGE (33).

Determination of serum concentrations of lipids, lipoproteins, and (apolipo)proteins, and plasma activities of LCAT, cholesteryl ester transfer protein, and phospholipid transfer protein
Serum concentrations of triglycerides and cholesterol were quantified with an autoanalyzer (Hitachi/Boehringer, Mannheim, Germany). HDL-cholesterol was measured in the supernatant obtained by precipitation of apoB-containing lipoproteins with phosphotungstic acid/MgCl₂ (Boehringer Mannheim). Concentrations of apoA-I, A-II, and B were determined with a modified commercially available turbidimetric assay (Boehringer Mannheim). Plasma activity of lecithin:cholesterol acyltransferase (LCAT) was assayed as the esterification of [³H]cholesterol (New England Nuclear, Boston, MA) that was incorporated into reconstituted, apoA-I-containing particles (31) (34). Cholesteryl ester transfer protein (CETP) activity was determined as the transfer of [³H]cholesteryl oleate (New England Nuclear) from artificial apoA-I-containing proteoliposomes to LDL as reported previously (31) (35). Phospholipid transfer protein (PLTP) activity was monitored as the transfer of [¹⁴C]phosphatidylcholine (New England Nuclear) from phospholipid vesicles to HDL₃ (36).

Measurement of glycerophospholipid transfers
In the course of this study we established assays to measure the activities of plasmas, cell culture homogenates, and cell culture media to transfer phosphatidylcholine (PC), phosphatidylinositol (PI), or phosphatidylethanolamine (PE) from donor vesicles onto immobilized acceptors. As the acceptors we used biotinylated apoA-I, biotinylated apoA-II, biotinylated human albumin (purchased from Sigma), or biotinylated rLpA-I that were conjugated to streptavidin-coated tubes (Boehringer Mannheim). Biotinylation of apoA-I, apoA-II, and albumin was done as recommended by the manufacturer of the biotinylation kit (Sigma). The solution containing biotinylated apoA-I was either directly incubated with streptavidin-coated tubes or used to prepare rLpA-I by the cholate-dialysis method (37). rLpA-I contained egg yolk PC (Sigma) and apoA-I at a weight per weight ratio of 7:1 (molar ratio approximately 250:1). Every streptavidin-coated tube was incubated for 24 h at 4°C with either 10 µg apoA-I, apoA-II, albumin, or rLpA-I that were solubilized in 300 µl assay buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1 mM EDTA.

As the donor particles we used vesicles containing PC from egg yolk (Sigma) as well as unlabeled and radiolabeled glycerophospholipids. The relative composition of glycerophospholipids was adapted to that of VLDL in human plasma. Generally, alcoholic solutions with the labeled and unlabeled phospholipids were dried under nitrogen and then dissolved in 1 ml assay buffer with 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1 mM EDTA. This suspension was sonicated three times for 60 sec using Soniprep 150 (MSE, England). Specifically, for the measurement of PC transfer onto rLpA-I, we prepared vesicles with 7 mg egg yolk PC and 3.2 µg (0.5 µCi) L--dipalmitoyl-di[1-¹⁴C]phosphatidylcholine (¹⁴C-PC; DuPont New England Nuclear). For the measurement of PI transfer, we prepared vesicles with 7 mg egg yolk PC, 70 µg PI from bovine liver (Sigma), and 13 ng (0.25 µCi) L-3-phosphatidyl-[2-³H-]inositol (³H-PI; Amersham). For the measurement of PE transfer, we prepared vesicles with 7 mg egg yolk PC, 70 µg PE from bovine brain (Sigma), and 3.5 ng (0.25 µCi) 1,2-dioleoyl-L-3-phosphatidyl[2-¹⁴C]ethanolamine (¹⁴C-PE, Amersham). The transfer of phospholipids onto lipid-free apolipoproteins or albumin was measured with vesicles that contained 5-fold higher amounts of radiolabeled phospholipids.

Shortly before the start of the phospholipid transfer assay, plasmas or media were thawed and cells were homogenized by sonicating three times for 5 min with Soniprep 150. For the assays, 20 µl emulsion of donor vesicles and plasmas, cell homogenates, or cell culture media at volumes or masses described in the results section were placed in tubes containing immobilized rLpA-I or apoA-I and filled up with assay buffer to a final volume of 300 µl. The mixtures were incubated with an overhead shaker at 37°C and for 1 h when not otherwise indicated. The solutions were subsequently removed and the tubes were washed three times with assay buffer. Finally, the tubes were filled with scintillation liquid (Ultima Gold XR, Packard, Groningen, NL) for counting radioactivity. Measurements were performed in duplicate. In some indicated instances we modified these tests by incubation at 4°C.

For the comparison of glycerophospholipid transfer activities in normal and patient plasmas the assays were performed at both 37°C and 4°C to correct for unspecific transfers. Fractional glycerophospholipid transfer rates were calculated by the following formula:

where cpm_{acceptor(37°C)} and cpm_{acceptor(4°C)} correspond to the radioactivity of ¹⁴C-PC, ¹⁴C-PE, or ³H-PI present in the tube after incubation of donor vesicles at 37°C and 4°C, respectively, and cpm_donor to the radioactivity present in the tube with donor vesicles before incubation with plasmas, cells, or media. The intraassay and interassay coefficients of variation of the assays were below 5% and 10%, respectively.

Other methods
Protein concentrations were determined by the method of Lowry et al. (38).

Statistics
All data are presented either as medians or means ± standard deviations. The levels of significance for differences between patients and controls were calculated by ANOVA. All calculations were done with Excel (Microsoft) and an add-in program for Excel (Astute, 1993 DDU Software, The University of Leeds).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Discussion REFERENCES

Assay characteristics
We established radiometric assays that monitor the activity of plasma, plasma fractions, cell homogenates, and cell culture media to transfer PC, PI, or PE from donor vesicles onto lipid-free apoA-I, apoA-II, albumin, or reconstituted apoA-I/PC complexes (rLpA-I). To separate donor particles from acceptor particles after incubation with the transfer activity, we immobilized lipid-free proteins or rLpA-I by the use of the streptavidin/biotin technology. The maximal binding of biotinylated acceptor particles was achieved by a 24-h incubation of 10 µg biotinylated apoA-I or rLpA-I with streptavidin-coated tubes (not shown). Subsequent washing removed 6–7 µg biotin apoA-I per tube. Another 0.5–1 µg biotinylated apoA-I (i.e., 15–25% of the biotin-apoA-I present in the assay) was removed during a 2-h assay incubation with vesicles and plasma, media, or cell homogenates.

Fractional transfer of PC from vesicles onto apoA-I by either plasma, LPDP, homogenates, or media of cultured fibroblasts peaked in the presence of 6–25 µl liposome emulsion that contained 40–225 µg PC ( Figure 1). Therefore, we used 20 µl of the liposome emulsion for all subsequently described experiments.

View larger version (18K): [in this window] [in a new window]   Figure 1. Effect of increasing donor concentrations on transfer of [14C]phosphatidylcholine onto lipid-free apoA-I by plasma, lipoprotein-depleted plasma, and homogenates and culture media of fibroblasts. Ten µg biotinylated, lipid-free apoA-I was immobilized in streptavidin-coated tubes. These were filled with increasing amounts of a PC vesicle-emulsion (7 mg/ml PC and 2.5 µCi/ml 14C-PC). These tubes were incubated for 1 h at 37°C with 2 µl plasma, 50 mg LPDP, 20 µg homogenate, or 400 µl medium of fibroblasts that were cultivated in the presence of 10% FCS. The curves represent the fractional transfer of PC from the donor vesicle onto immobilized apoA-I.

Transfer of PI, PC, and PE by normolipidemic and Tangier disease plasmas
Figure 2 demonstrates the time-dependent transfer of radiolabeled PC from vesicles onto immobilized apoA-I, apoA-II, or albumin by 2 µl plasma. The maximal transfer onto any acceptor was reached after 2 h incubation. However, at any time point, transfer rates onto apoA-I were 3- to 4-times higher than onto apoA-II and 8- to 10-fold higher than onto albumin.

View larger version (15K): [in this window] [in a new window]   Figure 2. Transfer of [14C]phosphatidylcholine from vesicles onto lipid-free apoA-I, apoA-II and albumin by normal plasma. Ten µg biotinylated lipid-free apoA-I, 10 µg biotinylated lipid-free apoA-II, or 10 µg biotinylated albumin was immobilized in streptavidin-coated tubes. These were filled with 20 µl of a PC vesicle-emulsion (7 mg/ml PC and 2.5 µCi/ml 14C-PC) and with 2 µl normolipidemic plasma. Incubations were performed for increasing time intervals. The curves represent the transfer of 14C-PC from the donor vesicle onto the immobilized proteins.

Figure 3A and 3B demonstrate the effects of increasing plasma volumes and incubation times on the transfer of radiolabeled PI from PI/PC vesicles onto either immobilized lipid-free apoA-I or immobilized rLpA-I. Maximal transfer of ³H-PI was reached after 1 h incubation with 2 µl plasma. Incubation with more than 4 µl plasma as well as incubations longer than for 1 h resulted in lower transfer rates. Moreover, significantly more ³H-PI was transferred onto lipidated rLpA-I as compared to lipid-free apoA-I (Figure 3A). Incubations with plasma at 4°C led to a transfer of ³H-PI onto rLpA-I that was much lower (Figure 3B). Similar observations were made for the activities of plasma to transfer radiolabeled PC or PE onto rLpA-I or apoA-I (not shown). At any volume, plasmas of three TD patients (T1, T2, and T3) were significantly less active in transferring ³H-PI onto either biotinylated rLpA-I or biotinylated apoA-I than normolipidemic plasmas (Figure 3C). Similar to normal plasma, incubation with 2 µl TD plasma resulted in maximal PI transfer rates. Larger volumes decreased PI transfer (Figure 3C). Maximal transfer by TD plasmas was reached by a 1-h long incubation at 37°C. This maximum, however, was 60% of that reached with normal plasma. Transfer of PI taking place in the presence of TD plasma at 4°C was as effective as in the presence of normal plasmas. Experiments on the transfer of PC or PE by TD plasma gave similar results (not shown).

View larger version (25K): [in this window] [in a new window]   Figure 3. Transfer of [3H]phosphatidylinositol from vesicles onto lipid-free apoA-I and reconstituted LpA-I by normal or TD plasmas. Ten µg biotinylated, lipid-free apoA-I (broken line and closed symbols) or 10 µg biotinylated rLpA-I (closed line and open symbols) was immobilized in streptavidin-coated tubes. These were filled with 20 µl of a PI/PC vesicle-emulsion (7 mg/ml PC, 70 µg/ml PI, and 0.25 µCi/ml 3H-PI). Increasing volumes of three normal (A) or three TD plasmas (patients T1, T2, and T3, C) were incubated in these tubes for 1 h at 37°C. Alternatively, tubes with immobilized rLpA-I were incubated for increasing time intervals with 2 µl plasma of three normal probands (B) or three TD patients (D) and at 37°C (closed line and open symbols) or 4°C (broken line and closed symbols). In all figures, symbols and bars represent mean values and standard deviations, respectively, of experiments on three normolipidemic plasmas and three TD plasmas.

Table 2 summarizes the data on transfers of PC, PI, and PE from donor vesicles onto rLpA-I by plasmas of ten normolipidemic probands, six patients with TD, and four patients with apoA-I deficiency. The results were corrected for the unspecific transfers taking place at 4°C (compare Figure 3B and Figure 3D). Plasmas of all six TD patients were less active in transferring glycerophospholipids onto rLpA-I than plasmas of normolipidemic controls. Transfers of ³H-PI, ¹⁴C-PE, and ³H-PC were reduced by 39–62% (mean 50%), 30–64% (mean 48%), and 37–56% (mean 43%), respectively (all P < 0.0001, Student's t -test). All three glycerophospholipid transfer activities were also significantly decreased in plasmas of apoA-I-deficient patients which, however, had still significantly higher glycerophospholipid transfer activities than TD plasmas (Table 2). When the PC transfer assay was carried out with lipid-free apoA-I or apoA-II, the transfer activities of TD plasma were about 50% lower than that of normal plasmas. The transfer onto albumin was close to background with any plasma.

To rule out the presence of an inhibitor in TD plasma that interferes with the transfer of glycerophospholipids, we mixed normolipidemic plasma with TD plasma at different volume ratios. At any ratio we recovered 100% transfer activity (not shown). By univariate regression analysis we tested the dependence of glycerophospholipid transfer activities of normal plasmas on one another and on other variables of lipid metabolism ( Table 3). Highly significant positive correlations were found among all three glycerophospholipid transfer activities. The positive correlation between PC transfer activity and apoA-I concentration, as well as negative correlations between all glycerophospholipid transfer activities with the plasma PLTP activity, were also significant (Table 3).

Transfer of PI, PC, and PE by plasma lipoproteins and lipoprotein-depleted plasmas
Table 4 as well as Figure 4 summarize data on the distribution of glycerophospholipid transfer activities among plasma lipoprotein subfractions. In normal plasma, transfer activities for PC, PI, and PE onto rLpA-I were encountered in every lipoprotein fraction. Similar to total plasma, increases in lipoprotein mass did not lead to equivalent increases in glycerophospholipid transfer when they exceeded threshold levels. The highest specific transfer activities were found in the presence of 1 µg protein of either VLDL, LDL, or HDL (Table 4) and 50 µg protein of LPDP (Figure 4).

View larger version (11K): [in this window] [in a new window]   Figure 4. Dosage- (A) and time-dependent transfers (B) of [14C]phosphatidylcholine from vesicles onto reconstituted LpA-I by lipoprotein- depleted plasmas of a normal proband and a TD patient. Ten µg biotinylated rLpA-I was immobilized in streptavidin-coated tubes which were then filled with 20 µl of vesicle emulsion containing either 14C-PC. In (A) increasing amounts of normal (closed line and open symbols) or TD LPDP (broken line and closed symbols, patient T1) were incubated for 1 h at 37°C. In (B) the tubes with immobilized rLpA-I were incubated for increasing time intervals with 50 µg LPDP protein of a normal proband (closed line and open symbols) or TD patient T1 (broken line and closed symbols). Similar results were obtained by experiments where vesicles contained 3H-PI or 14C-PE (not shown).

Similiar to total plasma, transfer of radiolabeled phospholipids mediated by lipoproteins from vesicles onto rLpA-I decreased during incubations that lasted longer than 1 h (not shown). Incubation with 50 µg LPDP resulted in maximal transfer of PC (Figure 4B) (and PI or PE, not shown) after 1 h long incubation. By contrast to incubations with plasma and lipoprotein fractions, this maximum persisted up to 16 h. Based on glycerophospholipid transfer activities measured in the presence of 1 µg VLDL, 1 µg IDL/LDL, and 1 µg HDL or 50 µg LPDP (all by protein) we estimate that in normal plasma about 54% of glycerophospholipid transfer activities reside in HDL, 25% in LPDP, 14% in IDL/LDL, and 7% in VLDL. With the exception of the IDL/LDL fraction, all plasma fractions of TD patient T1 contained lower glycerophospholipid transfer activities than the respective fractions of normal plasma (Table 4 and Figure 4). We estimated the following distribution of glycerophospholipid transfer activities: 24% in VLDL, 40% in IDL/LDL, 1% in the fraction which was isolated at the density of HDL, and 35% in LPDP.

We also measured the LPDP-mediated transfer of phospholipids onto lipid-free apoA-I, apoA-II, and albumin. Similar to the data shown for plasma in Figure 2, transfer of radiolabeled PC onto apoA-I was 3-fold higher than the transfer onto apoA-II and 6- to 7-fold higher than onto albumin.

Effect of LDL on the activity of normal fibroblasts to transfer radiolabeled PI, PC, and PE
We also monitored the activity of homogenized fibroblasts to transfer PC, PI, or PE onto rLpA-I. In initial experiments we analyzed the phospholipid transfer activity of homogenized fibroblasts that were cultivated in either the presence or absence of lipoproteins. Figure 5 shows the data on PE transfer as an example. Both cell homogenates (hatched lines in Figure 5A) and cell culture media (hatched lines in Figure 5B) exerted only moderate transfer activities that were close to the unspecific transfer activity measured at 4°C. After fibroblasts had been pre-incubated with medium containing either 10% FCS or 100 µg/ml LDL, both cell homogenates (solid lines in Figure 5A) and media (solid lines in Figure 5B) exerted time-dependent transfer activities. Maximal transfers of either ¹⁴C-PC, ³H-PI, or ¹⁴C-PE from phospholipid vesicles onto immobilized rLpA-I were reached by 60-min incubations (Figure 5). At this time point the transfer activities in either cell homogenates or media from LDL- or FCS-treated fibroblasts were 4- to 5-fold higher than in those of untreated fibroblasts.

View larger version (12K): [in this window] [in a new window]   Figure 5. Effect of LDL treatment on time-dependent transfers of [14H]phosphatidylethanolamine from vesicles onto reconstituted LpA-I by homogenates (A) and culture media of fibroblasts (B). Confluently grown fibroblasts of a normal proband were pre-incubated for 24 h either in lipoprotein-free medium (hatched line and closed symbols) or in the presence of 100 µg/ml LDL (closed line and open symbols). After extensive washing the cells were incubated for another 24 h in lipoprotein-free medium. Media were removed; cells were homogenized and solubilized. Aliquots with 20 µg cell protein (A) or 400 µl cell culture media (B) were incubated for increasing time intervals in streptavidin-coated tubes that contained 10 µg biotinylated rLpA-I and 20 µl of vesicle emulsion with 14C-PE. Similar results were obtained by experiments where vesicles contained 3H-PI or 14C-PC or where cells were pre-incubated for 24 h with 10% FCS instead of LDL (not shown).

Like plasma and LPDP, homogenates ( Figure 6A) and culture media (Figure 6B) of FCS-treated fibroblasts mediated time-dependent transfer of PC (and PE or PI) onto lipid-free apoA-I and, to a lesser degree, onto apoA-II. Again the transfer of phospholipids onto albumin was very low.

View larger version (23K): [in this window] [in a new window]   Figure 6. Transfer of [14C]phosphatidylcholine from vesicles onto lipid-free apoA-I, apoA-II, and albumin by homogenates (A) and culture media of fibroblasts (B). Ten µg biotinylated lipid-free apoA-I, 10 µg biotinylated lipid-free apoA-II or 10 µg biotinylated albumin were immobilized in streptavidin-coated tubes. These were filled with 20 µl of a PC vesicle emulsion (7 mg/ml PC and 2.5 µCi/ml 14C-PC) and with either 20 µg homogenized cell protein (A) or 400 µl medium (B) of fibroblasts that were cultivated in 10% FCS. Incubations were performed for increasing time intervals. The curves represent the transfer of 14C-PC from the donor vesicle onto the immobilized proteins.

Phospholipid transfer activities of homogenates and media from TD fibroblasts
Figure 7 compares dose- and time-dependent PI transfers by homogenates from fibroblasts of normal donors and TD patients that were pre-incubated with LDL. Transfers of PI (Figure 7A), PE, and PC (not shown) by cell homogenates increased dose-dependently until a dosage of 20 µg cell protein. Higher dosages decreased the amount of transferred ¹⁴C-PC, ³H-PI, or ¹⁴C-PE. At any amount of cell protein, TD cells were less active than control cells in transferring ¹⁴C-PC, ³H-PI, or ¹⁴C-PE from donor vesicles onto biotinylated rLpA-I (Figure 7A). The time-dependent transfer of PI (Figure 7B), PE, and PC (not shown) reached its maximum after 1 h. Longer incubations did not cause a decrease in radiolabel transfer. TD cells reached only about 50% of the maximum in transfer that was reached by normal fibroblasts, even after prolonged incubation (Figure 7B). Table 5 summarizes the data on transfers of radiolabeled PC, PI, and PE from donor vesicles onto rLpA-I by homogenates of LDL-loaded fibroblasts from eight healthy and normolipidemic donors and from four TD patients. Glycerophospholipid transfer activities in all four TD fibroblast cell lines were reduced by at least three standard deviations compared to the mean values of phospholipid transfer activities in normal cells. Compared to normal cells, the mean transfer activities of TD cells were reduced by 35%, 40%, and 45% for ³H-PI, ¹⁴C-PE, and ¹⁴C-PC, respectively (Table 5). As fibroblasts of TD patients and normal donors that were cultivated in lipoprotein-free medium did not differ in their residual phospholipid transfer activities (not shown), we subtracted these basal transfer activities from those of cholesterol-enriched cells. After this adjustment, the differences in mean transfer activities of normal and TD fibroblasts became even greater: Mean ³H-PI transfer in TD cells was reduced by 42% and mean transfers of ¹⁴C-PE and ³H-PC by 47%. When the assays were carried out with lipid-free apoA-I or apoA-II, the transfer activity of TD cells was 45% lower than that of normal cells. The very low transfer onto albumin did not differ among normal and TD cells.

View larger version (12K): [in this window] [in a new window]   Figure 7. Dose- (A) and time-dependent transfers (B) of [3H]phosphatidylinositol from vesicles onto reconstituted LpA-I by fibroblasts of normal donors and TD patients. Confluently growing fibroblasts of a normal proband (open symbols) and from a TD patient (crosses) were incubated for 24 h in the presence of 100 µg/ml LDL. After extensive washing the cells were incubated for another 24 h in lipoprotein-free medium. The cells were then removed, homogenized, and solubilized. In (A) increasing dosages of cell protein were incubated for 1 h in streptavidin-coated tubes that contained 10 µg biotinylated rLpA-I and 20 µl of vesicle emulsion with 3H-PI. In (B) aliquots with 20 µg cell protein were incubated for increasing time intervals.

Compared to media of normal cells, culture media of LDL-loaded TD fibroblasts were also significantly reduced in their activity to transfer glycerophospholipids onto rLpA-I ( Table 6), namely by 32% (³H-PI), 33% (¹⁴C-PE), and 30% (³H-PC). After correction for the basal transfer activities encountered in media of unloaded cells, which again did not differ between TD and normal fibroblasts, the differences increased to 49% (³H-PI), 51% (¹⁴C-PE), and 43% (³H-PC). The transfer of PC onto lipid-free apoA-I or apoA-II was about 50% lower in the presence of media from TD fibroblasts as compared to media of normal cells. The transfer onto albumin was close to background in the presence of either medium.

	Discussion

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Discussion REFERENCES

In this study we have demonstrated that normolipidemic plasma as well as cell homogenates and culture media of fibroblasts from normal volunteers transfer radiolabeled PI, PE, or PC in a dose- and time-dependent manner from donor vesicles onto rLpA-I, lipid-free apoA-I and, although to a much lesser degree, lipid-free apoA-II. Plasmas of TD patients as well as cell homogenates and culture media of TD fibroblasts had significantly lower glycerophospholipid transfer activities than the respective specimens from normolipidemic probands. The similar kinetics for phospholipid transfer in plasmas and cells (Figure 1 and Figure 4), in LPDP and cell culture media (Figure 3 and Figure 4) suggest that glycerophospholipid transfers are exerted by the same mechanism. This assumption is further supported by the fact that phospholipid transfer activities were decreased by a similiar extent in all four specimens when they were obtained from TD patients.

Several phenomena indicate that glycerophospholipid transfer occurred by a directed rather than a diffusional process. First, the fractional transfer onto rLpA-I was higher than the transfer onto lipid-free apoA-I and even more so than onto apoA-II or albumin. Thus, the assay monitors a specific transfer onto apolipoproteins rather than nonspecific transfer to the tube. Second, significant phospholipid transfer was observed at 37°C but not at 4°C where most enzymes are inactive (Figure 3B and Figure 3D). Third, in all specimens, phospholipid transfer occurred dependent on time and dosage. Fourth, phospholipid transfer activity in plasma was not dispersed throughout all lipoprotein subfractions but was found predominantly in HDL and LPDP. Fifth and finally, in cell homogenates and culture media significant transfer activities were only assessed after fibroblasts were pre-incubated with FCS or LDL, indicating that enrichment of cells with cholesterol induces the transfer activity (Figure 5).

Several intracellular and extracellular phospholipid transfer proteins have been identified (39) (40) (41) (42). As most of them translocate phospholipids from lipid-rich donor membranes or vesicles to lipid-poor acceptor membranes or particles, it is likely that several of them nonselectively contribute to the phospholipid transfer from vesicles onto rLpA-I or even lipid-free apoA-I, which was measured by our assay. Possibly they account for the about 50% residual phospholipid transfer activity encountered in plasmas and fibroblasts of TD patients. However, the reduced phospholipid transfer activity of TD plasmas as well as of cell homogenates and culture media of TD fibroblasts strongly suggests that a considerable proportion of the phospholipid transfer activity in our assay is exerted by a specific factor that is impaired in TD. In the plasma compartment, this specific proportion of glycerophospholipid transfer activity appears to be mediated independently of CETP and PLTP, as both activities were normal in TD plasma (Table 1). Moreover, in normal plasma, glycerophospholipid transfer activities did not correlate with CETP activity and even negatively with PLTP activity (Table 3). Finally, as apoA-I-deficient plasma had significantly higher phospholipid transfer activity than TD plasma, it is unlikely that the low phospholipid transfer activity in TD plasma is secondary to HDL deficiency.

Phospholipid transfer onto apoA-I was saturated at relatively low amounts of plasma or cell protein. Incubation with more than 2 µl plasma or 20 µg lysed cells even caused a decay of radioactivity from the immobilized acceptor particle. Several reasons may account for this phenomenon. First, plasmas and cells may contain a factor that inhibits the transfer of glycerophospholipids onto apoA-I. By mixing TD plasmas and normal plasmas, we have at least ruled out that such an inhibitor is responsible for defective phospholipid transfer in TD. Second, plasma lipoproteins or cell membranes may have served as secondary acceptors of radiolabeled phospholipids and have removed phospholipids from rLpA-I. Actually, small amounts of isolated lipoproteins (e.g., 10 µg VLDL) led to a more effective transfer of radiolabeled glycerophospholipids from donor vesicles than larger amounts (e.g., 100 µg VLDL) (Table 4). Third, endogenous phospholipids of either plasma or cells may compete with exogenous radiolabeled phospholipids for transfer. In agreement with this explanation, radioactivity did not disappear from the acceptor particles during prolonged incubation with either LPDP (Figure 2B), cell homogenates (Figure 3A and Figure 4B), or cell culture media (Figure 3B) when the amount of possible competitors did not increase. The disappearance of labeled phospholipids from the immobilized acceptor in the presence of plasma (Figure 1B and Figure 1D) may also be caused by the presence of excess apoA-I and several lipid transfer enzymes in plasma. In this case, immobilized rLpA-I will probably acquire not only glycerophospholipids but also additional apoA-I molecules that have a high affinity to phospholipids. Thus the de novo formed particles may be further processed by lipid transfer enzymes and thereby sequester radiolabeled phospholipids. It was previously shown that interconversions of mature HDL by phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) are associated with the release of apoA-I or preß₁-LpA-I (43) (44) (45) (46) (47) (48).

Is impaired transfer of glycerophospholipids onto apoA-I by cells and plasmas of TD patients consistent with previously described disturbances in Tangier disease? Unlike normal cells, TD fibroblasts do not release cholesterol and phospholipids in the presence of lipid-free apolipoproteins (6) (20) (21). Oram and colleagues have suggested that this defect interferes with the maturation of HDL (6) (17). We have previously reported that TD plasma is defective in converting lipid-free apoA-I or lipid-poor preß₁-LpA-I into lipid-rich -LpA-I (19). This conversion involves not only esterification of cholesterol by LCAT but also enrichment with phospholipids (4) (20) (21) (22) (23) (49) (50). In view of these cellular and plasmatic disturbances in TD we conclude from the data of this study that phospholipids of either cell membranes or plasma lipoprotein surfaces are transferred onto apoA-I by a transfer protein whose function is lost in TD. The mediation of phospholipid transfer by an identical protein in both intracellular and extracellular defect is not an unprecedented phenomenon. For example, the macrophage surface antigen CD14 exists as a soluble plasma isoform that assists lipopolysaccharide binding protein in the transfer of endotoxins and phospholipids (40) (51) (52). We hypothesize that defective phospholipid transfer interferes with maturation of HDL as well as efflux of cellular cholesterol and thereby causes deficiency of HDL cholesterol and foam cell formation, respectively. Abnormal plasma membrane phospholipid content observed in TD erythrocytes (53) as well as abnormalities in agonist-induced phospholipid breakdown by phospholipases (29) (54) observed in TD fibroblasts may occur secondary to an inadequate supply of phospholipid.

	ACKNOWLEDGMENTS

This project was supported by grants from Deutsche Forschungsgemeinschaft (Ec 116,2-2; Ec 116,3-2) to Dr. von Eckardstein and by a fellowship of Deutsche Infarktforschungshilfe to Dr. Lüttmann. We gratefully acknowledge the excellent technical assistance of Martina Plüster and Alois Roetrige. We are indebted to the patients for their participation in this study.

Manuscript received November 5, 1997; and in revised form December 11, 1997.

Abbreviations: apoA-I, apolipoprotein A-I; CETP, cholesteryl ester transfer protein; HDL, high density lipoprotein; LDL, low density lipoprotein; LpA-I, apoA-I-containing lipoprotein; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PLTP, phospholipid transfer protein; preß-LpA-I, prebeta-LpA-I; rLpA-I, reconstituted LpA-I; TD, Tangier disease; VLDL, very low density lipoprotein

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