High-quality RNA from cells isolated by laser capture microdissection.

Laser capture microdissection (LCM) provides a rapid and simple method for procuring homogeneous populations of cells. However, reproducible isolation of intact RNAfrom these cells can be problematic; the sample may deteriorate before or during sectioning, RNA may degrade during slide staining and LCM, and inadequate extraction and isolation methods may lead to poor recovery. Our report describes an optimized protocol for preparation of frozen sections for LCM using the HistoGene Frozen Section Staining Kit. This slide preparation method is combined with the PicoPure RNA Isolation Kitfor extraction and isolation of RNA from low numbers of microdissected cells. The procedure is easy to perform, rapid, and reproducible. Our results show that the RNA isolated from the LCM samples prepared according to our protocol is of high quality. The RNA maintains its integrity as shown by RT-PCR detection of genes of different abundance levels and by electrophoretic analysis of ribosomal RNA. RNA obtained by this method has also been used to synthesize probes for interrogating cDNA microarray analyses to study expression levels of thousands of genes from LCM samples.

Prebeta-1 HDL in plasma of normolipidemic individuals: influences of plasma lipoproteins, age, and gender.

Prebeta-1 HDL is a molecular species of plasma HDL of approximately 67 kDa mass that contains apolipoprotein A-I, phospholipids, and unesterified cholesterol. It participates in a cyclic process involved in the retrieval of cholesterol from peripheral tissues. In this cycle, unesterified cholesterol from cells is incorporated into prebeta-1 HDL, providing a substrate for esterification of cholesterol by lecithin:cholesterol acyltransferase. Prebeta-1 HDL then becomes incorporated into larger HDL species of alpha mobility as esterification proceeds and is regenerated during the transfer of cholesteryl esters from alpha HDL particles to acceptor lipoproteins. Thus the steady state level of prebeta-1 HDL in plasma reflects the relative efficiencies of the major metabolic processes involved in its generation and removal. We have used an isotope dilution technique to measure prebeta-1 HDL levels in the plasmas of 136 normolipidemic individuals (46 M, 90 F). The mean absolute concentration of prebeta-1 HDL as apolipoprotein A-I was 68 +/- 40 microg/ml for women, and 84 +/- 49 m/ml for men. Prebeta-1 HDL represented 5.5 +/- 3.3% of total apolipoprotein A-I in women, and 7.2 +/- 4.0% in men. The distributions of both absolute and percent prebeta-1 HDL are highly asymmetric, with skew toward higher values. However, the skew appears not to be attributable to either plasma cholesterol or triglyceride levels which are also skewed in population samples. The percent prebeta-1 HDL was negatively correlated with HDL cholesterol levels (P < 0.0001), whereas absolute levels of prebeta-1 HDL were positively correlated with apolipoprotein A-I and negatively correlated with HDL cholesterol (P, for both, < 0.0001). Multiple linear regression analysis revealed effects of age and gender, but no association with lipoprotein fractions other than HDL. Lower levels of prebeta-1 HDL were associated with female gender in all models.

Apolipoprotein L, a new human high density lipoprotein apolipoprotein expressed by the pancreas. Identification, cloning, characterization, and plasma distribution of apolipoprotein L.

In this study, we have identified and characterized a new protein present in human high density lipoprotein that we have designated apolipoprotein L. Using a combination of liquid-phase isoelectrophoresis and high resolution two-dimensional gel electrophoresis, apolipoprotein L was identified and partially sequenced from immunoisolated high density lipoprotein (Lp(A-I)). Expression was only detected in the pancreas. The cDNA sequence encoding the full-length protein was cloned using reverse transcription-polymerase chain reaction. The deduced amino acid sequence contains 383 residues, including a typical signal peptide of 12 amino acids. No significant homology was found with known sequences. The plasma protein is a single chain polypeptide with an apparent molecular mass of 42 kDa. Antibodies raised against this protein detected a truncated form with a molecular mass of 39 kDa. Both forms were predominantly associated with immunoaffinity-isolated apoA-I-containing lipoproteins and detected mainly in the density range 1.123 < d < 1.21 g/ml. Free apoL was not detected in plasma. Anti-apoL immunoaffinity chromatography was used to purify apoL-containing lipoproteins (Lp(L)) directly from plasma. Nondenaturing gel electrophoresis of Lp(L) showed two major molecular species with apparent diameters of 12.2-17 and 10.4-12.2 nm. Moreover, Lp(L) exhibited both pre-beta and alpha electromobility. Apolipoproteins A-I, A-II, A-IV, and C-III were also detected in the apoL-containing lipoprotein particles.

Isolation of subpopulations of high density lipoproteins: three particle species containing apoE and two species devoid of apoE that have affinity for heparin.

We have isolated and partially characterized five populations of lipoproteins from the pool of immunoisolated apoA-I-containing lipoproteins obtained from normal human plasma. The first three populations, each containing apoA-I and apoE, were isolated completely by sequential, selected affinity immunosorption against apoA-I and apoE. The lipoproteins isolated by this strategy fall into three morphologic groups; there are discs (LP-AI-E(1)), small spherical lipoproteins (LP-AI-E(2)), and large spherical lipoproteins (LP-AI-E(3)). The LP-AI-E(2) species was sufficiently abundant for detailed characterization. They have slightly larger diameters, and contain more lipid than the bulk of apoA-I-containing lipoproteins and they contain apoA-II:E heterodimers and apoE homodimers. Core lipids are enriched in triglyceride relative to cholesteryl esters. These lipoproteins compete with LDL equally, on a protein mass basis, for binding to human fibroblasts. After removal of apoE-containing lipoproteins from the pool of apoA-I-containing lipoproteins, we discovered two additional subpopulations of lipoproteins that bind to heparin. These lipoproteins, devoid of apoE, occur as populations of small, (LP-AI-HB(1)), and large, spherical lipoproteins, (LP-AI-HB(2). The heparin-binding lipoproteins were separated by gel permeation chromatography. The LP-AI-HB(1) population was of sufficient quantity for detailed study. These lipoproteins also had larger diameters than the bulk of HDL but their core lipids were enriched in cholesteryl esters rather than triglycerides. Three proteins associated with these lipoproteins were found to bind to heparin-Sepharose in the absence of lipid. The approximate molecular weights of these proteins are 40, 70, and 90 kDa. The 70 kDa molecule was found to be the SP 40,40 protein (apoJ).

Measurement of prebeta-1 HDL in human plasma by an ultrafiltration-isotope dilution technique.

Prebeta-1 HDL is a 67-kDa species of plasma high-density lipoproteins (HDL) that contains two copies of apolipoprotein A-I. It functions in a metabolic cycle of cholesterol retrieval and may be formed during lipolysis in plasma. We have found that centrifugal ultrafiltration using a membrane with a permeability limit of 100 kDa discriminates categorically between the 67-kDa species and larger HDL particle species. Thus, the ultrafiltrate samples the pool of prebeta-1 HDL in plasma. We have developed a technique using the dispersal of purified prebeta-1 HDL, labeled covalently with tritium, in plasma samples, to label the prebeta-1 HDL pool. Subsequent determination of the specific activity of prebeta-1 HDL in the ultrafiltrate provides a means of calculating the content of prebeta-1 HDL in plasma by the isotope dilution principle. We employ a modification of an enzyme-linked immunosorbent assay technique for apolipoprotein A-I that allows the equal detection of that protein in prebeta-1 HDL and in other HDL particle species for determination of the fraction of total apolipoprotein A-I that is present in the prebeta-1 HDL particle species. The mean level of prebeta-1 HDL-associated apolipoprotein A-I in plasma samples from 86 normolipidemic adults was 74 +/- 43 microg/ml (+/-SD), representing an average of 6.6% of the total apolipoprotein A-I in plasma.

Enumeration of CD34+ hematopoietic progenitor cells in peripheral blood and leukapheresis products by microvolume fluorimetry: a comparison with flow cytometry.

There is increasing interest in both standardization and simplification of methods for enumeration of CD34+ hematopoietic progenitor cells (HPC) to facilitate cellular therapies and to improve interinstitutional comparison of clinical and laboratory results. We evaluated a novel method for CD34+ cell enumeration based on microvolume fluorimetry (MVF) compared with our laboratory's routine flow cytometric method on samples of peripheral blood and leukapheresis products. The MVF method is semiautomated and uses a 633-nm light from a helium-neon laser to scan fluorochrome-labeled cells held in stasis in a capillary known volume. The performance of the MVF assay for enumeration of CD34+ cells was found to be comparable to our routine flow cytometric assay in linearity and accuracy in the range of 5-1500 cells per microliter. Precision of MVF for replicate assays on the same instrument was demonstrated by coefficient of variation (CV) values of 8.4% at a CD34+ cell concentration of 284/microliters for a sample volume of 0.8 microliters, and 15.7% at 12/microliters for a sample volume of 3.2 microliter. Precision among three different instruments was demonstrated, using sample volumes of 1.6 microliters, by CV values of 44% at 6 cells/microliters and 4.6% at 733 cells/microliters. In a field sample evaluation, precision of the entire assay system for paired measurements on 0.8-microliter sample volumes was demonstrated by CV values of 50%, 31%, and 15% for peripheral blood samples with concentrations of 0-10, 10-20, and 20-100 CD34+ cells/microliters, respectively, and 6.3%, 8.1% and 6.5% for leukapheresis samples with concentrations of 0-100, 100-1,000, and 1,000-2,500 CD34+ cells/microliters, respectively. The MVF assay was easy to perform, required minimal technical training time, and had a turnaround time of 40 min, of which less than 10 min was actual technical time. These observations suggest that the MVF method for CD34+ cell enumeration may prove useful to clinical laboratories providing support for HPC collection, processing, and transplantation services that require relatively simple, rapid assays for product quality control or to guide real-time clinical decisions.

Triglyceride-rich lipoproteins isolated by selected-affinity anti-apolipoprotein B immunosorption from human atherosclerotic plaque.

We isolated and characterized immunoreactive apolipoprotein B (apoB)-containing lipoproteins from human atherosclerotic plaque and plasma to determine whether very-low-density lipoprotein (VLDL) can enter and become incorporated into the atherosclerotic lesion and how plaque apoB-containing lipoproteins differ from apoB-containing lipoproteins isolated from plasma. Atherosclerotic plaques were obtained during aortic surgery and processed immediately. Lipoproteins were extracted from minced plaque in a buffered saline solution (extract A). In selected cases a second extraction was done after plaque was incubated with collagenase (extract B). Lipoproteins were then isolated from the extracts by anti-apoB immunosorption and separated into VLDL + intermediate-density lipoprotein (IDL) (d < 1.019 g/mL) and low-density lipoprotein (LDL) (1.019 < d < 1.070 g/mL) fractions by ultracentrifugation. The VLDL + IDL fractions from plaque contained more than one third of the total apoB-associated lipoprotein cholesterol in both extracts A and B. The lipid composition of VLDL + IDL in both extracts was related to that of plasma VLDL + IDL. By electron microscopy mean particle diameters of VLDL + IDL from extracts A and B were 9% and 23%, respectively, greater than VLDL + IDL diameters from plasma. Mean diameters of LDL from extracts A and B were 11% and 31% greater than LDL diameters from plasma. The apoE-apoB ratio of extract A VLDL + IDL was nearly twice that of plasma VLDL + IDL and severalfold higher than that of extract A LDL. Immunoblots of both VLDL + IDL and LDL from extract A demonstrated minimal fragmentation of apoB.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipopolysaccharide (LPS)-binding protein is carried on lipoproteins and acts as a cofactor in the neutralization of LPS.

Lipoproteins isolated from normal human plasma can bind and neutralize bacterial lipopolysaccharide (LPS) and may represent an important mechanism in host defense against gram-negative septic shock. Recent studies have shown that experimentally elevating the levels of circulating high-density lipoproteins (HDL) provides protection against death in animal models of endotoxic shock. We sought to define the components of HDL that are required for neutralization of LPS. To accomplish this we have studied the functional neutralization of LPS by native and reconstituted HDL using a rapid assay that measures the CD14-dependent activation of leukocyte integrins on human neutrophils. We report here that reconstituted HDL particles (R-HDL), prepared from purified apolipoprotein A-I (apoA-I) combined with phospholipid and free cholesterol, are not sufficient to neutralize the biologic activity of LPS. However, addition of recombinant LPS binding protein (LBP), a protein known to transfer LPS to CD14 and enhance responses of cells to LPS, enabled prompt binding and neutralization of LPS by R-HDL. Thus, LBP appears capable of transferring LPS not only to CD14 but also to lipoprotein particles. In contrast with R-HDL, apoA-I containing lipoproteins (LpA-I) isolated from plasma by selected affinity immunosorption (SAIS) on an anti-apoA-I column, neutralized LPS without addition of exogenous LBP. Several lines of evidence demonstrated that LBP is a constituent of LpA-I in plasma. Passage of plasma over an anti-apoA-I column removed more than 99% of the LBP detectable by ELISA, whereas 31% of the LBP was recovered by elution of the column. Similarly, the ability of plasma to enable activation of neutrophils by LPS (LBP/Septin activity) was depleted and recovered by the same process. Furthermore, an immobilized anti-LBP monoclonal antibody coprecipitated apoA-I. The results described here suggest that in addition to its ability to transfer LPS to CD14, LBP may also transfer LPS to lipoproteins. Since LBP appears to be physically associated with lipoproteins in plasma, it is positioned to play an important role in the neutralization of LPS.

Endotoxin and cytokines increase hepatic messenger RNA levels and serum concentrations of apolipoprotein J (clusterin) in Syrian hamsters.

Infection and inflammation induce alterations in hepatic synthesis and plasma concentrations of the acute phase proteins. Our results show that apolipoprotein (apo) J is a positive acute phase protein. Endotoxin (LPS), tumor necrosis factor (TNF), and interleukin (IL)-1 increased hepatic mRNA and serum protein levels of apo J in Syrian hamsters. Hepatic apo J mRNA levels increased 10- to 15-fold with doses of LPS from 0.1 to 100 micrograms/100 g body weight within 4 h and were elevated for > or = 24 h. Serum apo J concentrations were significantly increased by 16 h and further elevated to 3.3 times that of control, 24 h after LPS administration. Serum apo J was associated with high density lipoprotein and increased fivefold in this fraction, after LPS administration. Hepatic apo J mRNA levels increased 3.5- and 4.6-fold, with TNF and IL-1, respectively, and 8.2-fold with a combination of TNF and IL-1. Serum apo J concentrations were increased 2.3-fold by TNF, 79% by IL-1, and 2.9-fold with a combination of TNF and IL-1. These results demonstrate that apo J is a positive acute phase protein.

Identification of proteins associated with apolipoprotein A-I-containing lipoproteins purified by selected-affinity immunosorption.

The isolation of apolipoprotein A-I-containing lipoproteins [Lp(A-I)] by selected-affinity immunosorption minimizes the loss of associated proteins that occurs during the isolation of high-density lipoproteins (HDL) by sequential ultracentrifugation. We have used two-dimensional gel electrophoretic analysis to separate the proteins associated with Lp(A-I). Using a combination of amino acid sequencing of transblotted proteins and Western blotting with specific antisera, we have identified a number of associated proteins. The positions of the apolipoproteins (apo) A-I, A-II, A-IV, C-III, D, and E were located on the gels. Lecithin-cholesterol acyltransferase and cholesteryl ester transfer protein were identified in association with Lp(A-I) to a greater extent than found associated with HDL after centrifugation. In addition to those proteins previously identified in association with HDL, we detected a number of plasma proteins associated with Lp(A-I), namely, fibrinogen, haptoglobin, proline-rich protein (C4b-binding protein), and apolipoprotein J (SP40,40 sulfated glycoprotein). The co-isolation of these proteins with Lp(A-I) does not appear to be an artifact in that they have very low affinity for a sham column containing covalently bound preimmune goat IgG in place of the anti-apoA-I IgG. These findings suggest that in addition to apolipoproteins that exist largely in association with lipoproteins there is another class of proteins which exist in lipoprotein-associated form and in the dispersed state. Detection and identification of these lipoprotein-associated proteins may aid in the mechanistic determination of a number of observed functions attributed to HDL.

Apolipoprotein A-I-containing lipoproteins, with or without apolipoprotein A-II, as progenitors of pre-beta high-density lipoprotein particles.

Apolipoprotein A-I-(apoA-I-) containing lipoproteins isolated by immunoaffinity chromatography can be divided into two general subfractions on the basis of the presence [Lp(AI + AII)] or absence [Lp(AI - AII)] of apoA-II. The Lp(AI - AII) subfraction can be further subfractionated into two subgroups with pre-beta mobility as well as those of alpha mobility. We have characterized the Lp(AI - AII) and Lp(AI + AII) subfractions after the removal of pre-beta high-density lipoproteins (pre-beta-HDL) to compare only the two subfractions with alpha mobility. The Lp(AI - AII) and Lp(AI + AII) of alpha mobility, while both heterogeneous subfractions, share many gross features in common. Both subfractions were predominantly spherical in shape, had similar conformation of apoA-I as investigated by circular dichroism and specific endoproteases, and had similar contents of phospholipids, phospholipid species, triglycerides, and cholesterol ester. However, there was significantly less protein (-10%) and more free cholesterol (+46%) in the Lp(AI - AII) subfraction than in the Lp(AI + AII) subfraction. We investigated the generation of pre-beta-HDL from both the Lp(AI - AII) and Lp(AI + AII) subfractions during incubation with low-density lipoproteins and cholesteryl ester transfer protein. We found that both Lp(AI - AII) and Lp(AI + AII) subfractions were capable of generating pre-beta-HDL-like particles. Our results suggest that the formation of pre-beta-HDL involves dissociation of apoA-I from both Lp(AI - AII) and Lp(AI + AII) subfractions. These results refine a model describing the cycling of apoA-I between pre-beta-HDL and alpha-HDL linked to the movement of cholesteryl esters through HDL.

Interconversion between apolipoprotein A-I-containing lipoproteins of pre-beta and alpha electrophoretic mobilities.

Apolipoprotein (apo) A-I-containing lipoproteins can be separated into two subfractions, pre-beta HDL and alpha HDL (high density lipoproteins), based on differences in their electrophoretic mobility. In this report we present results indicating that these two subfractions are metabolically linked. When plasma was incubated for 2 h at 37 degrees C, apoA-I mass with pre-beta electrophoretic mobility disappeared. This shift in apoA-I mass to alpha electrophoretic mobility was blocked by the addition of either 1.4 mM DTNB or 10 mM menthol to the plasma prior to incubation, suggesting that lecithin:cholesterol acyltransferase (LCAT) activity was involved. There was no change in the electrophoretic mobility of either pre-beta HDL or alpha HDL when they were incubated with cholesterol-loaded fibroblasts. However, after exposure to the fibroblasts, the cholesterol content of the pre-beta HDL did increase approximately sixfold, suggesting that pre-beta HDL can associate with appreciable amounts of cellular cholesterol. Pre-beta HDL-like particles appear to be generated by the incubation of alpha HDL with cholesteryl ester transfer protein (CETP) and either very low density lipoproteins (VLDL) or low density lipoproteins (LDL). This generation of pre-beta HDL-like particles was documented both by immunoelectrophoresis and by molecular sieve chromatography. Based on these findings, we propose a cyclical model in which 1) apoA-I mass moves from pre-beta HDL to alpha HDL in connection with the action of LCAT and the generation of cholesteryl esters within the HDL, and 2) apoA-I moves from alpha HDL to pre-beta HDL in connection with the action of CETP and the movement of cholesteryl esters out of the HDL. Additionally, we propose that the relative plasma concentrations of pre-beta HDL and alpha HDL reflect the movement of cholesteryl esters through the HDL. Conditions that result in the accumulation of HDL cholesteryl esters will be associated with low concentrations of pre-beta HDL, whereas conditions that result in the depletion of HDL cholesteryl esters will be associated with elevated concentrations of pre-beta HDL. This postulate is consistent with published findings in patients with hypertriglyceridemia and LCAT deficiency.

Binding of transition metals by apolipoprotein A-I-containing plasma lipoproteins: inhibition of oxidation of low density lipoproteins.

We have found transition metals tightly bound to apolipoprotein A-I-containing lipoproteins [Lp(A-I)] isolated by selected affinity immunosorption from human serum. Prominent among the metal ions detected were iron and copper. By immunoblotting the proteins of Lp(A-I), we detected both transferrin and ceruloplasmin. The transferrin-containing Lp(A-I) particles, isolated by selected affinity immunosorption against transferrin, were larger (mean diameter of 14.2 nm) and had a higher protein content than most high density lipoproteins (HDL). Ultracentrifugally isolated HDL were found to contain much less transferrin, whereas transferrin was found associated with apolipoprotein A-I from the greater than 1.21-g/ml ultracentrifugal fraction. This suggests that the complex is not recovered in the classic HDL density interval because of its very high density. HDL inhibit copper-catalyzed oxidation of low density lipoproteins (LDL) in vitro. We have found that immunoisolated Lp(A-I) are an order of magnitude more effective in inhibiting the oxidation of LDL than ultracentrifugally isolated HDL, on the basis of protein mass. When the Lp(A-I) particles containing transferrin and ceruloplasmin were removed from the bulk of Lp(A-I), inhibition of the in vitro oxidation of LDL was significantly decreased.

Conformation of apolipoprotein B-100 in the low density lipoproteins of tangier disease. Identification of localized conformational response to triglyceride content.

The low density lipoproteins (LDL) from patients with Tangier disease are enriched in triglycerides, 27% of LDL mass versus 7% for normal LDL. To study whether this unique LDL core lipid composition affects the surface disposition of apolipoprotein (apo) B-100, we analyzed the LDL by protease digestion and in competitive radioimmunoassays. Limited proteolytic digestion of Tangier LDL by Staphylococcus aureus V8 protease generated a prominent fragment of 120 kDa (cleavage site at residue 1076), which was not visible in similarly digested normal LDL. In competitive radioimmunoassay, Tangier LDL bound weakly to the apoB-specific monoclonal antibody MB20, compared with control LDL. We localized the MB20 epitope between residues 1031 and 1084 of apoB-100, probably very near residue 1076. DNA sequencing of exon 21 of apoB genomic clones (coding for residues 1014-1084) from a Tangier patient revealed no difference from the normal DNA sequence, thus eliminating a protein polymorphism as a basis for the altered protease sensitivity and antibody binding. When the triglyceride contents of Tangier LDL were reduced to 10% of mass by incubation with normal high density lipoproteins, production of the 120-kDa fragment by proteolysis decreased and MB20 binding increased in affinity, implying a change toward normal conformation of apoB-100. Thus, using two independent techniques, proteolytic digestion and binding of monoclonal antibodies, we have demonstrated an alternative conformation of apoB-100 in the vicinity of residue 1076, which reflects the content of triglycerides in the LDL particle.

Pre-beta high density lipoprotein. Unique disposition of apolipoprotein A-I increases susceptibility to proteolysis.

Apolipoprotein A-I-containing lipoproteins (high density lipoproteins, HDL) can be separated into two subfractions, which have pre-beta and alpha electrophoretic mobilities, respectively. These fractions differ in both composition and structure. Some preparations of pre-beta-migrating HDL, but not alpha-migrating HDL, were found to contain two polypeptides with Mr of approximately 26 and 14 kDa, which are scission products of apolipoprotein (apo) A-I. They are recognized by monospecific antibodies to apo A-I and have N-terminal sequences identical to those of mature apo A-I. This proteolytic scission of apo A-I occurs primarily after venipuncture. Immediate addition of protease inhibitors minimized the appearance of the fragments in plasma. To study the relative susceptibilities of pre-beta and alpha HDL to proteolysis, the lipoproteins were incubated in vitro with plasmin. The apo A-I in pre-beta HDL was extensively degraded, but that in alpha-migrating HDL was degraded to a much lesser extent, indicating that the appearance of apo A-I fragments in pre-beta HDL was due to enhanced sensitivity to proteolysis. To varying degrees, thrombin, kallikrein, elastase, arginine C endoprotease, and chymotrypsin also appear to cleave pre-beta HDL faster than alpha HDL. Most of the proteases generated a 12 to 14 kDa peptide fragment under conditions of limited cleavage. These results suggest that the conformational state of apo A-I in pre-beta-migrating HDL or its spatial relationship to lipids is significantly different from that of apo A-I in alpha-migrating HDL.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-surface binding sites for high density lipoproteins do not mediate efflux of cholesterol from human fibroblasts in tissue culture.

The present investigation was designed to test the hypothesis that binding sites for high density lipoproteins (HDL3) on cell surfaces of peripheral tissues mediate cholesterol efflux from these cells. This hypothesis had been formulated to explain two observations: 1) HDL3 binding to peripheral cells and HDL3-mediated cholesterol efflux from these cells had both been found to saturate at similar unbound (free) HDL3 concentrations; and 2) both of these processes had been found to be similarly "up-regulated" by loading the cells with cholesterol. In the present study, however, we found that the "specific" binding of HDL3 to cholesterol-loaded human fibroblasts was saturated at a free HDL3 concentration of approximately 20 micrograms protein/ml, whereas efflux of cholesterol from these cells to HDL3 did not "saturate" even at a free HDL3 concentration of 2000 micrograms protein/ml. In addition, we found that the increase in cholesterol efflux caused by loading the fibroblasts with cholesterol was no greater when the acceptor particles were HDL3 than when albumin or phospholipid vesicles served as acceptors, despite a marked increase in HDL3 binding to these cells. Because HDL3 binding to these cells and HDL3-mediated cholesterol efflux from these cells do not saturate at similar free HDL3 concentrations, and because the cholesterol-induced increase in HDL3 binding is not accompanied by a similar increase in cholesterol efflux that is specific for HDL3, we conclude that the described HDL3 binding sites on human fibroblasts do not mediate cholesterol efflux.

Radiation inactivation of binding sites for high-density lipoproteins in human liver membranes.

High-density lipoproteins (HDL) are involved in 'reverse cholesterol transport'. Whether or not cell-surface receptors for HDL exist and participate in this process remains controversial, and part of this controversy has centered on the nature of the HDL binding sites. We therefore used radiation inactivation to determine the molecular mass of the HDL binding sites in human liver membranes in situ. These binding sites, which shared all the characteristics of previously described putative HDL receptors, had a molecular mass of less than 10 kDa, indicating that they are probably not proteins. In addition, the binding of HDL to protein-free liposomes was characterized and was found to display the same affinity (KD = 5 micrograms protein/ml approximately 5.10(-8) M) as that to cell membranes, indicating that HDL binding to cell membranes may not require membrane proteins. These observations highlight an important application of radiation inactivation: the ability to demonstrate that something - in this case, a high-molecular-weight protein that accounts for the majority of the HDL binding activity in human liver membranes - is absent.

Radiation inactivation of binding sites for high density lipoproteins in human fibroblast membranes.

Radiation inactivation and target analysis were used to determine the molecular mass of the binding sites for high density lipoproteins (HDL) on membranes prepared from human fibroblasts. These membrane binding sites shared characteristics with the previously described HDL binding sites on whole fibroblasts in tissue culture. They exhibited the same affinity for HDL, the same ligand specificity, and the same sensitivity to proteolytic agents. They were also up-regulated by cholesterol loading of the cells. Kinetics of HDL dissociation from membrane binding sites could not be described by a single exponential function, indicating that HDL probably bind to multiple classes of sites on fibroblast membranes. After exposure to ionizing radiation, these sites decreased in number as an apparent single exponential function of radiation dose, corresponding to an average molecular mass of 16,000 +/- 1,000 Da, which is smaller than any known cell-surface receptor protein. These data indicate that HDL binding sites on fibroblast membranes are not "classical" receptors in that they are kinetically heterogeneous and small in molecular mass.

Mechanism of the heparin-induced increase in the concentration of free thyroxine in plasma.

The iv administration of heparin causes an increase in the plasma free T4 concentration, as determined by equilibrium dialysis. The mechanism and physiological consequences of this action of heparin are unknown. To explore the possibility that the heparin-induced increase in plasma free T4 is an in vitro artifact due to generation of FFA during equilibrium dialysis, we studied plasma samples from 10 subjects treated with iv heparin. In plasma from 4 of these subjects, free T4 concentrations measured by equilibrium dialysis did not increase above baseline values after heparin administration. In incubations performed in parallel with the equilibrium dialysis measurements, FFA concentrations in these plasma samples were found to increase, but in no subject did they exceed 2.5 meq/L after incubation. In contrast, in plasma from the other 6 subjects treated with heparin, free T4 concentrations rose markedly (by 130-520%) above baseline values after heparin administration. In all of these postheparin plasma samples, FFA concentrations were less than 2.8 meq/L before incubation, but rose during incubation by 80-270% to more than 3.8 meq/L. Treatment of these plasma samples with protamine to inhibit lipoprotein lipase and with specific antiserum to inhibit hepatic triglyceride lipase before equilibrium dialysis or incubation prevented, in parallel, the heparin-induced increases in FFA and free T4 concentrations. From these findings we conclude that the heparin-induced increase in free T4 is usually an in vitro artifact, and that most subjects receiving heparin have a normal plasma free T4 concentration in vivo. We also conclude that this in vitro artifact may account for many of the findings that led to the postulate of an inhibitor of T4 binding to plasma and intracellular proteins in heparin-treated patients and perhaps in patients with nonthyroid illness as well.