European Journal of Clinical Investigation (1991) 21,638-643
Lipoproteins of human peripheral lymph. Apolipoprotein Al-containing lipoprotein with alpha-2 electrophoretic mobility D. REICHL, C. B. HATHAWAY, J. M. STERCHI* & N. E. MILLER, Section on Endocrinology and Metabolism, Department of Medicine, and *Division of Surgical Sciences, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina, USA Received 15 November 1990 and in revised form 14 March 1991 Abstract. Evidence from diverse sources has implicated a central role of apolipoprotein A; (apo AI), the most abundant protein of plasma high-density lipoproteins, in the transport of cholesterol from peripheral tissues to the liver (reverse cholesterol transport). Particles containing only apo A1 appear to be more effective as cholesterol acceptors in tissue culture than do particles which also contain apo AIL The apo AIcontaining lipoproteins of plasma have been extensively studied, but there is less information on those in tissue fluids, to which most peripheral cells are exposed. In the present study the heterogeneity of apo AI-containing particles in human peripheral lymph, collected from the dorsum of the foot, has been examined by starch block electrophoresis, exclusion chromatography and immunoelectrophoresis. The apo AI-containing particles of lymph were found to be more variable in both electrophoretic mobility and size than those of plasma from the same subjects. Of particular interest was a subpopulation which migrated on electrophoresis with the same mobility as alpha-2-macroglobulin. This fraction accounted for approximately 7% (range: 4-12%; n = 5) of lymph apo AI, contained no immunodetectable apo AII, and by exclusion chromatography was composed of particles the size of, or smaller than, albumin. Such physicochemical properties suggest that these alpha-:! migrating particles may function as the principal primary acceptors of cell cholesterol in the extracellular matrix of human peripheral tissues. By isoelectric focusing, lymph apo A1 was found to contain a higher proportion of more negatively charged isoforms than the apo A1 of plasma.
Keywords. Apolipoproteins, cholesterol, electrophoresis, exclusion chromatography, high-density lipoprotein, lymph. Introduction Most extra-hepatic cells continuously acquire cholesterol as a result of endogenous synthesis and/or Correspondence: D. Reichl, PhD, Section on Endocrinology and Metabolism, Department of Medicine, Bowman Gray School of Medicine, 300 South Hawthorne Road, Winston-Salem, NC 27103, USA.
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endocytosis of lipoproteins, but most cannot metabolize it. Hepatocytes differ from peripheral cells in that they can oxidize cholesterol to bile acids, and can secrete it directly into bile. Hence, under steady-state conditions, cholesterol must continuously be released from extra-hepatic cells and transported to the liver for elimination, a process commonly referred to as reverse cholesterol transport [I ,2]. Studies in uitro have shown that sustained efflux of cholesterol mass from cells takes place only in the presence of acceptors [3], and that high-density lipoprotein (HDL) and complexes of HDL apolipoproteins (apos) with phospholipids are among the most effective acceptors [4]. In v i m only a small proportion of cells are in direct contact with blood. For these cells plasma lipoproteins are available both as acceptors and as transporting agents of cholesterol to the liver. Recent studies have shown the presence in plasma of small particles containing apo A1 (the main protein component of HDL) that appear to be particularly effective cholesterol acceptors in tissue culture [5]. Notable features of these particles are their pre-beta mobility in the electrophoretic field and an absence of immunodetectable apo A11 [5]. In contrast, plasma HDL subclasses that contain both apo A1 and apo A11 appear to have little or no effect on cholesterol efflux from cultured cells [6,7]. Most cells of the human body are separated from blood by vascular endothelium, and usually also by the matrix of the interstitium, and for these cells lipoproteins of interstitial fluids must act as acceptors and initial transporters of cholesterol. Although interstitial fluid cannot be sampled in sufficient quantities for laboratory analysis, peripheral lymph resembles it in many respects [8], and can be collected from humans under physiologic conditions. In previous studies we have shown that the apo AI-containing particles of human lymph cover a broader range of sizes than do those of plasma [9-1 I]. Since the sieving characteristics of the interstitium dictate that the volumes of distribution of particles in tissues are inversely related to their diameters [121, the smallest lipoproteins are likely to play a prominent role in the first stage of reverse cholesterol transport. We have now further characterized the apo AI-
HUMAN PERIPHERAL LYMPH LIPOPROTEINS containing lipoproteins of human lymph, and have identified a population whose size and composition make them particularly suited to the role of initial acceptors of tissue cholesterol in vivo. These particles differ from others in having alpha-2 mobility on starch block electrophoresis. Materials and methods Clinical procedures
The study was approved by the institutional review board. Lymph was collected from five apparently healthy non-fasted subjects, each of whom gave informed written consent: four men aged 58,72,28 and 28 years with respective plasma cholesterol concentrations of 5.7,4.5,5.2 and 4.2 mmol I-', and one woman aged 32 years with a plasma cholesterol of 3.9 mmol 1- '. Cannulation of a lymphatic collecting trunk on the dorsum of the foot was performed after subcutaneous injection of Patent Blue, as previously described [ 131. Vials into which lymph was collected contained 500 pg of NazEDTA and 500 pg of 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) to inhibit lecithin :cholesterol acyltransferase (LCAT). Collecting vials were changed for each 0.5 ml of lymph. Total volumes of 0.25-1.50 ml were collected over 3 h. They were stored on ice until used for analyses (within 2 h). Blood was collected from an antecubital vein into Naz EDTA (1 mg ml-I), and centrifuged at 4°C within 20 min. Plasma was stored on ice after addition of solid DTNB (0.8 mg ml-I). Laboratory procedures
Lipoproteins were separated and analysed by combinations of high performance liquid chromatography (HPLC), starch block electrophoresis, isoelectric focusing and immunoelectrophoresis. Lymph and plasma were fractionated by HPLC using a Superose 12 column (Prepacked HR 10/30; Pharmacia LKB, Uppsala, Sweden) and a flow rate of 0.3 ml min- I. The eluting buffer was 0.15 M NaCI-0.05 M phosphate (PBS), pH 7.4, containing 0.1% (w/v) Na2EDTA and NaN3. When plasma was fractionated it was first diluted with 4 vols of elution buffer. Fractions from starch gel electrophoresis (see below) were fractionated on the same column using identical conditions. The elution pattern was monitored by continuously measuring the OD280 of the effluent. The column was standardized by measuring the elution times of human low-density lipoprotein (LDL) of density 1.018-1.063 g ml-', prepared by preparative ultracentrifugation [ 141, and crystalline bovine serum albumin (Sigma, St. Louis, MO, USA). Additionally, the concentrations of apo B (the major apolipoprotein of LDL) and albumin were determined by immunoelectrophoresis in all eluted fractions. Lymph and plasma were fractionated by starch block electrophoresis essentially according to Kunitake et al. 1151. Hydrated potato starch was formed
639
into a 1 1 x 29 x 0.5 cm block. The block was divided longitudinally into equal parts, and a transverse trough was cut into each part 4 cm from the cathodic end. One of the troughs was filled with 0.4 ml of lymph, the other with the same volume of 1 in 5 diluted plasma from the same subject. A drop of 5% (w/v) bovine serum albumin in 0-15M NaCI, stained with brompheno1 blue, was applied between the troughs as a marker. A constant current of 36 mA was then applied for 18 h, after which the block was cut transversely into sections 1 cm wide, and the sections transferred into glass tubes. PBS (1 50 pl), to which a trace of Na'*'I (approx. lo4 cpm ml-') had been added, was pipetted into each tube, and the slurry vortexed. The radioactivity in each tube was measured in a gamma-counter. The tubes were then centrifuged at 2000 rpm for 10 min; the supernatants were transferred into fresh tubes; another 150 p1 of PBS was added; the vortexing and centrifugation were repeated; and the second supernatant was added to the first. The volumes of the extracts were estimated by weighing, assuming a density of 1.006 g ml- ', and the radioactivity of the combined extracts was measured. It was assumed that the distribution of radioactivity between extracts and remaining hydrated starch reflected the distribution of all solutes. Known volumes of extracts were taken for measurement of solute concentrations, and the remainder stored at 4°C until the analyses were completed (within 48 h). Relevant fractions from starch block electrophoresis were combined, concentrated by centrifugation in Centricon tubes (cut-off 10 000; Amicon, Danvers, MA, USA), and submitted to HPLC on Superose 12 as described above. When plasma was electrophoresed in parallel with the same plasma sample to which Patent Blue had been added (final concentration 0.01 %), there was no difference between the samples in the distribution of apo A1 or apo AII. It was also shown that dilution of plasma had no effect on apolipoprotein distributions after starch block electrophoresis or HPLC. Isoelectric focusing on polyacrylamide gel containing 3 M urea and Pharmalite of pH range 5-6 (Pharmacia) was performed according to the Pharmacia specifications. Lymph and plasma were delipidated with ethano1:diethyl ether 2: 1 (v/v), and again with diethyl ether. The dry solids were re-dissolved in 0.1 M barbitone buffer, pH 8.6 (lymph solids in the original volume; plasma solids in five times the original volume). Five p1 of each were used for analysis. A 1 O/o (w/v) solution of Congo red in deionized water was used as marker. On completion of the focusing, relevant lanes were cut and submitted to crossed immunoelectrophoresis against anti-apo A1 antiserum. Immunoelectrophoresis was performed in 0.75% agarose in 0.2 M barbitone buffer, pH 8.6, containing 10% sorbitol and 3% polyethylene glycol [9]. Apo A1 was quantified after the samples had been made 6 M in respect to tetramethyl urea (TMU) [9].Apo AII, apo B, alpha-2-macroglobulin and albumin were analysed
D. REICHL et al.
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4
I
-
*
a - Macroglobulin 50 -
-E E E
-
v
0 .-
a,
I
x i o
Y
Plasma
0 0
U
M apobi
50 -
0
@ - - O apo A l l
5
10
15
20
Migration (cm) Figure 1. A representative example of the distributions of apo AI, apo AII, alpha-2-macroglobulin and albumin in lymph and
plasma samples submitted to starch block electrophoresis. Horizontal bars indicate the fractions in which alpha-2-macroglobulin and albumin were detectable, and the vertical lines those in which their respective concentrations were maximal. Apo B was detectable only in the first three fractions of lymph and plasma (nearest to the site of application). Each point represents the concentration of apo A1 or apo A11 (expressed as rocket height, corrected for recovery) eluted from a single 1-cm section of the starch block. The horizontal axis gives the distance of the trailing edge of each section from the origin. Lymph was electrophoresed undiluted; plasma was diluted 1:4 (v/v) with 0.15 M NaCl prior to electrophoresis. Laboratory details are given under Materials and Methods. Results from subject no. 1.
in native samples. All antisera were purchased from Boehringer Mannheim GmbH (Mannheim, Germany). Precinorm (Boehringer) made 6 M in respect to TMU was used as standard for quantification of apo AI. The limits of detection of the assays were as follows: apo AI, 0.05 pg ml- I ; apo AII, 0.05 pg ml-I; apo B, 2 pg ml- I ; alpha-2-macroglobulin, 2 pg m1-I; albumin, 5 pg ml-'.
Results When lymph was submitted to HPLC on Superose 12, apo A1 was detected in all fractions eluting at 26-5-49-0 min. Apo A11 was detected in all fractions eluting at 27.5-42.0 min. Under identical conditions immunodetectable apo B eluted at 25-28 min (peak at 26.6 4 0.02 (SD) min, n = 7), and immunodetectable albumin eluted at 38-46 min (peak at 40 +_ 0.1 min, n = 5). When plasma was fractionated under the same conditions, apo A1 and apo A11 were detected in all fractions between 28 and 42 rnin. These results demonstrated that the range of sizes of apo AI-containing particles in lymph exceeds that in plasma, and that both the largest
and smallest particles are Lp A1 w/o A11 (i.e. contain apo A1 but no immunodetectabfe apo AII). When lymph and plasma were submitted in parallel to starch block electrophoresis, the electrophoresed samples fractionated, and the distributions of apo A1 and apo A11 among the fractions determined, marked differences between the lipoproteins of the two fluids were seen. Lymph fractions that contained solutes with the slowest mobility (beta and pre-beta) contained no detectable apo A1 or apo AII, whereas in the corresponding fractions of plasma both apos could be detected. Apo A1 was detected in all fractions of both fluids that contained solutes of electrophoretic mobility greater than pre-beta, including those containing albumin (Fig. I). Only in the case of lymph, however, could apo A1 be detected also in fractions of greater mobility than that of albumin. Furthermore, the distribution of apo A1 concentration in electrophoresed fractions of lymph indicates that particles with alpha-2 mobility (i.e. fractions that contained alpha-2macroglobulin) contained predominantly apo AI. Apo A11 could be detected only in traces in this region, and only in those fractions in which particles of greater
HUMAN PERIPHERAL LYMPH LIPOPROTEINS
641
Fig. 3
Fig. 2
Elution Time (min)
Figures 2 and 3. Representative examples of the distributions of apo A1 and apo A11 in electrophoretic fractions of lymph and plasma submitted to HPLC on Superose 12. Figure 2. Elution patterns of: (A) combined fractions 4 7 of lymph; (B) combined fractions 8-13 of lymph; (C) combined fractions 14-20 of lymph. Figure 3. Elution patterns of: (A) combined fractions 4-7 of plasma; (B) combined fractions 8-1 3 of plasma; (C) combined fractions 14-20 of plasma. Horizontal bars indicate the fractions in which apo B and albumin were detectable, and the vertical lines those in which their respective concentrations were maximal. Samples of lymph and plasma (diluted 1 :4 (v/v) with 0.15 M NaCI) were subjected in parallel to starch block electrophoresis. The starch blocks were then divided into twenty I-cm sections, and the proteins in each section eluted as described under Materials and Methods. The eluates from Sections 4-7,8-13 and 14-20 were then pooled, each pool was concentrated and submitted to HPLC, and the HPLC fractions assayed for apo A1 and apo A11 by immunoelectrophoresis. Results are expressed as rocket heights, corrected for recoveries from starch gel. Subject no. 1.
Figure 4. Precipitin lines obtained when isoelectrically focused lymph (Panel A) and Plasma (Panel B)were immunoelectrophoresed against anti-apo A1 antiserum. The arrow indicates the inner edge of the anode wick. Laboratory details are given under Materials and Methods. Subject no. 3.
mobility than alpha-2-macroglobulin may have been trailing. Thus, the population of particles with alpha-2 mobility in lymph was predominantly Lp A1 w/o AIT. When the masses of apo A1 present in all fractions in which alpha-2-macroglobulin could be detected were summed, they represented on average approximately 7% (range: 4-12%) of total lymph apo AI. In order to obtain information on the particle size of the alpha-2-migrating LpAI w/o A11 of lymph, this subfraction was concentrated and submitted to HPLC on Superose 12. Apo A1 rockets were detected only in those chromatographic fractions that eluted between 42 and 47 min, with a maximum at 43 min (albumin maximum, 41 min) (Fig. 2A). Thus, the subfraction of lymph LpAI wjo A11 that migrated with alpha-2 electrophoretic mobility represented particles the size of, or smaller than, albumin. When alpha-2 fractions of plasma were analysed in the same way, apo A1 and apo A11 could be detected in chromatographic fractions that eluted at 40-46 min (Fig. 3A). When lymph fractions that contained apo A1 and had electrophoretic mobility greater than albumin were concentrated and submitted to HPLC, both apo A1 and apo A11 could be detected in fractions eluting between 30 and 44 min, revealing the presence of particles with a broad range of size (Fig. 2C). The presence in lymph of apo AI-containing particles with mobility greater than that of albumin could be due to several factors. Studies have shown the presence in plasma of several isoforms of apo AI, which differ in charge [ 161. Therefore, plasma and lymph were delipidated and submitted to isoelectric focusing in the pH range 5.0-6.0, and the focused
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D. REICNL et al.
samples submitted to crossed immunoelectrophoresis against anti-apo A1 antiserum. In the case of plasma a precipitin line was formed in the shape of single major rocket (Fig. 4). In the case of lymph, the precipitin line had the shape of two distinct rockets, the mid-point of the more cathodic of them being at the same position as that obtained with plasma (Fig. 4). The second of the rockets produced by lymph was nearer the anode, and therefore represented a more negatively (or less positively) charged form of apo AI. Thus, when compared to plasma, lymph contained a higher proportion of apo A1 isoform(s) that focus at a lower pH than its major mature isoform [16].
Discussion In previous studies we have shown that the apo AIcontaining lipoproteins of human peripheral lymph have a wider range of particle size distribution than do those of plasma [9,13]. The results of those studies suggested that compared with plasma, the concentration in lymph of small apo AI-containing particles is high relative to the total concentration of apo AI. Our present results extend these observations by identifying a population of apo AI-containing particles that is characterized by alpha-2 mobility on electrophoresis. These particles represent a proportion of the LpAI w/o A11 of lymph, are the size of, or smaller than, albumin, and comprise approximately 7% of the total apo A1 in lymph. Since the concentration of apo A1 in human lymph averages 16 mg dl-' 191, that of the alpha-2migrating particles is approximately 1.0 mg dl-I. These figures may be compared with those of Kunitake et al.1151 and Ishida et af.[ 161,who found that 14 k 5% (mean SD) and 4 & 2% respectively of plasma apo A1 in normolipidaemic subjects was present in pre-beta LpAI. LeFevre et af.[ 171found that 15& 4% of the apo A1 of canine peripheral lymph was present in pre-betamigrating particles. There is now abundant evidence that among human plasma lipoproteins the most avid acceptors of cholesterol from cultured fibroblasts [5], adipocytes [6] and macrophages [7] are members of the LpAI w/o A11 subclass of HDL. In contrast, LpAI w A11 particles in plasma have much less cholesterol-mobilizing activity [6,7]. If present also in tissue fluids, the smallest LpAI w/o A11 particles would be expected to be particularly important acceptors of cell cholesterol in vivo, owing to their predicted [I 11 large volume of distribution in the extracellular matrix. Thus, on the basis of their composition and size, it seems likely that the alpha-2migrating particles which we have identified in lymph play a major role as acceptors of cholesterol in human peripheral tissues. The electrophoretic mobility of these and other apo AI-containing particles in lymph may be determined in part by the net charge of their constituent apo AI. The mechanism underlying the higher proportion in lymph, as compared to plasma, of more negatively charged isoform(s) of apo A1 could not be elucidated
in the present study, due to the small quantities of lymph that were available. It may be that their presence reflects the presence of a higher proportion of deamidated isoforms of mature apo A1 [18], or of apo A1 covalently bound to fatty acid residues [19]. If such modifications of apo A1 occur in interstitial fluids, the small alpha-2-migrating LpAI w/o A11 particles of lymph may be a counterpart of the small pre-beta LpAI of plasma [5,15]. We have previously postulated two mechanisms by which small apo AI-containing particles may be generated at the vascular endothelium, within transport vesicles in vascular endothelial cells and/or in the interstitium itself [1,2]. These are the interaction of plasma HDL with excess phospholipids (e.g. the surface remnants of lipolysed triglyceride-rich lipoproteins) and the action on extravascular HDL of the phospholipase A-like activity of LCAT. Both of these reactions have been shown to generate small apo AIcontaining particles in vitro [20,21]. Studies are in progress to examine the kinetics of the alpha-2migrating LpAI w/o A11 of lymph, with a view to identifying the metabolic processes that regulate their production in vivo.
Acknowledgments We thank James King, MD for his assistance with the recruitment of subjects. References Reichl D, Miller NE. The anatomy and physiology of reverse cholesterol transport. Clin Sci 1986;70:221-31. Reichl D, Miller NE. Pathophysiology of reverse cholesterol transport: insights from inherited disorders of lipoprotein metabolism. Arteriosclerosis 1989;9:785-97. Werb Z, Cohn ZA. Cholesterol metabolism in the macrophage. Part 3. Ingestion and intracellular fate of cholesterol and cholesterol esters. J Exp Med 1972;135:21-44. Stein 0, Vanderhoek J, Stein Y. Cholesterol content and sterol synthesis in human skin fibroblasts and aortic smooth muscle cells exposed to lipoprotein-depleted serum and high density apoprotein/phospholipid mixtures. Biochim Biophys Acta 1976;431:347-58. Castro GR, Fielding CJ. Early incorporation of cell-derived cholesterol into pre-8-migrating high-density lipoprotein. Biochemistry 1988;27:25-9. Barbaras R, Puchois P, Fruchart J-C, Ailhaud G. Cholesterol efflux from cultured adipose cells is mediated by LpAI particles but not by LpA1:AII particles. Biochem Biophys Res Commun 1987;142~63-9. Nowicka G, Bruning T, Bottcher A, Kahl G , Schmitz G. The macrophage interaction of HDL subclasses separated by free flow isotachophoresis. J Lipid Res 1990;31:1947-63. Renkin EM. Lymph as a measure of the composition of interstitial fluid. In: Fishman AP, Renkin EM, eds. Pulmonary Edema. Bethesda: American Physiological Society 1979:145-59. Reichl D, M u g JJ. The concentration of apolipoprotein A1 in human peripheral lymph. Biochim Biophys Acta 1982;710:45663. 10 Reichl D, Rudra DN, Pflug J. The concentration of apolipoprotein A11 in human peripheral lymph. Biochim Biophys Acta 1989;1006:246-9. 11 Reichl D, Forte TM, Hong J-L. Rudra DN, Pflug J. Human lymphedema fluid lipoproteins. Particle size, cholesterol and
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14
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apoliprotein distributions and electron microscopic structure. J Lipid Res 1985;26:1399-411. Bell DR, Watson PD, Renkin EM. Exclusion of plasma protein in interstitium of tissues of the dog hind paw. Amer J Physiol 1980;239:H532-8. Reichl D, Simons LA, Myant NB, Pflug J, Mills GL. The lipids and lipoproteins of human peripheral lymph with observations on the transport of cholesterol from plasma and tissues into lymph. Clin Sci Mol Med 1973;45:313-29. Have1 RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 1955;34:1345-53. Kunitake ST, La Sala KJ. Kane JP. Apolipoprotein AIcontaining lipoproteins with pre-beta electrophoretic mobility. J Lipid Res 1985;26:549-55. Ishida BY, Frolich J, Fielding CJ. Prebeta-migrating high density lipoprotein: quantitation in normal and hyperlipidemic plasma by solid phase radioimmunoassay following electrophoretic transfer. J Lipid Res 1987;28:778-86.
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17 LeFevre M, Sloop CH, Roheim PS. Characterization of dog prenodal peripheral lymph lipoproteins. Evidence for the peripheral formation of lipoprotein-unassociated apo A-I with slow pre-P-electrophoretic mobility. J Lipid Res 1988;29:113948. 18 Bojanovski D, Gregg RE, Ghiselli G et al. Human apolipoprotein A1 isoprotein metabolism: proapo A1 conversion to mature apo AI. J Lipid Res 1985;26:185-93. 19 Hoeg JM, Meng MS, Ronan R, Fairwell T, Brewer HB. Human apolipoprotein AI. Post-translational modification by fatty acid acylation. J Biol Chem 1986;261:3911-14. 20 Nichols AV, Gong EL, Forte TM, Blanche PJ. Interaction of plasma high density lipoprotein HDL2b (d 1.063-1.100 g/ml) with single-bilayer liposomes of dimyristoylphosphatidylcholine. Lipids 1978;13:943-50. 21 Nichols AV, Gong EL, Blanche PJ, Forte TM, Shore VG. Pathways in the formation of human plasma high density lipoprotein subpopulation containing apolipoprotein A1 without apolipoprotein AIL J Lipid Res 1987;28:719-32.
Lipoproteins of human peripheral lymph. Apolipoprotein Al-containing lipoprotein with alpha-2 electrophoretic mobility D. REICHL, C. B. HATHAWAY, J. M. STERCHI* & N. E. MILLER, Section on Endocrinology and Metabolism, Department of Medicine, and *Division of Surgical Sciences, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina, USA Received 15 November 1990 and in revised form 14 March 1991 Abstract. Evidence from diverse sources has implicated a central role of apolipoprotein A; (apo AI), the most abundant protein of plasma high-density lipoproteins, in the transport of cholesterol from peripheral tissues to the liver (reverse cholesterol transport). Particles containing only apo A1 appear to be more effective as cholesterol acceptors in tissue culture than do particles which also contain apo AIL The apo AIcontaining lipoproteins of plasma have been extensively studied, but there is less information on those in tissue fluids, to which most peripheral cells are exposed. In the present study the heterogeneity of apo AI-containing particles in human peripheral lymph, collected from the dorsum of the foot, has been examined by starch block electrophoresis, exclusion chromatography and immunoelectrophoresis. The apo AI-containing particles of lymph were found to be more variable in both electrophoretic mobility and size than those of plasma from the same subjects. Of particular interest was a subpopulation which migrated on electrophoresis with the same mobility as alpha-2-macroglobulin. This fraction accounted for approximately 7% (range: 4-12%; n = 5) of lymph apo AI, contained no immunodetectable apo AII, and by exclusion chromatography was composed of particles the size of, or smaller than, albumin. Such physicochemical properties suggest that these alpha-:! migrating particles may function as the principal primary acceptors of cell cholesterol in the extracellular matrix of human peripheral tissues. By isoelectric focusing, lymph apo A1 was found to contain a higher proportion of more negatively charged isoforms than the apo A1 of plasma.
Keywords. Apolipoproteins, cholesterol, electrophoresis, exclusion chromatography, high-density lipoprotein, lymph. Introduction Most extra-hepatic cells continuously acquire cholesterol as a result of endogenous synthesis and/or Correspondence: D. Reichl, PhD, Section on Endocrinology and Metabolism, Department of Medicine, Bowman Gray School of Medicine, 300 South Hawthorne Road, Winston-Salem, NC 27103, USA.
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endocytosis of lipoproteins, but most cannot metabolize it. Hepatocytes differ from peripheral cells in that they can oxidize cholesterol to bile acids, and can secrete it directly into bile. Hence, under steady-state conditions, cholesterol must continuously be released from extra-hepatic cells and transported to the liver for elimination, a process commonly referred to as reverse cholesterol transport [I ,2]. Studies in uitro have shown that sustained efflux of cholesterol mass from cells takes place only in the presence of acceptors [3], and that high-density lipoprotein (HDL) and complexes of HDL apolipoproteins (apos) with phospholipids are among the most effective acceptors [4]. In v i m only a small proportion of cells are in direct contact with blood. For these cells plasma lipoproteins are available both as acceptors and as transporting agents of cholesterol to the liver. Recent studies have shown the presence in plasma of small particles containing apo A1 (the main protein component of HDL) that appear to be particularly effective cholesterol acceptors in tissue culture [5]. Notable features of these particles are their pre-beta mobility in the electrophoretic field and an absence of immunodetectable apo A11 [5]. In contrast, plasma HDL subclasses that contain both apo A1 and apo A11 appear to have little or no effect on cholesterol efflux from cultured cells [6,7]. Most cells of the human body are separated from blood by vascular endothelium, and usually also by the matrix of the interstitium, and for these cells lipoproteins of interstitial fluids must act as acceptors and initial transporters of cholesterol. Although interstitial fluid cannot be sampled in sufficient quantities for laboratory analysis, peripheral lymph resembles it in many respects [8], and can be collected from humans under physiologic conditions. In previous studies we have shown that the apo AI-containing particles of human lymph cover a broader range of sizes than do those of plasma [9-1 I]. Since the sieving characteristics of the interstitium dictate that the volumes of distribution of particles in tissues are inversely related to their diameters [121, the smallest lipoproteins are likely to play a prominent role in the first stage of reverse cholesterol transport. We have now further characterized the apo AI-
HUMAN PERIPHERAL LYMPH LIPOPROTEINS containing lipoproteins of human lymph, and have identified a population whose size and composition make them particularly suited to the role of initial acceptors of tissue cholesterol in vivo. These particles differ from others in having alpha-2 mobility on starch block electrophoresis. Materials and methods Clinical procedures
The study was approved by the institutional review board. Lymph was collected from five apparently healthy non-fasted subjects, each of whom gave informed written consent: four men aged 58,72,28 and 28 years with respective plasma cholesterol concentrations of 5.7,4.5,5.2 and 4.2 mmol I-', and one woman aged 32 years with a plasma cholesterol of 3.9 mmol 1- '. Cannulation of a lymphatic collecting trunk on the dorsum of the foot was performed after subcutaneous injection of Patent Blue, as previously described [ 131. Vials into which lymph was collected contained 500 pg of NazEDTA and 500 pg of 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) to inhibit lecithin :cholesterol acyltransferase (LCAT). Collecting vials were changed for each 0.5 ml of lymph. Total volumes of 0.25-1.50 ml were collected over 3 h. They were stored on ice until used for analyses (within 2 h). Blood was collected from an antecubital vein into Naz EDTA (1 mg ml-I), and centrifuged at 4°C within 20 min. Plasma was stored on ice after addition of solid DTNB (0.8 mg ml-I). Laboratory procedures
Lipoproteins were separated and analysed by combinations of high performance liquid chromatography (HPLC), starch block electrophoresis, isoelectric focusing and immunoelectrophoresis. Lymph and plasma were fractionated by HPLC using a Superose 12 column (Prepacked HR 10/30; Pharmacia LKB, Uppsala, Sweden) and a flow rate of 0.3 ml min- I. The eluting buffer was 0.15 M NaCI-0.05 M phosphate (PBS), pH 7.4, containing 0.1% (w/v) Na2EDTA and NaN3. When plasma was fractionated it was first diluted with 4 vols of elution buffer. Fractions from starch gel electrophoresis (see below) were fractionated on the same column using identical conditions. The elution pattern was monitored by continuously measuring the OD280 of the effluent. The column was standardized by measuring the elution times of human low-density lipoprotein (LDL) of density 1.018-1.063 g ml-', prepared by preparative ultracentrifugation [ 141, and crystalline bovine serum albumin (Sigma, St. Louis, MO, USA). Additionally, the concentrations of apo B (the major apolipoprotein of LDL) and albumin were determined by immunoelectrophoresis in all eluted fractions. Lymph and plasma were fractionated by starch block electrophoresis essentially according to Kunitake et al. 1151. Hydrated potato starch was formed
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into a 1 1 x 29 x 0.5 cm block. The block was divided longitudinally into equal parts, and a transverse trough was cut into each part 4 cm from the cathodic end. One of the troughs was filled with 0.4 ml of lymph, the other with the same volume of 1 in 5 diluted plasma from the same subject. A drop of 5% (w/v) bovine serum albumin in 0-15M NaCI, stained with brompheno1 blue, was applied between the troughs as a marker. A constant current of 36 mA was then applied for 18 h, after which the block was cut transversely into sections 1 cm wide, and the sections transferred into glass tubes. PBS (1 50 pl), to which a trace of Na'*'I (approx. lo4 cpm ml-') had been added, was pipetted into each tube, and the slurry vortexed. The radioactivity in each tube was measured in a gamma-counter. The tubes were then centrifuged at 2000 rpm for 10 min; the supernatants were transferred into fresh tubes; another 150 p1 of PBS was added; the vortexing and centrifugation were repeated; and the second supernatant was added to the first. The volumes of the extracts were estimated by weighing, assuming a density of 1.006 g ml- ', and the radioactivity of the combined extracts was measured. It was assumed that the distribution of radioactivity between extracts and remaining hydrated starch reflected the distribution of all solutes. Known volumes of extracts were taken for measurement of solute concentrations, and the remainder stored at 4°C until the analyses were completed (within 48 h). Relevant fractions from starch block electrophoresis were combined, concentrated by centrifugation in Centricon tubes (cut-off 10 000; Amicon, Danvers, MA, USA), and submitted to HPLC on Superose 12 as described above. When plasma was electrophoresed in parallel with the same plasma sample to which Patent Blue had been added (final concentration 0.01 %), there was no difference between the samples in the distribution of apo A1 or apo AII. It was also shown that dilution of plasma had no effect on apolipoprotein distributions after starch block electrophoresis or HPLC. Isoelectric focusing on polyacrylamide gel containing 3 M urea and Pharmalite of pH range 5-6 (Pharmacia) was performed according to the Pharmacia specifications. Lymph and plasma were delipidated with ethano1:diethyl ether 2: 1 (v/v), and again with diethyl ether. The dry solids were re-dissolved in 0.1 M barbitone buffer, pH 8.6 (lymph solids in the original volume; plasma solids in five times the original volume). Five p1 of each were used for analysis. A 1 O/o (w/v) solution of Congo red in deionized water was used as marker. On completion of the focusing, relevant lanes were cut and submitted to crossed immunoelectrophoresis against anti-apo A1 antiserum. Immunoelectrophoresis was performed in 0.75% agarose in 0.2 M barbitone buffer, pH 8.6, containing 10% sorbitol and 3% polyethylene glycol [9]. Apo A1 was quantified after the samples had been made 6 M in respect to tetramethyl urea (TMU) [9].Apo AII, apo B, alpha-2-macroglobulin and albumin were analysed
D. REICHL et al.
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4
I
-
*
a - Macroglobulin 50 -
-E E E
-
v
0 .-
a,
I
x i o
Y
Plasma
0 0
U
M apobi
50 -
0
@ - - O apo A l l
5
10
15
20
Migration (cm) Figure 1. A representative example of the distributions of apo AI, apo AII, alpha-2-macroglobulin and albumin in lymph and
plasma samples submitted to starch block electrophoresis. Horizontal bars indicate the fractions in which alpha-2-macroglobulin and albumin were detectable, and the vertical lines those in which their respective concentrations were maximal. Apo B was detectable only in the first three fractions of lymph and plasma (nearest to the site of application). Each point represents the concentration of apo A1 or apo A11 (expressed as rocket height, corrected for recovery) eluted from a single 1-cm section of the starch block. The horizontal axis gives the distance of the trailing edge of each section from the origin. Lymph was electrophoresed undiluted; plasma was diluted 1:4 (v/v) with 0.15 M NaCl prior to electrophoresis. Laboratory details are given under Materials and Methods. Results from subject no. 1.
in native samples. All antisera were purchased from Boehringer Mannheim GmbH (Mannheim, Germany). Precinorm (Boehringer) made 6 M in respect to TMU was used as standard for quantification of apo AI. The limits of detection of the assays were as follows: apo AI, 0.05 pg ml- I ; apo AII, 0.05 pg ml-I; apo B, 2 pg ml- I ; alpha-2-macroglobulin, 2 pg m1-I; albumin, 5 pg ml-'.
Results When lymph was submitted to HPLC on Superose 12, apo A1 was detected in all fractions eluting at 26-5-49-0 min. Apo A11 was detected in all fractions eluting at 27.5-42.0 min. Under identical conditions immunodetectable apo B eluted at 25-28 min (peak at 26.6 4 0.02 (SD) min, n = 7), and immunodetectable albumin eluted at 38-46 min (peak at 40 +_ 0.1 min, n = 5). When plasma was fractionated under the same conditions, apo A1 and apo A11 were detected in all fractions between 28 and 42 rnin. These results demonstrated that the range of sizes of apo AI-containing particles in lymph exceeds that in plasma, and that both the largest
and smallest particles are Lp A1 w/o A11 (i.e. contain apo A1 but no immunodetectabfe apo AII). When lymph and plasma were submitted in parallel to starch block electrophoresis, the electrophoresed samples fractionated, and the distributions of apo A1 and apo A11 among the fractions determined, marked differences between the lipoproteins of the two fluids were seen. Lymph fractions that contained solutes with the slowest mobility (beta and pre-beta) contained no detectable apo A1 or apo AII, whereas in the corresponding fractions of plasma both apos could be detected. Apo A1 was detected in all fractions of both fluids that contained solutes of electrophoretic mobility greater than pre-beta, including those containing albumin (Fig. I). Only in the case of lymph, however, could apo A1 be detected also in fractions of greater mobility than that of albumin. Furthermore, the distribution of apo A1 concentration in electrophoresed fractions of lymph indicates that particles with alpha-2 mobility (i.e. fractions that contained alpha-2macroglobulin) contained predominantly apo AI. Apo A11 could be detected only in traces in this region, and only in those fractions in which particles of greater
HUMAN PERIPHERAL LYMPH LIPOPROTEINS
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Fig. 3
Fig. 2
Elution Time (min)
Figures 2 and 3. Representative examples of the distributions of apo A1 and apo A11 in electrophoretic fractions of lymph and plasma submitted to HPLC on Superose 12. Figure 2. Elution patterns of: (A) combined fractions 4 7 of lymph; (B) combined fractions 8-13 of lymph; (C) combined fractions 14-20 of lymph. Figure 3. Elution patterns of: (A) combined fractions 4-7 of plasma; (B) combined fractions 8-1 3 of plasma; (C) combined fractions 14-20 of plasma. Horizontal bars indicate the fractions in which apo B and albumin were detectable, and the vertical lines those in which their respective concentrations were maximal. Samples of lymph and plasma (diluted 1 :4 (v/v) with 0.15 M NaCI) were subjected in parallel to starch block electrophoresis. The starch blocks were then divided into twenty I-cm sections, and the proteins in each section eluted as described under Materials and Methods. The eluates from Sections 4-7,8-13 and 14-20 were then pooled, each pool was concentrated and submitted to HPLC, and the HPLC fractions assayed for apo A1 and apo A11 by immunoelectrophoresis. Results are expressed as rocket heights, corrected for recoveries from starch gel. Subject no. 1.
Figure 4. Precipitin lines obtained when isoelectrically focused lymph (Panel A) and Plasma (Panel B)were immunoelectrophoresed against anti-apo A1 antiserum. The arrow indicates the inner edge of the anode wick. Laboratory details are given under Materials and Methods. Subject no. 3.
mobility than alpha-2-macroglobulin may have been trailing. Thus, the population of particles with alpha-2 mobility in lymph was predominantly Lp A1 w/o AIT. When the masses of apo A1 present in all fractions in which alpha-2-macroglobulin could be detected were summed, they represented on average approximately 7% (range: 4-12%) of total lymph apo AI. In order to obtain information on the particle size of the alpha-2-migrating LpAI w/o A11 of lymph, this subfraction was concentrated and submitted to HPLC on Superose 12. Apo A1 rockets were detected only in those chromatographic fractions that eluted between 42 and 47 min, with a maximum at 43 min (albumin maximum, 41 min) (Fig. 2A). Thus, the subfraction of lymph LpAI wjo A11 that migrated with alpha-2 electrophoretic mobility represented particles the size of, or smaller than, albumin. When alpha-2 fractions of plasma were analysed in the same way, apo A1 and apo A11 could be detected in chromatographic fractions that eluted at 40-46 min (Fig. 3A). When lymph fractions that contained apo A1 and had electrophoretic mobility greater than albumin were concentrated and submitted to HPLC, both apo A1 and apo A11 could be detected in fractions eluting between 30 and 44 min, revealing the presence of particles with a broad range of size (Fig. 2C). The presence in lymph of apo AI-containing particles with mobility greater than that of albumin could be due to several factors. Studies have shown the presence in plasma of several isoforms of apo AI, which differ in charge [ 161. Therefore, plasma and lymph were delipidated and submitted to isoelectric focusing in the pH range 5.0-6.0, and the focused
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samples submitted to crossed immunoelectrophoresis against anti-apo A1 antiserum. In the case of plasma a precipitin line was formed in the shape of single major rocket (Fig. 4). In the case of lymph, the precipitin line had the shape of two distinct rockets, the mid-point of the more cathodic of them being at the same position as that obtained with plasma (Fig. 4). The second of the rockets produced by lymph was nearer the anode, and therefore represented a more negatively (or less positively) charged form of apo AI. Thus, when compared to plasma, lymph contained a higher proportion of apo A1 isoform(s) that focus at a lower pH than its major mature isoform [16].
Discussion In previous studies we have shown that the apo AIcontaining lipoproteins of human peripheral lymph have a wider range of particle size distribution than do those of plasma [9,13]. The results of those studies suggested that compared with plasma, the concentration in lymph of small apo AI-containing particles is high relative to the total concentration of apo AI. Our present results extend these observations by identifying a population of apo AI-containing particles that is characterized by alpha-2 mobility on electrophoresis. These particles represent a proportion of the LpAI w/o A11 of lymph, are the size of, or smaller than, albumin, and comprise approximately 7% of the total apo A1 in lymph. Since the concentration of apo A1 in human lymph averages 16 mg dl-' 191, that of the alpha-2migrating particles is approximately 1.0 mg dl-I. These figures may be compared with those of Kunitake et al.1151 and Ishida et af.[ 161,who found that 14 k 5% (mean SD) and 4 & 2% respectively of plasma apo A1 in normolipidaemic subjects was present in pre-beta LpAI. LeFevre et af.[ 171found that 15& 4% of the apo A1 of canine peripheral lymph was present in pre-betamigrating particles. There is now abundant evidence that among human plasma lipoproteins the most avid acceptors of cholesterol from cultured fibroblasts [5], adipocytes [6] and macrophages [7] are members of the LpAI w/o A11 subclass of HDL. In contrast, LpAI w A11 particles in plasma have much less cholesterol-mobilizing activity [6,7]. If present also in tissue fluids, the smallest LpAI w/o A11 particles would be expected to be particularly important acceptors of cell cholesterol in vivo, owing to their predicted [I 11 large volume of distribution in the extracellular matrix. Thus, on the basis of their composition and size, it seems likely that the alpha-2migrating particles which we have identified in lymph play a major role as acceptors of cholesterol in human peripheral tissues. The electrophoretic mobility of these and other apo AI-containing particles in lymph may be determined in part by the net charge of their constituent apo AI. The mechanism underlying the higher proportion in lymph, as compared to plasma, of more negatively charged isoform(s) of apo A1 could not be elucidated
in the present study, due to the small quantities of lymph that were available. It may be that their presence reflects the presence of a higher proportion of deamidated isoforms of mature apo A1 [18], or of apo A1 covalently bound to fatty acid residues [19]. If such modifications of apo A1 occur in interstitial fluids, the small alpha-2-migrating LpAI w/o A11 particles of lymph may be a counterpart of the small pre-beta LpAI of plasma [5,15]. We have previously postulated two mechanisms by which small apo AI-containing particles may be generated at the vascular endothelium, within transport vesicles in vascular endothelial cells and/or in the interstitium itself [1,2]. These are the interaction of plasma HDL with excess phospholipids (e.g. the surface remnants of lipolysed triglyceride-rich lipoproteins) and the action on extravascular HDL of the phospholipase A-like activity of LCAT. Both of these reactions have been shown to generate small apo AIcontaining particles in vitro [20,21]. Studies are in progress to examine the kinetics of the alpha-2migrating LpAI w/o A11 of lymph, with a view to identifying the metabolic processes that regulate their production in vivo.
Acknowledgments We thank James King, MD for his assistance with the recruitment of subjects. References Reichl D, Miller NE. The anatomy and physiology of reverse cholesterol transport. Clin Sci 1986;70:221-31. Reichl D, Miller NE. Pathophysiology of reverse cholesterol transport: insights from inherited disorders of lipoprotein metabolism. Arteriosclerosis 1989;9:785-97. Werb Z, Cohn ZA. Cholesterol metabolism in the macrophage. Part 3. Ingestion and intracellular fate of cholesterol and cholesterol esters. J Exp Med 1972;135:21-44. Stein 0, Vanderhoek J, Stein Y. Cholesterol content and sterol synthesis in human skin fibroblasts and aortic smooth muscle cells exposed to lipoprotein-depleted serum and high density apoprotein/phospholipid mixtures. Biochim Biophys Acta 1976;431:347-58. Castro GR, Fielding CJ. Early incorporation of cell-derived cholesterol into pre-8-migrating high-density lipoprotein. Biochemistry 1988;27:25-9. Barbaras R, Puchois P, Fruchart J-C, Ailhaud G. Cholesterol efflux from cultured adipose cells is mediated by LpAI particles but not by LpA1:AII particles. Biochem Biophys Res Commun 1987;142~63-9. Nowicka G, Bruning T, Bottcher A, Kahl G , Schmitz G. The macrophage interaction of HDL subclasses separated by free flow isotachophoresis. J Lipid Res 1990;31:1947-63. Renkin EM. Lymph as a measure of the composition of interstitial fluid. In: Fishman AP, Renkin EM, eds. Pulmonary Edema. Bethesda: American Physiological Society 1979:145-59. Reichl D, M u g JJ. The concentration of apolipoprotein A1 in human peripheral lymph. Biochim Biophys Acta 1982;710:45663. 10 Reichl D, Rudra DN, Pflug J. The concentration of apolipoprotein A11 in human peripheral lymph. Biochim Biophys Acta 1989;1006:246-9. 11 Reichl D, Forte TM, Hong J-L. Rudra DN, Pflug J. Human lymphedema fluid lipoproteins. Particle size, cholesterol and
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apoliprotein distributions and electron microscopic structure. J Lipid Res 1985;26:1399-411. Bell DR, Watson PD, Renkin EM. Exclusion of plasma protein in interstitium of tissues of the dog hind paw. Amer J Physiol 1980;239:H532-8. Reichl D, Simons LA, Myant NB, Pflug J, Mills GL. The lipids and lipoproteins of human peripheral lymph with observations on the transport of cholesterol from plasma and tissues into lymph. Clin Sci Mol Med 1973;45:313-29. Have1 RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 1955;34:1345-53. Kunitake ST, La Sala KJ. Kane JP. Apolipoprotein AIcontaining lipoproteins with pre-beta electrophoretic mobility. J Lipid Res 1985;26:549-55. Ishida BY, Frolich J, Fielding CJ. Prebeta-migrating high density lipoprotein: quantitation in normal and hyperlipidemic plasma by solid phase radioimmunoassay following electrophoretic transfer. J Lipid Res 1987;28:778-86.
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17 LeFevre M, Sloop CH, Roheim PS. Characterization of dog prenodal peripheral lymph lipoproteins. Evidence for the peripheral formation of lipoprotein-unassociated apo A-I with slow pre-P-electrophoretic mobility. J Lipid Res 1988;29:113948. 18 Bojanovski D, Gregg RE, Ghiselli G et al. Human apolipoprotein A1 isoprotein metabolism: proapo A1 conversion to mature apo AI. J Lipid Res 1985;26:185-93. 19 Hoeg JM, Meng MS, Ronan R, Fairwell T, Brewer HB. Human apolipoprotein AI. Post-translational modification by fatty acid acylation. J Biol Chem 1986;261:3911-14. 20 Nichols AV, Gong EL, Forte TM, Blanche PJ. Interaction of plasma high density lipoprotein HDL2b (d 1.063-1.100 g/ml) with single-bilayer liposomes of dimyristoylphosphatidylcholine. Lipids 1978;13:943-50. 21 Nichols AV, Gong EL, Blanche PJ, Forte TM, Shore VG. Pathways in the formation of human plasma high density lipoprotein subpopulation containing apolipoprotein A1 without apolipoprotein AIL J Lipid Res 1987;28:719-32.
