Isolation of serum chylomicrons prior to density gradient ultracentrifugation of other serum lipoprotein classes

A method for the removal of serum chylomicrons before density gradient ultracentrifugation of the other serum lipoproteins using an SW 41 swinging bucket rotor is presented. In a preliminary spin, the chylomicrons with an Sf greater than 400 X 10(-13) s float to the top of the gradient, whereas the other lipoproteins are retained in the infranatant fraction. After removal of the chylomicrons, the other serum lipoproteins are subsequently fractionated by isopycnic density gradient ultracentrifugation. Analysis of the separated lipoprotein fractions suggested that this procedure permits isolation of a chylomicron fraction consisting solely of chylomicrons but that the very low density lipoprotein fraction subsequently isolated also contains chylomicrons or chylomicron remnants with an Sf less than 400 X 10(-13) s, and that there is considerable overlap in flotation rate and particle size of very low density lipoproteins and chylomicrons.     

Radioimmunoassay of human high density lipoprotein apo-protein A-1.

A double antibody radioimmunoassay technique was developed for the measurement of apolipoprotein A-I, the major apoprotein of human high density lipoproteins. Apolipoprotein A-I was prepared from human delipidated high density lipoprotein (d equal to 1.085-1.210) by gel filtration and ion-exchange chromatography. Purified apolipoprotein A-I antibodies were obtained by means of apolipoprotein A-I immunoadsorbent. Apolipoprotein A-I was radiolabeled with 125-I by the iodine monochloride technique. 65-80% of 125 I-labeled apolipoprotein A-I could be bound by the different apolipoprotein A-I antibodies, and more than 95% of the 125-I-labeled apolipoprotein A-I was displaced by unlabeled apolipoprotein A-I. The immunoassay was found to be sensitive for the detection of about 10 ng of apolipoprotein A-I in the incubation mixture, and accurate with a variability of only 3-5% (S.E.M.). This technique enables the quantitation of apolipoprotein A-I in whole plasma or high density lipoprotein without the need of delipidation. The quantitation of apolipoprotein A-I in high density lipoprotein was found similar to that obtained by gel filtration technique. The displacement capacity of the different lipoproteins and apoproteins in comparison to unlabeled apolipoprotein A-I was: very low density lipoprotein, 1.8%; low density lipoprotein, 2.6%; high density lipoprotein, 68%; apolipoprotein B, non-detectable; apolipoprotein C, 0.5%; and apolipoprotein A-II, 4%. The distribution of immunoassayable apolipoprotein A-I among the different plasma lipoproteins was as follows: smaller than 1% in very low density lipoprotein and low density lipoprotein; 50% in high density lipoprotein, and 50% in lipoprotein fraction of density greater than 1.21 g/ml. The amount of apolipoprotein A-I in the latter fraction was found to be related to the number of centrifugations.     

Chylomicron remnant cholesteryl esters as the major constituent of very low density lipoproteins in plasma of cholesterol-fed rabbits.

Feeding rabbits 500 mg of cholesterol daily for 4 to 15 days greatly increased the concentration of esterified cholesterol in lipoproteins of d less than 1.006 g/ml. The origin of hypercholesterolemic very low density lipoproteins was investigated by monitoring the degradation of labeled lymph chyomicrons administered to normal and cholesterol-fed rabbits. Chylomicrons were labeled in vivo by feeding either 1) [3H]cholesterol and [14C]oleic acid or 2) [14C]cholesterol and [3H]retinyl acetate. After intravenous injection of labeled chylomicrons to recipient rabbits, [14C]triglyceride hydrolysis was equally rapid in normal and cholesterol-fed animals. Normal rabbits rapidly removed from plasma both labeled cholesteryl and retinyl esters, whereas cholesterol-fed rabbits retained nearly 50% of doubly labeled remnants in plasma 25 min after chylomicron injection. Ultracentrifugal separation of plasma into subfractions of very low density lipoproteins showed that chylomicron remnants in cholesterol-fed animals are found among all subclasses of very low density lipoproteins. Analysis of cholesteryl ester specific activity-time curves for the very low density lipoproteins subfraction from hypercholesterolemic plasma showed that nearly all esterified cholesterol in large very low density lipoproteins and approximately 30% of esterified cholesterol in small very low density lipoproteins was derived from chylomicron degradation. Apparently, nearly two-thirds of the esterified cholesterol in total very low density lipoproteins from moderately hypercholesterolemic rabbits is of dietary origin.     

Distribution of cell-derived cholesterol among plasma lipoproteins: a comparison of three techniques

Three fractionation procedures (immunoaffinity chromatography, two-dimensional nondenaturing electrophoresis, and heparin-agarose affinity chromatography) have been compared in determining the kinetics of free and ester cholesterol transfer in normolipemic native plasma. Similar results were obtained in each case. Cell-derived free cholesterol is initially enriched in high density lipoproteins (HDL) (mainly HDL without apoE); at longer time periods (greater than 10 min) greater proportions are observed in very low density lipoproteins (VLDL) and low density lipoproteins (LDL). The major part of cholesteryl ester (about 90%) was retained in HDL, while VLDL and LDL, which contained about 75% of total cholesteryl ester mass, received only about 10% of cell-derived cholesteryl ester. Within HDL, almost all cholesteryl ester was in the apoE-free fraction. These data provide evidence that lipoprotein free and esterified cholesterol are not at chemical equilibrium in normal plasma, and that cell-derived cholesterol is preferentially directed to HDL. The techniques used had a comparable effectiveness for the rapid fractionation of labile lipoprotein lipid radioactivity.     

Plasma lipoproteins of lambs and sheep.

The major plasma lipoproteins of the adult sheep were high density lipoprotein (76%) having alpha-mobility on electrophoresis and low density lipoprotein (20%) having beta-mobility. Chylomicrons and very low density lipoproteins were minor constituents (less than 5%). The postabsorptive hyperlipidaemia in suckling lambs is mainly a result of increased concentration of low density and high density lipoproteins although the relative contribution of very low density lipoproteins was increased to 7-15% of the total lipoproteins. The hyperlipiaemia was markedly greater in an intact male lamb than in female or castrated male lambs. In suckling lambs a new lipoprotein (density 1-090 g/ml) appeared in the high density lipoprotein fraction but disappeared before weaning.     

Characterization of the Ehrlich ascites tumor plasma lipoproteins.

1. The lipoproteins of the Ehrlich ascites tumor plasma were separated into 3 distinct fractions, very low density, low density and high density lipoproteins by preparative ultracentrifugation combined with agarose column chromatography. 2. High density lipoproteins contained 74% of the total protein in the lipoproteins. By contrast, most of the lipids were present in the very low density lipoprotein fraction. 3. The fatty acid compositions of the cholesteryl esters were appreciably different in the very low, low and high density lipoproteins, whereas phospholipid and triacylglycerol fatty acid compositions were quite similar in the 3 lipoprotein fractions. 4. Very low and high density apoprotein electrophoretic patterns on sodium dodecyl sulfate-acrylamide gels were similar to those observed in the corresponding lipoprotein fractions obtained from other mammalian species. The low density fraction, however, contained 7 apoprotein bands, and 32% of the low density apoprotein was soluble in tetramethyl urea. 5. The average molecular weights as determined by analytical ultracentrifugation were 2-10(7) (very low density), 6-10(6) (low density) and 4.4-10(5) (high density).     

Plasma clearance of chylomicrons labeled with retinyl palmitate in healthy human subjects

To estimate hepatic uptake of chylomicron remnants in humans, chylomicrons and intestinal very low density lipoproteins (VLDL) were endogenously labeled with retinyl esters, harvested by plasmapheresis, and pulse-injected into the donor 44 hr after plasmapheresis. Plasma decay of retinyl palmitate was measured in eight healthy volunteers. Retinyl palmitate plasma disappearance obeyed an apparent first order function in seven studies and, in one study, a biexponential function with the second, slow exponential accounting for only 13% of the retinyl palmitate plasma decay. The mean fractional removal of rate was 0.037 +/- 0.037 min-1 (mean +/- SD) in a one-compartment model. The apparent volume of distribution, Vd, was 109 +/- 25% of the estimated plasma volume. Plasma clearance of retinyl palmitate was 130 +/- 97 ml/min calculated as Vd x Ke. Mean T 1/2 was 29 +/- 16 min. Both in vitro and in vivo the retinyl palmitate remained largely within chylomicrons and intestinal VLDL. Only 4.3% was transferred from chylomicrons to other lipoprotein classes during in vitro incubation for 5 hr. After plasma was stored for 42 hr, 5% was transferred to higher density lipoproteins. During 12 hr after a test meal containing retinyl palmitate, only 6.4 +/- 1.5% of the retinyl palmitate absorbed was found in the LDL fraction and 3.1 +/- 3.8% in the d 1.063 g/ml lipoproteins. We conclude that retinyl palmitate is a useful marker for chylomicrons and their remnants in humans and that the plasma clearance of retinyl palmitate-labeled chylomicrons is probably an estimate of chylomicron remnant plasma clearance in man.     

1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine: acetylhydrolase is associated primarily with low density lipoproteins in human blood

The distribution of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine: acetyl hydrolase acetyl hydrolase activity between different types of human plasma lipoproteins was studied. It was found that lipoprotein-depleted plasma is practically devoid of acetyl hydrolase and of almost all acetyl hydrolase activities recovered in the plasma lipoprotein fraction. Among different types of plasma lipoproteins the bulk of acetyl hydrolase is bound to low density lipoproteins; of those not more than 5-10% is associated with high density lipoproteins. Isolated plasma high density lipoproteins do not influence the activity of acetyl hydrolase associated with low density lipoproteins. It is suggested that low and high density lipoprotein acetyl hydrolase may play different functional roles in human plasma.     

Evidence for the presence of low density and very low density lipoproteins in human amniotic fluid

Previous analysis of amniotic fluid (AF) noted only the presence of high density lipoprotein (HDL). In this study AF lipoprotein profile was examined using gel filtration column chromatography and Ouchterlony gel diffusion. Unlike previous studies which showed only the presence of HDL, we found significant amounts of low density lipoprotein (LDL) and very low density lipoprotein (VLDL). AF-LDL and AF-VLDL were identified by reactions with anti-h-apolipoprotein AI and AII antiserum and anti-h-apolipoprotein B-antiserum, respectively. Furthermore, bulk of the cholesterol mass was carried in VLDL (53.6 +/- 7.7%) and LDL (32.5 +/- 4.3%) with minor amounts (13.9 +/- 1.3%) in HDL fraction. It is concluded that human AF contains all three lipoproteins with most of the cholesterol being carried in very low density lipoprotein fraction.     

Distribution of glycosphingolipids in the serum lipoproteins of normal human subjects and patients with hypo- and hyperlipidemias.

Five glycosphingolipids (GSL), glucosylceramide, lactosylceramide, trihexosylceramide, globoside, and hematoside (GM3) were studied in serum from normal human subjects and patients with dyslipoproteinemia and found to be exclusively associated with the various classes of serum lipoproteins. Based on a unit weight of lipoprotein protein, the total amount of GSL in serum normal subjects was twice as high in very low density lipoprotein (VLDL) (d less than 1.006 g/ml) and low density lipoprotein (LDL) (d 1.019-1.063 g/ml) as in high density lipoproteins HDL2 (d 1.063-1.125 g/ml) or HDL3 (d 1.125-1.21 g/ml). In abetalipoproteinemia the levels of serum GSL were slightly reduced when compared to normal serum and were all found in the only existing lipoprotein, HDL; this contained 2-3 moles of GSL/ mole of lipoprotein as compared to 0.5 GSL/mole in normal HDL. In hypobetalipoproteinemia and Tangier disease, the serum glycosphingolipids were 10 to 30% reduced in concentration compared to the 75% reduction in other lipids, and were again found to be associated only with the serum lipoproteins. The relative proportions of GSL did not vary substantially in the normo- and hypolipidemic subjects studied. Only in patients with homozygous familial hypercholesterolemia was there a significant (3-4-fold) elevation of all of the five GSL species and this elevation of all of the five GSL species and this elevation correlated well with that of the circulating cholesterol and LDL. On a molar basis the LDL of these patients contained the same amount of GSL as normal subjects (5 moles GSL/mole protein). It is concluded that: (1) glycosphingolipids are associated only with the major lipoprotein classes in both normal and dyslipoproteinemic serum; (2) the relative proportions of the five glycosphingolipids are not significantly affected by dyslipoproteinemia; (3) only in severe hypolipoproteinemia do the remaining serum lipoproteins carry a complement of glycosphingolipids greater than normal. Although our results establish that glycosphingolipids are intimately associated with serum lipoproteins, the mode of association or the structural and functional significance of such an association remains undetermined.     

Metabolism of lipoproteins in nonhuman primates. Studies on the origin of low density lipoprotein apoprotein in the plasma of the squirrel monkey.

The plasma of squirrel monkeys contains extremely low levels of very low density lipoproteins. The delipidated apoproteins from the different lipoprotein density classes of this species show a heterogeneity similar to that of man and the rat. The biosynthesis of the apoproteins of squirrel monkey lipoproteins was studied in fasted normal and Triton WR1339-treated animals. After intravenous injection of [3-H] leucine, maximal labeling of very low density lipoproteins occurred after 1 h, intermediate density lipoproteins (d 1.006--1.019) in 2 h, and low density lipoproteins after 3 h. At all times, however, low density lipoproteins had the greatest percentage of radioactivity. Polyacrylamide gel electrophoresis revealed that the apoprotein B moiety of very low density and intermediate density lipoproteins contained 62% and 81% of the total radioactivity in these lipoproteins whereas the fast-migrating peptides were minimally labeled. In monkeys injected with Triton WR1339, 70--80% of the radioactivity incorporated into d smaller than 1.063 lipoproteins was in very low density lipoproteins with only 10--15% in intermediate and low density lipoproteins. After injection of 3-H-labeled very low density lipoproteins and [14-C] leucine into Triton-treated monkeys, catabolism of 3-H-labeled very low density lipoprotein to intermediate and low density lipoproteins was small and was significantly less than corresponding values for the incorporation of [14-C] leucine. Thus, breakdown of very low density lipoproteins could not account for all the labeled apoprotein B present in the intermediate and low density lipoprotein fractions. The results indicate that most, but not all, of the newly synthesized apoprotein B enters plasma in very low density lipoproteins and that the low concentrations of this lipoprotein in squirrel monkey plasma are a consequence of its rapid turnover.     

Neutral glycosphingolipids of serum lipoproteins in Fabry's disease.

The neutral glycosphingolipid compositions of lipoprotein fractions of serum from eight healthy male volunteers and three patients with Fabry's disease were determined. Four fractions were studied: very low density lipoprotein (VLDL, d less than 1.006); low density lipoprotein (LDL, d 1.006-1.063); high density lipoprotein (HDL, d 1.063-1.21); and ultracentrifugal residue (Residue, d less than 1.21). All lipoprotein fractions contained the four major neutral glycosphingolipids (glucosylceramide, lactosylceramide, galactosylgalactosylglucosylceramide and N-acetylgalactosaminylgalactosylgalactosylglucosylceramide). The LDL and HDL, however, accounted for most of the total glycosphingolipid (69 and 20%, respectively); only small amounts were demonstrated in the VLDL and Residue. The relative distributions of the glycosphingolipids within the LDL and HDL fractions were similar to the distribution in unfractionated serum. Galactosylgalactosylglucosylceramide levels were 3-5 times normal in all three patients with Fabry's disease, and in two the distribution of the excess lipid among the major lipoprotein fractions was similar to that in the control group. In the third patient, who exhibited the presence of "sinking pre-beta lipoprotein", the amount of glycosphingolipid isolated with the HDL was greater than that in the LDL.     

Human chylomicron apolipoprotein metabolism.

Chylomicron apolipoprotein metabolism was studied utilizing chylomicrons isolated from the pleural fluid of a patient with a recurrent chylous pleural effusion. Chylomicrons contained apolipoproteins A-I, A-II, B, C-I, C-II, C-III, D, E, and albumin. Following intravenous injection of [¹²⁵I] chylomicrons, almost all of the A apolipoprotein radioactivity was recovered in high density lipoproteins, while only a small amount of the B apolipoprotein radioactivity was recovered in low density lipoproteins. These observations indicate that intestinal chylomicron A apolipoproteins serve as precursors for plasma high density lipoprotein A apolipoproteins and only a small fraction of chylomicron apolipoprotein B is metabolized to form low density lipoprotein apolipoprotein B.     

In vitro transfer of chylomicron retinol and retinyl esters

The redistribution of rat chylomicron retinoids following incubation with fasting- or postheparin human plasma was investigated. With fasting plasma, chylomicron retinol appeared among higher density lipoprotein acceptors and density greater than 1.21 gm/ml plasma proteins; only small amounts of retinyl ester were found therein. With postheparin plasma, retinyl ester-containing chylomicron remnants with densities spanning the low- and high density lipoprotein distributions were generated; appreciable quantities of retinyl esters appeared among rho greater than 1.019 lipoproteins only in the presence of postheparin plasma. These observations are consistent with the conservation of retinyl esters, but not retinol, among chylomicrons and their catabolic products.     

Role of plasma lecithin:cholesterol acyltransferase in the metabolism of high density lipoproteins

The role of the plasma lecithin:cholesterol acyltransferase reaction in the esterification of the cholesterol of human and baboon plasma high density lipoproteins has been studied. Human plasma was incubated in vitro, and the initial rate of cholesterol esterification in lipoprotein fractions obtained by chromatography on hydroxylapatite was determined. The rate of esterification was greater in the high density lipoprotein fraction than in the low density lipoprotein fraction. High density lipoproteins from human and baboon plasma were filtered through columns of Sephadex G 200, and the relative concentrations in the effluent of key lipids involved in the acyltransferase reaction were determined. The ratio of esterified to unesterified cholesterol varied across the lipoprotein peak obtained from either type of plasma. The relative concentration of lecithin compared to sphingomyelin also varied across the peaks obtained with human high density lipoproteins. When human or baboon plasma was incubated with cholesterol-(14)C and the high density lipoproteins were filtered through Sephadex, the specific activity of the esterified cholesterol varied across the lipoprotein peak. Similar results were obtained when plasma esterified cholesterol was labeled in vivo by the injection of labeled mevalonate into baboons. The data suggest that the acyltransferase reaction is the major source of the esterified cholesterol of the high density lipoproteins.     

Binding of chylomicron remnants and beta-very low density lipoproteins to hepatic and extrahepatic lipoprotein receptors. A process independent of apolipoprotein B48

The ability of apolipoprotein (apo-) B48 to interact with lipoprotein receptors was investigated using three different types of lipoproteins. First, canine chylomicron remnants, which contained apo-B48 as their primary apoprotein constituent, were generated by the hydrolysis of chylomicrons with milk lipoprotein lipase. These apo-B48-containing chylomicron remnants are deficient in apo-E and reacted very poorly with apo-E receptors on adult dog liver membranes and the low density lipoprotein (apo-B,E) receptors on human fibroblasts. Addition of normal human apo-E3 restored the receptor binding activity of these lipoproteins. Second, beta-very low density lipoproteins (beta-VLDL) from cholesterol-fed dogs were subfractionated into distinct classes containing apo-E along with either apo-B48 or apo-B100. Both classes bound to the apo-B,E and apo-E receptors. Their binding was almost completely mediated by apo-E, as evidenced by the ability of the anti-apo-E to inhibit the receptor interaction. Third, beta-VLDL from type III hyperlipoproteinemic patients were subfractionated by immunoaffinity chromatography into lipoproteins containing apo-E plus either apo-B48 or apo-B100. Both subfractions bound poorly to apo-B,E and apo-E receptors due to the presence of defective apo-E2. However, the residual binding of the apo-B48-containing and apo-B100-containing human beta-VLDL was inhibited by the anti-apo-E. After lipase hydrolysis, apo-B100 became a more prominant determinant responsible for mediating receptor binding to the apo-B,E receptor. By contrast, lipase hydrolysis did not increase the binding activity of the apo-B48-containing beta-VLDL. These results indicate that apo-B48 does not play a direct role in mediating the interaction of lipoproteins with receptors on fibroblasts or liver membranes.     

Lipoproteins: carriers of dehydroepiandrosterone fatty acid esters in human serum

The association between lipoproteins, cholesterol and cholesteryl esters is very well known to facilitate both the transport in plasma and the entry of these non-polar compounds into the cellular compartment. However, recent observations suggest that in addition to cholesterol, lipoproteins contain several other steroids in their lipoidal metabolite forms which may be transported in the very low, low and high density lipoproteins in human serum. Using the important androgen and oestrogen precursor, dehydroepiandrosterone (DHEA), the biosynthetic formation of lipoidal DHEA was demonstrated in human serum. Serum was also fractionated into its lipoprotein components during the course of its incubation with tritiated DHEA. A progressive movement of the label from the fraction containing the conventional steroid binding-proteins to the lipoproteins was observed with the fraction containing the low density lipoproteins demonstrating the greatest incorporation of the label. This displacement occurred simultaneous to an extensive esterification of the labelled DHEA in serum. After 6 h of incubation, approx. 90% of the radioactivity in all the lipoprotein fractions was in the lipoidal form. Very little labelled lipoidal DHEA was associated with the serum protein fraction throughout the duration of incubation. These data suggest that lipoproteins act as the carriers of lipoidal DHEA following its formation from the non-conjugate parent steroid in serum.     

Distribution and functions of lecithin:cholesterol acyltransferase and cholesteryl ester transfer protein in plasma lipoproteins. Evidence for a functional unit containing these activities together with apolipoproteins A-I and D that catalyzes the esterification and transfer of cell-derived cholesterol

The distribution of apolipoprotein A-I, apolipoprotein D, lecithin:cholesterol acyltransferase, and cholesteryl ester transfer protein in fasting normal human plasma was determined by two-dimensional electrophoresis followed by immunoblotting. The synthesis and transfer of labeled cholesteryl esters generated in plasma briefly incubated with [3H]cholesterol-labeled fibroblasts was followed in terms of the lipoprotein species containing these antigens. Following the early appearance of labeled free cholesterol in two pre beta-migrating apolipoprotein A-I species (Castro, G. R., and Fielding, C. J. (1988) Biochemistry 27, 25-29), labeled esters were first detected, after a 2-min delay, in a third pre beta-migrating species which also contained apolipoprotein D, lecithin:cholesterol acyltransferase, and cholesteryl ester transfer protein. Pulse-chase experiments determined that label generated in this fraction was the precursor of at least a major part of labeled cholesteryl esters in the bulk of alpha-migrating high density lipoprotein. Over the maximum time course of these experiments (15 min, 37 degrees C), less than 10% of labeled cholesteryl esters were recovered in low or very low density lipoproteins separated by electrophoresis, immunoaffinity, or heparin-agarose chromatography. These data suggest channeling of cell-derived cholesterol and cholesteryl esters derived from it through a preferred pathway involving several minor pre beta-migrating lipoproteins to alpha-migrating high density lipoprotein.     

Studies on the isolation and partial characterization of apolipoprotein D and lipoprotein D of human plasma.

This report describes further studies on the characterization of apolipoprotein D (ApoD), a recently recognized human plasma apolipoprotein, and presents results on the isolation and distribution of its lipoprotein form, lipoprotein D (LP-D). ApoD, isolated by a procedure combining hydroxylapatite and Sephadex G-100 column chromatography, migrated on 7% polyacrylamide gel as a single band with a mobility intermediate between those of A-II and C-II polypeptides. On double diffusion and immunoelectrophoresis, ApoD reacted only with antiserum to ApoD. It was characterized by the presence of all common amino acids including half-cystine. The amino terminal acid was blocked. Carbohydrate analysis demonstrated that ApoD is a glycoprotein with glucose, mannose, galactose, glucosamine, and sialic acid accounting for 18% of the dry weight of ApoD. The estimated molecular weight of ApoD IS 22 100. ApoD occurs in the serum as a lipoprotein which was isolated from high density lipoproteins3 by two different chromatographic procedures. In the first procedure, high density lipoproteins3 were treated with neuraminidase and chromatographed on concanavlin A. The retained fraction containing LP-D was purified by hydroxylapatite column chromatography. Alternatively, LP-D was isolated by a procedure combining chromatography of high density lipoproteins3 or whole serum on an immunosorber containing antibodies to ApoD, and hydroxylapatite column chromatography. LP-D displayed a single, symmetrical boundary in the analytical ultracentrifuge and a single band on 7% polyacrylamide gel electrophoresis. When injected into rabbits it produced antisera that reacted only with ApoD. On immunoelectrophoresis LP-D had a mobility different from that of lipoprotein A (LP-A). A direct immunological comparison of LP-D and LP-A showed a reaction of nonidentity. LP-D consists of 65-75% protein and 25-35% lipid. The lipid moiety contains cholesterol, cholesterol ester, triglyceride, and phospholipid. The phospholipid. composition is characterized by a relative high content of lysolecithin and sphingomyelin and a relatively low content of lecithin. We have concluded from these studies that ApoD is a unique apolipoprotein that exists in the form of a distinct lipoprotein family with a macromolecular distribution extending from very low density lipoproteins into very high density lipoproteins, but with a maximum concentration in high density lipoproteins3 and a minimum concentration in high density lipoproteins.