Apolipoprotein A-II/A-I ratio is a key determinant in vivo of HDL concentration and formation of pre-beta HDL containing apolipoprotein A-II

Overexpression of human apolipoprotein A-II (apo A-II) in mice induced postprandial hypertriglyceridemia and marked reduction in plasma HDL concentration and particle size [Boisfer et al. (1999) J. Biol. Chem. 274, 11564-11572]. We presently compared lipoprotein metabolism in three transgenic lines displaying plasma concentrations of human apo A-II ranging from normal to 4 times higher, under ad libitum feeding and after an overnight fast. Fasting dramatically decreased VLDL and lowered circulating human apo A-II in transgenic mice; conversely, plasma HDL levels increased in all genotypes. The apo A-I content of HDL was inversely related to the expression of human apo A-II, probably reflecting displacement of apo A-I by an excess of apo A-II. Thus, the molar ratios of apo A-II/A-I in HDL were significantly higher in fed as compared with fasted animals of the same transgenic line, while endogenous LCAT activity concomitantly decreased. The number and size of HDL particles decreased in direct proportion to the level of human apo A-II expression. Apo A-II was abundantly present in all HDL particles, in contrast to apo A-I mainly present in large ones. Two novel findings were the presence of pre-beta migrating HDL transporting only human apo A-II in the higher-expressing mice and the increase of plasma HDL concentrations by fasting in control and transgenic mice. These findings highlight the reciprocal modifications of VLDL and HDL induced by the feeding-fasting transition and the key role of the molar ratio of apo A-II/A-I as a determinant of HDL particle metabolism and pre-beta HDL formation.     

Stable isotope turnover of apolipoproteins of high-density lipoproteins in humans

Amino acid precursors labelled with stable isotopes have been successfully used to explore the metabolism of the apolipoproteins of HDL. Some methodological and mathematical modelling problems remain, mainly related to amino acid recycling in a plasma protein such as apolipoprotein A-I with a long residence time (the reciprocal of the fractional catabolic rate) of 4-5 days. Apolipoprotein A-I, apolipoprotein E, and apolipoprotein A-IV in triglyceride-rich lipoproteins (containing chylomicrons, VLDL, and remnants) exhibit more complex kinetics. The small amounts of apolipoprotein A-I and of apolipoprotein A-IV in the triglyceride-rich lipoproteins have a residence time similar to that of the apolipoprotein A-I of HDL. In contrast, the apolipoprotein E in triglyceride-rich lipoproteins has been found to have an average residence time of 0.11 days. Diets low in saturated fat and cholesterol, which lower HDL levels, do so by decreasing the secretion of apolipoprotein A-I, with apolipoprotein A-II kinetics unaffected. Individuals with impaired glucose tolerance have a decreased residence time of apolipoprotein A-I but no change in secretion rate or in apolipoprotein A-II kinetics. This suggests a link between insulin resistance and the risk of atherosclerosis. In heterozygous familial hypercholesterolemia, both the fractional catabolic rate and the secretion rate of apolipoprotein A-I are increased, resulting in no change in the plasma level. Stable isotope studies have strengthened the evidence that triglyceride enrichment of HDL increases its catabolism Laboratory.     

High density lipoprotein particle size distribution in subjects with obstructive jaundice

High density lipoproteins (HDL) from 14 patients with obstructive jaundice were examined by gradient gel electrophoresis to determine the effect of obstruction on particle size distribution. HDL from 7 of these patients were fractionated by gel permeation chromatography and further characterized by electron microscopy, SDS gel electrophoresis, apolipoprotein A-I and apolipoprotein A-II immunoturbidimetry, and analysis of chemical composition. In addition, lecithin:cholesterol acyltransferase (LCAT) activity was measured and correlated with plasma apolipoprotein A-I concentration and particle size distribution. HDL were abnormal in all patients regardless of severity, cause, or duration of obstruction. The major HDL subfraction in normal subjects, HDL3a (radius 4.1-4.3 nm) was either absent or considerably diminished, and HDL2b (radius 5.3 nm) was also frequently absent. Very small particles comparable in size to normal HDL3c (radius 3.8 nm) were prominent. In patients with a bilirubin concentration greater than 250 mumol/l, normal HDL had totally disappeared and were replaced by large discoidal particles of radius 8.5 nm and small spherical particles of radius 3.6-3.7 nm. Both populations of particles were markedly depleted of cholesteryl ester and enriched in free cholesterol and phospholipid. The discoidal particles were rich in apolipoproteins E, A-I, A-II, and C, while the small spherical particles contained predominantly apolipoprotein A-I. LCAT activity was diminished in all subjects to 8-54% of normal, and was strongly positively correlated (r = 0.91 P less than 0.05) with plasma apolipoprotein A-I levels.     

Subpopulations of apolipoprotein A-I in human high-density lipoproteins. Their metabolic properties and response to drug therapy

In this study immunological procedures were used to detect and quantify high-density lipoprotein (HDL) particles of differing apolipoprotein A composition. In the plasma of eight healthy female subjects, 45% of the total apolipoprotein A-I existed in particles (called '(AI)HDL') devoid of apolipoprotein A-II. The remainder circulated in association with apolipoprotein A-II at a molar ratio of approximately 1:1. Nicotinic acid selectively raised the plasma apolipoprotein A-I/A-II ratio by increasing the proportion of (AI)HDL particles. Probucol produced the opposite effect, lowering the plasma concentration of these particles. The kinetic properties of apolipoprotein A-I in total HDL and in the (AI)HDL particle were the same despite the fact that apolipoprotein A-I equilibration between these two species was incomplete. Therefore, there appear to be at least two apolipoprotein A-containing particle populations in HDL which are immunochemically and metabolically distinct.     

Expression of human apolipoprotein A-II and its effect on high density lipoproteins in transgenic mice.

Apolipoproteins A-I and A-II comprise approximately 70 and 20%, respectively, of the total protein content of HDL. Evidence suggests that apoA-I plays a central role in determining the structure and plasma concentration of HDL, while the role of apoA-II is uncertain. To help define the function of apoA-II and determine what effect increasing its plasma concentration has on HDL, transgenic mice expressing human apoA-II and both human apoA-I and human apoA-II were produced. Human apoA-II mRNA is expressed exclusively in the livers of transgenic animals, and the protein exists as a dimer as it does in humans. High level expression of human apoA-II did not increase HDL concentrations or decrease plasma concentrations of murine apoA-I and apoA-II in contrast to what was observed in mice overexpressing human apoA-I. The primary effect of overexpressing human apoA-II was the appearance of small HDL particles composed exclusively of human apoA-II. HDL from mice transgenic for both human apoA-I and human apoA-II displayed a unique size distribution when compared with either apoA-I or apoA-II transgenic mice and contain particles with both these human apolipoproteins. These results in mice, indicating that human apoA-II participates in determining HDL size, parallel results from human studies.     

Isolation and identification of HDL particles of low molecular weight

Small particles of high density lipoproteins (HDL) were isolated from fresh, fasting human plasma and from the ultracentrifugally isolated high density lipoprotein fraction by means of ultrafiltration through membranes of molecular weight cutoff of 70,000. These particles were found to contain cholesterol, phospholipids, and apolipoproteins A-I and A-II; moreover, they floated at a density of 1.21 kg/l. They contained 67.5% of their mass as protein and the rest as lipid. Two populations of small HDL particles were identified: one containing apolipoprotein A-I alone [(A-I)HDL] and the other containing both apolipoproteins A-I and A-II [A-I + A-II)HDL]. The molar ratio of apoA-I to apoA-II in the latter subclass isolated from plasma or HDL was 1:1. The molecular weights of these subpopulations were determined by nondenaturing gradient polyacrylamide gel electrophoresis and found to be 70,000; 1.5% of the plasma apoA-I was recovered in the plasma ultrafiltrate.     

Effect of high-density lipoproteins with varying ratios of apolipoprotein A-I to apolipoprotein A-II on steroidogenesis by cultured rat ovary granulosa cells

Plasma high-density lipoproteins (HDL) can provide rat ovary steroidogenic tissue with cholesterol for steroid hormone production, but the mechanism of cholesterol transfer is unknown. To test the importance of apolipoprotein A-I (the major HDL apolipoprotein) in HDL-cell interactions, we examined the ability of canine-human HDL hybrids containing various proportions of canine apolipoprotein A-I and human apolipoprotein A-II to stimulate steroidogenesis by cultured rat ovary granulosa cells. We observed that as the apolipoprotein A-II to apolipoprotein A-II ratio decreased, the ability of the hybrid particles to stimulate granulosa cell progestin (progesterone and 20 alpha-dihydroprogesterone) production diminished. However, granulosa cell progestin (progesterone and 20 alpha-dihydroprogesterone) production diminished. However, apolipoprotein A-I was not necessary for cholesterol transfer, since hybrids with less than 5% of their total apolipoprotein mass as apolipoprotein A-I stimulated progestin production 30% as effectively as canine HDL, which contained essentially only apolipoprotein A-I. These data indicate that the delivery of cholesterol from HDL into the rat ovary cell for steroidogenesis is not strictly dependent on the presence of a specific HDL apolipoprotein.     

Endothelial lipase is less effective at influencing HDL metabolism in vivo in mice expressing apoA-II

Endothelial lipase (EL) plays an important physiological role in modulating HDL metabolism. Data suggest that plasma contains an inhibitor of EL, and previous studies have suggested that apolipoprotein A-II (apoA-II) inhibits the activity of several enzymes involved in HDL metabolism. Therefore, we hypothesized that apoA-II may reduce the ability of EL to influence HDL metabolism. To test this hypothesis, we determined the effect of EL expression on plasma phospholipase activity and HDL metabolism in human apoA-I and human apoA-I/A-II transgenic mice. Expression of EL in vivo resulted in lower plasma phospholipase activity and significantly less reduction of HDL-cholesterol, phospholipid, and apoA-I levels in apoA-I/A-II double transgenic mice compared with apoA-I single transgenic mice. We conclude that the presence of apoA-II on HDL particles inhibits the ability of EL to influence the metabolism of HDL in vivo.     

The role of apolipoprotein A-I and apolipoprotein A-II in high-density lipoprotein binding to human adipocyte plasma membranes

Adipocyte plasma membranes purified from omental fat tissue biopsies of massively obese subjects possess specific binding sites for high-density lipoprotein (HDL3). This binding was independent of apolipoprotein E as HDL3 isolated from plasma of an apolipoprotein E-deficient individual was bound to a level comparable to that of normal HDL3. To examine the importance of apolipoprotein A-I, the major HDL3 apolipoprotein, in the specific binding of HDL3 to human adipocytes, HDL3 modified to contain varying proportions of apolipoproteins A-I and A-II was prepared by incubating normal HDL3 particles with different amounts of purified apolipoprotein A-II. As the apolipoproteins A-I-to-A-II ratio in HDL3 decreased, the binding of these particles to adipocyte plasma membranes was reduced. Compared to control HDL3, a 92 +/- 3.1% reduction (mean +/- S.E., n = 3) in maximum binding capacity was observed along with an increased binding affinity for HDL3 particles in which almost all of the apolipoprotein A-I had been replaced by A-II. The uptake of HDL cholesteryl ester by intact adipocytes as monitored by [3H]cholesteryl ether labeled HDL3, was also significantly reduced (about 35% reduction, P less than 0.005) by substituting apolipoprotein A-II for A-I in HDL3. These data suggest that HDL binding to human adipocyte membranes is mediated primarily by apolipoprotein A-I and that optimal delivery of cholesteryl ester from HDL to human adipocytes is also dependent on apolipoprotein A-I.     

Isolation of apolipoproteins A-I, A-II and E and their estimation by rocket immunoelectrophoresis in blood plasma of dis-alpha-lipoproteinemic patients

The methods for isolation of pure apolipoproteins A-I, A-II and E from the blood plasma of donors for preparation of monospecific rabbit antisera against these apolipoproteins and their estimation in human blood plasma using immunoelectrophoresis are described. It was found that the average content of apolipoprotein A-I (apo A-I) in the blood plasma of healthy males is 126.6 mg%, that of apolipoprotein A-II (apo A-II) is 56.8 mg%, that of apolipoprotein E (apo E) is 10.2 mg%. The apo A-I content in blood plasma is increased in hyper-alpha-lipoproteinemic patients and is decreased in hypo-alpha-lipoproteinemic ones, i. e. there is a direct relationship between the changes in concentration of high density lipoproteins (HDL) and apo A-I. The concentration of apo A-II in dis-alpha-lipoproteinemias varies within a narrow range. A considerable increase of the alpha-cholesterol/apo A-I ratio suggesting an increased capacity of HDL to transport cholesterol in hyper-alpha-lipoproteinemic patients is observed. There exists an indirect correlation between the changes in the contents of apo A-I and apo E in dis-alpha-lipoproteinemic patients.     

ApoA-I deficiency in mice is associated with redistribution of apoA-II and aggravated AApoAII amyloidosis

Apolipoprotein A-II (apoA-II) is the second major apolipoprotein following apolipoprotein A-I (apoA-I) in HDL. ApoA-II has multiple physiological functions and can form senile amyloid fibrils (AApoAII) in mice. Most circulating apoA-II is present in lipoprotein A-I/A-II. To study the influence of apoA-I on apoA-II and AApoAII amyloidosis, apoA-I-deficient (C57BL/6J.Apoa1^−/−) mice were used. Apoa1^−/− mice showed the expected significant reduction in total cholesterol (TC), HDL cholesterol (HDL-C), and triglyceride (TG) plasma levels. Unexpectedly, we found that apoA-I deficiency led to redistribution of apoA-II in HDL and an age-related increase in apoA-II levels, accompanied by larger HDL particle size and an age-related increase in TC, HDL-C, and TG. Aggravated AApoAII amyloidosis was induced in Apoa1^−/− mice systemically, especially in the heart. These results indicate that apoA-I plays key roles in maintaining apoA-II distribution and HDL particle size. Furthermore, apoA-II redistribution may be the main reason for aggravated AApoAII amyloidosis in Apoa1^−/− mice. These results may shed new light on the relationship between apoA-I and apoA-II as well as provide new information concerning amyloidosis mechanism and therapy.     

Isolation and characterization of simple and complex lipoproteins containing apolipoprotein F from human plasma

Apolipoprotein F (ApoF), one of the minor apolipoproteins in human plasma, has been recently isolated and partially characterized [Olofsson, S.O., McConathy, W.J., & Alaupovic, P. (1978) Biochemistry 17, 1032-1036]. In the present work, the interaction of ApoF with other apolipoproteins and lipids in human plasma was studied. By the successive use of immunosorbers specific for ApoF, apolipoprotein A-II (ApoA-II) and apolipoprotein A-I (ApoA-I), three different ApoF-containing lipoproteins were isolated from normolipidemic fasting human plasma. Their apolipoprotein content was determined by double immunodiffusion against monospecific antisera to all known serum apolipoproteins, electroimmunoassay, crossed immunoelectrophoresis, and polyacrylamide gel electrophoresis. Their lipid composition was determined by thin-layer chromatography. The three ApoF-containing lipoproteins were identified as LpF:A-I:A-II (lipoprotein containing ApoF, ApoA-I, and ApoA:II), LpF:A-I (lipoprotein containing ApoF and ApoA-I), and LpF (lipoprotein containing only ApoF). LpF:A-I:A-II was found to contain ApoF, ApoA-I, and ApoA-II in an apparent 2:1:1 molar ratio. Its lipid moiety was characterized by cholesterol ester (45%) and free cholesterol (28%) as the predominant lipids. LpF contained only ApoF, and in its major lipid components were also cholesterol esters (63%) and free cholesterol (21%). It is suggested that ApoF-containing lipoproteins may be involved in transport and/or esterification of cholesterol.     

Formation of high density lipoproteins containing both apolipoprotein A-I and A-II in the rabbit

Human plasma HDLs are classified on the basis of apolipoprotein composition into those that contain apolipoprotein A-I (apoA-I) without apoA-II [(A-I)HDL] and those containing apoA-I and apoA-II [(A-I/A-II)HDL]. ApoA-I enters the plasma as a component of discoidal particles, which are remodeled into spherical (A-I)HDL by LCAT. ApoA-II is secreted into the plasma either in the lipid-free form or as a component of discoidal high density lipoproteins containing apoA-II without apoA-I [(A-II)HDL]. As discoidal (A-II)HDL are poor substrates for LCAT, they are not converted into spherical (A-II)HDL. This study investigates the fate of apoA-II when it enters the plasma. Lipid-free apoA-II and apoA-II-containing discoidal reconstituted HDL [(A-II)rHDL] were injected intravenously into New Zealand White rabbits, a species that is deficient in apoA-II. In both cases, the apoA-II was rapidly and quantitatively incorporated into spherical (A-I)HDL to form spherical (A-I/A-II)HDL. These particles were comparable in size and composition to the (A-I/A-II)HDL in human plasma. Injection of lipid-free apoA-II and discoidal (A-II)rHDL was also accompanied by triglyceride enrichment of the endogenous (A-I)HDL and VLDL as well as the newly formed (A-I/A-II)HDL. We conclude that, irrespective of the form in which apoA-II enters the plasma, it is rapidly incorporated into spherical HDLs that also contain apoA-I to form (A-I/A-II)HDL.     

Lower plasma levels and accelerated clearance of high density lipoprotein (HDL) and non-HDL cholesterol in scavenger receptor class B type I transgenic mice

Recent studies have indicated that the scavenger receptor class B type I (SR-BI) may play an important role in the uptake of high density lipoprotein (HDL) cholesteryl ester in liver and steroidogenic tissues. To investigate the in vivo effects of liver-specific SR-BI overexpression on lipid metabolism, we created several lines of SR-BI transgenic mice with an SR-BI genomic construct where the SR-BI promoter region had been replaced by the apolipoprotein (apo)A-I promoter. The effect of constitutively increased SR-BI expression on plasma HDL and non-HDL lipoproteins and apolipoproteins was characterized. There was an inverse correlation between SR-BI expression and apoA-I and HDL cholesterol levels in transgenic mice fed either mouse chow or a diet high in fat and cholesterol. An unexpected finding in the SR-BI transgenic mice was the dramatic impact of the SR-BI transgene on non-HDL cholesterol and apoB whose levels were also inversely correlated with SR-BI expression. Consistent with the decrease in plasma HDL and non-HDL cholesterol was an accelerated clearance of HDL, non-HDL, and their major associated apolipoproteins in the transgenics compared with control animals. These in vivo studies of the effect of SR-BI overexpression on plasma lipoproteins support the previously proposed hypothesis that SR-BI accelerates the metabolism of HDL and also highlight the capacity of this receptor to participate in the metabolism of non-HDL lipoproteins.     

On the structural similarity of ApoA-I and ApoA-II of human high density lipoproteins.

The possible evolutionary origin of apolipoproteins was studied by comparing the primary structures of different plasma apolipoproteins and other phospholipid-binding proteins. Apolipoprotein A-I (ApoA-I) and apolipoprotein A-II (ApoA-II) of human high density lipoprotein (HDL) are related. The resemblance of these two HDL apolipoproteins are apparently restricted to the carboxyl terminal regions suggesting that these portions of the molecules are derived from the same ancestor. The homologous carboxyl terminal segments may be involved in the regulation of HDL metabolism or in the interaction with phospholipids.     

Formation of spherical, reconstituted high density lipoproteins containing both apolipoproteins A-I and A-II is mediated by lecithin:cholesterol acyltransferase

Previous studies have provided detailed information on the formation of spherical high density lipoproteins (HDL) containing apolipoprotein (apo) A-I but no apoA-II (A-I HDL) by an lecithin:cholesterol acyltransferase (LCAT)-mediated process. In this study we have investigated the formation of spherical HDL containing both apoA-I and apoA-II (A-I/A-II HDL). Incubations were carried out containing discoidal A-I reconstituted HDL (rHDL), discoidal A-II rHDL, and low density lipoproteins in the absence or presence of LCAT. After the incubation, the rHDL were reisolated and subjected to immunoaffinity chromatography to determine whether A-I/A-II rHDL were formed. In the absence of LCAT, the majority of the rHDL remained as either A-I rHDL or A-II rHDL, with only a small amount of A-I/A-II rHDL present. By contrast, when LCAT was present, a substantial proportion of the reisolated rHDL were A-I/A-II rHDL. The identity of the particles was confirmed using apoA-I rocket electrophoresis. The formation of the A-I/A-II rHDL was influenced by the relative concentrations of the precursor discoidal A-I and A-II rHDL. The A-I/A-II rHDL included several populations of HDL-sized particles; the predominant population having a Stokes' diameter of 9.9 nm. The particles were spherical in shape and had an electrophoretic mobility slightly slower than that of the alpha-migrating HDL in human plasma. The apoA-I:apoA-II molar ratio of the A-I/A-II rHDL was 0.7:1. Their major lipid constituents were phospholipids, unesterified cholesterol, and cholesteryl esters. The results presented are consistent with LCAT promoting fusion of the A-I rHDL and A-II rHDL to form spherical A-I/A-II rHDL. We suggest that this process may be an important source of A-I/A-II HDL in human plasma.     

Isolation and partial characterization of lipoprotein A-II (LP-A-II) particles of human plasma.

High density lipoproteins (HDL) consist of a mixture of chemically and functionally distinct families of particles defined by their characteristic apolipoprotein (Apo) composition. The two major lipoprotein families are lipoprotein A-I (LP-A-I) and lipoprotein A-I:A-II (LP-A-I:A-II). This study describes the isolation of a third minor HDL family of particles referred to as lipoprotein A-II (LP-A-II) because it lacks ApoA-I and contains ApoA-II as its main or sole apolipoprotein constituent. Because ApoA-II is an integral protein constituent of three distinct lipoprotein families (LP-A-I:A-II, LP-A-II: B:C:D:E and LP-A-II), LP-A-II particles were isolated from whole plasma by sequential immunoaffinity chromatography on immunosorbers with antisera to ApoA-II, ApoB and ApoA-I, respectively. In normolipidemic subjects, the concentration of LP-A-II particles, based on ApoA-II content, is 4-18 mg/dl accounting for 5-20% of the total ApoA-II not associated with ApoB-containing lipoproteins. The lipid composition of LP-A-II particles is characterized by low percentage of triglycerides and cholesterol esters and a high percentage of phospholipids in comparison with lipid composition of LP-A-I and LP-A-II: A-II. The major part of LP-A-II particles contain ApoA-II as the sole apolipoprotein constituent; however, small subsets of LP-A-II particles may also contain ApoD and other minor apolipoproteins. The lipid/protein ratio of LP-A-II is higher than those of LP-A-I and LP-A-I:A-II. In homozygous ApoA-I and ApoA-I/ApoC-III deficiencies, LP-A-II particles are the only ApoA-containing high density lipoprotein with levels found to be within the same range (7-13 mg/dl) as those of normolipidemic subjects. However, in contrast to normal LP-A-II, their lipid composition is characterized by higher percentages of triglycerides and cholesterol esters and a lower percentage of phospholipids and their apolipoprotein composition by the presence of ApoC-peptides and ApoE in addition to ApoA-II and ApoD. These results show that LP-A-II particles are a minor HDL family and suggest that, in the absence of ApoA-I-containing lipoproteins, they become an efficient acceptor/donor of ApoC-peptides and ApoE required for a normal metabolism of triglyceride-rich lipoproteins. Their other possible functional roles in lipid transport remain to be established in future experiments.     

Expression of serum amyloid A protein in the absence of the acute phase response does not reduce HDL cholesterol or apoA-I levels in human apoA-I transgenic mice

Plasma concentrations of high density lipoprotein (HDL) cholesterol and its major apolipoprotein (apo)A-I are significantly decreased in inflammatory states. Plasma levels of the serum amyloid A (SAA) protein increase markedly during the acute phase response and are elevated in many chronic inflammatory states. Because SAA is associated with HDL and has been shown to be capable of displacing apoA-I from HDL in vitro, it is believed that expression of SAA is the primary cause of the reduced HDL cholesterol and apoA-I in inflammatory states. In order to directly test this hypothesis, we constructed recombinant adenoviruses expressing the murine SAA and human SAA1 genes (the major acute phase SAA proteins in both species). These recombinant adenoviruses were injected intravenously into wild-type and human apoA-I transgenic mice and the effects of SAA expression on HDL cholesterol and apoA-I were compared with mice injected with a control adenovirus. Plasma levels of SAA were comparable to those seen in the acute phase response in mice and humans. However, despite high plasma levels of murine or human SAA, no significant changes in HDL cholesterol or apoA-I levels were observed. SAA was found associated with HDL but did not specifically alter the cholesterol or human apoA-I distribution among lipoproteins. In summary, high plasma levels of SAA in the absence of a generalized acute phase response did not result in reduction of HDL cholesterol or apoA-I in mice, suggesting that there are components of the acute phase response other than SAA expression that may directly influence HDL metabolism.     

Characterization of A-I-containing lipoproteins in subjects with A-I Milano variant

The A-I Milano variant of apolipoprotein A-I (A-IM), by virtue of its Arg-173----Cys substitution, is capable of forming a disulfide bond with the 77-amino-acid apolipoprotein A-II polypeptide (A-IIS) as well as with itself to produce dimers, A-IM/A-IIS and A-IM/A-IM, respectively. A-I-containing lipoproteins (Lp): particles with A-II (Lp(A-I with A-11)) and particles without A-II (Lp(A-I without A-II)) in the plasma of two nonhyperlipidemic A-IM carriers were investigated to determine the effect of A-IM on these lipoproteins. Despite the existence of abnormal apolipoprotein dimers and the unusually low HDL cholesterol (17 and 14 mg/dl), A-I (67 and 75 mg/dl), and A-II (18 and 18 mg/dl) levels in the two carriers, the plasma A-I of the carriers was distributed between Lp(A-I with A-II) and Lp(A-I without A-II) in a proportion comparable to that observed in normals. As expected, A-IM/A-IIS mixed dimer was found in carrier Lp(A-I with A-II). However, A-IM/A-IM dimer was located almost exclusively in carrier Lp(A-I without A-II). Chemical (dimethylsuberimidate) crosslinking of the protein moieties of the major subpopulations of Lp(A-I with A-II) and Lp(A-I without A-II) of normal and A-IM carriers showed that Lp(A-I with A-II), which is located predominantly in the 7.8-9.7 nm interval ((HDL2a + 3a + 3b)gge), had an apparent protein molecular weight equivalent to two molecules of A-I and one to two molecules of A-II per particle. Most of the Lp(A-I without A-II) particles, located predominantly in the size intervals of 9.7-12.9 nm (designated (HDL2b)gge) and 8.2-8.8 nm (HDL3a)gge) had protein moieties exhibiting a molecular weight equivalence predominantly of four and three molecules of A-I, respectively. A small quantity of particles with apparent protein content of two molecules of A-I in the 7.2-8.2 nm interval ((HDL3b + 3c)gge) was also detected. These studies showed that in nonhyperlipidemic A-IM carriers, the occurrence of apolipoprotein dimers had not markedly affected the protein stoichiometry of Lp(A-I with A-II) and Lp(A-I without A-II).