Immune complex hyperlipidemia induced by an apolipoprotein-reactive immunoglobulin A paraprotein from a patient with multiple myeloma. Characterization of this immunoglobulin.

An antibodylike paraprotein has been isolated from a patient with multiple myeloma and autoimmune hyperlipoproteinemia. The paraprotein bound to apolipoprotein B (apo B)-containing lipoproteins that formed macromolecular aggregates, and globules thought to be aggregated complexes of lipoproteins and reactive immunoglobulins were observed circulating within the retinal blood vessels of this patient. This binding specificity permitted purification of the paraprotein from both the agglutinated immune complexes and from the plasma. The protein is an IgA, kappa-immunoglobulin which exists primarily in a polymeric state. Capillary immunoprecipitation demonstrated reactivity with very low density lipoproteins (VLDL) and low density proteins (LDL), but not with high density lipoproteins (HDL). Delipidated apo B and apo E, but not apo A or apo C, formed precipitates with this immunoglobulin. In using a radioimmunoassay format, the affinity of the immunoglobulin was greatest for VLDL and decreases sequentially for intermediate density lipoproteins and LDL. No binding occurred with a dispersion of LDL lipids or with HDL. Deglycosylation did not change the binding to LDL. The apolipoproteins B and E bound with similar affinity, but no binding occurred with apo A-I or apo A-II. Weak binding appeared to occur with apo C. This paraprotein immunoprecipitated apo B-containing lipoproteins from all classes of vertebrates tested. Displacement of the lipids of LDL by Triton X-100 resulted in the formation of an apo B-Triton complex which, however, did not bind to the immunoglobulin; apparently the binding site on apo B was lost. Upon enzymatic digestion with the IgA-specific protease from Streptococcus sanguis the immunoglobulin was cleaved into Fc and Fab fragments, and the binding of LDL occurred only with the latter, consistent with the behavior of an immunoglobulin. The immunoreactivity of this paraprotein with apo B and apo E raises the interesting possibility that it may be binding to a site on these apolipoproteins which is reactive with the apo B, E receptor of the plasma membrane, a site which is conserved throughout the vertebrate phylum.

Lipodystrophic diabetes mellitus. Investigations of lipoprotein metabolism and the effects of omega-3 fatty acid administration in two patients.

We investigated the metabolic effects of omega-6 (safflower oil) and omega-3 (fish oil) fatty acid-enriched diets (65% carbohydrate, 20% fat) in two patients with a syndrome of diabetes mellitus, lipodystrophy, acanthosis nigricans, chylomicronemia, and abdominal pain. 3H-glycerol was used to evaluate triglyceride-rich lipoprotein-triglyceride (TRLP-TG) metabolism, and changes in glucose and insulin dynamics were also studied. On the omega-6 diet, both subjects demonstrated four- to five-times normal rates of TRLP-TG production and glycerol biosynthesis, and striking decrements in the fractional catabolic rate (FCR) for TRLP-TG and TRLP-particles. Both subjects had elevations in nonesterified fatty acid (NEFA) concentrations. In one patient, the omega-3 diet markedly decreased serum triglycerides and newly synthesized triglyceride glycerol production, in association with a fall in NEFA. In both subjects, plasma glycerol reutilization for triglyceride synthesis, normal on the omega-6 diet, was abolished on the omega-3 regimen. Plasma postheparin lipolytic activity was normal on both diets. On the omega-3 diet, xanthomas and hepatomegaly decreased and, in the patient who had no reduction in serum triglycerides, pancreatitis attacks virtually ceased. Mean 24-hour serum glucose levels were higher, and both basal and peak C-peptide responses to a carbohydrate meal were blunted on the omega-3 diet. One patient became ketonuric. We conclude the cause of hypertriglyceridemia in these patients was due to increased lipid synthesis and hypothesize that this is secondary to high plasma concentrations of NEFA. In addition, an omega-3 diet in these subjects inhibited insulin secretion and worsened glucose tolerance.

Immunologic comparison of the conformations of apolipoprotein B. Investigation of methodologies for the reconstitution of delipidated and denatured apolipoprotein B with nonionic surfactants.

Immunologic probes have been used to examine the conformation of apolipoprotein B (apo-B) as it exists within native low density lipoprotein (LDL) after lipid displacement with Triton X-100 and after denaturation with guanidine hydrochloride organic solvent delipidation and reconstitution with Triton X-100. Antigenic expression was assayed in two systems: by using either Triton X-100 or bovine serum albumin to maintain protein solubility. Apo-B delipidated by lipid displacement using Triton X-100 was virtually identical to LDL-apo-B in both systems, as assayed by polyclonal antisera prepared in rabbits against either antigen. Thus the native antigenic sites are preserved, although the displacement of the lipid core of LDL drastically alters the physical properties of the particle. Apo-B delipidated by solvent extraction in guanidine was reconstituted with Triton X-100 by several methods, and the products were examined immunologically. One method yielded a product that resembled apo-B as delipidated with Triton X-100, although full reconstitution could not be achieved. Nevertheless, Triton promoted refolding of apo-B to reform partial native structure as judged immunologically. By using both physical and immunologic methods for assessing structure, it is clearly evident that the perceptions of the conformational states of reconstituted apo-B can be very different, and multiple criteria need to be used to assess lipoprotein reconstitution.

Solubilization of apolipoprotein B and its specific binding by the cellular receptor for low density lipoprotein.

Low density lipoprotein (LDL) and very low density lipoprotein (VLDL) bind specifically to a receptor on fibroblasts, and it has been postulated that the apoprotein of LDL (apo B) confers the specificity of cellular binding. This hypothesis has been tested in the present study with a watersoluble apo B-bovine serum albumin complex. The binding of (125)I-labeled apo B to cultured fibroblasts was temperature-dependent. Specific binding ranged between 183 and 859 ng/mg of cell protein at a concentration of 5 mug/ml; at 37 degrees , 750-2199 ng/mg was bound and internalized. The binding of apo B greatly exceeded the amount of (125)I-labeled LDL bound at 4 degrees and 37 degrees in the same experiment. Fibroblasts from a subject homozygous for hyper-beta-lipoproteinemia showed minimal binding of (125)I-labeled LDL, consistent with the absence of the cellular LDL receptor. Such cells also had depressed binding of (125)I-labeled apo B. Lymphocytes grown in lipoprotein-deficient medium demonstrated specific binding of LDL; however, freshly isolated lymphocytes did not show such binding. The binding of (125)I-labeled apo B to lymphocytes paralleled the binding of (125)I-labeled LDL. Unlabeled LDL and apo B-albumin complex both competitively inhibited the binding of (125)I-labeled apo B and (125)I-labeled LDL to fibroblasts. When labeled LDL was incubated with fibroblasts for 6 hr at 37 degrees , it underwent cellular internalization and degradation, as measured by the release of (125)I-labeled fragments into the medium. This degradation was inhibited by unlabeled apo B. Conversely, (125)I-labeled apo B also was internalized and degraded by fibroblasts, and this process was inhibited by LDL. These findings demonstrate that apo B binds specifically to the LDL receptor and that the cellular binding of LDL is determined by this apoprotein.

Metabolic pathways of apolipoprotein B in heterozygous familial hypercholesterolemia: studies with a [3H]leucine tracer.

The kinetics of apolipoprotein B (apoB) were measured in seven studies in heterozygous, familial hypercholesterolemic subjects (FH) and in five studies in normal subjects, using in vivo tracer kinetic methodology with a [3H]leucine tracer. Very low density (VLDL) and low density lipoproteins (LDL) were isolated ultracentrifugally and LDL was fractionated into high and low molecular weight subspecies. ApoB was isolated, its specific radioactivity was measured, and the kinetic data were analyzed by compartmental modeling using the SAAM computer program. The pathways of apoB metabolism differ in FH and normal subjects in two major respects. Normals secrete greater than 90% of apoB as VLDL, while one-third of apoB is secreted as intermediate density lipoprotein IDL/LDL in FH. Normals lose 40-50% of apoB from plasma as VLDL/IDL, while FH subjects lose none, metabolizing all of apoB to LDL. In FH, there is also the known prolongation of LDL residence time. The leucine tracer, biosynthetically incorporated into plasma apoB, permits distinguishing the separate pathways by which the metabolism of apoB is channeled. ApoB synthesis and secretion require 1.3 h. ApoB is secreted by three routes: 1) as large VLDL where it is metabolized by a delipidation chain; 2) as a rapidly metabolized VLDL fraction converted to LDL; and 3) as IDL or LDL. ApoB is metabolized along two pathways. The delipidation chain processes large VLDL to small VLDL, IDL, and LDL. The IDL pathway channels nascent, rapidly metabolized VLDL and IDL particles into LDL. It thus provides a fast pathway for the entrance of apoB tracer into LDL, while the delipidation pathway is a slower route for channeling apoB through VLDL into LDL. LDL apoB is derived in almost equal amounts from both pathways, which feed predominantly into large LDL. Small LDL is a product of large LDL, and the major loss of LDL-apoB is from small LDL. Two features of apoB metabolism in FH, the major secretory pathway through IDL and the absence of a catabolic loss of apoB from VLDL/IDL, greatly facilitate measuring the metabolic channeling of apoB into LDL.

Structural studies on apolipoprotein B: controllable heterogeneity of the complex formed with the surfactant, Triton X-100.

Apolipoprotein B complexed with Triton X-100 (T-ApoB) has been isolated from human low density lipoprotein (LDL). Preparations are heterogeneous when analyzed by sedimentation velocity, with a major 12 S species and minor 17 S species present. The 12 S T-ApoB complex possesses a molecular weight of 880,000 containing 400,000 daltons of protein. Hydrodynamic measurements on this complex are consistent with a prolate ellipsoid model having an axial ratio of 13:1 and 0.22 g/g of bound water. Heterogeneity results from the irreversible aggregation of 12 S complexes into discrete 17 S and faster sedimenting components. A significant finding is that three determinants of this T-apoB heterogeneity could be elucidated and controlled. First, the initial state of aggregation is mainly influenced by the technique by which Triton and LDL are mixed. Second, once isolated, T-ApoB complexes slowly but spontaneously undergo further aggregation at 4 degrees C; the rate and extent of aggregation is enhanced remarkably with increasing temperature. Finally, reagents that unfold and expose protein structure (perchlorate, thiocyanate, and reducing reagents) lead to increased aggregation. The ability to control heterogeneity carries important implications for other studies concerning interactions of apoB with surfactants and lipids.

Nutritional regulation of cholesterol synthesis and apolipoprotein B kinetics: studies in patients with familial hypercholesterolemia and normal subjects treated with a high carbohydrate, low fat diet.

High carbohydrate, low fat diets decrease plasma low-density lipoprotein cholesterol (LDL-C) and apolipoprotein B (apoB) mass in normal subjects and in patients with familial hypercholesterolemia (FH). To investigate the mechanisms for these effects, four normal, four FH heterozygous, and one FH homozygous subjects were studied on a basal (45% carbohydrate, 40% fat) diet and during continuous nasogastric infusion of Vivonex (90% carbohydrate, 1% fat). For the entire group, the mean changes in total cholesterol, LDL-C, high-density lipoprotein cholesterol (HDL-C) and triglycerides were -90, -95, -14 (all P less than 0.01) and +114 (P less than 0.02) mg/dl, respectively. Fecal sterol balance measurements demonstrated a 24% decrease in whole body cholesterol synthesis in normals, from 8.4 +/- 4.4 (mean +/- SD) to 6.4 +/- 1.3 mg/kg per day and in FH subjects, a 58% decrease, from 11.4 +/- 5.6 to 4.8 +/- 1.7 mg/kg per day (both P less than 0.05). ApoB kinetic studies were performed using a [3H]leucine tracer in two normals and three FH heterozygotes on both basal and Vivonex regimens, and the results were analyzed by compartmental modeling using the SAAM program. Total apoB production was not altered in a consistent manner by carbohydrate feeding. ApoB secretion, however, was shifted from the production of small VLDL/IDL-like particles to large VLDL by Vivonex, with an accompanying increase in intrahepatic assemblage time before secretion. In the two normal subjects, Vivonex induced an increase in apoB loss as VLDL/IDL; however, in the FH patients no such loss occurred. A decrease (P less than 0.05) in the residence time of LDL-apoB occurred for all subjects and was the primary determinant of the fall in plasma LDL concentration, since LDL-apoB transport did not change consistently. Thus, in FH patients, a high carbohydrate, low fat diet results in suppression of cholesterol synthesis and a fall in plasma LDL concentration due to an increased plasma clearance rate for LDL.

The determination of specific radioactivity of proteins eluted intact from polyacrylamide gels, utilizing a fluorescamine assay.

The methodology described permits the measurement of the specific radioactivity of diverse proteins resolvable by separatory techniques using cylindrical polyacrylamide gels. Following separation, the proteins are electroeluted; eluted protein is quantitated in the microgram range using a fluorescamine assay, while the major portion of the recovered sample is used for radioactivity measurement. These procedures have been adapted for use in tracer studies of protein metabolism. Their utility in kinetic investigations is demonstrated with data on the time course of changing specific radioactivities of human plasma albumin and apolipoprotein B labeled in vivo with a [3H]leucine tracer.

Kinetic evidence for both a fast and a slow secretory pathway for apolipoprotein A-I in humans.

The kinetics of apolipoproteins A-I and A-II were examined in human subjects using leucine tracers administered intravenously. High density lipoproteins were separated and apoA-I and A-II were isolated. The specific activity or enrichment data for these apolipoprotein were analyzed by mathematical compartmental modeling. In 11 of 14 subjects studied with a bolus-injected [3H]leucine tracer, in 3 subjects studied similarly with [3H]leucine, and in one subject studied by primed dose, constant infusion of [3H]leucine, a rapidly turning-over apoA-I fraction was resolved. A similar component was observed in 7 of 10 studies of apoA-II. The apoA-I data were analyzed using a compartmental model (Zech, L.A. et al. 1983. J. Lipid Res. 24: 60-71) modified to incorporate plasma leucine as a precursor for apoprotein synthesis. The data permitted resolution of two apoA-I pools, one, C(2), turned-over with a residence time of less than 1 day, the other, C(1), a slowly turning-over pool, appeared in plasma after a delay of less than half a day. C(1) comprised the predominant mass of apoA-I and was also the primary determinant of the residence time of apoA-I. Although the mass of the fast pool, C(2), was considerably less than that of C(1), because of its rapid turnover, the quantities of apoA-I transported through this fast pathway were 2- to 4-fold greater. These kinetic studies indicate that apoA-I is secreted into both fast and slowly turning-over plasma pools. The latter is predominantly measured with radioiodinated apoA-I tracers. The data can be analyzed by postulating either separate input pathways to each of the pools or by assuming the fast pool is the precursor to the slow pool. Thus, apoA-I could be initially secreted as a family of particles that are rapidly cleared from plasma, and a portion of this apoprotein then reappears in a slowly turning-over pool that constitutes the major mass of apoA-I. The physiologic identity of these kinetically distinct apoA-I species is unknown; however, the fast pool of apoA-I demonstrated in these studies is strikingly similar to that seen in subjects with Tangier disease who lack the slow pool.