Laurence Wong

Two-dimensional electrophoresis of plasma lipoproteins: Recognition of new apo A-I-containing subpopulations

Two-dimensional electrophoresis has been used to resolve 12 distinct apo A-I-containing high-density lipoprotein (HDL) subpopulations in human plasma. The subpopulations were quantitated by 125I-labeled, monospecific antibody and phosphor-imaging. Modification and standardization of the agarose electrophoresis (first dimension) enabled us to recognize new HDL subpopulations. Lipoprotein mobilities in agarose were expressed relative to the mobility of the samples endogenous albumin. We demonstrated the presence of lipoproteins with mobilities faster than and similar to albumin, as well as subpopulations with mobilities slower than albumin. We refer to these as pre alpha, alpha and pre beta, respectively. Lipoprotein molecular sizes were determined with a non-denaturing polyacrylamide gradient gel electrophoresis (PAGE) (2% to 36%) in the second dimension. Internal standard of 125I-labeled proteins of known molecular size was run simultaneously in each gel permitting accurate size determination. We have demonstrated that ultracentrifugally-isolated lipoproteins are different from the native apo A-I-containing subpopulations. The major difference observed was the loss of pre beta 1 and pre beta 2 particles from the d < 1.21 g/ml fractions to the d > 1.21 g/ml fractions. Possible physiologic and pathologic implications of these findings are also discussed.

Role of Free Apolipoprotein A-I in Cholesterol Efflux: Formation of Pre–α-Migrating High-Density Lipoprotein Particles

This article characterizes products formed by the interaction of purified apolipoprotein (apo) A-I and human fibroblasts. Fibroblasts were incubated with different concentrations of purified apoA-I (1 to 30 micrograms/mL) in tissue culture medium for different periods of time (0 to 24 hours). The medium was then characterized by one- (agarose) and two-dimensional (agarose: polyacrylamide nondenaturing gradient gel) electrophoresis. At any given concentration of apoA-I, the rate of cellular cholesterol efflux appeared linear over 24 hours. Incubating purified apoA-I with fibroblasts for 4 hours, we detected five pre-alpha lipoproteins with particle sizes between 114 and 684 kDa. Formation of pre-alpha lipoproteins was concentration-dependent. At low concentrations (below 5 micrograms/mL apoA-I), all purified apoA-I (with pre-beta mobility) was converted to pre-alpha lipoproteins. At higher concentrations (greater than 5 micrograms/mL apoA-I), more apoA-I remained with pre-beta mobility. The pre-alpha lipoproteins were characterized by colocalization of apoA-I particles with 14C-cholesterol and 32P-phospholipids. Results showed that the pre-alpha particle of lowest molecular weight contained phospholipid and apoA-I but no cholesterol. The remaining pre-alpha particles contained all three substances. When pre-alpha particles were subjected to ultracentrifugation, all particles floated at d < 1.21 g/mL with some of the smallest phospholipid apoA-I only particles being present in the d > 1.21 g/mL fraction. Based on these results, we postulated that in the first stages of reverse cholesterol transport, pre-alpha lipoproteins are formed by the interaction of lipid free apoA-I and peripheral cells.

Comparison of apo A-I-containing subpopulations of dog plasma and prenodal peripheral lymph : evidence for alteration in subpopulations in the interstitial space

To study in vivo reverse cholesterol transport, dog plasma and lymph apo A-I-containing subpopulations were compared by two-dimensional electrophoresis. Charge and size of subpopulations were similar in plasma and lymph, but the distribution of subpopulations varied considerably. An increase in pre-beta and pre-alpha particles in lymph suggests these changes are a reflection of in vivo reverse cholesterol transport.

Normolipidemic Subjects With Low HDL Cholesterol Levels Have Altered HDL Subpopulations

Epidemiological studies have established that plasma concentration of HDL is inversely correlated with the risk of coronary heart disease, even in the absence of increased LDL cholesterol levels. We postulate that specific HDL subpopulations may be responsible for antiatherogenic properties of HDL. HDL subpopulations were quantitated by two-dimensional gel electrophoresis in 79 normolipidemic healthy male subjects. To eliminate the influence of diet, volunteers consumed an average American diet for 6 weeks. After the diet period, subjects were stratified according to their HDL cholesterol (HDL-C) levels to low HDL-C < 0.91 mmol/L (< 35 mg/dL), medium > 0.91 < 1.30 mmol/L (> 35 < 50 mg/dL), and high > or = 1.30 mmol/L (> or = 50 mg/dL) groups. Plasma triglycerides and insulin levels were in the normal range, but subjects with low HDL-C levels had higher concentrations of plasma triglycerides and insulin than subjects with medium or high HDL-C concentrations. The absolute concentration (mg/dL) of apoA-I in the largest alpha-migrating HDL subpopulation (alpha 1) was (P < .01) lower in the low HDL-C subjects compared with the medium and high HDL-C groups. The relative concentration (percent distribution) of apoA-I was decreased (P < .01) in alpha 1 and increased (P < .01) in alpha 3 subpopulations. A positive correlation between HDL-C and alpha 1 (P < .001) and a negative correlation between HDL-C and alpha 3 were observed. The inverse correlation of apoA-I distribution (relative concentration) between alpha 1 and alpha 3 suggests an interconversion of alpha 1 and alpha 3 subpopulations, possibly by cholesteryl ester transfer protein. Pre-beta subpopulations showed an inverse trend with HDL-C, while the pre-alpha subpopulation behaved similarly to the alpha-migrating subpopulation. Colocalization of apoA-I and apoA-II particles in the different HDL subpopulations demonstrated that alpha 1, pre-beta 1, and pre-beta 2 subpopulations are apoA-I-only particles rather than apoA-I:A-II particles.

Altered epitope expression of human interstitial fluid apolipoprotein A-I reduces its ability to activate lecithin cholesterol acyl transferase.

In human peripheral interstitial fluid, esterification of cholesterol by lecithin cholesterol acyltransferase (LCAT) was found to occur at a rate of only 10% of that in plasma (5.6 +/- 1.8 compared with 55.6 +/- 7.8 nmol/ml per h). Measurement of cholesterol esterification in the presence of excess reconstituted apoA-I HDL (rA-I HDL) revealed an LCAT activity in interstitial fluid of 24% of that in plasma, indicating that the low rate of esterification could not be caused by limiting mass of LCAT enzyme. When plasma was diluted to the same concentration as in interstitial fluid, the percent cholesterol esterification rate was the same as undiluted plasma and significantly higher than that of interstitial fluid. These findings led us to postulate that poor activation of LCAT in interstitial fluid may result from a change in conformation in apoA-I. To test this hypothesis, a monoclonal antibody AI-11 that inhibits apoA-I activation of LCAT was used to measure apoA-I in interstitial fluid and plasma. Antibody AI-11 recognized interstitial fluid apoA-I poorly, whereas a polyclonal antibody recognized interstitial fluid apoA-I normally. Incubation of antibody AI-11 with high density lipoprotein or rA-I HDL inhibited apoA-I activation of LCAT. We conclude that the altered conformation of apoA-I in interstitial fluid may render it a poor activator of LCAT.

Gradient acrylamide/agarose gels for electrophoretic separation of intact human very low density lipoproteins, intermediate density lipoproteins, lipoprotein a, and low density lipoproteins

An exponential gradient gel with 0-10% acrylamide and 0.5% agarose was developed for electrophoresis of intact high molecular weight lipoproteins. This system resolves very low density lipoproteins, intermediate density lipoproteins, lipoprotein a, and low density lipoproteins in a size-dependent fashion. The characteristic relative mobility of these species can be determined in relation to protein and colloidal gold reference materials. Electron microscopy of selected lipoprotein fractions confirmed that relative mobility was related to apparent lipoprotein diameter. The composite gel medium can be used with prestained lipoproteins and permits immunoelectroblotting for qualitative analysis of apolipoprotein constituents.

Lipid Composition of HDL Subfractions in Dog Plasma and Lymph

We report the lipid composition of dog plasma and peripheral lymph lipoproteins as separated into pre-beta, alpha, and pre-alpha fractions by agarose gel electrophoresis. Plasma lipoproteins with alpha mobility have a composition different from that of plasma lipoproteins with pre-alpha mobility, having 9% versus 11% free cholesterol, 21% versus 17% cholesterol ester, 1% versus 16% triacylglycerol, and 69% versus 56% phospholipid. On the other hand, lymph alpha and pre-alpha lipoproteins have compositions that are quite similar (9% versus 7% free cholesterol, 17% versus 17% cholesterol ester, 2% versus 4% triacylglycerol, and 71% versus 71% phospholipid). The lipid compositions of plasma and lymph alpha lipoproteins are quite similar (9% versus 9% free cholesterol, 21% versus 17% cholesterol ester, 1% versus 2% triacylglycerol, and 70% versus 72% phospholipid). The lipid compositions of plasma and lymph pre-alpha lipoproteins are different (11% versus 7% free cholesterol, 17% versus 17% cholesterol ester, 16% versus 4% triacylglycerol, and 56% versus 71% phospholipid). Peripheral lymph lipoproteins with pre-beta mobility contained 15% cholesterol, 13% cholesterol ester, 10% triacylglycerol, and 61% phospholipid. Compared with plasma, peripheral lymph lipoproteins are free cholesterol-enriched in all fractions. Calculated stoichiometric ratios of lipid to apoA-I per particle, alpha lipoproteins have two molecules of apoA-I per particle, and pre-alpha lipoproteins have four molecules of apoA-I per particle.

Comparison of the lipid and apolipoprotein composition of skeletal muscle and peripheral lymph in control dogs and in dogs fed a high fat, high cholesterol, hypothyroid-inducing diet

Most studies of peripheral interstitial fluid lipoprotein composition have been made on interstitial fluid-derived from skin and connective tissue. We developed techniques which allowed simultaneous comparison of lymph (a model of interstitial fluid) from skeletal muscle and skin in control (C) and cholesterol-fed (CF) dogs. Lipoprotein fractions were separated by ultracentrifugation. Skeletal muscle interstitial fluid HDL concentrations were approximately twice those of skin. However, the concentration of VLDL-LDL particles was similar in both interstitial spaces. HDL particles from both microvascular beds showed evidence of extensive remodelling when compared to plasma HDL from the same animal. Relative to apo A-I, skeletal muscle HDL was enriched in free cholesterol and apo E (C and CF dogs) and apo A-IV (CF dogs). Skin-derived HDL was consistently enriched in free cholesterol, apo E and A-IV in both C and CF dogs. These studies indicate that similar remodeling of plasma HDL occurs in widely different tissues which together constitute approximately 70% of the total interstitial space. The relatively high concentration of plasma-derived and remodeled HDL within the interstitial space of skeletal muscle is consistent with that tissues importance in reverse cholesterol transport.

Lipoprotein lipase and hepatic triacylglycerol lipase activities in peripheral and skeletal muscle lymph.

We studied the interstitial fluid concentration of two lipid-metabolizing enzymes (lipoprotein lipase and hepatic triacylglycerol lipase) to determine their importance in interstitial modification of filtered lipoproteins. Despite the use of a very sensitive lipase assay (1 nmol of fatty acid release/ml/hr), lipase activities in plasma and in peripheral and skeletal muscle lymph from control dogs were below the sensitivity of our assay. After heparin injection, hepatic triacylglycerol lipase and lipoprotein lipase activities in plasma were similar. However, the postheparin hepatic triacylglycerol lipase activities in peripheral and skeletal muscle lymph were only 1.4% and 1.1%, respectively, those of plasma. This concentration is considerably less than the lymph concentration of albumin, which has a similar size to the lipases but has a lymph concentration of 30% to 40% of plasma. Lipoprotein lipase activity in peripheral lymph and skeletal muscle lymph was 2.7% and 4.8%, respectively, of plasma activity. Since lipoprotein lipase has a similar size as hepatic triacylglycerol lipase, the disproportionate amount of lipoprotein lipase in lymph as compared to hepatic triacylglycerol lipase could be due to heparin crossing the capillary endothelium and displacing lipoprotein lipase from peripheral cells. Injection of radioactive heparin confirmed that it does cross into the interstitial space in sufficient concentrations to displace lipase from peripheral cells. We conclude that most of the lipase found in lymph after heparin injection is derived from peripheral cells and not from plasma. Furthermore, hepatic triacylglycerol lipase does not play a role in high density lipoprotein remodeling in interstitial fluid. Therefore, it seems likely that the considerable remodeling of high density lipoprotein that we found previously results from its interaction with peripheral cells.

Immunological heterogeneity of rat apolipoprotein B epitope expression. A study using monoclonal antibodies to rat apolipoprotein B

Epitope expression of rat apolipoprotein B on lipoproteins was investigated with the help of six monoclonal antibodies produced from mice. Through a variety of techniques, which include cotitrations, ELISAs and quantitative immunoadsorption precipitation, we concluded that the six monoclonal antibodies recognize five different epitopes. LRB 110 and LRB 260 recognize epitopes that may be overlapping. LRB 240 and LRB 250 recognize epitopes that are preferentially expressed in triacylglycerol-rich particles. LRB 220 recognizes an epitope that is expressed by all apolipoprotein-B-containing lipoproteins. We have also determined that apolipoprotein B epitope expression in rat lipoproteins is very similar to its human counterpart. Both rat and human apolipoprotein B epitope expression on lipoproteins showed heterogeneities even in homologous lipoprotein preparations. We concluded that a variety of techniques are necessary to fully characterize monoclonal antibodies to apolipoproteins. The possible implications of epitope expression in pathophysiology are also discussed.