Charles H. Sloop

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.

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.

Interstitial fluid (peripheral lymph) lipoproteins

Publisher Summary This chapter discusses the studies of interstitial fluid lipoproteins and their metabolism may provide the important link between tissue culture studies and in vivo studies of plasma lipoprotein metabolism. Perinodal peripheral lymph is an accepted model of interstitial fluid. The chapter also presents techniques, which allow collection of perinodal peripheral lymph in quantities sufficient for analytical or metabolic studies of interstitial fluid. The dog as an experimental model offers advantages in ease of surgical procedures, quantities of fluid obtained, and a well-characterized plasma lipoprotein profile. The experimental designs and data presented are examples of the potentially important information that can be obtained from studies of peripheral (or deep muscle) lymph lipoproteins. The chapter provides a link between studies on plasma lipoprotein metabolism and tissue culture studies using peripheral cell cultures (such as macrophage, smooth muscle cells, or endothelial cells) and thus provide an important contribution to the understanding of the function of lipoproteins.

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.

Portal and aortic blood sampling technique in unrestrained rats

Abstract A composite cannula has been designed to permit blood sampling from the distal aorta and portal vein in unanesthetized, unrestrained rats. The aortic and portal cannulas were constructed from polyvinylchloride (PVC) tubing drawn to a fine tip and fused to polyethylene tubing (PE 50). The portal vein was cannulated via a mesenteric vein. The abdominal aorta was cannulated non-occlusively. Both vascular cannulas were securely tied to the abdominal wall. A silicone rubber duodenal cannula was inserted into the membraneous portion of the stomach and passed into the duodenal lumen. Its end was tunnelled under the skin to the rats back and placed under a wound clip. The utility of these cannulas was illustrated by simultaneous sampling of aortic and portal vein blood during absorption of labelled amino acids. These chronic cannulas can be used to study behavioral or physiologic parameters while selectively infusing compounds into the duodenum or portal vein. The aortic cannula can be used to measure mean arterial blood pressure, to sample blood, or to infuse the caudal part of the rat.

Lipid absorption in unanesthetized, unrestrained rats effects of 4-aminopyrazolopyrimidine and ethinyl estradiol

Emulsified triacylglycerol containing [14C]palmitate was infused intraduodenally in unanesthetized, unrestrained animals treated with 4-aminopyrazolopyrimidine or pharmacologic doses of ethinyl estradiol. 4-Aminopyrazolo-pyrimidine practically eliminated the appearance of radioactivity in plasma but in ethinyl estradiol-treated animals the peak of radioactivity and shape of the plasma curve were similar to control, although lower in amplitude. A delayed appearance of radioactivity was also observed in 48-h- compared to 15-h-fasted controls, suggesting a requirement for induction of lipoprotein production prior to fat absorption.

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.