Abstract
A reproducible, fairly narrow-sized population of rat lymph chylomicrons, approximately 100 nm, was isolated by centrifugation and combined with low levels of [1-13C]oleic acid for NMR studies. The carboxyl chemical shift was monitored as a function of aqueous pH to characterize the ionization behavior of the fatty acid in these particles. The titration curves were very similar to those for oleic acid in equivalent-sized emulsion particles composed of egg phosphatidylcholine and triolein. A simple partition-ionization model was fitted to the data to derive values for apparent ionization constant, expressed as pKapp, of 7.4-7.5 and the "true" surface to core partition coefficient of approximately 7 for oleic acid in chylomicrons. The fatty acid in chylomicrons thus appeared to be largely associated with the surface regions of these particles. Addition of bovine serum albumin to the samples showed that near physiologic pH much of the fatty acid was bound to the albumin at fatty acid to albumin-binding stoichiometries as high as 5.1 and with mass ratios of greater than 2 in favor of the lipid or lipoprotein particles. Lowering the pH of the medium shifted the distribution of fatty acid away from albumin so that at pH 5 with the emulsion, virtually all the fatty acid was associated with the lipid. The behavior observed under physiologic conditions is consistent with the rapid clearance and redistribution of fatty acid generated in these particles by lipolytic processes. However, under conditions of severe acidosis, hyperlipidemia, and hypoalbuminemia a significant portion of fatty acids might be retained in triglyceride-rich lipoproteins and their remnants and affect subsequent metabolism.
Highlights
A reproducible, fairly narrow-sized population of rat To gain some insights into the factors important in fatty lymph chylomicrons, -100 nm, was isolated by cen- acid transport processes, we attempt tocharacterize here the trifugation and combined with low levels of [l-lsC] physical behavior of these species atthe source of their oleic acid for NMR studies
In the current work we physiologic pH much of the fattyacid wasbound to the examine how closely the ionization and distribution of fatty albumin at fatty acid to albumin-binding stoichiome- acid in chylomicrons might be mimickedusing a simple emultries as high as 5:1and withmass ratios of >2 in favor sion composed of phosphatidylcholine and triolein
Lowering the pH A major obstacle to achieving the above objectives is the of the medium shifted the distribution of fatty acid size variability within and between samples of the native and away from albuminso that atpH 5 with theemulsion, synthetic lipid dispersions, as well astheir generallypoor virtually all the fatty acid was associated lwipiitdh. thientegrity for controlled studies
Summary
Mechanisms for cellular uptake of fatty acids and the role of their binding with serum albumin. These lipids, used in emulsion preparation, were confirmed to be >99% pure by thin layer chromatography. Samples were centrifuged using an SW 28 rotor at 15,000 rpm to accumulate an w% of 2.7 X losrad's" For these calculations, the average particle density was taken as 0.939 g ~ m - d~e,termined for chylomicrons [20],and the overlay was assumed to have the viscosity of pure water (1.002cp). Centrifugation was conducted at 25,000 rpm to accumulate a n 0% of 1.75 X 10" rad%" or 2.11 X 108g.,s (-45 min), aforce estimated to spin-up particles of >100-nm diameter into the surface region These fractions were sliced from the tubes and concentrated to -300 mg of triglyceride ml-I by centrifugation into an overlay of the desired aqueous medium using a SW 41 rotor. Proton-decoupled "C NMR spectra were recorded at 50.3 MHz with a Bruker WP-200 spectrometer as described previously [23]
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