Anna M. Tercyak

Determination of Human Coronary Artery Composition by Raman Spectroscopy

BACKGROUND We present a method for in situ chemical analysis of human coronary artery using near-infrared Raman spectroscopy. It is rapid and accurate and does not require tissue removal; small volumes, approximately 1 mm3, can be sampled. This methodology is likely to be useful as a tool for intravascular diagnosis of artery disease. METHODS AND RESULTS Human coronary artery segments were obtained from nine explanted recipient hearts within 1 hour of heart transplantation. Minces from one or more segments were obtained through grinding in a mortar and pestle containing liquid nitrogen. Artery segments and minces were excited with 830 nm near-infrared light, and Raman spectra were collected with a specially designed spectrometer. A model was developed to analyze the spectra and quantify the amounts of cholesterol, cholesterol esters, triglycerides and phospholipids, and calcium salts present. The model provided excellent fits to spectra from the artery segments, indicating its applicability to intact tissue. In addition, the minces were assayed chemically for lipid and calcium salt content, and the results were compared. The relative weights obtained using the Raman technique agreed with those of the standard assays within a few percentage points. CONCLUSIONS The chemical composition of coronary artery can be quantified accurately with Raman spectroscopy. This opens the possibility of using histochemical analysis to predict acute events such as plaque rupture, to follow the progression of disease, and to select appropriate therapeutic interventions.

Effects of cholesterol content on the metabolism of protein-free emulsion models of lipoproteins

After intravenous injection, emulsions with compositions similar to chylomicrons behaved metabolically as described for chylomicrons, with faster removals of triacylglycerols than cholesteryl esters from the blood after injection into rats, and with greater uptakes of cholesteryl esters than triacylglycerols by the liver. In contrast, emulsions with a high content of free cholesterol showed equal removal rates from the blood of triacylglycerols and cholesteryl esters; and similar uptakes by the liver. This pattern of metabolism was that expected for a chylomicron core remnant particle. Emulsions poor in cholesteryl ester but rich in free cholesterol showed remnant-like behavior, whereas emulsions rich in cholesteryl ester but poor in free cholesterol were metabolized like nascent chylomicron particles. The amount of free cholesterol appeared to regulate metabolism by affecting the binding of apolipoproteins to the particle surface. Emulsions with a high content of free cholesterol bound less A-I, A-IV and C apolipoproteins, and the relative amount of apolipoprotein E was increased. All of these effects are consistent with the metabolic differences between chylomicrons and remnant particles, suggesting that the amount of free cholesterol plays a regulatory role in chylomicron metabolism.

Colorimetric assay for pluronic F-68 as measured in isolated rat liver perfusion systems.

A colorimetric assay has been developed for the quantitation of Pluronic F-68, a nonionic detergent (surfactant) which is a polyoxypropylene-polyoxyethylene (POP-POE) block copolymer. We measured this substance in organic extracts of rat liver perfusates from livers which had been perfused with an oxygenated perfluorocarbon, FC-43 Emulsion (Oxypherol).

Uptake of artificial model remnant lipoprotein emulsions by the perfused rat liver

In comparison with their precursor lipoproteins, the remnants of the triacylglycerol-rich lipoproteins are reduced in contents of triacylglycerols and apolipoproteins AI and AIV, whereas the contents of cholesterol (free and esterified) and apolipoprotein E are increased. In this study, lipid emulsion models of remnant lipoproteins were used to explore which of these factors are necessary for physiological rates of remnant uptake by the perfused rat liver. Uptake rates of lipid emulsion models of remnant lipoproteins in the presence of apolipoprotein E were similar to in vivo uptake rates.

In-situ histochemical analysis of human coronary artery by Raman spectroscopy compared with biochemical assay

We have developed a method to analyze quantitatively the biochemical composition of human coronary artery in situ using near infrared Raman spectroscopy. Human coronary arteries were obtained from explanted hearts after heart transplantation. Samples of normal intima/media, adventitia, non-calcified and calcified plaque were illuminated with 830 nm light from a CW Ti:Sapphire laser. The Raman scattered light was collected and coupled into a 1/4 meter spectrometer that dispersed the light onto a liquid nitrogen cooled, deep-depletion CCD detector. Raman spectra with sufficiently high S/N for extracting biochemical information could be collected in under one second. The spectra were analyzed using a recently developed model to quantitate the relative weight fractions of cholesterol, cholesterol esters, triacylglycerol, phospholipids, protein, and calcium salts. After spectral examination, the artery samples were biochemically assayed to determine the total lipid weight and the amount of the major lipid categories as a percentage of the total lipid content. The results of the lipid biochemical assay and the Raman spectral model compare favorably, indicating that relative lipid weights can be accurately determined in situ. Protein and calcium salts assays are underway. This in situ biochemical information may be useful in diagnosing atherosclerosis and studying disease progression.

The rate of transfer of unesterified cholesterol from rat erythrocytes to emulsions modeling nascent triglyceride-rich lipoproteins and chylomicrons depends on the degree of fluidity of the surface

Abstract We have measured the rate of transfer of unesterified cholesterol from rat erythrocyte to triolein emulsions modeling nascent triglyceride-rich lipoproteins. Emulsions (mean diameter ≈ 130 nm) were prepared with low cholesterol content (less than 2%) and various phosphatidylcholines that resulted in fluid (egg yolk phosphatidylcholine, dimyristoyl phosphatidylcholine) at transition (dipalmitoyl phosphatidylcholine) and solid (distearoyl phosphatidylcholine) surfaces at 37° C. Emulsions were incubated for 0, 20, 60, and 180 min with rat erythrocytes. Incubation mixtures initially contained approximately equal masses of phospholipid in the emulsion surfaces and the outer layer of plasma membrane of rat erythrocytes. There was a gradual and significant increase (P > egg yolk phosphatidylcholine-low cholesterol- > dipalmitoyl phosphatidylcholine-low cholesterol-triolein. There was no significant change in the composition of the surface phase of distearoyl phosphatidylcholine-low cholesterol-triolein emulsions. Therefore, transfer of unesterified cholesterol to the surface phase of emulsions during incubation with intact rat erythrocytes at 37° C in the absence of transfer proteins and plasma proteins is attributable to the degree of surface fluidity of emulsions.

The Lipid Surface of Triglyceride-Rich Particles Can Modulate (Apo)Protein Binding and Tissue Uptake

Plasma lipoproteins are aggregates varying in size from large chylomicrons to small HDL3. They are composed of complex combinations of apoproteins and lipids. The triglyceride-rich lipoproteins, secret ed by the intestine as chylomicrons or by the liver as VLDL, contain a core which is rich in triacylglycerols. Small but varying amounts of cholesterol esters also are contained in the core. The surface lipids are extremely complex and include a variety of phospholipids, including phosphatidylcholines, phosphatidylethanolamines and sphingoelins, as well as glycosphingolipids such as cerebrosides and gangliosides. Also present in the surface is cholesterol and small but significant amounts of triacylglycerols and cholesterol esters. In addition the surface contains insoluble and non-exchangeable apolipoproteins, specifically B100 or B43, and exchangeable soluble apolipoproteins such as A-I, A-II, A-IV, C-I, C-II, C-III, and E. The determinants of triglyceride-rich lipoprotein composition are: 1) the metabolism of the cell that secretes the nascent chylomicron or VLDL, 2) the physical exchanges of lipids and apoproteins that occur in plasma, 3) the transfer proteins mediated exchange between core molecules such as triacylglycerols and cholesterol ester and surface molecules such as phospholipids, and 4) the action of lipolytic enzymes such as lipoprotein lipase, hepatic lipase and lecithin-cholesterol acyltransferase, to produce metabolically important lipid products including fatty acids, monoacylglycerols, diacylglycerols, and lysophosphatides.

Physico-chemical properties of cholesterol-fed rabbit β-VLDL are not affected by different dietary oils

Various oils have been used as vehicles for cholesterol in diets used to produce atherosclerosis in rabbits. Because such oils may affect the physico-chemical properties of the beta-VLDL produced in response to cholesterol feeding, we have studied the physico-chemical characteristics of beta-VLDL isolated from cholesterol-fed rabbits using several different oils as vehicles (corn, safflower, cod liver, and peanut oils). All animals developed severe hypercholesterolemia by 2 weeks. During the second and third weeks on diet the apo beta-containing lipoproteins began to develop thermal transitions due to order-disorder cholesterol ester transitions in the lipoproteins. By 4 weeks the apo beta-containing lipoproteins (overwhelmingly beta-VLDL) had transitions which were quite similar (transition temperatures 41.0-42.5 degrees C). No statistical differences were noted between the transition temperatures or enthalpies of any of the dietary groups. Thus, the physico-chemical properties of the beta-VLDL of rabbits fed cholesterol appear to be quite similar, despite the vehicles used to carry the cholesterol in the diet. Thus other mechanisms must be looked for to explain the different atherogenicity of different oils used as cholesterol carrying vehicles.