Norman K. Freeman

STRUCTURE AND HOMOGENEITY OF THE LOW-DENSITY SERUM LIPOPROTEINS

Although human serum lipoproteins have been studied intensively over the past decade, we still lack complete information concerning the chemical composition and structure of these macromolecules. One difficulty with regard to such studies is the wide range of lipoprotein classes that exist as part of the total blood lipoprotein spectra. It might be estimated, for instance, that in a typical serum lipoprotein distribution (as shown schematically in FIGURE 1 for a normal 45-year-old male) there might easily be the order of 100 lipoprotein classes, each of which could be distinguished from the others by physical or chemical means or by some combination of analytical methodologies now available. In this presentation we shall examine variations in composition that occur within each broad lipoprotein group and consider further the evidence for homogeneity of lipoproteins isolated within one narrow Sf band. There is evidence that the larger low-density lipoproteins, particularly those above Sr 100, may be lipoprotein complexes of limited stability. Furthermore, these macromolecular units present in the blood stream may be in a state of constant transformation, possibly reflecting a relationship to the breakdown and transport of all classes of lipids within the blood compartment. Considering this, we shall present evidence bearing on a hypothesis of a lipoprotein complex model for these very large low-density lipoproteins.

Ultracentrifugal isolation of serum chylomicron-containing fractions with quantitation by infrared spectrometry and NCH elemental analysis.

An ultracentrifugal method for isolating chylomicron-containing fractions from serum by flotation, using either standard Spinco swinging-bucket rotors or a specially fabricated swinging-bucket rotor, is described. Lower limits of the Sf rates of the chylomicron fractions are evaluated using a computer technique to define lipoprotein flotation over a nonlinear NaCl density gradient. The latter is prepared by a special overlayering technique.Quantitation within a 9–50 μg region of mass assay is accomplished by both infrared spectrometry and elemental analysis for N, C and H. Results indicate that the chylomicron concentration in serum for a small population of nonfasting male adults ranges from approximately 0–50 mg %.

Applications of infrared absorption spectroscopy in the analysis of lipids

The uses of infrared spectroscopy in lipid chemistry are discussed, with main emphasis on quantitative analytical methods. In reviewing the infrared technique from an overall perspective, attention is also given to instrumental developments, sampling techniques and qualitative applications. Some features are brought out in the context of examples cited. These include some significant established methods as well as recent developments, such as the use of integrated band intensities and the involvement of computers and computer techniques in infrared analysis. Current trends and future directions are indicated, and some unrealized potentialities are suggested.

Use of an Infrared Laser in a Detection System for Liquid Chromatography

Abstract A liquid chromatography detection system based on an infrared laser has been constructed and some tentative evaluations of its performance have been made. Although restricted to use with a very few solvents (mainly halocarbons), this system should be applicable whenever chloroform or a halocarbon solvent of lower strength (polarity) can be used. Injected sample quantities as small as one microgram have been detected on elution.

Semiautomatic analysis of serum triglycerides and cholesteryl esters by infrared absorption

An instrument has been developed for the semiautomatic analysis of mixtures of triglycerides and cholesteryl esters. The method is based on high-resolution infrared spectrophotometry, and has previously been shown to be applicable to the determination of these components in the nonionic fraction of human serum lipids. A simple nonrecording grating spectrophotometer has been suitably modified to carry out this analysis; and appropriate computing circuitry has been coupled with it for performing the two-component calculation. The supplementary electronics consist of operational amplifiers, a logarithmic conversion circuit, a digital voltmeter, and a printer. Automatic operation is accomplished by a control mechanism, which programs the measurements, the steps in the calculations, and print-out of the results. Sample preparation consists of an extraction of lipids from serum in such a way as to exelude phospholipids. This may be done in a single step, although a two-step procedure—total lipid extraction followed by adsorption separation of the phospholipids—appears to be more reliable. Measurements are made on a solution of the neutral lipid fraction in carbon tetrachloride.

The Analysis of Human Serum Lipoprotein Distributions1 1This work was supported by Research Grants HE 01882-09 and HE 02029-09 from the National Heart Institute, Public Health Service, Bethesda, Maryland, and by the United States Atomic Energy Commission.

Publisher Summary This chapter reviews distributions of human serum lipoproteins. Macromolecular distributions such as the human lipoprotein system have for many years been analyzed by utilizing the analytical ultracentrifuge. A biochemically important macromolecular system, exhibiting these special physical characteristics, is the low- and high-density lipoprotein spectrum of human sera. Although the analytical difficulties are considerable, ultracentrifugal analysis provides one of the few available approaches for an extensive quantitative analysis of such macromolecular distributions. Largely because of technical limitations, previous ultracentrifugal methodology for lipoprotein analysis has considered only broad subtractions of the low-density spectra. In the case of the high-density lipoprotein spectrum, this lipoprotein distribution has been divided essentially into two main classes on the basis of hydrated density below and above approximately 1.125 gm/ml, corresponding to high-density lipoprotein HDL 2 and HDL 3 , respectively. Because of the elaborate calculations and data processing required for this kind of mathematical analysis, it has not been feasible to consider potentially useful techniques to accomplish this task manually.