Fat Absorption

Volume I

Arnis Kuksis

• Publisher: CRC Press
• Year: 2018
• Print ISBN: 9781315892894
• eBook ISBN: 9781351088893
• Topic: Medicine
• Subtopic: Nutrition, Dietics & Bariatrics

This book provides an authoritative and comprehensive source of information on the biochemical an metabolic aspects of digestion and absorption of different dietary fats and other lipids, with minimal discussion of the physical chemistry of the process, which has been covered in great detail in previous reviews. It is intended for both researchers and practitioners in the biomedical field who require detailed knowledge of the biomedical and metabolic transformations involed in the intestinal digestion and resynthesis of dietary fats and other lipids.

Chapter 1
ANALYTICAL METHODOLOGY IN THE FAT ABSORPTION AREA

A. Kuksis and J.J. Myher

Table of Contents

• I. Introduction
• II. Physicochemical Assays
  • A. Extraction of Lipids from Intestinal Mucosa and Lumen
    • 1. Preparation of Total Lipid Extracts
    • 2. Artifact Formation
  • B. Preliminary Separation of Lipid Classes
  • C. Resolution of Lipid Classes
    • 1. Separation of Neutral Lipid Classes
    • 2. Separation of Phospholipid Classes
    • 3. Separation of Glycolipid Classes
  • D. Resolution of Molecular Species
    • 1. Neutral Lipids and Free Fatty Acids
      • a. Argentation Thin-Layer Chromatography
      • b. Gas-Liquid Chromatography
      • c. High-Pressure Liquid Chromatography
    • 2. Glycerophospholipids and Sphingomyelins
      • a. Argentation Chromatography
      • b. High-Pressure Liquid Chromatography
    • 3. Glycolipids
      • a. Argentation Thin-Layer Chromatography
      • b. Gas-Liquid Chromatography
      • c. High-Pressure Liquid Chromatography
      • d. Mass Spectrometry
• III. Enzymatic Assays
  • A. Lipases and Acyltransferases
    • 1. Pancreatic Lipase
    • 2. Mono- and Diacylglycerol Acyltransferases
  • B. Positional and Stereospecific Analyses of Glycerolipids
    • 1. Glycerophospholipids
    • 2. Triacylglycerols, Diacylglycerols, and Monoacylglycerols
• IV. Radio- and Stable Isotope Tracer Methods
  • A. Utilization of Radioactive Tracers
    • 1. Radio-Thin-Layer Chromatography
    • 2. Radio-Gas-Liquid Chromatography
  • B. Utilization of Stable Isotope Tracers
    • 1. Gas-Liquid Chromatography/Mass Spectrometry
    • 2. High-Pressure Liquid Chromatography/Mass Spectrometry
• V. Summary and Conclusions
• Acknowledgments
• References

I. Introduction

It has been known for a long time that the various lipid classes and molecular species of dietary fats are digested and absorbed at different rates. For simplicity of analysis, however, much of the experimental work has been performed by means of model compounds and radiolabeled substrates of limited variety. This has led to the unwarranted assumption that all dietary lipids behave like the model compounds. Furthermore, the dependence on measurements of specific radioactivities in pools of lipid metabolites of unverified structure has led to ambiguous and often erroneous conclusions about the extent and/or metabolic course of the absorption process. The work with radioactive model compounds also has been unrewarding toward the understanding of the digestion and absorption of different dietary fats due to a lack of appropriately labeled substrates and to the difficulty of simultaneous studies of more than two labeled substrates in the absence of an extensive chromatographic prefractionation and chemical degradation. Recent work shows that an effective study of the absorption of dietary lipids requires systematic chromatographic and mass spectrometric monitoring of representative samples. With few exceptions, these techniques have been neglected in the studies of fat digestion and absorption in favor of the simpler and less elaborate approaches. Although both thin-layer and gas-liquid chromatographic methods have been utilized extensively in most areas of lipid chemistry and biochemistry, studies of fat digestion and absorption, with few exceptions, have remained largely at the level of such physicochemical routines as micellar solubilization and partition between aqueous and organic solvents of bulk lipids and micelles. The failure to combine radioisotopic studies with chromatography has led to inadequate identification of the re-esterification products of lumenal lipids. As a result, the contributions of the phosphatidic acid and monoacylglycerol pathways to the total mucosal triacylglycerol biosynthesis have remained shrewd guesses, as have the changes in these contributions with alterations in dietary lipid composition. Other areas of doubt due to analytical inadequacies are found in the relative contributions of the dietary, biliary, lumenal, and endogenous lipids to the mucosal biosynthesis of the triacylglycerols.

In addition to the chromatographic and mass spectrometric analyses, the review discusses the progress in stereospecific analyses of the acylglycerols. This is a subject that has remained controversial since the claim of enantiomeric and the counterclaim of racemic resynthesis of triacylglycerols via the monoacylglycerol pathway. The chapter stresses the potential advantages of the use of stable isotope labeling in combination with chromatography, mass spectrometry, and stereospecific analysis of prefractionated intestinal lipoproteins and cellular membrane components as potentially unambiguous indicators of the absorption mechanism.

II. Physicochemical Assays

An essential part of a modern study of fat digestion and absorption involves the determination of fatty acid composition, positional distribution, and molecular association in the dietary fats, their lumenal lypolysates, cellular assimilation intermediates, and the final lymphatic absorption products. This requires the extraction, separation, identification, and quantitation of the various component fatty acids, partial acylglycerols, triacylglycerols, glycerophospholipids, and other lipids. The most complete analyses have been obtained by capillary gas-liquid chromatography (GLC) and reversed-phase high-pressure liquid chromatography (HPLC) in combination with mass spectrometry (MS), but thin-layer chromatography (TLC) has also played a major role both as an effective preparative and analytical system, especially when combined with GLC. In most instances, the analyses have been limited to TLC, GLC, or combined TLC-GLC and more recently, to HPLC in combination with TLC or GLC.

The recent development of stereospecific analysis of acylglycerols allowing the recovery of pure enantiomers has greatly facilitated the identification and quantitation of the precursors and products of the various acyl transfer reactions involved in the acylglycerol lipolysis in the lumen and in their resynthesis in the villus cell. Furthermore, the enzymic resolution of enantiomers in combination with chromatographic and mass spectrometric analysis of stable isotope-labeled substrates has provided the first analytical system capable of effective assessment of the complexity of the digestion and absorption of natural fats.

A. Extraction of Lipids from Intestinal Mucosa and Lumen

Lipids exist in the intestinal lumen and mucosa in a variety of chemical and physical forms, including discrete droplets of neutral fat, mixed micelles, lipoproteins, and membranous vesicles, as well as monomeric molecules. Before these lipids can be analyzed, they must be isolated quantitatively by extraction with organic solvents, and the lipid extracts freed of nonlipid material by appropriate procedures. Ideally, the intestinal tissue preparations should be extracted as soon as possible after removal from the animal so that there is little opportunity for changes to occur, due to the introduction of artifacts of auto-oxidation, isomerization, and hydrolytic decomposition. When this is not possible, the tissues may be stored briefly in the presence of organic solvents, such as acetone or alcohol-free chloroform at −20°C. Methanol and chloroform-methanol should be avoided because of potential methanolysis. The presence of large amounts of free fatty acids is not necessarily an indication of lipolysis when dealing with intestinal tissue. The procedures described here are based on the experience reported elsewhere by the authors and their collaborators.^{1, 2 and 3}

1. Preparation of Total Lipid Extracts

Total lipid extracts are usually prepared by some variation of the chloroform-methanol routines originally described by Folch et al.⁴ and by Bligh and Dyer.⁵ Shaikh and Kuksis⁶ have described a modification of the method of Folch et al.⁴ in which approximately 1 mℓ of the intestinal tissue homogenate (about 2 to 4 mg protein) prepared in ice-cold 0.9% NaCl in a Potter-Elvehjem homogenizer is pipetted into a 40-mℓ centrifuge tube closed with a Teflon ®-lined screw cap containing 10 mℓ ice-cold methanol. The contents of the tube are mixed and 20 mℓ chloroform are added. After shaking the tube in a Buchler-Evapo-Mix for 15 min at 37°C, a biphasic mixture is produced by adding 6.5 mℓ of 0.9% NaCl. The tube is again briefly shaken and the two phases produced are separated by centrifugation. The lower lipid phase is withdrawn and the remaining upper phase containing interfacial residual proteins is re-extracted two times with 22.5-mℓ portions of “synthetic” lower phase (chloroform-methanol-0.58% NaCl, 86:14:1 by volume). The lower lipid phase is removed after centrifugation and the three extracts are pooled, passed through anhydrous sodium sulfate, and evaporated to small volume under nitrogen. The lipid residue is then transferred to a 7-mℓ glass vial with moist chloroform for further analyses. The recoveries of representative radioactive phospholipids from the above extractions ranged from 97 to 100%, including the lysophospholipids.^6,7 An improved procedure for the isolation of the acidic glycerophospholipids, including the phosphoinositides, has been described by Palmer.⁸

A simplified...

Table of contents

• Cover
• Title Page
• Copyright Page
• Preface
• The Editor
• Acknowledgments
• Contributors
• Table of Contents
• Chapter 1 Analytical Methodology in the Fat Absorption Area
• Chapter 2 Structure and Function of Enterocyte Membrane Lipids
• Chapter 3 Hydrolysis of Dietary Glycerides and Phosphoglycerides: Fatty Acid and Positional Specificity of Lipases and Phospholipases
• Chapter 4 Absorption of Glycerides Containing Short, Medium, and Long Chain Fatty Acids
• Chapter 5 Absorption of Deuterium-Labeled cis- and trans-Octadecenoic Acid Positional Isomers
• Chapter 6 The Role of Phosphatidylcholine in the Absorption and Transport of Dietary Fat
• Chapter 7 Role of Intestinal Hydrolases, Endogenous Substrates, and Chyloportal Partition in Fat Absorption
• Chapter 8 Biochemical Characterization and Purification of Intestinal Acylglycerol Acyltransferases
• Chapter 9 Clinical Aspects of Fat Malabsorption
• Index