Lipids and Related Compounds

355 Pages·1989·22.88 MB·English

by  Norman S. Radin (auth.), Alan A. Boulton, Glen B. Baker, Lloyd A. Horrocks (eds.)

NEUROMETHODS 0 7
Lipids and Related Compounds
NEUROMETHODS
Program Editors: Alan A. Boulton and Glen f3. Baker
1 General Neurochemical Techniques
Edlted by Alan A. Boulton and Glen B. Baker, 1985
2 Amines and Their Metabolites
Edited by Alan A. Boulton, Glen B. Baker, and Judith M. Baker, 1985
3 Amino Acids
Edlted by Alan A. Boulton, Glen B. Baker, and James D. Wood, 1985
4 Receptor Binding Techniques
Edlted by Alan A. Boulton, Glen B. Baker, and Pave1 D. Hrdina, 1986
5 Neurotransmitter Enzymes
Edited by Alan A. Boulton, Glen B. Baker, and Peter H. Yu, 1986
6 Peptides
Edlted by Alan A. Boulton, Glen B. Baker, and Quentin Pittman, 1987
7 Lipids and Related Compounds
Edited by Alan A. Boulton, Glen 8. Baker, and Lloyd A. Horrocks, 1988
8 Imaging and Correlative Physicochemical Techniques
Edited by Alan A. Boulton, Glen B. Baker, and Donald P. Bofsvert, 1988
9 The Neuronal Microenvironment
Edited by Alan A. Boulton, Glen B. Baker, and Wolfgang Walz, 1988
10 Analysis of Psychiatric Drugs
Edited by Alan A. Boulton, Glen B. Baker, and Ronald T. Coutts, 1988
11 Carbohydrates and Energy Metabolism
Edited by Alan A. Boulton, Glen B. Baker, and Roger F. Butterworth, 1989
12 Drugs as Tools in Neurotransmitter Research
Edlted by Alan A. Boulton, Glen B. Baker, and August0 V. Juorio, 1989
13 Psychopharmacology
Edlted by Alan A. Boulton, Glen B. Baker, and Andrew J. Greenshaw, 1989
14 Neurophysiology
Edlted by Alan A. Boulton, Glen B. Baker, and Case H. Vanderwolf
15 Neuropsychology
Edited by Alan A. Boulton, Glen B. Baker, and Merril Hiscock
16 Molecular Neurobiology
Edited by Alan A. Boulton, Glen B. Baker, and Anthony T. Campagnoni
NEUROMETHODS
Program Editors: Alan A. Boulton and Glen B. Baker
NEUROMETHODS 0 7
Lipids and
Related Compounds
Edited by
Alan A. Boulton
Univerxty of Saskatchewan, Saskatoon, Canada
Glen B. Baker
University of Alberta, Edmonton, Canada
and
Lloyd A. Horrocks
The Ohio State University, Columbus, Ohio
Humana Press l Clifton, New Jersey
Library of Congress Cataloging in Publication Data
Lipids and related compounds.
(Neuromethods ; 7)
Includes bibliographies and index.
1. Lipids-Analysis. 2. Neurochemistry-Methodology.
I. Boulton, A. A. (Alan A.) II. Baker, Glen B.,
Date. III. Horrocks, Lloyd A. IV. Series.
QP751. L55559 1988 612’ .8042 88-8909
ISBN 0-89603-124-l
0 1988 The Humana Press Inc.
Crescent Manor
PO Box 2148
Clifton, NJ 07015
All rights reserved
No part of this book may be reproduced, stored in a retrieval
system, or transmitted in any form or by any means, electronic,
mechanical, photocopying, microfilmmg, recording, or otherwise
without written permission from the Publisher.
Printed in the United States of America
Preface
Lipids m the nervous system are major components of the mem-
branes. The presence of glycolipids in high concentrations is
unique for the nervous system. Recent discoverres of the functronal
and pathological importance of lipids such as diacylglycerols,
polyphosphoinositides, prostaglandins, leukotrienes,
docosahexaenoic acid, platelet activating factor, and gangliosides
have markedly increased the number of publications on nervous
system lipids. Many new methods have been developed. Thus,
there IS a need for this volume that is dedicated to that methodolo-
gy. This volume places all of the methods for lipids into perspective
with recommendations concerning the selection of a method for a
specific purpose.
Lloyd A. Horrocks
vii
Contents
Preface to the Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preface.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . vi
List of Contributors . . . . . . . . . . . . . . . . ..*............................... xix
Lipid Extraction
Norman S. Radin
1. Introduction ...................................................... 1
2. Properties of Solvents ......................................... 5
2.1. Ethers ........................................................ 6
2.2. Alcohols ..................................................... 8
2.3. Halocarbons ................................................ 9
2.4. Hydrocarbons ............................................. 9
2.5. Miscellaneous Solvents ................................ 10
3. Storage of Lipid Extracts .................................... 10
4. Homogenizers .................................................. 14
5. Separating the Extract from the Tissue Residue ..... 15
5.1. Use of Filtration .......................................... 15
5.2. Centrifugation ............................................ 16
6. Removing Nonlipids from Lipid Extracts .............. 17
6.1. Removal of Nonlipids by Evaporation and
Reextraction ............................................... 17
6.2. Prewashing the Tissue Sample.. .................... 17
6.3. Removal of Nonlipids by Precipitating the
Lipids with the Proteins ............................... 18
6.4. Liquid/Liquid Partitioning ............................ 19
6.5. Removal of Nonlipids by Dialysis .................. 19
6.6. Separation from Nonlipids by Changing a
Partition Constant ....................................... 21
6.7. Liquid/Liquid Partitioning in a Column
with an Immobilizing Solid .......................... 21
ix
X Con tents
6.8. Removal of Nonlipids by Lipid Adsorbents ... 23
7. Extraction with Chloroform: Methanol Mixtures ... 24
7.1. The Methods of Folch et al. ........................ 24
7.2. Biphasic Chloroform: Methanol Methods ...... 26
7.3. Single-Phase Extraction with Other
Chloroform: Methanol Mixtures ................... 31
8. Extraction with Hexane/2-Propanol .................... 33
8.1. Initial Procedure ........................................ 33
8.2. Modified Hexane/2-Propanol Procedure ........ 34
9. Nondenaturing Extraction Methods .................... 36
9.1. Use of Diisopropyl Ether and l-Butanol........ 36
9.2. Use of l-Butanol Alone .............................. 37
9.3. Removal of Lipids from Serum Albumin.. ..... 37
9.4. Nondenaturing Extraction of Detergents
and Bile Salts ............................................ 39
9.5. Extraction with Acetone and Ethanol ............ 39
10. Extraction under Drying Conditions ................... 39
11. Selective Extractions ........................................ 40
11.1. Use of Acetone ........................................ 40
11.2. Extraction of Dispersed, Dry Tissues with
Sequential Solvents .................................. 41
11.3. Extraction with Isopropyl Ether .................. 41
11.4. Selective Extraction with Methanol ............. 41
11.5. Selective Extraction of Fatty Acids .............. 42
11.6. Extraction by Specifically Binding Column
Packings ................................................. 43
11.7. Extraction of Alkali-Stable Lipids after
Hydrolysis of Ester Lipids ......................... 43
11.8. Extraction of Alkali-Stable Lipids after
Tissue Saponification ................................ 44
11.9. Extraction of Gangliosides and Nonionic
Glycolipids with Buffered Tetrahydro-
furan ..................................................... 44
11.10. Extraction by Anion Exchange Resins.. ...... 45
11.11. Extraction of Fatty Acyl Coenzyme A.. ...... 45
12. Solid Phase Extraction ...................................... 46
12.1. Uptake from Physically Absorbed
Aqueous Samples .................................... 46
12.2. Reverse Phase Uptake of Lipids ................. 46
Contents xi
12.3. Uptake of Lipids by Adsorbents ................. 49
13. Special Problem of Labile Lipids ........................ 49
14. Pigment Problems ........................................... 50
15. Extraction with Simultaneous Dehydration .......... 50
15.1. Azeotropic Distillation with
1,2-Dichloroethane ................................... 50
15.2. Extraction during Chemical Removal
of Water ................................................. 51
15.3. Dehydration by Silica Gel on a TLC Plate .... 52
16. Miscellaneous Solvent Methods.. ....................... 52
16.1. Benzene + Methanol (1: 1) ........................ 52
16.2. Phenol + Water + Chloroform +
Petroleum Ether ...................................... 53
16.3. Thirty-one Miscellaneous Solvents .............. 53
16.4. Ethyl Acetate and Acetone ........................ 53
16.5. Ethyl Acetate .......................................... 54
16.6. A Low-Volatility Solvent ........................... 54
References .............................................. 54
Preparation and Analysis of Acyl and Alkenyl Groups of
Cilycerophospholipids from Brain Subcellular Membranes
Grace Y. Sun
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
2. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
2.1. Extraction of Lipids from Brain Tissue
Homogenates and Subcellular Membranes.. . . . . 64
2.2. Separation of Nonpolar Lipids, Glyco-
sphingolipids, and Phospholipids by Silicic
Acid (Unisil) Column Chromatography . . . . . . . , . . 65
2.3. Separation of Phospholipids and Neutral
Glycerides by Two-Dimensional TLC . . . . . . . . . . . . . 65
2.4. Determination of Phospholipids by Assay-
ing the Phosphorus Content . . . . . . . . . . . . . . . . . . . . . . . . . 67
2.5. Analysis of the Glycerolipid and Neutral
Glyceride Acyl Groups by Gas-Liquid
Chromatography . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
2.6. Analysis of the Alkenyl Group Composition
of Ethanolamine Plasmalogen . . . . . . . . . . . . . . . . . . . . . . . 70
xii Contents
3. Information on Phospholipid, Acyl, and Alkenyl
Group Composition in Brain and Subcellular
Membranes.. . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . 71
3.1. Phospholipid Composition . . . . . . . . . . . . . . ..*.......... 71
3.2. Alkenyl Group Composition of Ethanol-
amine Plasmalogen in Brain . . . . . . . . . . . . . . . . . . . . . . . . . 74
3.3. Acyl Group Composition of Phospholipids
in Brain Subcellular Membranes.. . . . . . . . . . . . . . . . . . . 76
4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
References ~............~...~,.~..,..~,~.........~~........~~..,~. 81
Quantitative Analysis of Acyl Group Composition
of Brain Phospholipids, Neutral Lipids,
and Free Fatty Acids
Victor L. Marcheselli, Burton L. Scott, T. Sanjeeva Reddy,
and Nicolas G. Bazan
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
2. Sampling of Brain . . . , . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . 84
3. Extraction of Lipids from Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
4. Separation of Phospholipids . . . . . . . ..a..................... 87
5. Separation of Free Fatty Acids (FFAs),
Diacylglycerols (DGs), and Triacylglycerols
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..*.......*.................*. 90
VW
6. Separation and Quantification of Fatty Acid
Methyl Esters by Gas-Liquid Chromatography . . . . . . 96
7. Separation of Fatty Acid Methyl Esters by
Capillary Gas-Liquid Chromatography . . . . . . . . . . . . . . . 100
8. Separation of Fatty Acid Methyl Esters by
High Performance Liquid Chromatography.. . . . . . . . 103
9. Conclusion . . . . . . ..*............*.............................. 105
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Steroids and Related lsoprenoids
Thomas J. Langan, Robert S. Rust, and Joseph J. Volpe
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
2. Neurochemical Strategies for Examination of
Steroid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113