The Vitamins: Chemistry, Physiology, Pathology, Methods

The Vitamins, Second Edition, Volume III: Chemistry, Physiology, Pathology, Methods covers an understanding of how each vitamin functions in animals and plants. The book describes the chemistry, industrial production, biogenesis, biochemistry, deficiency effects, requirements, pharmacology, and pathology of choline, vitamin D group, essential fatty acids, inositols, and vitamin K group. The text also describes the occurrence and effects of vitamin deficiency and the direct evidence of disease in humans. Chemists, physiologists, pathologists, and students taking related courses will find the book useful.

• Language: English
• Publisher: Academic Press
• Release date: Apr 24, 2014
• ISBN: 9781483220130

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THE VITAMINS

Chemistry, Physiology, Pathology, Methods

Second Edition

W.H. SEBRELL, JR.

Institute of Nutrition Science, Columbia University, New York, New York

ROBERT S. HARRIS

William F. Lasby Professor in the Health Sciences, University of Minnesota, Minneapolis, Minnesota

Table of Contents

Cover image

Title page

VOLUME I—VOLUME V

Copyright

Contributors to Volume III

Preface

Contents of Other Volumes

Chapter 6: CHOLINE

I: Nomenclature and Formulas

II: Chemistry

III: Industrial Preparation

IV: Biochemical Systems

V: Specificity of Action

VI: Biogenesis

VII: Estimation

VIII: Standardization of Activity

IX: Occurrence

X: Effect of Deficiency

XI: Man

Chapter 7: VITAMIN D GROUP

Publisher Summary

I: Nomenclature and Formulas

II: Chemistry

II: Chemistry

III: Industrial Preparation and Production

IV: Estimation in Foods and Food Supplements

V: Occurrence in Foods

VI: Standardization of Activity

VII: Biogenesis

VIII.: Active Compounds

IX.: Biochemical Systems

X.: Deficiency Effects in Animals

XI.: Deficiency Effects in Human Beings

XII.: Chemical Pathology and Pharmacology

XIII.: Requirements of Animals

XIV: Requirements of Human Beings

Chapter 8: ESSENTIAL FATTY ACIDS

I: Nomenclature and Formulas

II: Chemistry

III.: Bioassay and Active Compounds

IV: Occurrence in Foods

V: Biogenesis

VI: Biological Function

VII: Dietary Factors Affecting Essential Fatty Acid Metabolism

VIII: Deficiency Effects in Animals

IX: Deficiency Effects in Human Beings

X: Requirements of Animals and Human Beings

Chapter 9: INOSITOLS

I.: Nomenclature and Formula

II: Chemistry

III: Industrial Preparation

IV: Estimation

V: Occurrence

VI: Standardization of Activity

VII: Biogenesis

VIII: Antagonists

IX: Biochemical Systems

X: Deficiency Effects in Animals

XI: Deficiency Effects in Human Beings

XII: Pharmacology and Toxicology

XIII: Requirements of Animals

XIV: Requirements of Human Beings

Chapter 10: VITAMIN K GROUP

I: Nomenclature and Formulas

II: Chemistry

III: Industrial Preparation

IV: Estimation in Foods and Food Supplements

V: Occurrence in Foods

VI: Standardization of Activity

VII: Biogenesis

VIII: Active Compounds and Antagonists

IX: Biochemical Systems

X: Deficiency Effects in Animals and Human Beings

XI: Pharmacology and Toxicology

XII: Requirement of Microbes and Animals

XIII: Requirements of Human Beings

AUTHOR INDEX

SUBJECT INDEX

VOLUME I—VOLUME V

Edited by

W. H. SEBRELL, JR., and ROBERT S. HARRIS

VOLUME VI and VOLUME VII

Edited by

PAUL GYÖRGY and W. N. PEARSON

Copyright

COPYRIGHT © 1971, BY ACADEMIC PRESS, INC.

ALL RIGHTS RESERVED

NO PART OF THIS BOOK MAY BE BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by

ACADEMIC PRESS INC.(LONDON) LTD.

Berkeley Square House, London W1X 6BA

LIBRARY OF CONGRESS CATALOG CARD NUMBER: 66-26845

PRINTED IN THE UNITED STATES OF AMERICA

Contributors to Volume III

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

SYED Q. ALAM,     (356, 363, 368, 371, 377, 380), Department of Nutrition, Massachusetts Institute of Technology, Cambridge, Massachusetts

H.J. ALMQUIST,     (445, 447, 449, 466, 510), Pacific Vegetable Oil Corpora tion, San Francisco, California

LAURENS ANDERSON,     (341), Department of Biochemistry, College of Agriculture, University of Wisconsin, Madison, Wisconsin

S.J. ANGYAL,     (345), University of New South Wales, Kensington, N.S.W., Australia

JOHN W. BLUNT,     (213), Dyson Perrins Laboratory, South Parks Road, Oxford, England

T.J CUNHA,     (394, 410), Department of Animal Science, University of Florida, Gainesville, Florida

HECTOR F. DELUCA,     (213, 230, 240), Department of Biochemistry, University of Wisconsin, Madison, Wisconsin

DONALD GRIBETZ,     (259, 278, 290), Department of Pediatrics, The Mount Sinai Hospital School of Medicine, New York, New York

WENDELL H. GRIFFITH,     (43, 16, 63, 70, 76, 81), Beaumont House, 9650 Rockville Pike, Bethesda, Maryland

ROBERT S. HARRIS,     (2, 156, 417), William F. Lasby Professor in the Health Sciences, University of Minnesota, Minneapolis, Minne sota

W. STANLEY HARTROFT,     (123), Department of Pathology, University of Hawaii School of Medicine, Leahi Hospital, Honolulu, Hawaii

RALPH T. HOLMAN,     (304, 306, 313, 316, 319, 322, 324, 335), University of Minnesota, Hormel Institute, Austin, Minnesota

O. ISLER,     (418, 444), F. Hoffmann-La Roche & Company, Ltd., Basel, Switzerland

JAMES H. JONES,     (247, 285), Laboratories of Biochemistry, Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

BENJAMIN KRAMER,     (259, 278, 290), Department of Pediatrics, State University of New York, Downstate Medical Center, Brooklyn, New York

H. MAYER,     (418, 444), F. Hoffmann-La Roche & Company, Ltd., Basel, Switzerland

A.T. MILHORAT,     (398, 405, 412), Institute for Muscle Disease, Inc. New York, New York

JUAN M. NAVIA,     (158), Institute of Dental Research, University of Alabama in Birmingham, Birmingham, Alabama

JOSEPH F. NYC,     (3, 16, 63, 70, 76, 81), School of Medicine, University of California, Los Angeles, California

CHARLES A. OWEN, Jr.,     (470, 492, 521), Section of Clinical Pathology Mayo Clinic, Rochester, Minnesota

E.A. PORTA,     (123), The Hospital for Sick Children, Toronto, Ontario, Canada

JOSEPH J. RAHM,     (304, 306, 313, 316, 319, 322, 324, 335), Boise Cascade, International Falls, Minnesota

P. RIETZ,     (455), Department of Vitamin and Nutritional Research, F. Hoffmann-La Roche & Company, Ltd., Basel, Switzerland

HENRY RIKKERS,     (213), Department of Pediatrics, University of Wisconsin, Madison, Wisconsin

HENRY T. SCOTT,     (203, 206, 209, 211), Wisconsin Alumni Research Foundation, Madison, Wisconsin

JULIUS A. VIDA,     (180), Kendall Company, The Theodore Clark Labora tory, Cambridge, Massachusetts

F. WEBER,     (457), F. Hoffmann-La Roche & Company, Ltd., Basel, Switzerland

E.R. WEIDLEIN, Jr.,     (353), Tarrytown Technical Center, Union Carbide Corp., Tarrytown, New York

HILDA F. WIESE,     (327), Bruce Lyons Memorial Research Laboratory, Children’s Hospital Medical Center of Northern California, Oakland, California

O. WISS,     (457) F. Hoffmann-La Roche & Company, Ltd., Basel, Switzerland

Preface

We are pleased to present this second edition of The Vitamins. The fifteen years which have passed since publication of the first edition have been filled with diligent search by many scientists for an understanding as to how each vitamin functions in animals and plants. The content of The Vitamins has broadened and deepened, and the vast amount of new information has created a need for a nearly complete rewriting of the first edition. Since most of the recent advances have been concerned with chemistry, biochemistry, and physiology, it is understandable that these biodisciplines have received special emphasis in this second edition.

We have followed the same general principles as guided us in the first edition. The writing of each section of each chapter has been assigned to a scientist who is especially expert on the subject. Current knowledge concerning the chemistry, industrial production, biogenesis, biochemistry, deficiency effects, requirements, pharmacology, and pathology of each of the vitamins has been emphasized, and considerable space has been devoted to bibliographic material since this is essentially a reference work. Extensive discussion of clinical manifestations of vitamin deficiency or treatment has been omitted since this is well covered in clinical publications.

Little space was given in the first edition to methods of measurement and assay of the various vitamins. This important aspect of vitamin science has been consolidated and is presented in Volumes VI and VII of this treatise.

We hope that this critical summary of current vitamin knowledge will assist teachers, students, investigators, and practitioners toward a better understanding of the role of the vitamins in biology.

We take this opportunity to express our appreciation to the many authors who have contributed to these volumes, and to Academic Press for patient collaboration and cooperation in the production of these volumes.

March, 1971

W.H. Sebrell, Jr.

Robert S. Harris

Contents of Other Volumes

Volume I—Edited by W. H. Sebrell, Jr., and Robert S. Harris

Vitamins A and Carotene

J. Ganguly

Robert S. Harris

O. Isler

H. Kläui

D. McLaren

Thomas Moore

S. K. Murthy

Oswald A. Roels

U. Schwieter

U. Solms

Ascorbic Acid

G. C. Chatterjee

Robert S. Harris

G. W. Hay

B. A. Lewis

L. W. Mapson

Mamie Olliver

F. Smith

Richard W. Vilter

Volume II—Edited by W. H. Sebrell, Jr., and Robert S. Harris

Vitamin B6 Group

Robert S. Harris

Stanton A. Harris

G. Brubacher and O. Wiss

H. Meder and O. Wiss

H. Weiser, G. Brubacher, and O. Wiss

Ho werde E. Säuberlich

F. Weber, H. Weiser, and O. Wiss

P. György

Klaus R. Unna and George R. Honig

H. Weiser, P. Reusser, and O. Wiss

P. György

Vitamin Bl2

Robert S. Harris

Harold W. Moore and Karl Folkers

H. M. Wuest and D. Perlman

Harold L. Rosenthal

Harold W. Moore and Karl Folkers

H. A. Barker

M. E. Coates

E. H. Reisner

M. E. Coates

E. H. Reisner

Biotin

Robert S. Harris

Paul György and Bernhardt W. Langer, Jr.

Bernhardt W. Langer, Jr., and Paul György

Paul György and Bernhardt W. Langer, Jr.

Bernhardt W. Langer, Jr., and Paul György

Volume IV—Edited by W. H. Sebrell, Jr., and Robert S. Harris

Niacin

C. Gopalan

Robert S. Harris

O. Neal Miller

Pantothenic Acid

George M. Briggs

Alice M. Copping

Robert S. Harris

Sanford A. Miller

Juan M. Navia

Paul M. Newberne

G. D. Novelli

Elaine P. Ralli

Pteroylglutamic Acid

Robert S. Harris

E. L. R. Stokstad

Volume V—Edited by W. H. Sebrell, Jr., and Robert S. Harris

Riboflavin

Robert S. Harris

M. K. Horwitt

Theodor Wagner-Jauregg

Tocopherols

Stanley R. Ames

T. Brubacher

J. Green

Robert S. Harris

M. K. Horwitt

Otto Isler

Karl E. Mason

H. Mayer

Peter Schudel

Oswald Wiss

Thiamine

Gene M. Brown

Robert S. Harris

Merton P. Lamden

V. Ramalingaswami

Edward F. Rogers

W. H. Sebrell, Jr.

Klaus Unna

H. M. Wuest

Other Growth Factors

Annette Baich

G. S. Fraenkel

Stanley Friedman

R. A. Morton

Vernon H. Cheldelin

Volume VI—Edited by Paul György and W. N. Pearson

Animal Assays for Vitamins/C. I. Bliss and Paul György

Statistical Methods in Biological Assay of the Vitamins/C. I. Bliss and Colin White

Vitamin A/Oswald A. Roels and S. Mahadevan

Vitamin D/E Kodicek and D. E. M. Lawson

Vitamin K/Henrik Dam and Ebbe Sondergaard

Vitamin E/Raymond Howard Bunnell

Volume VII—Edited by Paul György and W. N. Pearson

Principles of Microbiological Assay/W. N. Pearson

Ascorbic Acid/Joseph H. Roe

Thiamine/W. N. Pearson

Riboflavin/W. N. Pearson

Niacin/Grace A. Goldsmith and O. Neal Miller

Vitamin B6/Howerde Säuberlich

Pantothenic Acid/Orson D. Bird and Robert Q. Thompson

Folic Acid/Victor Herbert and Joseph R. Bertino

Vitamin B12/Helen R. Skeggs

Biotin/Paul György

Clinical Evaluation of Malnutrition/Willard A. Krehl

CHAPTER 6

CHOLINE

Publisher Summary

This chapter discusses the isolation, physical and chemical properties, and synthesis of choline. Choline is widely distributed in biological materials as free choline, acetylcholine, and more complex phospholipids and their metabolic intermediates. Choline is characterized by a trimethyl quaternary nitrogen. Choline has been obtained from a variety of tissues and fluids since their original isolations. The earliest methods employed for isolating choline from biological extracts were dependent on the use of various sensitive though nonspecific precipitants. Choline is precipitated from alcoholic solutions as the double salt of platinum, gold, or mercury chlorides. The periodide separation is generally considered one of the most sensitive methods of precipitating choline. The chapter describes the industrial preparation of choline. The reaction between trimethylamine and either ethylene chlorohydrin or ethylene oxide is commonly used in the manufacture of choline. The studies on choline and its derivatives have emphasized the biochemical importance of these compounds as structural components of tissues, as intermediates in vital metabolic reactions, and as specific chemical reactants of marked biological potency.

I. Nomenclature and Formulas

Robert S. Harris

II. Chemistry

Wendell H. Griffith and Joseph F. Nyc

A. Isolation

B. Physical and Chemical Properties

C. Constitution and Synthesis

III. Industrial Preparation

Wendell H. Griffith and Joseph F. Nyc

IV. Biochemical Systems

Wendell H. Griffith and Joseph F. Nyc

A. Enzymes and Coenzymes

B. Acetylcholinesterase and Choline Acetylase

C. Biogenesis of Choline Phospholipids

D. Mechanism of Action of Choline

V. Specificity of Action

Wendell H. Griffith and Joseph F. Nyc

VI. Biogenesis

Wendell H. Griffith and Joseph F. Nyc

VII. Estimation 70Wendell H. Griffith and Joseph F. Nyc

A. Chemical Procedures

B. Microbiological Procedures

C. Biological Assays

D. Physical Estimation

VIII. Standardization of Activity

Wendell H. Griffith and Joseph F. Nyc

IX. Occurrence

Wendell H. Griffith and Joseph F. Nyc

X. Effects of Deficiency

Wendell H. Griffith and Joseph F. Nyc

A. General Manifestations

B. Rat

C. Avian Species

D. Dog

E. Other Species

XI. Man

W. Stanley Hartroft and E. A. Porta

A. Stainable Fat

B. Ceroid

C. Portal versus Nonportal Cirrhosis

D. Intracellular Hyalin (Mallory Bodies)

I

Nomenclature and Formulas

ROBERT S. HARRIS

Accepted name: Choline

Empirical formula: C5H15O2N

Chemical name: β-Hydroxyethyltrimethylammonium hydroxide

Structural formulas:

Free choline:

Combined choline (acetylcholine):

Combined choline (lecithin, one of the phospholipids):

II

Chemistry

WENDELL H. GRIFFITH and JOSEPH F. NYC

A Isolation

Choline is widely distributed in biological materials as free choline, as acetylcholine, and as more complex phospholipids and their metabolic intermediates. It is an integral part of the lecithins, which accounts for its occurrence, in combination at least, in all plant and animal cells. Certain of the plasmalogens are phosphatidalcholines. Choline is also one of the bases

of the sphingomyelins of animal tissues. Phosphorylcholine, glycerylphosphorylcholine, and the ester of phosphorylcholine with sphingosine have been reported to occur, but it is uncertain to what extent these components of phospholipids normally exist free in tissues. Choline is characterized by a trimethyl quaternary nitrogen. Substances related to choline in this respect include glycine betaine, carnitine, and ergothionine. With respect to lability of methyl groups, related compounds are methionine, dimethyl-β-propiothetin, and dimethylthetin. The latter is of considerable importance in laboratory studies but is not known to occur naturally.

Surprisingly, the first isolations of choline were not from materials rich in the complex lipids, but from hog bile by Strecker¹ in 1849 and from an alkaloid of white mustard seed (Sinapis alba) by von Babo and Hirschbrunn² in 1852. The latter workers named their product sinkaline, whereas Strecker³ in 1862 applied the name choline to the substance obtained from bile. Subsequently, Liebreich⁴ separated a fraction from hydrolyzed crude brain lecithin (protagon), which he named neurine. Dybkowsky⁵ soon found that Liebreich’s base was choline, not the vinyl compound known as neurine at the present time, and Claus and Keesé⁶ demonstrated the identity of sinkaline and choline.

Choline has been obtained from a great variety of tissues and fluids since these original isolations. Wrede and Bruch⁷ extracted various tissues with hot acidulated water, and the choline in these extracts was isolated and weighed as the chloroaurate. Bischoff et al.,⁸ using a reineckate precipitation, reported finding up to 45 mg of free choline (calculated as the chloride) per kilogram of muscle. Heesch⁹ prepared extracts of blood serum that had been treated with trichloracetic acid and found in these extracts 2.5–10 mg of choline per liter of blood. Strack et al.¹⁰ have presented data which suggest that much of the evidence for the presence of free choline in biological materials is unreliable owing to delay in the preparation of extracts, with resulting release of choline by autolysis. They found that dog’s liver contained 0–43 mg of choline if extracted immediately after death of the animal and 136-164 mg of choline per kilogram of liver if extracts were made 5 hours after death. A similar slow release of free choline occurred in experiments in which the fresh tissue was suspended in alcohol. Strack et al.¹¹ did not find free choline in rabbit, dog, or beef muscle. On careful investigation the substance in muscle that was precipitated as the reineckate and reported as choline by Bischoff et al.⁸ was found to be carnitine. On the other hand, Ackermann and his associates have isolated choline from cerebrospinal fluid,¹² from silkworm pupae,¹³ and from various marine organisms.¹⁴-¹⁷

Many solvents have been tested with respect to the thoroughness with which total choline, combined and free, is extracted from natural products. Among these are benzene, petroleum ether, ethyl ether, ethanol, methanol, acetone, chloroform, and mixtures thereof. None has proved to have any special advantage over methanol.¹⁸,¹⁹ Engel¹⁸ employed multiple extractions of samples with methanol in a Bailey-Walker extractor. The more convenient method of extraction with the Soxhlet apparatus is generally preferred. Glick²⁰ has recommended the mixing of powdered samples with No. 2 pulverized pumice, after weighing, to prevent caking of the sample and the resultant channeling of the extracting solvent

The residue of the methanol extracts of samples must be hydrolyzed if the total choline content is to be determined. Barium hydroxide has been favored as the alkali for the digestion process because there is no loss of choline when pure choline solutions are used.21-24 Gulewitsch²¹-²⁴ studied the effect of heating choline in aqueous baryta as well as in alcoholic solutions of sodium ethylate and found only a negligible breakdown of choline after boiling in baryta solution for 6 hours or after heating in a 5 % sodium alcoholate solution for 24 hours.

Beattie²⁵ studied the acid hydrolysis of a lecithin emulsion prepared from a commercial egg lecithin preparation and hydrolyzed in 7.8 % hydrochloric acid at 110°. The maximum value of free choline was obtained after hydrolysis for 21 hours. Acid hydrolysis has been used also in the liberation of choline from bound forms in tissues.²⁶-²⁸ Ducet and Kahane²⁸ refluxed animal and vegetable tissues with 30 % nitric acid until a clear solution was obtained. After neutralization of the solution with powdered calcium carbonate and dilution with several volumes of water, 10 ml of 50 % ferric sulfate and 5 gm of calcium carbonate were added for each gram of dry tissue originally taken. The mixture was heated to boiling and filtered. The filtrate and washings containing the choline were concentrated to a small volume, and the choline was precipitated by one of the reagents generally employed for this purpose. These workers found that no choline was destroyed during this procedure.

The earliest methods employed for isolating choline from biological extracts were dependent on the use of various sensitive though nonspecific precipitants. Choline may be precipitated from alcoholic solutions as the double salt of platinum, gold, or mercury chlorides.²¹,²⁹,³⁰ Precipitation as the reineckate or the periodide has been employed most extensively for the removal of choline from aqueous solution.

Beattie²⁵ observed that a quantitative precipitation of free choline as the reineckate can be obtained in solutions containing as little as 0.03 mg of choline chloride per milliliter and that the choline in about 7−10 ml of a solution of this concentration can be quantitatively determined. The slight extent to which other substances interfere with the reineckate precipitation and estimation of choline in animal tissues and fluids was demonstrated by Beattie by analysis of tissue extracts, a tryptic digest, and urine before and after the addition of known amounts of choline chloride (Table 1).

Table I

RECOVERY OF ADDED CHOLINE BY THE REINECKATEa

aFrom F. J. R. Beattie, Biochem. J. 30, 1554 (1936).

On the basis of a careful study of the reineckate method, as originally modified by Jacobi et al.²³ and Engel,¹⁸ Glick²⁰ has proposed the following procedure for the isolation of choline from natural materials.

A weighed sample, containing the equivalent of 2–5 mg of choline chloride, is placed in an alundum thimble of medium porosity (80 mm long and 22 mm in diameter) for extraction in a Soxhlet apparatus fitted with a 125-ml boiling flask. About 100 ml of methanol is used as the solvent, and the extraction is allowed to proceed for 24 hours. With some finely divided materials, such as flour, the tendency to form a hard cake makes it desirable to mix the sample intimately with No. 2 pulverized pumice to facilitate the extraction. The boiling flask containing the methanol extract is placed on a steam bath and, when only a few milliliters of solvent remains, 30 ml of a saturated solution of barium hydroxide is added and the heating is continued for 90 minutes. After the mixture is cooled, a drop of 1 % alcoholic thymolphthalein is added to the hydrolyzate and glacial acetic is introduced until the blue color is just discharged by 1 drop. The liquid is then filtered by suction through a sintered-glass filter tube of medium porosity (15− to 30–ml capacity) into a 125-ml suction flask. The boiling flask is rinsed with small portions of distilled water, and the rinsings are used to wash the filter, a total of about 15 ml of water being used. To the combined filtrate and washings is added 6 ml of a 2 % solution of reineckate salt in methanol, and the flask is placed in a refrigerator at about 5° for 2 hours. The choline reineckate precipitate is filtered with suction into a 30-ml sintered-glass filter tube of medium porosity. The dried precipitate is washed three times with 2.5-ml portions of n-propanol and again dried by means of the suction.

The above procedure avoids the precipitation of betaine reineckate, which is insoluble in acid solutions but soluble in slightly alkaline solutions. However, it has been noted ³¹ that dimethylaminoethanol appears to be carried down in part in the choline reineckate precipitate when a solution containing the two bases is treated with reineckate at a slightly alkaline pH.

According to Coujard,³² treatment of tissue sections with reineckate precipitates choline reineckate as birefringent crystals that are readily seen with a polarizing microscope. Keenan³³ has described microscopic procedures for the quantitative detection of traces of choline as the reineckate and as the chloroplatinate.

The periodide separation is generally considered to be one of the most sensitive methods of precipitating choline. Griess and Harrow³⁴ had utilized the insolubility of the periodide to isolate choline as early as 1885. In 1896

Florence³⁵ described a medicolegal test for semen stains based upon the typical crystals formed when this material was treated with iodine in potassium iodide solution. Bocarius³⁶isolated the typical Florence crystals and proved by chemical identification that choline was the substance which gave the insoluble periodide. Booth³⁷ estimated that in aqueous solutions potassium triiodide gives a precipitate with choline at a dilution of about 1:50,000. Staněk³⁸ studied the chemical composition of the choline periodide precipitate and the conditions under which it is formed. A detailed study of the periodide procedure for the isolation and subsequent estimation of choline was made by Kiesel.³⁹

Choline may also be precipitated from water with phosphotungstic, silicotungstic, and phosphomolybdic acids.²¹,⁴⁰ Ackermann⁴¹ used dipicrylamine as a precipitant, the choline salt being only slightly soluble in water (0.02 gm in 100 ml of water at 20°). The low solubility of the salt permitted the separation of choline from betaine and aminoethanol. Schoorl⁴² published descriptions and enlarged micrographs of the double salts of choline hydrochloride with the following reagents: platinum chloride, sodium gold chloride, mercuric chloride, mercuric iodide, potassium bismuth iodide, picric acid, and picrolonic acid

Several combined water-soluble forms of choline have been isolated from biological materials. In 1929 Dale and Dudley⁴³ succeeded in isolating acetylcholine from an extract of horse spleen in sufficient quantities for chemical identification. Since that time the acetylcholine in tissues has been widely studied. A summary of the early work on acetylcholine has been published by Gaddum.⁴⁴

Hunt⁴⁵ described a biological test for choline based on its conversion to acetylcholine and the demonstration of the effect of the ester in lowering blood pressure in cats or rabbits or in decreasing the amplitude of the beat of the frog’s heart. Ackermann and Mauer⁴⁶ prepared the acetylcholine salt of dipicrylamine, insoluble red crystals yielding a red solution with acetone suitable for colorimetric estimation. Rossi et al.⁴⁷ compared various derivatives and found that the formation of the crystalline silicotungstate was a useful method of distinguishing choline and acetylcholine.

As yet no methods exist for the accurate isolation and determination of the different forrns of choline in biological materials, particularly free choline and combined water-soluble choline. Kahane and Lévy⁴⁸ defined water-soluble choline as the total found in aqueous extracts of tissues after suitable precipitation and filtration. Ferric hydroxide formed within the mixture by addition of ferric sulfate, and calcium carbonate was recommended as the best precipitating agent. The choline of lecithin would not be included in the total water-soluble choline. Kahane and Simenauer have evaluated methods of degrading trimethylammonium compounds to trimethylamine.⁴⁹

Several attempts have been made to devise procedures for the separation of free choline from combined water-soluble choline as well as from other water-soluble substances which may interfere with its isolation and quantitative determination. Gebauer-Fuelnegg and Kendall⁵⁰ applied electrodialysis to the separation of histidine from histamine or choline, and also to the separation of an artificial mixture of protein or gelatin from histamine or choline. This is reported to be a suitable method for the separation of relatively strong, crystalloidal bases from mixtures with amphoteric or more weakly basic substances.

Horowitz and Beadle⁵¹ used Permutit columns to separate choline from nonbasic interfering substances. They found that a Permutit column measuring 110 × 0.6 mm, containing approximately 1 gm of Permutit, completely removes the choline from 5 ml of a solution containing up to 0.5 mg of choline per milliliter. Repeated tests showed that the absorbed choline is quantitatively eluted with 10 ml of 5 % sodium chloride. Ducet⁵² observed that free choline can be quantitatively adsorbed on silica gel whereas the combined water-soluble choline remains in the solvent.

The isolation of choline by paper chromatography was investigated by Munier and Macheboeuf.⁵³ These workers report that nonalkaloidal substances such as choline and betaine are readily separated from alkaloids because their partition coefficients in various solvent systems are different. Choline is detected on the paper strips by the blue color formed when the chromatograms are treated with solutions containing phosphomolybdic acid, acetic acid, and stannous chloride. Rf values are given by these workers for choline when it is chromatographed with various solvent mixtures containing n-butanol, acetic acid, and water. Separation of choline by paper chromatography has been used advantageously by a number of investigators.⁵³-⁵⁷a

Inukai and Nakahara⁵⁸ isolated phosphorylcholine from beef liver, and the amounts in rat tissues have been determined by Dawson.⁵⁹ Glycerylphosphorylcholine has been isolated from pancreas,⁶⁰ from semen,⁶¹ from sperm,⁶² and from the limpet Patella vulgata.⁶³ High levels found in the seminal vesicle secretion of rats decreased after castration and were restored to normal by the administration of androgens,⁶⁴ L-α-Glycerylphosphorylcholine was prepared in 40-65 % yield from pure lysolecithins by the action of an extract of Penicillium notatum.⁶⁵

Isolation from animal tissues of a water-soluble substance believed to be sphingosylphosphorylcholine has been reported by a number of investigators.¹⁰,, ⁶⁶-⁷⁰ However, Dawson was unable to demonstrate its presence in the free form in tissues.⁷¹ The compound has been prepared by Kaller from a hydrolyzate of the sphingomyelin of frog muscle.⁷²

Deoxycytidine diphosphate choline has been found in sea urchin eggs, microorganisms, and mammalian viscera by Sugino⁷³,⁷⁴ and from calf thymus by Potter and Buettner-Janusch.⁷⁵ As will be noted later, cytidine diphosphate choline is an active intermediate in the biogenesis of lecithins. Dumont⁷⁶ has shown that 87 % of the lipid of the Chinese crab, Eriocheir sinensis, is phospholipid which is mostly the plasmalogen phosphatidalcholine. Phosphatidalcholines have also been prepared from beef heart by Rapport⁷⁷ and by Klenk and Debuch,⁷⁸ from egg by Thiele,⁷⁹ from several rat tissues by Webster,⁸⁰ and from ram spermatozoa by Lovern et al.⁸¹ Choline was found in the hydrolyzate of an unidentified phospholipid isolated from malignant tissue by KöCsaki and co-workers,⁸²-⁸⁴ but the significance of the material has been questioned.⁸⁴a

Lysophosphatidylcholine has been isolated from hen’s egg by Rhodes and Lea⁸⁵ and by Hartree and Mann.⁸⁶ A choline-containing glycolipid, first isolated from oysters by Akiya and Nakazawa,⁸⁷ was shown by Nakazawa⁸⁸ to yield on hydrolysis equimolecular quantities of choline, 14-methylpentadec-4-enoic acid, lactic acid, taurine, and a trisaccharide.

Cyclic choline sulfate was isolated from Aspergillus sydowi by Woolley and Peterson⁸⁹ and from Penicillium chrysogenum by Stevens and Vohra⁹⁰ and by de Flines.⁹¹ It had been synthesized earlier by Schmidt and Wagner,⁹² and its enzymatic formation has been demonstrated by Kaji and McElroy.⁹³ Its use by Aspergillus oryzae⁹⁴ by P. chrysogenum⁹⁰ as a source of sulfur has been reported.

B Physical and Chemical Properties

Choline, hydroxyethyltrimethylammonium hydroxide, can be obtained with difficulty as a colorless crystalline mass by drying under high vacuum over P2O5.³⁴,⁹⁵ It is a strong base, decomposes ammonium salts, and has a marked tendency to absorb water and carbon dioxide from the air. Choline has no well-defined melting or boiling point but breaks down when heated into trimethylamine and glycol. Dimethylaminoethanol and dimethylvinylamine are also formed in lesser amounts by thermal decomposition of the base.⁹⁵ Dilute water solutions of the base are stable to heat, but concentrated solutions give off trimethylamine when boiled.⁹⁶

Choline is soluble in water, in formaldehyde, and in absolute methyl and ethyl alcohols. It is sparingly soluble in amyl alcohol, chloroform, dry acetone, and wet ether. Choline is insoluble in dry ether, carbon tetrachloride, carbon disulfide, toluene, benzene, and petroleum e ther.⁹⁷,⁹⁸

Edsall⁹⁹ reported the Raman spectrum for choline chloride, and the ultraviolet absorption spectrum of the base was described by Castille and Ruppal ¹⁰⁰ and by Graubner.¹⁰¹ Crystalline choline chloride is abnormally sensitive to decomposition by ionizing radiation.¹⁰¹-¹⁰⁴

C Constitution and Synthesis

The correct structure of choline was determined by Baeyer¹⁰⁵ and by Wurtz,¹⁰⁶who carried out the first syntheses, using the reaction of trimethylamine either on ethylene chlorohydrin or on ethylene oxide with the formation of the chloride or the free base, respectively.

Several of the early synthetic methods for choline were based on (2-bromoethyl)trimethylammonium bromide as the starting compound. This substance is easily prepared by allowing trimethylamine to react with ethylene bromide according to the following equation:(CH3)3N+BrCH2CH2Br→Br(CH3)3CH2CH2Br

Bode¹⁰⁷ converted the brominated product into choline by heating it in a solution of silver nitrate. Krüger and Bergell¹⁰⁸ effected the same conversion by heating its aqueous solution for 4 hours at 160° in a sealed tube. Lucius¹⁰⁹

heated the compound for 1 hour in an alcoholic solution of potassium hydroxide at 120° and obtained a mixture of choline and neurine (vinyltrimethylammonium hydroxide).

Choline has been synthesized also by the exhaustive methylation of aminoethanol with methyl iodide in a methanolic solution of potassium hydroxide,¹¹⁰ by modifications of the original methods of Wurtz¹⁰⁶ using trimethylamine,¹¹¹-¹¹³ by preparation of dimethylaminoethanol and its conversion to choline through the methiodide,¹¹⁴ and by hydrolysis of 2-(ethoxymethoxy)ethyltrimethylammonium formate formed from the corresponding dimethylamine derivative and methyl formate.¹¹⁵

The general problem involving the synthesis of hydroxy bases and of homologs of choline was studied by von Braun.¹¹⁴ This worker has shown that, by means of the compounds Br(CH2)xOBz and NHMe2, bases of the type Me2H(CH2)xOBz can be prepared. These are quantitatively converted to the hydroxy bases, Me2N(CH2)xOH, by alkaline hydrolysis. The methiodide of the product can then be treated with silver chloride to give Me3NCl(CH2)xOH.

The synthesis of choline with the hydrogens of the methyl groups replaced by deuterium was first undertaken by du Vigneaud1 ¹¹⁶ and his co-workers. Deuteriomethyl alcohol was converted with phosphorus and iodine to deuteriomethyliodide. The iodide with aminoethanol yielded choline with an overall yield of 64 % based on deuteriomethyl alcohol. Walz et al.¹¹⁷ synthesized choline and acetylcholine labeled in the ethylene chain with isotopic carbon-¹⁴. Acetylene ¹⁴-C, obtained from active carbonate in the usual manner,¹¹⁸ was reduced to ethylene by reaction with chromous chloride according to the method of Arrol and Glascock.¹¹⁹ The labeled ethylene was converted to ethylene bromohydrin-l,2-¹⁴C with N-bromoacetamide. The bromohydrin with excess trimethylamine in ether yielded choline bromide with an 83 % yield based on the bromohydrin. Dauben and Gee¹²⁰ have published an alternative procedure starting with carboxyl-labeled sodium acetate. This was converted to chloroacetic acid which was esterified with diazoethane.

The resulting chloroacetate was allowed to react with dimethylamine, and the product was reduced to N,N-dimethylaminoethanol with lithium aluminum hydride. The substituted ethanol was further methylated with methyl iodide, and the choline iodide was converted into choline chloride with ¹⁴C in the alcoholic carbon.

An improved synthesis of phosphorylcholine has been described by Baer.¹²¹,¹²² The compound was prepared by the catalytic hydrogenation of diphenylphosphorylcholine produced by the reaction of diphenylphosphoryl chloride and choline chloride in pyridine

Glycerylphosphoric acid esters of choline have been prepared by Ravazzoni and Fenaroli¹²³ and by Aloisi and Buffa¹²⁴ from bromocholine picrate and the silver α- and β-glycerylphosphates. These authors suggest that previous workers may have confused the choline salts of the glycerylphosphates with the choline esters. The choline salts form readily and block esterification. Baer and Kates¹²⁵,¹²⁶ prepared and studied the hydrolysis of L-α-glycerylphosphorylcholine and noted a reversible shifting of the phosphoric acid between the α- and β-carbons.

Salts of choline and of the common acids, including acetic, carbonic, hydrochloric, nitric, oxalic, picric, picrolonic, and sulfuric acids, are soluble in water and in ethanol, whereas the acid tartrate, chloroplatinate, monophosphate, and ruffinate are insoluble in ethanol. Double salts with cadmium chloride and with zinc chloride are also soluble in water and insoluble in ethanol. Double salts with gold chloride and with mercuric chloride are insoluble in water. Other water-insoluble salts include the hexaiodide, periodate, enneaiodide, phosphotungstate, phosphomolybdate, reineckate, and salts with Mayer’s reagent (potassium mercuric iodide) and with Kraut’s reagent (potassium bismuth iodide). The chloroplatinate is moderately soluble in water but very insoluble in ethanol. The flavianate is sparingly soluble in ethanol and is insoluble in n-butanol.

The properties of some of the more important salts are listed below:Chloride (C5H14ONCl): Soluble in water, methanol, ethanol, and formaldehyde; less soluble in carbon tetrachloride, chloroform, and acetone; insoluble in carbon disulfide, benzene, toluene, ether, and petroleum ether; deliquescent; stable up to 180°, decomposing on heating to give dimethylaminoethanol and methyl chloride.

Reineckate (C15H14ON·C4H7N6S4Cr): Melts above 250°,¹²⁷ soluble in water at 18° up to 0.02 %, in 10 % hydrochloric acid up to 0.03 %; in the presence of excess ammonium reineckate the solubility in water is greatly depressed ¹²⁸; insoluble in dilute ammonia, 0.1 N sodium hydroxide, ethanol, benzene, and ether, but has an appreciable solubility in acetone.¹²⁹,¹³⁰

Periodide: Periodides of choline are precipitated by iodine in potassium iodide solution, either as an insoluble oil or as a crystalline material, depending upon the conditions.³⁴,³⁸

Hexaiodide (C5H14ON·I·I5): Greenish black iridescent oil obtained when potassium triiodide solution is added to an excess of choline chloride; very insoluble in water and soluble in ethanol; converted to the enneaiodide by treatment with K¹³ solution or powdered iodine.

Enneaiodide (C5H14ON·I·I8): Green needles, soluble in alcohol but very insoluble in water; loses iodine rapidly in air and goes over to the hexaiodide.Mercuric chloride double salt (C5H14ON·Cl·6HgCl2): Melts at 249-251°,21 242-243° 28; insoluble in cold water and very insoluble in alcohol.

Chloroaurate (C5H14ON·Cl·AuCl3): Melts at 243-244° (slow heating), 259° (rapid heating),¹³¹ 257°, ¹³² 267-279°27; deep yellow needles from hot alcohol or octahedra and cubes from dilute alcohol; sparingly soluble in water, and very insoluble in alcohol.

Chloroplatinate (C5H14ON·Cl)2PtCl4: Quickly decomposes on heating at 241-242°95; dimorphous; crystallizes in cubes and octahedra from hot alcohol and water (1:1), but in six-sided pyramids or monoclinic rhombic crystals from water; both forms of crystals are orange-red in color; very insoluble in alcohol but moderately soluble in water.

Bromoplatinate (C5H14ON·Br)2PtBr4: Melts at 240° (decomp.); large dark-red prisms or octahedra; sparingly soluble in water.¹³³

Picrate (C5H14ON·C2H2O7N3): Melts at 240°; readily soluble in water and alcohol.¹³⁴

Complex with uranium [C5H14ON·UO2(NO3)2]2: Yellow, nonhygroscopic crystals insoluble in ethanol and ether and sparingly soluble in water; aqueous solution fluoresces in ultraviolet light.¹³⁵

III

Industrial Preparation

WENDELL H. GRIFFITH and JOSEPH F. NYC

The reaction between trimethylamine and either ethylene chlorohydrin ¹,² or ethylene oxide³ is used commonly in the manufacture of choline.

In Hopff and Vierling’s modification of the first reaction² gaseous trimethylamine is passed through ethylene chlorohydrin at 80°. In KöUrner’s procedure³ trimethylamine and ethylene oxide react in the presence of water and carbon dioxide and the resulting choline is transformed to other salts by treatment with various acids

Choline has been prepared more recently by a two-step synthesis.⁴ The quaternary salt, 2-(ethoxymethoxy)ethyltrimethylammonium formate, is formed by heating 2-(ethoxymethoxy)ethyldimethylamine with an excess of methyl formate at 140-150° under a pressure of 250 psi. The quaternary salt is then refluxed in a mixture of ethyl alcohol and hydrochloric acid, and the reaction mixture is taken to dryness at a reduced pressure. The crude choline chloride remaining in the residue is purified by crystallization from isobutyl alcohol.

IV

Biochemical Systems

WENDELL H. GRIFFITH and JOSEPH F. NYC

A Enzymes and Coenzymes

Studies on choline and its derivatives have emphasized the biochemical importance of these compounds as structural components of tissues, as intermediates in vital metabolic reactions, and as specific chemical reactants of marked biological potency. On the other hand, evidence for the participation of choline or of its derivatives in a specific manner as cofactors in enzymatic systems is meager, although a few reports have linked it or its phosphoric acid ester with phosphatases. Caution is needed in questioning the importance of choline as a component of coenzymes, because relatively little definite information is at hand regarding the functions and properties of the lipoproteins that contain choline phospholipids.

Kielley and Myerhof ¹ believe that a magnesium-activated adenosine-triphosphatase (ATPase) of muscle may consist of a lipoprotein with a choline-containing phospholipid as a constituent. The compound was devoid of myosin and actomyosin, and there was no indication that it was another form of myosin ATPase. Its pH optimum was 6.8, and it was strongly inhibited by calcium. Inactivation of the enzyme and hydrolysis of the phospholipid portion by lecithinase of Clostridium welchii paralleled each other. The occurrence of the pyrophosphoric acid ester of choline in the prosthetic groups of acid and alkaline phosphatases has been reported.² Other workers,³ have noted that this ester contains a labile phosphate group, hydrolyzable by crude, but not by purified, muscle pyrophosphatase; however, they are not of the opinion that it is a coenzyme of a phosphatase. Alkyl nitrogen-substituted derivatives of aminoethanol and of choline activate alkaline phosphomonoesterases.⁴ Activation of an ATPase system in rat submaxillary gland by acetylcholine in vitro has been reported.⁵

According to L. E. Hokin and M. R. Hokin, physiological concentrations of acetylcholine increased the in vitro secretion of enzymes by pigeon pancreas and also increased the incorporation of ³² P and inositol-2-3H into phosphoinositide and of ³²P and glycerol-1-¹⁴C into phosphatidic acid.⁶ They suggested that phosphoinositides, in particular, are involved in the transport of protein from inside of the pancreatic acinar cell into the lumen. Subsequently they observed that acetylcholine increased the secretion of epinephrine and the incorporation of ³² P into phosphoinositide and phosphatidic acid in slices of guinea pig adrenal medulla.⁷ In similar experiments they found that acetylcholine increased the formation of a monophosphoinositide in slices of guinea pig brain cortex and concluded that this result also represented a stimulation of transport of some substance in brain.⁸ Eserine sulfate was order to block its hydrolysis by tissue cholinesterase. Whether or not the effects described by the Hokins are due to the presence of acetylcholine in a coenzyme remains to be determined.

The enzymatic formation and breakdown of acetylcholine are discussed in Section B, below. Reference is made to other enzymes associated with the phosphorylation of choline, with its incorporation into various lipids, and with other aspects of its metabolism in Section C.

A functional role for phospholipid in an enzyme system has been described by Fleischer et al.⁸a-⁸c who have demonstrated a phospholipid requirement in each of three complexes pf the electron transport chain from succinate to oxygen, viz., succinate →CoQ, CoQH2→ cyt. c, and reduced cyt. c→O2. The experiments depended on the mild removal of lipid frommitochondria which were then unable to catalyze electron transfer unless phospholipid was restored. No specific phospholipid appeared to be involved, and the effect was not that of a coenzyme. The findings suggested that the requirement depended on the ability of the phospholipids to form micelles that acted as bridges between hydrophilic and hydrophobic areas and between functional groups. A soluble D-(—)-β-hydroxybutyric apodehydrogenase has been isolated from beef heart mitochondria by fractionation with cholate plus ammonium sulfate and by isoelectric precipitation; it has a specific and absolute requirement for unsaturated lecithin.⁸d

B Acetylcholinesterase and Choline Acetylase

Acetylcholine is the chemical agent responsible for the transmission of the nerve impulse from presynaptic to postsynaptic fibers in synapses of the sympathetic and parasympathetic nervous systems. In addition, acetylcholine is the effective agent causing a response in effector cells of organs innervated by postsynaptic fibers of the parasympathetic system. For this reason such fibers are called ‘cholinergic’ fibers in contrast to the postsynaptic ‘adrenergic’ fibers of the sympathetic system that initiate responses in effector cells by the liberation of epinephrine and norepinephrine. The principal physiological effects of acetylcholine are cardiac inhibition, peripheral vasodilation, and contraction of skeletal muscle. It appears to be concerned with the rhythmicity of the heart, and this action as well as its role in transmission of nerve impulses may be mediated through its influence on the transport of sodium and potassium ions across axonal membranes.⁸,⁹ The action of acetylcholine is terminated by its enzymatic hydrolysis to choline and acetic acid. The extreme physiological activity of acetylcholine makes it under-standable that a cholinesterase and a choline acetylase for its hydrolysis and resynthesis occur in all conducting nervous tissues.

Acetylcholine is hydrolyzed at various rates by miscellaneous tissue esterases and at a rapid rate by two cholinesterases, true acetylcholinesterase and pseudocholinesterase. The latter is more widely distributed in animal tissues but occurs with the true acetylcholinesterase in nerve tissue. Both enzymes are believed to participate in synaptic transmission. In contrast to the true cholinesterase, which is relatively specific for acetylcholine, the activity of the pseudocholinesterase increases with the chain length of the fatty acid acyl groups. An attempt at differentiation on the basis of substrate specificity has been made.¹⁰ Most of the true cholinesterase is found in the mitochondrial and microsomal fractions of brain homogenates whereas the pseudocholinesterase is highest in the nuclear and supernatant fractions.¹¹ Toschi¹² and Hanzon and Toschi¹³ have reported the association of cholinesterase with membranous structures. Both enzymes are inactivated by neostigmine and by the alkaloid eserine. Diisopropyl fluorophosphate is a specific inhibitor that has proved useful in the investigation of the active binding sites of pseudocholinesterase.¹⁴ Acetylcholine and cholinesterase are present in the erythrocyte where they may be involved in the membrane control of passage of sodium and potassium ions.

Acetylcholine is widely distributed in the animal kingdom and is a characteristic component of the nervous system of vertebrates.¹⁵ Its chemical and biological identification has been accomplished after perfusion of sympathetic ganglia with a solution containing ¹⁴C-labeled choline.¹⁶ It has been detected also in invertebrates, including some protozoans¹⁷ and bacteria.¹⁸ Feldberg,19¹⁹ Whittaker, ²⁰and Hebb²¹ have reviewed its functions and mode of action.

The presence of acetylcholine is commonly accepted as evidence of its biogenesis in the tissue. The mechanism of synthesis is the acetylation of choline catalyzed by a transacetylase, first named choline acetylase by Nachmansohn and Machado.²² Several groups contributed to the original demonstration of synthesis of acetylcholine in nervous tissue,²³-²⁵ and this finding was firmly established by Korkes et al.²⁶ The distribution of choline acetylase is in agreement with the postulated role of acetylcholine in nerve transmission.²¹ Relatively large amounts are found in the squid head ganglia27 and in the brain of the blowfly.²⁸ Other studies have shown its presence in the nervous system of man and other mammals,²⁹ in human placenta,³⁰ in the brain of the frog,²² in the muscle of goldfish,³¹ and in Lactobacillus plantarum ³²-³⁴ Bull et al. have described methods of analysis of the enzyme in small samples of nervous tissue.³⁵ Denervation of the submaxillary gland of the cat and rabbit reduced the acetylase activity.³⁶ Similarly, acetylase activity was markedly decreased following denervation of the Sachs electric organ of Electrophorus.³⁷ The properties of choline acetylase have been studied by Berman et al.,²⁷ by Kumagai and Ebashi,³⁸,³⁹ by Reisberg⁴⁰ and have been reviewed by Hebb.²¹

Coenzyme A (CoA) is the coenzyme for most, if not all, of the acetyltransferring systems, including the acetylation of choline to acetylcholine. The components of the coenzyme are adenosine 3’-phosphate 5’-pyrophosphate, the vitamin pantothenic acid, and β-mercaptoethylamine. The nucleoside is joined to the terminal hydroxyl of the pantothenate by the pyrophosphate bridge, and the sulfur component forms an acid amide linkage with the carboxyl of the β-alanine moiety of the pantothenate.⁴¹,⁴²

The free -SH group is the principal site of reactivity in the CoA molecule which is readily acetylated to acetyl-CoA (AcCoA) in the presence of ATP.

The acyl-mercaptide linkage is an energy-rich bond and the acetyl group is aptly described as active acetyl or active 2-carbon fragment. The application of these findings to the metabolism of choline is illustrated by the system which transfers acetyl from citrate to choline. Ochoa et al. isolated a condensing enzyme from heart muscle which catalyzes reversibly the reaction between AcCoA and oxaloacetate to give CoA and citrate.⁴³ If choline and a second acetyltransferring enzyme (choline acetylase) are present in addition to the condensing enzyme, citrate and CoA, acetylcholine is formed.⁴⁴ The acetyl group of acetylcholine may also arise from the decarboxylation of pyruvic acid and the β-oxidation of fatty acids.

C Biogenesis of Choline Phospholipids

Choline occurs as phosphatidylcholine in lecithin, as phosphatidalcholine in certain plasmalogens, and in the sphingomyelins. Kennedy and Weiss⁴⁵ have found that cytidine diphosphate choline (Cy-DPC) is the key intermediate in rat liver mitochondria which transfers its phosphorylcholine moiety to D-l,2-diglyceride with the formation of lecithin (reaction 3). Subsequently, it was demonstrated that D-l,2-diglyceride is also the intermediate in the synthesis of triglyceride (reaction 7)⁴⁶ and that Cy-DPC transfers phosphorylcholine to N-acylsphingosine (ceramide) with sphingomyelin as the product (reaction 8).⁴⁷ The steps in these reactions, according to Kennedy,⁴⁸ are listed in the reaction sequence (l)-(8).

Kennedy has described the purely chemical procedures for the synthesis of Cy-DPC⁴⁹ and Lieberman has prepared the pure compound from baker’s yeast.⁵⁰ The enzymatic synthesis is catalyzed by phosphorylcholinecytidyl transferase, an enzyme widely distributed in nature.⁵¹⁵² The enzymes involved in the transfer of the phosphorylcholine of Cy-DPC to diglyceride and to ceramide are named phosphorylcholine-glyceride transferase ⁵³-⁵⁵ and phosphorylcholine-ceramide transferase,⁵⁶ respectively. Particulate enzyme preparations of liver have been shown to cause a net synthesis of a plasmalogenic phosphatidylcholine from Cy-DPC and an aldehydogenic lipid resembling a diglyceride except that one fatty acid ester bond is replaced by an α,β-unsaturated ether linkage.⁵⁷ Ethanolamine phospholipids are formed in tissues by similar mechanisms with cytidine diphosphate ethanolamine as the key intermediate.⁵⁷,⁵⁸ Cytidine derivatives are also intermediate in the biogenesis of inositol monophosphatide,⁵⁹-⁶¹ phosphatidylserine,⁶² and phosphatidylglycerol.⁶³

The role of cytidine derivatives in the biogenesis of phospholipids has been considered to be uniquely specific for cytidine.⁵⁸ However, a modification of this view has been required by the isolation of deoxycytidine diphosphate choline (deoxy-Cy-DPC) by Sugino.⁶⁴ Deoxy-Cy-DPC and the corresponding ethanolamine derivative were found in calf thymus ⁶⁵ and in extracts of the Novikoff hepatoma.⁶⁶ Kennedy et al.⁶⁷ have found that the transferases active in the formation of the nucleotide diphosphates of choline and ethanolamine from cytidine triphosphate or deoxycytidine triphosphate, respectively, were equally effective. Deoxycytidine diphosphate ethanolamine was less effective as a donor of phosphorylethanolamine to diglyceride than the ribonucleotide derivative, whereas there was little difference between the two nucleotide diphosphate cholines in the formation of phosphatidylcholine. Schneider and Behki reported that the enzymatic formation of lecithin from deoxy-Cy-DPC was strongly inhibited by Cy-DPC.⁶⁷a

The biogenesis of Cy-DPC requires a supply of phosphorylcholine provided by the interaction of ATP, choline, and choline kinase. This kinase was recognized in liver and in yeast,⁶⁷b,⁶⁷c,R. E. McCaman, J. Biol. Chem. 237, 672 (1962). it has been partially purified from rapeseed,⁶⁸ and its microdetermination in nervous tissue has been reported.⁶⁹ The incorporation of phosphorylcholine into lecithin by guinea pig liver mitochondria has been demonstrated.⁷⁰ The enzymatic synthesis and hydrolysis of phosphorylcholine have been studied extensively.⁷¹-⁷³ Phosphorylcholine is a possible precursor of plant phospholipids.⁷³a

Although glycerylphosphorylcholine is not considered to be an intermediate in the formation of choline phospholipids, its occurrence in tissues has been noted. Lecithins are hydrolyzed to this molecule by rat intestine⁷⁴,⁷⁵and by a cell-free extract of pancreas.⁷⁶ Its formation from lysolecithins by extracts of Penicillium notatum has been reported.⁷⁷ On the basis of specific activities of ³² P-labeled products, Dawson concluded that glycerylphosphorylcholine could not be a precursor of phosphatidylcholine.⁷⁸ The findings were consistent with the assumption that the glycerylphosphorylcholine secreted by the rat epididymis was from lecithin or a choline plasmalogen.⁷⁸a An enzyme was found in rat liver which catalyzed its hydrolysis to glycerylphosphoric acid and choline.⁷⁹

In studies on the relation of the intracellular distribution of liver enzymes to the biogenesis of mitochondria Wilgram and Kennedy have found that phosphorylcholine-glyceride transferase and diglyceride acyltransferase have the same distribution as glucose-6-phosphatase and are microsomal enzymes.⁸⁰

Phosphatidic acid phosphatase is probably concentrated in the lysosomes. Phosphorylcholine-cytidyltransferase was found in both the microsomes and the soluble supernatant fraction. Schneider has suggested possible differences in the intracellular location of enzymes synthesizing lecithin from Cy-DPC and deoxy-Cy-DPC.⁶⁶,⁸⁰aThe properties of an enzyme in the particulate fraction of liver which catalyzes the net synthesis of triglyceride have been described.⁸

Biogenesis of phospholipids occurs not only by way of the reaction involving a cytidine diphosphate derivative, but also by an exchange reaction that is dependent on calcium ions. Incorporation of choline,⁸² ethanolamine,⁸³ dimethylethanolamine,⁸⁴ and serine by this method has been reported in studies using rat liver microsomal fractions. Hübscher, on the basis of inhibitor studies and on the basis of varying levels of calcium ions for the different substrates, concluded that a specific enzyme was required in the exchange incorporation of each base.⁸⁵ Borkenhagen et al. noted that ethanolamine and L-serine competed with each other for the same enzyme site but that choline did not displace either.86 Reference will be made later to the possibility that phosphatidyl monomethylaminoethanol and phosphatidyl dimethylaminoethanol are intermediates in the biogenesis of choline and of lecithin.

Research on the occurrence and metabolism of phospholipids has been markedly stimulated as a result of improved laboratory procedures and better understanding of the mechanisms of biogenesis. As a preliminary to further studies, the percentages of the phosphatides of choline and of aminoethanol, respectively, in the total phospholipid fraction of the following unusual tissues have been reported: house fly,⁸⁷ Musca domestica, 17 and 65; ciliated protozoan,⁸⁸ Tetrahymena pyriformis, 30 and 54; and slime mold,⁸⁹ Dictyostelium discoideum, 34 and 32. A complete description of the lipids in the canine adrenal gland has appeared.⁹⁰ Investigations of the formation of phospholipids in brain and nerve tissue are particularly important because of their role in membranes and in conduction. The synthetic reactions appear to involve phosphorylcholine and Cy-DPC in enzymatic systems similar to those in liver.⁹¹-⁹⁵ The inhibitory effect of chlorpromazine has been investigated in in vivo and in vitro experiments in rats.⁹⁶

D Mechanism of Action of Choline

1 DEVELOPMENT OF CONCEPT OF TRANSMETHYLATION

The first intimation that the dietary supply of choline might have nutritional significance resulted from survival studies on depancreatized dogs subsequent to the discovery of insulin by Banting and Best. Both Fisher⁹⁷ and Allan et al.⁹⁸ observed fatty and severely degenerated livers in animals deprived of the pancreas but supplied with insulin. In the latter study, survival was reported in the case of one animal that received raw pancreas in its diet. Six years passed before it was reported that the protective action of raw pancreas in depancreatized dogs was duplicated by the feeding of lecithin.⁹⁹-¹⁰¹ At this time Best et al.¹⁰² observed that fatty livers resulted from feeding rats mixed grains and fat and that dietary lecithin was lipotropic, i.e., it prevented the accumulation of hepatic fat under these conditions. Best and co-workers soon noted that the effective component in lecithin was choline.¹⁰³-¹⁰⁵ Betaine was also found to have lipotropic activity in rats.

The use of the rat as a test animal in place of the depancreatized dog facilitated greatly the extension of the investigations that comprised the first phase of the study of the role of choline as a dietary essential. During the next few years the study of the relation of dietary factors to choline-preventable fatty livers in rats was pursued vigorously. Best et al. noted the protective action of choline in diets containing added cholesterol¹⁰⁶,¹⁰⁷and the protective effect of protein.¹⁰⁸-¹¹¹ Channon and Wilkinson¹¹² and Beeston et al.¹¹³-¹¹⁵ examined the effect of protein and of amino acids. Following the demonstration of the antilipotropic effect of dietary cystine by Beeston and Channon,¹¹⁴ Tucker and Eckstein¹¹⁶ noted that supplements of methionine had an opposite effect and were lipotropic. This similarity in the anti-fatty liver action of choline and of methionine was the first observation in the second phase of studies of the nutritional importance of choline, a phase that was to place choline in a unique position in metabolism as a source of labile methyl groups as well as a component of biochemically important tissue constituents.

In 1932 Jackson and Block¹¹⁷ presented the first evidence of a mammalian requirement of methionine in experiments in which growth was improved in rats by the addition of this amino acid to a diet low in sulfur amino acids. In the same year Butz and du Vigneaud¹¹⁸ prepared homocystine, a demethylated product of methionine, by the action of strong sulfuric acid on methionine, and du Vigneaud et al.¹¹⁹ showed that homocystine supported the growth of rats on a cystine-poor diet. Later, Womack et al.¹²⁰ found that cystine was not an indispensable amino acid as had been believed since the evidence of its supplementary potency presented by Osborne and Mendel over 20 years earlier.¹²¹ The experiments in Rose’s laboratory showed the essential character of the methionine requirement¹²⁰ and that this amino acid was a precursor of cystine in vivo.¹²² It was evident also that cystine could spare the methionine requirement insofar as cystine was needed by the animal.¹²³

The demonstration of growth-stimulating activity of homocystine in rats on a methionine-poor diet was difficult to understand, inasmuch as cystine was ineffective.¹²⁴ The explanation of the methionine-like action of homocystine was soon found to depend upon the presence of choline in the diet. Homocystine replaced methionine as a growth factor in young rats if the water-soluble vitamins were provided in the form of concentrates of milk and rice bran, but not if purified vitamins were used.¹²⁵ In the latter instance markedly fatty livers were observed and the addition of choline to the mixture of purified vitamins permitted homocystine to function as a source of methionine.¹²⁶ Choline was isolated from the concentrates of milk and rice bran vitamins.

The concept of transmethylation or transfer of intact methyl groups was established by du Vigneaud and his collaborators in a series of experiments in which isotopically labeled compounds were employed. Choline containing deuterium-labeled methyl was isolated from the carcasses of rats fed a choline-deficient diet supplemented with methionine containing deuterium in the sulfur methyl¹²⁷ By the same procedure the methyl of creatine was shown to come from methionine.¹²⁸ In addition, the transfer of methyl from choline to creatine¹²⁸ and to methionine¹²⁹ occurred if labeled choline and homocystine replaced methionine in the diet. The transfer of methyl to guanidoacetic acid to form creatine, however, was irreversible.¹³⁰,¹³¹ This impressive evidence of transfer of intact methyls was given additional support by the experiment in which choline was isolated after feeding rats doubly labeled methionine. Within experimental error the same ratio of ¹⁴C to deuterium was found in choline and creatine methyl as in the sulfur methyl of the administered methionine.¹³² The methionine used in this study was intermolecularly labeled, i.e., a mixture of methionine-methyl-¹⁴C and methionine-methyl-D. Subsequently it was found that deuterium labeled methyls were oxidized more slowly than those containing protium.¹³³ The feeding of doubly labeled methionine in which the labeling was intramolecular confirmed the previous study and demonstrated clearly the transfer of the intact methyl group from methionine to choline and to creatine.¹³⁴

At the time these studies were in progress there was no reason to doubt the supposed inability of the animal organism to synthesize so-called labile methyl, the methyl of choline and of methionine. The ease with which either choline or methionine deficiency was produced and the ease of prevention of these deficiencies by methionine or by choline and homocystine, respectively, made the concept of a dietary deficiency of labile methyl very plausible. More recent findings, however, have made it necessary to revise this concept. The animal organism does have the ability to synthesize the methyl found in choline and methionine provided the diet is adequate. Nevertheless, transmethylation remains as an important and, probably, indispensable metabolic process for the transfer of labile methyls of both exogenous and endogenous origin. The identity of methyl donors, the mechanism of enzymatic transmethylation to methyl acceptors, the de novo biogenesis of choline in mammals, the oxidation of choline, and other aspects of methyl metabolism are described in following sections.

2 IDENTITY OF METHYL DONORS IN TRANSMETHYLATION

The process of transmethylation involves demethylation of a methyl donor, transfer of an intact methyl carbonium ion, and methylation of a methyl acceptor. Donors of intact methyl groups include some, but not all, methylated quaternary nitrogen and sulfur compounds. Lability of the methyl is related in part, at least, to its attachment to a nitrogen or sulfur atom which has or can acquire an additional covalent bond and positive charge.

The main nitrogen compounds in this category are betaine (glycine betaine) and compounds that are readily oxidized to betaine, such as choline and betaine aldehyde. When choline is described as a donor of labile methyl, the following reactions for its conversion to betaine and, in turn, to dimethylglycine are usually taken for granted:

Illustrative of the special character of the labile methyl group in the quaternary nitrogen compound betaine, is the fact that neither of the remaining two methyls attached to the trivalent nitrogen of dimethylglycine is labile. Substrate specificity is involved also because other betaines that have been tested in rats are not substitutes for glycine betaine.¹²⁶,¹³⁵,¹³⁶ In organisms having a requirement for choline, carnitine was ineffective in a cholineless mutant of Neurospora crassa, in the cockroach, in the flour beetle, and in the beetle

Lasioderma serricorne but was utilized by Drosophila melanogaster¹³⁷ and by the blowfly Phormia regina.¹³⁸ Of interest is the finding that the replacement of dietary choline by carnitine in this insect resulted in the production of a new phospholipid containing β-methylcholine instead of choline.¹³⁸a Carnitine did not exhibit lipotropic activity in rats on a choline-deficient diet.¹³⁹

As will be discussed later, an intermediate in the folic acid-tetrahydrofolic acid cycle, N⁵-methyltetrahydrofolic acid (prefolic A), is either a donor of labile methyl or is readily converted into such a donor.¹⁴⁰-¹⁴³ The possibility has been suggested that the actual donor may be a quaternary nitrogen compound such as adenosyl-N⁵-methyltetrahydrofolate.¹⁴² In any event the N⁵-methyltetrahydrofolate appears to be an essential intermediate in the methylation of homocysteine by the mechanism of formate-to-methyl synthesis.

The known sulfur compounds that are methyl donors in transmethylation are acetodimethylthetin (dimethylthetin), propio-β-dimethylthetin, α-amino-dimethyl-γ-butyrothetin (S-methylmethionine) and active methionine, the thetin S-adenosylmethionine (AMe). The thetins with the exception of AMe are not known to occur naturally in animal tissues, and, accordingly, AMe is the most important member of this group of methyl donors.

The discovery of AMe by Cantoni was an important step in the understanding of the role of methionine in transmethylation.¹⁴⁴,¹⁴⁵ Barrenscheen and von Valy-Nagi¹⁴⁶ had concluded previously that a sulfoxide of methionine was the first intermediate in the process of methyl transfer. Cantoni’s finding that AMe is a sulfonium derivative and an energy-rich compound removed the discrepancy involved in ascribing these properties to the thiol methionine:

Berg¹⁴⁷ found an enzyme in yeast which catalyzed the exchange of methionine for the phosphate of ATP. The activating enzyme has been prepared in high purified form from yeast and liver by Cantoni and Durell¹⁴⁵,¹⁴⁸,¹⁴⁹ and Mudd and Cantoni.¹⁵⁰ It is widely distributed in plant and animal tissues and is the key to the process responsible for most naturally occurring methyl transfers.¹⁵¹-¹⁵³ The level of the enzyme in the liver of female rats was found to be double that in male animals.¹⁵⁴

AMe–E–PPPi

AMe + E + PPi+ Pi

No evidence of other free intermediates was found.

The structures of S-adenosylmethionine and S-adenosylhomocysteine were established by Baddiley and

Reviews for The Vitamins

[aliaschase · Rating: 4 out of 5 stars]

This was a very radical and creative book, despite being difficult to read at times. Loved the political analysis and critique of homophobia within the Women's Liberation movement.