METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF John N. Abelson Melvin I. Simon DIVISION OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY PASADENA, CALIFORNIA FOUNDING EDITORS Sidney P. Colowick and Nathan 0. Kaplan Methods in Enzymology Volume 189 Ret&oids Part A Molecular and Metabolic Aspects EDITED BY Lester Packer DEPARTMENT OF MOLECULAR AND CELL BIOLOGY UNIVERSITY OF CALIFORNIA, BERKELEY BERKELEY, CALIFORNIA Editorial Advisory Board Frank Chytil Leonard Milstone DeWitt Goodman Concetta Nicotra Maria A. Livrea James A. Olson Stanley S. Shapiro ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers San Diego New York Boston London Sydney Tokyo Toronto Preface Spectacular progress and unprecedented interest in the field of reti- noids prompted us to consider this topic for two volumes in the Methods in Enzymology series: Volume 189, Retinoids, Part A: Molecular and Metabolic Aspects and Volume 190, Retinoids, Part B: Cell Differentia- tion and Clinical Applications. From a historical perspective we know that studies in the 1930s showed that vitamin A (retinol) and retinal had a role in the visual pro- cess. It was also recognized that some link between vitamin A and cancer incidence existed. Several decades ago it was discovered that retinoic acid had a dramatic effect on the chemically induced DMBA mouse skin carcinogenesis model in which enormous reductions in the tumor burden were observed. This led to the realization that retinoids had important effects on cell differentiation. This resulted almost immediately in the synthesis and evaluation of new retinoids. Indeed, the effects of retinoids on cell differentiation appear to be more universal and of greater importance than their light-dependent role in vision and microbial energy transduction. Progress has been rapid, and the importance of accurate methodology for this field is imperative to its further development. The importance of methodology applies to the use of retinoids in basic research in molecular, cellular, and developmental biology, and in clinical medicine. In medi- cine, applications have been mainly to cancer and in dermatology to the treatment of skin diseases and skin aging. As new retinoids are being tested in biological models and in clinical medicine, interest in the nutri- tion and pharmacology of retinoids has arisen. Moreover, the beneficial effects of retinoids in pharmacological treatment have led to a recognition of the "double-edged sword" of toxicity (teratogenicity). In Section I of this volume, Structure and Analysis, retinoid structure, stability, photosensitivity characteristics, and analytical procedures are included. Section II, Receptors, Transport, and Binding Proteins, focuses on protein isolation and the current status of methods for characteriza- tion, assay, and distribution. As the role of binding proteins is not com- pletely clear and new ones are still being identified, the methods for investigating their presence are of importance. In particular, different types of nuclear receptors for retinoic acid having sequence homologies with hormones have increased interest in this area, and the role of nuclear receptors in retinoid-modulated gene expression is an area that is under rapid development. In Section III, Enzymology and Metabolism, retinoid XV Contributors to Volume 189 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. PATRICIO ABARZOA (35), Department of On- cology and Virology, Roche Research Center, Nutley, New Jersey 07110 ALICE J. ADLER (21), Eye Research Insti- tute, Boston, Massachusetts 02114 N. R. AL-MALLAH (16), Laboratoire de Pharmacocin~tique et Toxicocindtique, Facultd de Pharmacie, 13385 Marseille, Cedex 5, France C. AUBERT (16), Laboratoire de Pharmaco- cin~tique et Toxicocindtique, Facultd de Pharmacie, 13385 MarseiUe, Cedex 5, France JOItN STUART BAILEY (37), Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G IL6, Canada MARK D. BALL (50), Department of Chemis- try, Rose-Hulman Institute of Technol- ogy, Terre Haute, Indiana 47803 FAAN WEN BANGERTER (44), Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 ARUN B. BARUA (13), Department of Bio- chemistry and Biophysics, Iowa State University, Ames, Iowa 50011 DAVID A. BERNLOHR (38), Department of Biochemistry, University of Minnesota, St. Paul, Minnesota 55108 PAUL S. BERNSTEIN (56), Jules Stein Eye Institute, School of Medicine, University of California at Los Angeles, Los Angeles, California 90024 HANS K. BIESALSKI (18), Department of Physiological Chemistry and Pathobio- chemistry, University of Mainz, D-6500 Mainz, Federal Republic of Germany WILLIAM S. BLANER (19, 27), Institute of Human Nutrition, Columbia University, New York, New York 10032 THEODORE R. BREITMAN (23), Laboratory of Biological Chemistry, Division of Can- cer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 C. D. B. BRIDGES (5, 20, 33), Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907 MELISSA K. BUELT (38), Department of Bio- chemistry, University of Minnesota, St. Paul, Minnesota 55108 H. BUN (16), Laboratoire de Pharmaco- cindtiqae et Toxicocindtique, Facaltd de Pharmacie, 13385 Marseille, Cedex 5, France CHRISTER BUSCH (32), Department of Pa- thology, Uppsala University, S-751 85 Uppsala, Sweden LOUISE M. CANFIELD (45), Department of Biochemistry, University of Arizona, Tuc- son, Arizona 85721 J. P. CANO (16), Sanofi Recherche, 34082 Montpellier, France GERALD J. CHADER (21), Laboratory of Ret- inal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892 RONALD F. CHILDS (l 1), Department of Chemistry, McMaster University, Hamil- ton, Ontario LSS 4L8, Canada LAURIE L. CHINANDER (38), Department of Biochemistry, University of Minnesota, St. Paul, Minnesota 55108 ANDREW J. CLIFFORD (9), Department of Nutrition, University of California at Davis, Davis, California 95616 DALE A. COOPER (55), Edible Oil Products Division, Procter & Gamble Company, Cincinnati, Ohio 45224 xi xii CONTRIBUTORS TO VOLUME 189 FgANS J. M. DAEMEN (43), Department of Biochemistry, University of N(imegen, 6500 HB Nijmegen, The Netherlands ANN K. DALY (24, 31), Department of Phar- macological Sciences, University of New- castle upon Tyne, Medical School, New- castle upon Tyne NE2 4HH, England M. I. DAWSON (2), Life Sciences Division, SRI International, Menlo Park, Califor- nia 94025 WILLEM J. DE GRIP (43), Department of Biochemistry, University of Nijmegen, 6500 HB Nijmegen, The Netherlands ANDREW P. DE LEENHEER (4, 10), Labora- toria voor Medische Biochemie en voor Klinische Analyse, Rijksuniversiteit Gent, B-9000 Gent, Belgium ULF ERIKSSON (32), Ludwig Institute for Cancer Research, Stockholm Branch, S-104 O1 Stockholm, Sweden RONALD M. EVANS (22), Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, California 92138 GORAN FEX (42), Department of Clinical Chemistry, University Hospital of Lund, S-221 85 Lund, Sweden SHAO-LING FONG (20, 33), Department of Ophthalmology, Indiana University, Indi- anapolis, Indiana 46202 THOMAS A. FRITZ (45), Department of Bio- chemistry, University of Arizona, Tucson, Arizona 85721 KEIKO FUNA (32), Ludwig Institute for Can- cer Research, University Hospital, S-751 85 Uppsala, Sweden HAROLD C. FURR (8, 9) Department of Bio- chemistry and Biophysics, Iowa State University, Ames, Iowa 50011 L. E. GERBER (47), Department of Food Sci- ence and Nutrition, University of Rhode Island, Kingston, Rhode Island 02881 VINCENT GIGU~,RE (22), Division of Endo- crinology, Research Institute, The Hospi- tal for Sick Children, Toronto, Ontario M5G 1](8, Canada DEWITT S. GOODMAN (19, 29), Department of Medicine, Columbia University, Col- lege of Physicians and Surgeons, New York, New York 10032 JEFFERY I. GORDON (58), Departments of Medicine, Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110 GEgHART GRAUPNER (26), La Jolla Cancer Research Foundation, La Jolla, Califor- nia 92126 JOSEPH F. GRIPPO (25), Department of Toxi- cology and Pathology, Hoffman-La Roche Ltd., Nutley, New Jersey 07110 MARY LOU GURLER (60), Department of Oncology and Virology, Roche Research Center, Nutley, New Jersey 07110 EARL H. HARRISON (52), Department of Physiology and Biochemistry, The Medi- cal College of Pennsylvania, Philadel- phia, Pennsylvania 19129 TIaOMAS HERMANN (26), La Jolla Cancer Research Foundation, La JoUa, Califor- nia 92126 DONALD L. HILt (3), Biochemistry Depart- ment, Southern Research Institute, Bir- mingham, Alabama 35255 P. D. HoBBs (2), Life Sciences Division, SRI International, Menlo Park, California 94025 ANTON M. JETTEN (25), Cell Biology Group, Laboratory of Pulmonary Patho- biology, National Institute of Environ- mental Health Sciences, Research Trian- gle Park, North Carolina 27709 GUNVOR JOHANNESSON (42), Department of Clinical Chemistry, University Hospital of Lund, S-221 85 Lund, Sweden A. DANIEL JONES (9), Facility for Advanced Instrumentation, University of California at Davis, Davis, California 95616 T. ALWYN JONES (28), Institute for Molecu- lar Biology, Biomedical Center, Uppsala University, S-751 24 Uppsala, Sweden PERE JULI)~ (48), Departament de Bioquimica i Biologia Molecular, Fa- cultat de CiOncies, Universitat AutOnoma de Barcelona, 08193 Barcelona, Spain CONTRIBUTORS TO VOLUME 189 xiii LAWRENCE A. KAPLAN (15), Department of Pathology, Bellevue Hospital Medical Center, New York, New York IOOI6 MICHIMASA KATO (34), First Department of Internal Medicine, Gifu University School of Medicine, Tsukasa-machi 40, Gifu 500, Japan MICHAEL KLAUS (1), Department of Phar- maceutical Research, Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland M. RAw LAKSHMAN (46), Lipid Research Laboratory, Veterans Administration Medical Center, Washington, D.C. 20422 WILLY E. LAMBERT (10), Laboratoria voor Medische Biochemie en voor Klinische Analyse, RUksuniversiteit Gent, B-9000 Gent, Belgium GARY M. LANDERS (6), Jules Stein Eye In- stitute, UCLA School of Medicine, Los Angeles, California 90024 JUERGEN M. LEHMANN (26), La Jolla Can- cer Research Foundation, La Jolla, Cali- fornia 92126 M. A. LEO (54, 59), Department of Medi- cine, Mount Sinai School of Medicine, City University of New York, New York, New York 10021 MARC S. LEVlN (58), Department of Medi- cine, Washington University School of Medicine, St. Louis, Missouri 63110 ELLEN LI (58), Departments of Medicine, Biochemistry and Molecular Biophysics, Washington University School of Medi- cine, St. Louis, Missouri 63110 C. S. LIEBER (54, 59), Department of Medi- cine and Pathology, Mount Sinai School of Medicine, City University of New York, New York, New York 10021 ZHENG-SHI LIN (33), Department of Neuro- biology and Behavior, State University of New York at Stony Brook, Stony Brook, New York 11794 MARIA A. LIVREA (39, 57), Istituto di Chimica Biologica, Universitd di Palermo Policlinico, 90127 Palermo, Italy WILLIAM A. MACCREHAN (17), Organic An- alytical Research Division, Center for An- alytical Chemistry, National Institute of Standards and Technology, Gaithers- burg, Maryland 20899 PAUL N. MACDONALD (51), Department of Biochemistry, University of Arizona, Tuc- son, Arizona 85724 BERNARD MARTIN (24), Centre Interna- tional De Recherches Dermatologiques, Sophia Antipolis, 06565 Valbomme, Ce- dex, France VALERIE MATARESE (38), Department of Biochemistry, University of Minnesota, St. Paul, Minnesota 55108 JUDITH A. MILLER (15), Medical Research Laboratories, Cincinnati, Ohio 45219 YASUTOSHI MUTO (34), First Department of Internal Medicine, Gifu University School of Medicine, Tsukasa-machi 40, Gifu 500, Japan JOSEPH L. NAPOLI (52, 53), Department of Biochemistry, School of Medicine and Biomedical Sciences, The State Univer- sity of New York at Buffalo, Buffalo, New York 14214 H. J. NELIS (4), Laboratoria voor Medische Biochemie en voor Klinische Analyse, Rijksuniversiteit Gent, B-9000 Gent, Belgium CLARA NERVI (25), Cell Biology Group, Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 MARCIA NEWCOMER (28), Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 HANS NORDLINDER (32), Department of Pa- thology, Uppsala University, S-751 85 Uppsala, Sweden MASATAKA OKUNO (34), First Department of Internal Medicine, Gifu University School of Medicine, Tsukasa-machi 40, Gifu 500, Japan JAMES ALLEN OLSON (46), Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011 DAVID E. ONG (51), Department of Bio- chemistry, Vanderbih University, Nash- ville, Tennessee 37232 xiv CONTRIBUTORS TO VOLUME 189 XAVIER PARI~S (48), Departament de Bioquimica i Biologia Molecular, Fa- cultat de Cidncies, Universitat Aut~Snoma de Barcelona, 08193 Barce- lona, Spain MAGNUS PFAHL (26), La Jolla Cancer Re- search Foundation, La Jolla, California 92126 ROBERT R. RANDO (44, 56), Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 CHRISTOPHER P. F. REDFERN (24, 31), De- partment of Dermatology, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 4LP, England A. CATHARINE ROSS (7, 49), Department of Physiology and Biochemistry, Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129 PUSPHA SAKENA (32), Department of Pa- thology, Uppsala University, Uppsala, Sweden BRAHMA P. SANI (3, 36), Biochemistry De- partment, Southern Research Institute, Birmingham, Alabama 35255 GARY S. SHAW (ll), Department of Bio- chemistry, University of Alberta, Edmon- ton, Alberta T6G 2H7, Canada MICHAEL I. SHERMAN (35, 60), Department of Oncology and Virology, Roche Re- search Center, Nutley, New Jersey 07110 FRIEORICH SIEBERT (12), Max-Planck-lnsti- tut ffir Biophysik, D-6000 Frankfurt am Main, Federal Republic of Germany GEORGES SIEGENTHALER (30, 61), Clinique Dermatologie, HOpital Cantonal Univer- sitaire, CH-1211 Geneva, Switzerland K. L. SIMPSON (47), Department of Food Science and Nutrition, University of Rhode Island, Kingston, Rhode Island O2881 CHI-HUNG SIU (37), Banting and Best De- partment of Medical Research, Univer- sity of Toronto, Toronto, Ontario MSG 1L6, Canada DIANNE ROBERT SOPRANO (29), Depart- ment of Biochemistry, Temple University School of Medicine, Philadelphia, Penn- sylvania 19140 MEIR J. STAMPFER (15), Department of Epi- demiology, Harvard Medical School of Public Health, and The Channing Labo- ratory, Harvard Medical School and Brigham and Woman's Hospital, Boston, Massachusetts 02115 EVAN A. STEIN (15), Medical Research Laboratories, Cincinnati, Ohio 45219 WILLIAM STILLWELL (40, 41), Department of Biology, Indiana University Purdue University at Indianapolis, Indianapolis, Indiana 46205 NORIKO TAKAHASHI (23), Laboratory of Bi- ological Chemistry, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 THOMAS E. TARARA (45), Department of Biochemistry, University of Arizona, Tuc- son, Arizona 85721 LUISA TESORIERE (39, 57), lstituto di Chimica Biologica, Universit6 di Palermo Policlinico, 90127 Palermo, Italy MATY TZUKERMAN (26), La Jolla Cancer Research Foundation, La Jolla, Califor- nia 92126 STEPHEN R. WASSALL (40, 41), Department of Physics, Indiana University Purdue University at Indianapolis, Indianapolis, Indiana 46205 BARBARA WIGGERT (21), Laboratory of Ret- inal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892 KEN N. WILLS (26), La Jolla Cancer Re- search Foundation, La Jolla, California 92126 RONALO WYSS (14), Department of Drug Metabolism, Pharmaceutical Research, Hoffmann-La Roche Ltd., CH-4002 Ba- sel, Switzerland XIAO-KUN ZHANG (26), La Jolla Cancer Re- search Foundation, La Jolla, California 92126 [1] STRUCTURE OF RETINOIDS 3 [1] Structure Characteristics of Natural and Synthetic Retinoids By MICHAEL KLAUS Introduction The term retinoids comprises the natural occurring vitamin A deriva- tives such as retinol, retinal, and retinoic acid as well as the large number of synthetic analogs prepared since the late 1960s, regardless of whether they have biological activity, i The goal of this intensive effort at synthesis in academia as well as in the pharmaceutical industry was to improve on the ratio of toxic effects (e.g., hypervitaminosis A) to therapeutic activity compared to retinoic acid. In addition, compounds with better selectivity toward the various therapeutic indications (dermatology, oncology, rheu- matology, immunology) have been sought. Structure of Retinoids Formally the molecule of retinoic acid consists of three main sections: a lipophilic part at one end, connected via a polyunsaturated chain as a spacer to a hydrophilic group at the other end of the molecule (Fig. 1). All three parts have been modified extensively. In the following a selection of compounds with typical variations is given. Changes in Hydrophilic Part Different Oxidation Level of Polar End Group. Compounds in which the oxidation level of the polar end group is different include the natural vitamin A derivatives retinol (1), retinal (2), retinoic acid (3), and close analogs such as esters (4) and amides (5). Because the free acid is gener- ally considered to be the active principle, for in vitro experiments only compounds with a carboxylic acid end group should be used. Esters and ~ OH 1 CHO 2 M. B. Sporn and A. B. Roberts, Ciba Found. Symp. 113, 1 (1985). Copyright © 1990 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 189 All rights of reproduction in any form reserved. 4 STRUCTURE AND ANALYSIS [1] ~ CO2H lipophilic spacer hydrophilic part part Fro. 1. Structural subunits of the retinoic acid molecule. amides may be inactive owing to their stability toward hydrolysis under the experimental conditions. Other Polar Groups. The carboxylic acid end group of retinoic acid (3) has been replaced by other acidic or polar moieties. Examples are sul- fonic and phosphonic acid derivatives 6 and 7, 2 sulfone 8, 2 diketo deriva- tive 9, 3 and the phenol 10. 4 3 0 ~ NHCaH5 5 0 7 9 ~ ~,~~O2CaH5 4 ~ SO2OC2H 5 6 ~ SO2C2H5 8 lO 2 M. Klaus, unpublished (1982 and 1984). 3 N. Acton, A. Brossi, D. L. Newton, and M. B. Sporn, J. Med. Chem. 23, 805 (1980). 4 B. Loev and W K. Chan, U.S. Patent 4,605,675 (1986). [1] STRUCTURE OF RETINOIDS 5 Changes in Lipophilic Part Substitution Pattern of Cyclohexenyl Ring. The group of analogs with an altered substitution pattern of the cyclohexenyl ring consists mainly of oxygenated metabolites of retinoic acid, for example, 4-hydroxyretinoic acid (11), 5 18-hydroxyretinoic acid (12), 6 and 5,6-epoxyretinoic acid (13), 7 but also comprises compounds such as 14 and 15. OH 11 OH 12 C02H 13 O~ CO2H 14 CO2H 15 Aromatization of Cyclohexenyl Ring. Replacement of the trimethylcy- clohexenyl group with a suitably substituted benzene ring leads to com- pounds such as etretinate (16) and motretinid (17) 8 having an increased therapeutic index. A certain lipophilicity in this part of the molecule seems to be necessary for biological activity. The unsubstituted analog 18 is virtually inactive in most of the relevant test systems. CH3~ cO2c2H5 16 0 CH30 ~ v x. 17 ~ C02H 18 5 M. Rosenberger, J. Org. Chem. 47, 1698 (1982). 6 M. Rosenberger and C. Neukom, J. Org. Chem. 47, 1782 (1982). 7 K. V. John, M. R. Lakshrnanan, and H. R. Cama, Biochem. J. 103, 539 (1967). 8 H. Mayer, W. Bollag, R. H~inni, and R. Rfiegg, Experientia 34, 1105 (1978). [...]... for analysis Curley reported superior resolution using these mobile phases on an irregular particle, nonend-capped C18 column (Ultrasil ODS, Beckman, San Ramon, CA), whereas a spherical particle, fully endcapped C18 column (Ultrasphere ODS, Beckman) gave improved resolution when aqueous methanol was used z2 A Novapak 4-/~m ODS radial compression cartridge (8 × 100 mm) in a RCM 8 × 10 Radial-PAK cartridge... ethyl acetate/hexane (HPLC grade) The ester is eluted in fractions 5-8 (200 ml) These fractions are pooled and concentrated under argon to give 4.17 g of crude ester as a yellow gum Analytical HPLC (Waters Associates, Milford, MA, ALC 210 equipped with a Radial-PAK B cartridge, detection at 260 nm at a flow rate of 2.0 ml/min of 1% ethyl acetate/hexane) indicated the presence of a minor, less-polar impurity... Instrumentation was as listed in Table I except that a Waters Associates 8 x 100 mm Nova-Pak C~s 4-p.m radial compression cartridge in a RCM 8 x l0 module was used The absorbance for E-8, 13Z-8, E-9, and E-10 was monitored at 325 nm; the others were monitored at 260 nm b Mobile phase at a flow rate of 2.0 ml/min: 30% aqueous a m m o n i u m acetate (1%)/ acetonitrile (A) , 2% water/acetonitrile (B), 5% water/acetonitrile... Retinoid Purification In cases where reaction procedures give more than one isomer a chromatographic separation is often necessary for purification rather than using crystallization This is especially true in the case of the polyolefinic retinoids Although preparative thin-layer chromatography on silica gel plates can often be used to separate olefinic bond isomers, oxidation on the plate can be a problem... when the attempt is made to run the plate in an inert atmosphere High-performance liquid chromatography (HPLC) is particularly effective for separating E and Z isomers, especially when the recycle technique is employed This technique has the added advantage that the chromatography is performed under an inert carrier gas such as nitrogen The separations are best performed on the esters of analogs of... RETINOIC ACID ANALOGS 17 extraction procedure The organic extract is washed twice with saturated brine, transferred to a 500-ml Erlenmeyer flask, dried over anhydrous Na2SO4, and concentrated at reduced pressure using a rotary evaporator (Btichi, Flawil, Switzerland) under argon The rotary evaporator is vented to the argon line The residue is chromatographed on a 4 × 40 cm column of silica gel 60 (EM... resolve satisfactorily in this system, the elutant was changed to 5-10% aqueous acetic acid (l%)/methanol Retinoid carboxylic acids (E-8, Eol0, E-15, 17, 18, 19, 21, 22, and 25) have been analyzed using 10% aqueous acetic acid (l%)/methanol This same solvent system may be employed for analyzing mixtures containing both esters and the parent carboxylic acids (e.g., 6 and 19) Trifluoroacetic acid cannot... visual process in the same manner as the natural retinoids do The synthetic retinoids have structural similarity to the natural compounds in that they possess the characteristic lipophilic head group and polar terminus separated by a spacer group 2 Generally the trend in the synthesis of analogs of (E)-retinoic acid has been the design of more stable, readily manipulatable species Therefore, the care... temperature of 80° in an oil bath over a period of 20 min and then maintained at this temperature for an additional 12 min The oil dissolves at 80° The reaction work-up is performed under argon The cooled reaction mixture is acidified with I0 ml of 50% aqueous acetic acid and then transferred to a 100-ml separatory funnel, where it is diluted with 50 ml of water The precipitated carboxylic acid is extracted... place of acetic acid for analysis of polyolefinic retinoids and other acid-sensitive compounds No improvement in resolution of the stable aromatic retinoidal carboxylic acids was found using 10% water/methanol containing 0.1% trifluoroacetic acid on the Novapak column Far inferior resolution for the carboxylic acids of both the polyolefinic and aromatic retinoid classes was observed when 30-45% aqueous . Aut~Snoma de Barcelona, 08193 Barce- lona, Spain MAGNUS PFAHL (26), La Jolla Cancer Re- search Foundation, La Jolla, California 92126 ROBERT R. RANDO (44, 56), Department of Biological. sylvania 19140 MEIR J. STAMPFER (15), Department of Epi- demiology, Harvard Medical School of Public Health, and The Channing Labo- ratory, Harvard Medical School and Brigham and Woman's. Health, Bethesda, Maryland 20892 KEN N. WILLS (26), La Jolla Cancer Re- search Foundation, La Jolla, California 92126 RONALO WYSS (14), Department of Drug Metabolism, Pharmaceutical Research,