Retinoid Protocols

422 Pages·1998·27.736 MB·English

by  Christopher Redfern

1
Properties of Retinoids
Structure, Handling, and Preparation
Arun 6. Barua and Harold C. Furr
1. Structure
Retinolds have been defined as a class of compounds consisting of four
isoprenoid units (H,C=C(CH3)-CH=CH*) joined in a head-to-tall manner. The
retinoid molecule can be divided into three parts: a trimethylated cyclohexene
ring, a conjugated tetraene side chain, and a polar carbon-oxygen functlonal
group. Retinol (I), retinaldehyde (II), and retinoic acid (III), as well as their
derivatives whose structures are shown in Structure 1 (I), are included by this
definition.
The conventional numbering of carbon atoms in the retmold molecule IS
shown in the structure of retmol (I). On the basis of this numbering scheme,
geometric isomers and substituted compounds can be named unambiguously,
e.g., 13-cis retinoic acid (IV), 3-hydroxyretinoic acid (V), and 9-cis retinoic
acid (VI).
To name retinoids systematically (IUPAC nomenclature), however, a dif-
ferent numbermg scheme must be used: the carbon atom bonded to the func-
tional group is given number 1. The numbering of carbon atoms by this scheme
is shown m structure III for all-truns retmoic acid. Accordingly, the systemic
name for all-trans retinoic acid is (all-trans) 3,7-dimethyl-9-(2,6,6-
tnmethylcyclohex- l-en- l-yl)-nona-2,4,6,8-tetraen- 1 -olc acid, or more
simply (all-truns) 3,7-dimethyl-9-(2,6,6-trimethylcyclohexene- l-yl)-2,4,6,8-
nonatetraenoic acid.
Also note that the terms E and Z are used frequently for tram and cis,
respectively. Thus all-trans retinoic acid is also known as all-E retmolc acid.
Other names for all-truns and 13-cis (or 13-Z) retinoic acid are tretinoin and
From Methods m Molecular Bfology, Vol 89 Retrnord Protocols
E&ted by C P F Redfern Q Humana Press Inc , Totowa, NJ
3
‘<
I
\
m
COOH
VI
\
1
COOH
Structure 1,
isotretinom, respectively. The same numbering system is also used for the
aromatic retinoids, such as etretmate (VII) and acetretm (VIII).
Retmoids are essential for several biological processes, mcludmg growth
and development, reproduction, and cellular differentiation. However, retinoids
are toxic when taken m excess, are irritating to the skin, and are highly terato-
gemc. In an attempt to produce retmoids that are efficacious, yet lack toxicity,
many new retmoids have been synthesized and tested biologtcally. Molecular
modifications have been made to all three units of the retinoid molecule: the
cyclohexene ring, the polyene chain, and the polar end group. Many such
retmolds, such as TTNPB (IX), TTNN (X), Ch-55 (XI), and Am-580 (XII)-
which are cychc, nonpolyisoprenoid compounds-have been shown to be more
active than retinol or retinoic acid in several accepted assays for retmoid activ-
ity. Hence the above defnution of retinoids has become obsolete. Sporn et al.
(2) recently redefined a retmoid as a substance that can elicit specific brologi-
cal responses by binding to and activatmg a specific receptor or set of recep-
tors, with the program for the biological response of the target cell residing m
the retinoid receptor rather than in the retinoid ligand itself. However, 13-cis
retmoic acid, which has no known unique receptor but has biological activity,
and anhydroretmol, which has little biological activity, are undisputably con-
Properties of Re tinoids
CH,O CHsO
VII
~cooH
X
,u pJ.&2fooH
0
xl XII
Structure 2.
srdered retinoids. We therefore suggest that retinoids be defined as compounds
that are structurally similar to retinol, or that can elrcrt specific brological
responses by binding to and activating a specific receptor or set of receptors.
In this chapter, emphasis will be given to the naturally occurrmg retinoids in
regard to their handling, stabihty, storage, and other physical and chemical
properties. The synthetic-aromatic retinoids, though they have similar brologt-
cal activity, are different from naturally occurring retinords in their structure
and chemical properties. There IS, however, no harm m treating them m the
same way as the naturally occurrmg retinoids. The reader IS also referred to
prevtous publications relative to chemistry, physical properties, storage, and
handling of retinoids (3-6).
Because of the presence of ConJugated double bonds, retinoids in general
are unstable compounds. They are readily oxidized and/or isomerized to altered
products, especially in the presence of oxidants (includmg air), hght, and
excessive heat. They are also labile toward strong acrds and solvents that have
dissolved oxygen or peroxides.
Barua and Furr
2. Physical Properties
Commercial preparations of retinoic acid, retinyl acetate, retinyl palmltate,
retinaldehyde, and retinol are pale-yellow-to-yellow crystalline or amorphous
solids. Retmol and its esters are low-melting (around 60°C) compounds, and if
the room temperature is very high, they may turn to 011. Pure preparations of
3,4-didehydroretinol (vltamm A, alcohol) can be crystallme or 011.
2.7. Handling and Storage
Some retmoids are very irritating to the skin, and some are highly teratoge-
mc. Hence when large amounts of retmoids are handled, adequate care should
be taken so that neither solid particles nor solutions come into contact with the
body, Because retmoids are lipid soluble, organic solvents are used to dissolve
them. If a solution of retinoid falls on the body, the solvent quickly evaporates,
and the retinold 1s absorbed by the skin. Therefore, it 1s recommended that
gloves be worn while working with retinoids.
The following three general rules apply towards handling, storage, and
analysis of retmolds:
1 Because retmolds are lsomerlzed and degraded by light, exposure of retmolds to
bright dayhght or bright electric light should be avoided. Black curtains should
be used to cover doors and windows in laboratories designated for working with
retinoids. Laboratories should be equipped with Gold fluorescent lights If that is
not possible, dim light should be used, and all labware should be amber colored,
or d transparent, wrapped with alumuuum foil.
2. Because retmolds are lsomerlzed or degraded by exposure to excessive heat, the
retmolds and samples contammg them should be kept frozen at or below -20°C
until time of analysis, and kept on ice (4°C) during handling.
3. Because retinolds are easily oxidized to inactive products, exposure of retmoids
or samples containing them to air or oxygen should be avoided at ail times
Retinoids, whether solid reference compounds or solutions and samples contain-
mg them, should always be stored under nitrogen or argon Because of their
unstable nature, retmolds should be stored m vacuum-sealed, amber-colored
ampules, vials, or bottles, protected from light and heat, at -20°C or below. When
stored unopened at -20°C or below, the retmolds keep well for a long time Once
containers are opened, the retmolds must be stored under nitrogen or argon,
protected well from light at -20°C or below If not all of the content from a large
sample 1s used immediately, it 1s recommended that allquots of solid or solution
be transfered to ampules, vials or bottles, the containers flushed with an inert gas
(nitrogen or argon) and sealed by torch under partial pressure of the inert gas
Argon may be preferable to nitrogen because of its lower residual oxygen content.
In blood and other tissues, retmoids are usually bound to proteins and/or
protected by natural antioxidants Therefore, when stored properly, retinoids
Properties of Retinoids 7
in plasma or serum and other tissues are usually safe for 15 mo to 8 y (6).
However, repeated freezing and thawing should be avoided. Once extracted
from blood or tissues, the freed retmolds are very susceptible to rapid decom-
position by any or all three of the above-mentioned agents. Therefore, extracted
samples should be analyzed immediately. Overnight storage of extracted
samples is not recommended.
The most frequently analyzed human sample is blood. The handling of blood
is of crucial importance. Immediately after withdrawal, blood should be pro-
tected from light, and centrifuged to remove the red blood cells. Hemolysis
should be avoided. Alternatively, the blood can be allowed to clot m a cool,
dark place. After centrifugation, the plasma or serum should be carefully
removed from the red blood cell mass, and transferred to a clean dry tube. The
size of the tube should be such that the sample of plasma or serum nearly fills
it. Thus, air will be displaced, preventing oxidation during storage. Thereafter,
the tube should be sealed securely and kept frozen at or below -20°C until time
of analysis. Some investigators prefer to flush the tube of plasma or serum with
nitrogen or argon before sealing it. However, analysis of the sample immedr-
ately after collection is ideal.
2.7.1. Radiolabeled Retinoids
Radiolabeled retinoids are more labile than their unlabeled analogs because
of radiolytic autolysis. Usually radiolabeled retinoids are stored dissolved in
toluene containing an antioxidant such as vitamin E or 2,5-di-tert-butyl
hydroxyquinone (DBHQ) under an atmosphere of nitrogen or argon at -70°C.
(Although it is chemically inert, benzene is not a satisfactory solvent for low-
temperature storage because of its high freezing point, 5°C.) Even when all
these precautions are taken, radioactive retinords degrade, and the degree of
degradation varies from retinord to retinoid. It is very important to check
sample purity and confirm the absence of degraded components in the labeled
retinoid before it is used. One way to check this is to subject an aliquot of the
solution to high-performance liquid chromatography (HPLC), and analyze the
collected fractions for radioactivity. Alternatively, the sample can be analyzed
by thin layer chromatography (TLC) (general procedure given below). If plas-
tic-backed TLC plates are used, the plate can be cut into small segments that
are placed m individual vials and the radioactivity m each segment can be
determined by lrqmd-scintillation counting. The degraded components are usu-
ally more polar, and either elute with the solvent front durmg reverse-phase
HPLC, or stay at the origin on TLC. If necessary, the fraction containing the
retinoid of interest should be collected, the solvent removed under an inert gas,
and the residue reconstituted in an appropriate solvent. The resulting prepara-
tion should be used immediately.
8 Barua and Furr
2.2. Solubility
All retinoids are fat soluble. The solubilities of individual retinoids m
organic solvents depend on the terminal group of the side chain. Retinol (which
has an alcoholic group) and retmoic acid (which has a carboxyhc group) are
soluble in alcohohc solvents such as methanol and ethanol, and also in acetom-
trile. On the other hand, retinyl palmnate, which has an esterified long-chain
fatty acid, is slightly soluble in methanol or ethanol, but highly soluble m hex-
ane. Therefore, if a mixture of retinotds, e.g., retmoids in a hver extract, is to
be dissolved, it is desirable to use a mixture of solvents such as isopropanoll
hexane (7). Chlorinated hydrocarbons (dichloromethane, BP 40°C;
dichloroethane, BP 83°C; chloroform, BP 61°C) all dissolve retinoids well:
however, chloroform should be avoided because rt may be acidic or may pro-
duce free radicals Dichloromethane is a very good solvent, but care must be
taken when this solvent is used because retinoids tend to isomerize m chlori-
nated solvents, especially in the presence of light. Acetone is also a good sol-
vent for the retinoids, but should not be used to work with retmaldehyde, which
reacts with it (aldol condensation to form a C-23 ketone). Dimethylsulfoxide is
often used to introduce retinoids mto ttssue culture systems; it is appropriate
for this use, but its low volatility (BP 189°C) precludes its use when solvent
must be evaporated. Ethers such as tetrahydrofuran and diethyl ether dissolve
retinoids well, but may contain peroxides that attack retmords. Ethyl acetate
has a convenient boiling point (77°C). it is sufficiently volatile to be removed
readily, but not so volatile as to be troublesome. It dissolves retmoids well, and
is a good diluent for solutions of retinoids m vegetable oils. If a solvent is to be
removed by evaporation, a solvent of low boiling point is preferred, i.e., metha-
nol 1s preferred over ethanol.
In summary: solutions or tissue extracts containing retmoids should always be
kept under mtrogen or argon, protected from light, preferably m amber-colored
glass contamers, m the cold at 4°C while working, and at -20°C while in storage.
2.3. Exposure to LJV Light, Acids, and Alkalis
When it is exposed to UV radiation, retinol exhibits strong yellow-green
fluorescence, while undergoing destruction. Measurement of absorption at
325 nm before and after destruction by exposure to UV hght was an old method
of quantitation of retmol. When retinol is treated with Lewis acids, including
sulfuric acid or phosphorus pentoxide, it passest hrough purple- or blue-colored
phases while undergoing rapid destruction (this is the basis of the traditional
Carr-Price assay for vitamin A). Anhydrous solvents containing even traces of
acid cause structural changes of retinoids. For example, retmol, when treated
wtth methanol containing 0.03 N HCl, is dehydrated to anhydroretinol (a
Properties of Retinoids 9
hydrocarbon with a r&o-double bond structure) m a matter of a few mm. Thus,
use of strong acid should be avoided. Alkali usually is not harmful to retmolds.
Indeed, samples containing esters of retinol (e.g., retmyl palmitate in hver or
milk) are routinely sapomfied in the presence of alkali to hydrolyze the esters
to free retinol. However, prolonged contact with alkali should be avoided.
Retinoid carboxylic acids form salts with alkali. If the free acids are to be ana-
lyzed, it is necessary to convert such salts by treatment with dilute acid (prefer-
ably acetic acid) to the free carboxylic acid. Retinoyl P-glucuromde (a
metabolite of retmoic acid, which is soluble m water) is easily hydrolyzed to
retinoic acid if it is treated with hydrochlorrc acid, but not with acetic acid
Retmords in solution degrade faster than in the solid state. It is customary to
add antioxidants such as vitamin E, butylated hydroxytoluene (BHT), tert-butyl
hydroxyanisole (BHA), pyrogallol, or ascorbic acid during workup and purifi-
cation of retmolds. If the sample to be extracted does not contam an antioxi-
dant naturally, it is recommended that an antioxidant is added during extraction
of retmoids from the sample. For example, some investigators add pyrogallol
or ascorbic acid to breast mtlk during extraction and analysis of retmolds. The
antioxidant selected should not mterfere with the retmolds during analysis. In
general, a lipid-soluble antioxidant (BHA, BHT, a-tocopherol) is preferred;
however, BHT may interfere with analysis of retmol under some reversed-
phase HPLC conditions.
2.4. Ultraviolet- Visible Spectroscopy
Because they have multiple, conjugated carbon-carbon double bonds,
retmoids absorb strongly m the ultraviolet-visible region of the spectrum
(1,4,6). For example, all-truns retinol, which possessesf ive conlugated double
bonds, absorbs maximally (h,,,) at 325 nm (Fig. l), whereas all-truns retinoic
acid, with an additional double bond (C=O of the carboxyl group) m conjuga-
tion, and 3,4didehydroretmol (vitamin A2 alcohol) with the additional carbon-
carbon double bond in conjugation in the cyclohexene rmg, absorb at 350 nm
in ethanol (Table 1). 5,6-Epoxy- and 5&epoxyretmoids, conversely, have
fewer conjugated double bonds and hence have lower-absorption maxima
(Table 1). Note that retmol, retinyl esters, and retinyl ethers have the same
absorption spectra because the polyene chromophore is not disturbed. In
an analogous manner, esters of retinoic acid (methyl retmoate, retmoyl
P-glucuronrde) have absorption spectra similar to that of unionized retinoic
acid. It 1st o be noted that the absorption spectrum of ionized compounds, such
as retinoic acid at pH above 7.0, is affected by the carbonyl oxygen that is
coqugated with the polyene system.T he absorption spectra of all-trans retinoic
acid at different pH are shown m Fig. 2. It is common to observe retinoic acid
IO Barua and Furr
60000
E 50000
a,
p
% 40000
s
E5 30000
5
w 20000
i
0
2 1OQQO
0
250 300 350 400 450
Wavelength (nm)
Fig. 1. Absorption spectra of all-trans retinol (vltamm A, alcohol, -) and all-
truns 3,4-dldehydroretmol (vitamm A2 alcohol, l **) m methanol
250 300 350 400 450
Wavelength (nm)
Fig. 2 Absorption spectra of all-truns retmoic acid under neutral (- ), alkalme
(a**), and acidic (- - -) condltlons
Properties of Retinoids II
showing h,,, at 337 nm instead of 350 nm owmg to traces of alkali present in
the test tube or cuvet. Axerophthene (vitamin A hydrocarbon) has an absorp-
tion spectrum nearly identical to that of retinol; anhydroretmol, which has a
retro double-bond structure, has an absorption spectrum reminiscent of that of
p-carotene (Fig. 3). The wavelengths of maximum absorption and the molar-
extinction coefficients of aporetinoids and apocarotenords increase almost lm-
early with an increasing number of double bonds for each chemical class
(alcohols, carboxylic acid, and aldehydes/ketones). Light-absorption spectros-
copy has also been used in conformational studies of retmals, other polyolefmic
retinoids, and aromatic retmoids (3), In general, cis isomers have lower molar-
extinction coefficients (E) and lower h,,, than the all-trans conformers (cf.,
Fig. 4 for spectra of 9-cis and all-trans retinoic acid). The aromatic analogs
TMMP-retinol and TMMP-retmoic acid have absorption spectra stmilar to
those of the natural compounds because of a similar polyene structure (Fig. 5).
The intensity of absorptron, expressed as either E1%rcmo r molar-extinctron
coefficient (E), IS characteristic for each retmord. Molar-extinction coefficients
and E’” Icm values of some of the commonly used retmords are given m Table
1. For example, EIB1,,,, (that IS, the absorptton at h,,, of a 1% solution mea-
sured in a cuvet of 1 cm length) IS 1845 for retinol at 325 nm, and 15 10 for
retinoic acid at 350 nm, both measured in ethanol. The corresponding molar-
extinction coefficients (calculated absorbance of a solution of concentration
1 mol/L, in a cuvet of l-cm pathlength) are 52770 and 45300, respectively.
(The use of molar units instead of mass units 1s encouraged.) This enables one
to quantify the amount of retmoid present m a solution of known volume. This
is very useful for quantification of amounts of retmoids too small to be weighed.
Moreover, retmotds are usually present in very small quantmes (picomoles or
nanomoles) m biological extracts. Even when milligram or gram quantities can
be weighed accurately, and the concentration can be determined by dissolving
accurately weighed amounts of retinoids m known volumes of solvent, the
quantrftcation may not be accurate, because of the presence of degraded com-
pounds, which usually would not absorb light at the wavelength of maximum
absorption of the retmoid. It 1s customary, therefore, to determine the exact
concentration from the extinction coefficient of the retmoid m solutron m a
partrcular solvent. Full-absorptron spectra (typrcally 250 nm-400 or 450 nm)
should be scanned; determmatlon of absorbance at only a single wavelength
will not disclose the presence of decomposrtion products. For example, if a
decomposed sample of retmol is scanned from 400 to 250 nm, rt will be
observed that although the solution absorbs considerably at 325 nm (typical for
retmol), the spectrum is not characteristic of retmol, but shows more absor-
bance at lower wavelengths. Hexane and methanol or ethanol are useful sol-
12 Barua and Furr
Table 1
Light Absorbances of Selected Retinoids
Retmord Solvent h,,, a El% Ref
Icm
All-trans retmol Ethanol 325 52770 1845 30
Hexane 325 51770 1810 30
13-czs retinol Ethanol 328 48305 1689 31
1 1-czs retmol Ethanol 319 34890 1220 30
Hexane 318 34320 1200 30
9-czs retmol Ethanol 323 42300 1477 31
11,13-dt-cu retmol Ethanol 311 29240 1024 32
9,13-dr-czs retmol Ethanol 324 39500 1379 31
All-truns retmyl acetate Ethanol 325 51180 1560 33
Hexane 325 52150 1590 34
All-trans retmyl palmnate Ethanol 325 49260 940 34
All-truns retinal Ethanol 383 42880 1510 30
Hexane 368 48000 1690 30
13-cu retinal Ethanol 375 35500 1250 30
Hexane 363 38770 1365 30
1 1-czs retinal Ethanol 380 24935 878 30
Hexane 365 26360 928 30
9-czs retinal Ethanol 313 36100 1270 35
11,13-dt-cu retinal Ethanol 373 19880 700 32
9,13-dt-czs retinal Ethanol 368 32380 1140 32
All-truns retmal oxtme (syn) Hexane 357 55600 1850 36
(anti) Hexane 361 51700 1723 36
11 -czs retinal oxime (syn) Hexane 347 35900 1197 36
(anti) Hexane 351 30000 1000 36
All-truns retmorc acid Ethanol 350 45300 1510 31
I3-czs retmoic actd Ethanol 354 39750 1325 31
9-czs retmorc acid Ethanol 345 36900 1230 31
11,13-dr-czs retinotc acid Ethanol 346 25890 863 34
9,13-dt-czs retinotc acid Ethanol 346 34500 1150 31
all-trans Methyl retmoate Ethanol 354 44340 1415 32
13-czs Methyl retmoate Ethanol 3.59 38310 1220 32
All-truns retmoyl P-glucuronide Methanol 360 50700 1065 23,37
13-w retmoyl P-glucuromde Methanol 369 a 14
9-czs retmoyl P-glucuromde Methanol 353 0 14
All-truns retmyl P-glucuromde Methanol 325 44950 973 24
a-Retmol Ethanol 311 47190 1650 31
a-Retmal Ethanol 368 48800 1720 35
cc-Retmolc acid Ethanol 340 33000 1100 31