Retinoids Ethanol

Retinoids are alcohols and accordingly soluble in ethanol, isopropanol, and polyethylenglycol. Major sources of natural retinoids are animal fats, fish liver oil (retinylesters) and yellow and green vegetables (carotenoids). Ingested retinylesters (RE) are hydrolyzed to retinol by enteral hydrolases in the intestine. ROL and carotenoids are absorbed by intestinal mucosa cells. 

Ethanol also inhibits ADH-catalyzed retinol oxidation in vitro, and ethanol treatment of mouse embtyos has been demonstrated to reduce endogenous RA levels. The inhibition of cytosolic RolDH activity and stimulation of microsomal RolDH activity could explain ethanol-mediated vitamin A depletion, separate from ADH isoenzymes. Although the exact mechanism of inhibition of retinoid metabolism by ethanol is unclear, these observations are consistent with the finding that patients with alcoholic liver disease have depletedhepatic vitamin A reserves [review see [2]. 

Unlike isotretinoin, acitretin (Soriatane) is not primarily sebosuppressive. Rather, it promotes normalization of dysregulated keratinocyte proliferative activity in the epidermis and is also antiinflammatory. Oral absorption is optimal when acitretin is taken with a fatty meal peak levels are reached approximately 3 hours after ingestion, while steady-state plasma levels are achieved after approximately 3 weeks of daily dosing. The mean terminal elimination half-life of the parent compound is 49 hours. However, when consumed with ethanol, acitretin may be transesterifled to form etretinate, a retinoid that is stored in adipose tissue, resulting in a much longer half-life (3-4 months or longer). 

For some agents for example valproic acid and ethanol, a threshold concentration must be reached before teratogenicity is induced and the effect is therefore related to the maximum plasma concentration For others such as retinoids and cyclophos-... 

Acitretin (Soriatane), a metabolite of the aromatic retinoid etretinate, is quite effective in the treatment of psoriasis, especially pustular forms. It is given orally at a dosage of 25-50 mg/d. Adverse effects attributable to acitretin therapy are similar to those seen with isotretinoin and resemble hypervitaminosis A. Elevations in cholesterol and triglycerides may be noted with acitretin, and hepatotoxicity with liver enzyme elevations has been reported. Acitretin is more teratogenic than isotretinoin in the animal species studied to date, which is of special concern in view of the drug s prolonged elimination time (more than 3 months) after chronic administration. In cases where etretinate is formed by concomitant administration of acitretin and ethanol, etretinate may be found in plasma and subcutaneous fat for many years. 

In contrast to retinoids, carotenoids were considered non-toxic, even when taken chronically in large amounts, until recently, when it was found that ethanol interacts with carotenoids, interfering with their conversion to retinol. In baboons, the consumption of ethanol together with beta-carotene resulted in more striking hepatic injury than consumption of either compound alone (98). This interaction occurred at a total dose of 7.2-10.8 mg of beta-carotene per Joule of diet. This dose is common in people who take supplements and is the same order of magnitude used in the Beta-Carotene and Retinol Efficacy Trial (CARET) (30 mg/day) (99) and in another study (20 mg/day for 12 weeks) (100). The amount of alcohol given to the baboons was equivalent to that taken by an average alcohohc. The well-known toxicity... 

Alltretinoln. Alitretinoin. 9-ci.s-reiinoic acid (Panretin). is a yellow powder that is slightly. soluble in ethanol and insoluble in water. Alitretinoin is a naturally occurring endogenous retinoid. 

Nonsteroidal anti-inflammatory drugs (naproxen and ibuprofen) Penicillins Phenylbutazone Probenecid Salicylates Sulfonamides Barbitu rates Phenytoin Probenecid Retinoids Salicylates Sulfonamides Sulfonylureas Tetracycline Ethanol Retinoids Dipyridamole... 

Retinoids are insoluble in water, but soluble in organic solvents, such as ethanol and dimethylsulfoxide (DMSO), which are the most commoidy used solvents for administration of tRA. Although one always performs control experiments, it is important to be aware of the fact that DMSO acts as a differentiating agent to embryonal carcinoma cell cultures in the same way that tRA does, so perhaps ethanol is a better solvent. The maximum solubility of tRA in DMSO is about 50 mg/mL. 

Lipofuscin harbours two unusual retinoids, the lipophilic cations N-retinyl-N-retinylidene ethanol-amine and its isoform, iso-N-retinyl-N-retinylidene ethanolamine, first isolated from the eyes of old... 

When measuring the level of nonpolar retinoids in blood, a modified method of Thompson et al, (1971) could be used (see also Varma and Beaton, 1972 Bieri et al., 1979 Ross, 1981). Briefly, the plasma or serum (100-200 xl) is mixed with 1 volume of internal standard solution (e.g., retinyl acetate) in ethanol. To the mixture is added 1-4 volumes of hexane the contents are mixed well and then centrifuged. The hexane (upper phase) is carefully removed and used for chromatography or other analyses. For added protection against oxidation, butylated hydroxytoluene (100 xg/ml) can be added to the hexane. Other solvents used for extraction of retinol and its esters from serum include chlo-roform methanol (see, for example, DeRuyter and De Leenheer, 1978) and hexane methylene chlorideiisopropanol (Besner /., 1980). Recoveries, when reported, range near 100%. 

Inasmuch as all-tran.s-retinol absorbs strongly at 325 nm ( of 1850 e of 52,995 in ethanol), direct measurement of the absorbancy of retinoids is very useful in those cases where the absorbancy due to retinoids is great relative to that provided by other compounds in the sample. Inasmuch as other retinoids also absorb strongly in the near-ultraviolet region (see Chapter 2), the procedure has general utility. Direct measurements of ultraviolet absorbancy are important... 

For further work, serum has been replaced by insulin and transferrin, and a routine assay for retinoids in serum-free medium is performed as follows (Breitman et al., 1980a) HL-60 cells are seeded at a density of 2 x 10 cells per ml in defined medium, which is a 1 1 mixture of Dulbecco s modified Eagle s minimum essential medium and Ham s F-12 medium supplemented with 3 x 10 M selenium dioxide, insulin (5 xg per ml), and transferrin (5 ig per ml). Retinoids are added to the HL-60 cells at the start of the assay, using ethanol as the vehicle final concentration of ethanol does not exceed 0.1%. The cells are incubated for 4 or 5 days, and differentiation is then measured by NBT reduction (Collins et al., 1979) of cytospin slide preparations. Results are expressed as percentage of NBT positive cells. Approximately 4-8% of the cells will differentiate spontaneously in the absence of added retinoid. As with the F9 system, dose-response curves have been determined for a large number of retinoids, and some of the more significant results are shown in Table III. 

In the procedures described above, the hexane extraction of the alkalinized-aqueous phase serves not only to recover the neutral retinoids, but to remove many neutral lipids that could interfere with the RA assay. If retinol and retinyl esters are the only target analytes, a simpler procedure can be used Extract 0 25 mL of human plasma by adding 0.5 mL ethanol, 100 ng of retinyl acetate (as internal standard), and then vortexing twice with 2 mL of hexane each time (6) Evaporate the solvents from the combined hexane extracts to dryness under a gentle stream of nitrogen. 

Retinoids 11-ci.y-retinaldehyde can be obtained through the National Eye Institute, Fundamental Retinal Processes Research Program, National Institute of Health, Bethesda, MD. 9-cw-retinaldehyde is obtainable from Sigma Chemical Company Both retinoids are stored dry, under argon at -70°C m the dark, and dissolved in ethanol for labeling purposes. 

Retinol and retinoids are particularly unstable in the aqueous medium However, a certain instability is also present in media (like ethanol and methanol) where they are generally dissolved before binding studies are carried out. Therefore, freshly prepared alcoholic solutions of retinoids, protected from light and kept at 0°C, should be used for such studies. 

Retinoid stocks of the appropnate concentration should be prepared in ethanol. Under conditions in which [CRABP] K, retinoid-stock concentrations should be 300- to 600-fold higher than [CRABP]. This is so that there will be less than 1% EtOH in the sample once the titration is completed, and will also minimize volume changes dunng the titration. If [CRABP] < K, then higher-retmoid stock concentrations will probably be necessary. Create retinoid stocks at several concentrations, for example 1000, 200, and 20 iM, allowing titrations under a variety of conditions. 

Quantify the retinoid stocks by UV spectroscopy. Use positive displacement pipets to accurately pipet ethanol (see Note 4) Discard the sample used in the spectrometer, as it may be photo-isomenzed... 

Solutions of retinoids are prepared in ethanol Dissolve several grains of the appropriate retinoid in 0.5-2 mL of ethanol in an amber vessel. Determine... 

Our stock solutions contain retinoids dissolved in either dimethy sulfoxide (DMSO) or absolute ethanol. Care must be taken to ensure that proper controls are performed in order to exclude any effects of the vehicle used to deliver the retinoid on the samples being examined... 

Fig. 13. Regeneration of rhodopsin in suspensions of frog ROS membranes. Enzyme and opsin source bleached ROS membranes in Ringer s (i mi), pH 7.4, temperature 25°. Additives were as foiiows 11-cw-retinol, 100 nmol 11-cu-retinaldehyde, 50 nmol NAD+ ( ) or NADP" (X), 200 nmol. Retinoids were added in about 10 p.1 ethanol. (From Bridges, 1976b.)...

Meyer et al. analyzed plasma retinol plus endogenous aW-trans and i-cis retinoic acid isocratically by normal-phase HPLC, using hexane 2-propanol acetic acid as mobile phase (90). Extraction of the retinoic acids required acidification of the sample however, too much acid can result in dehydration of retinol to anhydroretinol, and hydrolysis of endogenous retinoyl P-glucuronide (90). A synthetic retinoid sulfonic acid was used as internal standard. By using absorbance at 350 nm, limits of detection were 1.7 nM (0.5 (ig/L) for retinoic acid isomers, 35 nM (10 Xg/L) for retinol. 13-Demethyl retinoic acid has also been used as internal standard (142). Lanvers et al. used normal-phase HPLC with gradient elution (hexane 2-propanol glacial acetic acid) to analyze 13-c/x retinoic acid, 9-cis retinoic acid, aW-trans retinoic acid, retinol, and the 4-oxo metabolites of the retinoic acid isomers (143). Plasma samples were treated with ethanol to denature proteins, and then were extracted with hexane after addition of saturated ammonium sulfate solution (pH 5). 

Wyss pioneered use of automated column-switching techniques for analysis of retinoic acid and other polar retinoids (156-160). In general, a plasma sample is diluted with acetonitrile ethanol (95 5) containing an appropriate internal standard the supernatant is passed across a preconcentration column (such as Bonda-pak C18 Corasil, 14 mm X 4.6 mm ID), and the preconcentration column is rinsed with ammonium acetate acetonitrile. The retinoids were eluted from the preconcentration column with a gradient of ammonium acetate acetonitrile acetic acid, and analyzed on a Spherisorb ODS 1 column by gradient elution (159). 

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