Retinoic acid isomers

Gundersen T.E. and Blomhoff R., 1999. Online solid-phase extraction and isocratic separation of retinoic acid isomers in microbore column switching system. Meth Enzymol 299 430. 

Fig. 2.37. Gradient LC separation of the retinoid solution components and retinoic acid isomers by (A) UV-DAD detection (350 nm) and (B) fluorescence detection with on-line photoreactor switched (a) off and (b) on with irradiation at 366 nm. Peak identification 1 = 13-civ retinoic acid 2 = 9-civ retinoic acid 3 = all-fraws retinoic acid 4 = vitamin A palmitate 5 = /1-carotene. Reprinted with permission from R. Gatti el al. [85].

Isotretinoin is a retinoic acid isomer 20 h). It is used orally in acne (see p. 313). It is effective in preventing second primary tumours in patients who have been treated for squamous cell carcinoma of the head and neck. 

CRA has been detected in humans [34] and was the first RAR-RXR pan-agonist discovered [35-37] and may be classified as a retinoid/rexinoid. It is the only retinoic acid isomer not approved for the common dermatological diseases. However, it has recently been launched in the US as adjuvant topical treatment of AIDS-associated Kaposi s sarcoma [38-41]. This agent is the first RXR ligand to be approved for the treatment of a dermatological disease. In a randomized study with 268 AIDS-associated Kaposi s sarcoma patients, 35% treated with alitretinoin (0.1% gel) had a positive response, compared with 18% treated with vehicle gel irrespective of the number of concurrent antiretroviral therapies [41], 9-CRA is in clinical trials for the treatment of various cancers, including breast cancer [42], renal cell carcinoma [43,44] and squamous cell carcinoma [45—47]. 

Meyer, E. Lambert, W.E. De Leenheer, A.P. Simultaneous determination of endogenous retinoic acid isomers and retinol in human plasma by isocratic normal-phase HPLC with ultraviolet detection. Clin.Chem., 1994, 40, 48—51... 

However, the relevance of these transformations to retinoic acid homeostasis in target tissues is not clear. White et probed a panel of mRNAs from mammalian cell lines with a cDNA from a zebrafish P450 shown to be involved in retinoic acid-inducible retinoic acid oxidation and characterized P450 26A1 (ref [1391]). The heterologously expressed enzyme converted all-traws-retinoic acid to the 4-hydroxy-, and 4-oxo-, and 18-hydroxy products. The turnover numbers are unknown because the amount of P450 was not quantified, but the enzyme is clearly able to catalyze the oxidation of sub-p,M additions of all-fraris-retinoic acid. Apparently other retinoic acid isomers are not substrates. 

Zhang, Z. Y., J. E. Balmer, A. LovUe, S. H. Fromm, and R. Blomhoff. 1996. Specific Teratogenic Effects of Different Retinoic Acid Isomers and Analogs in the Developing Anterior Central Nervous System of Zebrafish. Dev Dynam 206, no 1 73-86. 

Retinoic acid isomers Partisil Isocratic methylene Puglisi and de Silva... 

As is apparent from Table II and for the reasons indicated above, reverse-phase chromatography has been used mainly for the separation of the more-polar retinoids, although an excellent separation of a mixture of retinyl esters has been obtained on an octyl reverse-phase column (Ross, 1981 Fig. 4). While straight-phase chromatography offers better resolution of the isomers of retinol and retinaldehyde (Stancher and Zonta, 1982a), reverse-phase HPLC is capable of partially separating a complex mixture of the more polar retinoic acid isomers (McKenzie et al., 1978a Fig. 5), as well as the isomers of the metabolites of... 

Retinoic acid isomers Partisil ODS-2 Isocratic acetonitrile 1 % Frolik et al. 

To isolate RARs, we routinely incubate cultured cells with radiolabeled retinoic-acid isomers, prepare nuclei, extract the ligand-bound receptors from the purified nuclei and fractionate them using either sucrose density-gradient centrifugation or high-performance size-exclusion chromatography (HPSEC). It is also possible to prepare nuclear extracts from untreated cells and incubate this material with radiolabeled retinoic acid prior to fractionation (3). How-... 

Radiolabeled retinoic acid isomers [11,12 H(N)] a -trans retinoic acid is available commercially from Dupont NEN (Stevenage, Hertfordshire, UK) This material can also be used for preparation of the 13-cis and 9-cis isomers [ H] 9-cis retinoic acid is available from Amersham Life Science (Little Chalfont, Buckinghamshire, UK)... 

Unlabeled retinoic acid isomers -trans, 13-cw and 9-cis retinoic acid are available from a range of suppliers including Sigma (Poole, UK) and ICN (Thame, UK). 

Al -trans retinoic-acid photo-isomerizes readily to give a mixture of stereoisomers with the 13-CJ5 and all-tmns forms predominating. Some 9-cis retinoic acid is also produced. For this reason, two important precautions are required for work with radioactive retinoic-acid isomers first, all manipulations should be done under darkroom conditions or m rooms equipped with yellow lights, and, second, the purity of the retinoic acid should be checked by HPLC analysis, and the required isomer repurified if necessary. A number of methods for the HPLC analysis of retinoic-acid isomers are available (ref. 8 see Chapter 1). We routinely use reversed-phase chromatography with a Cjg column. 

Apply the sample to the column (see Note 1) and elute with a linear gradient of 75 25 methanol/water increasing to 90 10 methanol/water buffered with 10 mAf sodium acetate, pH 7 4, over 20 mm at 2 mL/mm Retinoic-acid isomers are detected by measurement of the absorbance of the column effluent at 343 nm To avoid reisomerization during the purification of specific isomers, the lamp in the UV detector should be extinguished just before the peak of interest is eluted... 

Retinoic acid isomers undergo metabolism to more polar metabolites including 4-hydroxyretmoic acid in cultured cells. The rate of metabolism varies between different cell types (Redfern, CPF, unpublished data). We have obtained satisfactory results using incubation times of 2-4 h. Longer incubation times may present problems because of the absence of FCS and the possibility of retmoic-acid metabolism. 

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). 

Miyagi, M. Yokoyama, H. Shiraishi, H. Matsumoto, M. Ishii, H. Simultaneous quantification of retinol, retinal, and retinoic acid isomers by high-performtuice hquid chromatography with a simple gradiation, J.Chromatogr.B, 2001, 757, 365-368. 

Yamakoshi, Y. Fukasawa, H. Yamauchi, T. Waki, H. Kadowaki, T. Shudo, K. Kagechika, H. Determination of endogenous levels of retinoic acid isomers in type II diabetes melhtus patients. Possible correlation with HbAlc values, Biol.Pharm.BulL, 2002, 25, 1268-1271. 

Embryos depend on the maternal uptake of retinol which must be metabolized to active retinoic acid isomers in order to fulfill all of its roles in vertebrate development. During vertebrate embryogenesis RA is detected in distinct spatiotemporal patterns (32, 37) suggesting that... 

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