Biochemistry zyxwvu 1995,34, 1203- 1209 1203 Identification of zyxwvut 9-cis, 13-cis-Retinoic Acid as a Major Circulating Retinoid in Plasma+,* Ronald L. Horst,*,$ Timothy A. Reinhardt,$ Jesse P. Gaff,§ Brian J. Nonnecke,s Vineet K. Gambhir,$ Paul D. Fiorella," and Joseph L. Napoli" United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Metabolic Diseases and Immunology Research Unit, Ames, Iowa 50010-0070, and Department of Biochemistry, State University of New York at Buffalo, Buffalo, New York, 14214 Received July 7, 1994; Revised Manuscript Received September 12, 1994@ ABSTRACT: In the course of measuring the concentrations of retinoic acids (RA) in bovine plasma, a major peak was observed which comigrated with 9-cis-RA on normal-phase high-performance liquid chroma- tography. Rechromatography of this retinoic on reverse-phase high-performance liquid chromatography showed that it was distinct from 9-cis-, 13-cis-, and all-trans-RA, but comigrated with 9-cis,l3-cis-retinoic acid (9,13-di-cis-RA). This retinoid was identified as 9,13-di-cis-RA based on its chemical, spectral, and chromatographic properties. Plasma concentrations of 9,13-di-cis-RA increased from zyxw 50.5 ng/mL at birth to 5-6 ng/mL by 48 h of age in the calf. The 9,13-di-cis-RA was also a major circulating product of 9-cis-RA dosed intramuscularly to rats; conversely, intravenous administration of 9,13-di-cis-RA produced circulating 9-cis-RA in the rat. 9,13-Di-cis-RA had little or no affinity for cellular retinoic acid binding proteins types I and 11. This study establishes 9,13-di-cis-RA as a naturally-occurring retinoid under physiological conditions, shows that it undergoes interconversion with 9-cis-RA, and emphasizes a need for careful chromatography to resolve 9-cis-RA and 9,13-di-cis-RA. This is consistent with in vivo 13- cis isomerization operating to modify the concentration and perhaps the activity of 9-cis-RA in vivo. Retinoic acid (RA)' is an activated metabolite of retinol that modulates biological processes involved in embryogen- esis, skeletal development, cellular differentiation, and growth (Goss zyxwvutsrqp & McBumey, 1992; Morris-Kay, 1993; Rich- man, 1992; Summerbull & Maden, 1990; Wolf, 1984). Stereoisomersof RA induce transcription by binding to high- affinity intracellular receptors, which include two distinct families; RAR (Gigubre et al., 1987; Petrovich et al., 1987) and RXR (Mangelsdorf et al., 1990), each with multiple isoforms (Nagpal et zyxwvutsr al., 1992). Both all-trans-RA and 9-cis- RA can bind and activate RAR. The preferred ligand for RXR is 9-cis-RA (Heyman et al., 1992; Levin et al., 1992), which can transactivate RXR up to 40 times more efficiently than all-trans-RA (Heyman et al., 1992). Although the RAR and RXR act as ligand-activated transcription factors, there is evidence that RXR also acts as an accessory factor for other receptors. Through formation of heterodimers with RAR, vitamin D receptor, and thyroid hormone receptor, the RXR can promote high-affinity binding to respective hor- mone response elements (Glass et al., 1990; Liao et al., 1990; Murray & Towle, 1989; Yang et al., 1991). While this action requires the cognate ligand for RAR, vitamin D receptor, t This study was supported in part by NIH Grant DK47839 (J.L.N.). * Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable. * Address correspondence to this author. zyxwvuts 5 United States Department of Agriculture. 'I State University of New York at Buffalo. @Abstract published in zyxwvutsrqpo Advance ACS Abstracts, January 1, 1995. I Abbreviations: CRABP, cellular retinoic acid binding protein; HPLC, high-performance liquid chromatography; MeRA, methyl esters of retinoic acid; NMR, nuclear magnetic resonance; RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; 9,13-di-cis- RA, 9 4 s , 13-cis-retinoic acid. 0006-2960/95/0434- 1203$09.00/0 0 and thyroid hormone receptor, it is not dependent upon the RXR ligand 9-cis-RA. Because of their ability to activate receptor-mediated transcription events, recent attention has focused on the biosynthesis and metabolism of all-trans-RA and 9-cis-RA. Derivatives of all-trans-RA that circulate in the plasma under physiological conditions include 13-cis-RA and the glucu- ronides of all-trans- and 13-cis-RA (Barua & Olson, 1986; Eckhoff & Nau, 1990; Tang & Russel, 1990). Although the relationships of these metabolites to retinoid function have not been established, these modifications may provide reservoirs of slowly-metabolized, low-activity retinoids which contribute to maintaining steady-state concentrations of all- trans-RA (Barua et al., 1989; Gunning et al., 1993; McCor- mick et al., 1983; Napoli et al., 1982). Because its role as a ligand for RXR was perceived only recently, less is known about the metabolism of 9-cis-RA. The present study demonstrates that 9,13-di-cis-RA is a physiologically-oc- curring retinoid which can be produced from 9-cis-RA and can also support 9-cis-RA synthesis in vivo. Like 13-cis- RA, 9,13-di-cis-RA has less affinity for CRABP types I and 11, than its parent. These data suggest that 13-cis-isomer- ization in vivo may operate as a common mechanism for modifying the concentrations and activities of all-trans- and 9-cis-RA. Nobably 9,13-di-cis-RA, which exceeds the concentration of 9-cis-RA in newbom bovine plasma, migrates closely to 9-cis-RA during normal-phase HPLC. Unless care is taken, 9,13-di-cis-RA may be mistaken for 9-cis-RA. MATERIALS AND METHODS General. High-performance liquid chromatography was performed with a Waters ALCJGPC 204 liquid chromato- graph. Retinoids were detected at 340 nm. The analytical 1995 American Chemical Society