9-cw-Retinol

Physical Methods.—Separation and Assay. A range of isomers of astaxanthin (8) diacetate (9-cis, 13-cis, 15-cis, 9,9 -di-cis, 9,13-di-cw, 9,13 -di-cw, 13,13 -di-cw, 13,15-di-cw), prepared by thermal and iodine-catalysed isomerization of irans-(S) have been separated by h.p.l.c.126 A procedure has been developed for separation of bean leaf etioplast pigments, including carotenoids,127 by h.p.l.c. H.p.l.c. separations of esters of all-trans-, 9-cis-, 11-cw-, and 13-cw-retinol,128-130 and determinations of retinol in serum,131 retinol and 13-cw-retinoic acid,132 and the aromatic retinoid (195)133 in plasma have been described. A reversed-phase ion-pair... 

Alternatively, where samples are extracted into organic solvents, normal phase HPLC can also be used. This option is particularly valuable for the resolution of optical isomers of the A vitamins and, for example, 13-cw-retinol can be resolved using this system (Egberg et al., 1977). Retinoyl species may be converted to retinol prior to chromatography but the subsequent profile is difficult to interpret because of the number of possible intermediates that may also be generated. A computer-assisted analysis of vitamin A extracts from two species of tobacco leaf using non-aqueous reversed phase is shown in Fig. 11.8.2. 

Two methods were developed to stabilize retinol during GLC analysis, namely formation of the trimethylsilyl ethers (Vecchi et al., 1967, 1973) and formation of perhydro derivatives of retinol by catalytic hydrogenation (Fenton et al., 1973). Trimethylsilyl ethers of retinol were separated on columns of Chromosorb W-HP coated with 7% Silicone QF-1 and conditioned by repeated injections of V,0-bis(trimethylsilyl)acetamide. Some separation of isomers of retinol, i.e., all-rran5-retinol from 13-cw-retinol, was also possible. The retention times of various isomers, relative to the trimethylsilyl ether of all-fran -retinol (1.00), were 13-cw (0.64), 11,13-di-cw (0.28), and retro (0.72 and 1.00) (Vecchi etal., 1967). 

Noll found that the elution order of retinol isomers is dependent on the mobile phase in hexane (or heptane) modified with methyl t-butyl ether, the order is l3-cis < 11-cw < 9-cis < all-tran -refinol, whereas in hexane modified with dioxane, ll-cis elutes before 13-cw-retinol (in contradiction to peak identifications in some older literature) (135) peaks were identified by absorption spectroscopy and H-NMR. 

The highly hindered 9-cw,ll-ci5,13-cw-retinol (78) has been synthesized for the first time by a thermally induced [l,5]-sigmatropic rearrangement of the allenic retinol (79) treatment of the retinol with manganese dioxide furnishes... 

Synthetic 13-m-retinoic acid not only inhibited the incidence but also reduced the severity of bladder neoplasms induced by the intravesical administration of MNU of female Wister-Lewis rats [113]. In an experimental model with mice treated with TV-butyl-A -(4-hydroxybutyl)-nitrosamine (OH-BBN), retinoic acid (Re5), 13-cw-retinoic acid, and retinol acetate (Re2) show chemopreventive activity. In addition, a quite large number of synthetic -alkyl amide derivatives of Re2 have a greater activity to toxicity ratio than 13-cw-retinoic acid [113]. 

Retinoids include all natural and synthetic derivatives of vitamin A (retinol) (Figure 102.3). The naturally-occurring retinoids (aU-rntwr-retinoic acid (RA), 9- r-RA, and 13-cw-RA) are interconverted in vivo, and therefore have overlapping effects and toxicity. Synthetic retinoids, such... 

The successful synthesis of (all- )-retinoic acid (3) was reported by Arens and van Dorp (van Dorp and Arens, 1946) in 1946, but almost 10 years passed before Robeson et aL (1955a) were able to prepare 13-cw-retinoic acid (17). Between 1946 and 1956, a large number of papers were published on successful syntheses of the natural C20 retinoids retinol (1), retinaldehyde (2), and retinoic acid (3), and nine industrial processes for the synthesis of vitamin A were developed during this period (Isler, 1979). 

Fig. 1. Chromatogram of four cis-trans isomers of retinol. Column, Si60 (5 ixm) mobile phase, rt-hexane dioxane (95 5) flow rate, 1.1 ml/min peak (1) 1 l-cw-retinol peak (2) 13-c -retinol peak (3) 9-cw-retinol peak (4) all-rrfl .y-retinol. (Reprinted with permission from Paanakker and Groenen-dijk, 1979.)...

One of the most important phenomena for analysis of the mechanism of action of retinoids in prevention of cancer is their ability to suppress the transformation of nonneoplastic C3H/10T1/2 mouse fibroblasts that have been exposed to the chemical carcinogen 3-methylcholanthrene. This was first reported by Merriman and Bertram (1979) using retinyl acetate, retinol, and retinaldehyde as the retinoids. Similar findings were reported by Harisiadis et al. (1978), who showed that motretinid (F3) could suppress radiation-induced transformation of the same cell line. A series of retinoids has been tested by Bertram (1980 see also Bertram et al., 1982) for their ability to suppress methylcholanthrene-induced transformation effects were evaluated over a test range from approximately 10 to 10 M. Retinyl acetate, A -(4-hydroxyphenyl) retinamide (E16), and retinylidene di-medone (C6) were found to be most effective, and A -benzoylretinylamine (B2) was only slightly effective. Surprisingly, all-rran -retinoic acid and 13-cw-reti-noic acid were ineffective in these tests the reason for these results is not clear. 

As an alternative to the system described above, samples can be loaded onto a Dupont Zorbax-Sil column (0.62 x 25 cm, i.e., semipreparative although an analytical column would work as well), and retinol isomers resolved by elution with a mobile phase of 5% acetone in hexane. At a flow rate of 3 mL/mm retinol isomers are well-resolved 13-f/j-retinol (17 min) 9-cw-retinol (19.5 min) all-tran -retinol (25.3 min). 

Fig 1. Quantification of retinal and retinol by HPLC. Retinoids were resolved by a normal-phase Dupont Zorbax-Sil Reliance cartridge column (04x4 cm) eluted at 2 mL/min with a linear gradient from 4% tetrahydrofuran to 15% tetrahydrofuran in hexane for 5 min, followed by 5 mm of 4% tetrahydrofuran in hexane 13-as-retinal (not shown) 1, 9-ci5-retinal 2, all-tran -retinal 3, 3,4,-didehydroretinal 4, 9-cw-retinol 5, all-tran -retinol, 6, 3,4,-didehydroretinol. Retinoids were detected by UV-370 nm for retinals 325 nm for retinols. 

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.)...

Figure 1 Some naturally occuring retinoids (a) all-fra/ii -retinol (b) all-frans-retinal (c) all-fran5 -retinoic acid (d) 13-cw-retinoic acid (e) all-frani-3,4-didehydroretinol (vitamin Aj alcohol) (f) 11-cw-retinal (g) all-franj-5,6-epoxyretinol (h) all-irani-anhydroret-inol (i) all-rran5 -4-oxoretinol (j) all-irans-retinoyl 5-glucuronide (k) all-frani-retinyl i-glucuronide (1) all-franj-retinyl palmitate.

The predominant retinoid in the fasting circulation is retinol, all of which is bound to its specific plasma transport protein, retinol-binding protein (RBP) [1,2]. Although retinol accounts for approximately 95 to 99% of all retinoid in the circulation, other retinoids also are present. Fasting human and rodent blood contains very low levels of both sAhtrans- and 13-cw-retinoic acid (approximately 0.2 to 0.7% of those of retinol) [3], as well as low levels of retinyl esters in lipoprotein fractions, particularly very low-density lipoproteins (VLDL) and low density lipoproteins (LDL) [4]. Soluble glucuronides of both retinol and retinoic acid are also detectable in the circulation of humans and rodents [5], as are provitamin A carotenoids like P-carotene... 

Following consumption of a retinoid-rich meal, the circulation can also contain large amounts of retinyl esters in chylomicrons and/or their remnants [6,7]. Postprandial retinyl ester concentrations can easily exceed fasting retinol levels by several fold [7, 8]. Moreover, consumption of a retinoid-rich meal brings about an increase in circulating concentrations of all-trans- and 13-cw retinoic acid and of glucuronides of both retinol and retinoic acid [7, 9]. 

Figure 1. Formulas of major retinoids and of P-carotene. A, all-tranj retinol B, all-tranj retinal C, all-rran retinoic acid D, 11-cw retinal E, 13-cw retinoic acid F, all-tran retinyl palmtate G, ai -trans retinoyl p-glu-curonide H, the trimethyl methoxyphenol analog of all-rra 5 retinoic acid (etretin, acitretin) I, all-rra 5 p-carotene. From [5], p. 110, reprinted with the permission of the International Life Sciences Institute, Washington DC 20036-4810, Copyright 1996.

Retinoids can also modulate the production of TNF-a and nitric oxide (NO) by macrophages. TNF release was strongly induced in murine peritoneal macrophages by LPS and IFN-ybut was abrogated dose-dependently by RA, and less potently by 4-hydroxy-RA, 13-cw-RA, and retinol [52]. AW-trans-RA also reduced, but did not abolish, NO production in LPS + IFN-y-stimulated macrophages [52]. However, when murine RAW264.7 macrophage cells were pretreated with SiW-trans-RA, more NO was produced in response to stimulation with low concentrations of IFN-y or LPS [58]. 

An important observation is that endogenous levels of retinoids in human embryos are quantitatively and qualitatively similar to those in Xenopus embryos, whereby the following retinoids were detected 13-cw-RA, 9-cis-RA, dill-trans-RA, all-fran -retinol, all-fran -dd-retinol and all-trans-retinal [8, 37, 39]. Until this report, dd-retinol had been detected only in chick embryos [33], but not in rodent embryos. These retinoids were detected in Xenopus embryos at levels that can be accurately quantified with a sensitive HPLC detection system. In contrast, levels of the same endogenous retinoids in rodents are low [19, 20, 21], resulting in less reliable quantification. 

Creech Kraft J, Bui T, Juchau MR (1992) Elevated levels of all- ra 5-retinoic acid in cultured rat embryos 1.5 h after microinjections with 13-cw-retinoic acid or retinol and correlations with dysmorphogenesis. Biochem Pharmacol 44 R 21-24... 

Figure 4. Correlation between retinoid-induced decreases in saturation density and induction of gap junctional communication. Confluent cultures of 10 Tl/2 cells (panel A) or methylcholantlirene transformed 10 Tl/2 cells (panel B) were treated with various concentrations of retinoids for five days. At this time cells were probed by microinjection of Lucifer yellow and transfer of dye to adjacent cells was quantitated. Total cell counts were also performed at this time. Results are expressed as a percent of the untreated controls. Symbols retinol (A) retinyl acetate (A) diW-trans retinoic acid ( ) 13-cw retinoic acid ( ) TTNPB (O). The correlations were statistically highly significant, for 10 Tl/2 cells and for transformed cells, P < 0.001. Data from reference [28] with permission.

Retinoids include natural compounds and synthetic derivatives of retinol that exhibit vitamin A activity. First-generation retinoids include retinol, tretinoin (aU-ironi-retinoic acid), isotretinoin (13-cu-retinoic acid), and alitretinoin (9-cw-retinoic acid). Second-generation retinoids, also known as aromatic retinoids, were created by alteration of the cyclic end group and include acitretin. Third-generation retinoids contain further modifications and are called arotinoids. Members of this generation include tazarotene and bexarotene. Adapalene, a derivative of naphthoic acid with retinoid-like properties, does not fit precisely into any of the three generations. 

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