Retinol fluorescence

Milk Dilute 400 mg milk with water/MeOH / EtOH (55 9 36) and saponify in a culture tube. Extract unsaponifiables with hep- Perkin-Elmer HS-5-Silica 125 X 4 mm 6% 2-PrOH in heptane Retinol Fluorescence ex 344 nm em 472 nm 147... 

To follow the dissociation of a retinol (e.g., dX -trans- or 11-m-retinol) from IRBP, vesicles containing the fluorescent probe NBD-DPPE are used. The absorption spectrum of NBD extensively overlaps with the fluorescence emission spectrum of retinol Consequently, when both retinol and the probe are present within the same bilayer, energy transfer between the ligand and the NBD moiety of the probe results in quenching of retinol fluorescence Transfer of retinol from the protein to the vesicles can thus be monitored by following the time-dependent decrease in the fluorescence of the ligand. 

Fig 1 Transfer of all-rra s-retinol from IRBP to unilamellar vesicles of DOPC Vesicles containing 2 mol% NBD-DPPE (2 mil/) were mixed with all-/ra/j -retinol-IRBP complex (1 )M, ligand/protein mol ratio = 2). Transfer of all-tmns-retinol from IRBP to vesicles was followed by the time-dependent quenching of ail-traii5-retinol fluorescence upon its amval at the vesicles (excitation, 350 nm, emission, 480 nm) The insets shows the residuals of the fit of the trace to a single first-order reaction... 

Spectroscopic methods such as uv and fluorescence have rehed on the polyene chromophore of vitamin A as a basis for analysis. Indirectly, the classical Carr-Price colorimetric test also exploits this feature and measures the amount of a transient blue complex at 620 nm which is formed when vitamin A is dehydrated in the presence of Lewis acids. For uv measurements of retinol, retinyl acetate, and retinyl palmitate, analysis is done at 325 nm. More sensitive measurements can be obtained by fluorescence. Excitation is done at 325 nm and emission at 470 nm. Although useful, all of these methods suffer from the fact that the method is not specific and any compound which has spectral characteristics similar to vitamin A will assay like the vitamin... 

More specific methods involve chromatographic separation of the retinoids and carotenoids followed by an appropriate detection method. This subject has been reviewed (57). Typically, hplc techniques are used and are coupled with detection by uv. For the retinoids, fluorescent detection is possible and picogram quantities of retinol in plasma have been measured (58—62). These techniques are particularly powerful for the separation of isomers. Owing to the thermal lability of these compounds, gc methods have also been used but to a lesser extent. Recently, the utiUty of cool-on-column injection methods for these materials has been demonstrated (63). 

Although the majority of analytes do not possess natural fluorescence, the fluorescence detector has gained popularity due to its high sensitivity. The development of derivatization procedures used to label the separated analytes with a fluorescent compound has facilitated the broad application of fluorescence detection. These labeling reactions can be performed either pre- or post-separation, and a variety of these derivatization techniques have been recently reviewed by Fukushima et al. [18]. The usefulness of fluorescence detectors has recently been further demonstrated by the Wainer group, who developed a simple HPLC technique for the determination of all-trani-retinol and tocopherols in human plasma using variable wavelength fluorescence detection [19]. 

Figure B3.6.11 The binding of retinol to p-lactoglobutin that has been denatured by exposure to high pressure. The sample contaiining 270 pM p-lactoglobulin was pressurized to 400 MPa for 15 min. After release of pressure, retinol in ethanol was added and fluorescence was measured as a function of time. Parameters final protein concentration 8.5 pM in 20 mM phosphate buffer . ex = 330 nm k9m = 470 nm. Circles, native p-lactoglobulin triangles, pressurized p-lactoglobulin. Reprinted from Ikeuchi et al. (2001) with permission from the American Chemical Society.

Retinol and its esters and unesterified tocopherols and tocotrienols possess strong native fluorescence, but neither vitamin D nor vitamin K fluoresce. The carotenoids commonly associated with foods do not fluoresce to any significant extent, except notably phytofluene, which is found in considerable amounts in tomatoes (22) and in smaller amounts in carrots (130) and which fluoresces six times more intensely than retinyl acetate (131). 

The fluorescence excitation spectra of retinol and its esters correspond to their absorption spectra, with wavelength maxima in the 324-328-nm region emission takes place between 470 and 490 nm (Amax of 470 nm) (31). 

For many applications, fluorescence detection offers no real advantages over absorbance detection, and the linear response range is more limited. Moreover, the fluorescence response of 13-d.v-retinol is less than that of all-trans-retinol, the relative fluorescence depending upon the solvent (133,134). 

SK Jensen. Retinol determination in milk by HPLC and fluorescence detection. J Dairy Res 61 233 -240, 1994. 

FIGURE 5-52. Examples of normal phase separations, (a) Corn-oil tocopherols. Sample 10 /xL of corn oil in 100 /xL of mobile phase. Column Nova Pak Silica (4 jam), 3.9 mm ID x 150 mm. Mobile phase 0.3% isopropyl alcohol in isooctane. Flow rate 1.0 mL/min. Detection fluorescence 290 nm excitation and 335 nm emission. (b) Separation of vitamin E from vitamin A. Mobile phase 0.5% isopropyl alcohol in isooctane. Other conditions are the same as those in a with the exception that retinol was detected with 365 nm excitation and 510 nm emission, (c) Structures of compounds. 

A method has been presented for the determination of retinal (pug levels) by conversion into the intensely coloured and fluorescent derivative of 2-diphenylacetylindane-l,3-dione-l-hydrazone.82 The voltammetric oxidation of retinol at a carbon paste electrode has also been used to assay retinol.83... 

The foregoing conclusions have recently been shown to be also applicable to retinal and retinol for which the theoretically predicted (76,93) Ag state has been identified in the careful two-photon experiments of Birge and co-workers (118,119), with a transition observed on the low-energy side of the main Bj band (see Fig. 5). Moreover, Narva and Hochstrasser have recently detected a weak 410-nm absorption in a single crystal of all-trans retinal at 2°K which they attribute to the - Ag state (120). We shall also see in the following sections that substantial evidence with respect to the Ag state is also derived from fluorescence data. 

Retinol. The extensively investigated retinol molecule offers a relatively simple photophysical system due to the absence of a low-lying (n, tt ) state. Two main patterns characterize the fluorescence of all-trans retinol at 295°K (1) the break-... 

Rosenfeld et al. (144) have recorded the absorption spectrum of the fluorescent states of retinol, retinyl acetate, and retinyl-n-butylamine, using pulsed laser photolysis. Theoretical calculations (145) have closely reproudced the observed 435-nm band assuming that emission originates from the lowest - Ag state. Unfortunately, these results are not discriminative as far as identification of the lowest singlet state is concerned, since a strong absorption in the same region is also predicted for the 1B+ state (145). 

Retinol R C OH (ROH), Retinyl Acetate R CH20C0CH3 (RAc), and Retinyl-n-butylamine R Ch NHR" (RBA). These derivatives of the retinyl chromophore are distinguished by their relatively high room-temperature fluorescence yields (< >f = 0.025 for all three... 

Isomerization due to ionic photodissociation. The (all-trans) + (cis) photoisomerization of ROH, RAc, and RBA is markedly enhanced in polar solvents. Using pulsed laser photolysis it was also shown that ionic photodissociation is responsible for the polarity-induced shortening of the corresponding fluorescence lifetimes. An analysis of both effects led to the formulation of the following scheme (e.g., in the case of retinol)... 

The rate constants for reactions of all-trans retinal and retinol with have been measured in various solvents. Rate constants increase with dielectric constant, thereby suggesting that charge transfer is involved. Electronically excited triplet species generated during peroxide-catalysed aerobic oxidation of substrates gives rise to fluorescence from acceptors in micelles. Micelle-solubilized chlorophyll is an excellent detector of enzyme-generated triplet carbonyl species, and micelles make photochemistry without light possible for... 

Most current interest in u.v. spectra is concerned with retinal and its cis-trans isomers. Studies at 77 K include theoretical calculations, fluorescence, and the effect of protonation on the corresponding Schiff base. The singlet-triplet absorption spectrum of all-trans-retinol has been measured. Calculations on the absorption-emission spectra of rhodopsin suggest that there is little dependence on the angular twist in the polyene chain. Pulse radiolysis has made it... 

Similarly, the retinol acetate was taken up for dipole moment and fluorescence studies [69]. The change in dipole moment upon excitation was found to be 2.7D for retinol acetate. It was interpreted that the change in dipole moment upon excitation makes the retinol acetate molecule to interact more efficiently... 

In a review of the electronic structures and spectroscopy of linear polyenes, Hudson and Kohler have summarized the data available on the photochemical and photophysical parameters of a wide range of molecules, including aco-di-phenylpolyenes, retinals, and retinols.62 The role of an excited state lower in energy than the XBU state in polyenes does reconcile some of the anomalies in the fluorescence parameters of polyenes. There is clearly a need for a better theoretical 46 47 48... 

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