Retinoids extraction procedure

When attempting to extract and characterize the retinoids of vision, it is best to consider the above factors and then to follow Spom, Roberts Goodman s advice239 In conclusion, before any extraction procedure is applied to the removal of retinoids from tissue samples, it is essential that the method be thoroughly examined with the appropriate controls. Without this close examination, of methods used, the data obtained could be both difficult to reproduce and unrelated to physiological events. More specifically, the data will probably only apply to the residue, retinol, resulting from excessively aggressive extraction techniques. At best, it will apply to the Rhodonines in dilute solution. 

The higher concentration reported by Napoli et al. (22) may be due to hydrolysis of the glucuronide metabolite since retinoyl glucuronide is hydrolyzed by acid or base to retinoic acid (33.341. The extraction procedure of Napoli et al. (22) utilized 2N HC1 which could potentially convert most of the retinoyl glucuronide to retinoic acid. Therefore, the concentration of all trans retinoic acid as reported by this assay could represent the sum of these two retinoids, and not that of the free acid (22). Our assay uses less harsh acidic conditions for extraction which should prevent hydrolysis of the glucuronide, resulting in a lower plasma concentration of the all trans isomer as reported by Napoli et al. 22). 

When total vitamin A is to be measured regardless of whether it is free or esterified, a saponification step can be included. However, degradation of the retinoids occurs even under mild conditions and in the presence of antioxidants. Consequently, direct extraction procedures without any digestion step, allowing the separation and separate quantification of the ester forms and of retinol itself, have gained interest in the last two decades. 

In conclusion, before any extraction procedure is applied to the removal of retinoids from tissue samples, it is essential that the method be thoroughly examined with the appropriate controls. Without this close examination of methods used, the data obtained could be both difficult to reproduce and unrelated to physiological events. 

Zile and DeLuca reported the existence of a metabolite of retinoic acid that was found in rat liver and that was able to support the growth of retinoid-deficient rats. This metabolite was later identified as 13-a.r-retinoic acid (E6) and most, if not all, of it appeared to be generated from all-rrans-retinoic acid by the extraction procedure (Emerick et al., 1967). Sundaresan (1966) has likewise reported diat an acidic metabolite of retinoic acid extracted from normal rat liver was able to restore to near normal the activity of a sulfur-activating enzyme (ATP-sulliiiylase) whose activity was depressed in retinoid-deficient rat liver supernatants. Except for the 13-c/s-retinoic acid, none of these metabolites have been characterized further. 

An online filter was also used to protect the analytical column. A guard column was used before the T to prevent breakthrough. A restrictor was used to balance the pressure before, during, and after the valve switches. After sample transfer, the valve was switched back. With this method, both water-insoluble retinoids and water-soluble retinoic acid were extracted simultaneously. Because of the minimal light exposure of these light-sensitive analytes during the procedure, an extraction recovery of 97 to 100% was achieved with a quantitation range of 100 fmol to 3 nmol. 

Of the two primary methods of isolating the retinoids, saponification and direct extraction, saponification is almost bound to destroy any actual resonant retinoids present. Direct extraction promises more success. However, even in this method, care must be taken to avoid oxidation, or other modifications (including rearrangements) of the chromophores. Roberts, et. al.238 has shown that significant levels of hydrolysis occurred in both the direct extraction method, using the sodium sulfate procedure of Ames et. al., and the lyophilization method of Ito. It appears that hydrolysis leads to retinol as an end product. This accounts for the common assumption that retinol (which was found in the final residue) must have been the original chromophore (with or without an associated opsin). 

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. 

This chapter focuses on the extraction and handling of retinoids and carotenoids, their separation by various chromatographic techniques, and their detection and quantitation, primarily by absorption spectrophotometry, fluorescence, and mass spectrometry. A variety of other methods exist for their identification and characterization, including circular dichroism (333), infrared spectroscopy (334), resonance Raman spectroscopy (335), NMR spectroscopy (336), and x-ray crystallography (337). Although some of these procedures require substantial amounts of a retinoid or a carotenoid in an essentially pure form for study, others, such as resonance Raman spectroscopy, are extremely sensitive and can be used to detect the localization of carotenoids in single cells (338,339). 

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