Development and validation of a fast and sensitive chromatographic assay for all-trans-retinol and tocopherols in human serum and plasma using liquid-liquid extraction.

A sensitive HPLC assay for all-trans-retinol, alpha-tocopherol, and gamma-tocopherols in human serum and plasma is reported. Sample preparation is performed in one step and involves precipitation of proteins and extraction of lipids with two volumes of an ethanol-chloroform mixture (3:1, v/v) without I.S. addition. After removal of the precipitated protein, 20 microl aliquots of the supernatant (equivalent to 6.7 microl of serum or plasma) were injected into the HPLC system and analyzed using fluorometric detection. RP-HPLC was performed using a C(18) S3 ODS2 column with a methanol-water step gradient (97:3 to 100) at 1.0 ml/min. The quantification limit expressed as nanograms of analyte per milliliter of serum or plasma was approximately 30 ng for all-trans-retinol, 300 ng for alpha-tocopherol and 250 ng for gamma- and delta-tocopherol. The method was validated and applied to human serum and plasma from a total of 120 subjects. This procedure requires a small volume of serum or plasma and can therefore be a valuable tool for measuring low concentrations of these vitamins in preterm infants with sensitivity, precision and accuracy.

Xanthine oxidase catalyzes the synthesis of retinoic acid.

Milk xanthine oxidase (xanthine: oxygen oxidoreductase; XO; EC 1.1.3.22) was found to catalyze the conversion of retinaldehyde to retinoic acid. The ability of XO to synthesize all trans-retinoic acid efficiently was assessed by its turnover number of 31.56 min-1, determined at pH 7.0 with 1 nM XO and all trans-retinaldehyde varying between 0.05 to 2 microM. The determination of both retinoid and purine content in milk was also considered in order to correlate their concentrations with kinetic parameters of retinaldehyde oxidase activity. The velocity of the reaction was dependent on the isomeric form of the substrate, the all trans- and 9-cis-forms being the preferred substrates rather than 13-cis-retinaldehyde. The enzyme was able to oxidize retinaldehyde in the presence of oxygen with NAD or without NAD addition. In this latter condition the catalytic efficiency of the enzyme was higher. The synthesis of retinoic acid was inhibited 87% and 54% by 4 microM and 2 microM allopurinol respectively and inhibited 48% by 10 microM xanthine in enzyme assays performed at 2 microM all trans-retinaldehyde. The Ki value determined for xanthine as an inhibitor of retinaldehyde oxidase activity was 4 microM.

Micellar electrokinetic chromatography of polyamines and monoacetylpolyamines.

A selective procedure for qualitative and quantitative analysis of ten polyamines by micellar electrokinetic chromatography (MEKC) was developed. Benzoylated polyamines and acetylpolyamines in micellar phase of SDS (10 mM) were separated at 25 degrees C by 20 mM borate buffer pH 8.5, containing 8% ethanol, with an applied voltage of 25 kV (5 microA) and then detected at 198 nm. The experimental factors and operational parameters were optimized by performing analysis at different surfactant concentrations, pH, voltage and temperature with and without ethanol. The repeatibility of migration times and peak heights is a peculiarity of the method here described.

Rapid and simultaneous high-performance liquid chromatography assay of polyamines and monoacetylpolyamines in biological specimens.

A rapid, resolutive and reproducible reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for polyamines and acetylpolyamines by adopting pre-column derivatization with benzoyl chloride. In a single run lasting less than 15 min ten polyamines were separated as well as traces of benzoic acid, methylbenzoate and benzoic anhydride. These contaminants, produced during the derivatization reaction, were almost all eliminated by washing steps envisaged in the same procedure. This simple and sensitive method can be applied to routine determination of polyamines in biological samples. A fine application of this procedure to the determination of endogenous content of polyamines in chick embryo retina was reported.

Correlation between the effects of retinoic acid and dexamethasone on liver tyrosine aminotransferase.

A single dose of 50 microg of trans-retinoic acid administered to rats significantly raised the level of hepatic tyrosine after a few hours. This effect was compared with that of dexamethasone and a possible correlation between these effectors was also investigated. An equal increase in enzyme activity level caused by retinoic acid was observed in adrenalectomized rats, leading to the suggestion that the effect of retinoic acid on liver tyrosine aminotransferase is independent of glucocorticoids. However, the study of the binding activity of the liver nuclear glucocorticoid receptors vs dexamethasone showed that this activity is favoured by retinoic acid, whereas no variation was evidenced for retinoic acid receptors caused by dexamethasone. In the adrenalectomized rat, the synergistic effect produced by the association of retinoic acid and dexamethasone at the lowest doses used led us to conclude that retinoic acid is an efficient effector of liver tyrosine aminotransferase. It probably affects tyrosine aminotransferase activity in a dependent and an independent way, regulated respectively by the glucorticoid status and by the provision of retinoic acid.

Retinoid dynamics in chicken eye during pre- and postnatal development.

Changes in the steady state level of retinols, retinaldehydes and retinyl esters in the trans and 11-cis forms and trans retinoic acid were measured in whole chicken eye during development from day 6 in ovo to day 3 post-hatch. These retinoids, quantified by different HPLC systems, were detected in this time sequence: trans-retinol and trans-retinyl esters in the first week in ovo, 11-cis-retinol in the second week. The highest level of 11-cis-retinaldehyde and 11-cis-retinyl esters was reached at the end of development in ovo; however, their levels increased further after hatching. The retinoic acid level decreased at the end of the first week, rising again at the end of the second week. The enzyme activities involved in the metabolism of these retinoids-acyl-CoA: retinol acyltransferase, trans-retinol dehydrogenase, 11-cis-retinol dehydrogenase, trans-retinyl ester hydrolase and trans: 11-cis-retinol isomerase were also estimated and they were detectable already in the first week of development in ovo. At day 6 of the biosynthesis of retinoic acid by the retinaldehyde dehydrogenase activity from retina cytosol was also shown.

Retinyl ester hydrolases in retinal pigment epithelium.

In bovine retinal pigment epithelium membranes we have found three hydrolases which were active against trans-retinyl palmitate. This was possible by assaying different subcellular fractions as a function of pH in the range 3-9. Detection of these activities has been favored by the use in the enzyme assay of Triton X-100, which has an activating effect up to a concentration of 0.03% at a detergent-protein ratio of about 1.5-3.0. Apparent kinetic parameters for the retinyl ester hydrolases have been determined after a study of the optimization of assay conditions. Vmax values for hydrolases acting at pH 4.5, 6.0, and 7.0 were, respectively, 156, 55, and 70 nmol/h/mg. To identify the subcellular site for these hydrolytic activities, assays of marker enzymes from various organelles in each subcellular preparation were carried out, demonstrating the lysosomal origin of the pH 4.5 retinyl ester hydrolase and the microsomal origin of the pH 6.0 retinyl ester hydrolase and suggesting that the pH 7.0 retinyl ester hydrolase originates from the Golgi complex.