retinoyl-beta-glucuronide and Vitamin-A-Deficiency

Vitamin A is essential for multiple functions in mammals. Without vitamin A, mammals cannot grow, reproduce, or fight off disease. Because of its numerous functions in humans, biomarkers of vitamin A status are quite diverse. Assessment of liver reserves of vitamin A is considered the gold standard because the liver is the major storage organ. However, this measure is not feasible in human studies. Alternative biomarkers of status can be classified as biological, functional, histologic, and biochemical. Historically, signs of xerophthalmia were used to determine vitamin A deficiency. Before overt clinical damage to the eye, individuals who suffer from vitamin A deficiency are plagued by night blindness and longer vision-restoration times. These types of assessments require large population-based evaluations. Therefore, surrogate biochemical measures of vitamin A status, as defined by liver reserves, have been developed. Serum retinol concentrations are a common method used to evaluate vitamin A deficiency. Serum retinol concentrations are homeostatically controlled until liver reserves are dangerously low. Therefore, other biochemical methods that respond to liver reserves in the marginal category were developed. These included dose-response tests and isotope dilution assays. Dose-response tests work on the principle that apo-retinol-binding protein builds up in the liver as liver reserves become depleted. A challenge dose of vitamin A binds to this protein, and serum concentrations increase within a few hours if liver vitamin A concentrations are low. Isotope dilution assays use stable isotopes as tracers of total body reserves of vitamin A and evaluate a wide range of liver reserves. Resources available and study objectives often dictate the choice of a biomarker.

Topics: Biomarkers; Biopsy; Humans; Liver; Milk, Human; Night Blindness; Nutritional Status; Tretinoin; Vitamin A; Vitamin A Deficiency

The objective of the present study was to determine marginal vitamin A deficiency (VAD) by testing the hydrolysis of retinoyl glucuronide (RAG) to retinoic acid (RA) in children. Previous studies in rats showed that hydrolysis occurred when rats were vitamin A deficient. Children (n 61) aged 3-18 years, were divided into two groups, I and II. Blood was collected from the children in Group I (n 19) who were not dosed with RAG. Children in Group II (n 42) were administered all-trans retinoyl glucuronide (RAG) orally, and blood was collected 4 h after the dose. All serum samples were analysed for retinoids and carotenoids. RA was detected in serum only when serum retinol was < 0.85 micromol/l. Thus, hydrolysis of RAG to RA occurred in children with VAD or marginal VAD. Serum retinol was < 0.35 micromol/l in twenty-one children, 0.35-0.7 micromol/l in twenty-three children, 0.7-0.9 micromol/l in eleven children and >1 micromol/l in six children. Mean serum retinol in sixty-one children was 0.522 (sd 0.315) micromol/l. Mean beta-carotene (0.016 (sd 0.015) micromol/l) was far below normal compared to the level of lutein (0.176 (sd 0.10) micromol/l) in sixty-one children. A low beta-carotene level might be due to a low intake of carotene but high demand for vitamin A. The RAG hydrolysis test may prove to be a useful approach for the determination of marginal VAD with no clinical or subclinical signs of VAD. High prevalence of VAD amongst certain communities in Assam cannot be ruled out.

Topics: Adolescent; beta Carotene; Child; Child, Preschool; Chromatography, High Pressure Liquid; Developing Countries; Female; Humans; India; Linear Models; Lutein; Male; Nutritional Status; Spectrophotometry; Tretinoin; Vitamin A Deficiency

In keeping with the in vivo observation that the conversion of retinoyl beta-glucuronide (RBG) to retinoic acid (RA) is enhanced in vitamin A-deficient (A-) rats, the relative rates of hydrolysis of RBG to RA by various organelles of liver, kidney and intestine were found to be higher in A- rats than in vitamin A-sufficient (A+) rats (mean ratio 1.28; range 1.05-1.63). The lysosomal fraction of kidney and the microsomal fraction of liver showed the highest ratios for RBG hydrolysis; namely, 1.63 and 1.57, respectively (p < 0.005). The rates of hydrolysis of an ether glucuronide, p-nitrophenyl-beta-D-glucosiduronate (pNPG), were also enhanced in A- rats. The ratios of activities were again highest in kidney lysosomes and in liver microsomes; namely, 1.51 (p < 0.005) and 1.42 (p < 0.05), respectively. The non-ionic detergent, Triton X-100, increased RBG hydrolysis in organelles of A+ (19-27%) more than in those of A- rats (8-14%). The ratios of activities +/- SEM with 0.02% Triton X-100 in organelles of kidney, liver and intestine were 1.25 +/- 0.03, 1.22 +/- 0.03 and 1.24 +/- 0.03 in A+ rats and were 1.11 +/- 0.02, 1.07 +/- 0.02 and 1.13 +/- 0.03, respectively, in A- rats. Thus, Triton X-100 had a significantly greater effect (p < 0.005) on the membranes of A+ rats than on those of A- rats. In conclusion, the increased appearance of RA in the plasma after RBG administration in vivo seems to be caused by enhanced activity of beta-glucuronidases in tissue organelles, augmented in part by better access of the substrate to the membrane-bound enzymes.

Topics: Animals; Hydrolysis; Intestines; Kidney; Lysosomes; Male; Microsomes, Liver; Octoxynol; Organelles; Rats; Rats, Wistar; Tretinoin; Vitamin A Deficiency; Weaning

All-trans-retinoyl beta-glucuronide (RAG) was chemically synthesized in high yields (up to 79%) by a new procedure involving the reaction of the tetrabutylammonium salt of glucuronic acid with all-trans-retinoic acid (RA) via the imidazole or triazole derivative. When RAG was fed orally to vitamin A-deficient rats, RA was identified as the major metabolite in the serum within hours of administration of RAG. Very little or no RAG was detected in the serum. Thus RAG, which was not appreciably hydrolysed to RA in vitamin A-sufficient rats [Barua and Olson (1987) Biochem. J. 263, 403-409], was rapidly converted into RA in vitamin A-deficient rats.

Topics: Acitretin; Animals; Chromatography, High Pressure Liquid; Glucuronates; Molecular Structure; Quaternary Ammonium Compounds; Rats; Rats, Sprague-Dawley; Tretinoin; Vitamin A Deficiency

Administration of a single oral dose (10 micrograms/kg) of tetrachlorodibenzo-p-dioxin (TCDD) caused a 33% decrease in retinyl esters in the livers of male rats, but a 13-fold increase in retinyl esters in the kidney and a 3-fold increase in serum retinol. Liver and kidney microsomal uridine diphosphoglucuronosyltransferase (UDPGT) activity toward all-trans-retinoic acid was increased 3.7- and 2.6-fold, respectively, ten days following exposure to TCDD. Verification of the in vitro formation of [3H]retinoyl beta-glucuronide (RG) was by cochromatography with authenic RG on reversed phase high pressure liquid chromatography (HPLC), identification of retinoic acid as the hydrolysis product after beta-glucuronidase treatment, and the characterization of the all-trans-retinoyl glucuronide by negative fragment mass spectroscopy, fast atom bobardment. We conclude that increased retinoic acid glucuronidation may be a contributing factor to the hepatic depletion of vitamin A and the increased excretion of vitamin A metabolites following TCDD exposure.

Topics: Animals; Chromatography, High Pressure Liquid; Dioxins; Glucuronosyltransferase; Kidney; Kinetics; Liver; Male; Mass Spectrometry; Microsomes, Liver; Polychlorinated Dibenzodioxins; Rats; Rats, Inbred Strains; Reference Values; Tretinoin; Vitamin A; Vitamin A Deficiency

All-trans-retinoic acid is metabolized in vitro to a biologically active metabolite, retinoyl-beta-glucuronide. We have studied the synthesis of this metabolite in vitro. The identity of the product was established by cochromatography on reverse-phase high-performance liquid chromatography, beta-D-glucuronidase hydrolysis, and fast atom bombardment and collisionally activated decomposition/fast atom bombardment mass spectrometry. The formation of retinoyl-beta-glucuronide is catalyzed by a UDP-glucuronosyltransferase with apparent Km's of 54.7 microM for all-trans-retinoic acid and 2.4 mM for UDP-glucuronic acid. The reaction requires enzyme, UDP-glucuronate, and no other factor. It is strongly inhibited by millimolar concentrations of coenzyme A. The specific activity of UDP-glucuronosyltransferase is greatest in the liver and least in the kidney of those tissues examined. The specific activity of the enzyme is increased by vitamin A deficiency. The increased specific activity observed in the vitamin A-deficient rat liver is uncharacteristic of retinoic acid inactivation enzymes; therefore, retinoyl-beta-glucuronide may be of functional importance.

Topics: Animals; Chromatography, High Pressure Liquid; Coenzymes; Female; Glucuronosyltransferase; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Male; Microsomes, Liver; Rats; Temperature; Tretinoin; Vitamin A Deficiency

Biliary metabolites from physiological doses of all-trans-[10-3H]retinoic acid were examined in normal and vitamin A-deficient rats. The bile from normal and vitamin A-deficient rats contained approximately 60% of the administered dose following a 24-h collection period. However, vitamin A-deficient rats show a 6-h delay in the excretion of radioactivity compared to normal rats. Retinoyl-beta-glucuronide excretion was particularly sensitive to the vitamin A status of the rats. In normal rats, retinoyl-beta-glucuronide reached a maximum concentration of 235 pmol/ml of bile 2 h following the dose and then rapidly declined. Vitamin A-deficient rats show a relatively constant concentration of this metabolite (100-150 pmol/ml of bile) over a 10-h collection period. Retinoic acid excretion was low in both normal and deficient rats. The concentration of retinotaurine, a recently identified biliary metabolite, was approximately equal to retinoyl-beta-glucuronide in normal rats and appeared in the bile 2 h later than the glucuronide.

Topics: Animals; Bile; Kinetics; Male; Rats; Tretinoin; Vitamin A Deficiency

The metabolites of all-trans-[3H]retinoic acid appearing in the intestines of bile duct-cannulated rats were compared to those of similarly treated intact rats. 2.4% of administered radioactivity was found in the small intestines of bile duct-cannulated rats 2 H after dose, while a much larger proportion of the dose (7.2%) was found in the intestines of the intact rats. All-trans- and 3-cis-retinoic acids predominate in the intestinal mucosa of bile duct-cannulated rats shortly after dosing. Retinoyl glucuronide was the major metabolite occurring as a mixture of the all-trans and 13-cis forms. Highly polar metabolites of retinoic acid appear in mucosa at all times in both intact and bile duct-cannulated rats demonstrating rapid metabolism of retinoic acid in intestine. The finding of a similar proportion of the 13-cis isomers in retinoic acids and retinoyl glucuronides suggests that injected all-trans-retinoic acid is isomerized in vivo probably prior to conjugated with glucuronic acid.

Topics: Animals; Bile; Glucuronates; Intestinal Mucosa; Intestine, Small; Isotretinoin; Kinetics; Male; Rats; Tretinoin; Tritium; Vitamin A Deficiency

A method is described for measuring the relative activities of vitamin A active substances based on their direct effect on the cornified vaginal epithelium of vitamin A-deficient rats. The results obtained agree well with those found in the tracheal organ culture assay. The relative activities found for several test compounds were: all-trans-retinoyl-beta-glucuronide greater than all-trans-retinoic acid greater than all-trans retinol greater than all-trans-5,6-epoxyretinoic acid. The assay is simple and inexpensive to perform, and should find use in laboratories where the equipment and personnel required for the tracheal organ culture assay are not available.

Topics: Animals; Biological Assay; Castration; Dose-Response Relationship, Drug; Epithelium; Female; Glucuronates; Rats; Tretinoin; Vaginal Diseases; Vaginal Smears; Vitamin A; Vitamin A Deficiency

A description of the enzyme that produces 5,6-epoxyretinoic acid from all-trans-retinoic acid has been presented. This enzyme system is found in highest concentrations in the kidney followed by intestine, liver and spleen. The enzyme requires molecular oxygen, magnesium ions, ATP, and NADPH. In the kidney, it is found in the mitochondrial and microsomal fractions and has a Michaelis constant of 3.2 X 10(-6) M and 3.7 X 10(-6) M for 13-cis and all-trans-retinoic acid, respectively. The resultant product, 5,6-epoxyretinoic acid, has minimal activity in supporting growth of vitamin A-deficient rats, its activity estimated to be 0.5% that of retinoic acid. An investigation of the biliary excretion products of tritiated retinoic acid has revealed several unknown metabolites. A glucuronidase sensitive metabolite from these products has been isolated and identified as retinoyl-beta-glucuronide by ultraviolet absorption spectrometry and mass spectrometry. The retinoyl-beta-glucuronide originally discovered by Olson and collaborators accounts for only 12% of the total excreted biliary products of retinoic acid. At least four to six major unknown retinoic acid metabolites, in addition to retinoyl-beta-glucuronide, have been detected and will shortly be identified.

Topics: Animals; Bile; Chromatography, High Pressure Liquid; Epoxy Compounds; Glucuronates; Kidney; Rats; Tretinoin; Vitamin A Deficiency