A new approach to the assessment of marginal vitamin A deficiency in children in suburban Guwahati, India: hydrolysis of retinoyl glucuronide to retinoic acid.

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.

Elevation of retinyl ester level in the lungs of rats following repeated intraperitoneal injections of retinoic acid or retinoyl glucuronide.

Retinoic acid (RA) is involved in the development of both the conducting airway and alveolar portions of the lung. RA plays a key role in the induction of the formation of alveolar septa. Retinoyl beta-glucuronide (RAG), an endogenous retinoid, acts like RA, but is much less cytotoxic. We examined the effect of daily intraperitoneal doses of RA and RAG (1.66 micromol/kg) in dimethyl sulfoxide (DMSO) and cotton seed oil on the stores of retinol and retinyl ester (RE) in the lung of rats. In two separate experiments, one involving normal rats, and the other involving elastase-treated rats, there was up to a 9-fold increase in REs content in the lungs of rats that received RA or RAG for 12 days as compared to Control rats. The accumulation was most profound when RA was injected in DMSO and least when RAG was injected in cotton seed oil. The reason for the accumulation of REs in the lung is not clearly known.

Retinoyl beta-glucuronide: a biologically active interesting retinoid.

Numerous reports have indicated that the biological activity of all-trans retinoyl beta-glucuronide (RAG) is similar to that of all-trans-retinoic acid (RA), but without the toxic side effects of RA. In the present series of studies, we report new findings that support the contention that RAG can function as a nontoxic substitute for RA in a variety of clinic settings. One study on the effects of s.c. injected graded doses of RA and RAG (20-480 micromol/kg BW) into pregnant Sprague-Dawley rats showed that any differences between RAG and RA could be observed only at the highest dose levels of 360 and 420 micromol/kg BW, with RAG being much less toxic than RA. Similarly, daily topical application of RAG (0.16-1.6%) and RA (0.1-0.5%) to shaved swine dorsal skin for six mo resulted in redness and scabbing in RA-treated patches, and to a lesser extent in 1.6% RAG-treated, but not in other RAG-treated patches. Histological scores were significantly higher in the dermis and epidermis of RA-treated pigs than in RAG-treated pigs. Studies to document the pharmacokinetics of chronically administered RAG in mice indicated that, unlike RA, sustained blood levels of parent retinoid (RAG) can be achieved during at least 2 mo of daily administration. Another investigation to study the effects of RAG on the development and growth in nude mice of tumors derived from the human neuroblastoma cell line LA-N-5 showed that s.c. injection of RAG (30 micromol/kg BW) reduced tumor formation when the retinoid was first administered 3 d before tumor injection and continued daily for 30 d thereafter. In established tumors, RAG was shown to inhibit progressive tumor growth, the antitumor effects of RAG being comparable with RA. However, with RAG, as opposed to RA, there were no significant adverse physical side effects. Based on the results of these series of studies along with ample published reports over the last 15 y, we conclude that RAG may be a safe and effective alternative to RA and some other retinoids that are presently being utilized in the clinic.

Intestinal absorption and metabolism of retinoyl beta-glucuronide in humans, and of 15-[14C]-retinoyl beta-glucuronide in rats of different vitamin A status.

In order to prove the hypothesis that humans and animals with adequate vitamin A status do not absorb and metabolize orally administered all-trans retinoyl beta-glucuronide, unlabeled retinoyl glucuronide (0.1 mmol) was orally dosed to fasting well-nourished young men. Neither retinoyl glucuronide nor retinoic acid, a possible metabolite, appeared in the blood within 12 h after ingestion. Next, radiolabeled all-trans 15-[14C]-retinoyl beta-glucuronide was chemically synthesized by a new procedure, and fed orally to rats of different vitamin A status. Analysis of blood and other tissues 5 or 24 h after the dose, showed the presence of radioactivity ( approximately 0.5%) in the blood of vitamin A deficient rats, but not in sufficient rats. Livers of all rats contained small, but detectable amounts (0.3 to 1.1% of the dose) of radioactivity. The accumulation of radioactivity in the liver was highest in deficient rats. Analysis of the retinoids showed that the radioactivity in serum and liver was due to retinoic acid formed from retinoyl glucuronide. Within 24 h after the dose, 31 to 40% of the administered radioactivity was excreted in the feces, and 2 to 4.7% of the dose was excreted in the urine. Results of the present studies show that oral administration of retinoyl beta-glucuronide did not give rise to detectable changes in blood retinoyl glucuronide and/or retinoic acid concentrations in humans or rats with adequate vitamin A status.

A simple rapid method of sample preparation for LC analysis of retinoyl beta-glucuronide and retinoic acid in water-based creams.

A simple method of sample preparation for LC analysis of retinoic acid (RA) and retinoyl beta-glucuronide (RAG) in creams has been developed. Water-based cream of all trans-RAG, devoid of side effects but efficacious in the treatment of acne vulgaris, was found to be hydrolyzed to RA in a temperature dependant manner. The potential benefits of water-based RAG cream stored at room temperature for the treatment of acne and wrinkle is discussed.

Pharmacokinetics of all-trans retinoyl beta-glucuronide in rats following intraperitoneal and oral administration.

The purpose of this study was to examine the pharmacokinetics of a single dose (6.3 mumol, 3 mg) of all-trans retinoyl beta-glucuronide (RAG), when given either orally in corn oil or by intraperitoneal (i.p.) injection in dimethylsulfoxide (DMSO) to adult Sprague-Dawley rats. Following dosing, serial blood samples were collected at various times up to 48 hours from each rat via saphenous vein puncture. Retinoids were extracted from plasma samples and analyzed by high-performance liquid chromatography. In the plasma of i.p.-dosed rats (n = 6), a derivative of RAG, tentatively identified as the lactone of RAG (RAGL), was the major product found. RAGL persisted in the plasma for up to 48 hours. Much smaller concentrations of RAG and of retinoic acid (RA) were also present in the plasma at two to four hours, but generally not thereafter. In orally dosed rats (n = 6), neither RAG nor its products, except for occasional traces of the lactone, were detected. Plasma retinol levels decreased in both i.p.-injected and orally treated rats, the decrease being significant in orally dosed rats.

Absorption and conversion of a single oral dose of beta-carotene in corn oil to vitamin A in Sprague-Dawley rats with low reserve of vitamin A.

This study was carried out to determine how much of a single oral dose of beta-carotene in oil is absorbed and how much of the absorbed dose is converted to retinoids in rats having a vitamin A reserve at the lowest end of adequate status. Weanling rats raised on a vitamin A-deficient diet for four weeks were given a single oral dose of either corn oil or beta-carotene dissolved in corn oil (1.86 mumol). Serum, liver, and the entire digestive tract of the rats were analyzed for carotenoids and retinoids. Results showed that 4 hours after dosing, 1.64 mumol (88%) of the dose of beta-carotene was found intact, with 17.6% found in the stomach, 21% in the small intestine, and 49.3% in the large intestine. A total of 0.28 mumol of newly formed retinoids (expressed as retinyl palmitate) was present in serum, liver, and mucosa of small intestine. The results suggest that a single oral dose of beta-carotene might not be an effective way of raising vitamin A status in rats.

Absorption and conversion of 11,12-(3)H-beta-carotene to vitamin A in Sprague-Dawley rats of different vitamin A status.

The aim of this study was to determine the bioavailability and bioconversion to vitamin A of a single oral dose in oil or an aqueous dispersion of labeled beta-carotene in rats of different vitamin A status. Weanling Sprague-Dawley rats were fed a vitamin A-deficient diet and supplemented for 4 wk with 0, 7, 21 and 63 micro g/(rat. d) of retinyl acetate. The rats, of different vitamin A status, were then given a single oral dose of 11,12-(3)H-beta-carotene (0.15 micro mol) dissolved in corn oil or dispersed in aqueous Tween 80. The rats were killed 4 or 24 h after the dose, and serum, liver, the entire digestive tract, other tissues, urine and feces were analyzed for carotenoids, retinoids and associated radioactivity. At 4 h after the dose, 85 +/- 9% of the administered radioactivity was recovered. Almost 50% of the dose was present as intact beta-carotene in the large intestine where further absorption and conversion was ruled out. The absorption of beta-carotene was very low, and < 5% of the radioactive dose was converted to retinoids. The absorption and conversion to vitamin A did not differ among rats of different vitamin A status. The results suggest that a single oral dose of beta-carotene might not be an effective way of raising vitamin A stores in the body.

Effects of subcutaneously injected graded doses of all-trans retinoic acid and all-trans retinoyl beta-glucuronide on the outcome of pregnancy in Sprague-Dawley rats.

The effects of single subcutaneous injections (s.c.) of graded doses (20, 40, 80, 160, 320, and 480 mumol/kg body weight (BW) of all-trans retinoic acid (RA) and all-trans retinoyl beta-glucuronide (RAG) on day 8.5 of gestation on the outcome of pregnancy in Sprague-Dawley rats was studied. At dose levels of 20, 40, and 80 mumol/kg BW, neither RA nor RAG showed any adverse maternal or fetal effects. However, at dose levels of 160, 320, and 480 mumol/kg, RA was found to be much more toxic than RAG to both mother and fetus. Fetuses of animals receiving a 160 mumol/kg BW dose of RA were significantly reduced in weight and length, while animals receiving the same dose of RAG had fetuses of normal size. RA doses of 320 and 480 mumol/kg BW resulted in symptoms of maternal toxicity and even death. In contrast, RAG at these high levels produced no signs of maternal toxicity. RAG doses of 320 and 480 mumol/kg BW were also less toxic to fetuses. RA doses of 320 mumol/kg BW resulted in only 8% live births, while animals treated with an equivalent amount of RAG experienced 95% live births. Animals receiving a dose of 480 mumol/kg BW of RA had no live births, but similar doses of RAG resulted in 28% live births and pups of normal size.

Repeated intravenous doses of all-trans-retinoyl beta-D-glucuronide is not effective in the treatment of bacterial bronchopneumonia in lambs but is devoid of gross and acute toxicity.

BACKGROUND: All-trans-retinoyl beta-D-glucuronide, a water-soluble glucuronic acid conjugate of all-trans-retinoic acid, has demonstrated high biological activity and low toxicity in most in vitro and in vivo models. Since the reparative effects of retinoids on epithelium are well-known, our aim was to study the effect(s) of intravenously-administered all-trans-retinoyl beta-D-glucuronide in lambs with chronic bacterial bronchopneumonia. MATERIAL/METHODS: Two groups of lambs were inoculated intrabronchially with either pyrogen-free saline or Mannheimia haemolytica. Thirty-three days later, lambs were injected four times at five-day intervals with 2 mL of 116 mM all-trans-retinoyl beta-D-glucuronide (6.0-9.3 mmol/kg or 2.86-4.42 mg/kg animal body weight) in dimethyl sulfoxide, or dimethyl sulfoxide alone. Animal behavior and signs of clinical illness were logged daily. Lung and liver samples were assessed for histopathology, while serum and liver samples were extracted for retinoids and analyzed by reversed-phase gradient high-performance liquid chromatography. RESULTS: Repeated injections of highly concentrated all-trans-retinoyl beta-D-glucuronide did not cause changes in appetite, activity or other behaviors nor did it cause histologic lesions in liver and lung. However, it had no effect on resolution of lung lesions resultant of chronic Mannheimia haemolytica bronchopneumonia. CONCLUSIONS: Repeated intravenous administration of high amounts of all-trans-retinoyl beta-D-glucuronide to lambs did not elicit signs of gross or microscopic toxicity. However, administering all-trans-retinoyl beta-D-glucuronide too late in the progression of bacterial pneumonia is thought to be the main reason for its lack of effect in this model. A retinoid lactone derivative was detected in sheep serum and liver several days after treatment.

Comparative histologic effects of daily topical application of creams containing all-trans-retinoic acid or all-trans-retinoyl beta-glucuronide on pig skin.

The efficacy of all-trans-retinoic acid (tRA) and all-trans-retinoyl beta-glucuronide (RAG), a water-soluble metabolite of vitamin A, in the topical treatment of acne is comparable. However, whereas 3.3 mM tRA shows side effects, 3.3 mM RAG does not. To assess the relative toxic and histologic effects (dermal and epidermal changes) of long-term (24-week) daily applications of tRA and RAG on the skin, separate skin patches were measured and marked dorsally on the skin of six 21-day-old, castrated male pigs. Each skin patch area was treated daily with a cream formulation containing either 3.3 mM RAG, 16.5 mM RAG, 33 mM RAG, 3.3 mM tRA, 16.5 mM tRA or blank cream. To serve as controls, one patch received no treatment, one patch received blank cream only and for 5.3 weeks one 'washed' patch was given daily application of 33 mM RAG with routine cleansing using a mild soap typical of skin care. The amount of cream used per square centimeter remained the same during the course of the study. Biopsy tissue was collected at -1, 0, 2, 4, 8, 12 and 24 weeks from 7 test patches. The 'washed' patch was biopsied once at the 5.3-week mark. Topically applied RAG cream (3.3 mM) resulted in significantly lower histologic scores when compared with scores from tissue treated with an equimolar concentration of tRA. The highest concentration of RAG tested (33.3 mM) resulted in a response comparable to that observed in the lowest tRA (3.3 mM) treated patch area. Daily cleansing of the test area receiving 33.3 mM RAG completely eliminated any clinical signs or negative histologic changes. In conclusion, long-term topical tRA treatment in young pigs, as in humans, showed dose-dependent adverse effects on the skin, whereas RAG treatment had significantly lower histologic changes and less irritation and/or inflammation.