Retinoyl Beta-glucuronide Research Articles

Integrated Microbiome and Serum Metabolome Analysis Reveals Molecular Regulatory Mechanisms of the Average Daily Weight Gain of Yorkshire Pigs.

The average daily weight gain (ADG) is considered a crucial indicator for assessing growth rates in the swine industry. Therefore, investigating the gastrointestinal microbiota and serum metabolites influencing the ADG in pigs is pivotal for swine breed selection. This study involved the inclusion of 350 purebred Yorkshire pigs (age: 90 ± 2 days; body weight: 41.20 ± 4.60 kg). Concurrently, serum and fecal samples were collected during initial measurements of blood and serum indices. The pigs were categorized based on their ADG, with 27 male pigs divided into high-ADG (HADG) and low-ADG (LADG) groups based on their phenotype values. There were 12 pigs in LADG and 15 pigs in HADG. Feces and serum samples were collected on the 90th day. Microbiome and non-targeted metabolomics analyses were conducted using 16S rRNA sequencing and liquid chromatography-mass spectrometry (LC-MS). Pearson correlation, with Benjamini-Hochberg (BH) adjustment, was employed to assess the associations between these variables. The abundance of Lactobacillus and Prevotella in LADG was significantly higher than in HADG, while Erysipelothrix, Streptomyces, Dubosiella, Parolsenella, and Adlercreutzia in LADG were significantly lower than in HADG. The concentration of glutamine, etiocholanolone glucuronide, and retinoyl beta-glucuronide in LADG was significantly higher than in HADG, while arachidonic acid, allocholic acid, oleic acid, phenylalanine, and methyltestosterone in LADG were significantly lower than in HADG. The Lactobacillus-Streptomyces networks (Lactobacillus, Streptomyces, methyltestosterone, phenylalanine, oleic acid, arachidonic acid, glutamine, 3-ketosphingosine, L-octanoylcarnitine, camylofin, 4-guanidinobutyrate 3-methylcyclopentadecanone) were identified as the most influential at regulating swine weight gain. These findings suggest that the gastrointestinal tract regulates the daily weight gain of pigs through the network of Lactobacillus and Streptomyces. However, this study was limited to fecal and serum samples from growing and fattening boars. A comprehensive consideration of factors affecting the daily weight gain in pig production, including gender, parity, season, and breed, is warranted.

Esterase 22 and beta-glucuronidase hydrolyze retinoids in mouse liver

Excess dietary vitamin A is esterified with fatty acids and stored in the form of retinyl ester (RE) predominantly in the liver. According to the requirements of the body, liver RE stores are hydrolyzed and retinol is delivered to peripheral tissues. The controlled mobilization of retinol ensures a constant supply of the body with the vitamin. Currently, the enzymes catalyzing liver RE hydrolysis are unknown. In this study, we identified mouse esterase 22 (Es22) as potent RE hydrolase highly expressed in the liver, particularly in hepatocytes. The enzyme is located exclusively at the endoplasmic reticulum (ER), implying that it is not involved in the mobilization of RE present in cytosolic lipid droplets. Nevertheless, cell culture experiments revealed that overexpression of Es22 attenuated the formation of cellular RE stores, presumably by counteracting retinol esterification at the ER. Es22 was previously shown to form a complex with beta-glucuronidase (Gus). Our studies revealed that Gus colocalizes with Es22 at the ER but does not affect its RE hydrolase activity. Interestingly, however, Gus was capable of hydrolyzing the naturally occurring vitamin A metabolite retinoyl beta-glucuronide. In conclusion, our observations implicate that both Es22 and Gus play a role in liver retinoid metabolism.

Retinoyl β-Glucuronide: A Biologically Active Form of Vitamin A

Retinoyl beta-glucuronide is a naturally occurring, biologically active metabolite of vitamin A. Although retinoyl beta-glucuronide is regarded as a detoxification product of retinoic acid, it plays several roles in the functions of vitamin A. It can serve as a source of retinoic acid, and it may be a vehicle for transport of retinoic acid to target tissues. Topically applied retinoyl beta-glucuronide is comparable in efficacy to retinoic acid in the treatment of acne in humans, without the same side effects. Retinoyl beta-glucuronide may or may not be teratogenic, depending on the mode of administration and the species in which it is used. It may be a valuable therapeutic compound for the treatment of skin disorders and certain types of cancers.

Elevated serum concentrations of β-glucuronide metabolites and 4-oxoretinol in lactating sows after treatment with vitamin A: a model for evaluating supplementation in lactating women2–3

The effects of large doses of preformed vitamin A, such as those provided in supplementation programs for the prevention of deficiency, on total serum vitamin A have been inadequately investigated. This study characterized the time course of serum vitamin A metabolites in lactating sows after single high doses of retinyl ester. Lactating sows were fitted with jugular catheters and subsequently fed either 1.05 or 2.1 mmol retinyl ester (n = 6/group) or a corn oil vehicle (n = 3). Blood was collected at baseline and at intervals to 48 h and analyzed by gradient HPLC for retinol, retinyl esters, and metabolites. The mean (+/-SD) total serum vitamin A concentration peaked at 1 h (3.69 +/- 4.0 mumol/L) and 2 h (7.70 +/- 6.8 mumol/L) in the low- and high-dose groups, respectively (P < 0.05). Retinyl esters accounted for most of the serum vitamin A in both groups at peak time points. Mean serum retinol concentrations changed little and accounted for most of the serum vitamin A at baseline (94% and 97% for the low- and high-dose groups, respectively) but for only 22% and 14% at peak times for the low- and high-dose groups, respectively. Postdosage increases were observed for total vitamin A and retinyl esters, 4-oxoretinol, retinoyl beta-glucuronide, and retinyl beta-glucuronide but not for retinoic acid. Serum retinol concentration remains relatively static after a large dose of preformed vitamin A and therefore is not an appropriate measure of intervention efficacy. The increases in beta-glucuronide metabolites and 4-oxoretinol suggest a preventive role against a rise in retinoic acid and retinol.

Retinoyl β-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.

Assessing Vitamin A Status: Past, Present and Future

Xerophthalmia classification was traditionally used to identify populations with vitamin A deficiency. Currently, night blindness and dark adaptometry have been proposed as population assessment methods. While eye signs and function tests are still used in areas where vitamin A deficiency is severe, a subclinical vitamin A deficiency is more prevalent. Serum and breast milk retinol concentrations are used to identify vitamin A deficiency risk. However, in healthy individuals, serum retinol concentrations are homeostatically controlled and do not begin to decline until liver reserves of vitamin A are dangerously low. Moreover, serum retinol and retinol binding protein (RBP) concentrations fall during times of infection. The RBP:transthyretin ratio may help to determine if serum retinol concentrations are depressed by infection. Other methods better reflect liver reserves of vitamin A, the "gold" standard. The relative dose response and modified relative dose response tests involve giving a small dose of retinyl or dehydroretinyl ester, respectively, and determining a response in the serum at about 5 h. A new response test where retinoyl beta-glucuronide is administered and the degree of hydrolysis to retinoic acid is measured has been investigated. Unlike isotope dilution tests, the dose response tests lack utility in defining the total body reserve of vitamin A. The deuterated retinol isotope dilution test has been used in several different groups. Recently, a new isotope assay was developed using 13C-retinyl acetate and gas chromatography-combustion-isotope ratio mass spectrometry for analysis. Thus, having many choices of vitamin A assessment methods, laboratory sophistication and resources available will usually dictate which methods are chosen.

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.

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.

Bioactivities of N-(4-hydroxyphenyl) retinamide and retinoyl beta-glucuronide.

N-(4-Hydroxyphenyl) retinamide (4HPR) and retinoyl beta-glucuronide (RAG) are two derivatives of all-trans retinoic acid (RA) that show properties both similar to as well as different from their parent compound, RA. Both retinoids possess the important property of showing much-reduced toxicity relative to RA while maintaining significant biological activity. 4HPR, a synthetic derivative, is active in the prevention and treatment of a variety of neoplasms in animals, and by inducing apoptosis, shows growth inhibitory activity against many human tumor cell types in vitro. In humans, 4HPR reduces the incidence of new occurrences of leukoplakia and is currently being tested as a preventive agent for breast cancer. RAG, a naturally occurring metabolite of RA, effectively stimulates the growth of vitamin A-deficient animals, induces the differentiation of epithelial cells in vivo and in vitro, and is effective in the topical treatment of acne in humans. Unlike RA, RAG is nontoxic when applied to the skin and is nonteratogenic when given orally to rats. Possible mechanisms of action of both compounds are discussed. These two derivatives of retinoids show interesting physiologic effects and potentially beneficial pharmacologic actions.

Retinoic acid inhibition of transforming growth factor-beta-induced collagen production by human lung fibroblasts.

Transforming growth factor-beta (TGF-beta)-stimulated collagen production plays an important role in the pathogenesis of the fibrotic response seen in chronic inflammatory lung disorders. Retinoids are vitamin A analogues that are potent immunomodulators and have been shown to modulate stromal cell collagen production in a variety of nonpulmonary systems. We hypothesized that retinoids might also modulate lung fibroblast collagen production. To test this hypothesis, we determined whether all-trans retinoic acid (RA) and several other retinoid compounds regulate the production of types I and III collagen by unstimulated and TGF-beta 1-stimulated human lung fibroblasts. Unstimulated cells produced modest quantities of types I and III collagen, and TGF-beta 1 increased the production of these matrix molecules 2- to 4-fold. Preincubation with 10(-5) M RA caused a significant decrease in the basal levels of types I and III collagen produced by these cells. RA preincubation also totally abrogated the collagen inductive effects of TGF-beta 1. At 10(-5) M, RA preincubation caused a 97% decrease in the stimulation of type I collagen and a 115% decrease in the stimulation of type III collagen caused by TGF-beta 1. These inhibitory effects were dose dependent. Significant inhibition of type I and III collagen production was appreciated with doses of RA as low as 10(-9) and 10(-8), respectively. These inhibitory effects were not unique to RA since 13-cis-retinoic acid, 9-cis-retinoic acid, etretinate, all-trans etretin, and the water-soluble retinoids, retinoyl beta-glucuronide and retinyl-beta-glucuronide, also inhibited TGF-beta 1-stimulated type I collagen production.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse conceptuses have a limited capacity to elevate the mRNA level of cellular retinoid binding proteins in response to teratogenic doses of retinoic acid.

In these studies, we wished to determine the effect of teratogenic doses of retinoic acid on the expression of cellular retinoic acid binding protein I (CRABP-I) mRNA, cellular retinoic acid binding protein II (CRABP-II) mRNA, cellular retinol binding protein I (CRBP-I) mRNA, and cellular retinol binding protein II (CRBP-II) mRNA in mouse conceptuses. Levels of CRABP-II mRNA and CRBP-I mRNA were modestly elevated (2.5-fold and 1.5-fold, respectively) in 9-day gestation conceptuses following treatment of dams with 100 mg/kg b.w. of retinoic acid. These levels were elevated by 6 hr following treatment and remained elevated until 48 and 24 hr, respectively. Two other retinoids, etretinate and retinoyl beta-glucuronide, also moderately elevated CRABP-II mRNA and CRBP-I mRNA levels in conceptuses. In contrast, the levels of CRABP-I mRNA in the conceptuses remained unaffected by treatment with any of these three retinoids. These results demonstrate that conceptuses have a limited capacity to elevate the cellular retinoid binding proteins mRNA levels and presumably the synthesis of their respective proteins in response to high, teratogenic doses of retinoic acid. As a result, an excess of free retinoic acid becomes available to the nuclear retinoic acid receptors, which may lead to inappropriate gene expression and eventual maldevelopment.

Metabolism in vivo of all-trans-[11-3H]retinoic acid after an oral dose in rats. Characterization of retinoyl β-glucuronide in the blood and other tissues

Soon after [11-3H]retinoic acid (RA) (1.1 x 10(8) d.p.m.) was administered orally to rats either as a large dose (115 micrograms = 0.38 mumol/rat) or mixed with unlabelled RA as a huge dose (22 mg = 73.33 mumol/rat), retinoyl beta-glucuronide (RAG) was identified and characterized as a significant metabolite in the serum and small intestine. Of the administered dose, 70% remained unchanged as retinoic acid in the stomach up to 1 h. Significant amounts of 5,6-epoxyretinoic acid, 4-hydroxyretinoic acid, esters of retinoic acid and several polar retinoids, including 4-oxoretinoic acid, were also detected in the stomach. No significant difference was observed in the nature of the retinoids found after a large or a huge dose; however, the ratio of RAG/RA was higher after a huge dose than after a large dose. Thus RAG, which is biologically active in vivo and in vitro, is formed quickly in significant amounts in tissues after a dose of RA.

Effects of Retinoid Glucuronides on Mammary Gland Development in Organ Culture

Retinoyl beta-glucuronide (RAG) and retinyl beta-glucuronide (ROG) inhibit prolactin-induced morphological developments in cultured mouse mammary gland, and are equally effective in depressing prolactin and steroid hormone-induced DNA synthesis in the same tissue. Retinol is essentially inactive by both assay methods. Although RAG and ROG show little or no toxicity in several cell and animal models, some possibly toxic manifestations were seen at higher concentrations in cultured mammary tissue. Nonetheless, because of the strong correlation between the actions of chemopreventive agents in culture mouse mammary glands and their effectiveness in vivo, the retinoid glucuronides may well prove to be useful cancer chemopreventive agents.

Chemical synthesis of all-trans-[11-3H]retinoyl beta-glucuronide and its metabolism in rats in vivo.

All-trans-[11-3H]retinoyl beta-glucuronide (RAG) was synthesized in a single step from all-trans-[11-3H]retinoyl fluoride, with a 24% yield. After its intraperitoneal injection into rats, RAG was detected in the blood, liver, intestine and kidney during the following 24 h period. Although the concentration of radiolabelled metabolites decreased with time, RAG predominated at nearly all times in nearly all tissues. Small amounts of retinoic acid (RA) were also universally present, together with unidentified polar metabolites and small amounts of non-polar esters of RA. The major excretion products of RAG in faeces and urine were RA and polar metabolites. Thus RAG, although converted in part to RA in vivo, persists as a major component in blood and tissues for at least 24 h. These observations support the concept that the retinoid beta-glucuronides might serve a physiologically significant role in the function of vitamin A.

Characterization of retinyl beta-glucuronide in human blood

Retinyl beta-glucuronide has been identified as a normal endogenous component of human blood, as have retinoyl beta-glucuronide and retinoic acid. The mean serum concentration of retinyl beta-glucuronide in adult volunteers (n = 6) was 6.8 +/- 4.0 nmol/L (means +/- SD) with a range from 1 to 11 nmol/L. Purified serum retinyl beta-glucuronide was characterized by the mass spectrum of its methylated trimethylsilyl derivative, by its hydrolysis by beta-glucuronidase, and by its chromatographic behavior in high-performance liquid chromatography systems. During a single day, concentrations of retinyl beta-glucuronide, retinoyl beta-glucuronide, and retinoic acid varied approximately two-fold.

Effects of retinoid beta-glucuronides and N-retinoyl amines on the differentiation of HL-60 cells in vitro.

Retinoyl beta-glucuronide and retinyl beta-glucuronide, which are naturally occurring water-soluble metabolites of vitamin A, induce the granulocytic differentiation of HL-60 cells in vitro, as evidenced by an increased reduction of nitroblue tetrazolium. The relative effectiveness of various retinoids in differentiation is retinoic acid greater than retinoyl beta-glucuronide greater than retinyl beta-glucuronide. Under the selected assay conditions, retinol, hydroxyphenyl-retinamide, retinamide, and N-retinoyl-phenylalanine are essentially inactive in differentiation. At concentrations of retinoids from 10(-9) to 10(-5) M, cell viability was best with the retinoid beta-glucuronides and retinamide, less with retinoic acid and retinol, and poorest with the N-retinoyl aromatic amines. Cellular growth was depressed only slightly by retinyl beta-glucuronide and retinamide, but to a greater degree by the other derivatives. Retinoyl beta-glucuronide was hydrolyzed in part to retinoic acid, whereas retinyl beta-glucuronide was cleaved to retinol, if at all, at a very slow rate. Under the selected assay conditions, retinoic acid and the retinoid beta-glucuronides primarily induce the differentiation of HL-60 cells, whereas the N-retinoyl aromatic amines show cytotoxicity.

Chemical synthesis of all-trans retinoyl beta-glucuronide.

All-trans retinoyl fluoride reacts with the 3,6-lactone of glucuronic acid in slightly alkaline solution to give the lactone of retinoyl beta-glucuronide, along with other retinoyl glucurono-lactones, in about 60% yield. Hydrolysis of the lactone with very dilute alkali gives the free acid, retinoyl beta-glucuronide, in about 80% yield. Pure all-trans retinoyl beta-glucuronide (overall yield: 20-25%) was obtained free from other isomeric and anomeric forms by reverse-phase high pressure liquid chromatography. Retinoyl beta-glucuronide was characterized by UV-visible, infrared, and 1H-NMR spectra, by elementary analysis, by mass spectra, and by its susceptibility to hydrolysis by bacterial beta-glucuronidase.

Metabolism of 5,6-epoxyretinoic acid in vivo: isolation of a major intestinal metabolite.

The major metabolite in the small intestinal mucosa of vitamin A deficient rats dosed intrajugularly with 5,6-epoxy[3H]-retinoic acid has been identified as 5,6-epoxyretinoyl beta-glucuronide. The assignment was based on the metabolite's chemical, spectral, and chromatographic properties. Incubation of the metabolite with beta-glucuronidase released 5,6-epoxyretinoic acid. Incubation of 5,6-epoxyretinoic acid with rat liver microsomes in the presence of uridine-5'-diphospho-1 alpha-D-glucuronic acid produced the metabolite. 5,6-Epoxy[3H]retinoyl beta-glucuronide weas observed in the liver, small intestinal mucosa, and intestinal contents but not in kidney of vitamin a deficient rats. Its concentration was greatly diminished in liver and small intestinal mucosa, and it was not observed in kidney of vitamin A deficient rats dosed orally with retinoic acid for several days before administration of 5,6-epoxy[3H]retinoic acid. Generally, oral retinoic acid treatment accelerated 5,6-epoxyretinoic acid metabolism and enhanced accumulation of highly polar metabolites. Moreover, 5,6-epoxyretinoic acid metabolism was more rapid than that of retinoic acid and did not result in production of retinoic acid.

Characterization of retinoyl beta-glucuronide as a minor metabolite of retinoic acid in bile.

Several metabolites detected in the bile of rats given radioactive retinoic acid were separated by liquid/gel partition chromatography and purified by high-pressure liquid chromatography. One of these metabolites was found to be sensitive to beta-D-glucuronidase, yielding both 13-cis- and all-trans-retinoic acid. It had the characteristic ultraviolet absorption spectrum of retinoic acid esters. Trimethylsilyl ether and acetyl derivatives of the methylated metabolite were prepared and examined by mass spectrometry. The resulting mass spectra established the structure to be retinoyl beta-glucuronide. Retinoyl glucuronide was rapidly excreted into the bile: the excretion was complete by 12 hr after the administration of retinoic acid. At this time the metabolite represented 12% of bile radioactivity (10% of dose). These observations confirm the existence of retinoyl glucuronide but demonstrate that it represents only one of several retinoic acid metabolites in bile.

RETINOYL BETA-GLUCURONIC ACID: A MAJOR METABOLITE OF VITAMIN A IN RAT BILE.

The major metabolite in rat bile of injected C(14)-retinoic acid was purified by ion-exchange and silicic acid chromatography; it has the spectrum of methyl retinoate, releases retinoic acid upon basic hydrolysis or by treatment with beta-glucuronidase, and contains glucuronic acid. The metabolite was characterized by treatment with diazomethane followed by hexamethyl-disilazane, or with periodate followed by semicarbazide, and the products were chromatographed. The metabolite has been tentatively identified as retinoyl beta-glucuronide.