Biosynthesis of beta-glucuronides of retinol and of retinoic acid in vivo and in vitro

After the intraportal injection of retinol-6,7-(14)C to rats, the O-ether derivative of retinol, retinyl -glucosiduronate, appears in the bile. Both retinoyl -glucuronide and retinyl -glucosiduronate are also synthesized in vitro when washed rat liver microsomes are incubated with uridine diphosphoglucuronic acid (UDPGA) and either retinoic acid or retinol, respectively. The synthesis of retinoyl -glucuronide was also demonstrated in microsomes of the kidney and in particulate fractions of the intestinal mucosa. The glucuronides were characterized by their UV absorption spectra, by their quenching of UV light or fluorescence under it, by their thin-layer chromatographic behavior in two solvent systems, and by the identification of products released during their hydrolysis by -glucuronidase. With retinoic acid as the substrate, the UDP glucuronyl transferase of rat liver microsomes had a pH optimum of 7.0, a temperature optimum of 38 degrees C, and a marked dependence on the concentrations of both retinoic acid and UDPGA, but was unaffected by a number of possible inhibitors, protective agents, and competitive substrates. The conversion of retinal to retinoic acid and the synthesis of retinoyl -glucuronide from retinoic acid could not be detected in whole homogenates, cell fractions, or outer segments of the bovine retina.     

Multiple retinol and retinal dehydrogenases catalyze all-trans-retinoic acid biosynthesis in astrocytes

All-trans-retinoic acid (atRA) stimulates neurogenesis, dendritic growth of hippocampal neurons, and higher cognitive functions, such as spatial learning and memory formation. Although astrocyte-derived atRA has been considered a key factor in neurogenesis, little direct evidence identifies hippocampus cell types and the enzymes that biosynthesize atRA. Here we show that primary rat astrocytes, but not neurons, biosynthesize atRA using multiple retinol dehydrogenases (Rdh) of the short chain dehydrogenase/reductase gene family and retinaldehyde dehydrogenases (Raldh). Astrocytes secrete atRA into their medium; neurons sequester atRA. The first step, conversion of retinol into retinal, is rate-limiting. Neurons and astrocytes both synthesize retinyl esters and reduce retinal into retinol. siRNA knockdown indicates that Rdh10, Rdh2 (mRdh1), and Raldh1, -2, and -3 contribute to atRA production. Knockdown of the Rdh Dhrs9 increased atRA synthesis ～40% by increasing Raldh1 expression. Immunocytochemistry revealed cytosolic and nuclear expression of Raldh1 and cytosol and perinuclear expression of Raldh2. atRA autoregulated its concentrations by inducing retinyl ester synthesis via lecithin:retinol acyltransferase and stimulating its catabolism via inducing Cyp26B1. These data show that adult hippocampus astrocytes rely on multiple Rdh and Raldh to provide a paracrine source of atRA to neurons, and atRA regulates its own biosynthesis in astrocytes by directing flux of retinol. Observation of cross-talk between Dhrs9 and Raldh1 provides a novel mechanism of regulating atRA biosynthesis.     

Kinetic and thermodynamic analysis of 9-cis-retinoic acid binding to retinoid X receptor alpha.

The interaction of retinoid X receptor alpha with 9-cis-retinoic acid was studied using stopped-flow fluorescence spectroscopy. Transient kinetic analyses of this interaction suggest a two-step binding mechanism involving a rapid, enthalpically driven pre-equilibrium followed by a slower, entropically driven reaction that may arise from a conformational change within the ligand binding domain of the receptor. The assignment of this kinetic mechanism was supported by agreement between the overall equilibrium constant, Kov, derived from kinetic studies with that determined by equilibrium fluorescence titrations. Although these analyses do not preclude ligand-induced alteration in the oligomerization state of the receptor in solution, the simplest model that can be applied to these data involves the stoichiometric interaction of 9-cis-retinoic acid with retinoid X receptor alpha monomers.     

Substrate specificities and 13-cis-retinoic acid inhibition of human, mouse and bovine cis-retinol dehydrogenases

Recent studies of the human, mouse and bovine genes for 11-cis-retinol dehydrogenase (11cRDH) and human and mouse 9-cis-retinol dehydrogenase (9cRDH) suggest that they are homologs of the same enzyme. This conclusion is inconsistent with earlier literature indicating that 11cRDH is expressed solely in the eye and does not utilize 9-cis-retinol as a substrate. We have compared directly the kinetic properties of recombinant human and mouse 9cRDH with those of bovine 11cRDH for 9-cis- and 11-cis-retinol and investigated the inhibitory properties of 13-cis-retinoic acid on each of these enzymes. Human and mouse 9cRDH and bovine 11cRDH have very similar kinetic properties towards 9-cis- and 11-cis-retinol oxidation and they respond identically to 13-cis-retinoic acid inhibition. Our biochemical data are consistent with the conclusion that 9cRDH and 11cRDH are the same enzyme.     

Analysis, occurrence, and function of 9-cis-retinoic acid

Metabolic conversion of vitamin A (retinol) into retinoic acid (RA) controls numerous physiological processes. 9-cis-retinoic acid (9cRA), an active metabolite of vitamin A, is a high affinity ligand for retinoid X receptor (RXR) and also activates retinoic acid receptor (RAR). Despite the identification of candidate enzymes that produce 9cRA and the importance of RXRs as established by knockout experiments, in vivo detection of 9cRA in tissue was elusive until recently when 9cRA was identified as an endogenous pancreas retinoid by validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology. This review will discuss the current status of the analysis, occurrence, and function of 9cRA. Understanding both the nuclear receptor-mediated and non-genomic mechanisms of 9cRA will aid in the elucidation of disease physiology and possibly lead to the development of new retinoid-based therapeutics. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.     

Inactivation of alcohol and retinol dehydrogenases by acetaldehyde and formaldehyde.

The rapid and progressive inactivation of alcohol dehydrogenase from horse liver, rat liver and from human retina and of retinol dehydrogenase of rat liver by low concentrations of acetaldehyde or formaldehyde is illustrated. The inactivation of alcohol dehydrogenase can be largely prevented and partially reversed with glutathione. These findings are discussed as a model for better understanding of toxic effects of alcohol and in the context of the importance of protein turnover measurements for clarification of untoward alcohol effects.     

Functional analysis of Toxoplasma lactate dehydrogenases suggests critical roles of lactate fermentation for parasite growth in vivo

Glycolysis was thought to be the major pathway of energy supply in both fast‐replicating tachyzoites and slowly growing bradyzoites of Toxoplasma gondii. However, its biological significance has not been clearly verified. The genome of T. gondii encodes two lactate dehydrogenases (LDHs), which are differentially expressed in tachyzoites and bradyzoites. In this study, we knocked out the two LDH genes individually and in combination and found that neither gene was required for tachyzoite growth in vitro under standard growth conditions. However, during infection in mice, Δldh1 and Δldh1 Δldh2 mutants were unable to propagate and displayed significant virulence attenuation and cyst formation defects. LDH2 only played minor roles in these processes. To further elucidate the mechanisms underlying the critical requirement of LDH in vivo, we found that Δldh1 Δldh2 mutants replicated significantly more slowly than wild‐type parasites when cultured under conditions with physiological levels of oxygen (3%). In addition, Δldh1 Δldh2 mutants were more susceptible to the oxidative phosphorylation inhibitor oligomycin A. Together these results suggest that lactate fermentation is critical for parasite growth under physiological conditions, likely because energy production from oxidative phosphorylation is insufficient when oxygen is limited and lactate fermentation becomes a key supplementation.     

13-cis-retinoic acid metabolism in vivo. The major tissue metabolites in the rat have the all-trans configuration

The liver and intestinal metabolites of orally dosed 13-cis-[11-3H]retinoic acid were analyzed in normal and 13-cis-retinoic acid treated rats 3 h after administration of the radiolabeled retinoid. all-trans-Retinoic acid was identified as a liver and intestinal mucosa metabolite in normal rats given physiological doses of 13-cis-[3H]retinoic acid. all-trans-Retinoyl glucuronide was identified as the most abundant radiolabeled metabolite in mucosa and a prominent liver metabolite under the same conditions. Thus, the major 13-cis-retinoic acid metabolites retained in liver and mucosa, two retinoid target tissues, had the all-trans configuration. These data indicate that the isomerization of 13-cis-retinoic acid to all-trans-retinoic acid and the subsequent conversion to all-trans-retinoyl glucuronide are central events in the in vivo metabolism of 13-cis-retinoic acid in the rat. Moreover, the all-trans-retinoic acid detected in vivo could account for a significant fraction of the physiological activity of 13-cis-retinoic acid. The tissue disposition and metabolism of orally dosed 13-cis-[3H]retinoic acid are modulated by retinoid treatment. Chronic 13-cis-retinoic acid treatment apparently increased the intestinal accumulation of all-trans-retinoic acid, all-trans-retinoyl glucuronide, and 13-cis-retinoyl glucuronide. The liver concentrations of tritiated all-trans-retinoic acid and all-trans-retinoyl glucuronide were also elevated in 13-cis-retinoic acid treated rats.     

Simultaneous analysis of retinol,all-trans- and 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in plasma by liquid chromatography using on-column concentration after single-phase fluid extraction

A reversed-phase high-performance liquid chromatographic method for the simultaneous analysis of retinol, all-trans-retinoic acid, 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in human plasma and cell culture medium is described. Sample preparation involves precipitation of proteins and extraction of retinoids with 60% acetonitrile. After centrifugation, the acetonitrile content of the supernatant is reduced to 45%, allowing on-column concentration of analytes. Injection volumes up to 2.0 ml (equivalent to 0.525 ml of sample) can be used without compromising chromatographic resolution of all-trans-retinoic acid and 13-cis-retinoic acid. Retinoids were stable in this extract and showed no isomerization when stored in the dark in a cooled autosampler, allowing automated analysis of large series of samples. Recoveries from spiked plasma samples were between 95 and 103%. Although no internal standard was used, the inter-assay precision for all retinoids was better than 6% and 4% at concentrations of 30 nM and 100 nM, respectively. The method is a valuable tool for the study of cellular metabolism of all-trans-retinoic acid, as polar metabolites of this compound can be detected with high sensitivity in cell culture media.     

All-trans-retinoic acid and 13-cis-retinoic acid: pharmacokinetics and biological activity in different cell culture models of human keratinocytes.

Despite its known biological effect on epithelial cells, 13- CIS-retinoic acid shows low binding affinity to either cellular retinoic acid-binding proteins or nuclear retinoid receptors compared to its isomer all- TRANS-retinoic acid. We have postulated a prodrug-drug relation with 13- CIS-retinoic acid which isomerizes to all- TRANS-retinoic acid. On the other hand, the biological effects of these two compounds can differ in the widely used cell culture models of HaCaT and normal primary keratinocytes. In this study, we seeded HaCaT and normal keratinocytes at high densities leading to early confluence in order to imitate high keratinocyte proliferation, such as in acne and psoriasis, while to model decreased keratinocyte proliferation, as in aged and steroid-damaged skin, cells were seeded at a low density. High performance liquid chromatography was administered to examine retinoid uptake and metabolism in monolayer HaCaT and normal keratinocyte cultures and the 4-methylumbelliferyl heptanoate assay to estimate cell growth at different cell densities. Major qualitative and quantitative differences were detected in the two cell types regarding intracellular 13- CIS-retinoic acid isomerization to all- TRANS-retinoic acid. On the other hand, the two retinoic acid isomers showed similar effects on cell growth of both cell types tested with increasing proliferation at low cell densities, but being rather inactive at high ones in normal keratinocytes and exhibiting an antiproliferative effect in HaCaT keratinocytes. The missing effect of retinoids on cell proliferation in high seeding densities of normal keratinocytes may indicate that the normalizing activity of retinoids on hyperkeratotic diseases, such as acne or psoriasis, is likely to be carried out by modulation of cell differentiation than cell growth. On the other hand, induced keratinocyte proliferation in low seeding densities may provide an explanation for the acanthosis induced by topical retinoids in aged and steroid-damaged skin.     

The in vivo incorporation of mannose, retinol and mevalonic acid into phospholipids of hamster liver

Hamsters were injected intraperitoneally with [¹⁴C]mannose, [¹⁴C]retinol and [³H]mevalonic acid. The livers were removed, extracted with chloroform-methanol and the lipids chromatographed on DEAE-cellulose and silicic acid. The hamster liver lipid contained a component which could be labelled with mannose and mevalonic acid. The properties of this compound were in accord with it being dolichyl-mannosyl-phosphate, a possible lipid intermediate required for the biosynthesis of some glycoproteins. [¹⁴C]Retinol and [¹⁴C] mannose were incorporated into another phospholipid which was labile to mild alkali conditions commonly used for the preparation of dolichyl-mannosyl-phosphate. The retinol labelled compound had similar properties to $\text{in vitro}$ prepared mannosyl-retinyl-phosphate.     

Effects of isotretinoin (13-cis-retinoic acid) on the development of mouse limbs in vivo and in vitro.

Isotretinoin (13-cis-RA) is known to be teratogenic in humans and laboratory animals. The relatively low potency of 13-cis-RA in NRMI mice in comparison to the all-trans isomer has been proposed to be due to minimal transfer across the placenta (Creech-Kraft et al., '87). To further delineate the teratogenic potential of 13-cis-RA, a dose-response, temporal study was conducted in vivo and in vitro using submerged limb culture and image analysis evaluation of development. Dose-dependent embryotoxicity was produced by treatment on GD 7, while later treatments produced inconsistent effects on resorption rate and fetal weight. Treatment on either GD 7 or GD 8 produced a number of malformations in dose-dependent manner. Most common were tail and cleft palate defects, which were produced by 13-cis-RA on each of the days tested (GD 7-GD 11), with peak malformations occurring on GD 9 and GD 10 for tail and cleft palate, respectively. Most limb defects were produced after GD 10 and GD 11 exposure. The observed frequency of defects confirmed that in ICR mice 13-cis-RA is about 10-fold less potent than all-trans-RA as a limb teratogen (Kwasigroch and Kochhar, '80; Kochhar and Penner, '87). Effects observed via image analysis following maintenance of limbs in serum-free culture medium were dose dependent. Low dose treatment produced occasional polydactyly. The intermediate dose caused somewhat variable region-dependent increases in cartilaginous bone anlagen area. The high dose of 13-cis-RA produced irregular limb outlines, a reduction in bone anlagen area, and an inhibition of alcian blue staining of cartilage without affecting morphogenesis of bone anlagen. These results confirm that, when the effects of the administered doses are evaluated, 13-cis-RA is a much less potent teratogen in comparison to the all-trans isomer. More importantly, the results show that retinoids can enhance (at low and intermediate doses), depress (at high doses), or eliminate (high dose) chondrogenenic expression during limb morphogenesis in vitro. This indicates that retinoids such as 13-cis-RA can manipulate events in development in a variety of ways (i.e., produce malformations, interfere with chondrogenic expression without affecting morphogenesis, and stimulate growth) in a dose- and time-dependent manner. Although the ability of RA to act as a true morphogen has recently been questioned (Wanek et al., '91; Noji et al., '91), the results presented here support the position that RA can modulate the development of the limb (and probably other organ systems) in several vertebrate species.     

In vivo binding of retinol to chromatin. The binding is mediated by a lipoprotein

We have previously shown that exposure of responding cells to vitamin A leads to profound modifications of chromatin structure as revealed by an increased susceptibility to DNase I digestion, modified patterns of histone acetylation, and impaired synthesis of a nonhistone chromosomal protein (Ferrari, N., and Vidali, G. (1985) Eur. J. Biochem. 151, 305-310). The present results show that these effects are most probably due to the direct interaction between retinol and chromatin, and analysis of mononucleosomes and higher oligomers obtained from retinol-treated cells shows that retinol is indeed tightly bound to chromatin. Enzymatic digestions of vitamin A containing nucleosomes with proteinase K, phospholipase C, and phospholipase A2 support a model where the final binding of retinol to chromatin is mediated by a lipoprotein: the recognition of the binding sites on DNA being dictated by the proteic component while the hydrophobic retinol is solubilized in the fatty acid moiety.     

Biosynthesis of a nonphysiological sialic acid in different rat organs, using N-propanoyl-D-hexosamines as precursors.

In this study it could be shown that in rat the normally occurring N-acetyl neuraminic acid can be modified in its N-acyl moiety by in vivo administration of the chemically synthesized N-propanoyl precursors, N-propanoyl-D-glucosamine or N-propanoyl-D-mannosamine. It could be shown that each of these nonphysiological amino sugar analogues was incorporated into both membrane and serum glycoproteins. After treatment of rats with radiolabeled N-[acyl-1-14C]D-mannosamine, radioactivity could be removed from serum glycoprotein fractions by incubation with neuraminidase from Clostridium perfringens or from Arthrobacter ureafaciens. Mild acid hydrolysis removed 98% of the radioactivity after in vivo labeling with N-[acetyl-1-14C]D-mannosamine and 86% after labeling with N-[propanoyl-1-14C]D-mannosamine. Chromatographic analysis yielded two compounds, i.e. N-acetyl neuraminic acid and N-propanoyl neuraminic acid, the latter being identified by gas liquid chromatography/mass spectrometry studies. Measurement of protein-bound radioactivity in different rat organs revealed a different organotropy of the natural and the nonphysiological neuraminic acid precursor. Of the glucosamine derivatives, N-acetyl-D-glucosamine showed the higher rate of uptake and incorporation in most organs (except in the submandibulary gland), and especially in kidney cortex and Morris hepatoma 7777. Natural and the unphysiological mannosamine derivatives were incorporated at similar rates, except in liver, where N-acetyl-D-mannosamine was taken up and metabolized more effectively. This finding indicates that it is possible to modify the acyl group of N-acetyl neuraminic acid in vivo by the introduction of an N-propanoyl group and possibly other homologous N-acyl groups. This procedure may provide a tool for a further characterization of the biological function of sialic acids.