HPLC/UV quantitation of retinal, retinol, and retinyl esters in serum and tissues

We report robust HPLC/UV methods for quantifying retinyl esters (RE), retinol (ROL), and retinal (RAL) applicable to diverse biological samples with lower limits of detection of 0.7, 0.2, and 0.2 pmol, respectively, and linear ranges greater than 3 orders of magnitude. These assays function well with small, complex biological samples (10-20 mg tissue). Coefficients of variation range from 5.9 to 10.0% (intraday) and from 5.9 to 11.0% (interday). Quantification of endogenous RE, ROL, and RAL in mouse serum and tissues (liver, kidney, adipose, muscle, spleen, testis, skin, brain, and brain regions) reveals utility. Ability to discriminate spatial concentrations of ROL and RE is illustrated with C57BL/6 mouse brain loci (hippocampus, cortex, olfactory bulb, thalamus, cerebellum, and striatum). We also developed a method to distinguish isomeric forms of ROL to investigate precursors of retinoic acid. The ROL isomer assay has limits of detection between 3.5 and 4.5 pmol and has a linear range and coefficient of variation similar to those of the ROL/RE and RAL assays. The assays described here provide for sensitive and rigorous quantification of endogenous RE, ROL, and RAL to elucidate retinoid homeostasis in disease states such as Alzheimer’s disease, type 2 diabetes, obesity, and cancer.     

Role of nuclear receptors in breast cancer stem cells

The recapitulation of primary tumour heterogenity and the existence of a minor sub-population of cancer cells,capable of initiating tumour growth in xenografts on serial passages, led to the hypothesis that cancer stem cells(CSCs) exist. CSCs are present in many tumours, among which is breast cancer. Breast CSCs(BCSCs) are likely to sustain the growth of the primary tumour mass, as wellas to be responsible for disease relapse and metastatic spreading. Consequently, BCSCs represent the most significant target for new drugs in breast cancer therapy. Both the hypoxic condition in BCSCs biology and proinflammatory cytokine network has gained increasing importance in the recent past. Breast stromal cells are crucial components of the tumours milieu and are a major source of inflammatory mediators. Recently, the antiinflammatory role of some nuclear receptors ligands has emerged in several diseases, including breast cancer. Therefore, the use of nuclear receptors ligands may be a valid strategy to inhibit BCSCs viability and consequently breast cancer growth and disease relapse.     

RXR isoforms and endogenous retinoids in the fiddler crab, Uca pugilator

The pleiotropic effects of circulating ecdysteroids in the adult fiddler crab, Uca pugilator, during molting, regeneration, and reproduction are mediated by a limited number of receptor proteins. We hypothesize that hormonal effects in vivo may be the result of complex interactions between at least two receptor heterodimer conformations that differentially respond to multiple ecdysteroid/retinoid signals. Two splicing variants of the fiddler crab retinoid-X-receptor (UpRXR) differ from one another by the addition of a 33 amino acid insert in the ligand-binding domain. We show here that the ecdysteroid receptor in the fiddler crab (UpEcR) behaves differently depending upon the UpRXR isoform with which it is partnered. The two UpRXR variant partners for UpEcR confer slightly different responses in the binding of Ponasterone A (PA)—a naturally occurring ecdysteroid in the blood of Uca. UpRXR can bind 9-cis retinoic acid (9cRA) as well as terpenoids. 9cRA and the naturally occurring terpenoid, methyl farnesoate, influence the binding of PA to UpEcR/UpRXR dimers. Endogenous retinoids are found in the blastema of regenerating limbs of Uca and they (plus blood-borne terpenoids) may add additional levels of differential response by target tissues. Thus, the two sets of heterodimers tested here may represent different dynamic complexes whose properties are defined by the specific heterodimeric subunits involved and the specific ligands available.     

Structure-activity relationship of retinoids on the differentiation of cultured chick foot skin

Summary The influence of retinoids (vitamin A and its analogues) on epithelial differentiation was examined in explants of foot skin from chick embryos. In the presence of retinoic acid (10 M) keratinization and differentiation of scale-like structures, which occurred in tarsometatarsal skin explants, was inhibited and a mucous metaplasia developed. Retinoic acid caused club-shaped feathers in skin explants taken from the anterior surface of the tibia — skin which was determined to differentiate into feathers. In skin explants taken from a breed with feathered feet, the differentiation of tarsometatarsal skin was completely blocked; in tibial skin, club-shaped feathers resulted in response to retinoic acid. These findings indicated that skin of the two origins reacted differently to the retinoid, as was noted in the breed with scaly feet. The structure-activity relationship of 22 retinoids with marked differences in their biological activity was investigated in tarsometatarsal skin explants. Comparing the concentration of various retinoids needed to completely inhibit the differentiation of scale-like structures, retinoids containing tetramethylated indane or tetraline were 100 and 1,000 times more active than retinoic acid. Retinoids with a sulphur-containing end group were also active but less so than the corresponding compound with a carboxylic acid end group. The inactive ethyl, ester analogue, etretinate, was activated in the presence of esterase, indicating that the free carboxylic acid group was important for the activity of retinoids. The retinoid-induced inhibition of keratinization followed by mucous metaplasia in cultured chick embryo skin is a simple and useful model system to test new retinoids which may be helpful in the treatment of dermatological and oncological diseases.     

ι-Crystallin and vitamin A2 isomers in lenses of diurnal geckos

The ocular lenses of several genera of strictly diurnal dwarf geckos contain the monomeric ι-crystallin, which is closely related to the cellular retinol-binding protein type I (CRBP I). The contents of ι-crystallin vary between 2 and 12% of the total amount of crystallins depending on species. The endogenous ligand of ι-crystallin of all species investigated so far turns out to be 3,4-didehydroretinol (vitamin A₂). No other lenticular retinoids were detected. In lenses of Old World species (Lygodactylus, Pristurus, Quedenfeldtia), this ligand occurs exclusively in the all-trans form. In lenses of species of the neotropical genus Gonatodes, however, it occurs in two isomeric forms: all-trans and 11-cis. ι-Crystallin of Gonatodes is the first CRBP-like protein which naturally binds an 11-cis isomer of vitamin A. All-trans 3,4-didehydroretinol and its ester are present in eye cups of Lygodactylus. In contrast, eye cups of nocturnal geckos without ι-crystallin lack these retinoids. The retinal pigment epithelium is suggested to be the site of conversion of retinol to 3,4-didehydroretinol, which finally serves as ligand of ι-crystallin. Accepted: 13 March 1999     

13-Demethyl-13-substituted-13,14-dihydroretinols as potential affinity labels of retinol-binding proteins: syntheses and stability studies

13-Demethyl-13-substituted-13,14-dihydroretinols were synthesized and their stability under various conditions was measured in order to evaluate whether they would be useful as affinity labels of retinol binding proteins and retinol metabolizing enzymes. The 13-chloro analog could not be isolated because it eliminated HCl under the Wittig reaction conditions of its preparation. The trans- and cis-13,14-epoxy analogs are stable in non-protic organic solvents, but undergo an elimination reaction under various chromatographic conditions and in mixtures of organic solvents with water or alcohol. The 13-hydroxy and 13-methoxy analogs are stable in aqueous solutions and are therefore suitable for biological studies.     

Chromatographic analysis of endogenous retinoids in tissues and serum

We present a reliable, highly sensitive, and versatile method for the simultaneous determination of endogenous polar (acidic) and apolar (retinol, retinal, and retinyl esters) retinoids in various biological matrices. Following a single liquid extraction of retinoids from tissues or plasma with isopropanol, polar retinoids are separated from apolar retinoids and neutral lipids via automated solid-phase extraction using an aminopropyl phase. After vacuum concentration to dryness and reconstitution of the residue in appropriate solvents, the obtained fractions are injected onto two different high-performance liquid chromatography (HPLC)-systems. Polar retinoids are analyzed on a RP18 column (2.1mm ID) using a buffered gradient composed of methanol and water and on-column-focusing large-volume injection. Apolar retinoids are separated on a normal-bore RP18 column using a nonaqueous gradient composed of acetonitrile, chloroform, and methanol. Both HPLC systems are coupled with UV detection, and retinoids are quantitated against appropriate internal standards. The method was validated with regard to recovery, precision, robustness, selectivity, and analyte stability. Using 400 microl serum or 200mg tissue, the limits of detection for all-trans-retinoic acid were 0.15ng/ml or 0.3ng/g, respectively. The corresponding values for retinol were 1.2ng/ml or 2.4ng/g, respectively. This method was successfully applied to mouse, rat, and human tissue and serum samples.     

Hedgehog and retinoid signalling confines nkx2.2b expression to the lateral floor plate of the zebrafish trunk

The ventral neural tube of vertebrates consists of distinct neural progenitor domains positioned along the dorsoventral (DV) axis that develop different types of moto- and interneurons. Several signalling molecules, most notably Sonic Hedgehog (Shh), retinoic acid (RA) and fibroblast growth factor (FGF) have been implicated in the generation of these domains. Shh is secreted from the floor plate, the ventral most neural tube structure that consists of the medial (MFP) and the lateral floor plate (LFP). While the MFP is well characterized, organization and function of the LFP remains unclear. Here, we describe the novel homeobox gene nkx2.2b that is strongly expressed in the trunk LFP of zebrafish and thus represents a unique marker for the characterization of LFP formation and the identification of LFP deficient mutants. nkx2.2b and its paralog nkx2.2a (formerly known as nk2.2 and nkx2.2) arose by gene duplication in zebrafish. Both duplicates show significant differences in their expression patterns. For example, while prominent nkx2.2a expression has been described in the ventral brain [Barth, K.A., Wilson, S.W., 1995. Expression of zebrafish nk2.2 is influenced by sonic hedgehog/vertebrate hedgehog-1 and demarcates a zone of neuronal differentiation in the embryonic forebrain. Development 121, 1755-1768], hardly any expression can be found in the trunk LFP, which is in contrast to nkx2.2b. Overexpression, mutant and inhibitor analyses show that nkx2.2b expression in the LFP is up-regulated by Shh, but repressed by retinoids and ectopic islet-1 (isl1) expression. In contrast to previously described zebrafish trunk LFP markers, like e.g. tal2 or foxa2, nkx2.2b is exclusively expressed in the LFP. Thus, it represents a unique tool to analyse the mechanisms of ventral neural tube patterning in zebrafish.     

Synthesis of 11C-labeled retinoic acid, [11C]ATRA,via an alkenylboron precursor by Pd(0)-mediated rapid C-[11C]methylation

Retinoids are a class of chemical compounds which include both natural dietary vitamin A (retinol) metabolites and active synthetic analogs. Both experimental and clinical studies have revealed that retinoids regulate a wide variety of essential biological processes. In this study, we synthesized ¹¹C-labeled all-trans-retinoic acid (ATRA), the most potent biologically active metabolite of retinol and used in the treatment of acute promyelocytic leukemia. The synthesis of ¹¹C-labeled ATRA was accomplished by a combination of rapid Pd(0)-mediated C-[¹¹C]methylation of the corresponding pinacol borate precursor prepared by 8 steps and hydrolysis. [¹¹C]ATRA will prove useful as a PET imaging agent, particularly for elucidating the improved therapeutic activity of ATRA (natural retinoid) for acute promyelocytic leukemia by comparing with the corresponding PET probe [¹¹C]Tamibarotene (artificial retinoid).