Jörn Oliver Sass

Quantitation of total homocysteine in human plasma by derivatization to its N(O,S)-propoxycarbonyl propyl ester and gas chromatography-mass spectrometry analysis

Much evidence supports the hypothesis that mild or moderate hyperhomocysteinaemia represents an important and independent risk factor for occlusive vascular diseases. Therefore, the accurate and reliable determination of total plasma homocysteine has gained major importance for risk assessment. Furthermore, it can help in the detection of folate and vitamin B12 deficiency. This has prompted us to develop a sensitive gas chromatography-mass spectrometry (GC-MS) method in order to quantify total homocysteine in human plasma. Prior to chromatography, reduced homocysteine was released from disulfide bonds by incubation with excess dithiothreitol and converted into its N(O,S)-propoxycarbonyl propyl ester by derivatization with n-propyl chloroformate. Aminoethylcysteine served as internal standard. The method proved to be highly linear over the entire concentration range examined (corresponding to 0-266 microM homocysteine) and showed intra-assay and inter-assay variation (relative standard deviations) of approximately 5 and 5-10%, respectively. External quality control by comparison with duplicate analysis performed on a HPLC-based system revealed satisfactory correlation. The newly developed GC-MS based method provides simple, reliable and fast quantification of total homocysteine and requires only inexpensive chemicals, which are easy to obtain.

Glucuronidation and isomerization of all-trans- and 13-CIS-retinoic acid by liver microsomes of phenobarbital- or 3-methylcholanthrene-treated rats

Glucuronidation and isomerization of all-trans-retinoic acid (tr-RA) and 13-cis-retinoic acid (13-cis-RA) were investigated in an in vitro system using liver microsomes of differently pretreated rats. In agreement with their thermodynamic stability, more retinoic acid was isomerized from the 13-cis form to the all-trans form than vice versa. Also some 9-cis-retinoic acid (9-cis-RA) could be found. Isomerization was reduced, but in contrast to glucuronidation was still important if boiled microsomes were used. This supports the view that isomerization can proceed as a non-enzymatic process. 3-Methylcholanthrene (MC) pretreatment of the rats increased the microsomal glucuronidation of 13-cis-RA and tr-RA and the formation of 13-cis-retinoyl-beta-glucuronide was enhanced up to 7-fold by MC-induced rat microsomes. The rates of glucuronidation by uninduced and phenobarbital-induced rat microsomes differed only slightly. In addition to glucuronides of the applied retinoic acid isomers (13-cis-RA and tr-RA), 9-cis-RA and its glucuronide were found. Induction of retinoid glucuronidation by pretreatment with MC indicates that this metabolic reaction is catalysed by a MC-inducible UGT isozyme. After two recently described pathways (conversions of retinol to retinal and of retinyl methyl ether to retinol) this is a third step of retinoid metabolism, induced by pretreatment with MC. With human microsomes no more than traces of glucuronides were detected; also, incubations with human microsomes resulted in a lower degree of isomerization than with rat microsomal fractions.

Single-run analysis of isomers of retinoyl-β-D-glucuronide and retinoic acid by reversed-phase high-performance liquid chromatography

Reversed-phase HPLC methods capable of separating several retinoic acid isomers are generally not designed for simultaneous analysis of isomers of other classes of retinoids. A reversed-phase HPLC method is presented which allows the separation of at least four retinoic acid (RA) isomers (13-cis-RA, 9,13-dicis-RA, 9-cis-RA, all-trans-RA)and of all isomers of retinoyl-beta-D-glucuronide (RAG), which have been observed in vivo (13-cis-RAG, 9-cis-RAG,all-trans-RAG). The recovery of retinoids was generally between 80 and 90%. Intra-day reproducibility (expressed as relative standard deviation) was less than or equal to 7%. As little as 0.25 ng of RA isomers and of all-trans-RAG could be detected. This method allowed the study of the metabolism of 9-cis-retinoids, where isomerization reactions play a predominant role.

9-Cis-retinoyl-β-glucuronide is a major metabolite of 9-Cis-retinoic acid

Abstract The in vivo metabolism of 9-cis-retinoic acid (9-c-RA), an endogenous ligand of retinoid X receptors (RXRs), which can also bind to retinoic acid receptors (RARs), was examined in pregnant mice and rats following a single oral dose of 100 mg 9-cis- retinaldehyde (9-c-RAL) / kg body weight. 9-Cis-retinoyl-β-glucuronide (9-c-RAG), a metabolite not found in vivo before, was a major metabolite of 9-c-RA in mouse plasma and was also present in all mouse tissues examined as well as in rat plasma. In both species putative oxidation products of retinoic acids and high levels of retinyl esters were found. Concentrations of retinoic acid isomers and retinoyl-β-D-glucuronides in the mouse plasma greatly exceeded those of the rat plasma. The finding of high levels of 9-c-RAG underlines the importance of glucuronidation in the metabolism of retinoids.

Comparative studies on effects of all-trans-retinoic acid and all-trans-retinoyl-β-d-glucuronide on the development of foetal mouse thymus in an organ culture system

The thymus is a target organ of retinoid teratogens. Retinoids with a generally reduced teratogenic potency should therefore also exert reduced adverse effects on thymus development. The effects of all-trans-retinoic acid (a-tRA) and all-trans-retinoyl-beta-glucuronide (a-tRAG) on the in vitro development of thymic lobes of 15-day-old mouse foetuses were compared in an organ culture system. Both compounds were added to the medium at concentrations ranging from 10(-7) to 10(-5)m. The culture period was 6 days. The investigations showed a concentration-dependent effect of both substances on the proliferation of the lymphatic cells. At 10(-5)m the number of thymocytes was significantly reduced to values of about 70% of the controls by either of the retinoids (P 0.05). Results of flow cytometry showed significant differences concerning the differentiation markers CD4 and CD8 after the culture period. The presence of 10(-6)m a-tRA induced a significant increase in the percentages of CD4(+)CD8(-) cells and a significant decrease of CD4(+)CD8(+) cells. At 10(-5)m a-tRA an additional significant increase in the percentages of CD4(-)CD8(-) cells was found. In contrast, after treatment with a-tRAG, percentages of these populations were in the same range as the controls. Light and electron microscopic investigations revealed a depletion of lymphatic cells and an increase of intracytoplasmic vacuoles in the thymic epithelial cells at 10(-6) and 10(-5)m of either retinoid. HPLC analyses revealed a remarkable degree of retinoid isomerization and (in the case of a-tRAG) of hydrolysis. Compared with the culture medium, retinoids were accumulated in the thymic lobes. Possibly a-tRAG acts by way of limited hydrolysis to retinoic acid.

Biological effects and metabolism of 9-cis-retinoic acid and its metabolite 9,13-di-cis-retinoic acid in HaCaT keratinocytes in vitro: comparison with all-trans-retinoic acid

Abstract 9- cis -Retinoic acid (9cRA), a geometric isomer of all- trans -retinoic acid (atRA), is an endogenous high-affinity ligand for retinoid X receptors and retinoic acid receptors activating them with high potency. 9,13-di- cis -Retinoic acid (9,13dcRA) has been described as a major plasma metabolite of 9cRA. In this study, the biological activity and the metabolism of 9cRA and 9,13dcRA were investigated and compared with those of atRA in a retinol-free culture system of HaCaT keratinocytes. 9cRA exhibited a slightly weaker activity overall than atRA in inhibiting cell proliferation, inducing cellular retinoic acid binding protein II (CRABP II) mRNA levels and upregulating cytokeratin 19 expression. 9,13dcRA regulated HaCaT keratinocyte activity only at the highest concentration tested (10 –6 M ). In cultures of HaCaT keratinocytes with atRA and 9cRA, rapid intracellular accumulation of atRA was observed within 2 h, and atRA levels were higher with atRA treatment than with 9cRA treatment. 9,13dcRA remained relatively stable in the medium with intracellular 9,13dcRA levels below the level of detection. Taken together, 9cRA seems to be slightly less potent than atRA in regulating the biological activity of HaCaT keratinocytes, while its metabolite 9,13dcRA is effectively inactive at biologically relevant concentrations. Our data suggest a prodrug/drug relationship between 9cRA and atRA in human keratinocytes. 9,13dcRA seems to be a weaker prodrug of atRA or an inactive metabolic derivative.

Plasma retinoids after topical use of retinaldehyde on human skin.

BACKGROUND Retinaldehyde (RAL), a natural metabolite of beta-carotene and retinol (ROL), is tolerated by human skin after topical application. PURPOSE To see if topical application of a large quantity of RAL on human skin is associated with a detectable alteration of constitutive levels of plasma retinoids resulting from metabolism of RAL in the skin. METHODS Plasma retinoids [ROL, all-trans-retinoic acid (all-trans-RA), RAL, retinyl palmitate/oleate, 13-cis-RA and 4-oxo-13-cis-RA] were analyzed by high-pressure liquid chromatography. Determinations were done in 10 healthy male volunteers kept on a vitamin-A-poor diet before, during and after daily topical application of 7 mg of RAL to 40% of the body surface for 14 days. RESULTS The introduction of a restricted vitamin A diet before RAL application resulted in a decrease in the plasma levels of ROL, all-trans-RA and retinyl palmitate/oleate. Topical application of RAL did not induce an alteration of the plasma levels of retinoid metabolites. No RAL was detectable in any of the plasma samples. CONCLUSION The skin metabolism of topically applied RAL does not result in detectable alterations of constitutive levels of plasma retinoids in humans.

Transplacental pharmacokinetics of a synthetic retinoid which is not bound by mouse embryonic cellular retinoic acid-binding protein

Teratogenicity is a major side effect of retinoids, a class of compounds used in dermatology and oncology. The binding of retinoids to cellular retinoic acid-binding protein (CRABP) has been suggested to be important for the mechanism of retinoid embryopathy. Here data are presented on the transplacental pharmacokinetics of CD394 (4-[3-(1-adamantyl)-4-methoxybenzamido] benzoic acid) which does not bind to murine embryonic CRABP, although it is active in rat whole embryo culture and teratogenic in the rabbit in vivo. A single intragastric dose of CD394 (10 mg/kg) was administered to mice on day 11 of gestation. The extent of placental transfer of CD394, determined by HPLC, resembled more that of 13-cis-retinoic acid which also does not bind to CRABP, than that of the CRABP-binding all-trans-retinoic acid. CMax values of CD394 obtained after 1-2 h were: 1368 +/- 652 ng/ml for plasma, 203 +/- 132 ng/g for embryo and 856 +/- 563 ng/g for placenta. AUC (area-under-the-concentration-time-curve) values (0-12 h) were: 4319 ng x h/ml for plasma, 751 ng x h/g for embryo and 3163 ng x h/g for placenta. Thus, CD394 reached the embryo, although embryonic AUC values were less than one fifth of the maternal plasma AUC values. CD394 did not alter endogenous retinol concentrations in plasma, embryo, yolk sac or placenta. Our results indicate that CD394 reaches the embryo in vivo without binding to CRABP, although embryonic concentrations stayed well below plasma levels. This supports the opinion that binding to embryonic CRABP is not a prerequisite for reaching effective embryo concentrations and for the teratogenicity of retinoids.

Effects of all-trans-retinoyl-β-d-glucuronide and all-trans-retinoic acid on chondrogenesis and retinoid metabolism in mouse limb bud mesenchymal cells in vitro

Abstract Retinoids, derivatives of vitamin A, are essential for many vertebrate functions. Furthermore, several drugs of this class of compounds are valuable in the treatment of certain forms of skin disorders and cancer. However, the therapeutic application of retinoids is limited by their teratogenic potency. The limbs are important sites of retinoid-induced malformations in rodents. Therefore, organoid cultures of limb bud mesenchymal cells have been established for screening of the teratogenic potency of retinoids. We have now applied this system to compare the effects of all-trans-retinoyl-β-d-glucuronide (all-trans-RAG) with those of all-trans-retinoic acid (all-trans-RA) on chondrogenesis, as assessed by the Alcian blue binding assay and by electron microscopic evaluation including quantitative morphometric analysis. First data of retinoid toxicokinetics in the culture media as well as retinoid concentrations in the cultured mesenchymal limb bud cells were established. While all-trans-RA inhibited chondrogenesis at 10−7 M by ca. 50%, tenfold higher concentrations of all-trans-RAG were necessary to obtain the same effect. This difference reflects the ratio of RA isomers which were found in the medium after incubation with either all-trans-RAG or all-trans-RA. A pulse experiment (10−5 M all-trans-RAG or all-trans-RA for the first 2 h of a 6-day incubation period) demonstrated inhibition of chondrogenesis with all-trans-RA, but not with all-trans-RAG. The data indicate that RAG inhibits chondrogenesis upon hydrolysis to RA. Surprisingly, the rather polar RAG isoforms were extensively accumulated in the limb bud mesenchymal cells when compared to the medium. Both all-trans-RAG and all-trans-RA also induced a large increase of retinyl ester concentrations in the chondrocytes compared to vehicle-treated cells. This finding further supports a recent suggestion that RA regulates retinol metabolism via feedback inhibition of retinol oxidation and stimulation of the esterification of retinol.

Effects of all-trans-retinoic acid and all-trans-retinoyl glucuronide in two in vitro systems of distinct biological complexity.

Abstract. In vitro systems are widely used to evaluate the embryotoxic potential of retinoids. The effective concentrations of these retinoids, however, are not consistent in the various in vitro systems used in evaluating embryotoxicity. This may be explained by the different level of complexity for each individual system, which may lead to different concentrations of the substances in the target tissues. To verify this hypothesis we have compared two in vitro systems of distinct biological complexity: the rat whole embryo culture system, and the mouse limb bud organ culture system. The lipid soluble, teratogenic retinoid all-trans-retinoic acid (ATRA), and all-trans-retinoyl-β-D-glucuronide (ATRAG), an endogenous, water-soluble and biologically active retinoid with limited placental transfer, were compared with regard to their embryotoxic potential in vitro. In both in vitro systems, ATRAG showed a lower degree of embryotoxicity than ATRA. In the limb bud organ culture, ATRAG revealed only slightly less toxicity than ATRA, whereas the effective concentrations of the two compounds in the whole embryo culture system differed by almost two orders of magnitude. During incubation with ATRAG, ATRA is generated by hydrolysis and is found in culture media and exposed tissues. The presence of membrane barriers around the developing embryo in the whole embryo culture system possibly prevents the transfer of ATRAG to the embryo and, therefore, its exposure to the active hydrolysis product ATRA. From these results we conclude that analysis of retinoid concentrations in the culture media and in the exposed tissues is essential for the interpretation of results obtained from in vitro toxicity testing.