Arun B. Barua

New simplified procedures for the extraction and simultaneous high-performance liquid chromatographic analysis of retinol, tocopherols and carotenoids in human serum.

A short, simple extraction procedure and a sensitive reversed-phase high-performance liquid chromatographic assay, which utilizes isocratic elution and detection either by a photodiode-array detector or by two detectors set at 300 and 450 nm, have been developed to measure retinol, tocopherols and several carotenoids in human serum simultaneously. By relying on characteristic UV-visible spectra, seventeen carotenoids, retinol and alpha- and gamma-tocopherols were identified in concentrated serum extracts by use of the three-dimensional data mode of a photodiode-array detector. The presence of some recently reported carotenoids in human serum has been confirmed.

Properties of retinoids: Structure, handling, and preparation

Retinoids are unstable compounds being readily oxidized and/or isomerized to altered compounds, especially in the presence of oxidants including air, light, and excessive heat. They are labile toward strong acids and solvents that have dissolved oxygen or peroxides. In this review, procedures for handling and storage of retinoids and biological samples containing them have been described. The physical and chemical properties of retinoids have been reported. Simplified procedures for derivatizations and purification, and methods for quantitation of retinoids have been presented.

Induction of HL-60 cell differentiation by water-soluble and nitrogen-containing conjugates of retinoic acid and retinol.

Retinoids induce the promyelocytic cell line, HL‐60, to differentiate along the granulocytic pathway in vitro. A number of water‐soluble and nitrogen‐containing retinoids were synthesized in our laboratory [retinoyl‐glucose (RAGL), retinyl‐glucose (ROGL), retinoyl‐adenosine (RADS), retinoyl‐adenine (RAD), retinoyl‐β‐glucuronide (βRAG), and retinoyl‐α‐glucuronide (αRAG)]. These retinoids (10–5 to 10–8 M), as well as retinoic acid (RA) and retinol (ROL), were tested for their ability to induce the differentiation of HL‐60 cells in vitro and to affect cell growth and viability during a 24‐ to 72‐h incubation period. Differentiation was assessed by measuring the percentage of cells expressing the Mac‐1 antigen on their cell surfaces. RA and the conjugates of RA were all quite active in inducing HL‐60 cell differentiation, whereas ROL and ROGL had much less activity at equimolar concentrations. βRAG, αRAG, RADS, and RAD were less toxic, whereas the glucose conjugates of retinol and retinoic acid (ROGL and RAGL) were both considerably more toxic than either RA or ROL at equimolar concentrations. All retinoids affected cell growth in a dose‐dependent fashion. At 24 h, free RA or ROL was not detected in the cells after incubation with any of the retinoid conjugates.—Janick‐Buckner, D.; Barua, A. B.; Olson, J. A. Induction of HL‐60 cell differentiation by water‐soluble and nitrogen‐containing conjugates of retinoic acid and retinol. FASEB J. 5: 320–325; 1991.

Extraction and analysis by high-performance liquid chromatography of carotenoids in human serum

Publisher Summary This chapter discusses extraction and analysis by high performance liquid chromatography (HPLC) of carotenoids in human serum. The carotenoid pattern of human plasma or serum is not as complex as in vegetables and fruits. The major carotenoids occurring in human plasma are lutein, zeaxanthin, α-cryptoxanthin, β-cryptoxanthin, lycopene, α-carotene, and β-carotene. Although the trans forms predominate, cis forms of these carotenoids often occur in blood. The chapter discusses the criteria, which are important and critical in selecting the method for analysis. Continued work on the HPLC separation of carotenoids and retinoids during the past several years has made it possible to develop an extraction procedure and an isocratic HPLC procedure that enables simultaneous determination of all the major carotenoids and of retinol, retinyl esters, and the tocopherols in very small volumes of human serum. The details of the procedure are described in the chapter.

Improved normal-phase and reversed-phase gradient high-performance liquid chromatography procedures for the analysis of retinoids and carotenoids in human serum, plant and animal tissues.

Two high-performance liquid chromatography (HPLC) procedures, a rapid normal-phase isocratic method for the analysis primarily of retinol and retinoic acid on a 3 mu silica column, and a reversed-phase gradient method for the simultaneous analysis of retinoids and very polar to nonpolar carotenoids on a 3 mu C18 column, are described. The normal-phase isocratic HPLC procedure is rapid (12 min), requires a sample size of 100 microl or less of serum, and is suitable for routine analysis of retinol in any serum, and of retinol and retinoic acid in serum after administration of retinoic acid. The reversed-phase gradient method is suitable for the simultaneous analysis of very polar to nonpolar carotenoids such as epoxy-xanthophylls and xanthophyll esters, along with other carotenoids and retinoids that occur normally in human serum and other plant and animal tissues. A run time of 30-70 min is necessary, depending on the presence or absence of xanthophyll esters in the sample.

Properties of Retinoids

Retinoids are unstable compounds being readily oxidized and/or isomerized to altered compounds, especially in the presence of oxidants including air, light, and excessive heat. They are labile toward strong acids and solvents that have dissolved oxygen or peroxides. In this review, procedures for handling and storage of retinoids and biological samples containing them have been described. The physical and chemical properties of retinoids have been reported. Simplified procedures for derivatizations and purification, and methods for quantitation of retinoids have been presented.

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.

Formation of β-glucuronides and of β-galacturonides of various retinoids catalyzed by induced and noninduced microsomal UDP-glucuronosyltransferases of rat liver

The complex of microsomal UDP-glucuronosyl transferases (UGT; EC 2.4.1.17) of rat liver catalyzes the formation of retinoyl beta-glucuronide (RAG) from retinoic acid (RA) and retinyl beta-glucuronide (ROG) from retinol (ROL) in the presence of UDP-glucuronic acid (UDPGA). The relative rates of formation of beta-glucuronides in noninduced microsomes were: 9-cis RA > 13-cis RA > all-trans 4-oxo RA > TTNPB > all-trans RA > CD-367 > 13-cis ROL > 9-cis ROL > acitretin > all-trans ROL. Michaelis constants (KM) for all-trans RA, 13-cis ROL, TTNPB and UDPGA were 130 microM, 300 microM, 210 microM and 2.6 microM, respectively. Galacturonides of RA, but not detectably of ROL, were formed from UDP-galacturonic acid at 11-30% the rate of the beta-glucuronides, whereas UDP derivatives of nonionized sugars did not serve as substrates. Pretreatment of rats with 3-methylcholanthrene markedly increased RAG formation in the absence of detergent (Triton X-100), but less so in its presence. Clofibrate pretreatment doubled the rate of RAG formation, whereas phenobarbital pretreatment showed little effect. N-Ethylmaleimide (5 microM) minimized cis-trans isomerization without significantly affecting glucuronidation. Rates of glucuronidation of retinoids clearly depend both on their isomeric states and on their chemical structures. Different UGT enzymes might well act on different geometric isomers of RA.

Effects of Retinoid β-Glucuronides and N-Retinoyl Amines on the Differentiation of HL-60 Cells in Vitro

Abstract Retinoyl β-glucuronide and retinyl β-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 > retinoyl β-glucuronide > retinyl β-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 β-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 β-glucuronide and retinamide, but to a greater degree by the other derivatives. Retinoyl β-glucuronide was hydrolyzed in part to retinoic acid, whereas retinyl β-glucuronide was cleaved to retinol, if at all, at a very slow rate. Under the selected assay conditions, retinoic acid and the retinoid β-glucuronides primarily induce the differentiation of HL-60 cells, whereas the N-retinoyl aromatic amines show cytotoxicity.

Retinoyl β-glucuronide: A nontoxic retinoid for the topical treatment of acne

The purpose of this study was to evaluate the efficacy towards acne and the skin surface toxicity of all-trans-retinoyl β-glucuronide (RBG), a naturally occurring retinoid possessing low cytotoxicity and teratogenicity in animal and cell culture studies. In a double-blind study, 15 acne patients treated with 1.2% RBG cream for 16 weeks showed median reductions of 56%, 25% and 67% in total, inflammatory, and noninflammatory lesions, respectively, whereas the placebo group (14 patients) showed median reductions of 31%, 4% and 62%, respectively. The median reduction in total lesions was highly significant in the RBG group (P = 0.002) but not in the placebo group (P = 0.06). In a second initially double-blind study, ten acne patients treated topically for 26 weeks with 0.16–0.32% RBG cream and five patients with 0.1% all-trans-retinoic acid (Retin-A) showed similar responses, i.e. median reductions in total facial lesions of 70% (P = 0.001) and 75% (P = 0.01), respectively. Whereas all subjects treated topica...