Napoli J L
J Biol Chem. 1986 Oct 15;261(29):13592-7.
Specific assays, based on gas chromatography-mass spectrometry and high-performance liquid chromatography, were used to quantify the conversion of retinol and retinal into retinoic acid by the pig kidney cell line LLC-PK1. Retinoic acid synthesis was linear for 2-4 h as well as with graded amounts of either substrate to at least 50 microM. Retinoic acid concentrations increased through 6-8 h, but decreased thereafter because of substrate depletion (t1/2 of retinol = 13 h) and product metabolism (1/2 = 2.3 h). Retinoic acid metabolism was accelerated by treating cells with 100 nM retinoic acid for 10 h (t1/2 = 1.7 h) and was inhibited by the antimycotic imidazole ketoconazole. Feedback inhibition was not indicated since retinoic acid up to 100 nM did not inhibit its own synthesis. Retinol dehydrogenation was rate-limiting. The reduction and dehydrogenation of retinal were 4-8-fold and 30-60-fold faster, respectively. Greater than 95% of retinol was converted into metabolites other than retinoic acid, whereas the major metabolite of retinal was retinoic acid. The synthetic retinoid 13-cis-N-ethylretinamide inhibited retinoic acid synthesis, but 4-hydroxylphenylretinamide did not. 4'-(9-Acridinylamino)methanesulfon-m-anisidide, an inhibitor of aldehyde oxidase, and ethanol did not inhibit retinoic acid synthesis. 4-Methylpyrazole was a weak inhibitor: disulfiram was a potent inhibitor. These data indicate that retinol dehydrogenase is a sulfhydryl group-dependent enzyme, distinct from ethanol dehydrogenase. Homogenates of LLC-PK1 cells converted retinol into retinoic acid and retinyl palmitate and hydrolyzed retinyl palmitate. This report suggests that substrate availability, relative to enzyme activity/amount, is a primary determinant of the rate of retinoic acid synthesis, identifies inhibitors of retinoic acid synthesis, and places retinoic acid synthesis into perspective with several other known pathways of retinoid metabolism.
采用基于气相色谱 - 质谱联用和高效液相色谱的特定分析方法，对猪肾细胞系LLC - PK1中视黄醇和视黄醛向视黄酸的转化进行定量分析。视黄酸合成在2 - 4小时内呈线性，并且对于两种底物的分级量而言，至少在50微摩尔时也是线性的。视黄酸浓度在6 - 8小时内增加，但此后由于底物耗尽（视黄醇的半衰期 = 13小时）和产物代谢（半衰期 = 2.3小时）而下降。用100纳摩尔视黄酸处理细胞10小时可加速视黄酸代谢（半衰期 = 1.7小时），并且抗真菌药咪唑酮康唑可抑制视黄酸代谢。由于高达100纳摩尔的视黄酸并未抑制其自身合成，因此未显示出反馈抑制作用。视黄醇脱氢是限速步骤。视黄醛的还原和脱氢分别快4 - 8倍和30 - 60倍。超过95%的视黄醇转化为视黄酸以外的代谢产物，而视黄醛的主要代谢产物是视黄酸。合成类视黄醇13 - 顺式 - N - 乙基视黄酰胺抑制视黄酸合成，但4 - 羟基苯基视黄酰胺则不然。醛氧化酶抑制剂4' - (9 - 吖啶基氨基)甲磺酰基 - 间 - 茴香胺和乙醇不抑制视黄酸合成。4 - 甲基吡唑是一种弱抑制剂：双硫仑是一种强效抑制剂。这些数据表明，视黄醇脱氢酶是一种依赖巯基的酶，与乙醇脱氢酶不同。LLC - PK1细胞匀浆将视黄醇转化为视黄酸和视黄醇棕榈酸酯，并水解视黄醇棕榈酸酯。本报告表明，相对于酶活性/量的底物可用性是视黄酸合成速率的主要决定因素，确定了视黄酸合成的抑制剂，并将视黄酸合成与其他几种已知的类视黄醇代谢途径联系起来。
