Kishimoto M, Kajimoto Y, Kubota M, Watarai T, Shiba Y, Kawamori R, Inaba T, Kamada T, Yamasaki Y
First Department of Medicine, Osaka University, School of Medicine, Japan.
J Chromatogr B Biomed Sci Appl. 1997 Jan 10;688(1):1-10. doi: 10.1016/s0378-4347(97)88050-4.
One of the major mechanisms involved in diabetic microangiopathy is considered to be an altered polyol pathway. However, clarifying the pathophysiology is difficult due to the lack of a sensitive method for measuring the reduction of glucose to sorbitol in tissue. Here we report a sensitive and selective method for polyol measurement using trifluoroacetyl (TFA) derivatives of polyols and stable isotope-labeled D-sorbitol (U-[13C]sorbitol, 13C6H14O6, 98.7%) as an internal standard. Gas chromatography-mass spectrometry (GC-MS) using an SE-30 capillary column gave elution of TFA derivatives of sugars, polyols and U-[13C]sorbitol within 8 min, with clear separation of sorbitol. In the calibration study, the coefficients of correlation between the amount of sorbitol added and that determined in standard solutions containing 0.1-8.0 nmol sorbitol, erythrocyte mixture and liver cytosol mixture were r = 0.999, r = 0.997 and r = 0.997, respectively. The precision of the GC-MS measurement of standard solution was C.V. = 4.3%. Because glucose is used as a substrate, the method can clarify the polyol pathway under physiological conditions. With this method, Km and Vmax values of the reductase in erythrocytes were 115 +/- 19 mmol/l and 4.42 +/- nmol/min/g of hemoglobin. In human liver, on the other hand, they were 755 +/- 132 mmol/l and 0.773 +/- 0.090 nmol/min/mg of protein, respectively. This difference of Km values suggested that aldehyde reductase rather than aldose reductase is mainly responsible for reducing glucose to sorbitol in the liver. In conclusion, this newly developed method offers a highly sensitive and selective procedure for measuring low concentrations of sorbitol in various tissues and cells and should enable clarification of the kinetics of glucose reduction to sorbitol, which in turn can be used to evaluate the role of an altered polyol pathway in the pathophysiology of diabetic microangiopathy.
糖尿病微血管病变涉及的主要机制之一被认为是多元醇途径改变。然而，由于缺乏一种灵敏的方法来测量组织中葡萄糖向山梨醇的还原，阐明其病理生理学较为困难。在此，我们报告一种使用多元醇的三氟乙酰（TFA）衍生物和稳定同位素标记的D-山梨醇（U-[13C]山梨醇，13C6H14O6，98.7%）作为内标的灵敏且选择性的多元醇测量方法。使用SE-30毛细管柱的气相色谱-质谱联用（GC-MS）在8分钟内实现了糖、多元醇和U-[13C]山梨醇的TFA衍生物的洗脱，山梨醇分离清晰。在校准研究中，添加的山梨醇量与在含有0.1 - 8.0 nmol山梨醇的标准溶液、红细胞混合物和肝细胞溶胶混合物中测定的山梨醇量之间的相关系数分别为r = 0.999、r = 0.997和r = 0.997。GC-MS对标准溶液测量的精密度为变异系数（C.V.）= 4.3%。由于以葡萄糖为底物，该方法能够阐明生理条件下的多元醇途径。使用此方法，红细胞中还原酶的Km和Vmax值分别为115 ± 19 mmol/l和4.42 ± nmol/min/g血红蛋白。另一方面，在人肝脏中，它们分别为755 ± 132 mmol/l和0.773 ± 0.090 nmol/min/mg蛋白质。Km值的这种差异表明，在肝脏中，将葡萄糖还原为山梨醇的主要是醛糖还原酶而非醛还原酶。总之，这种新开发的方法为测量各种组织和细胞中低浓度山梨醇提供了一种高度灵敏且选择性的程序，应该能够阐明葡萄糖还原为山梨醇的动力学，进而可用于评估改变的多元醇途径在糖尿病微血管病变病理生理学中的作用。
