SENSITIVE, SELECTIVE GAS-CHROMATOGRAPHIC MASS-SPECTROMETRIC ANALYSIS WITH TRIFLUOROACETYL DERIVATIVES AND A STABLE-ISOTOPE FOR STUDYING TISSUE SORBITOL-PRODUCING ACTIVITY

One of the major mechanisms involved in diabetic microangiopathy is co nsidered to be an altered polyol pathway. However, clarifying the path ophysiology is difficult due to the lack of a sensitive method for mea suring the reduction of glucose to sorbitol in tissue. Here we report a sensitive and selective method for polyol measurement using trifluor oacetyl (TFA) derivatives of polyols and stable isotope-labeled D-sorb itol (U-[C-13]sorbitol, (C6H14O6)-C-13, 98.7%) as an internal standard . Gas chromatography-mass spectrometry (GC-MS) using an SE-30 capillar y column gave elution of TFA derivatives of sugars, polyols and U-[C-1 3]sorbitol within 8 min, with clear separation of sorbitol. In the cal ibration 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 wer e 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 p hysiological conditions. With this method, K-m and V-max values of the reductase in erythrocytes were 115+/-19 mmol/l and 4.42+/-0.26 nmol/m in/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. Th is difference of K-m values suggested that aldehyde reductase rather t han aldose reductase is mainly responsible for reducing glucose to sor bitol in the liver. In conclusion, this newly developed method offers a highly sensitive and selective procedure for measuring low concentra tions of sorbitol in various tissues and cells and should enable clari fication of the kinetics of glucose reduction to sorbitol, which in tu rn can be used to evaluate the role of an altered polyol pathway in th e pathophysiology of diabetic microangiopathy.