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 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.
Kishimoto
M
M
First Department of Medicine, Osaka University, School of Medicine, Japan.
Kajimoto
Y
Y
Kubota
M
M
Watarai
T
T
Shiba
Y
Y
Kawamori
R
R
Inaba
T
T
Kamada
T
T
Yamasaki
Y
Y
D000293:Adolescent; D000328:Adult; D000449:Aldehyde Reductase / Q000097:blood / Q000378:metabolism*; D001665:Binding Sites; D002138:Calibration; D002247:Carbon Isotopes; D004912:Erythrocytes / Q000201:enzymology / Q000378:metabolism*; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D005947:Glucose / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D012680:Sensitivity and Specificity; D013012:Sorbitol / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D013997:Time Factors; D014269:Trifluoroacetic Acid / Q000737:chemistry*
D002247:Carbon Isotopes; D013012:Sorbitol; D014269:Trifluoroacetic Acid; D000449:Aldehyde Reductase; D005947:Glucose