Determination of D-sorbitol in human erythrocytes by an enzymatic fluorometric method with an improved deproteinization procedure

Abstract

SUMMARY.We developed an improved enzymatic assay of D-sorbitol in human erythrocytes by employing highly speci®c D-sorbitol dehydrogenase from Pseudomonas sp. (EC 1.1.1.14) and replacing perchloric acid (HClO 4 ) and potassium carbonate (K 2 CO 3 ), generally used for deproteinization, with sodium hydroxide (NaOH) and zinc sulphate (ZnSO 4 ). In this assay, erythrocytes were separated from plasma by centrifugation and washed once with physiological saline. Subsequently, the erythrocytes were lysed with distilled water and proteins precipitated with NaOH and ZnSO 4 . After centrifugation, the resulting colourless supernatant was mixed with a glycine buffer (pH 9´0) containing NAD + and D-sorbitol dehydrogenase. After incubation for 30 min at 37°C, the NADH produced was measured uorimetrically. The¯uorescence intensities were corrected for sample blanks, and the values of D-sorbitol were normalized for haemoglobin content.The method had an analytical range of 1±180 mmol/L. The intra-and inter-assay precisions were < 3´3% and < 5´8%, respectively. The detection limit was 0´65 mmol/ L. In terms of the linearity, precision and sensitivity, the improved method using NaOH and ZnSO 4 was superior to the conventional method using HClO 4 and K 2 CO 3 .

Quantitative determination of endogenous sorbitol and fructose in human erythrocytes by atmospheric-pressure chemical ionization LC tandem mass spectrometry

“…The concentrations of unlabeled sorbitol and fructose in normal human red blood cells were shown in Table 9, which were the same as reported [33][34][35], indicating that the method is reliable and accurate. This validated assay has been successful in analyzing the clinical human red blood cell samples.…”

“…Analysis of monosaccharides by liquid chromatography is complicated by the fact that these compounds do not possess a UV chromophore; consequently they are usually analyzed by derivatization before detection with UV [1,2]. Other detectors are also used to analyze neutral carbohydrates, such as evaporative light-scattering detector [3], refractive-index, [4][5][6] and pulsed-amperometric detectors [7][8][9][10][11] and fluorometric detector [12]. Gas chromatography/mass spectrometry (GC/MS) has been used for analysis of monosaccharides but the methods require derivatization and laborious sample preparation [13][14][15][16][17].…”

HPLC with pulsed amperometric detection for sorbitol as a biomarker for diabetic neuropathy

“…In this regard, our method will be useful for monitoring sorbitol in DM patients because it is more specific and sensitive compared to current methods [6,12].…”

Quantitative determination of endogenous sorbitol and fructose in human nerve tissues by atmospheric‐pressure chemical ionization liquid chromatography/tandem mass spectrometry

“…Analysis of monosaccharides by liquid chromatography (LC) is complicated by the fact that these compounds do not possess a UV chromophore; consequently, they are usually analyzed by derivatization before detection with UV 1, 2. Other detectors are also used to analyze neutral carbohydrates, such as evaporative light‐scattering detectors,3 refractive‐index4–6 and pulsed‐amperometric detectors,7–11 and fluorometric detectors 12. Gas chromatography/mass spectrometry (GC/MS) has been used for analysis of monosaccharides but the methods require derivatization and laborious sample preparation 13–17…”