The Wassner's method (1980) for determination of Nτ-methylhistidine (τ-Meh) was revised. This method was applied to analyse τ-Meh in rat plasma and urine. Human plasma and urine were deproteinized with sulfosalicilic acid. Further hydrolysis (with 6N HCl, 100°C for 1 hr) was necessary in rat plasma and urine. Fluorescamine was added to the deproteinized samples and heated with perchloric acid according to the method of Nakamura and Pisano (1976). Reaction mixture (25-100 μl) was injected into HPLC equipped with Zorbax BP-ODS column (∅ 4 mm x 25 cm) and fluorometer. Solvent was 27% acetonitril-10 mM phosphate buffer, pH 7.5. Only two peaks of histidine and τ-Meh was detected. τ-Meh was eluted within 10 min. Regeneration of the column was not necessary. Linearity in standard curve from 33 pmol to 8.5 nmol of τ-Meh was observed. The sensitivity and separation were enough to estimate τ-Meh level in plasma and urine.
Eight healthy subjects (average age 27. 5 years old) were orally administered 200 ml of the control solution (containing 50 g of glucose) or the test solutions (containing 50 g of glucose and 5 g of glucomannan or cellulose), and compared with the suppressive effect on the enhancement of blood glucose level. Glucomannan delayed the intestinal absorption of glucose and could significantly suppress the maximal level of blood glucose, but cellulose could not. Addition of pullulan of 5 or 10 g to glucose solution also did not show the suppressive effect on blood glucose level.