Improvement of intradialytic hypotension in diabetic hemodialysis patients using vitamin E-bonded polysulfone membrane dialyzers.

Currently, there are no detailed reports on the effects of vitamin E-bonded polysulfone (PS) membrane dialyzers on intradialytic hypotension (IDH) in diabetic hemodialysis (HD) patients. This study was designed to evaluate changes in intradialytic systolic blood pressure (SBP) using "VPS-HA" vitamin E-bonded super high-flux PS membrane dialyzers. The subjects were 62 diabetic HD patients whose intradialytic SBP fell by more than 20%. Group A comprised patients who required vasopressors to be able to continue treatment or who had to discontinue therapy due to their lowest intradialytic SBP being observed at 210 min (28 patients). Group B comprised patients who showed no symptoms and required no vasopressors but showed a gradual reduction in blood pressure, with the lowest intradialytic SBP seen at the end of dialysis (34 patients). The primary outcome was defined as the lowest intradialytic SBP after 3 months using VPS-HA. Secondary outcomes included changes in the following: lowest intradialytic diastolic blood pressure, pulse pressure, pulse rate, plasma nitric oxide and peroxynitrite, serum albumin, and hemoglobin A1c. Group A's lowest intradialytic SBP had significantly improved at 3 months (128.0 ± 25.1 mm Hg vs. 117.1 ± 29.2 mm Hg; P = 0.017). Group B's lowest intradialytic SBP had significantly improved at 1 month (134.4 ± 13.2 mm Hg vs. 121.5 ± 25.8 mm Hg; P = 0.047) and 3 months (139.1 ± 20.9 mm Hg vs. 121.5 ± 25.8 mm Hg; P = 0.011). We conclude that VPS-HA may improve IDH in diabetic HD patients.

Structural and glycation site changes of albumin in diabetic patient with very high glycated albumin.

BACKGROUND: Glycated albumin (GA) has been utilized to monitor mid-term glycemic control, and reflects the status of blood glucose more rapidly and effectively than hemoglobin A(1c) (HbA(1c)). To examine the relationship between GA level and structural changes or glycation sites of albumin, we analyzed pre- and post-treatment samples from a diabetic patient with extraordinary increase of GA. METHOD: A female diabetic patient with poor glycemic control had a GA >94% and was treated with intensive insulin therapy to decrease blood glucose. We analyzed changes in fluorescence derived from tryptophan (Trp) and advanced glycation end product (AGE) of albumin isolated/purified from pre- and post-treatment samples. To determine the sites of glycation of albumin, samples were carboxymethylated and digested by Glu-C endoprotease, and peptides were analyzed using liquid chromatography/mass spectrometry. RESULTS: GA level decreased almost linearly and reflected the improved glycemic state well. Trp-related fluorescence of pre- and post-treated samples did not change while AGE-related fluorescence increased depending on GA level. Ten major glycation sites were detected in the pre-treatment sample, while 3 major glycation sites were detected in post-treated samples. CONCLUSIONS: GA level reflects the status of blood glucose more rapidly than HbA(1c). Since GA level was related to AGE-related fluorescence and number of glycation sites, it might be a good marker for not only glycemic control of diabetic patients but also structural and functional changes of albumin.

Alteration of substrate specificity of fructosyl-amino acid oxidase from Ulocladium sp. JS-103.

We showed by random mutagenesis that one-amino-acid substitution at Arg94 in fructosyl-amino acid oxidase from Ulocladium sp. JS-103 enhanced substrate specificity toward fructosyl valine (FV), a model compound of hemoglobin A(1c). Kinetic analysis showed that the specificity of the R94W mutant enzyme toward FV was 14-fold that of the wild-type enzyme. The mutant enzyme obtained will be useful in developing an enzymatic measurement method for hemoglobin A(1c).

Point of care testing system via enzymatic method for the rapid, efficient assay of glycated albumin.

The ratio of glycated albumin to albumin concentration in serum is termed the glycated albumin (GA) value. The GA value provides a time-averaged index of the state of glycemic control for the previous 2 weeks. In this study, a dry chemistry system (GA monitor) via an enzymatic method was proposed in order to provide a GA value measurement for point of care testing (POCT). The GA monitor was made from three devices a set of test-tapes, a test-strip and an optical analyzer. A GA test-tape, a ketoamine test-tape and an albumin test-tape were enclosed in the fabricated test-strip. Time-course changes of the optical characteristics were evaluated using the test-strip. It was found that the three test tapes must be enclosed in the test-strip to create a dry chemistry system for small sample volumes (20 microl). A temperature control unit, which could hold the temperature of the GA test-tape at 45 degrees C and at 25 degrees C for the other two types of test-tape was incorporated into the optical analyzer. With the GA test-tape held separately and controlled at 45 degrees C, the analytical time decreased to one-third of the time taken for the three tapes at 25 degrees C. The analytical accuracy of the three types of test-tape showed favorable results, with R2 values of 0.96-0.98 and coefficients of variation (CV) of 2.4-6.8%. Compared with a commercially available liquid chemistry system, the analytical accuracy of the GA monitor exhibited a relatively favorable linearity of R=0.82. According to these results, a new GA value analytical system was realized, in which the GA value could be assayed within five minutes using only 20 microl of blood sample with a disposable test-strip. This system could potentially be used for clinical purposes as test equipment for rapid and efficient POCT.

Study of glycated amino acid elimination reaction for an improved enzymatic glycated albumin measurement method.

BACKGROUND: In order to improve enzymatic glycated albumin measurement, we studied the endogenous glycated amino acid elimination reaction and the new bromocresolpurple (BCP) method for albumin measurement. METHODS: In the assay, endogenous glycated amino acids are first eliminated by oxidation by ketoamine oxidase. Second, glycated albumin is hydrolyzed to glycated amino acids by proteinase digestion, and glycated amino acids are oxidized to produce hydrogen peroxide, which is quantitatively measured. Third, albumin is measured by the new BCP method. Finally, glycated albumin value is calculated as the percentage of glycated albumin in total albumin. RESULTS: Glycated amino acid concentrations in prepared total parenteral nutrition products were increased in direct proportion to storage time and temperature. The glycated amino acid elimination reaction using ketoamine oxidase may be able to eliminate more than 15 mmol/l glycated amino acids. The glycated albumin values of samples calculated from the albumin concentrations using the new BCP method accorded with those calculated with the HPLC method. Fundamental performances (linearity, dilution test, analytical recovery, within-run and between-run CVs, interference study) of the present method were good. Detection of glycated albumin by the present method was significantly correlated with detection of glycated albumin by the high-performance liquid chromatography method (r(p) = 0.995). CONCLUSIONS: This new improved method is free of interference by endogenous glycated amino acids and is unaffected by albumin concentration, and enables more accurate analysis of glycated albumin.

An enzymatic method for the measurement of glycated albumin in biological samples.

BACKGROUND: In order to determine glycated albumin more easily and rapidly, we developed a new enzymatic method for glycated albumin in blood samples. METHODS: The method involves use of albumin-specific proteinase, ketoamine oxidase and serum albumin assay reagent. In the assay, glycated albumin is hydrolyzed to glycated amino acids by proteinase digestion, and ketoamine oxidase oxidizes the glycated amino acids to produce hydrogen peroxide, which is quantitatively measured. Glycated albumin is calculated as the percentage of glycated albumin in total albumin. RESULTS: The calibration curve for glycated albumin concentration was linear (r(p)=0.999) between 0.0 and 50.0 g/l and that for albumin concentration was linear (r(p)=0.999) between 0.0 and 60.0 g/l. The analytical recoveries of exogenous glycated albumin added to serum were 100-102.5%. The within-run and between-run CVs were 0.45-0.67% and 1.09-1.26%, respectively. This method was free from interference by bilirubin, chyle, glucose, globulins and labile intermediate. Weak interference by hemoglobin and ascorbic acid was observed. Glycated albumin detected by the present method was significantly correlated with glycated albumin detected by high-performance liquid-chromatographic (HPLC) method (serum: r(s)=0.989, plasma: r(p)=0.992). CONCLUSIONS: This new enzymatic method is simple, rapid, allows multiple determinations and enables quantitative analysis of glycated albumin.

Determination of urinary myo-inositol concentration by an improved enzymatic cycling method using myo-inositol dehydrogenase from Flavobacterium sp.

BACKGROUND: To determine myo-inositol more accurately, we improved the enzymatic cycling method. METHODS: We screened myo-inositol dehydrogenase (MIDH; EC.1.1.1.18) from Flavobacterium sp., which was highly specific to myo-inositol. We measured urinary myo-inositol/creatinine ratio 2 h after 75-g oral glucose tolerance test (2 h MI) of 71 volunteers, and investigated the relationship between diabetes and urinary myo-inositol concentration. RESULTS: The calibration curve was linear (r = 1.00) up to 2000 micromol/l, and the detection limit was 10 micromol/l. Within-run and between-run CVs were 0.5-1.1% and 0.4-1.3%, respectively. The 2 h MI of impaired fasting glycemia (IFG; 65.1 +/- 46.6 mg/g Cr, P < 0.005), impaired glucose tolerance (IGT; 85.0 +/- 73.7 mg/g Cr, P < 0.001) and diabetes (163.4 +/- 73.7 mg/g Cr, P < 0.0001) increased significantly compared with that of normal glucose tolerance (NGT; 24.0 +/- 14.4 mg/g Cr). From receiver operating characteristic analyses on 2 h MI, with 50 mg/g Cr as a tentative cutoff value to detect diabetes, the sensitivity and specificity were 100% and 77%, respectively. With 40 mg/g Cr as a tentative cutoff value to detect NGT, the sensitivity and specificity were 74% and 85%, respectively. CONCLUSIONS: The myo-inositol measurement method demonstrated high specificity and yielded accurate results. The results of clinical trials suggested that 2 h MI could not only determine diabetes but also distinguish IFG and IGT from NGT.

Alteration of substrate specificity of fructosyl-amino acid oxidase from Fusarium oxysporum.

Fructosyl-amino acid oxidase (FOD-F) from Fusarium oxysporum f. sp. raphani (NBRC 9972) is the enzyme catalyzing the oxidative deglycation of fructosyl-amino acids such as N(epsilon)-fructosyl N(alpha)-benzyloxycarbonyl-lysine (FZK) and fructosyl valine (FV), which are model compounds of the glycated proteins in blood. Wild-type FOD-F has high activities toward both substrates. We obtained a mutant FOD-F, which reacts with FZK but not with FV by random mutagenesis. One amino-acid substitution (K373R) occurred in the mutant FOD-F. In addition to K373R, K373W, K373M, K373T, and K373V, which were selected for optimization of the substitution at position K373, were purified and characterized. Kinetic analysis showed that the catalytic turnover for FV greatly decreased, whereas that for FZK did not. In consequence, the specificities toward FZK were increased in the mutant FOD-Fs. The relation between the substrate specificity of the mutant FOD-Fs and the position of the carboxyl group of the substrates was demonstrated using a series of the substrates having the carboxyl group at the different position. The mutant FOD-Fs are attractive candidates for developing an enzymatic measurement method for glycated proteins such as glycated albumin in serum. This study will be helpful to establish an easier and rapid clinical assay system of glycated albumin.