Development of glycated peptide enzyme sensor based flow injection analysis system for haemoglobin A1c monitoring using quasi-direct electron transfer type engineered fructosyl peptide oxidase.

Haemoglobin A1c (hemoglobin A1c, HbA1c) is an important long-term glycemic control marker for diabetes. The aim of this study was to develop an enzyme flow injection analysis (FIA) system using engineered fructosyl peptide oxidase (FPOx) based on 2.5th generation principle for an HbA1c automated analytical system. FPOx from Phaeosphaeria nodorum (PnFPOx) was engineered by introducing a Lys residue at the R414 position, to be modified with amine reactive phenazine ethosulfate (arPES) in proximity of FAD. The engineered PnFPOx mutant with minimized oxidase activity, N56A/R414K, showed quasi-direct electron transfer (quasi-DET) ability after PES-modification. The FIA system was constructed by employing a PES-modified PnFPOx N56A/R414K and operated at 0 V against Ag/AgCl. The system showed reproducible responses with a linear range of 20-500 µM for both fructosyl valine (FV) and fructosyl valylhistidine (FVH), with sensitivities of 0.49 nA µM-1 and 0.13 nA µM-1, and the detection limits of 1.3 µM and 2.0 µM for FV and FVH, respectively. These results indicate that the enzyme electrochemical FIA system covers the clinical range of HbA1c detection for more 200 consecutive measurements. Protease digested three different levels of HbA1c samples including healthy and diabetic range subjects were also measured with the FIA system. Thus, it will be possible to develop an integrated system consisting of sample pretreatment and sample electrochemical measurement based on an FIA system possessing quasi-DET type PnFPOx.

Use of fructosyl peptide oxidase for HbA1c assay.

ARKRAY, Inc developed the world's first automatic glycohemoglobin analyzer based on HPLC (1981). After that, ARKRAY developed enzymatic HbA1c assay "CinQ HbA1c" with the spread and diversification of HbA1c measurement (2007). CinQ HbA1c is the kit of Clinical Chemistry Analyzer, which uses fructosyl peptide oxidase (FPOX) for a measurement reaction. This report mainly indicates the developmental background, measurement principle, and future of the enzymatic method HbA1c reagent.

Determination of glycated hemoglobin in clinically silent hemoglobin variants.

BACKGROUND: Evaluation of glycated hemoglobin determination methods in patients with clinically silent hemoglobin variants. METHODS: HbA1c results were determined with various methods, including a new enzymatic assay, a boronate affinity HPLC, immunoassays and ion-exchange HPLC in patients with the clinically silent hemoglobin variants Hb Graz, Hb Sherwood Forest, Hb D and Hb O Padova. RESULTS: The effect of hemoglobin variants on glycated hemoglobin determination was method-dependent. The enzymatic and boronate affinity HPLC method did not interfere with any of the variants evaluated. In contrast, Hb Graz interfered with all immunoassay and ion-exchange HPLC methods evaluated. The Tosoh ion-exchange HPLC method HLC-723 did not detect the late migrating Hb O Padova in the chromatogram, but this hemoglobin variant still interfered causing artificially low HbA1c results. CONCLUSIONS: Our study underscores the need for clinical laboratories and physicians to be aware of the limitations of their HbA1c assay methods as well as the importance of visual inspection of ion-exchange chromatograms to detect abnormalities caused by the hemoglobin variants. Samples with clinically silent Hb variants should be analyzed by a second method with a different assay principle, preferably a boronate affinity HPLC or an enzymatic assay.

New enzymatic assay for glycohemoglobin.

BACKGROUND: Previous methods to measure glycohemoglobin (GHb) have been time-consuming or imprecise; we therefore developed a new enzymatic assay for GHb. METHODS: Blood cells were first hemolyzed, and hemoglobin was digested with protease to yield fructosyl amino acid. Fructosyl amino acid oxidase acts on the fructosyl amino acid and generates hydrogen peroxide, which reacts with chromogens in the presence of peroxidase. Total hemoglobin was measured spectrometrically in the same reaction tube. The results were reported as the ratio of the concentrations of GHb and hemoglobin. RESULTS: The measured values were comparable to those determined with a HPLC method and with an immunoassay in blood samples from 2854 patients with diabetes. Regression analysis for the enzymatic assay (y) vs the HPLC method (x) produced the following: r = 0.979; slope, 0.994 [95% confidence interval (CI), 0.986-1.001]; y-intercept, 0.04% (95% CI, -0.09% to 0.01%); n = 2854. For the enzymatic assay (y) vs the immunoassay (x), the regression statistics were as follows: r = 0.982; slope, 1.002 (95% CI, 0.995-1.009); y-intercept, 0% (95% CI, -0.05% to 0.05%); n = 2854. CONCLUSIONS: The values measured by the new enzymatic assay are sufficiently correlated with those of the conventional HPLC method and immunoassay, but the proposed assay for GHb is rapid and has high precision.