Estimation of the hemoglobin glycation rate constant

ParticipantsOne hundred and seven subjects including 31 patients with hemolytic anemia and 76 subjects without anemia were included in this study. All samples were prepared and analyzed in accordance with the protocols approved by the institutional committees at Kumamoto University and other collaborating institutions.Patients with hemolytic anemia were recruited from 115 patients who were older than 20 years old and required laboratory tests including complete blood counts and reticulocyte counts (Ret) for clinical reasons. Those who were suspected of having diabetes mellitus (DM) based on history, a low 1,5-Anhydroglucitol (1,5-AG) value (male, < 14.9 μg/mL; female, < 12.4 μg/mL), or had comorbid liver or renal diseases, were excluded, as liver and renal diseases affect HbA1c and GA. EC, HbA1c, GA, haptoglobin, and other biochemical screening items were measured using the existing plasma samples from these patients. Use of existing plasma samples from anemic patients without written consent was approved by the institutional review board.Participants without anemia were recruited from medical examination checkup recipients at Takagi Hospital. Those who had anemia, DM, liver disease, renal disease or who were pregnant were excluded to avoid confounding effects on HbA1c or GA value. We provided the healthy volunteers with detailed information about the study, and all participants without anemia provided written informed consent to participate.Data interpretationEC was measured enzymatically in accordance with a previous report4, HbA1c was measured by high performance liquid chromatography (HPLC) method15, and GA was measured by enzymatic method using albumin-specific protenase, ketoamine oxidase, and albumin assay reagent (Lucica GA-L; Asahi Kasei Pharma Co., Tokyo, Japan)16.HbA1c expressed in International Federation of Clinical Chemistry (IFCC) units (iA1c) was used for calculations in this study. While the National Glycohemoglobin Standardization Program (NGSP) is used to express HbA1c in many clinical research and medical care settings, NGSP is measured by an old standardized method and at the time of conception, HPLC was not able to distinguish true HbA1c from other products. HPLC technology later advanced, however the derived HbA1c value is adjusted to NGSP in the interest of consistency. IFCC provides a strict definition of iA1c as hemoglobin with a glycated valine in the N-terminal (beta )-chain. Thus, iA1c value is preferred value for estimation of hemoglobin glycation.To acquire iA1c from HbA1c expressed in NSGP unit, we used the following equation17:$$begin{aligned} mathrm{HbA1c}_{mathrm{NGSP}} , (%)= & {} 0.0915 times mathrm{iA1c} , (mathrm{mmol/mol}) + 2.153 (%) end{aligned}$$
$$begin{aligned} iff mathrm{iA1c} (mathrm{mmol/mol})= & {} 10.93 times mathrm{HbA1c}_{mathrm{NGSP}} , (% ) – 23.53 end{aligned}$$
(M_{RBC}) was acquired from EC by the aforementioned Eq. (3).An AG value of 100 mg/dL was substituted for plasma glucose values derived using CGM. This number was based on the average AG of non-diabetic participants and the previously reported findings from a study which showed the median AG in healthy subjects to be reported to be 101.0 (96.3–106.0) mg/dL18 and another ADAG (A1c-derived average glucose) study which found that the AG of the non-diabetic group of their study was similarly 100 mg/dL19,20.(M_{RBC}) was also determined using (^{51}hbox {Cr}) half-life. As the reference range for (^{51}hbox {Cr}) half-life was described as 28–30 days10, 30 ± 5 days11, and 26–40 days12, (M_{RBC}) was calculated by multiplying (^{51}hbox {Cr}) half-life and 2.14 (= 60/28), 60 days being the normal value for (M_{RBC}).Data analysisEC and (M_{RBC} ) data were analyzed using a spreadsheet software, Excel 365 (Microsoft Corporation, Redmond, WA, USA).Estimation of (k_g)
The following two methods were used to estimate (k_g). The slope method—the following Eq. (6) derived from Eq. (2) shows that the slope of the line connecting a point and the origin is (k_g mathrm{AG}).$$begin{aligned} frac{mathrm{iA1c}}{1000 – frac{2}{3} {mathrm{iA1c}} } = k_g mathrm{AG} times M_{RBC} end{aligned}$$
Estimating the slope of the regression line through the origin by the least square model:$$begin{aligned} frac{sum _i^n x_iy_i }{sum _i^n x_i^2 } end{aligned}$$
where (x_i), (y_i) are (M_{RBC}) and (frac{mathrm{iA1c}}{1000- frac{2}{3}{mathrm{iA1c}}}) of each participant, respectively.The direct method—the (k_g) of each participant was calculated by the following equation:$$begin{aligned} k_g = frac{mathrm{iA1c}}{(1000 – frac{2}{3} {mathrm{iA1c}}) M_{RBC} {mathrm{AG} }} end{aligned}$$
Then, average and standard deviation of each (k_g) was calculated.Confirmation of derived (k_g)
The method of obtaining (M_{RBC}) from AG and iA1c was applied to data from three patients with latent hemolysis who were presented in a previous case studies10,11,12.Data of Herranz10 and Ishii11 showed changes in HbA1c during the course of the study. Therefore, (M_{RBC}) was calculated separately for each period. For the Ishii case11, AG was calculated by averaging self-monitoring of blood glucose (SMBG) data for each period. The Hiratani study12 examined (^{51}hbox {Cr}) erythrocyte lifespan measurement during hospitalization in Oct 1999 and CGM in Feb 2016. While HbA1c and plasma glucose concentrations fluctuate routinely, RBC lifespan remain comparatively constant, especially when influenced by a certain diseases (stomatocytosis). Furthermore, supply of (^{51}hbox {Cr}) was ceased in Japan in 2015 and thus it can no longer be used to study erythrocyte lifespan.Ethical approval and consent to participateThe work was conducted in accordance with Ethical Guidelines for Medical and Health Research Involving Human Subjects in Japan and conformed to the Helsinki Declaration. All samples were prepared and analyzed in accordance with the protocols approved by the institutional committees at Kumamoto University and other collaborating institutions.

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