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A simple colorimetric assay for measuring fructosamine 3 kinase activity

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Abstract
Background: Fructosamine 3 kinase (FN3K) is a deglycating enzyme, which may play a key role in reducing diabetes- induced organ damage by removing bound glucose from glycated proteins. We wanted to develop a simple colorimetric method for assaying FN3K activity in human body fluids. Methods: Glycated bovine serum albumin (BSA) was obtained by glycation with a 10% glucose solution at 37 degrees C. After 72 h, glycated BSA was dialyzed against phosphate buffered saline (0.1 mol/L, pH 7.4). The dialyzed solution (containing +/- 1000 mu mol/L fructosamine) was used as an FN3K substrate. In the assay, 300 mu L of substrate was incubated with 50 mu L of serum and 100 mu L of MgCl 2 (0.7 mmol/L)/ATP (3.2 mmol/L). The fructosamine concentration was determined at the start and after incubation (120 min, 25 degrees C). The decrease in fructosamine concentration over time is a measure for the FN3K activity (1 U corresponding to 1 mu mol/min). Concomitantly, the FN3K SNP rs1056534 and the ferroportin SNP rs1156350 were genotyped. Results: Within-assay CV was 6.0%. Reference values for FN3K activity in serum were 14.2 +/- 1.6 U/L (n = 143). Reference values for FN3K were neither age-nor sex-dependent. The various FN3K SNP rs1056534 genotypes showed no significant differences in serum FN3K activity. In diabetics (n = 191), values (14.0 +/- 2.2 U/L) were comparable to those of the controls. FN3K activity in erythrocytes was significantly higher (170.3 +/- 7.6 U/L). The intra-erythrocytic FN3K activity makes the results prone to hemolysis. FN3K activity depended on the ferroportin Q248H genotypes, with the highest value for the wild type genotype. Neither transferrin saturation nor ferritin were confounders for the FN3K activity. FN3K activity was significantly (p < 0.0001) correlated with HbA(1c) values, although the correlation between FN3K and HbA(1c) was weak. Conclusions: The simple colorimetric method allows determining FN3K activity in human serum. The assay may be useful for studying the impact of deglycation processes in diabetes mellitus.
Keywords
diabetes mellitus, ferroportin Q248H mutation, fructosamine-3 kinase 900C/G polymorphism, fructosamine-3-kinase, glycation gap, CLINICAL-CHEMISTRY, GLYCOSYLATION GAP, DIABETIC-PATIENTS, SERUM FERRITIN, IRON OVERLOAD, GLYCATION GAP, 3-KINASE, PRODUCTS, HEMOCHROMATOSIS, POLYMORPHISMS

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Chicago
Cikomola, Justin C, Antoine S Kishabongo, Karl Vandepoele, Marieke De Mulder, Philippe B Katchunga, Bram Laukens, Loes van Schie, et al. 2017. “A Simple Colorimetric Assay for Measuring Fructosamine 3 Kinase Activity.” Clinical Chemistry and Laboratory Medicine 55 (1): 154–163.
APA
Cikomola, J. C., Kishabongo, A. S., Vandepoele, K., De Mulder, M., Katchunga, P. B., Laukens, B., van Schie, L., et al. (2017). A simple colorimetric assay for measuring fructosamine 3 kinase activity. CLINICAL CHEMISTRY AND LABORATORY MEDICINE, 55(1), 154–163.
Vancouver
1.
Cikomola JC, Kishabongo AS, Vandepoele K, De Mulder M, Katchunga PB, Laukens B, et al. A simple colorimetric assay for measuring fructosamine 3 kinase activity. CLINICAL CHEMISTRY AND LABORATORY MEDICINE. 2017;55(1):154–63.
MLA
Cikomola, Justin C, Antoine S Kishabongo, Karl Vandepoele, et al. “A Simple Colorimetric Assay for Measuring Fructosamine 3 Kinase Activity.” CLINICAL CHEMISTRY AND LABORATORY MEDICINE 55.1 (2017): 154–163. Print.
@article{8503194,
  abstract     = {Background: Fructosamine 3 kinase (FN3K) is a deglycating enzyme, which may play a key role in reducing diabetes- induced organ damage by removing bound glucose from glycated proteins. We wanted to develop a simple colorimetric method for assaying FN3K activity in human body fluids. 
Methods: Glycated bovine serum albumin (BSA) was obtained by glycation with a 10% glucose solution at 37 degrees C. After 72 h, glycated BSA was dialyzed against phosphate buffered saline (0.1 mol/L, pH 7.4). The dialyzed solution (containing +/- 1000 mu mol/L fructosamine) was used as an FN3K substrate. In the assay, 300 mu L of substrate was incubated with 50 mu L of serum and 100 mu L of MgCl 2 (0.7 mmol/L)/ATP (3.2 mmol/L). The fructosamine concentration was determined at the start and after incubation (120 min, 25 degrees C). The decrease in fructosamine concentration over time is a measure for the FN3K activity (1 U corresponding to 1 mu mol/min). Concomitantly, the FN3K SNP rs1056534 and the ferroportin SNP rs1156350 were genotyped. 
Results: Within-assay CV was 6.0%. Reference values for FN3K activity in serum were 14.2 +/- 1.6 U/L (n = 143). Reference values for FN3K were neither age-nor sex-dependent. The various FN3K SNP rs1056534 genotypes showed no significant differences in serum FN3K activity. In diabetics (n = 191), values (14.0 +/- 2.2 U/L) were comparable to those of the controls. FN3K activity in erythrocytes was significantly higher (170.3 +/- 7.6 U/L). The intra-erythrocytic FN3K activity makes the results prone to hemolysis. FN3K activity depended on the ferroportin Q248H genotypes, with the highest value for the wild type genotype. Neither transferrin saturation nor ferritin were confounders for the FN3K activity. FN3K activity was significantly (p < 0.0001) correlated with HbA(1c) values, although the correlation between FN3K and HbA(1c) was weak. 
Conclusions: The simple colorimetric method allows determining FN3K activity in human serum. The assay may be useful for studying the impact of deglycation processes in diabetes mellitus.},
  author       = {Cikomola, Justin C and Kishabongo, Antoine S and Vandepoele, Karl and De Mulder, Marieke and Katchunga, Philippe B and Laukens, Bram and van Schie, Loes and Grootaert, Hendrik and Callewaert, Nico and Speeckaert, Marijn and Delanghe, Joris},
  issn         = {1434-6621},
  journal      = {CLINICAL CHEMISTRY AND LABORATORY MEDICINE},
  keywords     = {diabetes mellitus,ferroportin Q248H mutation,fructosamine-3 kinase 900C/G polymorphism,fructosamine-3-kinase,glycation gap,CLINICAL-CHEMISTRY,GLYCOSYLATION GAP,DIABETIC-PATIENTS,SERUM FERRITIN,IRON OVERLOAD,GLYCATION GAP,3-KINASE,PRODUCTS,HEMOCHROMATOSIS,POLYMORPHISMS},
  language     = {eng},
  number       = {1},
  pages        = {154--163},
  title        = {A simple colorimetric assay for measuring fructosamine 3 kinase activity},
  url          = {http://dx.doi.org/10.1515/cclm-2016-0441},
  volume       = {55},
  year         = {2017},
}

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